Loss of Vps34 is associated with decreased eTreg survival but intact activation.

(A) Quantitative PCR analysis for Pik3c3 deletion efficiency in TCRβ⁺CD4⁺Foxp3-YFP⁺ Tregs that were sort-purified (pooled from spleen and PLNs) from 17- to 42-day-old control (n =  4) or Foxp3^CrePik3c3^fl/fl (n =  2) mice. PLNs, peripheral lymph nodes. (B) Quantification of frequency (left) and number (right) of total TCRβ⁺CD4⁺Foxp3-YFP⁺ Tregs derived from the spleen of 20- to 30-day-old control (n =  7) or Foxp3^CrePik3c3^fl/fl (n =  4) mice. (C) Flow cytometry analysis (left) and quantification (right) of frequencies and numbers of total TCRβ⁺CD4⁺Foxp3-YFP⁺ Tregs derived from the spleen of control mosaic (n =  13) or Foxp3^Cre/+Pik3c3^fl/fl mosaic (n =  15) mice. (D) Quantification of the frequency (left) and number (right) of total TCRβ⁺CD4⁺Foxp3-YFP⁺ Tregs derived from the lung of control mosaic or Foxp3^Cre/+Pik3c3^fl/fl mosaic mice (n =  6 per group). (E) CD4⁺Foxp3-YFP⁺ Tregs (CD45.2⁺) were sort-purified from the spleen of control (n =  4) or Foxp3^CrePik3c3^fl/fl (n =  5) mixed BM chimera mice and profiled by microarray analysis (see Materials and methods for details). Volcano plot depicting upregulated (red) and downregulated (blue) genes, with select eTreg-associated genes labeled. See also S1 Table. BM, bone marrow. (F) Quantification of frequencies (left) and numbers (right) of CD44^lowCD62L^high cTregs or CD44^highCD62L^low eTregs among total TCRβ⁺CD4⁺Foxp3-YFP⁺ Tregs derived from the spleen of 20- to 30-day-old control (n =  7) or Foxp3^CrePik3c3^fl/fl (n =  4) mice. (G) Quantification of frequencies (left) and numbers (right) of CD44^lowCD62L^high cTregs or CD44^highCD62L^low eTregs among total TCRβ⁺CD4⁺Foxp3-YFP⁺ Tregs derived from the spleen of control mosaic (n =  13) or Foxp3^Cre/+Pik3c3^fl/fl mosaic (n =  15) mice. (H) Quantification of relative (normalized to average control in each experiment) gMFIs of Foxp3 and CD25 in CD44^lowCD62L^high cTregs and CD44^highCD62L^low eTregs among total TCRβ⁺CD4⁺Foxp3⁺ (Foxp3) or TCRβ⁺CD4⁺Foxp3-YFP⁺ (CD25) Tregs (all pre-gated on CD45.2⁺ cells) derived from the spleen of control (n =  22 for Foxp3, 17 for CD25) or Foxp3^CrePik3c3^fl/fl (n =  20 for Foxp3, 15 for CD25) mixed BM chimera mice, as determined by flow cytometry analysis. gMFI, geometric mean fluorescence intensity. (I) Quantification of the frequencies of Ki67⁺ cells among total TCRβ⁺CD4⁺Foxp3⁺ Tregs, CD4⁺Foxp3⁺CD44^lowCD62L^high cTregs, or CD4⁺Foxp3⁺CD44^highCD62L^low eTregs (all pre-gated on CD45.2⁺ cells) derived from the spleen of control (n =  15) or Foxp3^CrePik3c3^fl/fl (n =  13) mixed BM chimera mice. (J) Spleen and PLNs of control mosaic or Foxp3^Cre/+Pik3c3^fl/fl mosaic mice were pooled, and TCRβ⁺CD4⁺Foxp3-YFP⁺CD44^lowCD62L^high cTregs were sort-purified and cultured in vitro in the presence of anti-CD3, anti-CD28, and IL-2 for 72 h. Quantification of the frequency (relative to the average of technical replicates in each experiment) of CD44^highCD62L^low eTreg-like cells (n =  4 biological replicates for control mosaic mice and 6 biological replicates for Foxp3^Cre/+Pik3c3^fl/fl mosaic mice). (K) Quantification of relative (normalized to average control in each experiment) frequencies of FVD⁺ non-viable CD44^lowCD62L^high cTregs or CD44^highCD62L^low eTregs among total TCRβ⁺CD4⁺YFP⁺Foxp3⁺ Tregs derived from the spleen of control mosaic or Foxp3^Cre/+Pik3c3^fl/fl mosaic mice (n =  6 per group). FVD, fixable viability dye. Data are shown as mean ±  s.e.m. (A–D, F–K). Two-tailed Student t test (A–D, F–K) or Wald test (E); NS, not significant. Data are compiled from 1 (A), 3 (B, F, J), 8 (C, G), 4 (D, K), or ≥5 (H, I) independent experiments. Numbers in plots indicate percentages of cells in gates (C). The numerical data underlying the graphs shown in this figure are found in S8 Data (A–D, F–K). Raw microarray data used for analysis in (E) have been deposited to GEO SuperSeries access number GSE279606.

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