Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: A) UMAP of scDNA and protein-seq data for pooled MNCs isolated from BM samples obtained from a CMML patient with pre-existing KRAS T58K and NRAS G12R mutations at diagnosis (n=1,826) and at BP after HMA therapy failure (n=4,001). BP was not associated with the clonal evolution of these mutations as they both had a VAF of approximately 50% at the onset of the disease. Each dot represents one cell. Cells are clustered based on immunophenotypic markers. Different colors represent cluster identity (left) or origin (right). Mono, monocytes; Ery, erythroblasts; cDC, classical dendritic cells; CD4T, CD4 + T-cells; MyHPC, myeloid hematopoietic progenitor cells; CD8T, CD8 + T-cells; NKC, natural killer cells. (B) Heatmap displaying DNA and protein reads from each sequenced cell type shown in . Colors for protein data correspond to antibody-oligonucleotide intensity signals. High protein expression is depicted in red and low protein expression is depicted in blue. DNA colors correspond to the genotypes for each individual mutation per cell read (wild-type=dark grey, mutant=red, missing=light grey) based on cluster. Percentages correspond to the frequencies of mutant reads within each cluster for a given mutation. C) UMAP of scDNA and protein-seq data for pooled MNCs isolated from BM samples obtained from a CMML patient at diagnosis (n=3,213) and at BP after HMA therapy failure (n=5,342). BP was associated with the clonal evolution of a pre-existing CBL F378Ifs mutation and the acquisition of a previously undetected CBL C384Y mutation. Each dot represents one cell. Cells are clustered based on immunophenotypic markers. Different colors represent cluster identity (left) or origin (right). Mono, monocytes; Ery, erythroblasts; DC, classical dendritic cells; CD4T, CD4 + T-cells; B-cell, B lymphocytes, myeloid hematopoietic progenitor cells; CD8T, CD8 + T-cells; NKC, natural killer cells. (D) Heatmap displaying DNA and protein reads from each sequenced cell type as shown in . Colors for protein data correspond to antibody-oligonucleotide intensity signals. Red indicates high protein expression, and blue indicates low protein expression. Colors for DNA data correspond to the genotype for each individual mutation per cell read (dark grey, wild type; red, mutant; light grey, missing) based on cluster. Percentages correspond to the frequencies of mutant reads within each cluster for a given mutation.

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Biomarker Discovery, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: (A) UMAP of scRNA-seq data for pooled single Lin − CD34 + cells isolated from BM samples of 2 HDs (n=895) and 5 CMML patients (n=3,161). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample origin (right). HSC, hematopoietic stem cells; eMyHPC, early myeloid progenitor cells; dMyHPC, differentiated myeloid progenitors; Ery/MkHPC, erythroid/megakaryocyte hematopoietic progenitor cells. Dashed lines indicate single clusters in each cell type population. (B) Distribution of HD (top) and CMML (bottom) Lin − CD34 + cell types among the clusters shown in . (C) Pathway enrichment analysis of the genes that were significantly upregulated in HSCs (left), eMyHPCs (middle), and dMyHPCs (right) from CMML samples compared with those from HD samples (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (D) UMAP of scRNA-seq data for pooled single MNCs isolated from BM samples of 3 HDs (n=9,896) and 5 CMML patients (n=9,319). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample origin (right). HSC, hematopoietic stem cells; MKP, megakaryocyte precursors; MyHPC, myeloid hematopoietic progenitor cells; Mono, monocytes; cDC, classical dendritic cells; pDC, plasmacytoid dendritic cells; Prog B, progenitor B-cells; PC, plasma cells; Pre-Ery, pre-erythrocytes; Ery-E, early erythroid precursors; Ery-L, late erythroid precursors; nCD4T, naïve CD4 + T cells; nmCD4T, naïve and memory CD4 + T cells; mCD4T, memory CD4 + T cells; nCD8T, naïve CD8 + T cells; nmCD8T, naïve and memory CD8 + T cells; eCD8T, effector CD8 + T cells; NKC, natural killer cells. Dashed lines indicate single clusters in each cell type population. (E) Distribution of HD (top) and CMML (bottom) cell types among the clusters shown in . (F) Pathway enrichment analysis of the genes that were significantly upregulated in the CMML monocyte clusters compared with those in the HD monocyte clusters shown in (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown.

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Clinical Proteomics

Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: (A) UMAP of scRNA-seq data for pooled single Lin − CD34 + cells isolated from BM samples of 5 CMML patients at diagnosis (n=1,840) and at BP after HMA therapy failure (n=1,711). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample origin (right). HSC, hematopoietic stem cells; eMyHPC, early myeloid hematopoietic progenitor cells; dMyHPC, differentiated myeloid hematopoietic progenitor cells; Ery/MkHPC, erythroid/megakaryocyte hematopoietic progenitor cells. Dashed lines indicate single clusters in each cell type population. (B) Distribution of Lin − CD34 + cell types at diagnosis (top) and BP (bottom) among the clusters shown in . (C) Pathway enrichment analysis of the genes that were significantly upregulated in HSCs (left) and dMyHPCs (right) at the time of BP after HMA therapy failure compared with those at diagnosis (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (D) UMAP of scATAC-seq data for pooled Lin − CD34 + cells isolated from BM samples obtained from a CMML patient at diagnosis (n=2,027) and at BP after HMA therapy failure (n=2,895). Each dot represents one cell. Different colors represent the cluster identity (left) or sample of origin (right). HSC, hematopoietic stem cells; MyHPC, myeloid progenitor cells; Ery/MkHPC, erythroid/megakaryocyte hematopoietic progenitor cells. (E) Pathway enrichment analysis of genes whose distal elements were enriched in open chromatin regions in HSCs (cluster 1, shown in ) at the time of BP as compared with those at diagnosis (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown.

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Biomarker Discovery

Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: (A) UMAP of scRNA-seq data for pooled single MNCs isolated from BM samples of 6 CMML patients at diagnosis (n=16,372) and at BP after HMA therapy failure (n=19,541). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample of origin (right). MyHPC, myeloid hematopoietic progenitors; My/MoP, myelo/monocytic progenitors; Mono, monocytes; cDC, classical dendritic cells; pDC, plasmacytoid dendritic cells; MKP, megakaryocyte precursors; Ery-E, early erythroid precursors; Ery-L, late erythroid precursors; B-cell, B lymphocytes; PC, plasma cells; nCD4T, naïve CD4 + T cells; mCD4T, memory CD4 + T-cells; eCD8T, effector CD8 T-cells, NKC, natural killer cells. Dashed lines indicate single clusters in each cell type population. (B) Distribution of MNC types at diagnosis (top) and (bottom) among the clusters shown in . (C) Pathway enrichment analysis of the genes that were significantly upregulated in the monocytic populations shown in the time of BP after HMA therapy failure compared with those at the time of diagnosis (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (D) Numbers of live Lin − CD34 + CD38 − HSCs and Lin − CD34 + CD38 + MyHPCs from CMML patients with BP after treatment with vehicle or 20 nM AMG-176 (n=4) for 48 h. Lines represent means ± SDs. Statistical significance was calculated using a two-tailed Student’s t- test (*** P <0.001; **** P <0.0001). (E) UMAP of scRNA-seq data for pooled single MNCs isolated from BM samples obtained from a representative CMML patient at the time of BP after HMA therapy failure (n=6,209) and after the failure of venetoclax-based therapy (n=6,795). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or the sample of origin (right). HSC, hematopoietic stem cells; MyHPC, myeloid hematopoietic progenitor cells; My/MoP, myelo/monocytic progenitors; Mono, monocytes; Ery/MkHPC, erythroid/megakaryocytic hematopoietic progenitor cells; Ery-E, early erythroid precursors; Ery-L, late erythroid precursors; Pre-E, pre-erythrocytes; mCD8T, memory CD8 + T cells; eCD8T, effector CD8 + T cells; NKC, natural killer cells. (F) Pathway enrichment analysis of the genes that were significantly upregulated in MyHPCs at the time of venetoclax failure compared with those at the time of HMA therapy failure (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (G) Distribution of myeloid cell types among the myeloid compartments at HMA therapy (top) and venetoclax-based therapy (bottom) failure. (H) Proposed working model of RAS pathway–mutated CMML initiation and progression after HMA and venetoclax-based therapies. Compared with physiological adult hematopoiesis (top left), RAS pathway–mutated CMML HSPCs undergo proliferation and monocytic differentiation in response to inflammatory responses while maintaining an intact apoptotic program. Inflammatory reprograming is exacerbated in downstream monocytic populations, which contributes to disease maintenance (bottom left). At BP after HMA therapy failure, RAS pathway–mutated CMML HSCs undergo epigenetic reprogramming and drive the expansion of downstream MyHPCs. MyHPCs and downstream monocytes rely on NF- K B signaling–mediated anti-apoptotic pathways to maintain survival and suppress the immune microenvironment (bottom right). NF- K B signaling–mediated survival pathway activation persists after venetoclax therapy and leads to treatment resistance and failure (top right).

