Fig 1.
Poor CD8 T cell response to intracellular bacterial pathogens in Xiap−/− mice.
(A-C) WT and Xiap−/− mice were infected with ST (200 CFU, i.v.) and the impact on host survival was monitored (A). Bacterial burden was measured in the spleens of infected mice at days 3, 4 and 5 post infection (B). Cytokine levels were measured in the serum at day 5 post infection (C). (D-I) WT and Xiap−/− mice were infected with ST-OVA (103 CFU, i.v.) and the spleens were harvested from the infected mice at various timepoints post-infection to evaluate the antigen specific CD8 T cell response to OVA257-264 peptide. D) Bacterial burden in the spleens. E) Numbers and F) percentage of OVA257-264 (SIINFEKL)-specific CD8 T cells, on day 7 post-infection were evaluated in the spleens of infected mice by staining with anti-CD8 antibody and H2-Kb-OVA257-264 Dextramer. (G-I) ELISPOT assay was performed in spleen cells stimulated with the OVA257-264 peptide in vitro. G) Representative IFN-γ positive spots in an ELISPOT assay plate. H) Number of OVA257-264 specific cells secreting IFN-γ in response to OVA257-264. I) Relative number of antigen specific cells in Xiap−/− mice in comparison to the number in WT mice. (J, K) WT and Xiap−/− mice were infected with LM-OVA (103 CFU, i.v.). J) Bacterial burden in the spleens of infected mice at various time intervals. K) Number of OVA257-264 specific cells secreting IFN-γ, in response to OVA257-264 peptide, evaluated by ELISPOT assay. Data is representative of 2 (A-C, J, K) or 3 (D-I) experiments. Each data point (B-E, H-K) represents a separate mouse. Statistical analysis was performed by log-rank test (A), unpaired student t-test (B, C, E, I), and 2-way ANOVA (H, J, K). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Fig 2.
WT CD8 T cells undergo poor expansion in infected Xiap−/− mice.
A) Schematic representation of the adoptive transfer protocol. B) CD8 T cells from WT OT1 mice (CD45.1+CD45.2+) were injected (106 cells, i.v.) into naïve WT mice (CD45.1- CD45.2+) or Xiap−/− mice (CD45.1+ CD45.2-) and the proportion of transferred cells evaluated at day 2 post cell transfer by flow cytometry. (C-G) Splenocytes from WT OT1 mice (CD45.1+CD45.2+) were injected (5 x 104 cells, i.v.) into WT mice (CD45.1- CD45.2+) or Xiap−/− mice (CD45.1+ CD45.2-). Two days later, the recipient mice were infected with ST-OVA (103 i.v.). On day 7 and 15 post-infection, the spleens of the recipient mice were harvested, and the donor OT1 cells were tracked by flow cytometry using antibodies against CD8, CD45.1 and CD45.2. (C) Representative dot plots and (D) numbers of the WT OT1 CD8 T cells in the recipient mice. (E, F) Representative dot plots and (G) percent distribution of various CD8 T cell subsets within the transferred OT1 populations after staining with various antibodies described in the methods. Data is representative of 3 (A-D) or 2 (E-G) experiments. Each data point (D, G) represents a separate mouse. Statistical analysis was performed by unpaired student t-test (*P<0.05, **P<0.01, ****P<0.0001).
Fig 3.
Xiap−/− DCs induce poor antigen presentation to WT CD8 T cells in vitro.
(A-F) Bone marrow derived DCs generated from WT and Xiap−/− mice were infected with ST-OVA as described in the methods section. Infected DCs were incubated with CFSE labeled purified WT OT1 CD8 T cells. At various time intervals, cells were harvested stained with Zombie Yellow, and antibodies against Ki67 and CD8 and evaluated for proliferation and viability via flow cytometry (A-D). Secretion of IFN-γ was evaluated by ELISA in cell supernatants collected at 72h (E). Cells were imaged at 72h post culture (F). Data is representative of 3 (A-D) or 2 (E, F) experiments. Each data point (B, D) represents a separate mouse. Statistical analysis was performed by 2-way ANOVA. (*P<0.05, **P<0.01).
Fig 4.
Impaired priming of CD8 T cells by Xiap−/− DCs is due to poor IL-6 expression.
