Decay accelerating factor can control T cell differentiation into IFN-gamma-producing effector cells via regulating local C5a-induced IL-12 production - PubMed (original) (raw)

Decay accelerating factor can control T cell differentiation into IFN-gamma-producing effector cells via regulating local C5a-induced IL-12 production

Peter N Lalli et al. J Immunol. 2007.

Abstract

A newly recognized link between the complement system and adaptive immunity is that decay accelerating factor (DAF), a cell surface C3/C5 convertase regulator, exerts control over T cell responses. Extending these results, we show that cultures of Marilyn TCR-transgenic T cells stimulated with DAF-deficient (Daf1(-/-)) APCs produce significantly more IL-12, C5a, and IFN-gamma compared with cultures containing wild-type APCs. DAF-regulated IL-12 production and subsequent T cell differentiation into IFN-gamma-producing effectors was prevented by the deficiency of either C3 or C5a receptor (C5aR) in the APC, demonstrating a link between DAF, local complement activation, IL-12, and T cell-produced IFN-gamma. Bone marrow chimera experiments verified that bone marrow cell-expressed C5aR is required for optimal differentiation into IFN-gamma-producing effector T cells. Overall, our results indicate that APC-expressed DAF regulates local production/activation of C5a following cognate T cell/APC interactions. Through binding to its receptor on APCs the C5a up-regulates IL-12 production, this in turn, contributes to directing T cell differentiation toward an IFN-gamma-producing phenotype. The findings have implications for design of therapies aimed at altering pathologic T cell immunity.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1

FIGURE 1

APC-expressed DAF regulates C3-dependent IL-12 production. A, Top, Concentration of IL-12p70 in supernatants of Mar T cells stimulated with Dby peptide-loaded WT, _Daf1_−/−, _C3_−/−, and _Daf1/C3_−/− peritoneal macrophage at 48 and 72 h. Results represent mean plus SD of three individual experiments. Bottom, Concentration of IL-12p70 in supernatants of BALB/c or C3H T cells stimulated with B6 WT, _Daf1_−/−, _C3_−/−, and _Daf1/C3_−/− spleen cell APCs at 72 h. No cytokines were detected in wells containing T cells alone, APCs alone, or mixtures of syngeneic T cells plus APCs (data not shown). *, p < 0.05 vs WT; †, p < 0.01 vs _Daf1_−/−. B, Relative quantification of mRNA for IL-12p40 (top) and p35 (bottom) in whole culture lysates of Mar T cells stimulated with Dby peptide-loaded WT, _Daf1_−/−, _C3_−/−, and _Daf1/C3_−/− peritoneal macrophage at 72 h. Data are normalized to WT APCs before stimulation. Results represent mean plus SE of at least two experiments for each group. *, p < 0.05 vs WT; †, p < 0.01 vs _Daf1_−/−.

FIGURE 2

FIGURE 2

APC-expressed DAF regulates C3-dependent T cell cytokine production. A, Left, Cytokines detected in 72 h (IFN-γ) or 48 h (IL-2/IL-4) culture supernatants of Mar T cells stimulated with WT, _Daf1_−/−, or _C3_−/− peritoneal macrophages. Right, Cytokines detected in 72 h culture supernatants of BALB/c or C3H T cells stimulated with B6 WT, _Daf1_−/−, or _C3_−/− spleen cell APCs. B, IFN-γ production by CFSE-labeled Mar T cells stimulated with WT, _Daf1_−/−, or _C3_−/− peritoneal macrophage. Representative flow plots from 3-day cultures (B) are depicted. Numbers in upper left quadrant represent percent IFN-γ+ T cells undergoing more than one division. C and D, In vivo effects of DAF on T cell IFN-γ production. Percentages of IFN-γ+ Mar T cells in the spleens (C) and the total numbers of Mar T cells in the spleens (D) 60 h after injection into WT, _Daf1_−/−, or _C3_−/− male mice (10 × 106 cells/mouse). Horizontal bars represent mean values for each group; n = 4/group. *, p < 0.05 vs WT.

FIGURE 3

FIGURE 3

DAF regulation of T cell IFN-γ production is dependent on APC-expressed IL-12 and T cell expressed IL-12R. A, IFN-γ detected in 72-h culture supernatants of Mar T cells mixed with WT, _Daf1_−/−, or _Daf1/IL12p40_−/− APCs ± anti-12 mAb (25 _μ_g/ml blocking anti-IL-12 mAb) or rIL-12. *, p < 0.05 vs WT; †, p < 0.05 vs _Daf1_−/−. B, Representative flow plots of CFSE-labeled Mar T cells stimulated in vitro for 3 days with HY_Dby_-loaded WT, _Daf1_−/−, or _Daf1/IL-12_−/− peritoneal macrophages ±25 _μ_g/ml blocking anti-IL-12 mAb or control Ab and stained for intracellular IFN-γ. Upper plots, Single-color histograms for IFN-γ (% IFN-γ+ in each panel); lower panels, CFSE dilution of responding T cells (the average number of cell divisions is shown in each panel, n = 2–3/group). Representative of more than three individual experiments per condition are shown. C, Flow cytometry plots of CFSE-labeled WT or _IL-12Rβ1_−/− Mar T cells stimulated with WT or _Daf1_−/− APCs in vitro on day 3. Upper plots, Single-color histograms for IFN-γ; lower panels, CFSE dilution of responding T cells. Percentages of IFN-γ+ cells or average number of cell divisions for each plot are shown. Representative of more than three individual experiments are shown. D, Percentages of IFN-γ+ WT or _IL-12Rβ1_−/− Mar T cells in recipient spleens at 60 h after injection into male WT, _Daf1_−/−, _IL-12p40_−/−, or _Daf1/IL-12p40_−/− recipients. Data represent mean values for groups plus SD n = 4/group. *, p < 0.05 vs WT T cells; †, p < 0.05 vs WT APCs; ‡, p < 0.05 vs _Daf1_−/− APCs.

