Imlifidase-generated Single-cleaved IgG: Implications for Transplantation.

BACKGROUND: Imlifidase is an immunoglobulin G (IgG)-specific protease conditionally approved in the EU for desensitization in highly sensitized crossmatch positive kidney transplant patients. Imlifidase efficiently cleaves both heavy chains of IgG in a 2-step process. However, low levels of the intermediate cleavage product, single-cleaved IgG (scIgG), may persist in the circulation. The study objective was to investigate Fc-mediated effector functions of scIgG and its potential impact on common clinical immunologic assays used to assess transplant eligibility. METHODS: Imlifidase-generated scIgG, obtained by in vitro cleavage of HLA-sensitized patient serum or selected antibodies, was investigated in different complement- and FcγR-dependent assays and models, including clinical tests used to evaluate HLA-specific antibodies. RESULTS: ScIgG had significantly reduced Fc-mediated effector function compared with intact IgG, although some degree of activity in complement- and FcγR-dependent models was still detectable. A preparation of concentrated scIgG generated from a highly HLA-sensitized individual gave rise to a positive signal in the anti-HLA IgG LABScreen, which uses anti-Fc detection, but was entirely negative in the C1qScreen. The same high-concentration HLA-binding scIgG preparation also generated positive complement-dependent cytotoxicity responses against 80%-100% of donor T and B cells, although follow-up titrations demonstrated a much lower intrinsic activity than for intact anti-HLA IgG. CONCLUSIONS: ScIgG has a significantly reduced capacity to mediate Fc-dependent effector functions. However, remaining HLA-reactive scIgG in plasma after imlifidase treatment can cause positive assay results equivalent to intact IgG in clinical assays. Therefore, complete IgG cleavage after imlifidase treatment is essential to allow correct decision-making in relation to transplant eligibility.

IgM single antigen bead HLA-assay is affected by imlifidase through the cleavage of IgG but not IgM.

AIM: The aim of this study was to investigate if human IgM is a cleavable substrate for imlifidase and to explain an observed effect in anti-HLA IgM single antigen bead (SAB) assays in sensitized patients. METHODS: Serum samples collected pre- and 24 h post-imlifidase administration from sensitized patients enrolled in a phase II trial were investigated for anti-HLA IgG and IgM using SAB assays, with and without in vitro IgG depletion using a CaptureSelect™ affinity matrix. In addition, pre-dose samples and purified human IgM samples were treated with imlifidase in vitro and evaluated by SDS-PAGE, Western blot (PE-conjugated anti-human IgM) and SAB (IgG, IgM) assays. RESULTS: By comparing the mean fluorescence intensity (MFI) of HLA-beads, pre- and post-imlifidase administration, three IgM-related patterns were observed; IgM-specific HLA-SABs with an increased MFI post-imlifidase, IgM-specific HLA-SABs with a decreased MFI post-imlifidase, and IgM-specific HLA-SABs with a marginal MFI difference between the pre- and post-imlifidase administration. These IgM signal patterns were observed despite neither purified IgM nor serum IgM could be cleaved by imlifidase. After removing IgG, the effects observed on anti-HLA IgM was largely eliminated with the biggest differences seen in patients with very high anti-HLA IgG in pre-dose samples. CONCLUSION: We demonstrate that imlifidase does not cleave human IgM, including HLA-specific IgM antibodies from highly sensitized subjects. Observed decreases of SAB-HLA IgM signals after imlifidase treatment may result from the cleavage of IgG-IgM complexes which are bound to SAB-HLA. Serum analysis of patients with high levels of anti-HLA IgG will result in a more accurate SAB-HLA IgM reading after IgG depletion.

ADAM12 is a costimulatory molecule that determines Th1 cell fate and mediates tissue inflammation.

