Recombinant T-cell receptor ligand RTL1000 limits inflammation and decreases infarct size after experimental ischemic stroke in middle-aged mice.

We have previously demonstrated that recombinant T-cell receptor ligand 1000 (RTL1000) reduces infarct size and improves long-term functional recovery after experimental stroke in young transgenic mice expressing human leukocyte antigen DR2 (DR2-Tg). In this study, we determined the effect of RTL1000 on infarct size in 12-month-old middle-aged DR2-Tg mice, and investigated its mechanism of action. Twelve-month-old male DR2-Tg mice underwent 60min of intraluminal reversible middle cerebral artery occlusion (MCAO). Vehicle or RTL1000 was injected 4, 24, 48 and 72h after MCAO. Cortical, striatal and total hemispheric infarcts were measured 96h after stroke. Spleen and brain tissues were collected 96h after stroke for immunological analysis. Our data showed that RTL1000 significantly reduced infarct size 96h after MCAO in middle-aged male DR2-Tg mice. RTL1000 decreased the number of activated monocytes/microglia cells (CD11b(+)CD45(hi)) and CD3(+) T cells in the ischemic hemisphere. RTL1000 also reduced the percentage of total T cells and inflammatory neutrophils in the spleen. These findings suggest that RTL1000 protects against ischemic stroke in middle-aged male mice by limiting post-ischemic inflammation.

Single-chain recombinant HLA-DQ2.5/peptide molecules block α2-gliadin-specific pathogenic CD4+ T-cell proliferation and attenuate production of inflammatory cytokines: a potential therapy for celiac disease.

Celiac disease (CD) is a disorder of the small intestine caused by intolerance to wheat gluten and related proteins in barley and rye. CD4(+) T cells have a central role in CD, recognizing and binding complexes of HLA-DQ2.5 bearing gluten peptides that have survived digestion and that are deamidated by tissue transglutaminase (TG2), propagating a cascade of inflammatory processes that damage and eventually destroy the villous tissue structures of the small intestine. In this study, we present data showing that recombinant DQ2.5-derived molecules bearing covalently tethered α2-gliadin-61-71 peptide have a remarkable ability to block antigen-specific T-cell proliferation and inhibited proinflammatory cytokine secretion in human DQ2.5-restricted α2-gliadin-specific T-cell clones obtained from patients with CD. The results from our in vitro studies suggest that HLA-DQ2.5-derived molecules could significantly inhibit and perhaps reverse the intestinal pathology caused by T-cell-mediated inflammation and the associated production of proinflammatory cytokines.

Progesterone treatment reduces disease severity and increases IL-10 in experimental autoimmune encephalomyelitis.

Ovarian hormones, including progesterone, are known to have immunomodulatory and neuroprotective effects which may alter the disease course of experimental autoimmune encephalomyelitis (EAE). In the current study, we examined the treatment potential of progesterone beginning at the onset of EAE symptoms. Progesterone treated animals showed reduced peak disease scores and cumulative disease indices, and decreased inflammatory cytokine secretion (IL-2 and IL-17). In addition, increased production of IL-10 was accompanied by increased numbers of CD19+ cells and an increase in CD8+ cells. Decreased chemokine and chemokine receptor expression in the spinal cord also contributed to decreased lesions in the spinal cord.

Effect of experimental stroke on peripheral immunity: CNS ischemia induces profound immunosuppression.

The profound damage to the CNS caused by ischemic lesions has been well documented. Yet, relatively little is known about the contribution to and effects on the immune system during stroke. We have focused on both early and late events in the peripheral immune system during stroke in mice and have observed an early activation of splenocytes that conceivably could result in immune-mediated damage in the developing CNS lesion, followed by global immunosuppression that affects the spleen, thymus, lymph nodes and circulation. While this second immunosuppressive phase may not directly enhance infarction size, it without doubt leads to an inability to respond to antigenic challenges, thereby enhancing the risk for crippling systemic infection and septicemia in stroke survivors. These novel findings advocate the need to develop or effectively utilize agents that can block early neural splenic activation and modulate immune cells specific for brain antigens as a means to prevent mobilization of T and B cells carrying a cytokine death warrant to the brain. Equally important for the recovering stroke patient are approaches that can derail the second phase of immune dysfunction and restore the ability to mount a defense against systemic infectious insults.

A new orally bioavailable synthetic androstene inhibits collagen-induced arthritis in the mouse: androstene hormones as regulators of regulatory T cells.

