Supplementary Figure 7: Role of TNFR-signaling in T cells in mice with chronic neonatal LCMV strain WE infection.

(a) Protocol used to determine the effect of TNF on T cells in cnLCMV-WE mice. 2 × 10⁶ CD8⁺ T cells from Thy1.2 congenic wild-type or TNFRI-TNFRII-deficient mice 8 days after acute infection with LCMV strain WEwere transferred to Thy1.1 mice with chronic neonatal LCMV infection and assessed after 10 days. (b,c) Left, sorting strategy to isolate Thy1.2⁺CD4⁺ and CD8⁺ T cells from acute LCMV strain WE-infected (b) wild-type and (c) TNFRI-TNFRII-deficient mice for adoptive transfer in cnLCMV-WE Thy1.1⁺ mice. Right, analysis of purities of isolated cell populations. (d,e) Flow cytometric analysis of gp33-specific Thy1.2⁺CD8⁺ T cells from mice receiving wild-type (n = 3) or TNFRI-TNFRII-deficient CD8⁺ T cells (n = 3). (d) Cumulative percentage of splenic gp33-specific CD8⁺ T cells. (e) Cumulative percentage of splenic PD-1⁺ gp33-specific CD8⁺ T cells. (f) ALT serum concentration in mice receiving no transfer (n = 4), wild-type (n = 3), or TNFRI-TNFRII-deficient CD8⁺ T cells (n = 3). (g) Quantification of LCMV titers in serum as in f. (h) Protocol used to determine the effect of TNF on T cells in cnLCMV-WE mice. 2 × 10⁶ CD8⁺ T cells and 2 × 10⁶ CD4⁺ from Thy1.2⁺ congenic wild-type or TNFRI-TNFRII-deficient mice 8 days after acute infection with LCMV strain WE were transferred to Thy1.1⁺ mice with chronic neonatal LCMV infection and assessed after 10 days. (i-k) Flow cytometric analysis of gp66-specific Thy1.1⁺CD4⁺ T cells from mice receiving no transfer (n = 4), wild-type (n = 4), or TNFRI-TNFRII-deficient CD4⁺ and CD8⁺ T cells (n = 4). (i) Cumulative percentage of splenic gp66-specific Thy1.1⁺CD4⁺ T cells. (j) Cumulative numbers of splenic gp66-specific Thy1.1⁺CD4⁺ T cells. (k) Cumulative percentage of splenic PD-1⁺ gp66-specific Thy1.1⁺ CD4⁺ T cells. (l) Left, representative flow cytometric analysis of PD-1 expression on Thy1.2⁺CD4⁺ T cells from mice receiving wild-type or TNFRI-TNFRII-deficient CD4⁺ and CD8⁺ T cells. Right, cumulative data. (m,n) Flow cytometric analysis of PD-1 expression on Thy1.1⁺CD4⁺ T cells as in i. (m) Cumulative percentage of splenic Thy1.1⁺CD4⁺PD-1⁺ T cells. (n) Cumulative numbers of splenic Thy1.1⁺CD4⁺PD-1⁺ T cells. (o) Left, representative flow cytometric analysis of PD-1 expression on Thy1.2⁺CD8⁺ T cells as in l. Right, cumulative data. (p,q) Flow cytometric analysis of PD-1 expression on Thy1.1⁺CD8⁺ T cells as in I. (p) Cumulative percentage of splenic Thy1.1⁺CD8⁺PD-1⁺ T cells. (q) Cumulative numbers of splenic Thy1.1⁺CD8⁺PD-1⁺ T cells. Data from 4 mice per group are shown. (d,e,i-q) Mean ± s.e.m. (d,e,l,o) *P < 0.05 (Student’s t-test). (i-k,m,n,p,q) *P < 0.05 vs. wild-type (one-way ANOVA with Bonferroni FDR correction). n.s. not significant. Data are representative of two independent experiments.