A disintegrin and metalloproteinase (ADAM)12 was previously found to be expressed in T cells in the inflamed brain. However, the function of ADAM12 in T-cell responses in general and in tissue inflammation has not been examined. Here, we studied the role of ADAM12 in T-cell responses, fate determination on activation, and its functions in T cells to mediate tissue inflammation. We identified ADAM12 as a costimulatory molecule that is expressed on naive T cells and downregulated on stimulation. ADAM12 mimics CD28 costimulatory signaling to activate and induce the proliferation of T-helper 1 (Th1) cells. Monoclonal ADAM12 Fab antibodies trigger T-cell activation by amplifying TCR signaling to stimulate T-bet-mediated IFNγ production. Lack of genomic ADAM12 and its knockdown in T cells diminished T-bet and IFNγ production in Th1 cells, whereas other T cells, including Th17 cells, were unaffected. ADAM12 had similar functions in vivo on myelin antigen (MOG35-55)-induced T-cell activation. We found that genetic loss of ADAM12 profoundly alleviated Th1-mediated neuroinflammation and thus disease severity in experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Transcriptomic profiling of MOG35-55-specific ADAM12-/- T cells revealed differentially expressed genes that are important for T-cell activation, proliferation, and costimulatory signaling and Th1 pathogenicity, consistent with their inability to cause T-cell-mediated skin inflammation in a model of adoptive delayed-type hypersensitivity. We conclude that ADAM12 is a T-cell costimulatory molecule that contributes to the pathogenesis of tissue inflammation and a potential target for the treatment of Th1-mediated diseases.

Pharmacological inhibition of carnitine palmitoyl transferase 1 inhibits and reverses experimental autoimmune encephalitis in rodents.

Multiple sclerosis (MS) is a neurodegenerative disease characterized by demyelination and inflammation. Dysregulated lipid metabolism and mitochondrial dysfunction are hypothesized to play a key role in MS. Carnitine Palmitoyl Transferase 1 (CPT1) is a rate-limiting enzyme for beta-oxidation of fatty acids in mitochondria. The therapeutic effect of pharmacological CPT1 inhibition with etomoxir was investigated in rodent models of myelin oligodendrocyte glycoprotein- and myelin basic protein-induced experimental autoimmune encephalitis (EAE). Mice receiving etomoxir showed lower clinical score compared to placebo, however this was not significant. Rats receiving etomoxir revealed significantly lower clinical score and lower body weight compared to placebo group. When comparing etomoxir with interferon-ß (IFN-ß), IFN-ß had no significant therapeutic effects, whereas etomoxir treatment starting at day 1 and 5 significantly improved the clinical scores compared to the IFN-ß and the placebo group. Immunohistochemistry and image assessments of brain sections from rats with EAE showed higher myelination intensity and decreased expression of CPT1A in etomoxir-treated rats compared to placebo group. Moreover, etomoxir mediated increased interleukin-4 production and decreased interleukin-17α production in activated T cells. In conclusion, CPT1 is a key protein in the pathogenesis of EAE and MS and a crucial therapeutic target for the treatment.

Desensitization using imlifidase and EndoS enables chimerism induction in allosensitized recipient mice.

Mixed hematopoietic chimerism induction as a way to foster tolerance to donor organs in recipients who have been sensitized to donor antigens is challenging. Donor-specific antibodies (DSA) are a dominant barrier toward successful donor bone marrow engraftment. Although desensitization methods are routinely used in recipients with allosensitization for allogeneic bone marrow transplantation, engraftment is frequently unsuccessful. To overcome the barrier of prior sensitization we tested enzymatic desensitization of donor-specific IgG using imlifidase and endoglycosidase of Streptococcus pyogenes (EndoS), which both partially block the function of DSA in mice, as a novel approach to improve murine bone marrow engraftment in primed hosts. We found that EndoS was capable of inhibiting antibody-mediated killing of donor cells in vivo. Furthermore, the effect of EndoS depended on the titer of DSA and the genetic background of the recipients. In combination with imlifidase, EndoS improved the survival of donor bone marrow cells. Together with cyclophosphamide, bortezomib, T cell depletion, and nonlethal irradiation, imlifidase in combination with EndoS allowed allogeneic bone marrow engraftment in sensitized recipients. We conclude that enzymatic inactivation of DSA, using the combination of imlifidase and EndoS, can be used for inducing donor hematopoietic chimerism in allosensitized recipient mice in combination with other desensitization strategies.

Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR.

The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation.

