Contribution of Myelin Autoantigen Citrullination to T Cell Autoaggression in the Central Nervous System

Abstract

Breakdown in immunological self tolerance, leading to autoimmune diseases such as multiple sclerosis, might arise from immune recognition of self proteins that have undergone heightened posttranslational modification under pathophysiological conditions. A posttranslational modification of particular interest is the deimination of Arg to citrulline, catalyzed by peptidylarginyl deiminase (PAD) enzymes. As a CD4+ T cell-driven model of multiple sclerosis, we used experimental autoimmune encephalomyelitis (EAE) induced with the immunodominant 35–55 peptide of myelin oligodendrocyte glycoprotein (pMOG) in C57BL/6 mice to test whether citrullination of a T cell epitope can contribute to disease etiopathology. Immunization with an altered peptide ligand (APL) of pMOG with an Arg→citrulline conversion at a TCR contact (residue 41) led to the activation of two populations of APL-responsive T cells that either did, or did not cross-react with the native pMOG peptide. This APL could induce EAE. However, this reflected the activation of T cells that cross-reacted with the native pMOG epitope, because prior tolerization of these T cells using pMOG prevented APL-induced EAE. Using a passive transfer model, we found that T cells that responded specifically to the citrullinated form of pMOG were neither necessary, nor sufficient to initiate the EAE lesion. Nevertheless, these cells could provoke exacerbation of pathology if transferred into mice with ongoing EAE. The PAD2 and PAD4 enzymes were markedly upregulated in the inflamed CNS. Therefore, once inflammation is established, citrullination of target autoantigens can allow an expanded repertoire of T cells to contribute to CNS pathology.

NADPH Oxidase Modifies Patterns of MHC Class II–Restricted Epitopic Repertoires through Redox Control of Antigen Processing

“…To achieve this, T cell hybridomas were first stably transfected with an NFA-T-enhanced IL-2 promoter-driven eGFP construct, followed by multiple rounds of selection by FACS to enrich those hybridoma clones that expressed eGFP only following presentation of their cognate peptide epitope by APCs (25). When added to BMMfs that were previously pulsed with HEL protein, we found that WT and Cybb 2/2 BMMfs were equally able to induce eGFP expression in two of the hybridomas (Hb1.9 and H46.13), indicating that the processing/presentation efficiency of HEL [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and HEL [48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] peptides were unaffected by NOX2 (Fig. 2D).…”

“…The relative abundance of the peptides was quantified using the area under corresponding peaks. Abundance of the cleavage product MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] relative to MOG (corresponding destruction of the I-A b binding region) was calculated using the following formula: RPA = (P x /S x )/(P 0 /S 0 ), where RPA indicates relative peptide abundance, P x indicates product (MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] ) of sample, S x indicates substrate (MOG ) of sample, P 0 indicates product (MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] ) without hydrogen peroxide, and S 0 indicates substrate (MOG ) without hydrogen peroxide.…”

“…To interrogate whether CNS-resident NOX2-deficient APCs are compromised in their ability to present endogenous MOG in preclinical EAE, the efficiency of reactivation of adoptively transferred MOG (49,50,55,56). Pertinently, both of these cleavage sites are located on a solvent-accessible loop in the MOG protein, rendering them potentially susceptible to hydrolysis in the early phagosome.…”

“…The inverse of the IC 50 by cysteine cathepsins using input sites from the MEROPS database. The illustration (see Fig.…”

“…To further interrogate the ability of NOX2 to alter processing patterns of a single Ag, we measured the efficiency of WT and Cybb 2/2 BMMfs to process four distinct HEL epitopes (HEL [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] , HEL [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] , HEL [48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] , and HEL 74-90 ) from whole HEL protein and present them to epitope-specific CD4 + T cell hybridomas (Hb1.9, H30.44, H46.13, and B04, respectively) (26). To achieve this, T cell hybridomas...…”