Longitudinal single-cell cytokine responses reveal recurrent autoimmune myelin reactivity in relapsing--remitting multiple sclerosis patients.

The relationship between multiple sclerosis (MS) disease activity and myelin protein-induced cytokine responses over time is not elucidated. We addressed this relationship by examining longitudinal cytokine responses to myelin proteins every three months for one year, in the context of gadolinium (gad)-enhancing brain lesions and of clinical relapses. The ELISPOT assay was used to determine the ex vivo cytokine production in response to nine amino acid long peptides spanning the entire proteolipid protein (PLP) and myelin basic protein (MBP) molecules in relapsing-remitting (RR) MS patients and matched healthy controls. We identified three longitudinal levels of myelin-induced cytokine secretion by adding up the positive responses for all PLP or MBP peptides obtained for five timepoints, at three-month intervals: low reactivity (< 200 cumulative cytokine-secreting cells), isolated peptide reactivity (201-450 cumulative cytokine-secreting cells) and recurrent protein-wide bursts of cytokine reactivity (> 451 cumulative cytokine-secreting cells). The majority of MS patients showed recurrent bursts to PLP and MBP. In contrast, controls showed a more even distribution between all levels of cytokine reactivity. The majority of patients with gad-enhancing lesions showed PLP/IFN gamma and MBP/IFN gamma recurrent burst responses. This is the first longitudinal study on MS patients in which nine amino acid long myelin peptides are used to reveal the broad range of PLP- and MBP-peptide cytokine reactivity across the whole molecule of these two major myelin proteins. This study also reveals the extremely dynamic nature of the immune reactivity to numerous regions of myelin, which can fluctuate dramatically over time. Such fluctuation could hamper the efficacy of antigen-based therapies for MS.

Immunomodulatory effects of interferon beta-1a in multiple sclerosis.

Several studies have established a role for interferon beta (IFNbeta) as a treatment for relapsing-remitting multiple sclerosis (MS). IFNbeta has been reported to decrease the relapse rate, relapse severity, progression of disability and development of new brain lesions. Its mechanisms of action, however, remain unclear. We hypothesize that immunomodulatory effects of IFNbeta may underlie its clinical efficacy. We used intracellular cytokine flow cytometry to analyze the effects of IFNbeta-1a on expression of the anti-inflammatory cytokine, IL-10, and its effects on major co-stimulatory molecules in MS patients. We found that peripheral blood mononuclear cells (PBMC) produced more IL-10 following in vitro or in vivo treatment with IFNbeta-1a. The primary cellular sources of IL-10 were monocytes and CD4(+) T lymphocytes. IL-10 production in response to IFNbeta-1a was increased in unseparated PBMC compared to purified lymphocyte cultures, indicating that interaction between monocytes and lymphocytes may influence IL-10 production in response to IFNbeta-1a. Using flow cytometry, we monitored the ex vivo expression of two major co-stimulatory pairs-B7/CD28 and CD40/CD40L-before and after intramuscular IFNbeta-1a treatment of MS patients. IFNbeta-1a lowered the expression of B7.1 on circulating B cells and increased B7.2 expression on monocytes. CD40 expression on B cells was down-regulated, but CD40 on monocytes was up-regulated by IFNbeta-1a treatment. These data suggest that co-stimulatory molecules are modulated by IFNbeta, providing a possible mechanism for its in vivo immune regulatory effects.

Quantification of self-recognition in multiple sclerosis by single-cell analysis of cytokine production.

Identifying and quantifying autoaggressive responses in multiple sclerosis (MS) has been difficult in the past due to the low frequency of autoantigen-specific T cells, the high number of putative determinants on the autoantigens, and the different cytokine signatures of the autoreactive T cells. We used single-cell resolution enzyme-linked immunospot (ELISPOT) assays to study, directly ex vivo, proteolipid protein (PLP)-specific memory cell reactivity from MS patients and controls. Overlapping 9-aa-long peptides, spanning the entire PLP molecule in single amino acid steps, were used to determine the frequency and fine specificity of PLP-specific lymphocytes as measured by their IFN-gamma and IL-5 production. MS patients (n = 22) responded to 4 times as many PLP peptides as did healthy controls (n = 22). The epitopes recognized in individual patients, up to 22 peptides, were scattered throughout the PLP molecule, showing considerable heterogeneity among MS patients. Frequency measurements showed that the number of PLP peptide-specific IFN-gamma-producing cells averaged 11 times higher in MS patients than in controls. PLP peptide-induced IL-5-producing T cells occurred in very low frequencies in both MS patients and controls. This first comprehensive assessment of the anti-PLP-Th1/Th2 response in MS shows a greatly increased Th1 effector cell mass in MS patients. Moreover, the highly IFN-gamma-polarized, IL-5-negative cytokine profile of the PLP-reactive T cells suggests that these cells are committed Th1 cells. The essential absence of uncommitted Th0 cells producing both cytokines may explain why therapeutic strategies that aim at the induction of immune deviation show little efficacy in the established disease.