Ag binding to the BCR is a critical step in B cell development and activation, initiating a cascade of signaling events ultimately leading to proliferation, differentiation, or cell death. A bacterial enzyme, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), was shown to specifically cleave IgG molecules below the hinge region of soluble IgG and when IgG is bound to Ag, resulting in one F(ab')2 molecule and one homodimeric Fc fragment. Whether IdeS could also cleave the IgG molecule when it is present in the BCR attached to the B cell membrane in a complex with CD79a and CD79b is unknown. In this article, we present human in vitro and ex vivo data showing that IdeS cleaves the IgG present in the BCR complex and very efficiently blocks Ag binding to the BCR. As a consequence of IdeS cleaving the BCR, signaling cascades downstream of the BCR are blocked, and memory B cells are temporarily silenced, preventing them from responding to antigenic stimulation and their transition into Ab-producing cells.

Complete Removal of Extracellular IgG Antibodies in a Randomized Dose-Escalation Phase I Study with the Bacterial Enzyme IdeS--A Novel Therapeutic Opportunity.

UNLABELLED: IdeS is a streptococcal protease that cleaves IgG antibodies into F(ab')2 and Fc fragments with a unique degree of specificity, thereby providing a novel treatment opportunity of IgG-driven autoimmune conditions and antibody mediated transplant rejection. Here we report the results from a first in man, double blinded and randomized study with single ascending doses of IdeS in healthy, male subjects. Twenty healthy subjects were given intravenous single ascending doses of IdeS. With impressive efficacy IdeS cleaved the entire plasma IgG-pool only minutes after dosing. IgG reached nadir 6-24 hours after dosing and then slowly recovered. The half-life of IdeS was 4.9 (±2.8) hours at 0.24 mg/kg with the main fraction eliminated during 24 hours. Already two hours after IdeS-dosing, the phagocytic capacity of IgG/IgG-fragments was reduced to background levels. Importantly, IdeS has the capacity to inactivate Fc-mediated effector function in vivo, was considered safe with no serious adverse events, and without dose limiting toxicity in this study. The complete, rapid, but temporary removal of IgG provides a new potent therapeutic opportunity in IgG-mediated pathogenic conditions. TRIAL REGISTRATION: ClinicalTrials.gov NCT01802697.

Influence of dietary components on regulatory T cells.

Common dietary components including vitamins A and D, omega-3 and probiotics are now widely accepted to be essential to protect against many diseases with an inflammatory nature. On the other hand, high-fat diets are documented to exert multiple deleterious effects, including fatty liver diseases. Here we discuss the effect of dietary components on regulatory T cell (Treg) homeostasis, a central element of the immune system to prevent chronic tissue inflammation. Accordingly, evidence on the impact of dietary components on diseases in which Tregs play an influential role will be discussed. We will review chronic tissue-specific autoimmune and inflammatory conditions such as inflammatory bowel disease, type 1 diabetes mellitus, multiple sclerosis, rheumatoid arthritis and allergies among chronic diseases where dietary factors could have a direct influence via modulation of Tregs homeostasis and functions.

Visualization and phenotyping of proinflammatory antigen-specific T cells during collagen-induced arthritis in a mouse with a fixed collagen type II-specific transgenic T-cell receptor ß-chain.

INTRODUCTION: The Vß12-transgenic mouse was previously generated to investigate the role of antigen-specific T cells in collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. This mouse expresses a transgenic collagen type II (CII)-specific T-cell receptor (TCR) ß-chain and consequently displays an increased immunity to CII and increased susceptibility to CIA. However, while the transgenic Vß12 chain recombines with endogenous α-chains, the frequency and distribution of CII-specific T cells in the Vß12-transgenic mouse has not been determined. The aim of the present report was to establish a system enabling identification of CII-specific T cells in the Vß12-transgenic mouse in order to determine to what extent the transgenic expression of the CII-specific ß-chain would skew the response towards the immunodominant galactosylated T-cell epitope and to use this system to monitor these cells throughout development of CIA. METHODS: We have generated and thoroughly characterized a clonotypic antibody, which recognizes a TCR specific for the galactosylated CII(260-270) peptide in the Vß12-transgenic mouse. Hereby, CII-specific T cells could be quantified and followed throughout development of CIA, and their phenotype was determined by combinatorial analysis with the early activation marker CD154 (CD40L) and production of cytokines. RESULTS: The Vß12-transgenic mouse expresses several related but distinct T-cell clones specific for the galactosylated CII peptide. The clonotypic antibody could specifically recognize the majority (80%) of these. Clonotypic T cells occurred at low levels in the naïve mouse, but rapidly expanded to around 4% of the CD4+ T cells, whereupon the frequency declined with developing disease. Analysis of the cytokine profile revealed an early Th1-biased response in the draining lymph nodes that would shift to also include Th17 around the onset of arthritis. Data showed that Th1 and Th17 constitute a minority among the CII-specific population, however, indicating that additional subpopulations of antigen-specific T cells regulate the development of CIA. CONCLUSIONS: The established system enables the detection and detailed phenotyping of T cells specific for the galactosylated CII peptide and constitutes a powerful tool for analysis of the importance of these cells and their effector functions throughout the different phases of arthritis.