Dehydroepiandrosterone (DHEA) has attracted much interest because of its many antiaging, metabolic and immune-modulating effects in rodents. Synthetic derivatives, such as 5-androstene-16alpha-fluoro-17-one (HE2500) and certain natural metabolites also provide benefit in various animal models of autoimmune and metabolic diseases. But, like DHEA, low potency and low oral bioavailability suggested limited usefulness of these compounds in humans. We hypothesized that HE3286, a novel 17-ethynyl derivative would be orally bioavailable, more potent, and chemically more useful in man than its parent compound. We found that on a dose/mass basis, HE3286 demonstrated up to 25% oral bioavailability in mice. In the DBA mouse model of collagen-induced arthritis (CIA), animals receiving oral treatment with HE3286 (50 mg/kg), beginning at onset of disease, significantly decreased CIA peak scores and daily severity of arthritis scores. Benefit was associated with decreases in: (1) production of TNF-alpha, IL-6, and IL-17; and (2) decreases in joint inflammation, erosion, and synovial proliferation as judged by histological analysis. HE3286 was not found to be immune suppressive in any of the classical models tested, including mitogen-induced proliferation, delayed-type hypersensitivity, or mixed lymphocyte reaction. Instead, benefit was associated with increases in numbers and function of CD4+CD25+FOXp3+CD127- regulatory T cells (T reg). To our knowledge, this is probably the first study to report that an orally bioavailable synthetic analogue of DHEA can ameliorate ongoing disease in a CIA mouse model with relevance to rheumatoid arthritis (RA) and to correlate that finding with decreases in proinflammatory cytokines and increases in T reg cells. Hormones targeting T reg cells hold the intriguing potential to treat autoimmune, infectious, and neoplastic diseases.

A highly immunogenic trivalent T cell receptor peptide vaccine for multiple sclerosis.

BACKGROUND: T cell receptor (TCR) peptide vaccination is a novel approach to treating multiple sclerosis (MS). The low immunogenicity of previous vaccines has hindered the development of TCR peptide vaccination for MS. OBJECTIVE: To compare the immunogenicity of intramuscular injections of TCR BV5S2, BV6S5 and BV13S1 CDR2 peptides in incomplete Freunds adjuvant (IFA) with intradermal injections of the same peptides without IFA. METHODS: MS subjects were randomized to receive TCR peptides/IFA, TCR peptides/saline or IFA alone. Subjects were on study for 24 weeks. RESULTS: The TCR peptides/IFA vaccine induced vigorous T cell responses in 100% of subjects completing the 24-week study (9/9) compared with only 20% (2/10) of those receiving the TCR peptides/saline vaccine (P =0.001). IFA alone induced a weak response in only one of five subjects. Aside from injection site reactions, there were no significant adverse events attributable to the treatment. CONCLUSIONS: The trivalent TCR peptide in IFA vaccine represents a significant improvement in immunogenicity over previous TCR peptide vaccines and warrants investigation of its ability to treat MS.

Anti-inflammatory and immune regulatory properties of 5-androsten-3beta, 17beta-diol (HE2100), and synthetic analogue HE3204: implications for treatment of autoimmune diseases.

5-Androsten-3beta, 17beta-diol (HE2100), and a synthetic analogue HE3204 are regarded as immune-regulating hormones, because both induce changes in the reporter antigen-popliteal lymph node assay (RA-PLNA). Mice were injected in the footpad with either HE2100 or HE3204 (0.01-3 mg), and a nonsensitizing dose of trinitrophenyl ovalbumin (TNP-OVA) was used as bystander reporter antigen. Seven days later, nodes were removed and numbers of cells (CD3, CD4, CD8, CD19; flow cytometry), TNP-specific IgM, IgG1, and IgG2a antibody-forming cells (AFCs; ELISPOT assay), and cytokines (interleukin-4 [IL-4], interferon-gamma [IFN-gamma]; ELISA) were measured. HE2100 and HE3204 increased cell numbers in a dose-dependent fashion. T (helper and suppressor) cells and B cells were increased (>5-fold). HE3204 was apparently twice as potent as HE2100. Both increased the B/T ratio (fivefold), increased TNP-specific IgM and IgG1 ( approximately 50-fold), and induced IgG2a AFCs. Both increased IL-4 and IFN-gamma secretion (up to threefold). Both displayed anti-inflammatory activity in the murine model of carrageenan-induced pleurisy, as evidenced by reduced neutrophil numbers and exudate volumes. Our observations suggest that both HE2100 and HE3204 are immune-regulating steroid hormones that exhibit anti-inflammatory properties. HE2100 (1 mg/mouse per day) provided significant benefit when given at disease onset in the SJL/J female mouse model of experimental autoimmune encephalomyelitis. These compounds and their analogues are candidates for further testing in autoimmune diseases.

