Myelin Basic Protein Epitope Research Articles

The myelin basic protein-specific T cell repertoire in Lewis rats: T cell receptor diversity is influenced both by intrathymic milieu and by extrathymic peptide presentation.

In the Lewis rat, the T lymphocyte response to guinea pig myelin basic protein (MBP) is focused almost exclusively on epitopes nested in the MBP peptide sequence p68-88, and is dominated by T cell receptors (TCR) using Vbeta8.2 gene elements, together with short N(D)N regions. Here we analyzed MBP-specific TCR from Lewis T cells differentiating in chimeric thymuses of Lewis rat/SCID mouse chimeras, in the absence of an intact rat thymic microenvironment (SCID(FL) mice). In these T cells, the TCR Vbeta repertoire is broad, N(D)N regions are significantly longer, and contain regular rates of template-independent N nucleotides. In striking contrast, a Vbeta8.2 biased TCR repertoire and few N-region inserts are seen in p68-88-specific, Lewis rat-derived T cells differentiating in the complete rat thymic microenvironment provided by chimeric SCID mice bearing embryonic Lewis thymus grafts (SCID(FL/FT) mice). A T cell repertoire resembling the one in SCID(FL) mice is used by T cells of intact Lewis rats following immunization with a truncated epitope of MBP, p69-86. Also this selection generates a broad TCR Vbeta pattern with long N(D)N regions, and higher numbers of N nucleotides. These results show that both intrathymic repertoire selection, and extrathymic peptide priming exert profound effects on the TCR usage in the anti-MBP response of Lewis rats.

Hierarchy in the ability of T cell epitopes to induce peripheral tolerance to antigens from myelin.

Nasal administration of peptide antigens has been shown to induce T cell tolerance. We have investigated the potential for peptide therapy of the autoimmune response to myelin antigens in experimental autoimmune encephalomyelitis (EAE). Three major encephalitogenic epitopes were studied for their ability to induce nasal tolerance to myelin antigens. These included epitopes Ac1-9 and 89-101 of myelin basic protein (MBP) and 139-151 from proteolipid protein (PLP). Peptide Ac1-9 from MBP effectively suppressed responses to both MBP epitopes, following immunization with whole myelin (linked suppression). The N-terminal epitope failed, however, to modify the response to epitope 139-151 of PLP. The second MBP epitope (89-101) was poorly tolerogenic for the immune response to any naturally processed myelin epitope. By contrast, PLP[139-151] was able to induce bystander suppression of T cells responsive to both itself and the two epitopes from MBP. Furthermore, this epitope suppressed EAE induced with peptides derived from MBP and was capable of treating ongoing disease. The mechanism of bystander suppression, mediated by PLP[139-151], did not correlate with an overt switch from the T helper 1 to the T helper 2 phenotype. These results demonstrate how a complex autoimmune disease may be controlled by treatment with a single peptide epitope and reveal a hierarchy in the suppressive properties of different myelin antigens.

Effects of vaccination with T cell receptor peptides: epitope switching to a possible disease-protective determinant of myelin basic protein that is cross-reactive with a TCR BV peptide.

Immunization of Lewis rats with guinea-pig myelin basic protein (Gp-MBP) induced T cell responses to primary and secondary encephalitogenic determinants, as well as to a third non-encephalitogenic epitope, residues 55-69. This sequence is of interest due to its protective activity against experimental autoimmune encephalomyelitis. Protection involved induction of MBP-55-69-specific T cells expressing cross-reactive TCR BV8S6 genes that activated regulatory T cells specific for TCR BV8S2 determinants expressed on encephalitogenic T cells. We here present and discuss new evidence suggesting a possible immunological cross-reactivity between the protective Gp-MBP-55-69 peptide and the regulatory BV8S2-39-59 peptide. This cross-reactivity, which may also occur between the human MBP-55-74 peptide and the BV12S2-38-58 sequence, has potentially important implications for human diseases such as multiple sclerosis.

Relapsing and remitting experimental allergic encephalomyelitis: a focused response to the encephalitogenic peptide rather than epitope spread.