DCs were generated from WT and Xiap−/− mice and infected with ST-OVA (A, B). Secretion of various cytokines by infected DCs was measured by ELISA in supernatants collected at 24hr post infection (A). Cell death of DCs was measured at 24hr post-infection by neutral red assay, and IL-1β secretion was measured by ELISA in the supernatants collected at 24h post infection (B). (C, D) DCs were infected with ST-OVA (25 MOI) and incubated with purified WT OT1 CD8 T cells in the presence or absence of IL-6 (50 ng/ml). Cell proliferation was measured by staining with Zombie Yellow, and antibodies against Ki67 and CD8 at 96hr. (E-G) WT OT1 CD8 T cells were injected (103/mouse) iv into WT (E, G) or Xiap−/− (F, G) recipient mice. After 24h recipient mice were infected with ST-OVA (103, ip). IL-6 was injected in several groups of mice (1 μg/mouse) ip on day 1, 3 and 5. Control mice received PBS. On day 7 post infection, spleens were removed from infected mice and spleen cells stained with labeled antibodies against CD45.1, CD45.2 and CD8. Stained cells were acquired on Flow cytometer and the relative numbers of adoptively transferred OT1 CD8 T cells evaluated. Data is representative of 3 (A-D) or 2 (E-G) experiments. Each data point (A, B, D, G) represents a separate mouse. Statistical analysis was performed by 2-way ANOVA (F) and unpaired student t-test (*P<0.05, **P<0.01, ***P<0.001).
Fig 5.
Xiap−/− CD8 T cells undergo increased contraction in WT mice.
(A) Schematic representation of the adoptive transfer protocol. Splenocytes from both WT OT1 (CD45.1-CD45.2+) and Xiap−/− OT1 (CD45.1+CD45.2+) were mixed 1:1 and injected (104 cells each, i.v.) into B6.SJL mice (CD45.1+ CD45.2-). After two days the recipient B6.SJL mice were infected with ST-OVA (103 i.v.). At day 7 and 15 post-infection, the spleens of the recipient mice were harvested, and the donor OT1 cell numbers evaluated by flow cytometry using antibodies against CD8, CD45.1 and CD45.2. Representative dot plots (B), and cell number (C) of WT and Xiap−/− OT1 CD8+ T cells in the WT recipient mouse is shown. Representative dot plots (D) and the distribution (E) of various OT1 CD8+ T cell subsets in the same host based on flow cytometric analysis following staining with antibodies is shown. Data is representative of 3 (A-C) or 2 (D, E) experiments. Each data point (C, E) represents a separate mouse. Statistical analysis was performed by unpaired student t-test (C) (****P<0.0001), and 2-way ANOVA (E).
Fig 6.
Cell intrinsic XIAP restricts cell cycling and death of activated CD8 T cells.
(A-D) Bone marrow derived dendritic cells were generated from WT mice and infected with ST-OVA. CD8 T cells were purified from WT OT1 and Xiap−/− OT1 spleens and labelled with CFSE and incubated with the infected WT DCs. At the indicated time intervals cells were stained with Zombie Yellow, and antibodies against Ki67, caspase-3 and CD8 and evaluated for proliferation and viability via flow cytometry. (A) Representative contour plot showing proliferation and cell death of OT1 CD8 T cells. (B) Representative histograms showing Ki67+ of OT1 CD8 T cells. (C) Graphs showing Ki67+ Zombielow OT1 CD8 T cells. (D, E) Expression of cleaved caspase-3 was evaluated by flow cytometry. (F) Supernatants were collected at 24hr post DC+OT1 CD8 co-culture and IFN-γ secretion evaluated by ELISA. (G-I) WT and Xiap−/− CD8 T cells were stimulated with plate-bound anti-CD3 (1 μg/ml) + anti-CD28 (10 μg/ml) antibodies. Expression of IL-2 was measured by qRT-PCR analysis at various time intervals (G). Activation of NFκB and BIM (H) and various caspases (I) was evaluated at various time intervals by western blotting of cell extracts. (J) WT OT1 and Xiap−/− OT1 spleen cells (107/mL) were incubated with ST-OVA (103) for 3h followed by culture in media containing gentamicin. Cells were diluted 3-fold daily after day 3 with supplementation of fresh IL-7 (1 ng/ml). Cells were harvested at various time intervals and counted. Data is representative of 3 (A-C, F, G) or 2 (D, E, H-J) experiments. Each data point (C, G) represents a separate mouse. Statistical analysis was performed by 2-way ANOVA. (**P<0.01, ***P<001).
Fig 7.
Cell intrinsic XIAP promotes functional CD8 T cell memory.
(A-C) WT and Xiap−/− OT1 cells were adoptively transferred into WT hosts and infected with ST-OVA as described in panel A. A group of ST-OVA infected mice were challenged with LM-OVA on day 30. On day 30 and day 35 post ST-OVA infection, the numbers of adoptively transferred OT-1 cells were enumerated in recipient mice. Dot plots (C) and the proportion (B) of WT and Xiap−/− OT1 cells among CD8 T cells are shown. (D, E) WT and Xiap−/− mice were infected with ST-OVA (103, iv). On day 30 post-infection mice were re-challenged (D) with a higher dose of ST-OVA (105, iv) and bacterial burden evaluated in the spleens of mice five days later (E). Data is representative of 3 (A, B) or 2 (C-E) experiments. Each data point (B, E) represents a separate mouse. Statistical analysis was performed by paired student t-test (*P<0.05, **P<0.01, ***P<0.001).