Rudimentary TCR signaling triggers default IL-10 secretion by human Th1 cells.

Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the alpha1 and beta1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85-99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR zeta-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-gamma; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases.

Diminished frequency of interleukin-10-secreting, T-cell receptor peptide-reactive T cells in multiple sclerosis patients might allow expansion of activated memory T cells bearing the cognate BV gene.

T cells responsive to T-cell receptor (TCR) determinants may regulate pathogenic Th1 responses in patients with multiple sclerosis (MS) through interleukin (IL)-10-dependent bystander suppression. In this study, innate IL-10- and interferon (IFN)-gamma-secreting T cells responsive to TCR peptides were quantified in peripheral blood mononuclear cells of MS patients and healthy controls (HC) using the ELISPOT assay. Most HC had vigorous IL-10 but low IFN-gamma frequencies to BV5S2 and BV6S1 peptides. In contrast, MS patients had significantly lower IL-10 frequency responses to the TCR peptides but normal responses to concanavalin A. Patients undergoing TCR-peptide vaccination had moderate responses that fluctuated in concert with vaccination. In an MS patient and HC, expression of BV6S1 by activated memory T cells was inversely associated with the presence of IL-10-secreting BV6S1-reactive T cells. These results suggest that MS patients have diminished frequencies of innate TCR-reactive T cells that may allow oligoclonal expansion of activated autoreactive Th1 effector cells expressing cognate V genes.

17 beta-estradiol inhibits cytokine, chemokine, and chemokine receptor mRNA expression in the central nervous system of female mice with experimental autoimmune encephalomyelitis.

Cytokines and chemokines govern leukocyte trafficking, thus regulating inflammatory responses. In this study, the anti-inflammatory effects of low dose 17 beta-estradiol were evaluated on chemokine, chemokine receptor, and cytokine expression in the spinal cords (SC) of BV8S2 transgenic female mice during acute and recovery phases of experimental autoimmune encephalomyelitis (EAE). In EAE protected mice, 17 beta-estradiol strongly inhibited mRNA expression of the chemokines RANTES, MIP-1 alpha, MIP-2, IP-10, and MCP-1, and of the chemokine receptors CCR1, CCR2 and CCR5 at both time points. Conversely, ovariectomy, which abrogated basal 17 beta-estradiol levels and increased the severity of EAE, enhanced the expression of MIP-1 alpha and MIP-2 that were over-expressed by inflammatory mononuclear cells in SC. 17 beta-estradiol inhibited expression of LT-beta, TNF-alpha, and IFN-gamma in SC, but had no effect on IL-4 or IL-10, indicating reduced inflammation but no deviation toward a Th2 response. Interestingly, elevated expression of CCR1 and CCR5 by lymph node cells was also inhibited in 17 beta-estradiol treated mice with EAE. Low doses of 17 beta-estradiol added in vitro to lymphocyte cultures had no direct effect on the activation of MBP-Ac1-11 specific T cells, and only at high doses diminished production of IFN-gamma, but not IL-12 or IL-10. These results suggest that the beneficial effects of 17 beta-estradiol are mediated in part by strong inhibition of recruited inflammatory cells, resulting in reduced production of inflammatory chemokines and cytokines in CNS, with modest effects on encephalitogenic T cells that seem to be relatively 17 beta-estradiol insensitive.

TCR peptide therapy in human autoimmune diseases.

