FIGURE 6.

Lack of Tregs favors progressive expansion of CD4⁺Nrp1⁺Foxp3⁻ T cells. (A–H) Flow cytometry–based characterization of CD4⁺Nrp1⁺Foxp3⁻ T cells in WT or Malt1PD cells isolated from blood, spleen, cLN, or mLN. (A) Dot plot displaying Foxp3 versus Nrp1 pregated on CD4⁺ T cells in cLN. (B) Frequency of CD4⁺Nrp1⁺Foxp3⁻ T cells in 8–12-wk-old WT or Malt1PD animals detected in the indicated organs. The results are expressed as the mean ± SEM; each dot represents a mouse. (C) Frequency of Nrp1⁺Foxp3⁻ T cells among CD4⁺ T cells in cLNs of 3–12-wk-old WT or Malt1PD animals. (D) Histograms showing expression of PD1, GITR, ICOS, CD25, and CD44 in WT or Malt1PD Nrp1⁺Foxp3⁻ among CD4⁺ T cells T cells from cLNs. (E) Frequency of IFN-γ⁺ cells within populations of CD4⁺Nrp1⁻Foxp3⁻ naive T cells, CD4⁺Nrp1⁺Foxp3⁻ T cells, CD4⁺Foxp3⁺ Tregs, and CD8⁺ T cells after 4 h PMA/Ionomycin stimulation of cLN cells isolated from WT or Malt1PD animals. The results are expressed as the mean ± SEM (n = 5). Data are representative of at least two independent experiments. (F) Frequency of Nrp1⁺Foxp3⁻ T cells among CD4⁺ T cells T cells in blood of 1:1 (WT/Malt1PD) mixed BM chimeras 9 wk after reconstitution compared with 10–12-wk-old nonchimeric WT or Malt1PD animals. (G) Dot plots showing the gating on anergic T cells in spleens of WT (upper panel) and Malt1PD (lower panel) animals at the age of 8–12 wk. CD4 T cells were gated on CD44⁺Foxp3⁻ cells, which were further divided into CD73⁺FR4⁺ anergic T cells (T_anergic) and CD73^lowFR4^low T_EM. Histograms display expression of Nrp1 in T_anergic versus T_EM. (H and I) Frequency of anergic T cells (H) and Nrp1⁺ cells within T_EM (I) in indicated organs of 8–12-wk-old WT or Malt1PD animals. The results are expressed as the mean ± SEM (n = 5). Data are representative of at least two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.