Extended Data Fig. 9: Transcriptomic analysis reveals that TH1-like T_reg cells inhibit CD8⁺ T cell memory during LCMV infection.

a-c, scRNA-seq of splenic pooled Tetramer⁺ (GP₃₃, GP₂₇₆, NP₃₉₆) CD8⁺ T cells from Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice (n = 3), 16 dpi with Cl. 13. a, UMAP embedding Tetramer⁺ CD8⁺ T cells into a two-dimensional space to generate 8 distinct clusters, with categories shown. b, Distribution of cells amongst clusters from individual mice. c, Percentage of cells in the memory cluster. d, e, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Arm. and the expression of effector CD8⁺ cells (d) (n = 18, control; n = 16, Ifngr1-deficient) and the ratio of MPECs to SLECs within splenic pooled CD44^hiCD62L^lo Tetramer⁺ CD8⁺ T cells (e) (n = 8) were determined by flow cytometry. f, g, Expression of Ifngr1 within the bulk population (f) and within the memory cluster (g) of the splenic pooled Tetramer⁺ CD8⁺ T cells from the scRNA-seq dataset in a-c (n = 3). h-j, Rag1^–/– mice were reconstituted with Ifngr1^–/– (n = 12) or control (WT) (n = 13) T_reg cells and infected with Arm. h, i, Frequency of splenic CD44^hiCD62L^lo (h) and CD44^hiCD62L^hi (i, left), and number of CD44^hiCD62L^hi (i, right) pooled Tetramer⁺ CD8⁺ T cells were determined by flow cytometry. j, Frequency of (left) and total number of (right) MPECs (upper) and SLECs (lower), and the ratio of MPECs to SLECs, splenic pooled Tetramer⁺ CD8⁺ T cells were determined by flow cytometry. k, heatmap of DE of the IL16/CD4/CCR5 signaling axis in T_reg cells from the scRNA-seq dataset in a-c. l-n, Foxp3^Cre-YFP and Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Arm. and transcripts of the IL16/CD4/CCR5 signaling axis in T_reg cells (l) (n = 11) or Il16 (m) (n = 11) and Ccl5 (n) (n = 6, control; n = 9, Ifngr1-deficient) expression in T_reg cells and CD44^hiCD62L^lo CD4⁺ and CD8⁺ T cells, were determined by qPCR. a-n, Data are presented as mean values and represent biologically independent mice and 1 (a-c, f, g), 2 (e, k, n) or 3 (d, h-j, l, m) independent experiments. Statistical significance and P values were determined by an unpaired two-tailed Student’s t-test relative to the Foxp3^Cre-YFP mice (c-g) or Rag1^–/– + WT T_reg cells (h-j), or by multiple unpaired Student t-tests relative to Foxp3^Cre-YFP mice (l) or by two-way ANOVA with multiple comparisons. (m,n), Adjusted P values were determined by one-way ANOVA (b) or Wilcoxon rank-sum test (k) relative to infected Foxp3^Cre-YFP mice (P values indicated when significant); NS, not significant.

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