Complement Component C4 Regulates the Development of Experimental Autoimmune Uveitis through a T Cell-Intrinsic Mechanism

Abstract

In addition to its conventional roles in the innate immune system, complement has been found to directly regulate T cells in the adaptive immune system. Complement components, including C3, C5, and factor D, are important in regulating T cell responses. However, whether complement component C4 is involved in regulating T cell responses remains unclear. In this study, we used a T cell-dependent model of autoimmunity, experimental autoimmune uveitis (EAU) to address this issue. We compared disease severity in wild-type (WT) and C4 knockout (KO) mice using indirect ophthalmoscopy, scanning laser ophthalmoscopy, spectral-domain optical coherence tomography, and histopathological analysis. We also explored the underlying mechanism by examining T cell responses in ex vivo antigen-specific recall assays and in in vitro T cell priming assays using bone marrow-derived dendritic cells, splenic dendritic cells, and T cells from WT or C4 KO mice. We found that C4 KO mice develop less severe retinal inflammation than WT mice in EAU and show reduced autoreactive T cell responses and decreased retinal T cell infiltration. We also found that T cells, but not dendritic cells, from C4 KO mice have impaired function. These results demonstrate a previously unknown role of C4 in regulating T cell responses, which affects the development of T cell-mediated autoimmunity, as exemplified by EAU. Our data could shed light on the pathogenesis of autoimmune uveitis in humans.

Keywords: C4; T cells; animal models; autoimmune uveitis; complement.

Figure 1

C4 KO mice show reduced disease severity in experimental autoimmune uveitis (EAU). (A) EAU clinical scores of wild-type (WT) and C4 KO mice after immunization to induce EAU (n = 22 in each group, mean ± SEM). Age- and sex-matched WT and C4 KO mice were immunized with interphotoreceptor retinoid binding protein peptide to induce EAU, then their eyes were examined by indirect ophthalmoscopy daily from day 7 to day 21 and the clinical score recorded. (B) EAU histopathological scores for the WT and C4 KO mice at day 21 after immunization to induce EAU (n = 12) mean ± SEM. (C) Representative histological analysis of retina sections from WT and C4 KO mice at day 21 after immunization. GCL, the ganglion cell layer; INL, the inner nuclear layer; ONL, the outer nuclear layer. *p < 0.05 by two-way ANOVA or unpaired t-test.

Figure 2

Representative pictures in wild-type (WT) and C4 KO mice with experimental autoimmune uveitis at day 15 after immunization using different ocular imaging techniques. (A,B) TEFI imaging showed linear and confluent chorioretinal lesions were common in WTs [(A), arrows] but significantly less in C4 KOs (B); (C–J) cSLO images showed hyper-reflective features to both inter (C,D,G,H) and outer layers (E,F,I,J) of the retina. Numerous foci in the inter retina can be found adjacent to retinal vessels in the WTs [(C,D), arrows]. These foci were significantly fewer in the KOs (G,H). In the outer retina, other hyper-reflective features emanating from or around the optical nerve can be seen in the WTs [(E,F), circles] but rarely found in the KOs (I,J); (K,L) SD-OCT images revealed more reflective foci and retinal infoldings in the WTs [(K), arrows] than with those in the KOs (L). A summary of the numbers of hyper-reflective foci in vitreous cavities was presented in panel (M).

Figure 3

Ex vivo immunological studies of wild-type (WT) and C4 KO mice in experimental autoimmune uveitis (EAU). At day 21 after EAU induction, splenocytes were collected from WT and C4 KO mice and cultured for 72 h without antigen (w/o) or with 20 µg/ml of the immunizing interphotoreceptor retinoid binding protein (IRBP) peptide or a non-relevant peptide (OVA), then, levels of interferon (IFN)γ (A) and IL-17 (B) were measured by ELISA (n = 22 in each group) mean ± SEM; *p < 0.05 by one-way ANOVA. On the same day, eyes were collected from five mice in each group and single cell suspensions prepared and pooled, then the percentage of CD4+ T cells in the infiltrating cells in the eye of WT (C) and C4 KO (D) mice was analyzed by flow cytometry.

Figure 4

Effects of C4 deficiency on DC differentiation and function. BM cells from naïve wild-type (WT) and C4 KO mice were cultured under DC differentiation conditions and differentiated double-positive cells (DCs) (MHCII+ CD11C+) analyzed by flow cytometry. (A,B) Representative flow cytometric analysis results for WT and C4 KO DC differentiation (A) and the summarized results (B) [n = 5 in each group, mean ± SEM]. (C,D) The same numbers of BM-derived WT or C4 KO DCs was then cultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells from OTII mice in the presence or absence (wo) of peptide OVA_323–339 for 72 h, then the proliferation of the activated T cells was assessed by flow cytometry (C) and levels of interferon (IFN)γ produced by the activated T cells measured by ELISA (D) (n = 5 in each group). The same T cell proliferation (E) and IFNγ (F) assays were done using splenic DCs isolated from WT and C4 KO mice (n = 3 in each group), no statistically significant difference was observed either (mean ± SEM).

Figure 5

Effect of C4 deficiency on T cell activation. CD4+ T cells purified from naïve wild-type (WT) and C4 KO mice were incubated with (w) or without (w/o) anti-CD3 and anti-CD28 mAbs for activation for 5 h, then the percentage of activated T cells (CD69+) in the total cells was quantitated by flow cytometry. (A) Representative flow cytometric analysis results. (B) Summarized results from three independent experiments. Mean ± SEM. *p < 0.05 by paired t-test.

Figure 6

Effect of C4 deficiency on the proliferation of activated T cells. CD4+ T cells purified from naïve wild-type (WT) and C4 KO mice were incubated for 24 h with anti-CD3 and anti-CD28 mAbs and BrdU, then the proliferation of the activated T cells was assessed by estimating the percentage of BrdU+ T cells in the total cells using flow cytometric analysis. (A) Representative results. (B) Summarized results from three independent experiments. Mean ± SEM. *p < 0.05 by paired t-test.

Figure 7

Effect of C4 deficiency on the survival of activated T cells. CD4+ T cells purified from naïve wild-type (WT) and C4 KO mice were incubated with anti-CD3 and anti-CD28 mAbs for 48 h, then the percentage of apoptotic or dead T cells was assessed, respectively, by Annexin V or PI staining, followed by flow cytometric analysis. (A) Representative results; (B,C) summarized results from three independent experiments. Mean ± SEM. *p < 0.05 by paired t-test.

Figure 8

Effect of C4 deficiency on cytokine production by activated T cells. CD4+ T cells purified from naïve wild-type (WT) and C4 KO mice were incubated for 48 or 72 h with anti-CD3 and anti-CD28 mAbs, then interferon (IFN)γ levels in the culture supernatants were measured by ELISA after 48 or 72 h. n = 3 in each group, mean ± SEM. *p < 0.05 by t-test.