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Biomarker Discovery, Clinical Proteomics, Two Tailed Test, Activation Assay

Journal: Stem Cells Translational Medicine

Article Title: Transient Inhibition of the JNK Pathway Promotes Human Hematopoietic Stem Cell Quiescence and Engraftment

doi: 10.1093/stcltm/szac019

Figure Lengend Snippet: Transient inhibition of the JNK pathway increased the HSC number in CB CD34 + cells. ( A ) Schematic of the experimental design. STF represents basic culture medium (StemSpan SFEM II supplemented with 100 ng/mL SCF, 50 ng/mL TPO, and 100 ng/mL Flt3L). Conditions that increased the frequency of Lin - CD34 + CD45RA − cells compared with culturing in basic culture medium were regarded as positive hits. ( B ) Bar plot showing the percentage of Lin − CD34 + CD45RA − cells in CB CD34 + cells cultured in medium supplemented with cytokines only (Ctrl), DMSO, AEG3482, SP600125, or JNK-IN-8 for 24 h. ( n = 3) ( C ) Representative FACS plots showing the expression of the indicated surface markers on DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. ( D and E ) Bar plot showing the frequencies of Lin − CD34 + CD45RA − ( D ) and Lin − CD34 + CD38 − CD45RA − ( E ) cells in DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. ( F ) Bar plot showing the Lin − CD34 + CD38 − CD45RA − cell count in 2 × 10 4 DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. ( G ) The frequency of engrafted human CD45 + cells in the PB of recipient mice receiving DMSO-treated, uncultured, or JNK-IN-8-treated CB CD34 + cells measured at 4-, 8-, and 12-weeks post-transplantation. ( H ) HSC frequencies presented as 1/uncultured CD34 + cell equivalent for DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells calculated with ELDA software at 8 weeks post-transplantation. The required CI was 95%. The cutoff for positive engraftment was set as more than 0.01% human CD45 + cells in the PB of the recipient ( n = 5 mice for each group). ( I ) Number of repopulating HSCs per 1 × 10 5 CD34 + cells in the DMSO-treated, uncultured, and JNK-IN-8-treated groups ( n = 5 mice for each group). See also ; . All data are shown as the mean value ± SD. Statistical significance was assessed using one-way ANOVA if not mentioned. ns, not significant; * P <.05; ** P < .01; *** P < .001.

Article Snippet: Human CB CD34 + cells were isolated with a CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s instructions and cultured in StemSpan SFEMII (Stem Cell Technologies) supplemented with recombinant human SCF (100 ng/mL, StemImmune LLC), recombinant human FLT3L (100 ng/mL, StemImmune LLC), recombinant human TPO (50 ng/mL, StemImmune LLC), and the indicated molecules.

Techniques: Inhibition, Cell Culture, Expressing, Cell Counting, Transplantation Assay, Software

Journal: Stem Cells Translational Medicine

Article Title: Transient Inhibition of the JNK Pathway Promotes Human Hematopoietic Stem Cell Quiescence and Engraftment

doi: 10.1093/stcltm/szac019

Figure Lengend Snippet: Transient inhibition of the JNK pathway increased the LT-HSC number during CB CD34 + cell isolation. ( A ) The frequency of engrafted human CD45 + cells in the PB of recipient mice receiving conventionally isolated (Ctrl) or JNK-IN-8-treated CB CD34 + cells measured at 4-, 8-, 12-, and 16-weeks post-transplantation. ( B – D ) Representative FACS plots showing human CD45 + cell engraftment in the PB ( B ), BM ( C ), and spleen ( D ) of recipient mice at 20 weeks after transplantation of conventionally isolated (Ctrl) or JNK-IN-8-treated CB CD34 + cells. ( E ) HSC frequencies presented as 1/uncultured CD34 + cell equivalent for conventionally isolated (Ctrl) and JNK-IN-8-treated CB CD34 + cells calculated with ELDA software at 20 weeks post-transplantation. The required CI was 95%. The cutoff for positive engraftment was set as more than 0.1% human CD45 + cells in the BM of the recipient ( n = 15 mice for each group with 3 independent experiments, **** P < .0001). ( F ) Number of LT-HSCs per 1 × 10 5 cells in conventionally isolated (Ctrl) and JNK-IN-8-treated CB CD34 + cells. See also and .( G ) Representative FACS plots showing human B (CD19 + ) and myeloid cell (CD33/CD14/CD11b + ) repopulation in recipient BM (gated in hCD45 + ) at 20 weeks post-transplantation. ( H ) Bar plot showing the lineage distribution of engrafted human CD45 + cells in recipient BM. B, CD19 + B cells; M, CD33 + /CD14 + /CD11b + myeloid cells; n = 5. ( I ) Representative FACS plots showing human T-cell (hCD45 + CD3 + ) repopulation in the recipient thymus at 20 weeks post-transplantation. ( J ) Level of human CD45 + cell engraftment in the PB of 2° recipients at the indicated doses of BM cells from primary recipients at 14 weeks post-transplantation. ( K ) 2° SRC frequencies in the BM of the primary recipients receiving conventionally isolated (Ctrl) and JNK-IN-8-treated CB CD34 + cells calculated with ELDA software at 14 weeks post-transplantation. The required CI was 95%. The cutoff for positive engraftment was set as more than 0.01% human CD45 + cells in the PB of the recipient ( n = 5 mice for each group, *** P < .001). ( L ) Number of 2° SRC per 1 × 10 7 cells in conventionally isolated (Ctrl) and JNK-IN-8-treated CB CD34 + cells. See also and . All data are shown as the mean value ± SD. Statistical significance was assessed using one-way ANOVA if not mentioned. ns, not significant; * P < .05; ** P < .01; *** P < .001; **** P < .0001.

Article Snippet: Human CB CD34 + cells were isolated with a CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s instructions and cultured in StemSpan SFEMII (Stem Cell Technologies) supplemented with recombinant human SCF (100 ng/mL, StemImmune LLC), recombinant human FLT3L (100 ng/mL, StemImmune LLC), recombinant human TPO (50 ng/mL, StemImmune LLC), and the indicated molecules.

