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: Immune Obese Mice Develop T Cell-Dependent Lethal Acute Pancreatitis upon Re-challengeCohorts of mice were fed lean or high-fat diet starting at 4–5 weeks of age until 13–15 weeks of age. Lean mice were <29 g (n = 10), and obese mice were >40 g (n = 13). Some of the mice were immunized with LCMV-Armstrong, and the rest were left LCMV naive. Sixty-five days later, the mice were challenged with LCMV-Clone13 (2 × 106 PFUs, i.p.) and analyzed 2 or 5 days later.(A) An illustration of the experimental approach.(B) Survival of naive (solid lines) and immune (dashed lines) mice after challenge, including mice requiring humane euthanasia. Among the re-challenged LCMV-immune mice, a Mantel-Cox log rank test revealed a significant difference (p = 0.0039) between the naive and obese mice. Data represent 7–16 mice/group.(C) Lipase activity in ascites (left) and serum (right) at 2 days post-challenge infection. Dots on figure represent individual mice (3–6 per group, two independent experiments).(D) Representative pancreas sections from the indicated four groups of mice at 2 days post-challenge infection. Sections were stained with H&E (upper two rows) or Von Kossa stain to reveal calcium deposition (dark gray, bottom row). The upper images show regions within the pancreas; the middle bottom images show the margin of the pancreas. Scale bar, 150 μm.(E) Concentrations of IL-6, IFNg, and TNF in blood. Symbols represent individual mice (3 or 4 per group, two independent experiments). Significance determined by unpaired Student’s t test: *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 4: In situ vaccination with single-dose NvH and NvIH enhanced antitumor immune responses and reduced immunosuppression in TME and systemically.(A) Experimental design. Balb/c mice were inoculated subcutaneously (s.c.) with 4.5 × 105 4T1 cells on the right flank. On day 21, mice were treated intratumorally with (NVs + αPD-1)-loaded in hydrogel (NvIH) or controls, and tumors and spleens were isolated for immune analysis on day 25. (B to E) qPCR results for cytokines and chemokines in 4T1 tumors indicate that NvH and NvIH enhanced the transcription of proinflammatory genes Tnfa, Il6, Nos2, Ifnb1, and Cxcl9 and reduced the transcription of tumor immunosuppressive genes Arg1 and Mrc1. (F to K) 4T1 tumor immune milieu analysis indicates that NvH and NvIH increased antitumor immune cells and reduced tumor immunosuppressive MDSCs, Treg, M2/M1-like macrophage ratios, and Treg/CD8+ T cell ratios. (L to N) NvH and NvIH enhanced CD40 and CD80 expression in splenic DCs (L), enhanced IFN-γ–expressing functional CD8+ T cells (M), and reduced M2/M1-like macrophage ratios (N), indicating systemic antitumor immunomodulation. Data: mean ± SEM (n = 4). P values were determined by two-way ANOVA followed by Tukey’s multiple-comparison test (n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig 5: CoH mice have increased frequency of “classical” Ly6Chi CD115+ monocytes at steady state that produce more TNF-α(A–D) CD11b+CD115+ monocytes were flow sort-purified from spleens of steady-state SPF and CoH mice, RNA was isolated, bulk RNA-seq analysis was performed, and comparison of gene expression was conducted.(A) Principal-component analysis from RNA-seq data showing unique clustering of CD115+ monocytes from steady-state SPF and CoH mice.(B) Volcano plot of log false discovery rate (FDR, y axis) by log fold change (x axis). Genes with an FDR < 0.01 with increased expression in CD115+ monocytes from spleens of steady-state CoH mice compared with SPF mice are shown in green, while genes with an FDR < 0.01 with decreased expression in CD115+ monocytes from spleens of steady-state CoH mice compared with SPF mice are shown in red.(C) Heatmap showing relative gene expression in CD115+ monocytes from spleens of steady-state SPF and CoH mice. Seven hundred and twenty genes were differentially expressed with an FDR < 0.01.(D) GO term analysis for pathways differentially regulated between CD115+ monocytes from spleens of steady-state CoH and SPF mice.(E) Representative histogram of MPO expression in SPF and CoH CD115+ monocyte/Mφ (left) and frequency of myeloperoxidase (MPO)+ cells among CD115+ monocytes in the spleen as measured by flow cytometry (right).(F) Number of Ly6c1 and Ly6c2 mRNA transcripts in CD115+ monocytes from steady-state SPF and CoH mice (left), representative histogram of Ly6C protein expression on SPF and CoH CD115+ monocytes (middle), and frequency of Ly6C+ cells among CD115+ monocytes in the spleens of CoH mice (right).(G) Frequency of TNF-α+ cells within CD11b−CD64+ resident macrophages, Ly6ChiCD64+ monocytes, and Ly6CloCD64+ monocytes in the spleens of SPF and CoH at steady-state and after in vitro LPS stimulation (100 ng/mL).(H) Geometric mean fluorescence intensity (gMFI) of TNF-α expression by unstimulated and LPS-stimulated TNF-a+CD11b−CD64+ resident macrophages (green), CD11b+Ly6ChiCD64+ classical (red), and CD11b+Ly6CloCD64+ non-classical (black) monocytes was determined by flow cytometry (left). Dashed lines, SPF; solid lines, CoH. TNF-α gMFI within TNF-α+CD11b−CD64+ resident macrophages, Ly6ChiCD64+ classical monocytes, and Ly6CloCD64+ non-classical monocytes in the spleens of SPF and CoH at steady-state and after in vitro LPS stimulation (right). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, and ****p < 0.0001 as determined by nonparametric Mann-Whitney test. Transcript (normalized copies per million [nCPM]) data in (F) were obtained from 3 to 4 mice per group. Flow data in (E) were combined from two experiments using a total of 9 mice per group, in (F) were combined from three experiments using a total of 25–26 mice per group, in (G) were combined from three experiments using a total of 8–9 mice per group, in (H) were combined from two experiments using 3 mice per group. Each symbol in (A and E–H) represents a mouse and bars indicate means with SEM.