Naïve CD4 ⁺ T cell heterogeneity. (a) Experimental strategy for profiling splenic naïve CD4⁺ T cells. TCRγδ⁻PBS57/CD1d tetramer⁻NK1.1⁻TCRβ⁺CD4⁺CD25⁻CD44^loCD62L^hi cells, were sorted from the spleen of C7 or Smarta tgTCR mice and adoptively transferred into CD45.2 B6 recipients. After 7 days, naïve host B6 and tgTCR CD4⁺ T cells were isolated and profiled by scRNA-seq. (b) UMAP visualization of 28,146 naïve CD4⁺ T cells, colored by Phenograph cluster. (c) Fraction of cells within each naïve CD4⁺ T cell cluster detected across strains and biological replicate samples colored by Phenograph cluster as shown in Fig. 1 b. C57Bl/6, 3,026 cells; C7_1, 2,675 cells; C7_2, 6,159 cells; Smarta_1, 7,834 cells; Smarta_2, 8,452 cells. (d) UMAP colored by MAGIC imputed expression of cluster-defining genes. (e) Quantitative real-time PCR analysis of ISGs in bulk splenic naïve CD4⁺ T cells from B6, Smarta, Irf9^−/−, or Ifnar1^−/− mice. (f) Naïve T cells, as shown in Fig. 4 b, visualized using diffusion map embedding of the first three DCs, with distinctive ISG⁺, quiescent, and CD5⁺ “self-reactive” cells labeled. Cells colored by Phenograph cluster as in Fig. 4 b. (g) Cells in the ISG⁺ naïve CD4⁺ T cell cluster (cluster 8, Fig. 4 b) and CD5⁺ naïve cells (cluster 10, Fig. 4 b), highlighted in distinct differentiation trajectories of early CD4⁺ T cell differentiation from Fig. 3 g. (h) Proportion of CD69⁺ cells among naïve C7 CD4⁺ T cells cultured for 36 h with irradiated T cell–depleted splenocytes (as antigen-presenting cells) and limiting concentrations of ESAT peptide. (i) IFNAR1 mean fluorescence intensity (MFI) on Mx1(GFP)⁺ versus Mx1(GFP)⁻ populations in uninfected mice. (j) Mx1(GFP)⁺ naïve CD4⁺ T cells are present in all lymphoid tissues, with varying frequencies across anatomically distinct LNs; PLN and MLN. (k) Representative MFI (histogram) (left) and summary bar graph (right) showing expression of pSTAT1 in sort-purified naïve Smarta CD4⁺ T cells treated in vitro with IFN-α or IFN-β for 4 h. (l) Naïve tdTomato⁻GFP⁻ CD4⁺ T cells from C7 × Mx1^GFP × tgMx1^CreRosa26^lsl-tdT mice were adoptively transferred into congenic B6 mice, administered the following day with poly(I:C) i.p. Representative flow cytometry plot showing expression of tdTomato and GFP in transferred splenic C7 naïve CD4⁺ T cells, 4 days after treatment (left) and quantification (right). (m) Mx1(GFP)⁺ or Mx1(GFP)⁻ naïve C7 CD4⁺ T cells were adoptively transferred into congenic B6 mice, subsequently infected intravenously with L.m.-ESAT and analyzed at 5 dpi. (n) Proportion of splenic pT_CM (CD62L⁺), T_H1 (T-bet⁺CXCR5⁻), and T_FH (T-bet⁻CXCR5⁺) T cells amongst transferred C7 T cells at 5 dpi. Results are from one experiment representative of 4 (j), 3 (m and n), 2 (e, i, k, and l) independent experiments with n = 3 (e, j, and l), n = 4 (i), n = 9 (n) mice per group and three replicate wells in h and k. Statistical significance by two-way ANOVA (h, i, and l); one-way ANOVA (j and k); unpaired t test (n); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Error bars: means ± SEM.