Techniques: Inhibition, Cell Isolation, Isolation, Transplantation Assay, Software

Journal: Stem Cells Translational Medicine

Article Title: Transient Inhibition of the JNK Pathway Promotes Human Hematopoietic Stem Cell Quiescence and Engraftment

doi: 10.1093/stcltm/szac019

Figure Lengend Snippet: Transient inhibition of the JNK pathway promoted HSC quiescence, preventing HSCs from undergoing cell cycle entry and metabolic activation. ( A ) Heatmap showing gene expression in DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. ( B ) Expression by RNA-seq of the indicated genes in DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. The fpkm value for uncultured cells is normalized to 1.0; n = 2. ( C and D ) GSEA plots showing enrichment of quiescent-HSC-enriched ( C ) and 120 HSC-associated ( D ) gene sets in the indicated groups. DMSO, DMSO-treated CB CD34 + cells; uncultured, uncultured CB CD34 + cells; JNK-IN-8, JNK-IN-8-treated CB CD34 + cells. Each group contained 2 replicates. ( E ) Bar plot showing the cell cycle status of DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells ( n = 3). ( F and G ) Representative FACS plots ( F ) and bar plot ( G ) showing glucose uptake activity (indicated by the fluorescence intensity of 2-NBDG) of DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells ( n = 3). ( H and I ) Representative FACS plots ( H ) and bar plot ( I ) showing ROS levels (indicated by the fluorescence intensity of DCFDA) of DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells ( n = 3). ( J ) Schematic of the proposed model demonstrating how transient JNK inhibition regulates CB HSPC engraftment through its roles in quiescence and stemness. All data are shown as the mean value ± SD. Statistical significance was assessed using one-way ANOVA if not mentioned. ns, not significant; ** P <.01; *** P < .001; **** P < .0001.

Article Snippet: Human CB CD34 + cells were isolated with a CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s instructions and cultured in StemSpan SFEMII (Stem Cell Technologies) supplemented with recombinant human SCF (100 ng/mL, StemImmune LLC), recombinant human FLT3L (100 ng/mL, StemImmune LLC), recombinant human TPO (50 ng/mL, StemImmune LLC), and the indicated molecules.

Techniques: Inhibition, Activation Assay, Gene Expression, Expressing, RNA Sequencing, Activity Assay, Fluorescence

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ cells from three SCD patient donors were electroporated with ABE8e-NRCH mRNA or RNP using an sgRNA targeting the SCD mutant HBB codon. (a) The edited region of HBB with the target A at protospacer position 7 shown in blue along with potential bystander edits in green (silent), brown (silent), and red (non-silent). (b) Editing efficiencies by HTS at target and bystander adenines, and indels after 6 days in stem-cell culture media following electroporation. (c) Proportion of β-like globin proteins by HPLC of reticulocyte lysates after 18 days in differentiation media following electroporation. (d) Representative phase-contrast images of reticulocytes derived from unedited or edited donor HSPCs incubated 8 hours in 2% O2. Nine images of >50 cells each were collected per sample. Scale bar=50 μm. (e) Quantification of sickled reticulocytes from counting >300 randomly selected cells by a blinded observer from images as in (d). (f) Venn diagram showing candidate off-target sites nominated by Cas-OFFinder and CIRCLE-seq, and nominated sites for which off-target editing was observed by targeted DNA sequencing in SCD patient CD34+ cells electroporated with ABE8e-NRCH mRNA. (g) Predicted genomic features of validated off-target sites. TTS, ≤1 kb from the transcription termination site; UTR, untranslated region. (h) ABE8e-NRCH-treated HSPCs from two different SCD patient donors were sequenced at 697 potential off-target sites. The histogram shows the number of validated off-target base editing sites binned by average percentage of sequencing reads for each site with any A•T-to-G•C mutations in protospacer nucleotides 4-10. Bar values in (b), (c), and (e) and error bars reflect mean±SD of three independent biological replicates, with individual values shown as dots.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Mutagenesis, Stem Cell Culture, Electroporation, Derivative Assay, Incubation, DNA Sequencing, Sequencing

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: Representative, immuno-flow cytometry for erythroid maturation stage markers42,43 at culture days 7 and 14. Top: gating strategy to identify single cells expressing the erythroid marker hCD235a. Bottom: gating strategy to track the progress of erythroid maturation based on expression of CD49D and Band3 in hCD235a+ cells. SSC-A: Side scatter area. SSC-W: Side scatter width. FSC-A: Forward scatter area.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Flow Cytometry, Expressing, Marker

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: Reverse-phase HPLC chromatograms of erythroid cell lysates at culture day 18, with β-like globins and their associated fractions marked near the associated peak. Data from the most efficiently edited donor is shown. Red arrows indicate the start and end of globin chain peaks.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: High Performance Liquid Chromatography, Derivative Assay, In Vitro

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CIRCLE-seq read counts obtained for each verified off-target site and the alignment of each site to the guide sequence are shown. Bar graphs show the percentage of sequencing reads containing A•T-to-G•C mutations within protospacer positions 4-10 at on-and off-target sites in genomic DNA samples from patient CD34+ HSPCs treated with ABE8e-NRCH mRNA, protein, or untreated controls (n=4). Note that the mutation frequency shown is summed across all reads with one or more A•T-to-G•C mutations in this window. Sequencing errors therefore accumulate in control samples compared to standard sequencing error frequencies for a single nucleotide. Bar values and error bars reflect mean±SD.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Sequencing, Mutagenesis, Control

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: Bar graph showing the percentage of sequencing reads containing alleles harboring indels at on-and off-target sites in genomic DNA samples from patient CD34+ HSPCs treated with ABE8e-NRCH mRNA, protein, or untreated controls (n=4). Bar values and error bars reflect mean±SD.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Sequencing

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ HSPCs from three HBBS/S SCD patient donors were electroporated with ABE8e-NRCH mRNA and sgRNA targeting the SCD mutant HBB codon. 2-5x105 treated cells were transplanted into NBSGW mice via tail-vein injection. Mice were analyzed 16 weeks after transplantation. (a) Experimental workflow. (b) Engraftment measured by percentage of human CD45+ (hCD45+) cells in recipient mouse bone marrow. (c) Human B-cells (hCD19+), myeloid cells (hCD33+), and T-cells (hCD3+) cells in recipient mouse bone marrow shown as percentages of the hCD45+ population. (d) Human erythroid precursors (hCD235a+) in recipient mouse bone marrow shown as percentage of human and mouse CD45− cells, (e) HBBS-to-HBBG editing efficiencies in human donor CD34+ cell-derived lineages from recipient bone marrow. Erythroid, myeloid, B-cell, and HSPC human lineages were collected using antibodies that recognize hCD235a, hCD33, hCD19, and hCD34, respectively, (f) Clonal editing outcomes determined by single-cell 5’ RNA-seq in CD235a+ cells from the bone marrow of two edited mice. (g) Proportions of β-like globin proteins by HPLC of human donor-derived reticulocytes isolated from recipient mouse bone marrow. (h) Representative phase-contrast images of human reticulocytes from bone marrow incubated 8 hours in 2% O2. Nine images of >50 cells each were collected per sample. Scale bar=50 μm. (i) Quantification of sickled cells as in Fig. 1e. n=14 mice receiving edited cells and n=13 mice receiving unedited cells in b-e, g, and i. Triangle, square, and circle symbols represent HSPCs from three different SCD donors. Plotted values and error bars reflect mean±SD. Statistical significance was assessed by one-way ANOVA in i and by two-tailed Student’s t-test elsewhere; “ns”, not significant.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Mutagenesis, Injection, Transplantation Assay, Derivative Assay, RNA Sequencing, Isolation, Incubation, Two Tailed Test