CD1d-dependent NKT cells play a protective role in acute and chronic arthritis models by ameliorating antigen-specific Th1 responses.

A protective and anti-inflammatory role for CD1d-dependent NKT cells (NKTs) has been reported in experimental and human autoimmune diseases. However, their role in arthritis has been unclear, with conflicting reports of CD1d-dependent NKTs acting both as regulatory and disease-promoting cells in arthritis. These differing modes of action might be due to genetic differences of inbred mice and incomplete backcrossing of gene-modified mice. We therefore put special emphasis on controlling the genetic backgrounds of the mice used. Additionally, we used two different murine arthritis models, Ag-induced arthritis (AIA) and collagen-induced arthritis (CIA), to evaluate acute and chronic arthritis in CD1d knockout mice and mice depleted of NK1.1(+) cells. CD1d-deficient mice developed more severe AIA compared with wild-type littermates, with a higher degree of inflammation and proteoglycan depletion. Chronic arthritis in CIA was also worse in the absence of CD1d-dependent NKTs. Elevated levels of Ag-specific IFN-gamma production accompanied these findings rather than changes in IL-17alpha. Depletion of NK1.1(+) cells supported these findings in AIA and CIA. This report provides support for CD1d-dependent NKTs being suppressor cells in acute and chronic arthritis, likely via inhibition of arthritogenic Th1 cells. These results make CD1d-dependent NKTs an attractive target for therapeutic intervention.

Structure and pathogenicity of antibodies specific for citrullinated collagen type II in experimental arthritis.

Antibodies to citrulline-modified proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical collagen type II (CII) epitope (position 359-369; ARGLTGRPGDA) with or without arginines modified by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients' synovial fluid demonstrates that cartilage-derived CII is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising beta-turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose that autoimmunity to CII, leading to the production of antibodies specific for both native and citrullinated CII, is an important pathogenic factor in the development of RA.

Alpha10 integrin expression is up-regulated on fibroblast growth factor-2-treated mesenchymal stem cells with improved chondrogenic differentiation potential.

Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to differentiate into various different cell lineages and can generate bone, cartilage and adipose tissue. MSCs are presently characterized using a broad range of different cell-surface markers that are not exclusive to MSCs and not sensitive to culture conditions or differentiation capacity. We show that the integrin subunits alpha10 and alpha11 of the collagen binding integrins alpha10beta1 and alpha11beta1 are expressed by human MSCs in monolayer cultures. We also demonstrate that the expression of alpha10 increases, while alpha1 and alpha11 decrease, during aggregate culture of MSCs in chondrogenic medium. Alpha10beta1 is expressed by chondrocytes in cartilage, whereas alpha11beta1 integrins are predominantly expressed by subsets of the fibroblastic lineage. In extensive monolayer cultures of MSCs, alpha10 expression is down-regulated. We show that this down-regulation is reversed by fibroblast growth factor-2 (FGF-2) treatment. Addition of FGF-2 to MSCs not only results in increased alpha10 expression, but also in decreased alpha11 expression. FGF-2 treatment of MSCs has been shown to keep the cells more multipotent and also induces cell proliferation and Sox-9 up-regulation. We demonstrate improved chondrogenecity as well as increased collagen-dependant migratory potential of FGF-2-treated MSCs having a high alpha10 expression. We also demonstrate expression of alpha10 and alpha11 integrin subunits in the endosteum and periosteum of mice, but very low or not detectable expression levels in freshly aspired human or mouse BM. We show that MSCs with high chondrogenic differentiation potential are highly alpha10 positive and propose alpha10 as a potential marker to predict the differentiation state of MSCs.

Fragmentation of two quantitative trait loci controlling collagen-induced arthritis reveals a new set of interacting subloci.

