Characterization of naïve CD4⁺ T cell heterogeneity. Related to Fig. 4. (a) Representative flow cytometry showing sorting strategy for isolation of naïve CD4⁺ Smarta or C7 cells on day 0, prior to transfer into CD45.2 recipient mice (upper and middle panel). On day 7 after transfer, tgTCR T cells and host B6 naïve CD4⁺CD25⁻CD44^loCD62L^hi T cells were sorted for scRNA-seq analysis (right panel). At day 7, tgTCR T cells retained their naïve cell surface phenotype. (b) Top: UMAP visualization of individual naive CD4⁺ T cell replicate samples, each colored by their collective Phenograph clustering from Fig. 4 b performed after batch-correction. Bottom: UMAP colored by donor cell origin sorted according to a: B6 (blue) from one donor mouse, C7 (orange) and SMARTA (green) each from two donor mice. (c) Heatmap showing imputed expression of top 50 DEGs across splenic naïve T cell clusters (log₂FC > 0.5, FDR < 0.01). The colored bar at the top of the heatmap shows the assignment of cells to clusters labeled in Fig. 4 b. Genes of interest are shown on the right. (d) Log-normalized expression values for comparison with imputed expression Fig. 4 d. (e) UMAP of naïve CD4⁺ T cells colored by imputed (left) or log normalized (right) expression of genes implicated in maintenance of naïve T cell quiescence. (f) Representative flow cytometry of naïve CD4⁺ T cells from the spleen (Sp) of Mx1^GFP mice. (g) Representative flow cytometry demonstrating gating strategy for analysis of CD4⁺ thymocyte populations from Mx1(GFP)⁺ mice. (h) Summary graph showing frequency of Mx1⁺ cells for each thymocyte population gated in g. Increased frequency of Mx1⁺ cells is observed as cells undergo progressive maturation from CD4⁺CD8⁺ DP thymocytes to mature single positive (SP) CD4⁺ T cells. Representative of two independent experiments, n = 4. Statistical significance was determined by one-way ANOVA; ****P < 0.0001. (i) Frequency of RAG2(GFP)⁺ cells within PLN, MLN, or spleen. (j) Expression of indicated ISGs in RAG2(GFP)⁺ or RAG2(GFP)⁻ naïve T cells, determined by qPCR. Representative of two independent experiments of n = 3. (k) Representative flow cytometric analysis of immune cell composition within the spleen of recipient mice, 5 days after infection, demonstrating frequency of pT_CM (CD62L⁺), T_H1 (T-bet⁺CXCR5⁻), and T_FH (T-bet⁻CXCR5⁺) cells amongst transferred C7 T cells. (l) CXCR5 geometric MFI (gMFI) in T cell subsets from k. Representative of two independent experiments. Statistical significance determined by two-way ANOVA; ****P < 0.0001.