T cell expansion in <i>Nfatc2</i> and <i>Tob1</i> KO mice is not due to Treg dysfunction.

<p>CD4 T cells were isolated from single cell suspensions of spleens and lymph nodes from age-matched WT, <i>Nfatc2</i> KO, and <i>Tob1</i> KO mice by negative immunomagnetic selection. CD4⁺CD25⁻ (Tconv) cells and CD4⁺CD25⁺ (Tregs) were enriched by sorting. Tconv cells were labeled with CFSE and Treg cells were labeled with PKH26. Tconvs (100,000/well) were then mixed 1∶1 with syngeneic AgPCs and stimulated with anti-CD3 in the presence of absence of 50,000 Treg cells as indicated. Proliferation was measured by CFSE dilution in CD4 T cells using flow cytometry after of 96 hr of culture. (A) Expression of CD4, CD25, and CTLA-4 was assessed by conventional cell surface staining; expression of Foxp3 was examined by intracellular staining Panels are representative two-dimensional contour plots showing CD4 and CD25 staining from WT, <i>Nfatc2</i> KO, and <i>Tob1</i> KO mice gated on CD3 T cells. Boxed areas represent CD4⁺CD25⁺ (Treg) cells and CD4⁺CD25⁻ (Tconv) cells. Small panels to the right represent two-dimensional contour plots showing CTLA-4 and Foxp3 staining, respectively for Tregs on top and Tconvs below. (B) Representative one-dimensional histograms of CFSE dilution from stimulated WT cells. Small panels to the right represent one-dimensional histograms of CFSE dilution for the same WT cells, respectively with the addition of WT Tregs, <i>Nfatc2</i> KO Tregs, <i>Tob1</i> KO Tregs, or WT Tregs treated with CsA as indicated. (C) Means ± S.D. of the percent of cells that underwent 0–7 divisions over 96 hr for each genotype with and without Tregs. The data summarize 8 experiments using WT Tregs, 4 experiments using <i>Nfatc2</i> KO Tregs, 2 experiments using <i>Tob1</i> KO Tregs, and 2 experiments using WT Tregs treated with CsA, each with pooled cells from 2 or 3 mice. The number of WT Tconv cells that did not undergo cell division was significantly greater (p<0.01), and the number of cells undergoing 2 or more divisions was significantly reduced (p<0.0001) when <i>Tob1</i> KO Tregs were present in the culture than when WT Tregs or <i>Nfatc2</i> KO Tregs were present in the culture.