mouse cd4 t cell isolation kit  (Miltenyi Biotec)

Journal: Materials Today Bio

Article Title: Targeting FN1 to overcome gemcitabine resistance in gallbladder cancer: Mechanistic insights and an iRGD-modified PEG-PLGA nanoparticle delivery strategy

doi: 10.1016/j.mtbio.2026.102877

Figure Lengend Snippet: Effect of FN1 knockdown on the immunosuppressive microenvironment in GBC-SD/GEM cells. Note: (A) Schematic of immunosuppressive cells and cytokines detection in tumor tissues; (B) FCM analysis of Tregs infiltration levels in GBC-SD/GEM cell xenograft tissues in various mouse groups; (C) FCM analysis of M2 and M1 macrophage infiltration levels in GBC-SD/GEM cell xenograft tissues from different mouse groups; (D-E) RT-qPCR analysis of immunosuppressive factors expression in GBC-SD/GEM cell xenograft tissues from various mouse groups; (F) Schematic of in vitro co-culture of GEM-resistant GBC cells with THP-1 and CD4 + T cells; (G) FCM analysis of Tregs levels in CD4 + T cells after co-culture with GBC-SD/GEM cells; (H) FCM analysis of M2 and M1 macrophage levels in THP-1 cells post co-culture with GBC-SD/GEM cells; (I) RT-qPCR analysis of IL-10 or CSF-1 expression in CD4 + T or THP-1 cells after co-culture with GBC-SD/GEM cells. In B-E, ∗ indicates p < 0.05 compared to the sh-NC + GEM group, each group consisting of 6 mice; in G-I, ∗ indicates p < 0.05 compared to the sh-NC group, # indicates p < 0.05 compared to the oe-NC group, experiments repeated three times.

Article Snippet: Human CD4 + T cells were isolated and enriched from mature lymphocytes (hPBMCs, PCS-800-011, ATCC, USA) using a Human CD4 + T Cell Enrichment Kit (8804-6814-74, Thermo Fisher Scientific, USA). hPBMCs were incubated with the enrichment reagents for 30 min to capture CD4 + T cells.

Techniques: Knockdown, Quantitative RT-PCR, Expressing, In Vitro, Co-Culture Assay

Journal: Materials Today Bio

Article Title: Targeting FN1 to overcome gemcitabine resistance in gallbladder cancer: Mechanistic insights and an iRGD-modified PEG-PLGA nanoparticle delivery strategy

doi: 10.1016/j.mtbio.2026.102877

Figure Lengend Snippet: Impact of NPs delivering si-FN1 on drug resistance and immune cell infiltration in GBC-SD/GEM cells. Note: (A) Schematic of the experimental setup for studying the impact of NPs delivering si-FN1 on GBC GEM resistance; (B-C) RT-qPCR (B) and Western Blot (C) analysis of FN1 and PI3K pathway protein expression in GBC-SD/GEM cells treated with NPs (si-FN1) and iRGD-NPs (si-FN1); (D) CCK-8 assay assessing the viability changes in GBC-SD/GEM cells after treatment with NPs (si-FN1) and iRGD-NPs (si-FN1); (E) Clonogenic assay evaluating colony formation in various groups of GBC-SD/GEM and NOZ/GEM cells; (F) FCM analysis of apoptosis in GBC-SD/GEM cells across different groups; (G) FCM analysis of Tregs levels in CD4 + T cells after co-culture with GBC-SD/GEM cells; (H) FCM analysis of M2 and M1 macrophage levels in THP-1 cells after co-culture with GBC-SD/GEM cells; (I) RT-qPCR analysis of IL-10 or CSF-1 expression in CD4 + T or THP-1 cells co-cultured with GBC-SD/GEM cells. ∗ indicates p < 0.05 compared to the NPs (si-NC) group, # indicates p < 0.05 compared to the NPs (si-FN1) group, experiments repeated three times.

Article Snippet: Human CD4 + T cells were isolated and enriched from mature lymphocytes (hPBMCs, PCS-800-011, ATCC, USA) using a Human CD4 + T Cell Enrichment Kit (8804-6814-74, Thermo Fisher Scientific, USA). hPBMCs were incubated with the enrichment reagents for 30 min to capture CD4 + T cells.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, Clonogenic Assay, Co-Culture Assay, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Activated PI3Kδ reshapes the HDM-induced immune response. (A–J) WT and Pik3cd E1020K/+ animals were sensitized intranasally with HDM extracts (200 μg on days 0 and 7; 50 μg on days 14, 16, and 18) and lungs examined on day 20. (A) Experimental outline. (B) H&E staining of paraffin-embedded lung sections. Top panel: Arrowheads indicate thickening of the alveolar septa; pound signs indicate iBALT. Bottom panel: Short arrows show lymphocytes, long arrows show macrophages, and yellow arrows show eosinophils. (C) CD4 T cell counts (liveCD45 + TCRβ + CD4 + CD8 − ) measured by flow cytometry. (D) Eosinophil numbers quantified from H&E-stained lung sections. (E) Neutrophil counts (liveLineage − CD11b + Ly6G + ) from lungs of the indicated animals. (F) Airway resistance (Rrs) was measured with a flexiVent instrument as cmH2O.s/ml using the indicated concentrations of methacholine. Data in E are representative of two independent experiments with n = 3–4 for each group per experiment. (G) UMAP showing clusters of lung CD45 + immune cells analyzed by scRNAseq from naïve WT, naïve Pik3cd E1020K/+ , HDM-treated WT, and HDM-treated Pik3cd E1020K/+ animals. Each cluster was assigned a cell type using SingleR and supervised analysis of gene expression specific to each cluster . Cells from three mice were analyzed per genotype and condition. (H) Individual lung CD45 + immune cells identified by scRNAseq analyzed for enrichment of response to IFNγ gene set. Left: UMAP visualization of enrichment P values in lung CD45 + immune cells from the indicated mice; P values described in color scale below. Right: Frequencies of cells with indicated magnitudes of enrichment P values. Statistical comparison of all CD45 + cells from WT and Pik3cd E1020K/+ HDM-treated groups was performed (chi-squared test), comparing frequencies of cells with P < 0.0005. (I) HDM-specific IgG2a, IgG3, IgE, and IgG1 from the indicated mice. (J) CCL3, CCL5, and CXCL10 (pg/ml) measured from lung homogenates from the indicated mice by Luminex. Data in C–E, I, and J are from n = 6–10 mice for each group, pooled from two independent experiments. Data in B are representative of two independent experiments. Unless otherwise indicated, statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Staining, Flow Cytometry, Gene Expression, Comparison, Luminex

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Aberrant Th1 responses at the expense of Th2 immunity in Pik3cd E1020K/+ lungs following HDM sensitization. (A) Scatter plot comparing gene expression in CD4 T cells from WT and Pik3cd E1020K/+ HDM-treated mice (cluster 1 from ). DEGs upregulated in WT CD4 T cells in red, and genes upregulated in Pik3cd E1020K/+ CD4 T cells in blue. (B) CD4 + T cells from scRNAseq of total CD45 + lung immune cells (cluster 1 from ) were re-clustered to identify five unique clusters (0–4) of CD4 + T cells. Left: UMAP showing distribution of clusters 0–4; identities of each cluster were assigned based on cluster-specific gene expression. Right: Proportion of cells from each cluster in indicated mice. (C) Seurat heatmap showing expression of cluster-defining genes for indicated populations. (A–C) Cells from three mice were analyzed per genotype and condition. (D) Representative flow cytometry plots of intracellular IFNγ and IL-5 expression in lung CD4 + T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from indicated mice. (E–H) Frequencies of lung CD4 + T cells expressing (E) IL-5; (F) IL-4; (G) IFNγ; and (H) IL-2 from indicated groups. (D–H) n = 9–10 for each group, pooled from two independent experiments. (I–L) TCRα-deficient recipient mice were injected with 1 × 10 6 WT or Pik3cd E1020K/+ naïve CD4 T cells 14 days prior to HDM sensitization. HDM was administered intranasally as indicated. n = 9–10 for each group, pooled from two independent experiments. (I) Experimental design. (J) Cell counts of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from the indicated groups. (K) Frequencies of IFNγ + (left) and IL-4/5/13 + (right) lung CD4 T cells from the indicated groups. Frequencies of IL-4/5/13 + cells were calculated using Boolean (or) gating. (L) Cell counts of eosinophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G − SiglecF + ) from lungs of the indicated mice. Statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Gene Expression, Expressing, Flow Cytometry, Injection

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Altered CD4 + T cell differentiation in Pik3cd E1020K/+ mice in vivo . Supporting data for . (A) Feature plots showing expression of Th2-defining genes Il1rl1 , Il4 , IL5 , and Il13 in scRNAseq analyses of lung CD45 + immune cells from the indicated mice. Cells from three mice were analyzed by scRNAseq per genotype and condition. (B) Flow cytometry of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) measuring GATA3 expression in the indicated populations. n = 9–10 for each group, pooled from two independent experiments. Left: Representative plots showing GATA3 and CD4 expression. Right: Frequencies (left) and cell counts (right) of GATA3 + CD4 T cells in the indicated groups. (C–E) Frequencies of (C) IL-13 + (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ); (D) IL-17A + (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ), and (E) Foxp3 + (Treg) lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ) in the indicated groups, measured by intracellular staining and flow cytometry. n = 9–10 for each group, pooled from two independent experiments. (F and G) Cytokine concentrations (pg/ml) measured from lung homogenates from the indicated mice by Luminex. Th2 cytokines IL-4 and IL-5 are shown in F, and Th1 cytokines GM-CSF and TNFα are shown in G. n = 9–10 for each group, pooled from two independent experiments. (H–L) WT and Pik3cd E1020K/+ animals were infected intranasally with C. neoformans , and were sacrificed 10 days after infection, and lung tissue was harvested for analysis. n = 6 for each group, pooled from two independent experiments. (H) Experimental design. (I) Cell counts of CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ) from lungs of the indicated mice. (J) Flow cytometry of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) measuring GATA3 and Tbet expression in the indicated populations. Left: Representative flow cytometry plots. Right: Frequencies of Tbet + and GATA3 + CD4 T cells from the indicated groups. (K) Frequencies of IL-4/5/13 + (left), IFNγ + (middle), and IL-2 + (right) lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ) in the indicated groups, measured by intracellular staining and flow cytometry. Frequencies of IL-4/5/13 + cells were calculated using Boolean (or) gating. (L) Cell counts of eosinophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G − SiglecF + ) and neutrophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G + ) from lungs of the indicated mice. (M–O) Supplemental data for CD4 + T cell transfer experiments in . n = 9–10 mice for each group, pooled from two independent experiments. (M) Cell counts (left) and frequencies (right) of GATA3 + CD4 T cells from the indicated groups. (N) Representative flow cytometry plots showing IFNγ and IL-4 expression in lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from the indicated mice. (O) Cell counts of neutrophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G + ) from lungs of the indicated mice. Statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Cell Differentiation, In Vivo, Expressing, Flow Cytometry, Staining, Luminex, Infection

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Hyperactivated PI3Kδ disrupts Th2 lineage restriction. (A–E) Naïve CD4 T cells were activated with αCD3 + αCD28 in the presence of WT T-depleted APCs under Th2-polarizing conditions (IL-4 + αIL-12) for 72 h. (A) Left: Representative flow cytometry plots showing IL-4 and IL-13 staining in Th2-polarized live CD4 + cells. Right: Percentages of IL-4 + and IL-13 + cells from the indicated mice. n = 15 for each group, from 15 independent experiments. (B) Left: Representative flow cytometry histograms showing GATA3 expression in Th2-polarized live CD4 + cells from the indicated mice. Right: Fold GATA3 expression (MFI normalized to WT) in Th2-polarized live CD4 + cells from the indicated mice. n = 14 for each group, from 14 independent experiments. (C) Left: Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4 + cells. Right: Percentages of IFNγ + cells. (D) Left: Representative flow cytometry histograms showing Tbet expression in Th2 and WT control Th1-polarized live CD4 + cells. Right: Fold Tbet expression (MFI normalized to WT) in Th2-polarized live CD4 + cells from the indicated mice. (C and D) n = 14 for each group, from 14 independent experiments. (E) Percentages of IFNγ + (left) and IL-13 + (right) cells over a time course of Th2 differentiation (0, 24, 48, and 72 h). n = 10 for each group, from 10 independent experiments. (F) Bulk RNAseq of WT and Pik3cd E1020K/+ naïve CD4 T cells and in vitro polarized Th2 cells. n = 3 biological replicates for each group. Volcano plots showing DEGs in red (WT upregulated) and blue ( Pik3cd E1020K/+ upregulated); DEGs defined using fold change >1.5, P < 0.05. (G) Enrichment of hallmark pathways among DEGs comparing WT and Pik3cd E1020K/+ Th2 cells. (H) Time course of fold induction of pAKT(T308), pS6(S240/44), and pAKT(S473) in WT and Pik3cd E1020K/+ live CD4 + cells during Th2 differentiation (0, 24, 48, and 72 h), measured by flow cytometry. Fold induction calculated using MFIs of the indicated readouts normalized to the 0 time point of the corresponding genotype. n = 5–9 for each group, from five to nine independent experiments. Statistical comparisons were made using ratio paired t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Flow Cytometry, Staining, Expressing, Control, RNA sequencing, In Vitro

