Treatment of multiple sclerosis with T-cell receptor peptides: results of a double-blind pilot trial.

A T-cell receptor (TCR) peptide vaccine from the V beta 5.2 sequence expressed in multiple sclerosis (MS) plaques and on myelin basic protein (MBP)-specific T cells boosted peptide-reactive T cells in patients with progressive MS. Vaccine responders had a reduced MBP response and remained clinically stable without side effects during one year of therapy, whereas nonresponders had an increased MBP response and progressed clinically. Peptide-specific T helper 2 cells directly inhibited MBP-specific T helper 1 cells in vitro through the release of interleukin-10, implicating a bystander suppression mechanism that holds promise for treatment of MS and other autoimmune diseases.

Selective depletion of myelin-reactive T cells with the anti-OX-40 antibody ameliorates autoimmune encephalomyelitis.

The OX-40 protein was selectively upregulated on encephalitogenic myelin basic protein (MBP)-specific T cells at the site of inflammation during the onset of experimental autoimmune encephalomyelitis (EAE). An OX-40 immunotoxin was used to target and eliminate MBP-specific T cells within the central nervous system without affecting peripheral T cells. When injected in vivo, the OX-40 immunotoxin bound exclusively to myelin-reactive T cells isolated from the CNS, which resulted in amelioration of EAE. Expression of the human OX-40 antigen was also found in peripheral blood of patients with acute graft-versus-host disease and the synovia of patients with rheumatoid arthritis during active disease. The unique expression of the OX-40 molecule may provide a novel therapeutic strategy for eliminating autoreactive CD4+T cells that does not require prior knowledge of the pathogenic autoantigen.

Analysis of T cell receptor beta chains in Lewis rats with experimental allergic encephalomyelitis: conserved complementarity determining region 3.

This study explores the usage of T cell antigen receptor (TCR) beta chain elements in Lewis rats with experimentally induced allergic encephalomyelitis (EAE). TCRs from 15 different T cell clones and hybridomas derived from animals immunized with myelin basic protein (MBP), and all having specificity for the 21-mer encephalitogenic fragment MBP 68-88, utilized V beta 8.2. In addition, there was a marked conservation of the first two amino acid residues of the junctional complementarity determining region 3 (CDR3) associated with the V beta 8.2 receptors. 12 of 15 contained an aspartic acid followed by serine regardless of the associated J beta element. At the nucleotide level, this conservation of AspSer residues was accomplished with few or no nongermline-encoded nucleotide (N) additions. A similar pattern of AspSer usage and N region nucleotide additions was observed in a number of V beta 8.2 isolates derived from MBP-immunized lymph nodes. In contrast, V beta 8.2 polymerase chain reaction amplified isolates from Lewis T cells activated with concanavalin A or from lymph nodes of complete Freund's adjuvant-immunized animals showed no AspSer utilization (0/31) in the CDR3, and four to nine N region nucleotide additions. We conclude from this finding that AspSer residues in the CDR3, limited N region nucleotide additions, along with V beta 8.2 sequences, contribute to TCR specificity for MBP 68-88. This raises the possibility that encephalitogenic, disease-causing T cells either represent a population that derives from late fetal life or alternatively, that they are rare cells with this particular TCR phenotype contributed to the T cell pool throughout adulthood and are selected by antigen. In either case, the CDR3 AspSer sequences as well as V beta 8.2 sequences are candidates for the receptor target structures recognized by regulator T cells in recovery from and resistance to active EAE. In this respect, a preliminary analysis of TCR utilization in three T cell clones specific for MBP 68-88 isolated from animals recovered from active EAE indicates that while all three use V beta 8.2, only one contains AspSer in the CDR3.

Both rat and mouse T cell receptors specific for the encephalitogenic determinant of myelin basic protein use similar V alpha and V beta chain genes even though the major histocompatibility complex and encephalitogenic determinants being recognized are different.

Prospects for specific immune intervention in T cell-mediated autoimmune disease via anti-idiotypic regulation depend on the degree of diversity of the responder cell antigen receptor repertoire. A highly heterogenous response against self epitopes offers little chance for such regulation. We report here that the Lewis rat autoimmune disease experimental allergic encephalomyelitis, generally considered to be a model of human multiple sclerosis, is caused by T cells that use a limited set of TCR V genes. We have cloned the rat TCR alpha and beta chain cDNAs from the Lewis rat x mouse T cell hybridoma 510, which retains the rat specificity for the encephalitogenic determinant of myelin basic protein (MBP). Using Northern blot analysis of T cell RNA with the cloned V region probes, we have found a specific, and near perfect, correlation between expression of TCR message hybridizing to the V alpha 510 and VB510 probes and specificity for the encephalitogenic determinant of MBP in both T cell hybridomas and encephalitogenic T cell clones. This restricted V gene usage provides a basis for observed idiotypic regulation of auto-reactive T cells, and possible therapy for autoimmune disease. A curious and unexplained observation is that the Lewis rat V alpha/V beta combination that dominates the encephalitogenic response to the 68-88 peptide of MBP is precisely the same V alpha/V beta combination used by the B10.PL mouse response to the encephalitogenic response to the 1-9 peptide of MBP.

