magnetic selection  (Miltenyi Biotec)

Journal: iScience

Article Title: The ionotropic AMPA receptor contributes to autoimmunity via altered regulatory T cell differentiation

doi: 10.1016/j.isci.2025.114267

Figure Lengend Snippet: T ΔAMPAR mice are protected from developing severe autoimmune disease, and this is associated with an enhanced effector Treg presence WT and T ΔAMPAR mice were immunized with CFA-MOG and injected with PTX to induce EAE. (A) EAE paralysis scores and (B) weight change over the course of disease of WT and T ΔAMPAR mice (mean ± SEM, ANOVA, representative of more than 3 independent experiments with 4–6 biological replicates per group). (C–E) Spinal cord infiltrating immune cells were isolated and evaluated by flow cytometry on day 15. (C) Quantification of CD45 + frequency and total CD45 + cell number, (D) quantification of frequency of the CD4 + TNF⍺, GMCSF, IFN γ , IL17, and (E) Foxp3 (mean ± SEM, ANOVA, representative of more than 3 independent experiments with 4–6 biological replicates per group). (F–I) Live CD4 T cells were isolated and pooled from the spinal cords of WT and T ΔAMPAR mice with EAE at day 14, and scRNAseq was performed. (F) 2-D UMAP projection of WT and T ΔAMPAR CD4 T cells. Unique clusters are color coded and annotated with cluster names (left). Heatmap depicts the relative expression of the top 5 differential genes for each cluster (right). (G) Proportion of WT and T ΔAMPAR CD4 T cells found within the Treg cluster. (H and I) GSEA plots of T ΔAMPAR versus WT cells in the Treg cluster. (H) Hallmark (Hm) Tgf beta signaling (top) and IL2 Stat5 signaling (bottom), (I) Hm mTORC1 signaling (left) and Hm glycolysis (right). (J) Volcano plot highlights effector Treg associated genes upregulated in T ΔAMPAR Treg cluster 3 cells. Statistical significance is represented as ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: CD4 T cells from WT (GluA1/2/ 3 fL/fL Cre negative control) and T ΔAMPAR (GluA1/2/ 3 fL/fL Cre positive) were isolated and enriched from pooled lymph nodes and spleens by negative selection total CD4 magnetic bead kit (Miltenyi Biotech, 130-104-454).

Techniques: Injection, Isolation, Flow Cytometry, Expressing

Journal: iScience

Article Title: The ionotropic AMPA receptor contributes to autoimmunity via altered regulatory T cell differentiation

doi: 10.1016/j.isci.2025.114267

Figure Lengend Snippet: AMPAR deficiency preferentially polarizes CD4 T cells to Tregs (A–D) Naive CD4 T cells were activated with 5 μg/ml anti-CD3 and 2 μg/ml anti-CD28 under Th1, Th17, and iTreg polarizing conditions. Cells were analyzed on day 3. Representative histogram of lineage defining transcription factor expression with gMFI and frequency annotation (left) and frequency quantification (right) for (A) Th1, (B) Th17, and (C) iTreg cells (mean ± SEM, unpaired t test, representative of more than 3 independent experiments). (D) Foxp3 expression over the course of iTreg differentiation in vitro (mean ± SEM, ANOVA, representative of 2 independent experiments). (E) Naive CD4 T cells were isolated and cultured under iTreg polarizing conditions and a titration of IL6. Quantification of CD4 + Foxp3 + frequency (mean ± SEM, ANOVA, representative of 2 independent experiments). (F) Representative histogram of Foxp3 expression (left) and quantification (right) for Th17 cells (mean ± SEM, unpaired t test, representative of more than 3 independent experiments). (G) Naive CD4 T cells were isolated from WT and T ΔAMPAR mice and adoptively transferred to Rag2 −/− mice. Representative flow plots of Foxp3 + CD4 + expression (left) and quantification (right) of week 3 harvested mLN (top) and pLN (bottom) (mean ± SEM, ANOVA, representative of 3 independent experiments). (H) WT and T ΔAMPAR mice were immunized with CFA-MOG, and their dLN were isolated and restimulated with 50 μg/ml MOG peptide under iTreg culture conditions. Representative flow plot (left) and quantification (right) of CD4 + Foxp3 + frequency (mean ± SEM, t test, representative of 2 independent experiments). (I) Quantification of CD4 + GITR and OX40 frequency among iTreg cells (mean ± SEM, ANOVA, representative of 2–3 independent experiments). (J) WT and T ΔAMPAR CD4 T cells derived from iTreg day 3 culture were co-cultured with CTV-labeled B6 WT CD8 T cells at varying ratios. Representative histograms of CTV by decreasing ratios of CD4:CD8 (left). Gating is of undivided CD8 T cells. Quantification of undivided CD8 T cell percentage by decreasing the ratio of CD4:CD8 (right). CTV = cell trace violet. (mean ± SEM, ANOVA, representative of 2 independent experiments). Statistical significance represented as ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: CD4 T cells from WT (GluA1/2/ 3 fL/fL Cre negative control) and T ΔAMPAR (GluA1/2/ 3 fL/fL Cre positive) were isolated and enriched from pooled lymph nodes and spleens by negative selection total CD4 magnetic bead kit (Miltenyi Biotech, 130-104-454).