Inflammatory Th1 cells reacting to tissue/myelin derived antigens likely contribute to the pathogenesis of diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and psoriasis. One regulatory mechanism that may be useful for treating autoimmune diseases involves an innate second set of Th2 cells specific for portions of the T cell receptor of clonally expanded pathogenic Th1 cells. These Th2 cells are programmed to respond to internally modified V region peptides from the T cell receptor (TCR) that are expressed on the Th1 cell surface in association with major histocompatibility molecules. Once the regulatory Th2 cells are specifically activated, they may inhibit inflammatory Th1 cells through a non-specific bystander mechanism. A variety of strategies have been used by us to identify candidate disease-associated TCR V genes present on pathogenic Th1 cells, including BV5S2, BV6S5, and BV13SI in MS, BV3, BV14, and BV17 in RA, and BV3 and BV13S1 in psoriasis. TCR peptides corresponding to the mid region of these BV genes were found to be consistently immunogenic in vivo when administered either i.d. in saline or i.m. in incomplete Freund's adjuvant (IFA). In MS patients, repeated injection of low doses of peptides (100-300 microg) significantly boosted the number of TCR-reactive Th2 cells. These activated cells secreted cytokines, including IL-10, that are known to inhibit inflammatory Th1 cells. Cytokine release could also be induced in TCR-reactive Th2 cells by direct cell-cell contact with Th1 cells expressing the target V gene. These findings indicate the potential of regulatory Th2 cells to inhibit not only the target Th1 cells, but also bystander Th1 cells expressing different V genes specific for other autoantigens. TCR peptide vaccines have been used in our studies to treat a total of 171 MS patients (6 trials), 484 RA patients (7 trials), and 177 psoriasis patients (2 trials). Based on this experience in 824 patients with autoimmune diseases, TCR peptide vaccination is safe and well tolerated, and can produce significant clinical improvement in a subset of patients that respond to immunization. TCR peptide vaccination represents a promising approach that is well-suited for treating complex autoimmune diseases.

Estrogen treatment down-regulates TNF-alpha production and reduces the severity of experimental autoimmune encephalomyelitis in cytokine knockout mice.

A shift toward Th2 cytokine production has been demonstrated during pregnancy and high dose estrogen therapy and is thought to be the primary mechanism by which estrogen suppresses the development of experimental autoimmune encephalomyelitis. However, low dose estrogen treatment is equally protective in the absence of a significant shift in cytokine production. In this study cytokine-deficient mice were treated with estrogen to determine whether a shift in Th2 cytokine production was required for the protective effects of hormone therapy. Estrogen effectively suppressed the development of experimental autoimmune encephalomyelitis in IL-4 and IL-10 knockout mice and in wild type littermate mice with a similar potency of protection. Significant disease suppression was also seen in IFN-gamma-deficient mice. The decrease in disease severity was accompanied by a concomitant reduction in the number of proinflammatory cytokine- and chemokine-producing cells in the CNS. Although there was no apparent increase in compensatory Th2 cytokine production in cytokine-deficient mice, there was a profound decrease in the frequency of TNF-alpha-producing cells in the CNS and the periphery. Therefore, we propose that one mechanism by which estrogen protects females from the development of cell-mediated autoimmunity is through a hormone-dependent regulation of TNF-alpha production.

Low-dose estrogen therapy ameliorates experimental autoimmune encephalomyelitis in two different inbred mouse strains.

It has been proposed that homeostatic levels of estrogen can enhance female susceptibility to autoimmunity, whereas the heightened levels of estrogen associated with pregnancy are protective. This hypothesis was tested using the mouse model of experimental autoimmune encephalomyelitis (EAE). Diestrus (<100 pg/ml in serum) levels of 17beta-estradiol were found to significantly reduce the clinical manifestations of active EAE in both male and female mice. Estriol was also effective but at doses below those previously established for pregnancy. The reduction in disease severity was accompanied by a coincident reduction in the number and size of inflammatory foci in the CNS of estrogen (17beta-estradiol or estriol)-treated mice. Recipients of encephalitogenic T cells from low-dose estrogen-treated mice developed less severe paralysis than mice receiving T cells from placebo-treated mice. A modest shift in Th1/Th2 balance suggested that low dose estrogen therapy could bias the immune reaction toward a protective anti-inflammatory cytokine response. However, estrogen treatment at the onset of active EAE failed to reduce disease severity, a result that is consistent with the hypothesis that naive cells are more sensitive to sex hormones than differentiated effector cells. These data suggest that treatment with low doses of estrogen can reduce the capacity of developing myelin-reactive T cells to initiate disease and challenges the idea that increased susceptibility to autoimmunity in females is dependent on homeostatic levels of estrogen.

Rat RT1.B-transfected fibroblast lines process and present myelin antigens and activate T cells to induce experimental autoimmune encephalomyelitis.