T cell determinants of myelin basic protein include a unique encephalitogenic I-E-restricted epitope for Lewis rats.

The major encephalitogenic epitope for Lewis rats is the 72-89 sequence of guinea pig basic protein (GP-BP) or rat basic protein (Rt-BP). T cells responsive to this epitope are I-A restricted and preferentially express the V alpha 2:V beta 8 gene combination in their TCR. In this work, we describe for the first time the delayed appearance of T cells specific for additional discrete determinant of BP, the nonencephalitogenic 55-68 sequence of GP-BP restricted by I-A, and the encephalitogenic 87-99 sequence of Rt-BP restricted by I-E. The TCR V alpha 2:V beta 8 gene combination was expressed by both encephalitogenic GP-BP S72-89 and Rt-BP S87-99 T cell specificities but not by GP-BP 44-68-specific T cells. This is the first demonstration of I-E-restricted encephalitogenic T cells in Lewis rats and supports the conclusion that the I-E class II locus is involved in autoimmune diseases.

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.

T cell receptor peptide therapy triggers autoregulation of experimental encephalomyelitis.

Encephalitogenic T cells specific for myelin basic protein share common V beta 8 peptide sequences in their T cell receptor (TCR) that can induce autoregulatory T cells and antibodies that prevent clinical signs of experimental autoimmune encephalomyelitis (EAE). It is not known, however, if TCR peptides can treat established disease. To test its therapeutic value, TCR-V beta 8-39-59 peptide was injected into rats with clinical signs of EAE. This treatment reduced disease severity and speeded recovery, apparently by boosting anti-V beta 8 T cells and antibodies raised naturally in response to encephalitogenic V beta 8+ T cells. These results demonstrate that synthetic TCR peptides can be used therapeutically, and implicate the TCR-V beta 8-39-59 sequence as a natural idiotope involved in EAE recovery. Similarly, human TCR peptides may be effective in enhancing natural regulation of autoreactive T cells that share common V genes.

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.

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.

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.

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.

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.

Lymphocyte vaccination against experimental autoimmune encephalomyelitis: evaluation of vaccination protocols.

Repeated vaccination with encephalitogenic but not other T cell lines could effect marked resistance to 'active' experimental autoimmune encephalomyelitis (EAE) induced by injection of GP-BP in adjuvant. Partial resistance to active EAE was observed in rats recovered from 'passive' line-mediated EAE and in rats vaccinated with T cells attenuated by irradiation or ganglioside treatment. However, no resistance was observed in animals given low doses of activated encephalitogenic T cells. Treatment with hydrostatic pressure alone was found to be ineffective as a means of attenuation, and vaccination with pressure-treated encephalitogenic T cells actually induced mild signs of EAE. However, vaccination with cells that were first pressure treated and then irradiated prevented both clinical and histologic signs of active EAE. In contrast, protection against passive EAE appeared to be clonotypic. Lymphocyte vaccination induced delayed type hypersensitivity (DTH) reactions against autologous T cells, mostly to shared antigens, demonstrating the immunogenicity of multiple antigens on the vaccinating cells.

The synthetic 87-99 peptide of myelin basic protein is encephalitogenic in Buffalo rats.

A synthetic peptide corresponding to residues 87-99 (S87-99) of myelin basic protein (BP) induced the proliferation of an encephalitogenic, BP-specific T cell line selected in vitro from inbred Buffalo-strain rats (RT1b). Active immunization with guinea pig (GP)-BP or S87-99 in complete Freund's adjuvant (CFA) and intravenous pertussigen induced acute experimental autoimmune encephalomyelitis (EAE) 10-12 days after immunization. Fifty percent of recovered rats developed a single relapse 17-21 days after immunization. T lymphocytes selected in vitro with S87-99 transferred acute, non-relapsing EAE into naive recipients. Histological examination during acute EAE revealed foci of inflammatory cells associated with demyelination in the spinal cords and peripheral nerve roots. Thus, as in several other rodent strains, the 87-99 region of BP is antigenic and encephalitogenic in the inbred Buffalo-strain rat. Additionally, the 87-99 sequence of GP-BP was predicted to be antigenic by two different methods. These results suggest that the 87-99 region of BP, which is highly conserved among mammalian species, may be widely encephalitogenic due to antigen-intrinsic properties.

