Functional and genetic analyses of the MHC and its impact on autoimmunity in the rat

The major histocompatibility complex (MHC) is an allele-rich and exceptionally gene dense region on human chromosome 6. Over 40% of the genes in this region have immune-related functions, including genes encoding MHCI and MHCII molecules. These molecules, which are found in nearly all vertebrates, present antigenic peptides to CD4 and CD8 T cells. Alleles of MHCI and MHCII are believed to be strong risk factors in autoimmune disorders, such as rheumatoid arthritis (RA), as well as in infectious diseases. However, the differentiation between haplotype and allele associations in the MHC is not straightforward. Strong linkage disequilibrium exists between gene segments throughout the region and impedes identification of disease associated variants. These gene segments can be isolated and studied individually in congenic mice and rats. We produced for this thesis an extensive number of intra-MHC congenic rats to study the association between MHC genes and experimental arthritis, T cell selection and MHC regulation. Study I describes a genome-wide approach in heterogenous stock rats to identify quantitative trait loci (QTLs) associated with variations in MHC levels and CD4 and CD8 T cell numbers. A total of 10 QTLs were identified, of which 3 mapped to the MHC. We showed by congenic mapping that two minimal haplotypes of ~0.2 Mb explained the associations to the MHC. We further identified two allelic variants of the gene Tap2 that contributed to the variation in T cell numbers. Study II describes the effect of these minimal haplotypes on arthritis development and positions the MHCII region for the first time in an adjuvant model. We show that genes in the MHCII regulate onset, progression and severity of arthritis but not chronicity. Comparative analyses of different congenic MHCII haplotypes showed an inverse correlation between arthritis severity and proportion of recent thymic emigrants. Study III shows an MHCII associated T cell response to the cartilage protein collagen type XI in chronic pristane-induced arthritis (PIA) and the corresponding antibody response to the same antigen in human RA. Study IV describes the adoptive transfer of PIA in DA rats and outlines the conditions necessary for the model

Natural Polymorphisms in Tap2 Influence Negative Selection and CD4 : CD8 Lineage Commitment in the Rat

Contains fulltext : 136368.pdf (publisher's version ) (Open Access)Genetic variation in the major histocompatibility complex (MHC) affects CD4ratioCD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4ratioCD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4ratioCD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of approximately 0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells

Animal Models of Rheumatoid Arthritis (I) : Pristane-Induced Arthritis in the Rat

BACKGROUND: To facilitate the development of therapies for rheumatoid arthritis (RA), the Innovative Medicines Initiative BTCure has combined the experience from several laboratories worldwide to establish a series of protocols for different animal models of arthritis that reflect the pathogenesis of RA. Here, we describe chronic pristane-induced arthritis (PIA) model in DA rats, and provide detailed instructions to set up and evaluate the model and for reporting data. METHODS: We optimized dose of pristane and immunization procedures and determined the effect of age, gender, and housing conditions. We further assessed cage-effects, reproducibility, and frequency of chronic arthritis, disease markers, and efficacy of standard and novel therapies. RESULTS: Out of 271 rats, 99.6% developed arthritis after pristane-administration. Mean values for day of onset, day of maximum arthritis severity and maximum clinical scores were 11.8±2.0 days, 20.3±5.1 days and 34.2±11 points on a 60-point scale, respectively. The mean frequency of chronic arthritis was 86% but approached 100% in long-term experiments over 110 days. Pristane was arthritogenic even at 5 microliters dose but needed to be administrated intradermally to induce robust disease with minimal variation. The development of arthritis was age-dependent but independent of gender and whether the rats were housed in conventional or barrier facilities. PIA correlated well with weight loss and acute phase reactants, and was ameliorated by etanercept, dexamethasone, cyclosporine A and fingolimod treatment. CONCLUSIONS: PIA has high incidence and excellent reproducibility. The chronic relapsing-remitting disease and limited systemic manifestations make it more suitable than adjuvant arthritis for long-term studies of joint-inflammation and screening and validation of new therapeutics

Pristane, a non-antigenic adjuvant, induces MHC class II-restricted, arthritogenic T cells in the rat.

Pristane-induced arthritis (PIA) in rats, a model for rheumatoid arthritis (RA), is a T cell-dependent disease. However, pristane itself is a lipid and unable to form a stable complex with a MHC class II molecule. Therefore, the specificity and function of the T cells in PIA are as unclear as in rheumatoid arthritis. In this study, we show that activated CD4+ alphabetaT cells, which target peripheral joints, transfer PIA. The pristane-primed T cells are of oligo or polyclonal origin as determined by their arthritogenicity after stimulation with several mitogenic anti-TCRVbeta and anti-TCRValpha mAbs. Arthritogenic cells secreted IFN-gamma and TNF-alpha (but not IL-4) when stimulated with Con A in vitro, and pretreatments of recipient rats with either anti-IFN-gamma or a recombinant TNF-alpha receptor before transfer ameliorated arthritis development. Most importantly, we show that these T cells are MHC class II restricted, because treatment with Abs against either DQ or DR molecules ameliorates arthritis development. The MHC class II restriction was confirmed by transferring donor T cells to irradiated recipients that were syngenic, semiallogenic, or allogenic to MHC class II molecules, in which only syngenic and semiallogenic recipients developed arthritis. These data suggest that the in vivo administration of a non-antigenic adjuvant, like pristane, activates CD4+ alphabetaT cells that are MHC class II restricted and arthritogenic