T cell recognition of myelin proteolipid protein and myelin proteolipid protein peptides in the peripheral blood of multiple sclerosis and control subjects.

Myelin proteolipid protein (PLP) is a prime candidate autoantigen for multiple sclerosis. In order to define potential immunodominant epitopes, T cell lines (TCL) from the peripheral blood of HLA-DR 15(2) MS patients were established which responded to the intact molecule of PLP. These TCL were then tested in individual proliferation assays with a variety of PLP peptides spanning most of the PLP molecule. Multiple peptides were recognized by TCL from the MS population, with more than one peptide often recognized by lines from the same individual. Three immunodominant peptides were identified which were recognized by the majority of MS patients. Estimated frequency analyses were then performed on the peripheral blood of HLA-DR15(2)-positive MS and control subjects using TCL initiated by the three immunodominant peptides, 40-60, 95-117, and 185-206. TCL from HLA-DR15 MS subjects recognized peptide 95-117 significantly more often than TCL from control subjects.

CD8+ myelin peptide-specific T cells can chemoattract CD4+ myelin peptide-specific T cells: importance of IFN-inducible protein 10.

The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is due, in part, to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. While it is thought that the inflammatory and demyelination process in MS is the product of Th1-associated cytokines secreted by CD4+ myelin protein-specific T cells present in the CNS, the mechanisms that are responsible for the recruitment and maintenance of these myelin-reactive CD4+ T cells in the CNS have not been elucidated. We have shown previously that CD8+ CTL that recognize peptides derived from sequences of the myelin proteolipid protein (PLP) presented by HLA class I molecules can be generated in vitro, and that these PLP-specific CD8+ CTL secrete the proinflammatory chemokines macrophage-inflammatory protein-1alpha and -1beta, IL-16, and IP-10. In this study, we demonstrate that soluble products of these PLP-specific CD8+ CTL can chemoattract CD4+ T cells that are specific for a myelin basic protein peptide and a PLP peptide, and that the majority of this chemotactic activity is mediated by IFN-inducible protein 10. These results demonstrate that PLP-specific CD8+ T cells can play a role in the recruitment and retention of myelin-derived peptide-specific CD4+ T cells, and indicate that they may play a proinflammatory role in the pathogenesis of MS.

VLA-4 expression on peripheral blood lymphocytes is downregulated after treatment of multiple sclerosis with interferon beta.

Adhesion molecules are likely to play a critical role in the immunopathogenesis of multiple sclerosis (MS). The interaction of vascular cell adhesion molecule-1 (VCAM-1) with its lymphocyte ligand very late antigen-4 (VLA-4) may mediate migration of lymphocytes into the CNS. We have previously demonstrated that MS patients treated with interferon beta (IFN-beta) have a significant increase in soluble VCAM-1 (sVCAM-1) soon after the initiation of treatment, and this effect correlated with the resolution of contrast-enhancing MRI lesions. We studied the cell surface expression of VLA-4 by flow cytometry in 10 MS patients before and during IFN-beta treatment. We found a significant decrease in mean VLA-4 fluorescence of MS patients' lymphocytes on treatment and no change in untreated controls. In vitro treatment of lymphocytes with IFN-beta did not reproduce this effect, but the addition of sVCAM-1 did result in a decrease in VLA-4 expression. These data indicate that the previously identified increase in sVCAM-1 may lead to a decrease in VLA-4 expression and that this effect may partially explain the mechanism of action of IFN-beta.

HPRT mutant T-cell lines from multiple sclerosis patients recognize myelin proteolipid protein peptides.

Mutation of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in a T-cell is believed to be an indication that the T-cell has been activated and has proliferated in vivo. HPRT mutant T-cell lines were generated from peripheral blood mononuclear cells from patients with MS and control subjects. More lines were isolated from the MS patients than from the control subjects. Using stringent criteria for recognition, none of the lines from MS-affected or control subjects recognized intact myelin basic protein (MBP) or myelin proteolipid protein (PLP) molecules. Using stringent criteria, two of the 10 MS patients harbored mutant lines each recognizing distinct PLP peptides (PLP peptide 40-60 recognized by 3 lines from one patient and PLP peptide 178-191 recognized by 2 lines from the other patient). A single line recognizing PLP peptide 89-106 was derived from 1 of 7 normal controls. HPRT mutant lines recognizing multiple epitopes of PLP which spanned much of the molecule could be isolated from MS patients, and to a lesser extent, normal subjects.