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ HSPCs from three HBBS/S SCD patient donors were electroporated with ABE8e-NRCH RNP using a single guide RNA (sgRNA) targeting the SCD mutant codon, followed by transplantation of 2-5x105 treated cells into NBSGW mice via tail-vein injection. Mice were sacrificed and analyzed 16 weeks after transplantation, (a) Experimental workflow, (b) Engraftment measured by the percentage of human donor CD45+ cells (hCD45+ cells) in recipient mouse bone marrow, (c) Human B-cells (hCD19+), myeloid cells (hCD33+), and T-cells (hCD3+) cells in recipient mouse bone marrow, shown as percentages of the total hCD45+ population. (d) Human erythroid precursors (hCD235a+) in recipient mouse bone marrow shown as percentage of total human and mouse CD45−cells. (e) On-target (A7, Fig. 1a) editing efficiencies in human donor CD34+ cell-derived lineages purified from recipient bone marrow by fluorescence-activated cell sorting. Erythroid, myeloid, B-cell, and HSPC human lineages were collected using antibodies that recognize hCD235a, hCD33, hCD19, and hCD34+, respectively. Statistical significance was assessed by one-way ANOVA to compare groups; “ns”, not significant. (f) Percentages of β-like globin proteins determined by reverse-phase HPLC analysis of human donor-derived reticulocytes isolated from recipient mouse bone marrow. (g) Representative phase contrast images of human reticulocytes purified from bone marrow and incubated for 8 hours in 2% O2. Nine images of >50 cells per image were collected per sample. Scale bar=50 μm. (h) Quantification of sickled cells calculated by counting images after incubation for 8 hours in 2% O2 such as in (g). More than 300 randomly selected cells per sample were counted by a blinded observer. n=14 total mice analyzed in panels b-f; triangle, square, and circle symbols represent samples from three different SCD CD34+ HSPC donors. Negative control data is shared with Figure 2. Bar values and error bars reflect mean±SD. Statistical significance between treated and untreated samples was assessed by a two-tailed Student’s t-test; “ns”, not significant.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Mutagenesis, Transplantation Assay, Injection, Derivative Assay, Purification, Fluorescence, FACS, Isolation, Incubation, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: Ponatinib inhibits LCK and PI3K signaling and promotes CD8 + T stem cell memory cell development

doi: 10.1038/s41467-026-71375-2

Figure Lengend Snippet: a , b Proliferation inhibition and apoptosis induction by ponatinib. CD8 + T cells and regulatory T (Treg) cells purified from the peripheral blood mononuclear cells (PBMC) of healthy individuals were stimulated with anti-CD3/CD28 monoclonal antibodies (mAb) along with graded doses of ponatinib for 4 days. Proliferation and apoptosis were assessed by Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution and by Annexin V and Fixable Viability Dye staining, respectively. a Experimental scheme. Created in BioRender. https://BioRender.com/cxg8twg . b Representative histograms (left) and frequencies of proliferating cells and apoptotic cells (right) ( n = 3 donors per group). The gray dashed lines indicate the trough level of ponatinib. c–f PBMCs purified from healthy individuals were cultured with dimethyl sulfoxide (DMSO; control), imatinib, or ponatinib and analyzed by flow cytometry on Days 3 and 9. c Experimental scheme. Created in BioRender. https://BioRender.com/cxg8twg . d Representative contour plots (CD45RA and FOXP3) of CD4 + T cells in the DMSO (left, black), imatinib (left, blue), and ponatinib (left, red) groups and cell numbers of effector Treg (eTreg, CD4 + CD45RA − FOXP3 hi ) cells ( n = 6 donors per group). e Cell numbers of CD8 + T cells ( n = 6 donors per group). f Representative contour plots (CCR7 and CD45RA) of CD8 + T cells treated with DMSO (left, black), imatinib (left, blue), or ponatinib (left, red) and frequencies of naive (CCR7 + CD45RA + ) and effector memory (T EM : CCR7 − CD45RA − ) subsets among CD8 + T cells (right) ( n = 6 donors per group). Data in ( d – f ) were normalized to those of the DMSO control (set to 1.0). Data are presented as the mean ± SEM ( b , d – f ). Significance was assessed using two-sided paired t tests ( b , d – f ). All experiments were performed independently at least two times with similar results.

Article Snippet: CD8 + T cells and Treg cells (CD4 + CD25 + ) were purified from the PBMCs of healthy individuals using a CD8 MicroBeads (Miltenyi Biotec, #130-045-201, Bergisch Gladbach, Germany) and a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, #130-091-301), respectively, with an autoMACS separator (Miltenyi Biotec) according to the manufacturers’ instructions.

Techniques: Inhibition, Purification, Bioprocessing, Staining, Cell Culture, Control, Flow Cytometry

Journal: Nature Communications

Article Title: Ponatinib inhibits LCK and PI3K signaling and promotes CD8 + T stem cell memory cell development

doi: 10.1038/s41467-026-71375-2

Figure Lengend Snippet: a – d Murine CD3 + T cells purified from splenocytes were stimulated with anti-CD3/CD28 monoclonal antibodies (mAb) for 48 h along with dimethyl sulfoxide (DMSO; control) or ponatinib and analyzed by flow cytometry. a Experimental scheme. Created in BioRender. https://BioRender.com/cxg8twg . b t-SNE plots were generated to reduce all the phenotypic data to 2 dimensions (left), and DMSO (right, black) and ponatinib (right, red) treatments were applied. tSNE: t-distributed stochastic neighbor embedding ( n = 3 mice per group). c Representative contour plots (CD62L and CD44) of CD8 + T cells treated with DMSO (left, black) or ponatinib (left, red) and frequencies of naive (CD44 − CD62L + ), central memory (T CM : CD44 + CD62L + ), and effector memory (T EM : CD44 + CD62L − ) subsets among CD8 + T cells (right) ( n = 3 mice per group). d Absolute numbers of regulatory T (Treg) cells (CD4 + Foxp3 + CD25 high ) and frequencies of CTLA-4 + cells among Treg cells (right) ( n = 3 mice per group). e – j Murine naive CD8 + T cells purified from splenocytes by magnetic separation were stimulated with anti-CD3/CD28 mAbs for 48 h along with DMSO or ponatinib and subjected to RNA sequencing analysis (RNA-seq) and flow cytometry analysis. e Experimental scheme. Created in BioRender. https://BioRender.com/cxg8twg . f Gene set enrichment analysis (GSEA) plots of gene sets upregulated in naive versus effector CD8 + T cells (left) and in memory versus effector CD8 + T cells (right) for the ponatinib-treated group compared with the DMSO group ( n = 3 mice per group). ES: enrichment score; FDR: false discovery rate. g Heatmap of the expression of CD8 + T cell phenotype related genes, such as naive/memory, activation/effector, exhaustion, and cytotoxicity genes ( n = 3 mice per group). h Gating strategy for murine CD8 + T stem cell memory (T SCM : CD8 + CD44 − CD62L + Sca-1 + ) cell (left) and absolute numbers of T SCM cells ( n = 7 mice per group) (right). i Frequencies of IL-7Rα + naive subset in CD8 + T cells ( n = 6 mice per group). j Expression of Tcf1 and Tox on CD8 + T cells ( n = 6 mice per group) MFI, mean fluorescence intensity. Data are presented as the mean ± SEM ( c , d , h – j ). Significance was assessed using two-sided unpaired t tests ( c , d , h–j ). All experiments were performed independently at least two times with similar results.

Article Snippet: CD8 + T cells and Treg cells (CD4 + CD25 + ) were purified from the PBMCs of healthy individuals using a CD8 MicroBeads (Miltenyi Biotec, #130-045-201, Bergisch Gladbach, Germany) and a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, #130-091-301), respectively, with an autoMACS separator (Miltenyi Biotec) according to the manufacturers’ instructions.

