CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs - PubMed (original) (raw)

CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs

Jie Sun et al. Nat Immunol. 2011 Apr.

Abstract

Interleukin (IL)-10 is an important regulatory cytokine that can modulate excessive immune mediated injury. Several distinct cell types have been demonstrated to produce IL-10, including most recently CD8+ cytotoxic T lymphocytes (CTLs) responding to respiratory virus infection. Here we report that CD4+ T cell help in the form of IL-2 is required for IL-10 production by CTLs, but not for the induction of CTL effector cytokines. We show that IL-2 derived from CD4+ helper T cells cooperates with innate immune cell-derived IL-27 to amplify IL-10 production by CTLs through a Blimp-1-dependent mechanism. These findings reveal a previously unrecognized pathway that coordinates signals derived from innate and helper T cells to control the production of a regulatory cytokine by CTLs during acute viral infection.

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Figures

Figure 1

Figure 1. Induction of IL-10 producing CTL in vivo requires IL-27 and CD4+ T cells

(a) WT or _p35_−/− mice were infected with influenza. At d7 p.i., the production of IL-10 and IFN-γ by CTL from MLN or lungs was measured by ICS. (b, c) WT or _Ebi3_−/− mice were infected with influenza. At d7 p.i., the production of IL-10 and IFN-γ by CTL from MLN or lungs was measured by ICS. (c) The normalized percentages of IL-10+ cells in influenza-specific CTL (IFN-γ+) from MLN or lungs of infected WT and _Ebi3_−/− mice are depicted. (d) WT or _Il21r_−/− mice were infected with influenza. At d7 p.i., the production of IL-10 and IFN-γ by CTL from MLN or lungs was measured by ICS. (e, f, g) WT, _Ebi3_−/− or CD4+ T cell-depleted (α-CD4) _Ebi3_−/− mice were infected with influenza. At d7 p.i., the production of IL-10 and IFN-γ by CTL from MLN or lungs was measured by ICS. (f) The normalized percentages of IL-10+ cells in influenza-specific CTL (IFN-γ+) from MLN or lungs of WT and _Ebi3_−/− mice are depicted. (g) The mean fluorescence intensity (MFI) of IL-10 in IL-10+ cells from infected lungs is depicted. Numbers are the percentages of cells in gated population. *, P <= 0.05; **, P <= 0.01. Data are from one experiment, but are typical of those obtained from at least two.

Figure 2

Figure 2. CD4+ T cell “help” is selectively required for the induction of IL-10 producing CTL in vivo

(a, b) WT mice were injected with Rat IgG or anti-CD4 (α-CD4) depleting Ab and infected with influenza. At d8 p.i., the production of IL-10, IFN-γ and TNF by CTL from MLN or lungs was measured by ICS. (b) The normalized percentages of IL-10+ or TNF+ cells in influenza-specific CTL (IFN-γ+) from MLN or lungs are depicted. (c) WT or _MhcII_−/− mice were infected with influenza. At d7 p.i., the normalized percentages of IL-10+ or TNF+ cells in influenza-specific CTL (IFN-γ+) from lungs of WT and _MhcII_−/− are depicted. (d) Vert-X mice were injected with Rat IgG or anti-CD4 Ab and infected with influenza. At d7 p.i., the percentages of IL-10-eGFP+ cells in influenza-specific PA224 or NP366 tetramer+ cells are depicted. (e) MhcII+/−-Vert-X or _MhcII_−/−-Vert-X mice were infected with influenza. At d7 p.i., the percentages of IL-10-eGFP+ cells in influenza-specific lung PA224 or NP366 tetramer+ cells are depicted. (f) Yeti mice were injected with Rat IgG or α-CD4 Ab and infected with influenza. At d7 p.i., the percentages of IFN-γ-eYFP+ cells in influenza-specific lung PA224 or NP366 tetramer+ cells are depicted. Numbers are the percentages of cells in gated population. *, P <= 0.05; **, P <= 0.01. (a-d, f) Data are from one experiment, but are typical of three. (e) Data are pooled from total of four experiments.

Figure 3

Figure 3. IL-2 provides the “help” from CD4+ T cell to CTL for IL-10 production in vitro

(a) CFSE labeled CD8+ CL-4 cells were stimulated with influenza-infected DC in the presence or absence of CD4+ TS-1 cells for 4d. The cultured cells were treated with indicated conditions. Then the production of IL-10 and IFN-γ by CL-4 cells were measured through ICS. (b, c) CD8+ Vert-X-OT-I (V-OT-I) cells were stimulated with influenza-OVA-infected DC in the absence or presence of OT-II cells. (b) The percentages of IL-10-eGFP+ cells in V-OT-I cells are depicted. (c) The release of IL-2 into medium after 2d in culture was measured by ELISA. (d, e) V-OT-I or OT-II cells were activated separately by influenza-OVA-infected DC for 4d. The activated V-OT-I cells were unmanipulated or co-cultured with the activated OT-II cells. Cells were then either left unstimulated or stimulated with plate-bound anti-CD3 (α-CD3). The cultured cells were treated as indicated. (d) The expression of IL-10-eGFP by V-OT-I cells after 2d in culture was measured by flow cytometry. (e) The release of IL-2 into medium after overnight in culture was measured by ELISA. (f) OT-I cells were stimulated with influenza-OVA-infected DC. After 4 d in culture, OT-I cells were treated with hIL-2 or hIL-2 plus plate-bound α-CD3 for 4h. The expression of IL-10 was measured by quantitative RT-PCR. Numbers are the percentages of cells in the gated population. Data are from one experiment, but are representatives of at least 3 replicates.

