Figure 3.
Emergence of pTCMfrom naïve CD4+T cells. (a) Experimental strategy to study in vivo naïve C7 TCR transgenic CD4+ T cell differentiation during acute L.m.-ESAT infection. (b–i) Force-directed layout, following Harmony normalization, of naïve and effector C7 CD4+ T cells, sampled 16 and 40 h after infection with L.m.-ESAT, and overlaid by different coloring schemes: (b) time of sampling. (c) Expression of a cell cycle (G1/S and G2/M) gene signature (d) Palantir differentiation potential (left panel) and pseudotime (right panel) using a quiescent (Ccr7hiIl7rhi) naïve start cell, demonstrating two regions of reduced differentiation potential that indicate lineage specification and commitment. (e) Expression of genes associated with TH1 CD4+ T cell lineage and average expression of TH1 signature genes with delineation of a proposed Branch 1. (f) Expression of genes associated with TFH or pTCM CD4+ T cell lineages and average expression of TFH or pTCM signature genes with delineation of a proposed Branch 2 (g) Pseudotemporal ordering of alternative differentiation trajectories for naïve CD4+ T cells, showing selected start and end points on the force directed layouts (top panels). Heatmaps depict inferred temporal gene expression trends along the two differentiation pathways; left represents cells with high TCR signaling-dependent gene expression (“TCR-hi”), right represents “TCR-lo” pTCM differentiation. (h) Force-directed layouts colored by expression of genes associated with IL-2 -STAT5 signaling. (i) Type I IFN and IFN-γ receptor gene expression, overlaid on CD4+ T cell force-directed layout. (j) C7 × Mx1GFP × tgMx1CreRosa26lsl-tdT mice were infected with L.m.-gp66 and analyzed 7 days post infection (dpi). (k) Representative flow cytometric analysis of Listeriolysin O (LLO) peptide-specific CD4+ T cells demonstrating increased proportion of Mx1 fate-mapped cells amongst CD62L+ pTCM cells. (l) Proportion of LLO:I-Ab- (left) and gp66:I-Ab-specific effector memory CD4+ T cell subsets that are Mx1 fate-mapped. Each symbol represents an individual mouse (l). Data from one of two experiments (l) Error bars: means ± SEM of replicates. Statistical significance determined by one-way ANOVA (l); *P < 0.05; **P < 0.01; ***P < 0.001.

Emergence of pT CM from naïve CD4 + T cells. (a) Experimental strategy to study in vivo naïve C7 TCR transgenic CD4+ T cell differentiation during acute L.m.-ESAT infection. (b–i) Force-directed layout, following Harmony normalization, of naïve and effector C7 CD4+ T cells, sampled 16 and 40 h after infection with L.m.-ESAT, and overlaid by different coloring schemes: (b) time of sampling. (c) Expression of a cell cycle (G1/S and G2/M) gene signature (d) Palantir differentiation potential (left panel) and pseudotime (right panel) using a quiescent (Ccr7hiIl7rhi) naïve start cell, demonstrating two regions of reduced differentiation potential that indicate lineage specification and commitment. (e) Expression of genes associated with TH1 CD4+ T cell lineage and average expression of TH1 signature genes with delineation of a proposed Branch 1. (f) Expression of genes associated with TFH or pTCM CD4+ T cell lineages and average expression of TFH or pTCM signature genes with delineation of a proposed Branch 2 (g) Pseudotemporal ordering of alternative differentiation trajectories for naïve CD4+ T cells, showing selected start and end points on the force directed layouts (top panels). Heatmaps depict inferred temporal gene expression trends along the two differentiation pathways; left represents cells with high TCR signaling-dependent gene expression (“TCR-hi”), right represents “TCR-lo” pTCM differentiation. (h) Force-directed layouts colored by expression of genes associated with IL-2 -STAT5 signaling. (i) Type I IFN and IFN-γ receptor gene expression, overlaid on CD4+ T cell force-directed layout. (j) C7 × Mx1GFP × tgMx1CreRosa26lsl-tdT mice were infected with L.m.-gp66 and analyzed 7 days post infection (dpi). (k) Representative flow cytometric analysis of Listeriolysin O (LLO) peptide-specific CD4+ T cells demonstrating increased proportion of Mx1 fate-mapped cells amongst CD62L+ pTCM cells. (l) Proportion of LLO:I-Ab- (left) and gp66:I-Ab-specific effector memory CD4+ T cell subsets that are Mx1 fate-mapped. Each symbol represents an individual mouse (l). Data from one of two experiments (l) Error bars: means ± SEM of replicates. Statistical significance determined by one-way ANOVA (l); *P < 0.05; **P < 0.01; ***P < 0.001.

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