Supplementary Figure 5: Costimulation and IFN-γ contribute to the increased differentiation of Ptpn2-deleted cells into Tim-3+ cells.
From: PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity
(a) Quantification of CD25 MFI on CD8+ CD25+ control or Ptpn2-deleted CD8+ T cells following in vitro stimulation (αCD3/CD28) in the presence of indicated cytokines or blocking antibodies. Representative of two pooled experiments, n ≥ 4 technical replicates. (b) Representative flow cytometry plots of Slamf6 and Tim-3 expression on control or Ptpn2-deleted CD8+ T cells following in vitro stimulation (αCD3/CD28) in the presence of IL-2 and IFN-α. Representative of two independent experiments, n ≥ 2 technical replicates. (c) Quantification of Slamf6+ Tim-3–, Slamf6+ Tim-3+, Slamf6– Tim-3+ subsets in control or Ptpn2-deleted CD8+ T cells following in vitro stimulation (αCD3 without αCD28) in the presence of IL-2 and IFN-α. Representative of one experiment, n ≥ 2 technical replicates. (d) Quantification of Slamf6+ Tim-3+ (left graph) and Slamf6– Tim-3+ (right graph) subsets in control cells following in vitro stimulation (αCD3/CD28) in the presence of indicated cytokines or blocking antibodies and addition of pre-conditioned supernatant from stimulated control or Ptpn2-deleted CD8+ T cells. Representative of one experiment, n ≥ 2 technical replicates. (e) Representative histograms of IFNAR1 expression on naive control or Ptpn2-deleted CD8+ T cells. Representative of one experiment, n = 1 mouse. (f) Quantification of frequencies of co-transferred control or Ptpn2-deleted CD8+ T cells day 4 post LCMV Clone 13 infection following treatment with isotype (left graph) or IFN-γ blocking antibody (right graph). Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 5 mice. (g) Quantification of Slamf6+ Tim-3–, Slamf6+ Tim-3+, and Slamf6– Tim-3+ subsets in Ptpn2-deleted P14 CD8+ T cells day 4 post LCMV Clone 13 infection in mice that received isotype or IFN-γ blocking antibody as in (f). Representative of two independent experiments, n = 5 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-way ANOVA (a, g) or two-sided Student’s paired t-test (f) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001).