Techniques: Purification, Bioprocessing, Control, Flow Cytometry, Generated, RNA Sequencing, Expressing, Activation Assay, Fluorescence

Journal: Nature Communications

Article Title: Ponatinib inhibits LCK and PI3K signaling and promotes CD8 + T stem cell memory cell development

doi: 10.1038/s41467-026-71375-2

Figure Lengend Snippet: a , b Wild-type C57BL/6 J mice were subcutaneously (s.c.) inoculated with MC38 cells or B16F10 cells and treated with ponatinib or 1% 2-hydroxypropyl-beta-cyclodextrin (Dex; control) and/or an anti-PD-1 monoclonal antibodies (mAb). a Experimental scheme. p.o., oral administration. Created in BioRender. https://BioRender.com/cxg8twg . b Tumor growth curves of MC38 tumor-bearing mice (left) and B16F10 tumor-bearing mice (right) (MC38: Dex, n = 9; ponatinib, n = 9; Dex + anti–PD-1 mAb, n = 8; ponatinib + anti-PD-1 mAb, n = 8; B16F10: Dex, n = 8; ponatinib, n = 9; Dex + anti–PD-1 mAb, n = 9; ponatinib + anti-PD-1 mAb, n = 8; all n -values represent the number of individual mice). c RAG2 knockout (KO) mice or nude mice were subcutaneously inoculated with MC38 cells and treated with Dex or ponatinib. Tumor growth curves of RAG2 KO mice (left; Dex, n = 8 mice; ponatinib, n = 7 mice) and nude mice (right; Dex, n = 10 mice; ponatinib, n = 11 mice). d Wild-type C57BL/6 J mice were subcutaneously inoculated with MC38 cells and treated with Dex or ponatinib. Some mice received intraperitoneal injections of anti-CD8β mAbs (CD8 + T cell depletion model) or anti-CD4 mAbs (CD4 + T cell depletion model) ( n = 5 mice per group). Tumor growth curves of mice depleted of CD8 + T cells (left) or CD4 + T cells (right). Dex + Anti-CD8β mAb, mice treated with Dex and anti- CD8β mAb; Ponatinib + Anti-CD8β mAb, mice treated with ponatinib and anti-CD8β mAb; Dex + Anti-CD4 mAb, mice treated with Dex and anti- CD4 mAb; Ponatinib + Anti-CD4 mAb, mice treated with ponatinib and anti-CD4 mAb. e Wild-type or Foxp3-DTR C57BL/6 J mice were subcutaneously inoculated with MC38 cells and treated with Dex or ponatinib in addition to diphtheria toxin (DT) administration (regulatory T (Treg) cell depletion model). Tumor growth curves of the Treg cell depletion model ( n = 4 mice per group). Dex-Foxp3-DTR: Foxp3-DTR mice treated with Dex. Ponaitnib-Foxp3-DTR: Foxp3-DTR mice treated with ponatinib. Dex-WT: wild-type mice treated with Dex. Ponatinib-WT: wild-type mice treated with ponatinib. The average tumor size of the groups on a certain day is shown as a dot ( b–e ). Data are presented as the mean ± SEM ( b – e ). Significance was assessed using two-way ANOVA with Tukey’s multiple comparison test ( b – e ). All experiments were performed independently at least two times with similar results.

Article Snippet: CD8 + T cells and Treg cells (CD4 + CD25 + ) were purified from the PBMCs of healthy individuals using a CD8 MicroBeads (Miltenyi Biotec, #130-045-201, Bergisch Gladbach, Germany) and a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, #130-091-301), respectively, with an autoMACS separator (Miltenyi Biotec) according to the manufacturers’ instructions.

Techniques: Control, Bioprocessing, Knock-Out, Comparison

Journal: Nature Communications

Article Title: Ponatinib inhibits LCK and PI3K signaling and promotes CD8 + T stem cell memory cell development

doi: 10.1038/s41467-026-71375-2

Figure Lengend Snippet: a – h Wild-type C57BL/6 J mice were subcutaneously (s.c.) inoculated with MC38 cells and treated with ponatinib or 1% 2-hydroxypropyl-beta-cyclodextrin (Dex; control). On Day 8, the mice were euthanized, and the tumor-infiltrating lymphocytes (TIL) were collected for the flow cytometry analysis. a Experimental scheme. p.o., oral administration. Created in BioRender. https://BioRender.com/cxg8twg . b Absolute numbers (per 1 g of tumor) of CD8 + TILs with the T stem cell memory (T SCM ) phenotype (CD8 + CD44 − CD62L + Sca-1 + ) ( n = 12 mice per group). c Frequencies of Tcf1 + and Bcl-2 + cells among CD8 + TILs ( n = 8 mice per group). d Ratios of Eomes to T-bet in CD8 + TILs ( n = 8 mice per group). MFI, mean fluorescence intensity. e Frequencies of Tox + cells in CD8 + TILs ( n = 6 mice per group). f Ratios of T SCM cells to regulatory T (Treg; CD4 + CD25 hi Foxp3 hi ) cells ( n = 12 mice per group). g Representative contour plots (IFN-γ and TNF) of CD8 + TILs (left, Dex in black; right, ponatinib in red). h Frequencies of TNF- and IFN-γ-, TNF- and IL-2-, IFN-γ- and granzyme B (GZMB)-producing cells among CD8 + TILs ( n = 11 mice per group). i – l Wild-type C57BL/6 J mice were subcutaneously inoculated with MC38-OVA cells and treated with ponatinib or Dex. The mice were euthanized on Day 8 (early phase) or when the tumor volume reached 1000 mm 3 (late phase), and the TILs were collected for the flow cytometry analysis. i Experimental scheme. Created in BioRender. https://BioRender.com/cxg8twg . j Representative contour plots; left, CD8 and OVA dextramer (total CD8 + TILs); middle, CD62L and CD44 (antigen-specific CD8 + TILs (CD8 + OVA dextramers + )); right, CD62L and Sca-1 (antigen-specific naive CD8 + TILs, CD8 + OVA dextramers + CD44 − CD62L + ) in the DMSO group (black) and ponatinib group (red). k Absolute numbers (per 1 g of tumor) of antigen-specific T SCM cells (CD8 + OVA dextramers + CD44 − CD62L + Sca-1 + ) in the early phase (Dex, n = 7 mice; ponatinib, n = 8 mice). l Representative contour plots (CD8 and OVA dextramer) of effector CD8 + TILs (CD8 + CD44 + ) in the Dex (black) and ponatinib groups (red) (left) and absolute numbers (per 1 g of tumor) of antigen-specific effector CD8 + TILs (CD8 + CD44 + OVA dextramers + ) in the late phase ( n = 6 mice per group). In box plots ( b , f , h ), the center line indicates the mean, the box represents ± SEM, and whiskers indicate the minimum and maximum values. In dot plots ( c – e ), horizontal lines indicate the mean. In bar graphs ( k , l ), bars represent the mean ± SEM. Significance was assessed using two-sided Mann–Whitney U -test ( b – f , h , k , l ). All experiments were performed independently at least two times with similar results.

Article Snippet: CD8 + T cells and Treg cells (CD4 + CD25 + ) were purified from the PBMCs of healthy individuals using a CD8 MicroBeads (Miltenyi Biotec, #130-045-201, Bergisch Gladbach, Germany) and a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, #130-091-301), respectively, with an autoMACS separator (Miltenyi Biotec) according to the manufacturers’ instructions.