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Analyses of in vitro polarized Th2 cells. Supporting data for , , and . (A) WT and Pik3cd E1020K /+ naïve CD4 T cells were Th2-polarized in the presence or absence of an αIFNγ blocking antibody. Left: Percentages of IFNγ + cells from the indicated groups. Right: Fold Tbet expression (MFI normalized to control WT) in live CD4 + cells from the indicated groups. n = 6–8 for each group, from six to eight independent experiments. (B) Gene expression (RPKM) of Il4ra , Il12rb1 , and Il12rb2 in WT and Pik3cd E1020K /+ Th2-polarized cells. n = 3, bulk RNAseq. (C) Naïve CD4 T cells from the indicated mice were Th2-polarized for 24 h in the absence of inhibitors. At 24 h of culture, PI3Kδ inhibitor Cal101 (10 nM) or mTOR inhibitor rapamycin (200 nM) was added to polarizing media and cells were cultured for an additional 48 h. Representative flow cytometry plots showing IFNγ and IL-4 staining in live CD4 + cells. Data are representative of three independent experiments, n = 3. (D) GSEA comparing WT and Pik3cd E1020K/+ transcriptomes for expression of TFT gene sets. (E) Fold induction of Foxo1 expression in Th2-polarized live CD4 + cells, measured by flow cytometry and calculated by normalizing Foxo1 MFIs to the 0 time point of the corresponding genotype. n = 5 for each group, from five independent experiments. (F) Naïve CD4 T cells from the indicated mice were Th2-polarized in the presence or absence of αIFNγ blocking antibody, and pFoxo1(S256) was measured in live CD4 + cells from the indicated groups by flow cytometry. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 5 for each group, from five independent experiments. (G) Naïve CD4 T cells were nucleofected with Cas9-gRNA complexes containing NC or Foxo1 -targeting gRNAs and underwent Th2 polarization. Representative flow cytometry histogram showing Foxo1 expression from the indicated cells, one example from nine independent experiments. (H) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were transduced with GFP only–, Foxo1-WT– or Foxo1-AAA–encoding retroviruses under Th2-polarizing conditions. Top: Representative flow cytometry plots showing IFNγ and IL-4 expression in live GFP + CD4 + T cells cultured under Th2-polarizing conditions from the indicated groups. Bottom, percentages of IL-4 + (left), IL-13 + (middle), and IFNγ + (right) Th2-polarized liveCD4 GFP + T cells from the indicated groups. n = 6 for each group, from six independent experiments. (I) WT Naïve CD4 T cells were nucleofected with NC or Foxo1 -targeting gRNA-Cas9 complexes and polarized under Th2 conditions in the presence or absence of an IL-2 blocking antibody. Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + cells. Data are representative of two independent experiments, n = 5. (J) Comparison of transcriptomes from Foxo1 KO Treg cells and Pik3cd E1020K/+ Th2 cells. Genes upregulated (FC >1.5, P < 0.05) in Foxo1 KO Treg 36 (versus WT Treg) and Pik3cd E1020K/+ Th2 (versus WT Th2) were compared (left), and pathway analysis (Enrichr; ) was performed on common DEGs (right). (K–M) Supplemental data related to , transfer of Foxo1 gRNA-treated cells. (K) Cell counts of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from the indicated groups. (L) Frequencies of IL-4 + lung CD4 T cells from the indicated groups. (M) Cell counts of lung eosinophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G − SiglecF + ) from the indicated groups. n = 6–9 for each group, pooled from two independent experiments. Statistical comparisons were made using ratio paired t tests (A, B, E, F, and H) or unpaired t tests (K–M). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: In Vitro, Blocking Assay, Expressing, Control, Gene Expression, RNA sequencing, Cell Culture, Flow Cytometry, Staining, Transduction, Comparison, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Dysregulated IL-2 signaling rewires Th2 differentiation of Pik3cd E1020K /+ CD4 T cells. (A) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in Th2-polarized cells from the indicated mice. Right: Percentages of IL-2 + Th2 cells. n = 9 for each group, from nine independent experiments. (B, C, and E) Time course analysis (0, 24, 48, 72 h) of IL-2 production, and pSTAT5(Y694) and CD25 during Th2 polarization, measured by flow cytometry. n = 7–10 for each group, from 7–10 independent experiments. (B) Percentages of IL-2 + cells over time from the indicated mice. (C) Fold induction of pSTAT5(Y694) over time from the indicated mice. Fold induction was calculated using pSTAT5(Y694) MFIs normalized to the 0 time point of the corresponding genotype. (D) Schematic describing experiments shown in F and G. (E) Fold induction of CD25 expression from the indicated mice. Fold induction was calculated using CD25 MFIs normalized to the 0 time point of the corresponding genotype. (F and G) Th2-polarized CD4 T cells from WT and Pik3cd E1020K/+ were rested in serum-free media for 4 h and subsequently stimulated with hIL-2 over the indicated time course (0, 15, 60, 120 min). n = 4–5 for each group, from four to five independent experiments. (F) Fold induction of pSTAT5(Y694) over time from the indicated mice, measured by flow cytometry. (G) Fold induction of pS6(S240/44) over time from the indicated mice, measured by flow cytometry. Fold induction was calculated using MFIs (pSTAT5(Y694) or pS6(S240/44)) normalized to the 0 time point of the corresponding genotype. (H–J) Naïve CD4 T cells were Th2-polarized in the presence or absence of αIL-2 blocking antibody (20 μg/ml). (H) Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4 + cells. (I) Percentages of IL-4 + (left), IL-13 + (middle), and IFNγ + (right) cells from the indicated mice, in the presence or absence of αIL-2. n = 11 for each group, from 11 independent experiments. (J) Left: Representative flow cytometry plots showing pAKT(T308) in Th2-polarized live CD4 + cells in the presence or absence of αIL-2. Right: Fold induction of pAKT(T308) in Th2-polarized live CD4 + cells from the indicated groups. Fold induction was calculated by normalizing pAKT(T308) MFIs to WT control cells. n = 5 for each group, from five independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Flow Cytometry, Expressing, Blocking Assay, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Inactivation of Foxo1 in Pik3cd E1020K/+ CD4 + T cells impairs Th2 lineage restriction. (A) Pathway enrichment of TF perturbations followed by expression gene sets performed using Enrichr : significantly enriched gene sets colored in blue. Genes upregulated in HDM-treated Pik3cd E1020K/+ CD4 T cells relative to WT counterparts were used as input for pathway enrichment. (B) Time course (0, 24, 48, 72 h) of pFoxo1(S256) in Th2-polarized live CD4 + cells from indicated mice. Left: Representative flow cytometry plots. Right: Fold induction of pFoxo1(S256) over time. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to the 0 time point of the corresponding genotype. n = 8 for each group, from eight independent experiments. (C) GSEA comparing Th2-polarized WT and Pik3cd E1020K/+ transcriptomes for the expression of Foxo1-activated (left) and Foxo1-repressed (right) gene sets. (D) Gene expression heatmap (row z-score) showing normalized RPKM values of leading edge genes from Foxo1-activated and Foxo1-repressed GSEA described in . (E) Naïve CD4 + T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody. Left: Representative flow cytometry plots showing pFoxo1(S256). Right: Fold induction of pFoxo1(S256). Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 10 for each group, from 10 independent experiments. (F) GSEA comparing control and αIL-2–treated Th2-polarized CD4 T cell transcriptomes for the expression of Foxo1-activated (top) and Foxo1-repressed (bottom) gene sets in the indicated groups. (G and H) Naïve CD4 T cells were nucleofected with gRNA-Cas9 complexes containing NC or Foxo1 -targeting gRNAs and differentiated under Th2 conditions. n = 9 for each group, from nine independent experiments. (G) Left: Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + T cells. Right: Percentages of IFNγ + and IL-4 + cells in Th2-polarized cells. (H) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in live CD4 + T cells. Right: Percentages of IL-2 + cells in Th2-polarized cells from the indicated groups. (I and J) TCRα-deficient recipient mice were injected with 1 × 10 6 NC or Foxo1 gRNA-Cas9–nucleofected naïve CD4 T cells 14 days prior to HDM sensitization. n = 6–9 for each group, pooled from two independent experiments. (I) Experimental design. (J) Frequencies of IFNγ + (left) and IL-2 + (right) lung CD4 T cells. Statistical comparisons used ratio paired t tests (B and E–G) or unpaired t tests (I). **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Expressing, Flow Cytometry, Gene Expression, Blocking Assay, Control, Injection, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Pik3cd E1020K reshapes the epigenetic landscape of CD4 + T cells. (A and B) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were polarized under Th2 conditions and evaluated by ATACseq ( n = 3). A total of 71,040 peaks were detected. (A) Venn diagram of WT-specific, Pik3cd E1020K/+ -specific, and common peaks. (B) WT and Pik3cd E1020K/+ -specific peaks examined by motif enrichment analysis. Enrichment P values were plotted for both groups. Red: motifs specifically enriched in WT peaks; blue: motifs specifically enriched in Pik3cd E1020K/+ peaks; orange: motifs enriched in both groups. (C) Peak heatmap of CTCF CUT&Tag peaks from WT and Pik3cd E1020K/+ naïve, Th1, and Th2 cells organized into six clusters (1–6) specific to each indicated population, as described. (D) CTCF motif enrichment P value (top) and fold enrichment (bottom) in clusters 1–6. (E) DEGs (WT vs Pik3cd E1020K/+ ) and non-DEGs from bulk RNAseq data were compared in the indicated populations for percentages of genes showing differential CTCF peaks (WT versus Pik3cd E1020K/+ ). (F) Frequencies of CTCF peaks repressed, induced, or both induced and repressed in Pik3cd E1020K/+ Th2 cells (versus WT Th2) near DEGs (WT Th2 versus Pik3cd E1020K/+ Th2). (G) Th2 DEGs (WT Th2 versus Pik3cd E1020K/+ Th2) were organized into two categories: DEGs showing no change in CTCF (WT versus Pik3cd E1020K/+ ) and DEGs showing repressed CTCF peaks in Pik3cd E1020K/+ relative to WT. Pathway enrichment analysis (Enrichr; ) of TFTs (ChEA; ) was performed using these two categories of DEGs. Adjusted P values (−log 10 ) of the top 25 enriched TF signatures in each category plotted against each other. (H) Western blot evaluating CTCF and Zap70 in lysates from WT and Pik3cd E1020K/+ Th2-polarized cells, cultured in the presence or absence of Cal101 (10 nM) or rapamycin (200 nM). Data are representative of three independent experiments ( n = 3), quantified in . (I) Western blot evaluating CTCF and Zap70 in lysates from Th2-polarized NC and Foxo1 gRNA-Cas9–nucleofected WT CD4 T cells, compared with Pik3cd E1020K/+ cells. Data are representative of three independent experiments ( n = 3), . NC, negative control. Source data are available for this figure: .