The genes encoding the Lewis rat RT1.B molecule (MHC Class II I-A equivalent) were transfected and expressed in mouse DAP.3 fibroblast cells together with the gene encoding the mouse ICAM-1 molecule. Both molecules were stably expressed on the cell surface of DAP.3 cells under longterm culture conditions. The RT1.B/mICAM-1 transfectants presented antigen in a specific manner to a RT1. B-restricted rat T cell hybridoma specific for the 69-89 peptide of myelin basic protein (BP). In addition, the transfectants were able to present antigen to a BP69-89-specific rat T cell line. Presentation to a RT1.D (MHC Class II I-E equivalent)-restricted BP87-99-specific T cell line was minimal. Production of the Th1 cytokine IFN-gamma by BP69-89-specific T cells when stimulated by RT1.B/mICAM-1 transfectants correlated very well with proliferation to specific antigen. Moreover, RT1.B-transfected DAP.3 cells sufficiently stimulated BP69-89-specific T cells such that they were able to transfer experimental autoimmune encephalomyelitis (EAE) to Lewis rat recipients. Thus, the RT1.B molecule is functionally expressed on the surface of transfected Dap.3 fibroblasts and is capable of MHC Class II-restricted, antigen-specific presentation to rat T cells.

T lymphocytes promote the development of bone marrow-derived APC in the central nervous system.

Certain cells within the CNS, microglial cells and perivascular macrophages, develop from hemopoietic myelomonocytic lineage progenitors in the bone marrow (BM). Such BM-derived cells function as CNS APC during the development of T cell-mediated paralytic inflammation in diseases such as experimental autoimmune encephalomyelitis and multiple sclerosis. We used a novel, interspecies, rat-into-mouse T cell and/or BM cell-transfer method to examine the development and function of BM-derived APC in the CNS. Activated rat T cells, specific for either myelin or nonmyelin Ag, entered the SCID mouse CNS within 3-5 days of cell transfer and caused an accelerated recruitment of BM-derived APC into the CNS. Rat APC in the mouse CNS developed from transferred rat BM within an 8-day period and were entirely sufficient for induction of CNS inflammation and paralysis mediated by myelin-specific rat T cells. The results demonstrate that T cells modulate the development of BM-derived CNS APC in an Ag-independent fashion. This previously unrecognized regulatory pathway, governing the presence of functional APC in the CNS, may be relevant to pathogenesis in experimental autoimmune encephalomyelitis, multiple sclerosis, and/or other CNS diseases involving myelomonocytic lineage cells.

Epitope recognition and T cell receptors in recurrent autoimmune anterior uveitis in Lewis rats immunized with myelin basic protein.

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Rats recover and become resistant to further reinduction of EAE. We investigated whether the resistance to reinduction of EAE was associated with the resistance to AU in LEW rats reinjected with MBP. We demonstrated that while rats remained resistant to EAE, they become susceptible to uveitis after recovery, and suffered a second episode of disease. The susceptibility to reinduced disease was associated with the recognition of new MBP epitopes. In contrast to the initial episode of AU, TCR Vbeta8.2 predominance was not observed in the iris/ciliary body. Our results suggest that T cells specific for MBP, which are rapidly reactivated when re-exposed to antigen, are sufficient to induce clinical uveitis in LEW rats. This process may involve a shifting of T cell specificity from the major encephalitogenic peptide utilizing the Vbeta8.2 receptor to a more diverse cell repertoire.

Regulation of encephalitogenic T cells with recombinant TCR ligands.

We have previously described recombinant MHC class II beta1 and alpha1 domains loaded with free antigenic peptides with potent inhibitory activity on encephalitogenic T cells. We have now produced single-chain constructs in which the peptide Ag is genetically encoded within the same exon as the linked beta1 and alpha1 domains, overcoming the problem of displacement of peptide Ag from the peptide binding cleft. We here describe clinical effects of recombinant TCR ligands (RTLs) comprised of the rat RT1.B beta1alpha1 domains covalently linked to the 72-89 peptide of guinea pig myelin basic protein (RTL-201), to the corresponding 72-89 peptide from rat myelin basic protein (RTL-200), or to cardiac myosin peptide CM-2 (RTL-203). Only RTL-201 possessed the ability to prevent and treat active or passive experimental autoimmune encephalomyelitis. Amelioration of experimental autoimmune encephalomyelitis was associated with a selective inhibition of proliferation response and cytokine production by Ag-stimulated lymph node T cells and a drastic reduction in the number of encephalitogenic and recruited inflammatory cells infiltrating the CNS. The exquisitely selective inhibition could be observed between molecules that differ by a single methyl group (the single amino acid residue difference between RTL-200 (threonine) and RTL-201 (serine) at position 80 of the myelin basic protein peptide). These novel RTLs provide a platform for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease.

