A vital role for interleukin-21 in the control of a chronic viral infection

Abstract

Understanding the factors that regulate the induction, quality, and longevity of antiviral T cell responses is essential for devising rational strategies to prevent or combat infections. In this study, we show that interleukin-21 (IL-21), likely produced by CD4+ T cells, directly influences the generation of polyfunctional CD8+ T cells and that the number of CD4+ T cells that produce IL-21 differs markedly between acute and chronic infections. IL-21 regulates the development of CD8+ T cell exhaustion and the ability to contain chronic lymphocytic choriomeningitis virus infection. Thus, IL-21 serves as a critical helper factor that shapes the functional quality of antiviral CD8+ T cells and is required for viral control.

Fig. 1

Diminished IL-21⁺ CD4⁺ T cell responses during the initial phase of LCMV-Cl 13 infection. IL-21 and IFN-γ production by LCMV GP_61-80 CD4⁺ T cells was determined eight days following LCMV-Arm or Cl 13 infections of B6 mice. (A) Flow cytometric analysis of intracellular staining for IL-21 and IFN-γ in splenocytes from LCMV infected Il21^−/− and Il21^+/+ mice after stimulation with GP_61-80 peptide. Gated total CD4⁺ T cells are shown. (B) Enumeration of IL-21-producing CD4⁺ T cells at eight days after LCMV-Arm or Cl 13 infection. Graphs represent mean ± SD; ***P < 0.001. Representative results are shown from two independent experiments (n = 8–9 for Il21^+/+ cohorts and n = 2 for Il21^−/− mice).

Fig. 2

Severe CD8⁺ T cell exhaustion and viral persistence in the absence of IL-21. Splenic CD8⁺ T cell responses and viral titers were evaluated following LCMV-Cl 13 infection of Il21^+/+, ^+/−, and ^−/− mice. (A) Flow cytometric analysis of intracellular cytokine staining for IFN-γ and IL-2 production by CD8⁺ T cells at eight days following infection after restimulation without or with the indicated peptide epitopes. Gated CD8⁺ T cells are shown and the percentages of CD8⁺, IFN-γ⁺ cells that co-produce IL-2 are reported in parentheses. (B) Percentages of epitope-specific CD8⁺, IFN-γ⁺ cells that coproduce IL-2 at eight days following infection. Error bars are SEM; * P<0.05 by comparison with Il21^+/+ group. (C) Serum viral titers over time following LCMV-Cl 13 infection of Il21^+/+, ^+/−, ^−/−, and Cd4^−/− mice. Results from individual mice are shown; the dotted line represents the limit of detection. (D) IFN-γ and IL-2 production by LCMV-specific CD8⁺ T cells at 136 days following infection. Gated CD8⁺ T cells are shown. (E and F) CD43 and PD-1 expression by GP33 tetramer⁺ CD8⁺ T cells from Il21^+/+ (shaded), ^+/− (dashed line), and ^−/− (bold line) mice at eight (E) and 136 days (F) post-infection. The Il21^+/− data shown in (D) and (F) are from mice that were aviremic at the time of analysis. Representative or composite data are shown from two independent experiments (n=3–6).

Fig. 3

IL-21 acts directly to sustain virus-specific CD8⁺ T cells during an ongoing infection. Cohorts of control Il21r^+/+/IL21r^+/+ (CD45.1/CD45.2) and experimental IL21r^+/+/IL21r^−/− (CD45.1/CD45.2) mixed bone-marrow chimeras were infected with LCMV-Cl 13 and CD8⁺ T cell responses evaluated over time. (A) PBMCs were evaluated by flow cytometry to check reconstitution of CD8⁺ T cells in Il21r^+/+/Il21r^+/+ or Il21r^+/+/Il21r^−/− mixed bone-marrow chimeras prior to infection. Gated CD8⁺ T cells are shown. (B) Flow cytometric analysis of GP₃₃- and GP₂₇₆-specific CD8⁺ T cell responses in the circulation at days eight and 16 after infection. Gated tetramer⁺ CD8⁺ T cells are shown. (C) Flow cytometric analysis of splenic CD8⁺ T cells and GP₃₃- and GP₂₇₆-specific responses at three weeks following infection. Gated CD8⁺ (left panel) or CD8⁺ tetramer⁺ (right panels) cells are shown. (D) Absolute numbers of GP₃₃- and GP₂₇₆-specific CD8⁺ T cells in mixed bone-marrow chimeras three weeks following infection. Graphs represent average + SD of Il21r^+/+ CD45.1 CD8⁺ T cells (black), Il21r^+/+ CD45.2 CD8⁺ T cells (gray) and Il21r^−/− CD45.2 CD8 T cells (white). **P < 0.01, ***P < 0.001. Representative results are shown from one of two similar experiments (n= 7 and 8 for the Il21r^+/+/IL21r^+/+ and IL21r^+/+/IL21r^−/− cohorts, respectively)

Fig. 4

IL-21 treatment enhances CD8⁺ T cell responses and reduces viral titers in Cd4^−/− mice. LCMV-Cl 13 infected Cd4^−/− mice were either left untreated or administered daily doses of 10μg recombinant IL-21 for eight days. At day nine after infection CD8⁺ T cell responses and viral loads were analyzed. (A) Flow cytometric analysis of intracellular staining of IFN-γ and IL-2 production in splenic virus-specific CD8⁺ T cells from control or treated cohorts. Gated CD8⁺ T cells are shown. The mean-fluorescence-intensity (MFI) of IFN-γ producing CD8⁺ T cells are reported in parentheses. (B) Flow cytometric analysis of GP₃₃ and NP₃₉₆ tetramer⁺ CD8⁺ T cells. Plots show gated CD8⁺ T cells. (C) Viral titers were assessed in the serum, lungs, and liver of control and IL-21-treated mice. Dotted line indicates the limit of detection (50 pfu/mL) for serum samples. *P < 0.05. Representative results from one of two independent experiments are shown (n= 7 and 6 for control and treated groups, respectively).