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: RNA sequencing, Western Blot, Cell Culture, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Altered chromatin accessibility and CTCF activity in Pik3cd E1020K/+ Th2 cells. Supporting data for . (A and B) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice underwent Th2 polarization and were examined by ATACseq. n = 3. Volcano plot showing WT-specific peaks in red and Pik3cd E1020K/+ -specific peaks in blue (fold change >1.5, P < 0.05) (B) CTCF CUT&Tag tracks of Id3 , Bcl2 , Tbx21 , and Eomes loci. (C) Quantification of CTCF protein expressed as a ratio of CTCF/Zap70. Supporting data for . n = 3 for each group, from three independent experiments. (D) Quantification of CTCF protein expressed as a ratio of CTCF/Zap70. Supporting data for . n = 3 for each group, from three independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-FasL signaling potentiates T cell activation. Supporting data for , , and . (A) Naïve CD4 + T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody, and surface FasL expression was measured by flow cytometry. Left: Representative flow cytometry histograms of surface FasL. Right: Fold FasL expression (MFI normalized to WT control) on Th2-polarized cells from the indicated groups. n = 5 for each group, from five independent experiments. (B and C) Supplemental data for . (B) Frequencies of FasL low , FasL mid , and FasL high live CD4 T cells from the indicated groups. n = 7 for each group, from seven independent experiments. (C) Frequencies of IFNγ + cells in the indicated FasL expression category from the indicated mice. n = 7 for each group, from seven independent experiments. (D) Frequencies of dead Th2-polarized cells (gated as total CD4 + ) from the indicated groups. Left: Frequencies of dead cells within FasL low , FasL mid , and FasL high . Right: Frequencies of dead Th2 cells among total CD4 + cells. n = 7 for each group, from seven independent experiments. (E) Linear regression analyses comparing FasL MFIs with percentages of dead cells from the indicated groups. Correlation R 2 and P values are indicated. (F and G) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas - or Fasl -targeting gRNAs and underwent Th2 polarization. (F) Representative flow cytometry histograms showing surface Fas expression in the indicated groups. (G) Representative flow cytometry histograms showing surface FasL expression in the indicated groups. (H) Representative flow cytometry histograms showing pAKT(T308), pFoxo1(S256), and pS6(S240/44) staining in NC, Fas , and Fasl gRNA-targeted Th2-polarized live CD4 + T cells from the indicated mice. Supporting data for . n = 8–10 for each group, from 8 to 10 independent experiments. (I) Fold induction of p-p65(S529) in Th2-polarized live CD4 T cells from the indicated groups, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. n = 7 for each group, from seven independent experiments. (J) WT (CD45.1 + CD45.2 + ) and Pik3cd E1020K/+ (CD45.1 − CD45.2 + ) naïve CD4 T cells were Th2-polarized either alone (top) or cocultured (bottom) at a 1:1 ratio. Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + cells. Data are representative of six independent experiments. (K) Supporting data for . Representative flow cytometry histograms showing FADD staining in NC and Fadd gRNA-Cas9–treated cells from the indicated groups. (L and M) Naïve CD4 + T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas -targeting gRNAs and underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (L) Fold induction of pAKT(T308) and (M) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. (N and O) Naïve CD4 + T cells from WT and Pik3cd E1020K/+ mice underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (N) Fold induction of pAKT(T308) and (O) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001. AUC, area under the curve; NC, negative control.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Activation Assay, Blocking Assay, Expressing, Flow Cytometry, Control, Staining, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-FasL signaling potentiates T cell activation, exacerbating CD4 + T cell dysregulation in the presence of activated PI3Kδ. (A) Fold surface FasL expression (MFI normalized to WT) on Th2-polarized live CD4 + T cells, measured by flow cytometry (gated on live CD4 + ). n = 8 for each group, from eight independent experiments. (B) Fold surface FasL expression (MFI normalized to NC) on NC and Foxo1 gRNA-targeted naïve CD4 T cells cultured under Th2-polarizing conditions. n = 4 for each group, from four independent experiments. (C) Left: Th2 polarized cells (live CD4 + ) from WT and Pik3cd E1020K/+ animals were gated as FasL low , FasL mid , and FasL high . Right: Linear regressions comparing FasL MFIs in each gate with percentages of IFNγ + cells from the indicated groups. Correlation R 2 and P values are indicated. n = 7 for each group, from seven independent experiments. (D–F) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas - or Fasl -targeting gRNAs and polarized under Th2 conditions. n = 8–10 for each group, from 8 to 10 independent experiments. (D) Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + T cells. (E) Percentages of IFNγ + (left), IL-4 + (middle), and IL-13 + (right) in Th2-polarized live CD4 + T cells. (F) Fold induction of pAKT(T308) (left), pFoxo1(S256) (middle), and pS6(S240/44) (right) in Th2-polarized live CD4 T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. (G and H) NC, Fas , and Fasl gRNA-treated naïve CD4 T cells underwent Th2 polarization in the presence or absence of recombinant multimeric FasL (FasL-LZ), and phosphorylation of AKT(T308), Foxo1(S256), and S6(S240/44) was analyzed by flow cytometry. n = 6 for each group, from six independent experiments. (G) Representative flow cytometry histograms showing pS6(S240/44) staining in Th2-polarized live CD4 + T cells. Dashed lines represent cells cultured without FasL-LZ; solid lines show cells cultured with FasL-LZ. (H) Fold induction of pAKT(T308), pFoxo1(S256), and pS6(S240/44) in Th2-polarized (–/+ FasL-LZ) live CD4 + T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Activation Assay, Expressing, Flow Cytometry, Cell Culture, Recombinant, Phospho-proteomics, Staining, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions in the presence or absence of FasL-LZ (25 ng/ml). Top: Representative flow cytometry histograms showing pS6(S240/44) staining in live CD4 + cells. Bottom: Fold induction of pS6(S240/44) in Th2-polarized (−/+ FasL-LZ) live CD4 + T cells. Fold induction was calculated by normalizing MFIs to NC WT cells. n = 4–5 for each group, from four to five independent experiments. (B and C) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions. n = 6 for each group, from six independent experiments. (B) Frequencies of IFNγ + Th2-polarized cells. (C) Th2-polarized cells were gated as FasL low , FasL mid , and FasL high . Frequencies of IFNγ + cells were measured in the indicated groups. (D–G) Fas was tagged with BioID2 on its intracellular C terminus (Fas-BioID) and stably expressed in Fas-deficient Jurkat cells. Fas-BioID Jurkat cells were cultured in the presence or absence of recombinant multimeric FasL (FasL-LZ). Jurkat cells expressing BioID alone were used as a control. (D) Venn diagram showing proteins identified following mass spectrometry analysis of biotinylated proteins (streptavidin pull-down) that were common between or specific to BioID-alone Jurkat cells versus Fas-BioID + FasL-LZ (P < 0.05, n = 3 for all groups). (E) Heatmap (row z-score) showing % normalized spectral abundance of proteins specifically upregulated in Fas-BioID ± FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells. (F) Pathway enrichment of Reactome gene sets was performed using Enrichr , with significantly enriched gene sets colored in blue; proteins specifically upregulated in Fas-BioID + FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells were used as input for pathway enrichment. (G) Normalized spectral abundance (%) of the indicated proteins in BioID-alone, Fas-BioID, and Fas-BioID + FasL-LZ Jurkat cells, measured by mass spectrometry. Statistical comparisons were made using ratio paired t tests (G). *P < 0.05, **P < 0.01. NC, negative control.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Activation Assay, Flow Cytometry, Staining, Stable Transfection, Cell Culture, Recombinant, Expressing, Control, Mass Spectrometry, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas interacts with the TCR complex and costimulates TCR signaling. (A) Confocal imaging of CD3ε and Fas in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 (green) and αFas-AF555 (red), and colocalization (yellow) was measured. Data are representative of three individual experiments, n = 19–24 unique images for each group. Left: Representative images of CD3ε and Fas costaining from the indicated groups. Right: Quantification of % colocalization between CD3ε and Fas in the indicated groups. (B and C) FLIM-FRET microscopy of CD3ε-Fas interactions in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 alone (donor control) or costained with αCD3ε-AF488 (donor) and αFas-AF555 (acceptor), and FL was measured. Results are representative of three independent experiments. (B) Representative images from the indicated groups. Scale bars in images indicate 4 μm. (C) Left: FL (ns) measurements of individual pixels from ROIs on cells from indicated groups. Right: FRET efficiency (%) within ROI from individual cells from the indicated groups. n = 18 for each group. (D) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas -targeting gRNAs and stimulated with αCD3/CD28 in the presence of hIL-2 for 72 h. Cells were rested in serum-free media, treated with αCD3 (1 μg/ml) (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses for the indicated groups. (E) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, treated with αCD3 (1 μg/ml) in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time for the indicated groups. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses. Statistical comparisons were made using ratio paired t tests (D and E) and unpaired t tests (A and C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AUC, area under the curve; NC, negative control; ROIs, regions of interest.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Imaging, Staining, Microscopy, Control, Flow Cytometry, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: PI3Kδ regulates CD4 + T cell differentiation through integration of TCR, IL-2 receptor, and Fas signaling, driving Foxo1 inactivation and transcriptional reprogramming.

Article Snippet: Following culture, activated CD4 T cells were purified using a Miltenyi mouse CD4 T cell isolation kit.

Techniques: Cell Differentiation

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Activated PI3Kδ reshapes the HDM-induced immune response. (A–J) WT and Pik3cd E1020K/+ animals were sensitized intranasally with HDM extracts (200 μg on days 0 and 7; 50 μg on days 14, 16, and 18) and lungs examined on day 20. (A) Experimental outline. (B) H&E staining of paraffin-embedded lung sections. Top panel: Arrowheads indicate thickening of the alveolar septa; pound signs indicate iBALT. Bottom panel: Short arrows show lymphocytes, long arrows show macrophages, and yellow arrows show eosinophils. (C) CD4 T cell counts (liveCD45 + TCRβ + CD4 + CD8 − ) measured by flow cytometry. (D) Eosinophil numbers quantified from H&E-stained lung sections. (E) Neutrophil counts (liveLineage − CD11b + Ly6G + ) from lungs of the indicated animals. (F) Airway resistance (Rrs) was measured with a flexiVent instrument as cmH2O.s/ml using the indicated concentrations of methacholine. Data in E are representative of two independent experiments with n = 3–4 for each group per experiment. (G) UMAP showing clusters of lung CD45 + immune cells analyzed by scRNAseq from naïve WT, naïve Pik3cd E1020K/+ , HDM-treated WT, and HDM-treated Pik3cd E1020K/+ animals. Each cluster was assigned a cell type using SingleR and supervised analysis of gene expression specific to each cluster . Cells from three mice were analyzed per genotype and condition. (H) Individual lung CD45 + immune cells identified by scRNAseq analyzed for enrichment of response to IFNγ gene set. Left: UMAP visualization of enrichment P values in lung CD45 + immune cells from the indicated mice; P values described in color scale below. Right: Frequencies of cells with indicated magnitudes of enrichment P values. Statistical comparison of all CD45 + cells from WT and Pik3cd E1020K/+ HDM-treated groups was performed (chi-squared test), comparing frequencies of cells with P < 0.0005. (I) HDM-specific IgG2a, IgG3, IgE, and IgG1 from the indicated mice. (J) CCL3, CCL5, and CXCL10 (pg/ml) measured from lung homogenates from the indicated mice by Luminex. Data in C–E, I, and J are from n = 6–10 mice for each group, pooled from two independent experiments. Data in B are representative of two independent experiments. Unless otherwise indicated, statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Staining, Flow Cytometry, Gene Expression, Comparison, Luminex

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Aberrant Th1 responses at the expense of Th2 immunity in Pik3cd E1020K/+ lungs following HDM sensitization. (A) Scatter plot comparing gene expression in CD4 T cells from WT and Pik3cd E1020K/+ HDM-treated mice (cluster 1 from ). DEGs upregulated in WT CD4 T cells in red, and genes upregulated in Pik3cd E1020K/+ CD4 T cells in blue. (B) CD4 + T cells from scRNAseq of total CD45 + lung immune cells (cluster 1 from ) were re-clustered to identify five unique clusters (0–4) of CD4 + T cells. Left: UMAP showing distribution of clusters 0–4; identities of each cluster were assigned based on cluster-specific gene expression. Right: Proportion of cells from each cluster in indicated mice. (C) Seurat heatmap showing expression of cluster-defining genes for indicated populations. (A–C) Cells from three mice were analyzed per genotype and condition. (D) Representative flow cytometry plots of intracellular IFNγ and IL-5 expression in lung CD4 + T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from indicated mice. (E–H) Frequencies of lung CD4 + T cells expressing (E) IL-5; (F) IL-4; (G) IFNγ; and (H) IL-2 from indicated groups. (D–H) n = 9–10 for each group, pooled from two independent experiments. (I–L) TCRα-deficient recipient mice were injected with 1 × 10 6 WT or Pik3cd E1020K/+ naïve CD4 T cells 14 days prior to HDM sensitization. HDM was administered intranasally as indicated. n = 9–10 for each group, pooled from two independent experiments. (I) Experimental design. (J) Cell counts of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from the indicated groups. (K) Frequencies of IFNγ + (left) and IL-4/5/13 + (right) lung CD4 T cells from the indicated groups. Frequencies of IL-4/5/13 + cells were calculated using Boolean (or) gating. (L) Cell counts of eosinophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G − SiglecF + ) from lungs of the indicated mice. Statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Gene Expression, Expressing, Flow Cytometry, Injection