Techniques: Control, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: Nature Communications

Article Title: Ponatinib inhibits LCK and PI3K signaling and promotes CD8 + T stem cell memory cell development

doi: 10.1038/s41467-026-71375-2

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMC) from patients with chronic myeloid leukemia (CML) treated with either imatinib, dasatinib, or ponatinib were collected at trough levels and analyzed by flow cytometry (imatinib, n = 6 samples; dasatinib, n = 4 samples; ponatinib, n = 5 samples). a Experimental scheme. Created in BioRender. https://BioRender.com/cxg8twg . b Representative contour plots (left, CCR7 and CD45RA; right, CD95 and CD62L) of CD8 + T cells and naive CD8 + T cells (CD8 + CD45RA + CCR7 + ), respectively, of the imatinib (blue), dasatinib (green), and ponatinib (red) groups. c Frequencies of T SCM cells (CCR7 + CD45RA + CD62L + CD95 + ) among CD3 + T cells. d Representative contour plots (CD45RA and FOXP3) of CD4 + T cells in the imatinib (blue), dasatinib (green), and ponatinib (red) groups (left) and frequencies of effector Treg cells (eTreg, CD4 + CD45RA − FOXP3 hi ) among CD3 + T cells (right). e Ratios of T SCM cells to Treg cells. f Ratios of CD8 + T cells to Treg cells. Data are presented as the mean ± SEM ( c – f ). Significance was assessed using two-sided Mann–Whitney U -test ( c – f ).

Article Snippet: CD8 + T cells and Treg cells (CD4 + CD25 + ) were purified from the PBMCs of healthy individuals using a CD8 MicroBeads (Miltenyi Biotec, #130-045-201, Bergisch Gladbach, Germany) and a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, #130-091-301), respectively, with an autoMACS separator (Miltenyi Biotec) according to the manufacturers’ instructions.

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A . Pulmonary CT scans from SAVI patients (SAVI 1 and SAVI 5) showing fibrotic regions. Additional scans are shown in Figure S1A. B . Pathological characterization of SAVI lung tissue. Left panels: H&E and Masson-Trichrome staining of alveolar regions (SAVI 5) and controls (Ctrl), highlighting alveolar wall thickening and alveolar capillary fibrosis (orange box and magnified insets). Scale bar: 150 µm. Right panel: Quantification of inflammation and fibrosis scores (n=7 SAVI patients, n=5 controls). Mann-Whitney test; *, 0.01

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: Staining, MANN-WHITNEY, Multiplex Assay, Control, Labeling, Fluorescence, Two Tailed Test, Double Staining, Gene Expression

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Experimental workflow and group design (created with BioRender.com ). B. Flow cytometric analysis of endothelial surface markers in HC, SAVI, and isogenic–SAVI (iso-SAVI) iECs. SAVI iECs progressively lost CD144 (VE-cadherin) and CD31 beginning at P3. Data summarize four HC- and SAVI-derived iEC lines and two iso-SAVI lines (mean ± SEM; ***p < 0.001, 2-way ANOVA). Representative flow cytometry profiles are shown in Figure S2C. C. Morphology and tube-formation capacity of iECs. Upper: HC and iso-SAVI iECs maintained cobblestone morphology from P1 to P5, whereas SAVI iECs transitioned to elongated fibroblast-like cells. Scale bar: 100 µm. Lower: Tube-formation assays performed at passages 1, 3, and 5; representative P5 images are shown (P1 images in Figure S2B). Total branch length and mesh area comparisons across groups are shown on the right (mean ± SEM; ***p < 0.001, **p < 0.01, *p < 0.05; 2-way ANOVA). Scale bar: 200 µm. D. Western blot analysis of endothelial (VE-cadherin, CD31) and mesenchymal (SMA, SM22) markers in iECs at P5. iECs were generated from three HC and three SAVI donors, with two iso-SAVI lines (three clones total). Representative blots and quantification (mean ± SEM; Mann–Whitney test) are shown. GAPDH served as a loading control. E. Schematic illustration summarizing panel D.

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: Derivative Assay, Flow Cytometry, Western Blot, Generated, Clone Assay, MANN-WHITNEY, Control

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Top pathways enriched in SAVI P5 vs. P1 (iso-SAVI_SAVI cohort) by Ingenuity Pathway Analysis (IPA). Positive z-scores indicate pathway activation. Additional HC vs. SAVI analysis is shown in Figure S3A–B. B . EndMT signature heatmap in parental SAVI and isogenic iECs, with box-and-whisker plots of representative endothelial, mesenchymal, and previously reported EndMT-associated genes. Asterisks mark genes upregulated under TGFβ-induced EndMT but downregulated in SAVI iECs. C. IPA-derived transcription factor network of significant upstream regulators in the iso-SAVI_SAVI cohort (orange = activated; blue = inhibited). D . Heatmap of activation z-scores for the same transcription factors in panel C, showing similar patterns in SAVI vs. HC iECs and SAVI vs. HC lung tissue. E . Constitutive STAT3 nuclear translocation in SAVI iECs. STAT3 immunofluorescence (red) with DAPI (blue) at P5 from one SAVI and matched isogenic control line; white arrows indicate nuclear STAT3 (scale bar: 20 µm). Quantification reflects STAT3⁺ nuclei per total DAPI⁺ cells across 7–11 fields from 3–4 wells (mean ± SEM; *p < 0.05, two-tailed unpaired t-test). F . STAT3 activation in SAVI lung biopsies. Lung sections from 4 HC and 3 SAVI patients were stained for p-STAT3 Y705 (red) and CD31 (green) with DAPI (blue). Representative images (scale bar: 20 µm) and quantification of p-STAT3 Y705 /DAPI ratios (≥5 images per patient) show increased STAT3 activation in SAVI (mean ± SEM; ***p < 0.001, two-tailed unpaired t-test).

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: Activation Assay, Whisker Assay, Derivative Assay, Translocation Assay, Immunofluorescence, Control, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Workflow showing ATAC-seq, motif enrichment and pathway analyses. B . Heatmap of differentially accessible regions in HC and SAVI iECs at P1 and P3 (ATAC-seq; chi-square with Yates correction). C. Motif enrichment in regions differentially accessible between SAVI P1 and SAVI P3. Motifs enriched in the 43,995 SAVI P1–open regions appear on the left; motifs enriched in the 69 SAVI P3–open regions appear on the right. Additional comparisons are in Figure S5B. D. ATAC-seq in HLMECs stimulated with 2’3’-cGAMP or IFNβ for 8 h. Sankey plot shows the number of gained and lost accessible regions relative to non-treated (NT) cells. E. The enrichment of transcription factor binding motifs within the differentially accessible regions between the non-stimulated (NT), 2’3’-cGAMP or IFNβ stimulated HLMEC ATAC-seq libraries. Motif enrichment in regions more accessible after cGAMP stimulation in HLMECs (1,868 regions; none were more open in NT cells). Motif enrichment within the 1025 regions with increased accessibility in the IFNβ stimulated condition is shown on the right side of the y = 0 line. Cloud color as in B grey cloud indicates IRF TF motifs that are becoming increasingly accessible. F. These plots show the results of permutation tests from the regioneR package for the overlap between SOX18 ChIP-seq peaks from the Overman et al dataset and the SAVI P1 to SAVI P3 closing regions, or the HC P1 to HC P3 closing regions. A differential permutation test was also conducted to compare the relative enrichment of SOX18 ChIP-seq peaks in these two sets of regions. The green bar indicates the observed number of overlaps between the datasets. The black bar indicates the mean value of overlaps between the transcription factor ChIP-seq peaks and the random permuted regions. the distribution of overlaps from the permutations are shown as the gray histogram). The red bar indicates the number of overlaps at the threshold of significance p = 0.05. Differential enrichment analysis (bottom plot) showed significant overrepresentation of SOX18 binding sites in SAVI closing CRs at P3 (273 regions, z = 29.54 ; p < 0.001). G . The pathway enrichment of genes annotated by GREAT to differentially accessible regions from the SAVI iEC P1 to SAVI iEC P3 conditions that exhibited a decrease in accessibility in the P3 cells. These regions (and annotated genes) were filtered by the regions that overlapped SOX18, JUN, FOS, and GATA2 ChIP-seq peaks from the HUVEC ChIP-seq data sets listed in the methods section. H. Gene expression changes of endothelial lineage transcription factors across SAVI vs. iso-SAVI iECs (P1, P5) and cGAMP-treated vs. NT HLMECs (d1, d5) by RNAseq analysis. Red points indicate higher expression in SAVI or cGAMP conditions; endothelial TFs are starred. I. Reduced SOX18 protein in SAVI iECs at P5 compared with HC and iso-SAVI lines (3 individual iEC lines for each group), and partial restoration (∼50%) following STING inhibitor treatment (IFM35883, 2.5 μM, P2–P5) in SAVI iECs (SAVI1, n = 3). GAPDH served as loading control. Data are represented as mean ± SEM; ***p < 0.001, **p < 0.01, two-tailed unpaired t-test. J. SOX18 downregulation in HC iECs following 20 μg/ml 2’3’-cGAMP stimulation (30 min–24 h), peaking at 8 h (∼50% reduction). Quantification from three HC donors (mean ±SEM, ***p < 0.001, two-tailed unpaired t-test). K. SOX18 overexpression preserves endothelial surface markers CD144 and CD31 in SAVI iECs at P5. SAVI iECs were transduced at P2 with control or SOX18 cDNA; Flow cytometry analysis at P5 shows increased CD144⁺ and CD31⁺cells in SOX18 overexpression group. Data are represented as mean ± SEM; ***p < 0.001, **p < 0.01, two-tailed unpaired t-test. (n = 4, SAVI1 iEC line).

