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. 2013 Sep 19;39(3):548-59.
doi: 10.1016/j.immuni.2013.08.010. Epub 2013 Aug 29.

Cell-intrinsic IL-27 and gp130 cytokine receptor signaling regulates virus-specific CD4⁺ T cell responses and viral control during chronic infection

James A Harker  1 , Aleksandr Dolgoter, Elina I Zuniga
Affiliations

Cell-intrinsic IL-27 and gp130 cytokine receptor signaling regulates virus-specific CD4⁺ T cell responses and viral control during chronic infection

James A Harker et al. Immunity. .

Abstract

The outcome of chronic viral infections, which affect millions of people worldwide, is greatly dependent on CD4⁺ T cells. Here we showed that T cell-specific ablation of the common interleukin-6 (IL-6) family receptor, gp130, profoundly compromised virus-specific CD4⁺ T cell survival, T follicular helper responses, and IL-21 production at late stages of chronic lymphocytic choriomeningitis virus (LCMV) infection. These effects were cell intrinsic for CD4⁺ T cells and were accompanied by a reduction of CD8⁺ T cells, antibodies, and a severe failure in viral control. We identified IL-27 as a gp130 cytokine that promoted antiviral CD4⁺ T cell accumulation in vivo and that rapidly induced IL-21 ex vivo. Furthermore, IL-27R was critical for control of persistent LCMV in vivo. These results reveal that gp130 cytokines (particularly IL-27) are key regulators of CD4⁺ T cell responses during an established chronic viral infection, empowering both humoral and cytotoxic immunity.

