Extended Data Fig. 5: scRNAseq reveals transcriptionally unique clusters and functions of Cre Control versus Lag3^∆TM CD8⁺ T cells.

scRNAseq assessment of intra-islet CD8⁺ T cells. (a) The Lag3^L/L-YFP (Extended Data Fig. 2a) construct crossed to a Cre recombinase is shown. Upon crossing Lag3^L/L-YFP to a Cre recombinase, exon 7 (the transmembrane domain) is deleted (Lag3^∆TM). The result is the generation of only the soluble form of LAG3 protein. (b) qPCR determining deletion efficiency of the CD8 specific LAG3^ΔTM mouse. Ratio of Exon 7 to Exon 3 was quantified in Cre Control (E8I^CRE/CRE-GFP.NOD), vs Lag3^∆TM (Lag3^L/L-YFPE8I^CRE/CRE-GFP.NOD) experimental mice. Cells derived from spleens of five 8-week-old females for 1 experiment (n = 5). (c-g) CD8⁺ T cells from the islets and ndLN were isolated from 4 Cre Control and 4 Lag3^∆TM 8-week-old NOD female mice and were subjected to 5’ paired single cell RNAseq (scRNAseq) and single cell T cell receptor sequencing (scTCRseq). (c) Cells were visualized by UMAP and colored by tissue, genotype, or individual sample. (d) Quantification of specific cell types in each DRAGON cluster (Fig. 3b). (e) Overrepresentation analyses on gene signatures characterizing the Cre Control (6) and Lag3^∆TM dominated clusters (3 + 4) was performed using KEGG pathways and the top 10 overrepresented in each genotype are shown. Enrichment ratio and –log₁₀FDR (false discovery rate) are portrayed. (f) Heatmap of gene expression levels in the over-represented KEGG pathways.

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