Linkage analysis of F(2) crosses has led to identification of large numbers of quantitative trait loci (QTL) for complex diseases, but identification of the underlying genes has been more difficult. Reasons for this could be complications that arise from separation of interacting or neighboring loci. We made a partial advanced intercross (PAI) to characterize and fine-map linkage to collagen-induced arthritis in two chromosomal regions derived from the DBA/1 strain and crossed into the B10.Q strain: Cia7 on chromosome 7 and a locus on chromosome 15. Only Cia7 was detected by a previous F(2) cross. Linkage analysis of the PAI revealed a different linkage pattern than the F(2) cross, adding multiple loci and strong linkage to the previously unlinked chromosome 15 region. Subcongenic strains derived from animals in the PAI confirmed the loci and revealed additional subloci. In total, no less than seven new loci were identified. Several loci interacted and three loci were protective, thus partly balancing the effect of the disease-promoting loci. Our results indicate that F(2) crosses do not reveal the full complexity of identified QTLs, and that detection is more dependent on the genetic context of a QTL than the potential effect of the underlying gene.

Type II collagen without adjuvant induces eosinophilic arthritis.

Eosinophilia is a characteristic feature of many inflammatory diseases including inflammatory bowel disease and asthma. It also occurs in a subtype of rheumatoid arthritis but the role of eosinophils has been unclear and animal models have been lacking. Here, we introduce a new mouse model to study the role of eosinophilia in arthritis. Intraperitoneal injection of type II collagen alone, without any adjuvant, was sufficient to induce chronic arthritis in a mouse with transgenic T cells specific for type II collagen. The arthritis was accompanied by infiltration of eosinophils into the synovial tissue and the disease could be blocked with neutralizing anti-IL-5 antibodies. To our knowledge, this is the first description of an eosinophilic disease form of destructive arthritis.

Side-chain and backbone amide bond requirements for glycopeptide stimulation of T-cells obtained in a mouse model for rheumatoid arthritis.

Collagen induced arthritis (CIA) is the most studied animal model for rheumatoid arthritis and is associated with the MHC class II molecule Aq. T-cell recognition of a peptide from type II collagen, CII256-270, bound to Aq is a requirement for development of CIA. Lysine 264 is the major T-cell recognition site of CII256-270 and CIA is in particular associated with recognition of lysine 264 after posttranslational hydroxylation and subsequent attachment of a beta-D-galactopyranosyl moiety. In this paper we have studied the structural requirements of collagenous glycopeptides required for T-cell stimulation, as an extension of earlier studies of the recognition of the galactose moiety. Synthesis and evaluation of alanine substituted glycopeptides revealed that there are T-cells that only recognise the galactosylated hydroxylysine 264, and no other amino acid side chains in the peptide. Other T-cells also require glutamic acid 266 as a T-cell contact point. Introduction of a methylene ether isostere instead of the amide bond between residues 260 and 261 allowed weaker recognition by some, but not all, of the T-cells. Altogether, these results allowed us to propose a model for glycopeptide recognition by the T-cells, where recognition from one or the other side of the galactose moiety could explain the different binding patterns of the T-cells.

Induction of a B-cell-dependent chronic arthritis with glucose-6-phosphate isomerase.

Antibodies specific for glucose-6-phosphate isomerase (G6PI) from T-cell receptor transgenic K/BxN mice are known to induce arthritis in mice, and immunization of DBA/1 mice with G6PI led to acute arthritis without permanent deformation of their joints. Because rheumatoid arthritis is a chronic disease, we set out to identify the capacity of G6PI to induce chronic arthritis in mice. Immunization with recombinant human G6PI induced a chronically active arthritis in mice with a C3H genomic background, whereas the DBA/1 background allowed only acute arthritis and the C57BL/10 background permitted no or very mild arthritis. The disease was associated with the major histocompatibility region sharing an allelic association similar to that of collagen-induced arthritis (i.e. q > p > r). All strains developed a strong antibody response to G6PI that correlated only in the C3H.NB strain with arthritis severity. Similarly, a weak response to type II collagen in a few mice was observed, which was associated with arthritis in C3H.NB mice. Mice on the C3H background also developed ankylosing spondylitis in the vertebrae of the tail. Both C3H.Q and B10.Q mice deficient for B cells were resistant to arthritis. We conclude that G6PI has the ability to induce a chronic arthritis, which is MHC associated and B-cell dependent. Thus, there are striking similarities between this and the collagen-induced arthritis model.