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: Binding Assay, ChIP-sequencing, Gene Expression, RNA sequencing, Expressing, Control, Two Tailed Test, Over Expression, Flow Cytometry

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Schematic of drug testing in the SAVI in-vitro disease model. B. Partial rescue of SAVI iEC EndMT by the FDA-approved JAK/STAT inhibitor Baricitinib. SAVI iECs were treated with Baricitinib (1 μM) from P2 to P3–4 (early harvest due to toxicity). Flow cytometry analysis of CD144 (VE-cadherin) and CD31 showed modest preservation of endothelial markers versus DMSO. Data from three SAVI donors (mean ± SEM; *p < 0.05; two-tailed t-test). Effects in cGAMP-treated HLMECs are shown in Fig. S6A–C. C. Impact of STING inhibition and FDA-approved antifibrotic drugs on EC surface markers. SAVI iECs were treated from P2 with IFM35883 (2.5 μM), pirfenidone (10 μM), nintedanib (1 μM), or DMSO. STING inhibition nearly fully preserved CD144 (VE-cadherin) and CD31, pirfenidone had no effect, and nintedanib markedly worsened EndMT. Representative flow cytometry profiles in Fig. S6E. Data from ≥3 experiments in the SAVI1 line (mean ± SEM; *****p < 0.0001; 2-way ANOVA). D. STING inhibition normalizes EndMT markers in SAVI iECs. SAVI iECs treated with IFM35883 (2.5 μM) or DMSO from P2–P5 were analyzed by Western blot. VE-cadherin and CD31 increased, while SMA and SM22 decreased with STING inhibition. Quantification from three experiments in SAVI1; representative blots shown (mean ± SEM; ***p < 0.001, **p < 0.01; two-tailed unpaired t-test).

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: In Vitro, Flow Cytometry, Preserving, Two Tailed Test, Inhibition, Western Blot

Journal: Pharmaceutics

Article Title: Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy

doi: 10.3390/pharmaceutics15041074

Figure Lengend Snippet: Thalidomide and its analogs inhibit Treg expansion mediated by TNF in vitro. MACS was used to purify CD4 + T cells from lymphocytes. CD4 + T cells were stimulated in the presence of IL-2 (10 ng/mL), with or without TNF (20 ng/mL), for 72 h. Thalidomide and its analogs with two concentrations, 50 μM and 100 μM, with or without TNF (20 ng/mL), for 72 h. Flow cytometry was used to analyze proportion of Foxp3 + Tregs. ( A ) Typical flow cytometry data of CD4 + Foxp3 + Treg cells proportion. ( B – D ) Summarized data of CD4 + Foxp3 + Treg cells proportion. Data (means ± SEM, n = 9), pooled from 2 or 3 separate experiments with similar results. * p < 0.05, as compared with IL-2 alone group. # p < 0.05, ## p < 0.01, as compared with IL-2 plus TNF group.

Article Snippet: CD4 (L3T4) MicroBeads mouse isolation kit (#130-097-054) were purchased from Miltenyl (Bergisch Gladbach, Germany).

Techniques: In Vitro, Flow Cytometry

Journal: Pharmaceutics

Article Title: Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy

doi: 10.3390/pharmaceutics15041074

Figure Lengend Snippet: Thalidomide and its analogs inhibit Treg proliferative expansion mediated by TNF in vitro. CFSE was used to label CD4 + T cells purified by MACS from lymphocytes. CFSE-labeled CD4 + T cells were stimulated in the presence of IL-2 (10 ng/mL), with or without TNF (20 ng/mL), for 72 h. Thalidomide and its analogs with two concentrations, 50 μM and 100 μM, were added. Flow cytometry was used to analyze Treg proliferation by gating on CD4 + Foxp3 + cells. ( A ) Typical flow cytometry data of CD4 + Foxp3 + Treg cells proliferation. ( B – D ) Summarized data of CD4 + Foxp3 + Treg cells proliferation. Data (means ± SEM, n = 9), pooled from 2 or 3 separate experiments with similar results. *** p < 0.001, as compared with IL-2 alone group. # p < 0.05; ## p < 0.01, as compared with IL-2 plus TNF group.

Article Snippet: CD4 (L3T4) MicroBeads mouse isolation kit (#130-097-054) were purchased from Miltenyl (Bergisch Gladbach, Germany).

Techniques: In Vitro, Purification, Labeling, Flow Cytometry

Journal: Pharmaceutics

Article Title: Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy

doi: 10.3390/pharmaceutics15041074

Figure Lengend Snippet: HE-THD inhibits Treg expansion and proliferation mediated by TNF in vitro. CellTrace violet-labeling lymphocyte were stimulated in the presence of IL-2 (10 ng/mL), with or without TNF (20 ng/mL) for 72 h. HE-THD was added with concentration of 50 μM. Flow cytometry was used to analyze proportion and proliferation of Foxp3 + Tregs. ( A ) Typical Flow cytometry data of CD4 + Foxp3 + Treg cells proportion. ( B , D ) Summarized data of CD4 + Foxp3 + Treg cells proportion. ( C ) Typical flow cytometry data of CD4 + Foxp3 + Treg cells proliferation. Data (means ± SEM, n = 9), pooled from 2 or 3 separate experiments with similar results. ** p < 0.01, *** p < 0.001, as compared with IL-2 plus TNF group.

Article Snippet: CD4 (L3T4) MicroBeads mouse isolation kit (#130-097-054) were purchased from Miltenyl (Bergisch Gladbach, Germany).

