Figure 5.

Distinct kinetic profiles of PD-1⁺ and PD-1⁻ Tfr cells in cGVHD Cd38^−/− versus cGVHD WT mice. (A) Gating strategy to detect PD-1^hiCXCR5⁺ FoxP3⁺ or PD1⁻CXCR5⁺FoxP3⁺ Tfrs cells in spleen cells from cGVHD mice. Left panel: FoxP3 versus CD4 plot of gated CD4⁺CXCR5⁺PD-1⁻ cells, showing the frequency of the PD-1⁻FoxP3⁺ Tfr cells. Middle panel: PD-1 versus CXCR5 plot on gated CD4⁺ cells showing the gated CXCR5⁺PD-1^hi and CXCR5⁻PD-1⁻ subpopulations. Right panel: FoxP3 versus CD4 plot showing the frequencies of PD-1⁺FoxP3⁺ Tfr cells and PD-1⁻FoxP3⁻ Tfr cells. (B) Kinetics of the frequencies of PD1⁺ (open red circles) and PD-1⁻ (open black circles) Tfr cells relative to total Tfr cells in spleen cells from cGVHD Cd38^−/− mice. (C) Kinetics of the frequencies of PD1⁺ (closed red circles) and PD-1⁻ (closed black circles) Tfr cells relative to total Tfr cells in spleen cells from cGVHD WT mice. (D) Kinetics of total numbers of PD1⁺ (open red circles) and PD-1⁻ (open black circles) Tfr cells relative to total Tfr cells in spleen cells from cGVHD Cd38^−/− mice. (E) Kinetics of total numbers of PD1⁺ (closed red circles) and PD-1⁻ (closed black circles) Tfr cells relative to total Tfr cells in spleen cells from cGVHD WT mice. The symbols are the mean values and the vertical bars represent ± SEM. P-values are shown for Welch’s t-test. The results are cumulative data from two to three different experiments per time point and mouse type, each with three to four mice per experiment. *P < 0.05, **P < 0.01, ****P < 0.0001, *****P < 0.00001.