Critical role for IL-21 in both primary and memory anti-viral CD8+ T-cell responses

Abstract

While it is well established that CD8(+) T cells generated in the absence of CD4(+) T cells mediate defective recall responses, the mechanism by which CD4(+) T cells confer help in the generation of CD8(+) T-cell responses remains poorly understood. To determine whether CD4(+) T-cell-derived IL-21 is an important regulator of CD8(+) T-cell responses in help-dependent and -independent viral infections, we examined these responses in the IL-21Rα(-/-) mouse model. We show that IL-21 has a role in primary CD8(+) T-cell responses and in recall CD8(+) T-cell responses in help-dependent viral infections. This effect is due to a direct action of IL-21 in enhancing the proliferation of virus-specific CD8(+) T cells and reducing their TRAIL expression. These findings indicate that IL-21 is an important mediator of CD4(+) T-cell help to CD8(+) T cells.

Figure 1

IL-21Rα^−/− mice generate reduced antigen-specific primary CD8⁺ T-cell responses to virally encoded antigens. (A) WT and IL-21Rα^−/− mice were inoculated with rAd-gp140 and p18-specific CD8⁺ T cells in the peripheral blood of individual mice were quantitated with an H-2D^d/p18 tetramer. Data are presented as the percentages of CD8⁺ T cells that bind tetramer and represent the means of six mice per group ± SE and are representative of eight experiments. (B) WT and IL-21Rα^−/− mice were inoculated with rVac-gp160 and p18-specific CD8⁺ T cells in the peripheral blood of individual mice were quantitated with an H-2D^d/p18 tetramer. Data are presented as the percentages of CD8⁺ T cells that bind tetramer and represent the means of six mice per group ± SE and are representative of two experiments. (C) WT and IL-21Rα^−/− mice were inoculated with rAd-gp140 and sacrificed 44 days post-inoculation. Lymphocytes were isolated from peripheral blood, mesenteric lymph nodes and spleen, and p18-specific CD8⁺ T cells of individual mice were quantitated with an H-2D^d/p18 tetramer. Splenocytes from individual mice were counted to allow enumeration of the number of tetramer-positive cells per mouse. Data represent the means ± SE of 11 mice per group and are representative of three experiments. (D) In vivo expression of the luciferase protein from a recombinant adenovirus (rAd). WT and IL-21Rα^−/− mice were inoculated with rAd-luciferase. The levels of luciferase expression were measured over time in the inoculated mice using IVIS. Upper panel: Representative images of luciferase expression in the mice following priming inoculation. Lower panel: The mean values of the amount of luciferase expressed by groups of four mice ± SE following inoculation and are representative of two experiments. The Mann–Whitney test was used for statistical comparisons.

Figure 2

IL-21Rα^−/− mice make similar memory phenotype responses and produce similar cytokines as wildtype mice during primary responses. (A) WT and IL-21Rα^−/− mice were inoculated with rAd-gp140 and p18-specific CD8⁺ T cells in the peripheral blood of individual mice were divided into effector (CD62L^lo CD127^lo), effector memory (CD62L^lo CD127^hi), central memory (CD62L^hiCD127^hi), and CD62L^hiCD127^locell subsets. Percentage of CD27⁺ p18-specific CD8⁺ T cells in peripheral blood of individual mice was also measured. Data represent the means of six mice per group ± SE and are representative of eight experiments. (B) WT and IL-21Rα^−/− mice were inoculated with rVac-gp160 and p18-specific CD8⁺ T cells in the peripheral blood of individual mice were divided into effector, effector memory, central memory, and CD62L^hiCD127^locell subsets. Percentage of CD27⁺ p18-specific CD8⁺ T cells in peripheral blood of individual mice was also measured. Data represent the means of six mice per group ± SE and are representative of two experiments. (C) WT and IL-21Rα^−/− mice were inoculated with rAd-gp140 and sacrificed 44 days post-inoculation. Splenocytes were subjected to intracellular cytokine staining. Data are presented as the percentages of tetramer-positive CD8⁺ T cells staining positively for IFN-γ, TNF-α, or IL-2 and represent the means ± SE of 11 mice per group and are representative of three experiments. The Mann–Whitney test was used for statistical comparisons.

Figure 3

IL-21Rα^−/− mice generate reduced antigen-specific secondary CD8⁺ T-cell responses to virally encoded antigens. (A) WT and IL-21Rα^−/− mice were inoculated with rAd-gp140 and then re-inoculated with rAd-gp140 eight weeks later and p18-specific CD8⁺ T cells were quantitated with an H-2D^d/p18 tetramer. Data are presented as the percentages of CD8⁺ T cells that bind tetramer and represent the means of six mice per group ± SE and are representative of four experiments. (B) The fold increase of cells undergoing secondary expansion after secondary rAd-gp140 inoculation was calculated by dividing the tetramer percentage for each mouse at the peak time point by the tetramer percentage for the same mouse at the day of boost. Data represent the means ± SE of six mice per group and are representative of four experiments. (C) CD8⁺ T cells from rAd-gp140 primed WT or IL-21Rα^−/− mice were transferred into Nude/SCID mice and then reconstituted mice were challenged intranasally with rVac-gp160. Plaque forming units in the ovaries of these mice were quantitated 6 days after challenge. (D) WT and IL-21Rα^−/− mice were inoculated with rVac-gp160 and then re-inoculated with rVac-gp160 nine weeks later. p18-specific CD8⁺ T cells in the peripheral blood of individual mice were quantitated with an H-2D^d/p18 tetramer. Data are presented as the percentages of CD8⁺ T cells that bind tetramer and represent the means of six mice per group ± SE and are representative of two experiments. (E) WT and IL-21Rα^−/− mice were inoculated with rAd-gp140 and then re-inoculated with rAd-gp140 eight weeks later and then sacrificed 21 days post-secondary inoculation. Lymphocytes were isolated from peripheral blood, inguinal lymph nodes, and spleen, and p18-specific CD8⁺ T cells of individual mice were quantitated with an H-2D^d/p18 tetramer. Splenocytes from individual mice were counted to allow enumeration of the number of tetramer-positive cells per mouse. Data represent the mean ± SE of four mice per group and are representative of three experiments. The Mann–Whitney test was used for statistical comparisons.