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Altered CD4 + T cell differentiation in Pik3cd E1020K/+ mice in vivo . Supporting data for . (A) Feature plots showing expression of Th2-defining genes Il1rl1 , Il4 , IL5 , and Il13 in scRNAseq analyses of lung CD45 + immune cells from the indicated mice. Cells from three mice were analyzed by scRNAseq per genotype and condition. (B) Flow cytometry of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) measuring GATA3 expression in the indicated populations. n = 9–10 for each group, pooled from two independent experiments. Left: Representative plots showing GATA3 and CD4 expression. Right: Frequencies (left) and cell counts (right) of GATA3 + CD4 T cells in the indicated groups. (C–E) Frequencies of (C) IL-13 + (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ); (D) IL-17A + (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ), and (E) Foxp3 + (Treg) lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ) in the indicated groups, measured by intracellular staining and flow cytometry. n = 9–10 for each group, pooled from two independent experiments. (F and G) Cytokine concentrations (pg/ml) measured from lung homogenates from the indicated mice by Luminex. Th2 cytokines IL-4 and IL-5 are shown in F, and Th1 cytokines GM-CSF and TNFα are shown in G. n = 9–10 for each group, pooled from two independent experiments. (H–L) WT and Pik3cd E1020K/+ animals were infected intranasally with C. neoformans , and were sacrificed 10 days after infection, and lung tissue was harvested for analysis. n = 6 for each group, pooled from two independent experiments. (H) Experimental design. (I) Cell counts of CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ) from lungs of the indicated mice. (J) Flow cytometry of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) measuring GATA3 and Tbet expression in the indicated populations. Left: Representative flow cytometry plots. Right: Frequencies of Tbet + and GATA3 + CD4 T cells from the indicated groups. (K) Frequencies of IL-4/5/13 + (left), IFNγ + (middle), and IL-2 + (right) lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ) in the indicated groups, measured by intracellular staining and flow cytometry. Frequencies of IL-4/5/13 + cells were calculated using Boolean (or) gating. (L) Cell counts of eosinophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G − SiglecF + ) and neutrophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G + ) from lungs of the indicated mice. (M–O) Supplemental data for CD4 + T cell transfer experiments in . n = 9–10 mice for each group, pooled from two independent experiments. (M) Cell counts (left) and frequencies (right) of GATA3 + CD4 T cells from the indicated groups. (N) Representative flow cytometry plots showing IFNγ and IL-4 expression in lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from the indicated mice. (O) Cell counts of neutrophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G + ) from lungs of the indicated mice. Statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Cell Differentiation, In Vivo, Expressing, Flow Cytometry, Staining, Luminex, Infection

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Hyperactivated PI3Kδ disrupts Th2 lineage restriction. (A–E) Naïve CD4 T cells were activated with αCD3 + αCD28 in the presence of WT T-depleted APCs under Th2-polarizing conditions (IL-4 + αIL-12) for 72 h. (A) Left: Representative flow cytometry plots showing IL-4 and IL-13 staining in Th2-polarized live CD4 + cells. Right: Percentages of IL-4 + and IL-13 + cells from the indicated mice. n = 15 for each group, from 15 independent experiments. (B) Left: Representative flow cytometry histograms showing GATA3 expression in Th2-polarized live CD4 + cells from the indicated mice. Right: Fold GATA3 expression (MFI normalized to WT) in Th2-polarized live CD4 + cells from the indicated mice. n = 14 for each group, from 14 independent experiments. (C) Left: Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4 + cells. Right: Percentages of IFNγ + cells. (D) Left: Representative flow cytometry histograms showing Tbet expression in Th2 and WT control Th1-polarized live CD4 + cells. Right: Fold Tbet expression (MFI normalized to WT) in Th2-polarized live CD4 + cells from the indicated mice. (C and D) n = 14 for each group, from 14 independent experiments. (E) Percentages of IFNγ + (left) and IL-13 + (right) cells over a time course of Th2 differentiation (0, 24, 48, and 72 h). n = 10 for each group, from 10 independent experiments. (F) Bulk RNAseq of WT and Pik3cd E1020K/+ naïve CD4 T cells and in vitro polarized Th2 cells. n = 3 biological replicates for each group. Volcano plots showing DEGs in red (WT upregulated) and blue ( Pik3cd E1020K/+ upregulated); DEGs defined using fold change >1.5, P < 0.05. (G) Enrichment of hallmark pathways among DEGs comparing WT and Pik3cd E1020K/+ Th2 cells. (H) Time course of fold induction of pAKT(T308), pS6(S240/44), and pAKT(S473) in WT and Pik3cd E1020K/+ live CD4 + cells during Th2 differentiation (0, 24, 48, and 72 h), measured by flow cytometry. Fold induction calculated using MFIs of the indicated readouts normalized to the 0 time point of the corresponding genotype. n = 5–9 for each group, from five to nine independent experiments. Statistical comparisons were made using ratio paired t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Flow Cytometry, Staining, Expressing, Control, RNA sequencing, In Vitro

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Analyses of in vitro polarized Th2 cells. Supporting data for , , and . (A) WT and Pik3cd E1020K /+ naïve CD4 T cells were Th2-polarized in the presence or absence of an αIFNγ blocking antibody. Left: Percentages of IFNγ + cells from the indicated groups. Right: Fold Tbet expression (MFI normalized to control WT) in live CD4 + cells from the indicated groups. n = 6–8 for each group, from six to eight independent experiments. (B) Gene expression (RPKM) of Il4ra , Il12rb1 , and Il12rb2 in WT and Pik3cd E1020K /+ Th2-polarized cells. n = 3, bulk RNAseq. (C) Naïve CD4 T cells from the indicated mice were Th2-polarized for 24 h in the absence of inhibitors. At 24 h of culture, PI3Kδ inhibitor Cal101 (10 nM) or mTOR inhibitor rapamycin (200 nM) was added to polarizing media and cells were cultured for an additional 48 h. Representative flow cytometry plots showing IFNγ and IL-4 staining in live CD4 + cells. Data are representative of three independent experiments, n = 3. (D) GSEA comparing WT and Pik3cd E1020K/+ transcriptomes for expression of TFT gene sets. (E) Fold induction of Foxo1 expression in Th2-polarized live CD4 + cells, measured by flow cytometry and calculated by normalizing Foxo1 MFIs to the 0 time point of the corresponding genotype. n = 5 for each group, from five independent experiments. (F) Naïve CD4 T cells from the indicated mice were Th2-polarized in the presence or absence of αIFNγ blocking antibody, and pFoxo1(S256) was measured in live CD4 + cells from the indicated groups by flow cytometry. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 5 for each group, from five independent experiments. (G) Naïve CD4 T cells were nucleofected with Cas9-gRNA complexes containing NC or Foxo1 -targeting gRNAs and underwent Th2 polarization. Representative flow cytometry histogram showing Foxo1 expression from the indicated cells, one example from nine independent experiments. (H) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were transduced with GFP only–, Foxo1-WT– or Foxo1-AAA–encoding retroviruses under Th2-polarizing conditions. Top: Representative flow cytometry plots showing IFNγ and IL-4 expression in live GFP + CD4 + T cells cultured under Th2-polarizing conditions from the indicated groups. Bottom, percentages of IL-4 + (left), IL-13 + (middle), and IFNγ + (right) Th2-polarized liveCD4 GFP + T cells from the indicated groups. n = 6 for each group, from six independent experiments. (I) WT Naïve CD4 T cells were nucleofected with NC or Foxo1 -targeting gRNA-Cas9 complexes and polarized under Th2 conditions in the presence or absence of an IL-2 blocking antibody. Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + cells. Data are representative of two independent experiments, n = 5. (J) Comparison of transcriptomes from Foxo1 KO Treg cells and Pik3cd E1020K/+ Th2 cells. Genes upregulated (FC >1.5, P < 0.05) in Foxo1 KO Treg 36 (versus WT Treg) and Pik3cd E1020K/+ Th2 (versus WT Th2) were compared (left), and pathway analysis (Enrichr; ) was performed on common DEGs (right). (K–M) Supplemental data related to , transfer of Foxo1 gRNA-treated cells. (K) Cell counts of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from the indicated groups. (L) Frequencies of IL-4 + lung CD4 T cells from the indicated groups. (M) Cell counts of lung eosinophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G − SiglecF + ) from the indicated groups. n = 6–9 for each group, pooled from two independent experiments. Statistical comparisons were made using ratio paired t tests (A, B, E, F, and H) or unpaired t tests (K–M). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: In Vitro, Blocking Assay, Expressing, Control, Gene Expression, RNA sequencing, Cell Culture, Flow Cytometry, Staining, Transduction, Comparison, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Dysregulated IL-2 signaling rewires Th2 differentiation of Pik3cd E1020K /+ CD4 T cells. (A) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in Th2-polarized cells from the indicated mice. Right: Percentages of IL-2 + Th2 cells. n = 9 for each group, from nine independent experiments. (B, C, and E) Time course analysis (0, 24, 48, 72 h) of IL-2 production, and pSTAT5(Y694) and CD25 during Th2 polarization, measured by flow cytometry. n = 7–10 for each group, from 7–10 independent experiments. (B) Percentages of IL-2 + cells over time from the indicated mice. (C) Fold induction of pSTAT5(Y694) over time from the indicated mice. Fold induction was calculated using pSTAT5(Y694) MFIs normalized to the 0 time point of the corresponding genotype. (D) Schematic describing experiments shown in F and G. (E) Fold induction of CD25 expression from the indicated mice. Fold induction was calculated using CD25 MFIs normalized to the 0 time point of the corresponding genotype. (F and G) Th2-polarized CD4 T cells from WT and Pik3cd E1020K/+ were rested in serum-free media for 4 h and subsequently stimulated with hIL-2 over the indicated time course (0, 15, 60, 120 min). n = 4–5 for each group, from four to five independent experiments. (F) Fold induction of pSTAT5(Y694) over time from the indicated mice, measured by flow cytometry. (G) Fold induction of pS6(S240/44) over time from the indicated mice, measured by flow cytometry. Fold induction was calculated using MFIs (pSTAT5(Y694) or pS6(S240/44)) normalized to the 0 time point of the corresponding genotype. (H–J) Naïve CD4 T cells were Th2-polarized in the presence or absence of αIL-2 blocking antibody (20 μg/ml). (H) Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4 + cells. (I) Percentages of IL-4 + (left), IL-13 + (middle), and IFNγ + (right) cells from the indicated mice, in the presence or absence of αIL-2. n = 11 for each group, from 11 independent experiments. (J) Left: Representative flow cytometry plots showing pAKT(T308) in Th2-polarized live CD4 + cells in the presence or absence of αIL-2. Right: Fold induction of pAKT(T308) in Th2-polarized live CD4 + cells from the indicated groups. Fold induction was calculated by normalizing pAKT(T308) MFIs to WT control cells. n = 5 for each group, from five independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Flow Cytometry, Expressing, Blocking Assay, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Inactivation of Foxo1 in Pik3cd E1020K/+ CD4 + T cells impairs Th2 lineage restriction. (A) Pathway enrichment of TF perturbations followed by expression gene sets performed using Enrichr : significantly enriched gene sets colored in blue. Genes upregulated in HDM-treated Pik3cd E1020K/+ CD4 T cells relative to WT counterparts were used as input for pathway enrichment. (B) Time course (0, 24, 48, 72 h) of pFoxo1(S256) in Th2-polarized live CD4 + cells from indicated mice. Left: Representative flow cytometry plots. Right: Fold induction of pFoxo1(S256) over time. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to the 0 time point of the corresponding genotype. n = 8 for each group, from eight independent experiments. (C) GSEA comparing Th2-polarized WT and Pik3cd E1020K/+ transcriptomes for the expression of Foxo1-activated (left) and Foxo1-repressed (right) gene sets. (D) Gene expression heatmap (row z-score) showing normalized RPKM values of leading edge genes from Foxo1-activated and Foxo1-repressed GSEA described in . (E) Naïve CD4 + T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody. Left: Representative flow cytometry plots showing pFoxo1(S256). Right: Fold induction of pFoxo1(S256). Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 10 for each group, from 10 independent experiments. (F) GSEA comparing control and αIL-2–treated Th2-polarized CD4 T cell transcriptomes for the expression of Foxo1-activated (top) and Foxo1-repressed (bottom) gene sets in the indicated groups. (G and H) Naïve CD4 T cells were nucleofected with gRNA-Cas9 complexes containing NC or Foxo1 -targeting gRNAs and differentiated under Th2 conditions. n = 9 for each group, from nine independent experiments. (G) Left: Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + T cells. Right: Percentages of IFNγ + and IL-4 + cells in Th2-polarized cells. (H) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in live CD4 + T cells. Right: Percentages of IL-2 + cells in Th2-polarized cells from the indicated groups. (I and J) TCRα-deficient recipient mice were injected with 1 × 10 6 NC or Foxo1 gRNA-Cas9–nucleofected naïve CD4 T cells 14 days prior to HDM sensitization. n = 6–9 for each group, pooled from two independent experiments. (I) Experimental design. (J) Frequencies of IFNγ + (left) and IL-2 + (right) lung CD4 T cells. Statistical comparisons used ratio paired t tests (B and E–G) or unpaired t tests (I). **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Expressing, Flow Cytometry, Gene Expression, Blocking Assay, Control, Injection, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Pik3cd E1020K reshapes the epigenetic landscape of CD4 + T cells. (A and B) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were polarized under Th2 conditions and evaluated by ATACseq ( n = 3). A total of 71,040 peaks were detected. (A) Venn diagram of WT-specific, Pik3cd E1020K/+ -specific, and common peaks. (B) WT and Pik3cd E1020K/+ -specific peaks examined by motif enrichment analysis. Enrichment P values were plotted for both groups. Red: motifs specifically enriched in WT peaks; blue: motifs specifically enriched in Pik3cd E1020K/+ peaks; orange: motifs enriched in both groups. (C) Peak heatmap of CTCF CUT&Tag peaks from WT and Pik3cd E1020K/+ naïve, Th1, and Th2 cells organized into six clusters (1–6) specific to each indicated population, as described. (D) CTCF motif enrichment P value (top) and fold enrichment (bottom) in clusters 1–6. (E) DEGs (WT vs Pik3cd E1020K/+ ) and non-DEGs from bulk RNAseq data were compared in the indicated populations for percentages of genes showing differential CTCF peaks (WT versus Pik3cd E1020K/+ ). (F) Frequencies of CTCF peaks repressed, induced, or both induced and repressed in Pik3cd E1020K/+ Th2 cells (versus WT Th2) near DEGs (WT Th2 versus Pik3cd E1020K/+ Th2). (G) Th2 DEGs (WT Th2 versus Pik3cd E1020K/+ Th2) were organized into two categories: DEGs showing no change in CTCF (WT versus Pik3cd E1020K/+ ) and DEGs showing repressed CTCF peaks in Pik3cd E1020K/+ relative to WT. Pathway enrichment analysis (Enrichr; ) of TFTs (ChEA; ) was performed using these two categories of DEGs. Adjusted P values (−log 10 ) of the top 25 enriched TF signatures in each category plotted against each other. (H) Western blot evaluating CTCF and Zap70 in lysates from WT and Pik3cd E1020K/+ Th2-polarized cells, cultured in the presence or absence of Cal101 (10 nM) or rapamycin (200 nM). Data are representative of three independent experiments ( n = 3), quantified in . (I) Western blot evaluating CTCF and Zap70 in lysates from Th2-polarized NC and Foxo1 gRNA-Cas9–nucleofected WT CD4 T cells, compared with Pik3cd E1020K/+ cells. Data are representative of three independent experiments ( n = 3), . NC, negative control. Source data are available for this figure: .