T cell response to two immunodominant proteolipid protein (PLP) peptides in multiple sclerosis patients and healthy controls.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system in which autoimmune T lymphocytes reacting with myelin antigens are believed to play a pathogenic role. Since HLA binding is involved in the selection of T cell responses, we have examined PLP peptide binding to HLA DR2, an HLA allele frequently found in MS patients. Both PLP 40-60 and PLP 89-106 show significant, high affinity binding to HLA DR2. We then tested whether responses to PLP peptides 40-60 and 89-106 are elevated in multiple sclerosis patients compared to matched controls. We also analysed T cell responses to MBP 87-106, which is considered to be the immunodominant region of MBP in humans. Here we demonstrate heterogenous T cell responses to PLP 40-60, PLP 89-106 and MBP 87-106 in both MS patients and controls. The overall number of TCL and the HLA restriction of those TCL did not vary significantly in the two groups. PLP 40-60 specific cytolytic TCL were increased in MS patients, whereas healthy controls had increased percentages of cytolytic TCL responding to PLP 89-106 and MBP 87-106. Although the data presented here shows heterogenous responses in T cell numbers, differences in numbers and specificity of cytolytic cells could be involved in the pathogenesis of autoimmune demyelinating disease.

Purification of immunologically active recombinant 21.5 kDa isoform of human myelin basic protein.

We have designed and expressed in bacteria a recombinant fetal form of human myelin basic protein (21.5 kDa isoform; rhMBP21.5), a candidate autoantigen in multiple sclerosis. An exon 2 insertion, carboxy-terminal histidine tag and preferred bacterial codons differentiate the MBP21.5 gene from that encoding the adult, brain-derived form of human MBP (18.5 kDa isoform; hMBP18.5). MBPs were expressed at high levels in E. coli and extracted from whole cells by simultaneous acid solubilization and mechanical disruption. A nearly two-fold increase in recombinant protein was detected in strains harboring MBP genes with bacterial preferred codons compared to genes containing human codons. The recombinant molecules were purified in two steps, first by reversed-phase chromatographic separation and then by metal affinity chromatography. Dimeric forms of recombinant MBP21.5 were detected under physiological conditions, however, substitution of a serine for the single cysteine at amino acid residue 81 resulted in only monomer formation. All forms of recombinant MBPs induced proliferative responses of human T lymphocytes specific for epitopes in MBP18.5 kDa. In contrast, human T cell lines that recognize an exon 2-encoded epitope of MBP responded to the 21.5 kDa isoform of MBP, but not the 18.5 kDa isoform.

Two mechanisms of antigen-specific apoptosis of myelin basic protein (MBP)-specific T lymphocytes derived from multiple sclerosis patients and normal individuals.

Several stimuli induce mature T cells to undergo apoptosis or programmed cell death (PCD) including specific Ag. We have demonstrated previously that Ag induces the death of encephalitogenic T cells in vitro and deletion in vivo, leading to amelioration of autoimmune encephalomyelitis. We have now examined whether activated, myelin basic protein (MBP)-specific human T cells may be eliminated by Ag-induced PCD. We demonstrate that activated MBP-specific T cell lines (TCL) undergo the classic nuclear morphologic changes and DNA fragmentation characteristic of apoptosis when given a TCR challenge. We found evidence that two mechanisms led to apoptosis: a propriocidal mechanism that was highly Ag-specific and dependent on the dose of exogenously added rIL-2, and a cytolytic mechanism in which MBP-specific TCL lysed B cell targets and engaged in considerable "fratricidal" cytolysis of other MBP-specific T cells. These two pathways leading to MBP-specific apoptotic death could be distinguished by their glucocorticoid sensitivity. Glucocorticoid treatment significantly blocked MBP-induced propriocidal apoptosis but had no effect on T cell cytolysis of B cell targets. Although it has been proposed that autoimmune disease could result from the failure of normal deletional mechanisms, this preliminary survey of MBP-reactive mature TCL from multiple sclerosis patients revealed that such cells are highly susceptible to TCR-induced PCD and comparable with TCL from normal subjects. Thus, therapeutic strategies based on Ag-induced PCD of T lymphocytes may be feasible in man.

Identification of a second T cell epitope of human proteolipid protein (residues 89-106) recognized by proliferative and cytolytic CD4+ T cells from multiple sclerosis patients.