The progression of experimental allergic encephalomyelitis (EAE) in certain mouse strains has been reported to involve a broadening of the response to myelin antigens, apparently resulting from priming to endogenous determinants of the myelin sheath. The phenomenon has been termed determinant spread. Interest in this effect has centered on the mechanism it offers to explain the progressive, relapsing and remitting course of EAE and indeed of multiple sclerosis. We have conducted a systematic, longitudinal study in SJL mice to look for determinant spread during relapsing and remitting EAE, correlating epitope recognition and cytokine production with disease severity. Disease was induced using three of the four encephalitogenic proteolipid protein or myelin basic protein epitopes, and responses to each of four epitopes recognized by SJL T cells were tracked through acute disease, remission and relapse. The responses of lymph node cells, splenocytes and central nervous system (CNS)-infiltrating T cells were analyzed. While marginal, transient responses to secondary epitopes were detectable in splenocytes, CNS-infiltrating cells showed a dominant response to the original disease-inducing epitope without evidence of a shift to other determinants during relapse. Disease relapse was correlated with an increase in CNS-infiltrating cells and a high proliferative and interferon (IFN)-gamma response to the disease-inducing peptide. During remission, there was a decrease in numbers of cells infiltrating the CNS. These cells were down-regulated, showing low if any response to the myelin peptides tested as measured by proliferation, production of IFN-gamma or production of IL-4. Our findings argue strongly against a causal role for determinant spread in disease relapse as observed in these models of EAE.

Interstrain variability of autoimmune encephalomyelitis in rats: multiple encephalitogenic myelin basic protein epitopes for DA rats

We investigated T cell epitopes of guinea pig myelin basic protein (MBP) that induce experimental autoimmune encephalomyelitis (EAE) in DA rats, using synthetic peptides that correspond to regions of the guinea pig MBP molecule that are homologous to rat MBP. Four peptides were encephalitogenic when tested in DA rats. MBP63–81, which partially overlaps the dominant encephalitogenic MBP epitope for Lewis (LEW) rats, caused severe EAE in the DA strain but did not elicit EAE in LEW rats. MBP66–81 and MBP63–76 were also encephalitogenic for DA but not LEW rats. MBP79–99 also induced EAE in DA rats, although MBP87–99, the minor encephalitogenic LEW epitope, was inactive. This indicates that part of the 79–86 sequence is necessary for encephalitogenic activity in the DA strain. MBP101–120, and MBP142–167 were also encephalitogenic for DA rats. T cells from DA rats immunized with intact MBP proliferated in response to the whole protein and to MBP79–99, but were not stimulated to a significant extent by the other encephalitogenic peptides, suggesting that these may represent cryptic or subdominant epitopes. However, MBP63–81-specific T cell lines could be isolated by repeated restimulation with peptide, indicating that the peptide-specific T cells were present in DA rats at low frequency.

Differential expression of cyclic nucleotide phosphodiesterase 3 and 4 activities in human T cell clones specific for myelin basic protein.

Abstract Little is known concerning the relative distribution and function of the different cyclic nucleotide phosphodiesterases (PDEs) in lymphocytes. Recent reports, however, have indicated that specific PDE4 inhibitors were effective in treatment of experimental allergic encephalomyelitis, an animal model of multiple sclerosis. The therapeutic effect of PDE4 inhibitors is thought to be related to inhibition of autoreactive CD4+ T cells specific for myelin basic protein (MBP) or other myelin proteins. Human autoreactive CD4+ T lymphocyte clones (TCC), specific for the immunodominant MBP epitope (amino acids 83-99), contain PDE3 and PDE4, two PDEs that exhibit a high affinity for cAMP. Amplification of TCC mRNA by reverse transcription-PCR indicated that TCC PDE3 mRNA was of the PDE3B, not PDE3A, subtype. Different TCC contained different proportions of PDE3 and PDE4, and their activities increased during Ag (MBP) stimulation. Specific PDE3 (cilostamide) and PDE4 (rolipram) inhibitors suppressed [3H]thymidine incorporation in TCC. Since it is believed that many autoimmune diseases are at least partially mediated by autoreactive CD4+ T cells, these observations may have important implications not only for the treatment of multiple sclerosis but also for other autoimmune diseases.

Immunodominance of a low-affinity major histocompatibility complex-binding myelin basic protein epitope (residues 111-129) in HLA-DR4 (B1*0401) subjects is associated with a restricted T cell receptor repertoire.