Techniques: Expressing, In Vitro, Isolation, Cell Culture, Titration, Derivative Assay, Labeling

Journal: iScience

Article Title: The ionotropic AMPA receptor contributes to autoimmunity via altered regulatory T cell differentiation

doi: 10.1016/j.isci.2025.114267

Figure Lengend Snippet: AMPAR-deficient CD4 T cells changed cytokine sensitivity and metabolically shifted toward glycolysis and lipid metabolism (A) Quantification of Foxp3 gMFI flow staining of WT and T ΔAMPAR iTregs cultured under the titration of IL-2 (mean ± SEM, ANOVA, representative of 3 independent experiments). (B) Representative flow plot of pSTAT5 + Foxp3 + expression (left) and quantification (right) from WT and T ΔAMPAR iTreg cultures (mean ± SEM, t test, representative of 3 independent experiments). (C) Quantification of CD4 + CD25 + frequency over the course of iTreg differentiation (mean ± SEM, ANOVA, representative of 3 independent experiments). (D) Western blot of WT and T ΔAMPAR CD4 T cells collected on day 1, 2, and 3 of iTreg differentiation, immunoblotted for phosphorylated mTOR (pmTOR), total mTOR, and β-Actin expression (top to bottom, representative of 3 independent experiments). (E) Quantification of CD71 + cells among CD4 + T cells on day 3 of iTreg differentiation. (mean ± SEM, t test, representative of 3 independent experiments). (F–I) Seahorse metabolic flux analysis of iTreg cells. (F) extracellular acidification rate (ECAR) measurement over time (minutes) and (G) quantification of glycolysis (left) and glycolytic capacity (right) measurements. (H) oxygen consumption rate (OCR) measurement over time (minutes) and (I) quantification of maximum respiratory capacity (left) and reserve capacity (right) measurements (mean ± SEM, ANOVA, representative of 3 independent experiments). (J and K) WT and T ΔAMPAR CD4 were cultured under iTreg polarizing conditions and subjected to bulk RNA sequencing and analysis. (J) GSEA plot of cholesterol metabolism pathway enrichment in T ΔAMPAR versus WT cells. (K) Heatmap of genes associated with cholesterol metabolism in T ΔAMPAR versus WT. (L) Quantification of Srebf1 , Srebf2 , Hmgcr , Acaca , Fasn , and Sqle mRNA relative expression in WT and T ΔAMPAR CD4 from iTreg culture determined by qPCR (mean ± SEM, ANOVA, representative of 2 independent experiments). (M) Western blot images (left) of WT and T ΔAMPAR immunoblotted for Srebf1 and Srebf2 (image is representative of 2 independent experiments) and quantification (right) of cleaved to precursor ratios analyzed by densitometry. (N and O) Cell staining for Lipid and cholesterol content. (N) Bodipy and (O) Filipin III gMFI flow staining of WT and T ΔAMPAR CD4 from day 2 iTreg cultures (mean ± SEM, t test, representative of 3 independent experiments). Statistical significance represented as ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: CD4 T cells from WT (GluA1/2/ 3 fL/fL Cre negative control) and T ΔAMPAR (GluA1/2/ 3 fL/fL Cre positive) were isolated and enriched from pooled lymph nodes and spleens by negative selection total CD4 magnetic bead kit (Miltenyi Biotech, 130-104-454).

Techniques: Metabolic Labelling, Staining, Cell Culture, Titration, Expressing, Western Blot, RNA Sequencing

Journal: iScience

Article Title: The ionotropic AMPA receptor contributes to autoimmunity via altered regulatory T cell differentiation

doi: 10.1016/j.isci.2025.114267

Figure Lengend Snippet: AMPA receptor antagonists enhance Treg generation and provide protection from severe autoimmune disease (A–C) Naive CD4 T cells were isolated from B6 WT mice and cultured in vitro under iTreg polarizing conditions in the presence of PBS (vehicle) or 50 μM of NBQX. Cells were analyzed on day 3. (A) Quantification of CD4 + Foxp3 + frequency. (B and C) Quantification of 41BB, GITR (B), and CD71 (C) frequency among CD4 + Foxp3 + cells (mean ± SEM, ANOVA, representative of 3 independent experiments with 3–4 biological replicates per group). (D–G) B6 WT mice were immunized with CFA-MOG and injected with PTX to induce EAE. Disease monitoring began on day 7, and mice were separated into PBS and NBQX treatment groups. On day 10, treatment began with the q12 h dosing of PBS or NBQX (30 mg/kg) daily until day 17. (D) EAE paralysis scores over the course of disease in PBS and NBQX-treated mice. Quantification of (E) CD45 proportion of spinal cord infiltrates, (F) IFN γ , (G) IL17, (H) Foxp3 + Rorgt + , and (I) Foxp3 + proportion among live CD4 cells from the spinal cords of PBS and NBQX-treated mice (mean ± SEM, ANOVA, representative of 3 independent experiments with 5–9 biological replicates per group). Statistical significance represented as ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: CD4 T cells from WT (GluA1/2/ 3 fL/fL Cre negative control) and T ΔAMPAR (GluA1/2/ 3 fL/fL Cre positive) were isolated and enriched from pooled lymph nodes and spleens by negative selection total CD4 magnetic bead kit (Miltenyi Biotech, 130-104-454).

Techniques: Isolation, Cell Culture, In Vitro, Injection