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.
Highlights
Complement is an important component of the innate immune system, the primary role of which is to fight infections [1]
We examined T cell responses using ex vivo T cell recall assays and carried out in vitro T cell activation assays using bone marrow-derived dendritic cells (BM-Double-positive cells (DCs)), isolated splenic dendritic cells, and CD4+ T cells from naïve WT and C4 KO mice to further dissect the underlying mechanism involved in this process
We examined the potential role of C4 in EAU by comparing disease severity in age- and sex-matched WT and C4 KO mice after EAU induction using various ocular imaging, Figure 3 | Ex vivo immunological studies of wild-type (WT) and C4 KO mice in experimental autoimmune uveitis (EAU)
Summary
Complement is an important component of the innate immune system, the primary role of which is to fight infections [1]. For each of the immunized WT and C4 KO mice, 4 × 105 splenocytes were cultured in 96-well round-bottomed microtiter plates in 100 μl of complete RPMI 1640 medium in the presence or absence of 20 μg/ml of peptide IRBP651–670 or a non-relevant peptide (ovalbumin OVA323–339, Genscript, NJ, USA) for 72 h, the supernatants were collected and interferon (IFN)γ and interleukin (IL)-17 levels measured using ELISA kits (BioLegend, San Diego, CA, USA). T cells were enriched from splenocytes of OTII mice (Jackson Laboratory) using nylon wool and labeled with carboxyfluorescein succinimidyl ester (CFSE) according to the manufacturer’s instruction (Thermo Fisher Scientific, Waltham, MA, USA), were cocultured with the same numbers of differentiated WT or C4 KO DCs (10:1 ratio) in the presence or absence of 2 μg/ml of peptide OVA323–339 for 72 h. Two sets of data were compared by Student’s t-test and three sets of data were analyzed by one-way ANOVA. p-Values less than 0.05 were considered significant
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