The pathogenesis of multiple sclerosis (MS) is currently ascribed in part to a T cell-mediated process targeting myelin components. The T cell response to one candidate autoantigen, myelin basic protein (MBP), in the context of HLA-DR15Dw2, has been previously studied in detail. However, the characteristics of cellular immunity in the context of other MS-associated HLA-DR haplotypes are scarcely known. MBP-specific T cell lines (TCL) were generated from HLA-DR4 (B1*0401)-positive MS subjects. Out of 275 MBP-specific TCL, 178 (64. 7%) specifically recognized region MBP(111-129), predominantly in the context of DRB1*0401. The major T cell epitope for MBP recognition corresponded to residues MBP(116-123). These TCL expressed disparate profiles of cytokine secretion and cytotoxicity. T cell receptor analysis, on the other hand, revealed a strikingly limited heterogeneity of rearrangements. In contrast to MBP(81-99), which binds with high affinity to HLA-DR15 and is recognized by a diverse T cell repertoire, MBP(111-129) binds weakly to DRB1*0401, suggesting that only high affinity T cell receptors might be able to efficiently engage such unstable MHC/peptide complexes, thus accounting for the T cell receptor restriction we observed. This study provides new insight about MBP recognition and proposes an alternative mechanism for immunodominance of self-antigen T cell epitopes in humans.

Binding of copolymer 1 and myelin basic protein leads to clustering of class II MHC molecules on antigen-presenting cells.

Copolymer 1 (Cop 1), a synthetic copolymer of amino acids, effective in suppression of experimental allergic encephalomyelitis (EAE) and myelin basic protein (MBP), was shown to bind extensively and promiscuously to the class II MHC molecules on antigen-presenting cells (APC) without prior processing. In the case of human APC, binding has earlier been demonstrated to DR but not DQ or class I molecules. In the present study, we examined whether binding of Cop 1 and MBP affects MHC class II expression on the cell membrane. Biotinylated derivatives of these antigens were used to monitor their direct binding to MHC molecules on living APC by flow cytometry using phycoerythrin-streptavidin, while the levels of MHC surface expression were monitored by staining with FITC-conjugated anti-class I- and class II-specific antibodies. When Cop 1 or MBP were incubated with the APC, intensity of cell staining with anti-DR, but not with anti-DQ or anti-class I antibodies, was significantly increased, compared to the staining of control APC not reacted with these antigens. In contrast, staining intensity was unaffected when p84-102, a human immunodominant epitope of MBP, or ovalbumin (OVA), a protein which undergoes proteolytic degradation prior to binding, were incubated with the APC. Cycloheximide, a protein synthesis inhibitor, had no effect on the enhanced staining intensity with anti-DR antibody of cells treated with Cop 1 or MBP, whereas it inhibited the enhanced staining of both DR and DQ molecules caused by the respective antibodies, in the absence of these antigens. Brefeldin A, a protein transport inhibitor, lowered the levels of staining intensity with anti-DR and anti-DQ antibodies in both cases, with and without antigen added to the APC. Fluorescence microscopic analysis revealed that cells incubated with Cop 1 or MBP, but not with p84-102 or OVA, exhibit both bright staining of the cell membrane and clusters produced by the aggregation of DR molecules with these antigens. Taken together, these observations indicate that Cop 1 and MBP, due to their polyvalent character, lead to increased fluorescence intensity of their complexes with HLA-DR, possibly due to recruitment and clustering of previously synthesized DR molecules. This can explain the efficient binding of these antigens to the MHC class II molecules.

Inhibition of nitric oxide synthase for treatment of experimental autoimmune encephalomyelitis.

Aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase, prevented the clinical development of experimental autoimmune encephalomyelitis (EAE) with a reduction in inflammation and demyelination. Administration of AG reduced the expression of nitrosotyrosine in inflammatory lesions in the central nervous system. Cytokine expression, determined by semiquantitative PCR, revealed increased expression of IFN-gamma, IL-10, and TGF-beta, which was associated with protection from EAE, and reduced TNF-alpha, associated with the development of EAE. Furthermore, AG blocked the secretion of nitric oxide, TNF-alpha, and PGE2 in astrocyte cultures. AG did not influence the proliferation response of T cells to a pathogenic epitope of myelin basic protein. Down-regulation of nitric oxide by AG has widespread consequences for cytokine production in central nervous system inflammation and prevents EAE.