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Figures

Figure 1
Figure 1. IL-6 independent gp130 signaling is crucial for control of chronic viral infection
Wildtype (C57B/6 or Il6stfl/fl) and Cd4-cre Il6stfl/fl mice were infected with 2 × 106 pfu of LCMV Cl13 i.v. (A–B) Viral load was monitored in the serum throughout infection (A) and the indicated tissues at day 135 p.i. by immunofocus assay (B). Data is representative of 2 experimental repeats n ≥ 4 mice per group with mean ± S.E.M. depicted. This figure is supported by supplementary figure 1.
Figure 2
Figure 2. T cell specific gp130 signaling is required for accumulation of virus specific T cell responses and viral control during chronic infection
Wildtype (C57B/6 or Il6stfl/fl) and Cd4-cre Il6stfl/fl mice were infected with 2 × 106 pfu of LCMV Cl13 i.v. (A) PBMCs were analyzed to determine the number of GP276–284 CD8+ T cells. (B) At days 9 and 30 p.i. splenocytes were analyzed by flow cytometry to determine the number of H2-Db GP276–284 and GP33–41 CD8+ T cells. (C) As in (A) PBMC were analyzed to determine the number of PD-1+ CD4+ T cells. (D) As in (B) I-Ab GP67–77 + CD4+ T cells numbers were determined in the spleen, the fold increase from CD4-cre Il6fl/fl to WT cells at day 30 p.i. is indicated. Data is representative of 3 experimental repeats n ≥ 4 mice per group with mean ± S.E.M. depicted. This figure is supported by supplementary figure 2.
Figure 3
Figure 3. T cell specific gp130 signaling is required for IL-21 production during chronic LCMV infection
Wildtype (C57B/6 or Il6stfl/fl) and Cd4-cre Il6stfl/fl mice were infected with 2 × 106 pfu of LCMV Cl13 i.v. At days 9 and 30 p.i. splenocytes were stimulated with (A) LCMV GP67–77 peptide or (B) PMA and ionomycin and the number of IFN-γ secreting cells (GP67–77 specific only), and the proportion of IFN-γ+ cells that were also secreting IL-21, were determined by flow cytometry. (C) At day 30 p.i. CD4+ and I-Ab LCMV GP67–77 + cells were isolated by flow cytomtery and Il21 expression, relative to Gapdh, was determined by qPCR. n.d = not detectable, data is representative of 3 experimental repeats n ≥ 4 mice per group with mean ± S.E.M. depicted. This figure is supported by supplementary figure 3.
Figure 4
Figure 4. CD4+ intrinsic gp130 signaling is required for CD4+ T cell numbers, accumulation and IL-21 production
Congenic wildtype (CD45.1) mice were lethally irradiated and reconstituted with a 50:50 mix of CD45.1 and Cd4-cre Il6stfl/fl (CD45.2) bone marrow cells. 8 weeks later chimeras were infected with 2 × 106 pfu of LCMV Cl13. At day 30 p.i. the percentages of splenic CD8+ T cells that were H2-Db GP33–41 + (A) and the % and number of splenic CD4+ T cells that were I-Ab GP67–77 + (B) in CD45.1+ or CD45.2+ compartments was determined by flow cytometry, the fold increase between WT and Il6st−/− is depicted. (C) Splenocytes were stimulated ex vivo with GP67–77 peptide and the proportion of IFN-γ producing CD4+ T cells that were also producing IL-21 was determined by flow cytometry and (D) Il21 expression determined in I-Ab GP67–77 + CD4+ T cells by qPCR. N.d. = not detectable. Data are representative of 4 independent repeats from pooled samples of n ≥ 4 mice, with mean ± S.E.M. depicted. This figure is supported by supplementary figure 4.
Figure 5
Figure 5. Virus specific CD4 T cells exhibits an intrinsic survival defect in the absence of gp130 signaling
WT, Cd4-cre Il6stfl/fl (A&D) or WT:Cd4-cre Il6stfl/fl mixed chimeric (B,C,E&F) mice were infected with 2 × 106 pfu of LCMV Cl13 i.v. and splenocytes analyzed by flow cytometry at day 30 p.i. A&B) The % Annexin V binding in I-Ab GP67–77 + CD4+ T cells was determined. C) The % of PD1+ CD4+ T cells expressing cleaved Caspase-3+ was determined. D&E) The proportion of I-Ab GP67–77 + CD4+ T cells that had incorporated BrdU incorporation was quantified. F) The proportion of Ki67+ was analyzed within PD1+ CD4+ T cells. Data are representative of 2 or 3 independent experiments, of n ≤ 4 mice per group, with mean ± S.E.M. depicted. This figure is supported by supplementary figure 5.
Figure 6
Figure 6. IL-27 signaling on virus specific CD4+ T cells is vital for survival during chronic viral infection
A) LCMV specific CD45.1+ transgenic CD4+ T cells (Smarta) were transferred i.v. 1 day prior to LCMV Cl13 infection and splenocytes isolated at 18 days p.i.. The amount of pSTAT1 and pSTAT3 were then determined in naïve and Smarta CD4+ T cells 30 minutes after ex vivo stimulation with 50 ng/ml of the indicated recombinant cytokines by flow cytometry. B&C) WT:Il27ra−/− mixed bone marrow chimeras were generated and infected with LCMV Cl13, 30 days p.i. the proportion of splenic CD4+ T cells that were I-Ab Gp67–77 + (B) and CD4+ T cells that were IFN-γ+ IL-21+ after GP67–77 peptide stimulation (C) were determined by flow cytometry. D&E) At day 18 post LCMV Cl13 infection PD1+ CD4+ T cells (D) or Smarta cells (E) prepared as in (A) were isolated by flow cytometry. Expression of Il21 relative to Gapdh was determined 6, 12 and 24 hours (D) or 6 hours (E) after ex vivo stimulation with IL-6, IL-27 or PBS. A is representative of 2 independent experiments with pools of 5 mice per group. B, C and D–E are representative of 5, 3 and 2 independent experiments, respectively with mean ± S.E.M. depicted. This figure is supported by supplementary figure 6.
Figure 7
Figure 7. IL-27 is critical in controlling viral load at both early and late stages of infection
WT or Il27ra−/− animals were infected with LCMV Cl13. (A & B) Splenic I-Ab GP67–77+ CD4+ T cells at days 9 and 30 p.i. were enumerated by flow cytometry (A). Example flow plots are shown and the proportion of Db GP67–77 + CD4+ T cells at day 30 p.i. compared to day 9 p.i. was compared to that observed in Cd4-cre Il6stfl/fl animals (B). (C) PD-1+ CD4+ T cells were enumerated in the blood by flow cytometry. (D & E) Viral load was determined in the blood (D) at various timepoints and in tissues (E) at day 130 post infection. Data is representative of n = 2 experimental repeats of n = 5 mice per group, with mean ± S.E.M. depicted.
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