Figure 4

IL-15Rα^−/− mice generate WT antigen-specific primary and secondary CD8⁺ T-cell responses to virally encoded antigens yet more knockout cells have a memory phenotype. (A) WT and IL-15Rα^−/− mice were inoculated with rAd-Ova; SIINFEKL-specific CD8⁺ T cells in the peripheral blood of individual mice were quantitated with an H-2K^b/SIINFEKL tetramer. Data are presented as the percentages of CD8⁺ T cells that bind tetramer and represent the means of five mice per group ± SE and are representative of two experiments. (B) WT and IL-15Rα^−/− mice were inoculated with rAd-Ova and SIINFEKL-specific CD8⁺ T cells in the peripheral blood of individual mice were divided into effector, effector memory, central memory, and CD62L^hiCD127^locell subsets. Percentage of CD27⁺ p18-specific CD8⁺ T cells in peripheral blood of individual mice was also measured. Data represent the means of five mice per group ± SE and are representative of two experiments. (C) WT and IL-15Rα^−/− mice were inoculated with rAd-Ova and then re-inoculated with rAd-Ova nine weeks later and responses were quantitated with an H-2K^b/SIINFEKL tetramer. Data are presented as the percentages of CD8⁺ T cells that bind tetramer and represent the means of five mice per group ± SE and are representative of two experiments. The Mann–Whitney test was used for statistical comparisons.

Figure 5

Defective secondary responses in IL-21Rα^−/− mice are due to abnormalities in CD8⁺ T cells (A) WT and IL-21Rα^−/− mice were inoculated with rAd-gp140 and sacrificed 44 days post-inoculation. Splenocytes were subjected to intracellular cytokine staining. Data are presented as the percentages of CD4⁺ T cells staining positively for IL-17A, IL-2, TNF-α, or IFN-γ and represent the means ± SE of 11 mice per group and are representative of three experiments. (B) WT and IL-21Rα^−/− mice were inoculated with rAd-gp140 and then re-inoculated with rAd-gp140 eight weeks later and then sacrificed 21 days post-secondary inoculation. Splenocytes were subjected to intracellular cytokine staining. Data are presented as the percentages of CD4⁺ T cells staining positively for IL-17A, IL-2, TNF-α, and IFN-γ and represent the means ± SE of four mice per group and are representative of three experiments. (C) Irradiated mice were reconstituted with either WT Thy1.1⁺ or IL-21Rα^−/− Thy 1.2⁺ bone marrow cells and then inoculated with rAd-gp140 six weeks later. These mice were then re-inoculated with rAd-gp140 eight weeks later and p18-specific CD8⁺ T cells were quantitated with an H-2D^d/p18 tetramer. Data are presented as the percentages of Thy1.1⁺ or Thy 1.2⁺ CD8⁺ T cells that bind tetramer and represent the means of six mice per group ± SE and are representative of two experiments. (D) Irradiated mice were reconstituted with WT Thy1.1⁺ and IL-21Rα^−/− Thy 1.2⁺ bone marrow cells and then inoculated with rAd-gp140 six weeks later. These mice were then re-inoculated with rAd-gp140 eight weeks later and p18-specific CD8⁺ T cells were quantitated with an H-2D^d/p18 tetramer. Data are presented as the percentages of Thy1.1⁺ or Thy1.2⁺ CD8⁺ T cells that bind tetramer and represent the means of six mice per group ± SE and are representative of two experiments. The Mann–Whitney test was used for statistical comparisons.

Figure 6

Mechanism underlying deficient IL-21Rα^−/− CD8⁺ T-cell responses. WT and IL-21Rα^−/− mice were inoculated with rAd-gp140 and sacrificed 21 days post-inoculation. Lymphocytes were isolated from spleens and CD8⁺ T cells were enriched by negative selection. (A) CD8⁺ T cells were labeled with CFSE and placed in culture either unstimulated or stimulated with p18 peptide for seven days. Data are presented as the percentages of CD8⁺ T cells that have divided and represent the means ± SE of four mice per group. This study is representative of two experiments. The Mann–Whitney test was used for statistical comparisons. (B) WT and IL-21Rα^−/− CD8⁺ T cells were stimulated with p18 peptide for 4 h, followed by RNA extraction. RNA was then subjected to RT-PCR-based array to measure the expression of 84 apoptosis-related genes. A heat map showing the relative fold increase in gene expression in knockout cells relative to WT cells is shown, and genes that are up- or downregulated more than two-fold in knockout cells relative to WT cells is shown in the table. Data represent RNA extracted from four mice per group.