Self-reactive T cells induce and perpetuate chronic relapsing arthritis

Background: CD4+ T cells play a central role during the early stages of rheumatoid arthritis (RA), but to which extent they are required for the perpetuation of the disease is still not fully understood. The aim of the current study was to obtain conclusive evidence that T cells drive chronic relapsing arthritis. Methods: We used the rat pristane-induced arthritis model, which accurately portrays the chronic relapsing-remitting disease course of RA, to examine the contribution of T cells to chronic arthritis. Results: Rats subjected to whole-body irradiation and injected with CD4+ T cells from lymph nodes of pristane-injected donors developed chronic arthritis that lasted for more than 4 months, whereas T cells from the spleen only induced acute disease. Thymectomy in combination with irradiation enhanced the severity of arthritis, suggesting that sustained lymphopenia promotes T cell-driven chronic inflammation in this model. The ability of T cells to induce chronic arthritis correlated with their expression of Th17-associated transcripts, and while depletion of T cells in rats with chronic PIA led to transient, albeit significant, reduction in disease, neutralization of IL-17 resulted in almost complete and sustained remission. Conclusion: These findings show that, once activated, self-reactive T cells can sustain inflammatory responses for extended periods of time and suggest that such responses are promoted in the presence of IL-17

Genetic analysis of neuropathic pain-like behavior following peripheral nerve injury suggests a role of the major histocompatibility complex in development of allodynia

Neuropathic pain is a common consequence of damage to the nervous system. We here report a genetic analysis of development of neuropathic pain-like behaviors after unilateral photochemically-induced ischemic sciatic nerve injury in a panel of inbred rat strains known to display different susceptibility to autoimmune neuroinflammation. Pain behavior was initially characterized in Dark-Agouti (DA; RT1(avl)), Piebald Virol Glaxo (PVG; RT1(c)), and in the major histocompatibility complex (MHC)-congenic strain PVG-RT1(avl). All strains developed mechanical hypersensitivity (allodynia) following nerve injury. However, the extent and duration of allodynia varied significantly among the strains, with PVG displaying more severe allodynia compared to DA rats. Interestingly, the response of PVG-RT1(avR1) was similar to that of DA, suggesting regulation by the MHC locus. This notion was subsequently confirmed in an F2 cohort derived from crossing of the PVG and PVG-RT1(avl) strains, where allodynia was reduced in homozygous or heterozygous carriers of the RT1(avl) allele in comparison to rats homozygous for the RT1(c) allele. These results indicate that certain allelic variants of the MHC could influence susceptibility to develop and maintain neuropathic pain-like behavior following peripheral nerve injury in rats

The rheumatoid arthritis-associated autoantigen hnRNP-A2 (RA33) is a major stimulator of autoimmunity in rats with pristane-induced arthritis

A single intradermal injection of the mineral oil pristane in susceptible DA.1F rats induces erosive arthritis closely mimicking rheumatoid arthritis (RA). Pristane-induced arthritis (PIA) is driven by autoreactive T cells but no autoantigen has been identified to date. We therefore analyzed B and T cell responses to autoantigens potentially involved in the pathogenesis of RA, including IgG, citrullinated proteins, stress proteins, glucose-6-phosphate isomerase, and heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (RA33). IgG and lgM autoantibodies to hnRNP-A2 were detectable in sera of pristane-primed DA.1F rats already 1 wk before disease onset, reached maximum levels during the acute phase, and correlated with arthritis severity. Apart from rheumatoid factor, autoantibodies to other Ags were not observed. CD4(+) lymph node cells isolated 10 days after pristane injection produced IFN-gamma but not IL-4 in response to stimulation with hnRNP-A2, whereas none of the other candidate Ags elicited cytokine secretion. Surprisingly, hnRNP-A2 also stimulated lymph node cells of naive animals to produce inflammatory cytokines in a MyD88-dependent manner. Furthermore, hnRNP-A2 was highly overexpressed in the joints of rats injected with pristane. Overexpression coincided with the appearance of anti-RA33 Abs and preceded the onset of clinical symptoms of PIA by several days. Taken together, these data suggest hnRNP-A2 to be among the primary inducers of autoimmunity in PIA. Therefore, this Ag might play a pivotal role in the pathogenesis of PIA and possibly also human RA