Fig 2: UTX-deficient CD8+ T cells fail to control disseminated virus infection(A) Approach: WT and UTX-TCD mice were depleted of CD4+ T cells followed by LCMV-A22 infection. The contribution of UTX to CD8+ T cell-dependent immune control of infection was assessed at day 21 post-infection.(B and C) Titers of infectious virus in sera (B) and in livers, lungs, and kidneys (C) at day 21 post-infection.(D) Dot plots show CD8+ T cell expression of CD44 and CD62L, and the graphs show the number of CD44hi T cells in the spleen or their percentage among CD8+ T cells at day 21.(E) Dot plots show examples of CD8+CD44hi T cells bound to DbGP33 tetramer, and the graphs show the number of DbGP33+ T cells per spleen or their percentage among splenic CD8+ T cells.(F) Dot plot shows CD8+ T cells and their expression of IFN? and TNF following ex vivo stimulation with GP33–41 peptide; the graphs depict the total number of IFN?+CD8+ T cells per spleen or the gMFI of IFN? among IFN?-producing CD8+ T cells.Data were combined from two independent experiments with 5–6 mice per group. Error bars display mean ± SEM. Significance was determined using an unpaired Student’s t test (*p < 0.05, **p < 0.01).See also Figures S1 and S2.

Fig 3: UTX limits CD8+ T cell accumulation and maintenance(A–G) WT and UTX-deficient P14+CD8+ T cells were introduced into the same recipients, followed by CD4 depletion and infection with LCMV-A22.(A) The two donor CD8+ T cell populations and host CD8+ T cells were identified by surface expression of Ly5a and Ly5b.(B) Infectious virus in livers, lungs, and kidneys at day 35 post-infection.(C) The frequency of donor cells among all CD8+ T cells in the blood at days 10, 22, and 35 post-infection. For illustrative purposes, donor populations are graphed separately yet were in the same mice.(D) The frequency and number of donor cells in the spleen at days 9, 22, and 35 post-infection. Lines connect pairs of WT and UTX-deficient P14+ cells present in each recipient.(B)–(D) show one representative experiment of two independent experiments (n = 3–6).(E and F) Recipient mice were treated with 2 mg of BrdU via intraperitoneal injection on days 4, 5, and 6 post-infection.(E) Illustration of the approach.(F) The percentage of donor cells staining positive for BrdU in the blood or spleen day 7 post-infection.(G) The percentage of donor cells staining positive for Ki-67 in the spleen as determined by flow cytometry.(F) and (G) show one representative experiment of two independent experiments (n = 3).(H–J) WT and UTX-deficient P14+CD8+ T cells were introduced into separate mice, followed by CD4 depletion and infection with LCMV-A22. Cells were isolated at day 9 post-infection. Anti-BIM and anti-BCL2 fluorescence intensity were assessed directly ex vivo, and cleaved caspase-3 and annexin V staining were measured after brief culture.(H) Representative plots display the expression of Bim and Bcl-2 in donor cells. Graphs show gMFI for BIM and BCL-2, as well as the ratio of BIM gMFI to BCL-2 gMFI. Data are from one of three independent experiments with 2–3 mice per group.(I) Donor cells were cultured with GP33–41 peptide for 5 h followed by quantification of intracellular cleaved caspase-3.(J) Surface binding of annexin V following GP33–41 stimulation.Graphs in (I) and (J) show data compiled from three independent experiments with 6–8 mice per group.Error bars display mean ± SEM. Significance was determined by a paired (C, D, F, and G) or unpaired (H and J) Student’s test (*p < 0.05, **p < 0.01, ***p < 0.001).See also Figures S3 and S4.

Fig 4: Inhibition of Either Lipid Uptake or Lipid Oxidation Prevents Immunosuppressive Capabilities of Regulatory T Cells(A) Sorted and expanded Tregs were cultured for 72 h in the presence of the fatty acid oxidation inhibitor etomoxir (200 µM) or the fatty acid uptake inhibitor SSO (200 µM), and the expression of various Tregs markers was assessed via flow cytometry.(B and E) Sorted Tregs were cultured for 72 h in the presence of etomoxir (Eto; 200 µM; B) or SSO (200 µM; E), and Foxp3 retention was determined.(C and D) Percent CD8 T cell proliferation with co-culture of Eto pre-treated Tregs shown as a histogram (C) and analyzed as bar graphs with reducing Treg ratios (D) that were enumerated via flow cytometry.(F and G) Percent CD8 T cell proliferation with co-culture of SSO pre-treated Tregs shown as a histogram (F) and analyzed as bar graphs with reducing Treg ratios (G) that were enumerated via flow cytometry.(H and I) Sorted and expanded WT or HIF-1a KO Tregs were pretreated with (H) Eto (200 µM) or (I) SSO (200 µM) for 24 h before a suppressor assay was run under 1% O2. After 72 h, expansion indexes were determined via flow cytometry.Flow cytometry statistics were calculated and shown as percent positive or MFI ± SEM; n = 5 per group in (A), and n = 3 per ratio in (B)–(I), representative of 2–3 independent experiments. A one-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.See also Figure S3.

Fig 5: A Distinct Subset of Memory T Cells in WAT(A and B) B6 recipient mice (Ly5a–) were given TCR-transgenic (Tg+) 2 × 104 P14 CD8+ (Ly5a+) T cells or SMARTA CD4+ (Ly5a+) T cells and then infected with LCMV-Armstrong 1–2 days later. At day 38 post-infection, intravascular (i.v.) staining was performed to label circulating donor CD4+ or CD8+ T cells and distinguish them from T cells within the spleen or adipose tissue.(A) An illustration of the method whereby intravenous staining is coupled to ex vivo staining that labels all T cells in suspension.(B) Representative dot plots showing the proportion of T cells protected from intravenous staining because of their location within the parenchyma of the spleen or WAT. Note that the vast majority of T cells purified from WAT are not contaminants from circulating subsets. Data represent three independent experiments with three or four mice per group. (C and D) B6 recipient mice (Ly5a–) were given TCR-transgenic (Tg+) splenic 2 × 104 P14 (Ly5a) or SMARTA (Ly5a+) T cells and then infected with LCMV-Arm 1–2 days after engraftment. At day 38, spleen and WAT cells were isolated and co-stained to identify the donor T cells and their expression of phenotypic markers.(C) The histograms are gated on P14 memory cells (top) or SMARTA memory cells (bottom) and show their surface expression of the indicated molecules. Memory donor cells from spleen (red) or WAT (blue) are shown with naive splenic Tg+ cells (black). Data represent two experiments with four to six mice per group.(D) Memory P14 and SMARTA donor cells from day 50 post-infection were FACS-purified from the spleen or adipose tissue and subjected to RNA sequencing to identify changes in gene expression. The dot plots show donor cells from adipose tissue before and after FACS. The graphs show EdgeR analysis of the RNA-seq data with log fold change (logFC) in RNA expression in WAT T cells relative to splenic T cells plotted against theamount of RNA per cell, expressed as log counts per million reads (logCPM). The red circles identify significant (false discovery rate [FDR] < 0.05) differences. These data are from two replicated experiments. Each replicate used pooled RNA isolated from ten (P14) or five (SMARTA) recipient mice.See also Figures S2 and S3 and Tables S1, S2, and S3.