No difference in numbers and percentages of wild type and GPR109A^−/− splenic T cells. (A) Immunofluorescence of magnetically sorted WT B6 macrophages, WT and KO T cells showing GPR109A (green) and DAPI (blue). Bar = 100 μm. (B) Magnetically sorted WT CD4⁺ and CD8⁺ T cells were stimulated with anti-CD3/CD28 beads for 72 hours and RNA was extracted to measure expression of GPR109A mRNA by qRT-PCR. Positive control (PC) was magnetically sorted F4-80⁺ cells. (C) FACS-sorted WT CD4⁺ and CD8⁺ T cells isolated from BALB/c irradiated mice transplanted with TCD BM and 10 × 10⁶ WT T cells at 24-, 48-, and 72-hours post allo-HCT. RNA was extracted to measure expression of GPR109A mRNA by qRT-PCR and expression is shown as fold change compared with expression in naïve FACS-sorted CD4⁺ and CD8⁺ WT T cells, respectively. Expression levels were assessed as biological triplicates. Positive control (PC) was FACS sorted CD11b⁺ cells from a naïve BALB/c mouse spleen; expression of PC as fold change to naïve FACS-sorted CD4⁺ WT T cells. (D) Percentage and numbers of splenic CD4⁺ and CD8⁺ T cells from GPR109a^+/+ (WT) and GPR109a^−/− (KO) littermate mice at baseline. (E) Gating strategy for effector memory (CD44⁺CD62L⁻), central memory (CD44⁺CD62L⁺), and naïve (CD44⁻CD62L⁺) T cell populations. (F-H) Percent and numbers of effector memory (F), central memory (G), and naïve T cells (H), previously gated on live, CD45⁺, CD3⁺, and/or CD4⁺ and CD8⁺. (I) Representative flow cytometric analysis of percent and number of regulatory T cells (CD25⁺Foxp3⁺), previously gated on live, CD45⁺, CD3⁺, and CD4⁺. (J-K) Magnetically sorted WT and KO CD4⁺ and CD8⁺ T cells were labeled with CFSE and stimulated with anti-CD3/CD28 for 72 hours. Representative concatenated CFSE line graphs and quantification showing percentages of CFSE^medium/low WT and KO T cells. (K) CD4⁺ and CD8⁺ T cells were stimulated with PMA/ionomycin for 4 hours and mean fluorescence intensity (MFI) of intracellular IFNγ measured. Comparisons in (B) and (C) were performed by two-tailed unpaired Mann-Whitney Test versus unstimulated or naïve cells. *P < .05, **P < .01, ***P < .001. For (C) significance was calculated by comparing to naïve CD4⁺ or CD8⁺ T cells using a one-sample T-test. Differences in pairs in (D) to (K) are not significant, measured by two-tailed unpaired Mann-Whitney test, n = 6-10 mice per group. All values are means ± standard deviation. All results from three independent experiments.