Journal: The Journal of Experimental Medicine

Article Title: A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8 + T cell tolerance

doi: 10.1084/jem.20210759

Figure Lengend Snippet: STAT3 +/K392R drives expansion of the CD8 + effector compartment with up-regulation of cytokine and chemokine genes. (A) UMAP projection of reclustered T cells (CD3 + cells) from islet immune infiltrate used in scRNA-seq experiment (see ) with associated cell counts per cluster and comparison of cluster frequencies between the two genotypes (STAT3 +/K392R versus WT). γδT, γδ T cells. (B) Volcano plot showing STAT3 +/K392R versus WT differential gene expression in CD8 + clusters. FDR, false discovery rate. (C) Flow cytometry of Ccl5 protein expression in CD8 + memory T cells from the pLN in nondiabetic males at 14 wk ( n = 3 per group). (D) UMAP projection of reclustered CD8 + T cells. (E and F) Marker gene (E) or module score (F) expression in CD8 + T cell clusters. In F, genes used to calculate module scores for terminally exhausted T cells were Cd101 , Cd200r2 , Cd7 , Cd200r1 , and Il10 , and for transitory T cells, they were Cx3cr1 , Klrg1 , Il2ra , Il18rap , and S1pr5 , as previously described . (G) Relative population frequencies among nonnaive CD8 + cell clusters. (H) Volcano plot showing differential gene expression in CD8 + effector and transitory cell clusters relative to all CD8 + cells. Differential gene expression (B and H) based on nonparametric Wilcoxon rank-sum test. Volcano plots (B and H) use log N fold cutoff 0.25, and genes of interest are labeled. Data in C are representative of two independent experiments. Student’s t test (C) was used. Data are shown as mean ± SD. *, P ≤ 0.05.

Article Snippet: NOD cells were CD4 enriched using MACS EasySep CD4-negative selection kits (STEMCELL Technologies), and 8.3Tg + cells were naive CD8 enriched using MACS EasySep naive CD8-negative selection kits (STEMCELL Technologies).

Techniques: Expressing, Flow Cytometry, Marker, Labeling

Journal: The Journal of Experimental Medicine

Article Title: A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8 + T cell tolerance

doi: 10.1084/jem.20210759

Figure Lengend Snippet: scATAC-seq of infiltrating islet immune cells, additional characterizations of scTCR-seq data, and functional T cell adoptive transfer experiments. (A) UMAP projection of scATAC-seq profiles of T cells (CD3 + ) subclustered from CD45 + islet infiltrates of 8–10-wk-old male nondiabetic mice, WT ( n = 3, pooled), and STAT3 +/K392R ( n = 3, pooled). γδT, γδ T cells. (B) Normalized pseudobulk ATAC-seq tracks of CD8 + T cell clusters split by genotype around genes identified as up-regulated in corresponding scRNA-seq CD8 + T cell clusters. Peaks significantly up-regulated (from C) are shown in red. (C) Volcano plot showing differential peaks whose nearest gene is present in the significantly up-regulated genes between CD8 + T cell clusters (WT versus STAT3 +/K392R ). FDR, false discovery rate. (D) Volcano plot showing differential transcription factor motifs between CD8 + T cell clusters (WT versus STAT3 +/K392R ). (E) Transcription factor motif activity for CD8 + T cell clusters split by genotype. Motif sequence is shown above each violin plot. (F) TCR expression in UMAP clusters from subset of CD3 + T cells identified in scRNA-seq analysis. (G) Gini index showing clonal expansion in STAT3 +/K392R broken down by cluster and genotype. (H) Diabetes incidence after adoptive transfer of CD4 + BDC2.5 + Teff cells from mice without ( n = 6) and with STAT3 +/K392R ( n = 9) into NOD.Rag1 −/− mice (solid lines) and diabetes incidence after adoptive transfer of naive CD8 + T cells from WT 8.3Tg + mice with polyclonal CD4 + T cells from mice without ( n = 10) and with STAT3 +/K392R ( n = 5) into NOD.SCID mice (dotted lines). Results are pooled from two independent experiments for each set of incidence curves (solid lines and dotted lines), and a log-rank (Mantel-Cox) test was used.

Article Snippet: NOD cells were CD4 enriched using MACS EasySep CD4-negative selection kits (STEMCELL Technologies), and 8.3Tg + cells were naive CD8 enriched using MACS EasySep naive CD8-negative selection kits (STEMCELL Technologies).

Techniques: Functional Assay, Adoptive Transfer Assay, Activity Assay, Sequencing, Expressing

Journal: The Journal of Experimental Medicine

Article Title: A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8 + T cell tolerance

doi: 10.1084/jem.20210759

Figure Lengend Snippet: STAT3 +/K392R drives clonal expansion of diabetogenic CD8 + T cells. (A) Top 20 CD8 clones by count in each sample. (B) Top 10 specific CD8 clones with corresponding CDR3 sequences displayed. The CDR3 sequence for clone 6158 in red is nearly identical to that of the CD8-restricted TCR specific for islet-specific antigen IGRP (TCR-8.3; sequence shown below table). The only difference between clone 6158 and TCR-8.3 is a serine, denoted in blue, that replaces an alanine of the TCR-β chain. (C) Diabetes incidence of 8.3Tg + (IGRP-specific TCR) mice with ( n = 7) and without ( n = 11) STAT3 +/K392R confirms increased diabetogenicity. (D) Diabetes incidence after adoptive transfer of polyclonal WT CD4 + T cells with naive CD8 + T cells from 8.3Tg + mice with ( n = 5) and without STAT3 +/K392R ( n = 10) into NOD.SCID mice. Results are pooled from two independent experiments. A log-rank (Mantel-Cox) test (C and D) was used.

Article Snippet: NOD cells were CD4 enriched using MACS EasySep CD4-negative selection kits (STEMCELL Technologies), and 8.3Tg + cells were naive CD8 enriched using MACS EasySep naive CD8-negative selection kits (STEMCELL Technologies).

Techniques: Clone Assay, Sequencing, Adoptive Transfer Assay