Supplementary Figure 4: Neutralization of TNF in mice with chronic neonatal LCMV strain WE infection restores immunity to LCMV and reverts CD4⁺PD-1⁺ T cell gene expression.

(a) Model for TNF neutralization in 8 week old cnLCMV-WE mice. (b) Left, representative, right, cumulative flow cytometric analysis of np396 expression on splenic CD8⁺ T cells after vehicle treatment or TNF neutralization (each n = 5). (c) Total numbers of splenic np396⁺CD8⁺ T cells (each n = 5). (d) PD-1 expression on splenic np396⁺CD8⁺ T cells (each n = 5). (e) Left, representative, right, cumulative flow cytometric analysis of PD-1 expression on splenic CD4⁺ T cells after vehicle treatment (n = 5) or TNF neutralization (n = 4). (f) Left, representative, right, cumulative flow cytometric analysis of PD-1 expression on splenic CD8⁺ T cells from animals after vehicle treatment (n = 6) or TNF neutralization (n = 5). (g-k) Role of TNFR-signaling in acute murine LCMV strain WE infection. Wild-type mice were acute infected with LCMV strain WE (2 × 10⁴ pfu) and analyzed after 10 days. (g,h) Immunoblot analysis of pIkkα/β (Ser176/180) (top) and β-actin (bottom) in (g) CD4⁺ and (h) CD8⁺ T cells from animals after vehicle treatment or TNF neutralization during acute LCMV strain WE infection (n = 3). Data shown are representative of three mice each. (i-k) Left, representative, right, cumulative flow cytometric analysis of (i) PD-1 expression on CD4⁺ T cells, (j) PD-1 expression on CD8⁺ T cells, and (k) gp33-specific CD8⁺ T cells from animals after vehicle treatment or TNF neutralization during acute LCMV strain WE infection (n = 3). (l) Heatmap of z-transformed gene expression data for genes expressed in low amounts in at least one of the inhibitory conditions in human CD4⁺ T cells, up-regulated in CD4⁺PD-1⁺ T cells from mice after TNF neutralization. (m) Heatmap of z-transformed gene expression data for genes highly expressed under at least one of the inhibitory conditions in human CD4⁺ T cells, down-regulated in CD4⁺PD-1⁺ T cells from mice after TNF neutralization. (n) Fold-change-fold-change plot showing the influence of TNF-neutralization on gene expression in CD4⁺PD-1⁻ and CD4⁺PD-1⁺ T cells. The y-axis compares the expression profiles between CD4⁺PD-1⁻ T cells from mice after vehicle treatment or TNF neutralization, whereas the x-axis compares the expression profiles of CD4⁺PD-1⁺ T cells. Highlighted in red are genes assessed in o. (o) Relative mRNA expression of CD4⁺PD-1⁻ and CD4⁺PD-1⁺ T cells from mice after vehicle treatment or TNF neutralization for Ly6c1 and Klrd1 by qPCR. Mean ± s.e.m. of at least triplicates, representative of two independent experiments. *P < 0.05 (Student’s t-test). (p) GSEA using the murine cnLCMV-WE CD4⁺ T-cell RNA fingerprint as the gene set in CD4⁺PD-1⁺ T cells from mice after vehicle treatment or TNF neutralization. (q) Heatmap of z-transformed gene expression data for transcription factors associated with CD4⁺ T cell exhaustion in LCMV clone 13 infection²⁵ in CD4⁺PD-1⁺ T cells from mice after vehicle treatment or TNF neutralization. (r) GSEA using a murine TNF RNA fingerprint (GSE2504)²⁷ as the gene set in CD4⁺PD-1⁺ and CD4⁺PD-1⁻ T cells in mice after vehicle treatment (left) or TNF neutralization (right). (s) GSEA using the human TNF RNA fingerprint genes defined in CD4⁺ T cells as the gene set in CD4⁺PD-1⁺ T cells in mice after vehicle treatment or TNF neutralization. (b-f,i-k) Mean ± s.e.m.*P < 0.05 (Student’s t-test). Data are representative of two independent experiments. n.s. not significant. (p,r,s) ES: enrichment score, FDR: false-discovery rate.