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: RNA sequencing, Western Blot, Cell Culture, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Altered chromatin accessibility and CTCF activity in Pik3cd E1020K/+ Th2 cells. Supporting data for . (A and B) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice underwent Th2 polarization and were examined by ATACseq. n = 3. Volcano plot showing WT-specific peaks in red and Pik3cd E1020K/+ -specific peaks in blue (fold change >1.5, P < 0.05) (B) CTCF CUT&Tag tracks of Id3 , Bcl2 , Tbx21 , and Eomes loci. (C) Quantification of CTCF protein expressed as a ratio of CTCF/Zap70. Supporting data for . n = 3 for each group, from three independent experiments. (D) Quantification of CTCF protein expressed as a ratio of CTCF/Zap70. Supporting data for . n = 3 for each group, from three independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-FasL signaling potentiates T cell activation. Supporting data for , , and . (A) Naïve CD4 + T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody, and surface FasL expression was measured by flow cytometry. Left: Representative flow cytometry histograms of surface FasL. Right: Fold FasL expression (MFI normalized to WT control) on Th2-polarized cells from the indicated groups. n = 5 for each group, from five independent experiments. (B and C) Supplemental data for . (B) Frequencies of FasL low , FasL mid , and FasL high live CD4 T cells from the indicated groups. n = 7 for each group, from seven independent experiments. (C) Frequencies of IFNγ + cells in the indicated FasL expression category from the indicated mice. n = 7 for each group, from seven independent experiments. (D) Frequencies of dead Th2-polarized cells (gated as total CD4 + ) from the indicated groups. Left: Frequencies of dead cells within FasL low , FasL mid , and FasL high . Right: Frequencies of dead Th2 cells among total CD4 + cells. n = 7 for each group, from seven independent experiments. (E) Linear regression analyses comparing FasL MFIs with percentages of dead cells from the indicated groups. Correlation R 2 and P values are indicated. (F and G) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas - or Fasl -targeting gRNAs and underwent Th2 polarization. (F) Representative flow cytometry histograms showing surface Fas expression in the indicated groups. (G) Representative flow cytometry histograms showing surface FasL expression in the indicated groups. (H) Representative flow cytometry histograms showing pAKT(T308), pFoxo1(S256), and pS6(S240/44) staining in NC, Fas , and Fasl gRNA-targeted Th2-polarized live CD4 + T cells from the indicated mice. Supporting data for . n = 8–10 for each group, from 8 to 10 independent experiments. (I) Fold induction of p-p65(S529) in Th2-polarized live CD4 T cells from the indicated groups, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. n = 7 for each group, from seven independent experiments. (J) WT (CD45.1 + CD45.2 + ) and Pik3cd E1020K/+ (CD45.1 − CD45.2 + ) naïve CD4 T cells were Th2-polarized either alone (top) or cocultured (bottom) at a 1:1 ratio. Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + cells. Data are representative of six independent experiments. (K) Supporting data for . Representative flow cytometry histograms showing FADD staining in NC and Fadd gRNA-Cas9–treated cells from the indicated groups. (L and M) Naïve CD4 + T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas -targeting gRNAs and underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (L) Fold induction of pAKT(T308) and (M) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. (N and O) Naïve CD4 + T cells from WT and Pik3cd E1020K/+ mice underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (N) Fold induction of pAKT(T308) and (O) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001. AUC, area under the curve; NC, negative control.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Activation Assay, Blocking Assay, Expressing, Flow Cytometry, Control, Staining, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-FasL signaling potentiates T cell activation, exacerbating CD4 + T cell dysregulation in the presence of activated PI3Kδ. (A) Fold surface FasL expression (MFI normalized to WT) on Th2-polarized live CD4 + T cells, measured by flow cytometry (gated on live CD4 + ). n = 8 for each group, from eight independent experiments. (B) Fold surface FasL expression (MFI normalized to NC) on NC and Foxo1 gRNA-targeted naïve CD4 T cells cultured under Th2-polarizing conditions. n = 4 for each group, from four independent experiments. (C) Left: Th2 polarized cells (live CD4 + ) from WT and Pik3cd E1020K/+ animals were gated as FasL low , FasL mid , and FasL high . Right: Linear regressions comparing FasL MFIs in each gate with percentages of IFNγ + cells from the indicated groups. Correlation R 2 and P values are indicated. n = 7 for each group, from seven independent experiments. (D–F) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas - or Fasl -targeting gRNAs and polarized under Th2 conditions. n = 8–10 for each group, from 8 to 10 independent experiments. (D) Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + T cells. (E) Percentages of IFNγ + (left), IL-4 + (middle), and IL-13 + (right) in Th2-polarized live CD4 + T cells. (F) Fold induction of pAKT(T308) (left), pFoxo1(S256) (middle), and pS6(S240/44) (right) in Th2-polarized live CD4 T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. (G and H) NC, Fas , and Fasl gRNA-treated naïve CD4 T cells underwent Th2 polarization in the presence or absence of recombinant multimeric FasL (FasL-LZ), and phosphorylation of AKT(T308), Foxo1(S256), and S6(S240/44) was analyzed by flow cytometry. n = 6 for each group, from six independent experiments. (G) Representative flow cytometry histograms showing pS6(S240/44) staining in Th2-polarized live CD4 + T cells. Dashed lines represent cells cultured without FasL-LZ; solid lines show cells cultured with FasL-LZ. (H) Fold induction of pAKT(T308), pFoxo1(S256), and pS6(S240/44) in Th2-polarized (–/+ FasL-LZ) live CD4 + T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Activation Assay, Expressing, Flow Cytometry, Cell Culture, Recombinant, Phospho-proteomics, Staining, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions in the presence or absence of FasL-LZ (25 ng/ml). Top: Representative flow cytometry histograms showing pS6(S240/44) staining in live CD4 + cells. Bottom: Fold induction of pS6(S240/44) in Th2-polarized (−/+ FasL-LZ) live CD4 + T cells. Fold induction was calculated by normalizing MFIs to NC WT cells. n = 4–5 for each group, from four to five independent experiments. (B and C) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions. n = 6 for each group, from six independent experiments. (B) Frequencies of IFNγ + Th2-polarized cells. (C) Th2-polarized cells were gated as FasL low , FasL mid , and FasL high . Frequencies of IFNγ + cells were measured in the indicated groups. (D–G) Fas was tagged with BioID2 on its intracellular C terminus (Fas-BioID) and stably expressed in Fas-deficient Jurkat cells. Fas-BioID Jurkat cells were cultured in the presence or absence of recombinant multimeric FasL (FasL-LZ). Jurkat cells expressing BioID alone were used as a control. (D) Venn diagram showing proteins identified following mass spectrometry analysis of biotinylated proteins (streptavidin pull-down) that were common between or specific to BioID-alone Jurkat cells versus Fas-BioID + FasL-LZ (P < 0.05, n = 3 for all groups). (E) Heatmap (row z-score) showing % normalized spectral abundance of proteins specifically upregulated in Fas-BioID ± FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells. (F) Pathway enrichment of Reactome gene sets was performed using Enrichr , with significantly enriched gene sets colored in blue; proteins specifically upregulated in Fas-BioID + FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells were used as input for pathway enrichment. (G) Normalized spectral abundance (%) of the indicated proteins in BioID-alone, Fas-BioID, and Fas-BioID + FasL-LZ Jurkat cells, measured by mass spectrometry. Statistical comparisons were made using ratio paired t tests (G). *P < 0.05, **P < 0.01. NC, negative control.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Activation Assay, Flow Cytometry, Staining, Stable Transfection, Cell Culture, Recombinant, Expressing, Control, Mass Spectrometry, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas interacts with the TCR complex and costimulates TCR signaling. (A) Confocal imaging of CD3ε and Fas in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 (green) and αFas-AF555 (red), and colocalization (yellow) was measured. Data are representative of three individual experiments, n = 19–24 unique images for each group. Left: Representative images of CD3ε and Fas costaining from the indicated groups. Right: Quantification of % colocalization between CD3ε and Fas in the indicated groups. (B and C) FLIM-FRET microscopy of CD3ε-Fas interactions in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 alone (donor control) or costained with αCD3ε-AF488 (donor) and αFas-AF555 (acceptor), and FL was measured. Results are representative of three independent experiments. (B) Representative images from the indicated groups. Scale bars in images indicate 4 μm. (C) Left: FL (ns) measurements of individual pixels from ROIs on cells from indicated groups. Right: FRET efficiency (%) within ROI from individual cells from the indicated groups. n = 18 for each group. (D) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas -targeting gRNAs and stimulated with αCD3/CD28 in the presence of hIL-2 for 72 h. Cells were rested in serum-free media, treated with αCD3 (1 μg/ml) (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses for the indicated groups. (E) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, treated with αCD3 (1 μg/ml) in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time for the indicated groups. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses. Statistical comparisons were made using ratio paired t tests (D and E) and unpaired t tests (A and C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AUC, area under the curve; NC, negative control; ROIs, regions of interest.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Imaging, Staining, Microscopy, Control, Flow Cytometry, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: PI3Kδ regulates CD4 + T cell differentiation through integration of TCR, IL-2 receptor, and Fas signaling, driving Foxo1 inactivation and transcriptional reprogramming.