Figure 4

Figure 4. IL-2 is required for the induction IL-10-producing CTL in vivo

(a, b). IL-2 production by gated Thy1+ cells from influenza infected lung is measured by ICS. (a) IL-2 production by Thy1+CD4+ and Thy1+CD4− cells depicted (b) with the indicated IL-2 MFI (c) IL-2 production by cultured MLN and lung cells from influenza infected mice treated with indicated antibodies is determined by ELISA. (d) Following transfer of WT or _Il2ra_−/− T cells into Thy1.1 mice and influenza infection, the normalized percentages of IL-10+ cells among influenza-specific CTL (IFN-γ+) from infected lungs at d8 p.i. is determined. (e, f) WT:_Il2ra_−/− chimeric mice were infected with influenza. At d7 p.i., the production of IL-10, TNF and IFN-γ by CTL was measured by ICS. (f) The normalized percentages of IL-10+ or TNF+ cells among lung influenza-specific CTL (IFN-γ+) are depicted. (g) Following infection of WT:_Il2ra_−/− chimeric mice the normalized percentages of lung IL-10+ cells among specific IFN-γ+ CD8+ T cells at d7 p.i., are depicted. (h, i) Following α-CD4 Ab treatment and influenza infection Vert-X mice received rmIL-2 or PBS at d5 and 6 p.i.,. The expression of IL-10-eGFP by CTL was measured by flow cytometry. (i) At d7 p.i, the percentages of IL-10-eGFP+ cells among gated CTL are depicted. Numbers represent percentages of cells in gated population. *, P <= 0.05; **, P <= 0.01. Data are representative of at least two experiments.

Figure 5

Figure 5. IL-2 and IL-27 synergistically induce IL-10 production by both murine and human CTL

(a, b) CFSE labeled CD8+ CL-4 cells were stimulated with influenza-infected DC in the presence or absence of CD4+ TS-1 cells for 4d. The cultured cells were treated with indicated conditions. Then the production of IL-10 and IFN-γ by CL-4 cells were measured through ICS. (b) The MFI of IL-10 in IL-10+ cells is depicted. (c) WT mice were infected with influenza and various cell types of the infected lungs were sorted out as described in methods. The expression of IL-27 EBI-3 and p28 subunits was measured through quantitative RT-PCR. (d) WT mice were injected with Rat IgG or α-CD4 Ab and infected with influenza. At d6 p.i., the levels of IL-27 p28 in BALF were measured through ELISA. (e) Purified human CD8+ T cells were stimulated with α-CD3 plus α-CD28 with indicated conditions for 3d. Then the levels of IL-10 in the medium were measured by ELISA. Numbers are the percentages of cells in gated population. (a-d) Data are representative of at least three separate experiments. (e) Data are representative of at least two separate experiments employing an additional donor.

Figure 6

Figure 6. Induction of IL-10 producing CTL by IL-2 and IL-27 is Blimp-1 dependent

(a) Vert-X mice were infected with influenza. At d7 p.i., lung CD8+ CD44hiIL-10-eGFP− cells and CD8+ CD44hiIL-10-eGFP+ cells were FACS sorted. The expression of IL-10 and Blimp-1 were measured by quantitative RT-PCR. (b) MhcII+/− or _MhcII_−/− mice were infected with influenza. At d7 p.i., lung CD8+ cells were isolated and the expression of IL-10, Blimp-1, IFN-γ and T-bet was determined by quantitative RT-PCR. (c) WT or _Ebi3_−/− mice were infected with influenza. At d7 p.i., lung CD8+ cells were isolated and the expression of IL-10, Blimp-1, IFN-γ and T-bet was determined by quantitative RT-PCR. (d) OT-I cells were cultured with influenza-OVA infected DC in the absence or presence of hIL-2, IL-27 or hIL-2 plus IL-27 for 4d. The expression of Blimp-1 was measured by quantitative RT-PCR. (e) OT-I cells were transduced with control vector (mock) or Blimp-1 expressing retrovirus (Blimp-1). Then cells were cultured for additional 3 days and IL-10 production by OT-I cells measured by ICS. hCD2− (untransduced cells), hCD2+ (transduced cells). (f) CFSE labeled CD8+ T cells from WT or CD4-Cre _Prdm_fl/fl (Prdm1 cKO) mice were stimulated with DC plus soluble α-CD3 for 4 days under indicated conditions and IL-10 production by CTL then measured by ICS. Numbers are the percentages of cells in the gated population. Data are representative of at least two independent experiments.

Figure 7

Figure 7. Blimp-1 deficiency in T cells results in diminished IL-10 production and enhanced pulmonary inflammation

CD4-Cre _Prdm1_fl/+ or CD4-Cre _Prdm1_fl/fl mice were infected with influenza. (a, b) At d7 p.i., the production of IL-10 and IFN-γ by CTL was measured by ICS. (b) The normalized percentages of IL-10+ or TNF+ cells in influenza-specific CTL (IFN-γ+) in CD4-Cre _Prdm1_fl/+ or CD4-Cre _Prdm1_fl/fl mice are depicted. (c) At d7 p.i., the levels of IL-10 and IFN-γ in BALF were determined through ELISA. (d) At d9 p.i., the numbers of lung monocytes and neutrophils were measured through flow cytometry. Numbers are the percentages of cells in gated population. *, P <= 0.05; **, P <= 0.01. (b, c) Data are from three pooled experiments. (e) WT or CD4-Cre _Prdm1_fl/fl T cells were transferred into Thy1.1+ WT mice and infected with influenza. At d7 p.i., the production of IL-10 and IFN-γ by CTL was measured by ICS following restimulation with influenza-infected BMDC. The normalized percentages of IL-10+ cells in influenza-specific CTL (IFN-γ+) from infected lungs are depicted. Pooled data from two experiments are represented.

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