Suppression of experimental allergic encephalomyelitis in the Lewis rat, by administration of an acylated synthetic peptide of myelin basic protein

A Myelin Basic Protein (MBP) epitope encephalitogenic for the Lewis rat (amino acid residues 68–86) was synthesized and acylated by the attachment of a palmitoyl residue. Lewis rats treated intravenously (i.v.) with the palmitoylated peptide alone were better protected against clinical manifestations of experimental allergic encephalomyelitis (EAE) than rats treated with the peptide inserted into liposomes or with the native peptide at similar doses. The administration of the acylated peptide (PAL68 86) conferred excellent protection against a challenge with the encephalitogenic peptide (p68–86) or with the intact MBP molecule, both before and after induction of active disease, and also when administered to recipients after the transfer of lymphocytes from MBP-challenged donors. Histological manifestations were also reduced to a statistically significant degree. Treatment with a palmitoylated peptide from a non-encephalitogenic region of the MBP molecule (PAL44–62) or with a palmitoylated unrelated peptide were ineffective. In vitro Ag-specific proliferative responses as well as the ability to transfer disease to syngeneic recipients, by lymph node lymphocytes from PAL68–86-treated donors, were considerably reduced. Addition of IL-2 to these cultures failed to restore either Ag-specific responsiveness or the ability of the cells to transfer disease. The results suggest that the administration of acylated peptides induces a profound state of unresponsiveness, and thus may provide an effective means for treating T cell-mediated autoimmune inflammatory disorders.

Prior exposure to superantigen can inhibit or exacerbate autoimmune encephalomyelitis: T-cell repertoire engaged by the autoantigen determines clinical outcome

Experimental allergic encephalomyelitis (EAE) is inducible in experimental animals immunized with myelin basic protein (MBP), proteolipid protein (PLP) or their peptides. We compared T-cell responses to encephalitogenic epitopes of PLP(43–64) and MBP(Ac1–11) in a single mouse strain, (PL/J × SJL)F1. MBP(1–11)-specific T-cell hybridomas expressed predominantly TCR Vβ8 or Vβ4, while PLP(43–64)-specific hybridomas expressed a diverse TCR repertoire. To analyze the biologic significance of the TCR repertoire (limited vs. diverse) to disease susceptibility, we pretreated mice with a superantigen (SEB), and then induced disease with these autoantigens. Mice injected with SEB and immunized with MBP(Ac1–11) showed significant inhibition of EAE, whereas SEB-pretreated mice immunized with PLP(43–64) had an increased severity of EAE and developed a chronic disease. These data demonstrate that prior exposure to microbial superantigens can significantly alter the autoimmune disease course depending upon the TCR repertoire used by the autoantigen.

A study of human T-cell lines generated from multiple sclerosis patients and controls by stimulation with peptides of myelin basic protein

We generated T-cell lines from the peripheral blood of controls and of patients with multiple sclerosis (MS) by stimulation with overlapping synthetic peptides representing the entire sequences of all four isoforms of human myelin basic protein (MBP). The T-cell lines reacted to a wide range of epitopes in the major isoforms of MBP and to epitopes that were present only in the minor isoforms. Many MS patients and controls had T-cells responding to one or more cryptic MBP epitopes, as indicated by the generation of a peptide-specific T-cell line(s)by stimulation with synthetic peptides but not by stimulation with whole MBP. About one-third of the peptide-generated lines were cytotoxic. Although we have shown that this technique of peptide stimulation is effective in generating human antiviral cytotoxic CD8 + T-cell lines, all the cytotoxic MBP-specific lines generated by this method were predominantly CD4 +. Our study did not reveal any significant differences, between MS patients and controls, in reactivity to epitopes within any of the isoforms of MBP.