TCR peptide therapy decreases the frequency of encephalitogenic T cells in the periphery and the central nervous system.

The V beta 8 CDR2 consensus peptide, residues 44-54, is highly effective in the treatment of clinical experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To monitor immunological changes during EAE resulting from TCR peptide therapy, the frequencies of encephalitogenic and regulatory T cells were quantitated in lymph nodes, blood, and spinal cord. The frequency of T cells specific for basic protein and its major encephalitogenic epitope, residues 72-89, increased during EAE to about 1 cell per 100,000 lymph node or blood cells at the peak of clinical disease, and then declined. In contrast, the frequency of these T cells in spinal cord was highest, 50 per 100,000, prior to onset of clinical signs, and then decreased rapidly prior to spontaneous recovery. Injection of 100 micrograms of TCR V beta 8-44-54 peptide caused a decrease within 1-5 days in the frequencies of guinea pig basic-protein (GP-BP) and 72-89-reactive T cells in blood and spinal cord, and in the total number of infiltrating cells in spinal cord. In lymph nodes, 72-89-reactive T cells decreased as T cells specific for a protective epitope, residues 55-69 of GP-BP increased, suggesting epitope switching at the site of GP-BP immunization. Conversely, the frequency of T cells specific for the V beta 8-44-54 peptide increased, especially in blood and spinal cord, whereas T cell frequencies to control antigens were unchanged. These data document the critical presence of encephalitogenic T cells within the spinal cord during clinical EAE, and demonstrate that rapid and profound changes in T cell frequencies in the periphery and spinal cord are triggered by TCR peptide therapy.

Delayed type hypersensitivity to gangliosides in the Lewis rat.

Systematic study of the immunologic properties of gangliosides has been hampered by the lack of a suitable assay. In this study, significant delayed type hypersensitivity reactions to gangliosides were observed in Lewis rats immunized with whole guinea pig spinal cord (GP-SC) in complete Freund's adjuvant (CFA). The reaction was manifested by an increase in ear thickness after intradermal injection of a mixture of gangliosides and methylated bovine serum albumin (mBSA). No responses were observed to either gangliosides or mBSA alone. The reaction to gangliosides increased after immunization, persisted for 48 h, and was characterized by perivascular infiltration of mononuclear cells. Further evidence for a cellular response was demonstrated by the transfer of ganglioside-specific ear swelling by cultured spleen cells. The response to gangliosides was not due to contamination with myelin basic protein (BP) since no reaction to gangliosides was observed in GP-BP/CFA-immunized rats, and no reaction to BP was observed in ganglioside/CFA-immunized rats. In BP-immunized rats, responsiveness to BP persisted after recovery from clinical EAE for at least 60 days. However, no response to gangliosides was observed in BP-immunized animals after recovery from clinical EAE, suggesting the lack of autosensitization to gangliosides due to the disease process itself.

Myelin basic protein-specific T cells induce demyelinating experimental autoimmune encephalomyelitis in Buffalo rats.

This is the first description of acute demyelinating experimental autoimmune encephalomyelitis (EAE) induced in rats by myelin basic protein (BP)-specific T lymphocytes without the administration of demyelinating antibodies. BP-specific T cell lines were selected from inbred Buffalo-strain rats (Rt-1b) following techniques used to develop similar lines from Lewis rats (Rt-1l). Unlike those of Lewis rats, the spinal cords of Buffalo rats with T cell line-mediated EAE had prominent perivascular demyelination associated with mononuclear inflammation. Like Lewis rat lines. Buffalo rat BP-specific T cell lines transferred acute, non-relapsing EAE into syngeneic recipients, demonstrating that demyelination in passive acute EAE can occur without subsequent clinical relapses.

Characterization of basic protein-specific T cell lines selected from Lewis rats with relapsing experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by intraperitoneal immunization with guinea pig whole central nervous system tissue. Basic protein (BP)-specific T cell lines selected from rats with relapsing EAE proliferated in response to BP, the 44-89 peptidase fragment of BP and the synthetic peptide, S72-89, as did lines selected from rats with non-relapsing EAE induced by immunization with guinea pig BP. BP-specific T cell lines selected from rats with relapsing EAE transferred acute but not relapsing EAE. BP-specific T cell lines selected from Lewis rats with relapsing EAE appear not to differ from those selected from rats with non-relapsing EAE.

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.

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.