Extended Data Fig. 9: CD4⁺ T cells acquire partial anergic-like phenotype after activation by efferocytosis-associated islet macrophages both ex vivo and in vivo.

a. Peritoneal macrophages isolated from NOD mouse using magnetic-activated cell sorting (MACS). CD11b^pos macrophages are enriched into Tim-4^hi large peritoneal macrophages (LPM, expressing low level of MHC-II) and Tim-4^low small peritoneal macrophages (SPM, MHC-II^hi). Representative of N = 3 independent experiments. b. Antigen presentation assay in which naive TCR transgenic BDC2.5 CD4⁺ T cells were co-cultured for 3 days with SPM or LPM at indicated concentrations of antigenic peptide; N = 2. c. Gene set enrichment analysis (GSEA) plot showing enrichment of gene signature of CD4⁺ T cells activated by “e-Mac enriched” macrophages interrogated against publicly available transcriptional dataset comparing anergic and naive CD4⁺ T cells (GSE143739⁴³). d. Summary plot of GSEA using CD4⁺ T cells gene signature induced by islet macrophages from STZ^1-low- or vehicle- treated mice (“e-Mac enriched” and “Vehicle” correspondingly). Differential expression comparisons between anergic cells and either naive or antigen-experienced CD4⁺ T cells were used as reference ⁴³ (GSE143739). The CD4⁺ T cells in the reference dataset were exposed to different levels of antigen. e. GSEA pathways comparing gene expression pathways upregulated in CD4⁺ T cells activated by islet macrophages from STZ^1-low- versus vehicle- treated mice. Gene Ontology MSigDB (GO:BP). f. Heatmap showing differentially expressed genes among CD4⁺ T cell subsets in the islets of NOD mice 10 days after the treatment with either STZ^1-low- or control vehicle. g. GSEA plot showing islet anergic-like CD4⁺ T cells gene signature interrogated against bulk transcriptional dataset⁴³ (GSE143739⁴³) comparing anergic and naive cells. NES, normalized enrichment score. Statistics: weighted Kolmogorov-Smirnov test (c, e, g).

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