Article Snippet: Naïve CD4 T cells were isolated from spleen and lymph nodes of WT and Pik3cd E1020K/+ mice using Miltenyi mouse naïve CD4 T cell isolation kits.

Techniques: Cell Differentiation

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Activated PI3Kδ reshapes the HDM-induced immune response. (A–J) WT and Pik3cd E1020K/+ animals were sensitized intranasally with HDM extracts (200 μg on days 0 and 7; 50 μg on days 14, 16, and 18) and lungs examined on day 20. (A) Experimental outline. (B) H&E staining of paraffin-embedded lung sections. Top panel: Arrowheads indicate thickening of the alveolar septa; pound signs indicate iBALT. Bottom panel: Short arrows show lymphocytes, long arrows show macrophages, and yellow arrows show eosinophils. (C) CD4 T cell counts (liveCD45 + TCRβ + CD4 + CD8 − ) measured by flow cytometry. (D) Eosinophil numbers quantified from H&E-stained lung sections. (E) Neutrophil counts (liveLineage − CD11b + Ly6G + ) from lungs of the indicated animals. (F) Airway resistance (Rrs) was measured with a flexiVent instrument as cmH2O.s/ml using the indicated concentrations of methacholine. Data in E are representative of two independent experiments with n = 3–4 for each group per experiment. (G) UMAP showing clusters of lung CD45 + immune cells analyzed by scRNAseq from naïve WT, naïve Pik3cd E1020K/+ , HDM-treated WT, and HDM-treated Pik3cd E1020K/+ animals. Each cluster was assigned a cell type using SingleR and supervised analysis of gene expression specific to each cluster . Cells from three mice were analyzed per genotype and condition. (H) Individual lung CD45 + immune cells identified by scRNAseq analyzed for enrichment of response to IFNγ gene set. Left: UMAP visualization of enrichment P values in lung CD45 + immune cells from the indicated mice; P values described in color scale below. Right: Frequencies of cells with indicated magnitudes of enrichment P values. Statistical comparison of all CD45 + cells from WT and Pik3cd E1020K/+ HDM-treated groups was performed (chi-squared test), comparing frequencies of cells with P < 0.0005. (I) HDM-specific IgG2a, IgG3, IgE, and IgG1 from the indicated mice. (J) CCL3, CCL5, and CXCL10 (pg/ml) measured from lung homogenates from the indicated mice by Luminex. Data in C–E, I, and J are from n = 6–10 mice for each group, pooled from two independent experiments. Data in B are representative of two independent experiments. Unless otherwise indicated, statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Staining, Flow Cytometry, Gene Expression, Comparison, Luminex

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Aberrant Th1 responses at the expense of Th2 immunity in Pik3cd E1020K/+ lungs following HDM sensitization. (A) Scatter plot comparing gene expression in CD4 T cells from WT and Pik3cd E1020K/+ HDM-treated mice (cluster 1 from ). DEGs upregulated in WT CD4 T cells in red, and genes upregulated in Pik3cd E1020K/+ CD4 T cells in blue. (B) CD4 + T cells from scRNAseq of total CD45 + lung immune cells (cluster 1 from ) were re-clustered to identify five unique clusters (0–4) of CD4 + T cells. Left: UMAP showing distribution of clusters 0–4; identities of each cluster were assigned based on cluster-specific gene expression. Right: Proportion of cells from each cluster in indicated mice. (C) Seurat heatmap showing expression of cluster-defining genes for indicated populations. (A–C) Cells from three mice were analyzed per genotype and condition. (D) Representative flow cytometry plots of intracellular IFNγ and IL-5 expression in lung CD4 + T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from indicated mice. (E–H) Frequencies of lung CD4 + T cells expressing (E) IL-5; (F) IL-4; (G) IFNγ; and (H) IL-2 from indicated groups. (D–H) n = 9–10 for each group, pooled from two independent experiments. (I–L) TCRα-deficient recipient mice were injected with 1 × 10 6 WT or Pik3cd E1020K/+ naïve CD4 T cells 14 days prior to HDM sensitization. HDM was administered intranasally as indicated. n = 9–10 for each group, pooled from two independent experiments. (I) Experimental design. (J) Cell counts of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from the indicated groups. (K) Frequencies of IFNγ + (left) and IL-4/5/13 + (right) lung CD4 T cells from the indicated groups. Frequencies of IL-4/5/13 + cells were calculated using Boolean (or) gating. (L) Cell counts of eosinophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G − SiglecF + ) from lungs of the indicated mice. Statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Gene Expression, Expressing, Flow Cytometry, Injection

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Altered CD4 + T cell differentiation in Pik3cd E1020K/+ mice in vivo . Supporting data for . (A) Feature plots showing expression of Th2-defining genes Il1rl1 , Il4 , IL5 , and Il13 in scRNAseq analyses of lung CD45 + immune cells from the indicated mice. Cells from three mice were analyzed by scRNAseq per genotype and condition. (B) Flow cytometry of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) measuring GATA3 expression in the indicated populations. n = 9–10 for each group, pooled from two independent experiments. Left: Representative plots showing GATA3 and CD4 expression. Right: Frequencies (left) and cell counts (right) of GATA3 + CD4 T cells in the indicated groups. (C–E) Frequencies of (C) IL-13 + (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ); (D) IL-17A + (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ), and (E) Foxp3 + (Treg) lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ) in the indicated groups, measured by intracellular staining and flow cytometry. n = 9–10 for each group, pooled from two independent experiments. (F and G) Cytokine concentrations (pg/ml) measured from lung homogenates from the indicated mice by Luminex. Th2 cytokines IL-4 and IL-5 are shown in F, and Th1 cytokines GM-CSF and TNFα are shown in G. n = 9–10 for each group, pooled from two independent experiments. (H–L) WT and Pik3cd E1020K/+ animals were infected intranasally with C. neoformans , and were sacrificed 10 days after infection, and lung tissue was harvested for analysis. n = 6 for each group, pooled from two independent experiments. (H) Experimental design. (I) Cell counts of CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ) from lungs of the indicated mice. (J) Flow cytometry of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) measuring GATA3 and Tbet expression in the indicated populations. Left: Representative flow cytometry plots. Right: Frequencies of Tbet + and GATA3 + CD4 T cells from the indicated groups. (K) Frequencies of IL-4/5/13 + (left), IFNγ + (middle), and IL-2 + (right) lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − CD4 + ) in the indicated groups, measured by intracellular staining and flow cytometry. Frequencies of IL-4/5/13 + cells were calculated using Boolean (or) gating. (L) Cell counts of eosinophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G − SiglecF + ) and neutrophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G + ) from lungs of the indicated mice. (M–O) Supplemental data for CD4 + T cell transfer experiments in . n = 9–10 mice for each group, pooled from two independent experiments. (M) Cell counts (left) and frequencies (right) of GATA3 + CD4 T cells from the indicated groups. (N) Representative flow cytometry plots showing IFNγ and IL-4 expression in lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from the indicated mice. (O) Cell counts of neutrophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G + ) from lungs of the indicated mice. Statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Cell Differentiation, In Vivo, Expressing, Flow Cytometry, Staining, Luminex, Infection

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Hyperactivated PI3Kδ disrupts Th2 lineage restriction. (A–E) Naïve CD4 T cells were activated with αCD3 + αCD28 in the presence of WT T-depleted APCs under Th2-polarizing conditions (IL-4 + αIL-12) for 72 h. (A) Left: Representative flow cytometry plots showing IL-4 and IL-13 staining in Th2-polarized live CD4 + cells. Right: Percentages of IL-4 + and IL-13 + cells from the indicated mice. n = 15 for each group, from 15 independent experiments. (B) Left: Representative flow cytometry histograms showing GATA3 expression in Th2-polarized live CD4 + cells from the indicated mice. Right: Fold GATA3 expression (MFI normalized to WT) in Th2-polarized live CD4 + cells from the indicated mice. n = 14 for each group, from 14 independent experiments. (C) Left: Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4 + cells. Right: Percentages of IFNγ + cells. (D) Left: Representative flow cytometry histograms showing Tbet expression in Th2 and WT control Th1-polarized live CD4 + cells. Right: Fold Tbet expression (MFI normalized to WT) in Th2-polarized live CD4 + cells from the indicated mice. (C and D) n = 14 for each group, from 14 independent experiments. (E) Percentages of IFNγ + (left) and IL-13 + (right) cells over a time course of Th2 differentiation (0, 24, 48, and 72 h). n = 10 for each group, from 10 independent experiments. (F) Bulk RNAseq of WT and Pik3cd E1020K/+ naïve CD4 T cells and in vitro polarized Th2 cells. n = 3 biological replicates for each group. Volcano plots showing DEGs in red (WT upregulated) and blue ( Pik3cd E1020K/+ upregulated); DEGs defined using fold change >1.5, P < 0.05. (G) Enrichment of hallmark pathways among DEGs comparing WT and Pik3cd E1020K/+ Th2 cells. (H) Time course of fold induction of pAKT(T308), pS6(S240/44), and pAKT(S473) in WT and Pik3cd E1020K/+ live CD4 + cells during Th2 differentiation (0, 24, 48, and 72 h), measured by flow cytometry. Fold induction calculated using MFIs of the indicated readouts normalized to the 0 time point of the corresponding genotype. n = 5–9 for each group, from five to nine independent experiments. Statistical comparisons were made using ratio paired t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Flow Cytometry, Staining, Expressing, Control, RNA sequencing, In Vitro