Human T lymphocytes specific for the immunodominant 83-99 epitope of myelin basic protein: Recognition of golli MBP HOG 7

Myelin basic protein (MBP) is a candidate autoantigen in the disease multiple sclerosis. Although MBP was thought to be sequestered behind the blood-brain barrier, isoforms of MBPs have recently been demonstrated in lymphoid tissues. These isoforms, termed golli MBPs, contain sequences that are shared with "classic" MBP within the CNS. In the present study, we have determined that epitopes within golli MBP isoforms may be recognized by human T lymphocyte clones specific for classic MBP. Ten of 12 T-cell clones recognized golli MBP. Although 11 clones were specific for the immunodominant 83-99 sequence, the clones differed with respect to human leukocyte antigen (HLA) restriction, T-helper phenotype, cytolytic activity, and T-cell receptor usage. Greater responses to classic MBP than to golli MBP suggested a difference in the ability of the two proteins to be processed and to present epitopes therein. These data advance the hypothesis that golli MBP sequences expressed within lymphoid tissues may be recognized by classic MBP-specific T lymphocytes during central or peripheral tolerance.

Male SJL mice do not relapse after induction of EAE with PLP 139-151.

SJL mice immunized with proteolipid protein (PLP) develop relapsing experimental autoimmune encephalomyelitis (R-EAE). R-EAE is a CD4+, Th1 cell-mediated demyelinating disease of the central nervous system (CNS) that is used as a model for the human disease multiple sclerosis (MS). Previous studies showed that young (< 8 weeks) male SJL mice were resistant to active induction of EAE with CNS homogenate, while female mice were susceptible. We have recently observed that young male SJL mice immunized with a major encephalitogenic peptide of myelin, PLP 139-151, developed initial clinical and histological symptoms of EAE with a severity similar to age-matched females; however, unlike females, male mice did not relapse. Significant T cell proliferation to PLP 139-151, but not to other PLP and myelin basic protein (MBP) epitopes, was observed in both males and females during the initial episode, recovery, and first relapse of clinical disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of lymphokine mRNA revealed differences in IFN-gamma and IL-4 synthesis consistent with the hypothesis that Th2 T cells develop in young male SJL mice that regulate the relapsing phase of the disease. These data suggest that immunization of young male SJL mice with PLP 139-151 overrides a defect in antigen presentation responsible for the previously observed resistance to EAE, and that natural processing and presentation of neuroantigens during the course of acute EAE induces Th2 cells that prevent the relapse of disease.

The major myelin protein genes are expressed in the human thymus.

Demyelinating diseases, such as multiple sclerosis (MS) in man or experimental allergic encephalomyelitis (EAE) in rodents, may include an associated immune response directed against myelin protein antigens such as the proteolipid protein (PLP) and the myelin basic protein (MBP). Development of an immune response has been attributed, in part, to the sequestration of central nervous system antigens behind the blood-brain barrier. Recently, we identified a novel gene, the golli gene, which overlaps the mbp gene. The Golli transcription unit produces a family of mRNAs, and their corresponding proteins possess MBP epitopes known to be encephalitogenic in EAE. Transcription of the golli gene was detected in immune system tissue. Therefore, we wished to determine whether genes that encode the two major myelin protein components, PLP and MBP, were expressed in the human thymus. Our data demonstrate that both the plp and golli genes are transcribed in the fetal human thymus. Moreover, both the PLP and DM-20 transcripts are produced from the plp gene, and the HOG 7 and HOG 5 transcripts are produced from the golli gene. Confocal fluorescent immunohistochemistry using antibodies for the PLP/DM-20 and Golli proteins, co-localized expression of these antigens to thymic macrophages. Thus, the plp and golli genes are expressed, and their corresponding protein produced, in an antigen presenting cell in the human immune system.

Encephalitogenic T cells are present in Lewis rats protected from autoimmune encephalomyelitis by coimmunization with MBP73-84 and its analog.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) that is mediated by T helper 1 (Th1) CD4+ T cells. Lewis rats can be protected from actively induced EAE by coimmunization with the encephalitogenic myelin basic protein (MBP) epitope 73-84 and its single alanine-substituted analog 1028. Although analog 1028 cannot induce either active or passive EAE, it does elicit a Th1-like response that is cross-reactive with MBP73-84. Analog 1028 can effectively inhibit clinical EAE in a dose-dependent manner when rats are coimmunized with the encephalitogenic peptide MBP73-84 and 1028 in complete Freund adjuvant (CFA). Stimulation of cells from MBP73-84:1028-coimmunized protected rats proliferate and secrete interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in vitro in response to MBP73-84. Furthermore, coimmunized protected rats harbor a population of MBP73-84-reactive potentially encephalitogenic T cells, because splenocytes from these rats can be stimulated to transfer passive EAE to naive recipients. Thus, the protection of coimmunized rats by analog 1028 is not due to the inhibition of priming of MBP73-84-reactive T cells or alteration of the cytokine secretion profile of the MBP73-84-reactive cell population. Rather, MBP73-84-reactive potentially encephalitogenic T cells are primed in these protected animals.

