Fig 1: PTPN2 Deficiency Enhances Programmed Expansion of Briefly Stimulated Effector T Cells(A–D) PTPN2-deficient or WT OT-I T cells were activated in vitro with SIINFEKL, H-2Kb, and CD80 expressing artificial APCs (MEC.B7.SigOVA) for 1 (A and B), 2 (C), and 7 (D) days. Activated T cells (105) were then transferred into antigen-free CD45-congenic C57BL/6J host mice. (A and B) Frequency of OT-I among total CD8 T cells was determined 7 days post-transfer (A). Bar graphs in (B) show the representative phenotype of five individual mice of the recovered OT-I T cells. (C and D) Frequency of OT-I among total CD8 T cells was determined 5 days post-transfer.(E) Hosts were grafted as in (A), but 7 days after the transfer they received 106 CD45-congenic, carboxyfluorescein succinimidyl ester (CFSE)-labeled target splenocytes that were pulsed with SIINFEKL peptide and 106 CD45-congenic unpulsed control splenocytes which served as a reference population. The plots show the calculated frequency of residual peptide-pulsed target cells at 6 h post-injection in the spleen.(F) Same setup as (E), but 105 Ova- and GFP-expressing RMA cells and antigen-negative mCherry-expressing RMA cells were intraperitoneally (i.p.) injected. The ratio of GFP versus mCherry RMA cells in the peritoneal fluid was determined by flow cytometry 2 days after the transfer.(G) The left plot shows the OT-I T cell numbers recovered per spleen at 20 h post-transfer and at 7 days post-transfer of 105 activated WT versus KO OT-I T cells. The plot to the right shows the ratio of KO/WT OT-I T cells at 6 h post-transfer of 106 naive T cells. The data are representative of five (A and B) or two (C and G) independent experiments with 3–5 mice each.Dots in all panels represent data from a mouse and horizontal lines the mean. Statistical analysis: unpaired t test, ****p = 0.00001, ***p = 0.0001, **p = 0.001, *p = 0.01, nsp = 0.05.
Fig 2: RelA and RelB increase RANKL expression by MPCs. Calvarial pre-osteoblasts from 7-day-old WT mice were infected with GFP control, RelA, or RelB retroviruses for 48 h. a RelA and b RelB mRNA expression tested by real-time PCR to confirm successful over-expression. Mean ± SD (n = 3 biologically independent samples; **p < 0.01; unpaired Student's t test). c RANKL mRNA expression tested by real-time PCR. Mean ± SD (n = 3; *p < 0.05, **p < 0.01; one-way ANOVA with Tukey’s post-hoc test). d Culture media collected from culture wells and RANKL protein levels measured by ELISA. Mean ± SD (n = 3 biologically independent samples; no significant difference; one-way ANOVA with Tukey’s post-hoc test). e Membrane-bound RANKL levels in 50,000 CD45-Sca-1+ MPCs tested by flow cytometry and expressed as mean fluorescence intensity (MFI). Average of 2 biologically independent samples from two individual experiments. All the in vitro experiments were repeated twice with similar results
Fig 3: Activation of cGAS-STING suppressed bladder cancer in cisplatin-treated C57 mice. (A) Establish of bladder cancer transplantation models in C57 mice, left. Tumor volumes of different groups, right. (B–E) Infiltration percentages of (B) CD8+, CD45+ T cells, (C) CD11b+, MHC?+ DCs, (D) F4/80+, CD11c+ Macrophage, (E) CD3+, CD45+ cells in MB49 transplantation tumors. (F–J) CYBERSORT predicted infiltration percentages of CD8+ T cells (F), DCs activated cells (G), DCs resting cells (H), Macrophages M1 cells (I), and Macrophages M2 cells (J) in response group (n = 50, CR or PR) was significantly higher than non-response group (n = 30, PD or SD) after cisplatin-based chemotherapy. * p < 0.05, ** p < 0.01, *** p < 0.001. DDP, cisplatin.
Fig 4: T cell differentiation is disrupted in p40-/- and p35-/- mice(A–H) p40-/- (open circles, blue), p35-/- (open squares, green), and WT (open triangles, black) mice were challenged with 106 dsRed L. major parasites by intradermal injection in the footpad.(A) Biweekly measurement of footpad thickness following appearance of lesions for p40-/- (open circles, blue), p35-/- (open squares, green), and WT (open triangles, black) through the duration of dsRed L. major infection (n = 3).(B) Footpad thickness of p40-/- (open circles, blue), p35-/- (open squares, green), and WT (open triangles, black) mice 28 days after dsRed L. major infection (unpaired t test, n = 3).(C and D) Infected animals were bled 4 h and at days 1, 2, 3, 4, 7, 14, and 21 after L. major infection. Serum (C) p40 and (D) IL-12 levels for p40-/- (open circles, blue), p35-/- (open squares, green), and WT (open triangles, black) animals were measured by ELISA (n = 3).(E–H) Splenocytes harvested 28 days after L. major infection were restimulated and analyzed via flow cytometry. Gating strategy used in these panels is shown in Figure S1A.(E) Percentage of IFN?-producing CD4+ T cells within the CD44hi subset in the spleen measured 28 days after dsRed L. major infection (n = 3).(F) Number of CD44hiIFN?+CD4+ T cells in the spleen of p40-/- (open circles, blue), p35-/- (open squares, green), and WT (open triangles, black) animals measured 28 days after dsRed L. major infection (unpaired t test, n = 3).(G) Similar to (D), except CD8+ T cells (n = 3).(H) Similar to (E), except CD8+ T cells (unpaired t test, n = 3).(I) Cells from the spleen, draining popliteal lymph nodes (dLNs), and footpad were harvested 28 days after dsRed L. major infection and restimulated with soluble Leishmania antigen (SLA). Concentration of IFN? in supernatant was measured by ELISA collected from p40-/- (blue), p35-/- (green), and WT (black, shaded) after restimulation for 48 h (unpaired t test, n = 3).(J and K) SMARTA T cells from CD45.1+ donor animals were sorted and transferred into CD45.2+ p40-/- or CD45.1+CD45.2+ WT recipient animals and challenged 24 h later with GP61–80 and LPS. 4 days after challenge, harvested cells from the spleen and LNs were restimulated with GP61–80 in vitro for 24 or 48 h. Concentrations of IFN? in the supernatant of (J) splenocyte or (K) LN SMARTA T cells transferred into p40-/- (open bars, blue gradient) or WT (open bars, gray gradient) animals 24 or 48 h after restimulation (n = 3) are shown.Data in this figure are representative of two independent experiments where n refers to the number of biological replicates in each experiment. (A)–(D), (F), and (H)–(K) are displayed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Colored asterisks indicate relative significance to different groups (blue is significance to p40-/- and green is significance to p35-/-). Anything unmarked is considered not significant.
Fig 5: The Expansion and Activation of Non-classical CD8+ T Cells is MCMV Dependent(A) Representative cell proliferation dye (CPD) labeling of donor non-classical CD8+ T cells (CD45.2+) in the spleen and liver of KbDb-/-.SJL (CD45.1+) recipients on day 4 post-transfer. Recipients were left naive (black), treated with Poly(I:C) + CpG (gray), or infected with MCMV (blue). Histograms are gated on total donor non-classical CD8+ T cells.(B–D) Frequencies of donor CD8+ T cells that (B) have undergone more than one cycle of proliferation, (C) are CD69+, or (D) are KLRG1+ (n = 5–6).Data are pooled from two independent experiments and represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
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