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Analyses of in vitro polarized Th2 cells. Supporting data for , , and . (A) WT and Pik3cd E1020K /+ naïve CD4 T cells were Th2-polarized in the presence or absence of an αIFNγ blocking antibody. Left: Percentages of IFNγ + cells from the indicated groups. Right: Fold Tbet expression (MFI normalized to control WT) in live CD4 + cells from the indicated groups. n = 6–8 for each group, from six to eight independent experiments. (B) Gene expression (RPKM) of Il4ra , Il12rb1 , and Il12rb2 in WT and Pik3cd E1020K /+ Th2-polarized cells. n = 3, bulk RNAseq. (C) Naïve CD4 T cells from the indicated mice were Th2-polarized for 24 h in the absence of inhibitors. At 24 h of culture, PI3Kδ inhibitor Cal101 (10 nM) or mTOR inhibitor rapamycin (200 nM) was added to polarizing media and cells were cultured for an additional 48 h. Representative flow cytometry plots showing IFNγ and IL-4 staining in live CD4 + cells. Data are representative of three independent experiments, n = 3. (D) GSEA comparing WT and Pik3cd E1020K/+ transcriptomes for expression of TFT gene sets. (E) Fold induction of Foxo1 expression in Th2-polarized live CD4 + cells, measured by flow cytometry and calculated by normalizing Foxo1 MFIs to the 0 time point of the corresponding genotype. n = 5 for each group, from five independent experiments. (F) Naïve CD4 T cells from the indicated mice were Th2-polarized in the presence or absence of αIFNγ blocking antibody, and pFoxo1(S256) was measured in live CD4 + cells from the indicated groups by flow cytometry. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 5 for each group, from five independent experiments. (G) Naïve CD4 T cells were nucleofected with Cas9-gRNA complexes containing NC or Foxo1 -targeting gRNAs and underwent Th2 polarization. Representative flow cytometry histogram showing Foxo1 expression from the indicated cells, one example from nine independent experiments. (H) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were transduced with GFP only–, Foxo1-WT– or Foxo1-AAA–encoding retroviruses under Th2-polarizing conditions. Top: Representative flow cytometry plots showing IFNγ and IL-4 expression in live GFP + CD4 + T cells cultured under Th2-polarizing conditions from the indicated groups. Bottom, percentages of IL-4 + (left), IL-13 + (middle), and IFNγ + (right) Th2-polarized liveCD4 GFP + T cells from the indicated groups. n = 6 for each group, from six independent experiments. (I) WT Naïve CD4 T cells were nucleofected with NC or Foxo1 -targeting gRNA-Cas9 complexes and polarized under Th2 conditions in the presence or absence of an IL-2 blocking antibody. Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + cells. Data are representative of two independent experiments, n = 5. (J) Comparison of transcriptomes from Foxo1 KO Treg cells and Pik3cd E1020K/+ Th2 cells. Genes upregulated (FC >1.5, P < 0.05) in Foxo1 KO Treg 36 (versus WT Treg) and Pik3cd E1020K/+ Th2 (versus WT Th2) were compared (left), and pathway analysis (Enrichr; ) was performed on common DEGs (right). (K–M) Supplemental data related to , transfer of Foxo1 gRNA-treated cells. (K) Cell counts of lung CD4 T cells (liveCD45 + TCRβ + CD4 + CD8 − ) from the indicated groups. (L) Frequencies of IL-4 + lung CD4 T cells from the indicated groups. (M) Cell counts of lung eosinophils (liveCD45 + CD3 − NK1.1 − CD19 − CD11b + Ly6G − SiglecF + ) from the indicated groups. n = 6–9 for each group, pooled from two independent experiments. Statistical comparisons were made using ratio paired t tests (A, B, E, F, and H) or unpaired t tests (K–M). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: In Vitro, Blocking Assay, Expressing, Control, Gene Expression, RNA sequencing, Cell Culture, Flow Cytometry, Staining, Transduction, Comparison, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Dysregulated IL-2 signaling rewires Th2 differentiation of Pik3cd E1020K /+ CD4 T cells. (A) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in Th2-polarized cells from the indicated mice. Right: Percentages of IL-2 + Th2 cells. n = 9 for each group, from nine independent experiments. (B, C, and E) Time course analysis (0, 24, 48, 72 h) of IL-2 production, and pSTAT5(Y694) and CD25 during Th2 polarization, measured by flow cytometry. n = 7–10 for each group, from 7–10 independent experiments. (B) Percentages of IL-2 + cells over time from the indicated mice. (C) Fold induction of pSTAT5(Y694) over time from the indicated mice. Fold induction was calculated using pSTAT5(Y694) MFIs normalized to the 0 time point of the corresponding genotype. (D) Schematic describing experiments shown in F and G. (E) Fold induction of CD25 expression from the indicated mice. Fold induction was calculated using CD25 MFIs normalized to the 0 time point of the corresponding genotype. (F and G) Th2-polarized CD4 T cells from WT and Pik3cd E1020K/+ were rested in serum-free media for 4 h and subsequently stimulated with hIL-2 over the indicated time course (0, 15, 60, 120 min). n = 4–5 for each group, from four to five independent experiments. (F) Fold induction of pSTAT5(Y694) over time from the indicated mice, measured by flow cytometry. (G) Fold induction of pS6(S240/44) over time from the indicated mice, measured by flow cytometry. Fold induction was calculated using MFIs (pSTAT5(Y694) or pS6(S240/44)) normalized to the 0 time point of the corresponding genotype. (H–J) Naïve CD4 T cells were Th2-polarized in the presence or absence of αIL-2 blocking antibody (20 μg/ml). (H) Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4 + cells. (I) Percentages of IL-4 + (left), IL-13 + (middle), and IFNγ + (right) cells from the indicated mice, in the presence or absence of αIL-2. n = 11 for each group, from 11 independent experiments. (J) Left: Representative flow cytometry plots showing pAKT(T308) in Th2-polarized live CD4 + cells in the presence or absence of αIL-2. Right: Fold induction of pAKT(T308) in Th2-polarized live CD4 + cells from the indicated groups. Fold induction was calculated by normalizing pAKT(T308) MFIs to WT control cells. n = 5 for each group, from five independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Flow Cytometry, Expressing, Blocking Assay, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Inactivation of Foxo1 in Pik3cd E1020K/+ CD4 + T cells impairs Th2 lineage restriction. (A) Pathway enrichment of TF perturbations followed by expression gene sets performed using Enrichr : significantly enriched gene sets colored in blue. Genes upregulated in HDM-treated Pik3cd E1020K/+ CD4 T cells relative to WT counterparts were used as input for pathway enrichment. (B) Time course (0, 24, 48, 72 h) of pFoxo1(S256) in Th2-polarized live CD4 + cells from indicated mice. Left: Representative flow cytometry plots. Right: Fold induction of pFoxo1(S256) over time. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to the 0 time point of the corresponding genotype. n = 8 for each group, from eight independent experiments. (C) GSEA comparing Th2-polarized WT and Pik3cd E1020K/+ transcriptomes for the expression of Foxo1-activated (left) and Foxo1-repressed (right) gene sets. (D) Gene expression heatmap (row z-score) showing normalized RPKM values of leading edge genes from Foxo1-activated and Foxo1-repressed GSEA described in . (E) Naïve CD4 + T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody. Left: Representative flow cytometry plots showing pFoxo1(S256). Right: Fold induction of pFoxo1(S256). Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 10 for each group, from 10 independent experiments. (F) GSEA comparing control and αIL-2–treated Th2-polarized CD4 T cell transcriptomes for the expression of Foxo1-activated (top) and Foxo1-repressed (bottom) gene sets in the indicated groups. (G and H) Naïve CD4 T cells were nucleofected with gRNA-Cas9 complexes containing NC or Foxo1 -targeting gRNAs and differentiated under Th2 conditions. n = 9 for each group, from nine independent experiments. (G) Left: Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + T cells. Right: Percentages of IFNγ + and IL-4 + cells in Th2-polarized cells. (H) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in live CD4 + T cells. Right: Percentages of IL-2 + cells in Th2-polarized cells from the indicated groups. (I and J) TCRα-deficient recipient mice were injected with 1 × 10 6 NC or Foxo1 gRNA-Cas9–nucleofected naïve CD4 T cells 14 days prior to HDM sensitization. n = 6–9 for each group, pooled from two independent experiments. (I) Experimental design. (J) Frequencies of IFNγ + (left) and IL-2 + (right) lung CD4 T cells. Statistical comparisons used ratio paired t tests (B and E–G) or unpaired t tests (I). **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Expressing, Flow Cytometry, Gene Expression, Blocking Assay, Control, Injection, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Pik3cd E1020K reshapes the epigenetic landscape of CD4 + T cells. (A and B) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were polarized under Th2 conditions and evaluated by ATACseq ( n = 3). A total of 71,040 peaks were detected. (A) Venn diagram of WT-specific, Pik3cd E1020K/+ -specific, and common peaks. (B) WT and Pik3cd E1020K/+ -specific peaks examined by motif enrichment analysis. Enrichment P values were plotted for both groups. Red: motifs specifically enriched in WT peaks; blue: motifs specifically enriched in Pik3cd E1020K/+ peaks; orange: motifs enriched in both groups. (C) Peak heatmap of CTCF CUT&Tag peaks from WT and Pik3cd E1020K/+ naïve, Th1, and Th2 cells organized into six clusters (1–6) specific to each indicated population, as described. (D) CTCF motif enrichment P value (top) and fold enrichment (bottom) in clusters 1–6. (E) DEGs (WT vs Pik3cd E1020K/+ ) and non-DEGs from bulk RNAseq data were compared in the indicated populations for percentages of genes showing differential CTCF peaks (WT versus Pik3cd E1020K/+ ). (F) Frequencies of CTCF peaks repressed, induced, or both induced and repressed in Pik3cd E1020K/+ Th2 cells (versus WT Th2) near DEGs (WT Th2 versus Pik3cd E1020K/+ Th2). (G) Th2 DEGs (WT Th2 versus Pik3cd E1020K/+ Th2) were organized into two categories: DEGs showing no change in CTCF (WT versus Pik3cd E1020K/+ ) and DEGs showing repressed CTCF peaks in Pik3cd E1020K/+ relative to WT. Pathway enrichment analysis (Enrichr; ) of TFTs (ChEA; ) was performed using these two categories of DEGs. Adjusted P values (−log 10 ) of the top 25 enriched TF signatures in each category plotted against each other. (H) Western blot evaluating CTCF and Zap70 in lysates from WT and Pik3cd E1020K/+ Th2-polarized cells, cultured in the presence or absence of Cal101 (10 nM) or rapamycin (200 nM). Data are representative of three independent experiments ( n = 3), quantified in . (I) Western blot evaluating CTCF and Zap70 in lysates from Th2-polarized NC and Foxo1 gRNA-Cas9–nucleofected WT CD4 T cells, compared with Pik3cd E1020K/+ cells. Data are representative of three independent experiments ( n = 3), . NC, negative control. Source data are available for this figure: .