Techniques: In Vitro, Labeling, Concentration Assay, Flow Cytometry

Journal: Pharmaceutics

Article Title: Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy

doi: 10.3390/pharmaceutics15041074

Figure Lengend Snippet: Upregulation of TNFR2 expression on Tregs induced by TNF is abrogated by HE-THD. Lymphocytes were cultured in the presence of IL-2 (10 ng/mL), or IL-2 + TNF (20 ng/mL), with medium alone or with HE-THD (50 μM). The cells were cultured for 72 h. The proportion of surface expression of TNFR2 in CD4 + Foxp3 + Tregs was analyzed with FACS. ( A ) Typical flow cytometry data of surface TNFR2 expression on CD4 + Foxp3 + Treg cells. ( B ) Summarized data of proportion of TNFR2 on CD4 + Foxp3 + Treg cells. ( C ) Mean fluorescence intensity (MFI) for TNFR2 expression. Representative FACS data from at least three separate experiments with similar results are shown on the upper panel. Summarized data (mean ±SEM), pooled from 3 to 4 separate experiments ( n = 9~12). * p < 0.05, ** p < 0.01, as compared with “TNF + IL-2” group (without HE-THD).

Article Snippet: CD4 (L3T4) MicroBeads mouse isolation kit (#130-097-054) were purchased from Miltenyl (Bergisch Gladbach, Germany).

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: FOXP3 expression in CD4+CD25+ T cells induced by optimal and suboptimal stimulation with SEC1 in a Vβ-specific way. Human PBMCs depleted of CD25+ cells were stimulated with SEC1 at concentrations inducing optimal (1 µg/ml) or suboptimal (1 ng/ml) stimulation for up to 8 d. FOXP3 expression before and during stimulation was measured using flow cytometry. (A) The percentage (mean ± SEM) of CD4+CD25+FOXP3+ T cells was obtained with data combined from three donors. Data shown are a single representative of three donors and gated on live/CD4+ T cells. (B) Expression of Vβ2 and Vβ14, representing nonspecific and specific Vβ subsets to SEC1, respectively, was measured by flow cytometry before and after stimulation with SEC1 (1 ng/ml) for 6 d. The percentage of Vβ-positive population (dark gray, upper histogram) was measured on the basis of fluorescence minus one control (light gray, lower histogram) in CD4+ T cells. (C) The percentage of CD25+FOXP3+ cells in Vβ positive population was measured after suboptimal stimulation for 6 d. Data shown are combined from three independent experiments (n = 9), and statistical difference was analyzed using the Student t test (**p < 0.001).

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Fluorescence, Control

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: CD4+CD25+ T cells induced by suboptimal stimulation are immunosuppressive predominantly mediated by cell-to-cell contact and partly by soluble factors. (A and B) For the MLR assay, CFSE-stained naive CD4+CD25− T cells were stimulated with anti-CD3/CD28 beads alone or cocultured with CD4+CD25+ T cells induced by suboptimal (1 ng/ml) or optimal (1 µg/ml) stimulation stained with CellTrace FarRed dye for 4 d. (B) Percentage (mean ± SEM) of nonproliferating responder cells from three independent experiments (n = 9). (C and D) CD4+CD25+ T cells induced by suboptimal stimulation with SEC1 (1 ng/ml) were stained with CellTrace FarRed and treated with mitomycin C (indicated as M) for the contact-dependent suppression assay or placed in the top chamber of a Transwell (indicated as T) for the soluble factor–mediated suppression assay. CFSE-labeled naive CD4+CD25− T cells were cocultured as responder cells and stimulated with anti-CD3/CD28 beads. (D) The percentage (mean ± SEM) of nonproliferating CD4+ T cells was obtained by combining results from three independent experiments (n = 9). (E and F) Neutralizing mAb (10 µg/ml) for galectin-1 (indicated as G) was added in an MLR. In some experiments, CD4+CD25+ T cells were treated with mitomycin C (indicated as M) or placed in the top chamber of the Transwell (indicated as T). (F) The percentage (mean ± SEM) of nonproliferating CD4+ T cells was obtained from combined results from three independent experiments. Statistical difference was determined by one-way ANOVA followed by Tukey honestly significant difference test (**p < 0.01, ***p < 0.001).

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Mlr Assay, Staining, Suppression Assay, Labeling

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: Differential activation of PI3K-Akt-mTOR signaling pathway by optimal and suboptimal stimulation with SEC1. PBMCs depleted of CD25+ T cells were stimulated with optimal or suboptimal concentrations of SEC1 for up to 6 d, and total protein was extracted at the indicated time points. Immunoblot analysis of total and/or phosphorylated S6, Akt, mTOR, PTEN, and β-actin was performed. (A) Representative immunoblot data are shown, and band intensity was measured using ImageJ software. (B) Relative band intensity was calculated by normalizing to its unphosphorylated form (for S6 and Akt) or β-actin (for mTOR and PTEN). Results were combined from two independent experiments. (C) PTEN mRNA expression was measured by quantitative PCR, analysis and relative expression to day 0 was calculated after normalization to β-actin. Statistical significance between optimal and suboptimal stimulation was determined by two-way ANOVA with Tukey honestly significant difference test (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Software, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: Differential subcellular localization of FOXP3 isoforms by optimal and suboptimal stimulation with SEC1. (A) Two anti-FOXP3 Ab clones were used to detect FOXP3 isoforms. For (B)–(D), PBMCs depleted of CD25+ cells were stimulated with optimal (4 d; Op) or suboptimal (6 d; Sub) stimulation with SEC1. (B) Confocal microscopic images were taken after staining with anti-FOXP3 Ab conjugated with Alexa Fluor 488 (green). Nucleus was counterstained with DAPI (blue). Original magnification ×40. (C) Cell lysates were fractionated and blotted against anti-FOXP3 Abs and control markers (Erk1/2 for cytoplasmic fraction, histone H3 for nucleic fraction). Cell lysates from day 0 (unstimulated; Un) were used as a negative control. (D) Quantification of Western blot bands was performed using ImageJ software. The bar graph represents the relative expression levels of FOXP3 proteins normalized to unstimulated sample. Results were combined from two independent experiments (**p < 0.01, ***p < 0.001).

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Clone Assay, Staining, Control, Negative Control, Western Blot, Software, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: Blockade of PI3K signaling induced suppressive functionality by optimal stimulation with SEC1. PBMCs depleted of CD25+ T cells were stimulated with optimal stimulation concentration of SEC1 (1 µg/ml) in the presence of vehicle (DMSO; Veh) or PI3K inhibitor (LY294002, 10 µM; PI3Ki) for 4 d. (A) Expression of CD4+CD25+FOXP3+ T cells was analyzed using flow cytometry. A representative flow plot gated in live/CD4+ T cells (left) and the percentage (mean ± SEM) of CD4+CD25+FOXP3+ T cells (right). (B) Percentage of CD25+FOXP3+ cells in Vβ14+ T cells. Data shown are combined from three independent experiments (n = 9), and statistical difference was analyzed using the Student t test (*p < 0.05). (C) After 4 d of stimulation, CD4+CD25+ T cells were isolated, labeled with CellTrace FarRed dye, and cocultured with CFSE-labeled naive CD4+CD25− T cells isolated from same donor. Anti-CD3/CD28 beads were added as a stimulant, and dilution of CFSE signal was measured on day 4 after coculture using flow cytometry. Single representative flow data are shown (left), and the percentage (mean ± SEM) of nonproliferating CD4+ T cells was obtained by combining results from two independent experiments (n = 5) (right). Statistical significance was determined by ANOVA followed by Tukey honestly significant difference test (*p < 0.05, ***p < 0.001). (D) Confocal microscopic images were taken after staining with anti-FOXP3 Ab conjugated with Alexa Fluor 488 (green). The nucleus was counterstained with DAPI (blue). Original magnification ×40.

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Expressing, Flow Cytometry, Isolation, Labeling, Staining