Research into the pathogenesis of multiple sclerosis (MS) has focused on myelin antigens as potential targets of autoimmune attack. Proteolipid protein (PLP) is the most abundant myelin protein comprising more than 50% of central nervous system myelin. Although PLP is a hydrophobic membrane protein which has made it difficult to study, the use of synthetic peptides based on the PLP sequence provides an alternative method for studying the immunological properties of PLP. Using peripheral blood lymphocytes from MS patients, long-term TCL established in the presence of PLP reacted weakly to PLP in proliferation assays; however, these same lines were much more reactive to synthetic peptides of PLP. Thus, we established short-term T cell lines (TCL) from the peripheral blood lymphocytes (PBL) of MS patients in the presence of five separate synthetic PLP peptides. In 6/7 MS patients, proliferative responses were elicited most often to PLP 40-60 compared to four other PLP peptides (PLP 89-106, 103-120, 125-143, and 139-154) (Pelfrey et al., 1993). Interestingly, however, the magnitude of the proliferative response was greatest in response to PLP 89-106. Characterization of PLP 89-106-responsive TCL from several MS patients, indicated that TCL proliferating to the peptide also lysed PLP 89-106 pulsed autologous targets. The majority of cytolytic PLP 89-106 TCL were CD4+ and MHC class II restricted and the predominant restriction elements were those most commonly found in MS patients. These findings suggest that the use of synthetic peptides represents a viable alternative approach to the study of PLP reactivity in humans. We report here that MS PBL recognize several PLP peptides, with the predominant responses to PLP 40-60 and PLP 89-106. Since these cells have both helper (CD4+) and cytolytic capabilities, it is possible that they may play a role in the pathogenesis or progression of MS.

Identification of a novel T cell epitope of human proteolipid protein (residues 40-60) recognized by proliferative and cytolytic CD4+ T cells from multiple sclerosis patients.

Research into the pathogenesis of multiple sclerosis (MS) has focused on myelin antigens as potential targets of autoimmune attack. Proteolipid protein (PLP), which makes up more than 50% of central nervous system myelin, is a hydrophobic membrane protein with many properties that historically have made it difficult to study. The use of synthetic peptides based on the PLP sequence provides an alternative method for studying the immunological properties of PLP. Using peripheral blood lymphocytes from MS patients, long-term TCL established in the presence of PLP reacted weakly to PLP in proliferation assays; however, these same lines were much more reactive to synthetic peptides of PLP. Thus, we established short-term T cell lines (TCL) from the peripheral blood lymphocytes (PBL) of MS patients in the presence of five separate synthetic PLP peptides. In six out of seven MS patients, proliferative responses were elicited most often to PLP 40-60 compared to four other PLP peptides (PLP 89-106, 103-120, 125-143, and 139-154). Characterization of PLP 40-60-responsive TCL from a single MS patient, MS1, indicated that six out of seven TCL proliferating to the peptide also lysed PLP 40-60 pulsed autologous targets. All cytolytic PLP 40-60 TCL were CD4+ and MHC class II restricted and further analysis of MS1 TCL showed that the PLP 40-60 TCL were restricted by DR4 whereas the MBP TCL from MS1 were restricted by DR6. These findings suggest that difficulties in examining the immune response to PLP have been due to the poor response generated in vitro using the whole molecule and that the use of synthetic peptides may represent an alternative approach to the study of PLP. These results also suggest that MS PBL recognize several PLP peptides, with the predominant response to PLP 40-60. Since these cells phenotypically resemble T cells known to mediate experimental autoimmune encephalomyelitis, it is possible that they may play a role in the pathogenesis of MS.

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins.

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.

Genetic resistance in experimental autoimmune encephalomyelitis. I. Analysis of the mechanism of LeR resistance using radiation chimeras.

Experimental autoimmune encephalomyelitis (EAE) is a cell-mediated autoimmune disease of the central nervous system that has been extensively studied in the rat. The Lewis rat is highly susceptible to the induction of EAE, while the Lewis resistant (LeR) rat is known to be resistant. In this paper, we demonstrate that the LeR rat, which was derived from the Lewis strain by inbreeding of fully resistant animals, is histocompatible with the Lewis strain. Radiation chimeras, a tool for distinguishing between immunologic and nonimmunologic resistance mechanisms, were utilized to analyze the cellular mechanisms involved in genetic resistance to EAE. By transplanting bone marrow cells from LeR rats into irradiated Lewis recipients, Lewis rats were rendered resistant to EAE induction. Likewise, transplanting Lewis bone marrow cells into irradiated LeR recipients rendered LeR rats susceptible. Mixed lymphoid cell chimeras using bone marrow, spleen, and thymus cells in Lewis recipient rats revealed individual lymphoid cell types and cell interactions that significantly affected the incidence and severity of EAE. Our results suggest that LeR resistance is mediated by hematopoietic/immune cells, and that cells located in the spleen appear to play a critical role in the resistance/susceptibility to EAE induction. Depletion of splenic adherent cells did not change the patterns of EAE resistance. In vivo cell mixing studies suggested the presence of a suppressor cell population in the LeR spleen preparations which exerted an inhibitory effect on Lewis autoimmune responses. Thus, the mechanism of LeR resistance appears to be different from that in other EAE-resistant animals.