Interleukin 7 is a potent co-stimulator of myelin specific T cells that enhances the adoptive transfer of experimental autoimmune encephalomyelitis.

Interleukin 7 (IL-7), originally described as a B cell growth factor, has recently been found to play a critical role in T and B lymphocyte development and function. This study evaluated the effects of IL-7 on myelin specific T cells. IL-7 strongly enhanced proliferation of proteolipid protein (PLP) 139-151 specific T cells in association with elevated secretion of the T cell growth factor IL-2. Co-stimulation with IL-7 preferentially increased the levels of pro-inflammatory cytokines secreted by PLP 139-151 specific T cells and adoptive transfer of these cells into naive recipients induced a profound enhancement of experimental autoimmune encephalomyelitis, an animal model for the human disease multiple sclerosis. These results suggest that IL-7 may be a critical co-stimulatory factor that enhances the extrathymic expansion of inflammatory T cells and may play an important role in the pathogenesis of a number of inflammatory autoimmune disorders.

Estrogen potentiates treatment with T-cell receptor protein of female mice with experimental encephalomyelitis.

Transgenic mice expressing the BV8S2 chain, which is specific for the myelin basic protein determinant Ac1-11, possess a naturally induced set of regulatory T cells directed against BV8S2. Further activation of anti-BV8S2 T cells in male mice with recombinant BV8S2 protein can inhibit IFN-gamma release by Ac1-11-specific T cells through a cytokine-driven mechanism and prevent induction of experimental autoimmune encephalomyelitis (EAE). In contrast, naive female mice possess fewer anti-BV8S2-reactive T cells, and treatment with BV8S2 delayed but did not prevent EAE. We here demonstrate that combining T-cell receptor (TCR) vaccination with supplemental estrus doses of estrogen potentiated IL-10 production by anti-BV8S2-reactive T cells and induced Ac1-11-specific T cells to produce IL-10 and TGF-beta. This combined treatment resulted in full protection against EAE, which was not observed with either therapy alone. These findings imply that supplemental estrogen can enhance the efficacy of TCR-based immunotherapy for autoimmune diseases that predominate in females.

Human TCR as antigen: homologies and potentially cross-reactive HLA-DR2-restricted epitopes within the AV and BV CDR2 loops.

The major function of the T-cell receptor is to confer antigen specificity to T cells. However, nascent TCR proteins that are not assembled into functional heterodimers may be processed and displayed with self MHC molecules on the T-cell surface, and are thought to be the genesis of autoregulatory T cells that can limit inflammatory responses through T-T network interactions. In previous work, we and others have exploited this natural regulatory system using TCR peptides to amplify regulatory T cells that potentially can treat human autoimmune diseases such as multiple sclerosis (MS) and arthritis. The development of this approach is limited by the diversity of human TCR V gene sequences, and by lack of knowledge of exactly which regions of the V gene proteins are immunogenic in association with various MHC alleles. To identify similar amino acid sequences within and among human V gene families that might have immunologic cross reactivity, we aligned 74 known AV and 109 known BV protein sequences into homologous groups using the ClustalX program. Moreover, with a focus on CDR2 peptides that have previously been used to induce regulatory T cells in clinical trials, we established homologous peptide groups, and then identified the optimal amino acid motifs for binding to two alleles, HLA-DRB1*1501 and DRB5*0101, that have been associated with susceptibility to MS. From this analysis, > 75% of AV and BV CDR2 sequences were predicted to bind with at least moderate avidity to each of the DR2 alleles, thus enhancing the likelihood that they could be antigenic. Further ordering of putative TCR contact residues revealed a different set of homology groupings, including many intrafamily sequence matches and some interfamily matches that might allow immunological cross reactivity. Particularly striking were DRB1*1501-restricted IH-S and IY-S motifs shared by BV11, BV12, and BV13 and BV3, BV12, BV13, and BV17 family members, respectively, and DRB5*0101-restricted RL-H and RL-Y motifs shared by BV11, BV12, and BV13 and BV13 and BV17 family members, respectively. This analysis may be useful in designing an array of clinically useful homologous peptides with optimal MHC binding properties and highly cross-reactive TCR binding motifs.