Figure 1.
Qualitative and quantitative differences in IFN-γ production induced by combinations of IL-12, IL-18, and/or IL-21.
Splenocytes from LCMV-Arm infected B6 mice (acute infection) were prepared at 9 days post-infection and stimulated with the indicated cytokines. (A) Representative flow cytometry plots show IFN-γ production by gated CD8 T cells following stimulation with the indicated cytokines. The numbers in parentheses indicate the percentage of IFN-γ-producing DbGP33+ cells. (B) The MFI of IFN-γ production by gated IFN-γ+ tetramer+ CD8 T cells is shown in response to stimulation with the various cytokines. Error bars are SD. Representative or composite results are shown from five experiments analyzing a total of 11-16 mice.
Figure 2.
Exhausted CD8 T cells do not produce IFN-γ in response to antigen-independent stimulation.
Splenocytes were stimulated for 5.5 hr with the indicated cytokines and IFN-γ production by DbGP33 tetramer binding cells was determined by intracellular cytokine analysis at days 9 (effector) or 35-36 (memory/exhausted) following infection. (A–C) Representative flow cytometry plots show gated CD8 T cells, and numbers in parentheses indicate the percentage of LCMV GP33-specific CD8 T cells that produce IFN-γ in response to activation with IL-12+IL-18+IL-21 following (A) acute, (B) protracted, or (C) chronic infection. (D) Mean percentage ± SD of LCMV GP33-specific CD8 T cells that produce IFN-γ in response to the indicated cytokines at days 9 and 35-26. Acute: LCMV-Arm, black circles; protracted: LCMV-cl13 infected B6 mice, white squares; and chronic: LCMV-cl13 infected CD4-/- mice, black triangles. Representative (A) or composite (B) results are shown from five independent day 9 experiments with a total of 14-19 mice per group and three independent day 35-36 experiments with a total of 8-9 mice per group.
Figure 3.
Exhausted CD8 T cells do not upregulate CD25 expression following IL-12, IL-18, and/or IL-21 exposure.
(A) Representative flow cytometry plots show the expression of CD25 by gated CD8 T cells following acute (B6 Arm), protracted (B6 cl13), and chronic (CD4-/- cl13) LCMV infections in response to a 5.5 hr incubation with or without IL-12, IL-18, and IL-21 in the absence of BFA. Numbers in parentheses indicate the percentage of CD25+ LCMV GP33-specific CD8 T cells. (B) The MFI± SD of CD25 expression on LCMV DbGP33-specific CD8 T cells following stimulation with the indicated sets of cytokines. Acute: LCMV-Arm, black bars; protracted: LCMV-cl13 infected B6 mice, white bars; and chronic: LCMV-cl13 infected CD4-/- mice, striped bars. Representative or composite data are shown from two independent experiments at 38-39 days post-infection with a total of 7-8 mice per group.
Figure 4.
Cognate cytokine receptor expression on effector, memory, and exhausted CD8 T cells.
Splenic LCMV DbGP33-specific CD8 T cells were analyzed at days 9 or 35-36 following acute, protracted, and chronic infections for the expression of IL-12Rβ2 (A, D), IL-21R (B, E), and IL-18Rα (C, F). (A–C) Representative histograms depict the expression of the cytokine receptors on GP33-specific CD8 T cells. Light shaded peak: isotype control, dark shaded peak: B6 Arm (acute), solid line: B6 cl13 (protracted), dotted line: CD4-/- cl13 (chronic). (D-F) Show the MFI of cytokine receptor expression on the virus-specific CD8 T cells from individual mice. Mean values are indicated by the horizontal bars. The results are shown from a total of 6-9 individual mice per group, derived from 2-3 independent experiments.
Figure 5.
Rh123 efflux in memory and exhausted CD8 T cells.
Splenocytes prepared from mice following acute (B6 Arm), protracted (B6 cl13), and chronic (CD4-/- cl13) LCMV infections were loaded with Rh123 and then left untreated or treated with CsA or vinblastine. The efflux of Rh123 was determined 1hr later. (A) The Rh123 efflux profiles of gated CD8 T cells, without or with CsA treatment, are shown in conjunction with IL-18Rα staining. (B) The Rh123 efflux of gated LCMV GP33-specific CD8 T cells is shown. Shaded line: without inhibitor, solid line: with CsA, dashed line: with vinblastine. Data are representative of two independent experiments at day 38-39 post-infection with a total of 7-8 mice analyzed per group.
Figure 6.
LCMV-specific IL-18Rα-/- CD8 T cells are preferentially lost from day 7-16 following cl13 infection.
The maintenance of IL-18Rα+/+ and IL-18Rα-/- LCMV GP33-specific CD8 T cells was monitored in mixed bone marrow chimeras following cl13 infection. Mice from control (CD45.1, IL-18Rα+/+ & CD45.2, IL-18Rα+/+) and experimental (CD45.1, IL-18Rα+/+ & CD45.2, IL-18Rα-/-) cohorts were bled and analyzed at the indicated days post-infection. (A) The percentage of CD45.2 LCMV GP33-specific CD8 T cells over time and (B) the fold change in the CD45.2+GP33+ population normalized to the levels detected at day 7 post infection in the control (IL-18Rα+/+, open squares) and experimental (IL-18Rα-/-, filled triangles) cohorts. (C) The MFI of IL-18Rα expression on CD45.1+ and CD45.2+ GP33-specific cells in the control and experimental cohorts. Data are shown from two independent experiments with (A and B) 8-11 mice per group and (C) 3-10 mice per group at each time-point; *** p<0.001.
Figure 7.
Memory but not exhausted T cells express IFN-γ and CD25 in response to LM infection.
Cohorts of mice were challenged with wild-type LM 59-81 days following acute (LCMV-Arm), protracted (LCMV-cl13 infected B6 mice) and chronic (LCMV-cl13 infected CD4-/- mice) infections. Splenocytes were prepared 20 hr after the bacterial infection and cultured in the presence of BFA for 3hr prior to surface MHC tetramer staining and intracellular staining for IFN-γ accumulation. (A) Overview of the experimental design. (B) Representative flow cytometry plots show IL-18Rα expression and IFN-γ production by gated CD8 T cells are shown with and without LM infection. (C) IL-18Rα and (D) CD25 expression in conjunction with IFN-γ production are shown for gated LCMV DbGP33+ CD8 T cells. The percentages of (E) IFN-γ-producing and (F) CD25-expressing IFN-γ+ LCMV Db(GP33) and Db(GP276) epitope-specific CD8 T cells are shown for individual mice, and mean values are indicated by the horizontal bars. (G) and (H) Bacterial titers in the spleens and livers, respectively, following LM challenge of the cohorts of mice that were previously infected with LCMV. Flow cytometry plots show representative data from either (B, C) three or (D) two independent experiments analyzing a total of 11-12 LM challenged mice per group. Graphs show results from (E) 4–5 experiments with 15-18 mice per group, from (F) two experiments with 6-8 mice per group, from (G) four experiments with 14-15 mice per group, and from (H) three experiments with 10–12 mice per group. *** p<0.001, ** p<0.01, * p<0.05.