Humoral immune response to citrullinated collagen type II determinants in early rheumatoid arthritis.

Collagen type II (CII) is a relevant joint-specific autoantigen in the pathogenesis of rheumatoid arthritis (RA). Whereas the reasons for the breakage of self tolerance to this major cartilage component are still enigmatic, T cell responses to glycosylated CII determinants in RA patients indicate that post-translational modifications play a role. Since the conversion of arginine into citrulline by peptidylarginine deiminases (PAD) in some non-joint-specific antigens such as filaggrin or fibrin has been shown to give rise to RA-specific humoral immune responses, we investigated whether PAD modification of cartilage-specific CII might affect its recognition by circulating autoantibodies in early RA. In vitro treatment with purified PAD led to arginine deimination of native CII or of synthetic CII peptides as evidenced by amino acid analysis. The citrullination resulted in modified recognition of the immunodominant CII epitope C1(III) (amino acid residues 359-369) by murine and human antibodies. In a cohort of early RA patients (n=286), IgG antibodies directed toward a synthetic citrullinated C1(III) peptide (citC1(III)-P) were detectable with a prevalence of 40.4%. The partial autoantibody cross-reactivity between citC1(III)-P and citrullinated peptides mimicking epitopes of the cytoskeletal autoantigen filaggrin suggests that autoimmunity to cartilage-specific modified self might be a critical intermediate bridging recognition of PAD-modified extra-articular autoantigens with the disruption of tolerance to native cartilage constituents.

A transient post-translationally modified form of cartilage type II collagen is ignored by self-reactive T cells.

Lysine residues in type II collagen (CII) are normally hydroxylated and subsequently glycosylated in the chondrocyte. The immunodominant T cell epitope of CII involves such post-translationally modified lysine at position 264 that has been shown to be critical in the pathogenesis of murine collagen-induced arthritis and also in human rheumatoid arthritis. In this study we identified a line of transgenic mice expressing a TCR specific for hydroxylated rat CII epitope. They were crossed with transgenic mice expressing the rat CII epitope, either specifically in cartilage (MMC mice) or systemically (TSC mice), to analyze T cell tolerance to a post-translationally modified form of self-CII. The mechanism of T cell tolerance to the hydroxylated CII epitope in TSC mice was found to involve intrathymic deletion and induction of peripheral tolerance. In contrast, we did not observe T cell tolerance in the MMC mice. Analysis of CII prepared from rat or human joint cartilage revealed that most of the lysine 264 is glycosylated rather than remaining hydroxylated. Therefore, we conclude that the transient post-translationally modified form of cartilage CII does not induce T cell tolerance. This lack of T cell tolerance could increase the risk of developing autoimmune arthritis.

CD1-dependent regulation of chronic central nervous system inflammation in experimental autoimmune encephalomyelitis.

The existence of T cells restricted for the MHC I-like molecule CD1 is well established, but the function of these cells is still obscure; one implication is that CD1-dependent T cells regulate autoimmunity. In this study, we investigate their role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, using CD1-deficient mice on a C57BL/6 background. We show that CD1-/- mice develop a clinically more severe and chronic EAE compared with CD1+/+ C57BL/6 mice, which was histopathologically confirmed with increased demyelination and CNS infiltration in CD1-/- mice. Autoantigen rechallenge in vitro revealed similar T cell proliferation in CD+/+ and CD1-/- mice but an amplified cytokine response in CD1-/- mice as measured by both the Th1 cytokine IFN-gamma and the Th2 cytokine IL-4. Investigation of cytokine production at the site of inflammation showed a CNS influx of TGF-beta1-producing cells early in the disease in CD1+/+ mice, which was absent in the CD1-/- mice. Passive transfer of EAE using an autoreactive T cell line induced equivalent disease in both groups, which suggested additional requirements for activation of the CD1-dependent regulatory pathway(s). When immunized with CFA before T cell transfer, the CD1-/- mice again developed an augmented EAE compared with CD1+/+ mice. We suggest that CD1 exerts its function during CFA-mediated activation, regulating development of EAE both through enhancing TGF-beta1 production and through limiting autoreactive T cell activation, but not necessarily via effects on the Th1/Th2 balance.