The myelin basic protein‐specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment

In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68-88, and use the V beta 8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15-16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B1), and none by I-Ad of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68-88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of V beta genes without a bias for V beta 8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of V beta 8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68-88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.

Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein.

Following induction of experimental encephalomyelitis with a T-cell clone, L10C1, that is specific for the myelin basic protein epitope p87-99, the inflammatory infiltrate in the central nervous system contains a diverse collection of T cells with heterogeneous receptors. We show here that when clone L10C1 is tolerized in vivo with an analogue of p87-99, established paralysis is reversed, inflammatory infiltrates regress, and the heterogeneous T-cell infiltrate disappears from the brain, with only the T-cell clones that incited disease remaining in the original lesions. We found that antibody raised against interleukin-4 reversed the tolerance induced by the altered peptide ligand. Treatment with this altered peptide ligand selectively silences pathogenic T cells and actively signals for the efflux of other T cells recruited to the site of disease as a result of the production of interleukin-4 and the reduction of tumour-necrosis factor-alpha in the lesion.

OX-40 antibody enhances for autoantigen specific V beta 8.2+ T cells within the spinal cord of Lewis rats with autoimmune encephalomyelitis.

The V beta 8.2 T cell receptor (TCR) component is the predominant V beta gene product associated with antigen specific CD4+ T cell response to the major encephalitogenic epitope of myelin basic protein (MBP) in Lewis rats. Lewis rats were actively immunized with MBP in complete Freund's adjuvant and the V beta 8.2 positive and negative cells were analyzed for IFN-gamma mRNA production and OX-40 cell surface expression during the onset of EAE. The V beta 8.2+ T cells isolated from the spinal cord produced the majority of mRNA for IFN-gamma and also showed a marked enhancement for OX-40 expression compared to V beta 8.2+ T cells isolated from the lymph nodes. Only a fraction of IL-2 receptor positive T cells examined ex vivo from the inflammatory compartments co-expressed the OX-40 antigen. These results suggested that OX-40 cell surface expression could be used to identify and isolate the most recently activated T cells ex vivo. OX-40+ T cells isolated from the spinal cord were highly enriched for the V beta 8.2 T cell receptor component compared to OX-40- or unsorted spinal cord lymphocytes. OX-40+ T cells isolated from the spinal cord had an enhanced response to MBP, whereas OX-40+ cells isolated from the lymph nodes responded to both MBP and purified protein derivative. These data suggest that activated T cells can be isolated and characterized with the OX-40 antibody which only respond to the antigens present at the local site. The data also imply that isolation of OX-40+ T cells will be useful in identifying V beta biases and autoantigen specific cells within inflamed tissues even when the antigen specificity is unknown.

Administration of myelin basic protein synthetic peptides to multiple sclerosis patients

A double blind Phase 1 clinical research project was conducted in vivo in multiple sclerosis (MS) patients to determine the effect of myelin basic protein (MBP) synthetic peptides on free (F) and bound (B) titers of anti-MBP in cerebrospinal fluid (CSF). Intrathecal administration of peptide MBP75–95, either as a single dose, or as repeated injections for periods up to 10 weeks, produced complete binding-neutralization of F anti-MBP with no change in B levels. A control peptide MBP35–58 had no effect on F and B anti-MBP levels. Intravenous administration of MBP75–95 resulted in significant decline of F and B CSF anti-MBP levels over a period of one month. Administration of MBP synthetic peptides to MS patients either intrathecally or intravenously did not have any adverse neurological effects and systemic complications did not occur. The MBP epitope for MS anti-MBP was further localized to an area between Pro85 and Pro96.