Fig. 3: Lpar5 signaling on CD8 T cells promotes phenotypic tolerogenic states through exhaustive-like differentiation. | Nature Communications

Fig. 3: Lpar5 signaling on CD8 T cells promotes phenotypic tolerogenic states through exhaustive-like differentiation.

From: Lysophosphatidic acid modulates CD8 T cell immunosurveillance and metabolism to impair anti-tumor immunity

Fig. 3

A Schematic of in vitro chronic stimulation. Effector CD8 T cells are persistently cultured with either anti-CD3 + LPA or LPA alone until Day 15. B–D Flow cytometric analysis and quantification of percent PD1+ Tim3+ from the CD8 T cell population. B, C Representative contour plots for (B) OT-I or (C) Lpar5−/− OT-I CD8 T cells persistently cultured with LPA. D Quantification of percent of CD8+ T cells that are PD1+ Tim3+ (n = 3 mice per group). Exact p-values are as follows, OT-I CD3 + LPA vs OT-I LPA p = 0.0049; OT-I CD3 + LPA vs Lpar5−/− OT-I LPA p < 0.0001; Lpar5−/− OT-I CD3 + LPA vs Lpar5−/− OT-I LPA p < 0.0001; OT-I LPA vs Lpar5−/− OT-I LPA p = 0.0012. (E–G) Flow cytometric analysis of Tim3 expression on OT-I or Lpar5−/− OT-I CD8 T cells cultured in either anti-CD3 + LPA or LPA. E, F Representative flow cytometric histograms and (G) quantification the geometric mean fluorescence intensity (gMFI) of PD1 (n = 3 mice). Exact p-values are as follow, OT-I CD3 + LPA vs Lpar5−/− OT-I CD3 + LPA p < 0.0001; OT-I CD3 + LPA vs OT-I LPA p < 0.0001; OT-I CD3 + LPA vs Lpar5−/− OT-I LPA p < 0.0001; Lpar5−/− OT-I CD3 + LPA vs OT-I LPA p = 0.0007; Lpar5−/− OT-I CD3 + LPA vs Lpar5−/− OT-I LPA p = 0.0003. H–J Flow cytometric analysis of Tim3 expression on OT-I or Lpar5−/− OT-I CD8 T cells cultured in either anti-CD3 + LPA or LPA. H, I Representative flow cytometric histograms and (J) quantification the geometric mean fluorescence intensity (gMFI) of Tim3 (n = 3 mice). Exact p-values are as follows, OT-I CD3 + LPA vs Lpar5−/− OT-I p = 0.0012; OT-I CD3 + LPA vs OT-I LPA p < 0.0001; OT-I CD3 + LPA vs Lpar5−/− OT-I LPA p < 0.0001; Lpar5−/− OT-I CD3 + LPA vs OT-I LPA p = 0.0044; Lpar5−/− OT-I CD3 + LPA vs Lpar5−/− OT-I LPA p = 0.0031. K Schematic of study design where B16.cOVA tumor cells and OT-I CD8 T cells are co-transferred in a 1:1 ratio into the mice on day 1. Mice were harvested on day 20 and evaluated for tumor burden and flow cytometric analysis for exhaustion markers. L Quantified tumor burden in the lung after intravascular injection of B16.cOVA cells. Tumor burden is presented as the number of tumor nodules in the lung where n = 5 mice per OT-I group and n = 7 mice per Lpar5−/− OT-I group and p = 0.0124. (M,N) Flow cytometric quantification of Lag3 expression with a (M) representative histogram and (N) quantification of CD45.1+ CD8+ T cells represented as gMFI where n = 5 mice per OT-I group and n = 7 mice per Lpar5−/− OT-I group and p = 0.0216. O, P Flow cytometric quantification of Tox expression with a (O) representative histogram and (P) quantification of CD45.1+ CD8+ T cells represented as gMFI where n = 5 mice per OT-I group and n = 7 mice per Lpar5−/− OT-I group and p = 0.0151. Statistics for panels (D, G, J) were performed using a Two-way ANOVA with a Tukey’s post-hoc analysis where *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. Statistics for panels (L, N, P) were performed using the unpaired two-sided Student’s t-test analysis where *p < 0.05. Error bars for panels (D, G, J, L, N, P) represent standard error of the mean.

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