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: RNA sequencing, Western Blot, Cell Culture, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Altered chromatin accessibility and CTCF activity in Pik3cd E1020K/+ Th2 cells. Supporting data for . (A and B) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice underwent Th2 polarization and were examined by ATACseq. n = 3. Volcano plot showing WT-specific peaks in red and Pik3cd E1020K/+ -specific peaks in blue (fold change >1.5, P < 0.05) (B) CTCF CUT&Tag tracks of Id3 , Bcl2 , Tbx21 , and Eomes loci. (C) Quantification of CTCF protein expressed as a ratio of CTCF/Zap70. Supporting data for . n = 3 for each group, from three independent experiments. (D) Quantification of CTCF protein expressed as a ratio of CTCF/Zap70. Supporting data for . n = 3 for each group, from three independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-FasL signaling potentiates T cell activation. Supporting data for , , and . (A) Naïve CD4 + T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody, and surface FasL expression was measured by flow cytometry. Left: Representative flow cytometry histograms of surface FasL. Right: Fold FasL expression (MFI normalized to WT control) on Th2-polarized cells from the indicated groups. n = 5 for each group, from five independent experiments. (B and C) Supplemental data for . (B) Frequencies of FasL low , FasL mid , and FasL high live CD4 T cells from the indicated groups. n = 7 for each group, from seven independent experiments. (C) Frequencies of IFNγ + cells in the indicated FasL expression category from the indicated mice. n = 7 for each group, from seven independent experiments. (D) Frequencies of dead Th2-polarized cells (gated as total CD4 + ) from the indicated groups. Left: Frequencies of dead cells within FasL low , FasL mid , and FasL high . Right: Frequencies of dead Th2 cells among total CD4 + cells. n = 7 for each group, from seven independent experiments. (E) Linear regression analyses comparing FasL MFIs with percentages of dead cells from the indicated groups. Correlation R 2 and P values are indicated. (F and G) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas - or Fasl -targeting gRNAs and underwent Th2 polarization. (F) Representative flow cytometry histograms showing surface Fas expression in the indicated groups. (G) Representative flow cytometry histograms showing surface FasL expression in the indicated groups. (H) Representative flow cytometry histograms showing pAKT(T308), pFoxo1(S256), and pS6(S240/44) staining in NC, Fas , and Fasl gRNA-targeted Th2-polarized live CD4 + T cells from the indicated mice. Supporting data for . n = 8–10 for each group, from 8 to 10 independent experiments. (I) Fold induction of p-p65(S529) in Th2-polarized live CD4 T cells from the indicated groups, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. n = 7 for each group, from seven independent experiments. (J) WT (CD45.1 + CD45.2 + ) and Pik3cd E1020K/+ (CD45.1 − CD45.2 + ) naïve CD4 T cells were Th2-polarized either alone (top) or cocultured (bottom) at a 1:1 ratio. Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + cells. Data are representative of six independent experiments. (K) Supporting data for . Representative flow cytometry histograms showing FADD staining in NC and Fadd gRNA-Cas9–treated cells from the indicated groups. (L and M) Naïve CD4 + T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas -targeting gRNAs and underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (L) Fold induction of pAKT(T308) and (M) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. (N and O) Naïve CD4 + T cells from WT and Pik3cd E1020K/+ mice underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (N) Fold induction of pAKT(T308) and (O) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001. AUC, area under the curve; NC, negative control.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Activation Assay, Blocking Assay, Expressing, Flow Cytometry, Control, Staining, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-FasL signaling potentiates T cell activation, exacerbating CD4 + T cell dysregulation in the presence of activated PI3Kδ. (A) Fold surface FasL expression (MFI normalized to WT) on Th2-polarized live CD4 + T cells, measured by flow cytometry (gated on live CD4 + ). n = 8 for each group, from eight independent experiments. (B) Fold surface FasL expression (MFI normalized to NC) on NC and Foxo1 gRNA-targeted naïve CD4 T cells cultured under Th2-polarizing conditions. n = 4 for each group, from four independent experiments. (C) Left: Th2 polarized cells (live CD4 + ) from WT and Pik3cd E1020K/+ animals were gated as FasL low , FasL mid , and FasL high . Right: Linear regressions comparing FasL MFIs in each gate with percentages of IFNγ + cells from the indicated groups. Correlation R 2 and P values are indicated. n = 7 for each group, from seven independent experiments. (D–F) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas - or Fasl -targeting gRNAs and polarized under Th2 conditions. n = 8–10 for each group, from 8 to 10 independent experiments. (D) Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4 + T cells. (E) Percentages of IFNγ + (left), IL-4 + (middle), and IL-13 + (right) in Th2-polarized live CD4 + T cells. (F) Fold induction of pAKT(T308) (left), pFoxo1(S256) (middle), and pS6(S240/44) (right) in Th2-polarized live CD4 T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. (G and H) NC, Fas , and Fasl gRNA-treated naïve CD4 T cells underwent Th2 polarization in the presence or absence of recombinant multimeric FasL (FasL-LZ), and phosphorylation of AKT(T308), Foxo1(S256), and S6(S240/44) was analyzed by flow cytometry. n = 6 for each group, from six independent experiments. (G) Representative flow cytometry histograms showing pS6(S240/44) staining in Th2-polarized live CD4 + T cells. Dashed lines represent cells cultured without FasL-LZ; solid lines show cells cultured with FasL-LZ. (H) Fold induction of pAKT(T308), pFoxo1(S256), and pS6(S240/44) in Th2-polarized (–/+ FasL-LZ) live CD4 + T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Activation Assay, Expressing, Flow Cytometry, Cell Culture, Recombinant, Phospho-proteomics, Staining, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions in the presence or absence of FasL-LZ (25 ng/ml). Top: Representative flow cytometry histograms showing pS6(S240/44) staining in live CD4 + cells. Bottom: Fold induction of pS6(S240/44) in Th2-polarized (−/+ FasL-LZ) live CD4 + T cells. Fold induction was calculated by normalizing MFIs to NC WT cells. n = 4–5 for each group, from four to five independent experiments. (B and C) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions. n = 6 for each group, from six independent experiments. (B) Frequencies of IFNγ + Th2-polarized cells. (C) Th2-polarized cells were gated as FasL low , FasL mid , and FasL high . Frequencies of IFNγ + cells were measured in the indicated groups. (D–G) Fas was tagged with BioID2 on its intracellular C terminus (Fas-BioID) and stably expressed in Fas-deficient Jurkat cells. Fas-BioID Jurkat cells were cultured in the presence or absence of recombinant multimeric FasL (FasL-LZ). Jurkat cells expressing BioID alone were used as a control. (D) Venn diagram showing proteins identified following mass spectrometry analysis of biotinylated proteins (streptavidin pull-down) that were common between or specific to BioID-alone Jurkat cells versus Fas-BioID + FasL-LZ (P < 0.05, n = 3 for all groups). (E) Heatmap (row z-score) showing % normalized spectral abundance of proteins specifically upregulated in Fas-BioID ± FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells. (F) Pathway enrichment of Reactome gene sets was performed using Enrichr , with significantly enriched gene sets colored in blue; proteins specifically upregulated in Fas-BioID + FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells were used as input for pathway enrichment. (G) Normalized spectral abundance (%) of the indicated proteins in BioID-alone, Fas-BioID, and Fas-BioID + FasL-LZ Jurkat cells, measured by mass spectrometry. Statistical comparisons were made using ratio paired t tests (G). *P < 0.05, **P < 0.01. NC, negative control.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Activation Assay, Flow Cytometry, Staining, Stable Transfection, Cell Culture, Recombinant, Expressing, Control, Mass Spectrometry, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas interacts with the TCR complex and costimulates TCR signaling. (A) Confocal imaging of CD3ε and Fas in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 (green) and αFas-AF555 (red), and colocalization (yellow) was measured. Data are representative of three individual experiments, n = 19–24 unique images for each group. Left: Representative images of CD3ε and Fas costaining from the indicated groups. Right: Quantification of % colocalization between CD3ε and Fas in the indicated groups. (B and C) FLIM-FRET microscopy of CD3ε-Fas interactions in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 alone (donor control) or costained with αCD3ε-AF488 (donor) and αFas-AF555 (acceptor), and FL was measured. Results are representative of three independent experiments. (B) Representative images from the indicated groups. Scale bars in images indicate 4 μm. (C) Left: FL (ns) measurements of individual pixels from ROIs on cells from indicated groups. Right: FRET efficiency (%) within ROI from individual cells from the indicated groups. n = 18 for each group. (D) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas -targeting gRNAs and stimulated with αCD3/CD28 in the presence of hIL-2 for 72 h. Cells were rested in serum-free media, treated with αCD3 (1 μg/ml) (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses for the indicated groups. (E) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, treated with αCD3 (1 μg/ml) in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time for the indicated groups. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses. Statistical comparisons were made using ratio paired t tests (D and E) and unpaired t tests (A and C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AUC, area under the curve; NC, negative control; ROIs, regions of interest.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Imaging, Staining, Microscopy, Control, Flow Cytometry, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: PI3Kδ regulates CD4 + T cell differentiation through integration of TCR, IL-2 receptor, and Fas signaling, driving Foxo1 inactivation and transcriptional reprogramming.

Article Snippet: For adoptive transfer experiments, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Pik3cd E1020K/+ mice using Miltenyi naïve CD4 T cell isolation kits and 1 × 10 6 cells per mouse were injected intraperitoneally into TCRα KO mice.

Techniques: Cell Differentiation

Journal: Journal of Nanobiotechnology

Article Title: MiR-1290 in natural killer cell derived extracellular vesicles: a pathogenic mediator of lupus nephritis and therapeutic target for th17 regulation

doi: 10.1186/s12951-026-04212-9

Figure Lengend Snippet: Characterization of NK-EVs and their effect on Th17 cell differentiation in vitro. (a) Schematic of the experimental procedure for treating primary T cells with NK-EVs. (b) Purity analysis of sorted CD4 + T cells by flow cytometry. (c) Purity analysis of sorted NK cells, showing the proportion of CD3 − CD56 + NK cells exceeded 94%. (d) Size distribution of NK-EVs as determined by NTA. (e) Western blot analysis of exosomal markers and specific cargos in NK-EVs, with 293T-derived EVs as a control. (f) TEM analysis of NK-EVs showing cup-shaped bilayer membrane structures (indicated by black arrows, scale bar: 200 nm). (g) Immunofluorescence staining showing the uptake of NK-EVs by T cells (indicated by black arrows, scale bar: 10 μm) and (h) fluorescence intensity analysis of the local region indicated by white arrows. (i) Gating strategy and representative flow cytometry plots for CD4 + IL-17 A + T cells under Th17-polarizing conditions after EVs treatment. (j) LN-EVs significantly reduced the proportion of CD4 + IL-17 A + cells ( N = 3). (k) Schematic of the procedures for generating Protein-free NK-EVs via protease treatment and RNA-free NK-EVs via RNase treatment. (l) Effects of LN-derived NK-EVs, Protein-free NK-EVs, and RNA-free NK-EVs on the proportion of Th17 cell differentiation. (m) Effects of LN-derived NK-EVs, Protein-free NK-EVs, and RNA-free NK-EVs on the IL17A mRNA level in T cells. (n) Effects of LN-derived NK-EVs, Protein-free NK-EVs, and RNA-free NK-EVs on the levels of inflammatory cytokines IFN-γ, TNF-α, IL-17 A, and IL-10 in T cell culture supernatants. Data are presented as mean ± SD ( N = 3). * P < 0.05, ** P < 0.01 (One-way ANOVA)

Article Snippet: Briefly, CD4 + T cells were isolated from PBMCs using CD4 + microbeads (Miltenyi Bio, #130-045-101).

Techniques: Cell Differentiation, In Vitro, Flow Cytometry, Western Blot, Derivative Assay, Control, Membrane, Immunofluorescence, Staining, Fluorescence, Cell Culture

Journal: Journal of Nanobiotechnology

Article Title: MiR-1290 in natural killer cell derived extracellular vesicles: a pathogenic mediator of lupus nephritis and therapeutic target for th17 regulation

doi: 10.1186/s12951-026-04212-9

Figure Lengend Snippet: MiR-1290 drives Th17/Treg imbalance by directly targeting NR4A2. (a) Integrated analysis of sequencing data and the GEO database to predict potential target genes of miR-1290. (b) Effect of miR-1290 mimic transfection on the secretion of IL-17 A and IFN-γ under Th17-polarizing conditions ( N = 6). (c) Impact of miR-1290 mimic transfection on the Th17/Treg ratio under respective polarizing conditions ( N = 3). (d) Gating strategy for identifying CD4 + IL-17 A + Th17 cells after transfection under Th17-polarizing conditions. (e) Gating strategy for identifying CD4 + Foxp3 + Treg cells after transfection under Treg-polarizing conditions. (f) Dual-luciferase reporter assay verifying the direct targeting of miR-1290 to the 3’UTR of NR4A2 ( N = 3). (g) Western blot analysis showing the effect of miR-1290 mimic or inhibitor on NR4A2 protein expression in T cells. (h) Modulation of NR4A2 protein levels in Jurkat cells via lentiviral transduction (overexpression, OE; knockdown, KD). (i) Effect of NR4A2 levels on the proportion of CD4 + Foxp3 + Jurkat cells under Treg-polarizing conditions ( N = 3). (j) KEGG pathway analysis of RNA sequencing data from NR4A2-overexpressing Jurkat cells versus controls. (k) GSEA of the same RNA-seq data, showing inflammatory signaling pathways suppressed by NR4A2 overexpression. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01 (One-way ANOVA)

Article Snippet: Briefly, CD4 + T cells were isolated from PBMCs using CD4 + microbeads (Miltenyi Bio, #130-045-101).

Techniques: Sequencing, Transfection, Luciferase, Reporter Assay, Western Blot, Expressing, Transduction, Over Expression, Knockdown, RNA Sequencing, Protein-Protein interactions

Journal: Journal of Nanobiotechnology

Article Title: MiR-1290 in natural killer cell derived extracellular vesicles: a pathogenic mediator of lupus nephritis and therapeutic target for th17 regulation

doi: 10.1186/s12951-026-04212-9

Figure Lengend Snippet: NR4A2 inhibited the AKT/NF-κB pathway. (a) Confocal fluorescence microscopy images showing the effect of NR4A2 on the nuclear localization of p65 in Jurkat cells (sacle bar: 10 μm). (b) Western blot analysis of the effects of NR4A2 on the phosphorylation of p65 and AKT under inflammatory stimulation. (c) Grayscale analysis of the p-p65/p65 ratio ( N = 3). (d) Grayscale analysis of the p-AKT/AKT ratio ( N = 3). (e) Effects of miR-1290 mimics and inhibitor on NR4A2 protein expression in mice CD4 + T cells and (f) corresponding grayscale analysis ( N = 3). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01 (One-way ANOVA)

Article Snippet: Briefly, CD4 + T cells were isolated from PBMCs using CD4 + microbeads (Miltenyi Bio, #130-045-101).

Techniques: Fluorescence, Microscopy, Western Blot, Phospho-proteomics, Expressing

Journal: Journal of Nanobiotechnology

Article Title: MiR-1290 in natural killer cell derived extracellular vesicles: a pathogenic mediator of lupus nephritis and therapeutic target for th17 regulation

doi: 10.1186/s12951-026-04212-9

Figure Lengend Snippet: Engineered NK-EVs reshape T cell balance and cytokine profile in lupus nephritis. (a) Experimental timeline of EVs administration in MRL/ lpr mice. (b) Measurement and analysis of urine protein-to-creatinine ratios, reflecting renal function impairment ( N = 8). (c) Detection of inflammation-related mRNAs in the renal cortex of mice ( N = 8). (d) Gating strategy for flow cytometric analysis of the effects of consecutive administration of therapeutic antagomir or pathogenic agomir loaded NK-EVs on splenic T cell function. (e) Changes in the proportion of CD4 + IL-17 A + cells ( N = 6). (f) Changes in the proportion of CD4 + CTLA-4 + FOXP3 + cells. (g) Representative images of CD4 + and CD8 + T cell infiltration in the kidneys of LN mice (scale bar, 50 μm) and (h) quantitative analysis of cell counts ( N = 6). (i) Expression levels of inflammatory cytokines in mouse serum ( N = 6). (j) Confocal microscopy images showing T cell infiltration and IL-17 A expression around kidney-resident macrophages (scale bar, 20 μm), and (k) quantitative analysis of cell counts ( N = 3). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01 (One-way ANOVA)

Article Snippet: Briefly, CD4 + T cells were isolated from PBMCs using CD4 + microbeads (Miltenyi Bio, #130-045-101).

Techniques: Cell Function Assay, Expressing, Confocal Microscopy