FIGURE 4

FIGURE 4

Diminished production of IFN-γ by T cells stimulated with _C3_−/− APCs is overcome by exogenous IL-12. A, Flow cytometry plots of CFSE-labeled WT Mar T cells stimulated with WT or _C3_−/− APCs for 3 days in the presence or absence of 10 ng/ml rIL-12 and stained for intra-cellular IFN-γ. Upper plots, Single-color histograms for IFN-γ; lower panels, CFSE dilution of responding T cells. Percentages of IFN-γ+ cells or average number of cell divisions for each plot are shown. Representative of more than three individual experiments are shown. B, IFN-γ in 72-h culture supernatants of Mar T cells responding to WT or _C3_−/− APC with or without 10 ng/ml rIL-12. Results are representative of two individual experiments. *, p < 0.05 vs WT.

FIGURE 5

FIGURE 5

C5a is produced by cognate interactions and promotes production of APC IL-12. A, Western blot of culture supernatants for C5a from cultures of Mar T cells with male WT, _Daf1_−/−, and _C3_−/−APCs. rC5a is shown as a positive control. B, IL-12p70 ELISA of culture supernatants taken from cultures of Mar T cells stimulated with WT, _Daf1_−/−, _C5aR_−/−, or _Daf1/C5aR_−/− APCs at 48 and 72 h. Results represent mean plus SD of duplicate wells of two individual experiments. *, p < 0.05 vs WT; †, p < 0.01 vs _Daf1_−/−. C, IL-12p70 protein in supernatants of unstimulated (top) or LPS stimulated (bottom, 10 ng/ml LPS) WT APCs in the presence of increasing concentrations of rC5a as determined by ELISA. No IL-12p70 was detected after stimulation of _C5aR_−/− macrophages with the same concentrations of C5a (data not shown). Data represents mean plus SD for each group. *, p < 0.05 vs no C5a control.

FIGURE 6

FIGURE 6

APC-expressed C5aR is required for optimal effector T cell production of IFN-γ. A, IFN-γ detected in 72-h culture supernatants of WT Mar T cells (■) or IL-12R−/− Mar T cells (□) 3 days after stimulation with WT, _Daf1_−/−, _C5aR_−/−, or _Daf1/C5aR_−/− HY_Dby_-loaded APCs in the presence or absence of 10 ng/ml rIL-12. Data represent mean plus SD for each group. *, p < 0.05 vs WT T cells; †, p < 0.01 vs _Daf1_−/− APCs. B, Percentages of IFN-_γ_-producing WT (■) or _IL-12Rβ1_−/− (□) Mar T cells on day 3 of cultures with each APC type as depicted in the panel. C, IFN-γ ELISPOTs produced by WT polyclonal C3H (H-2k) T cells stimulated in vitro with WT, _Daf1_−/−, _C5aR_−/−, or _Daf1/C5aR_−/− (H-2b) splenocytes. Data represent mean plus SD for each group. *, p < 0.05 vs WT; †, p < 0.01 vs _Daf1_−/−. D, Percentages of IFN-_γ_-producing WT (■) or _IL-12Rβ1_−/− (□) Mar T cells 60 h after injection into WT, _Daf1_−/−, _C5aR_−/−, or _Daf1/C5aR_−/− male recipients. Data represent mean values for groups plus SD; n = 4/group. *, p < 0.05 vs WT T cells.

FIGURE 7

FIGURE 7

BM-derived cell expression of C5aR is required for optimal T cell IFN-γ production in vivo. A, Representative flow cytometry plots of CFSE-labeled CD4+ Mar T cells of Mar T cells injected into WT (Thy1.1)→_C5aR_−/− (Thy1.2) or _C5aR_−/− (Thy1.2)→WT (Thy1.1) BM chimeric mice. Chimerism was determined to be >90% donor BM based on Thy1.1/Thy1.2 polymorphisms (inset). B, Percentages of injected IFN-γ+ Mar T cells 72 h after injection into WT→WT, _C5aR_−/−→ _C5aR_−/−, WT→_C5aR_−/−, or _C5aR_−/−→ WT BM chimeric mice. Data represent mean values for groups plus SD; n = 3/group. *, p < 0.05 vs WT→WT.

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