Jia-Bin Sun

T Cells Recognizing Multiple Peptides of Myelin Basic Protein are Found in Blood and Enriched in Cerebrospinal Fluid in Optic Neuritis and Multiple Sclerosis

The cause of multiple sclerosis (MS) is unknown. Recently reported abnormal T‐cell responses to several myelin proteins and myelin basic protein (MBP) peptides in peripheral blood constitute one line of evidence that autoimmune mechanisms could be involved in the pathogenesis of the disease. Monosymptomatic unilateral optic neuritis (ON) is a common first manifestation of MS and important to examine for a possible restriction of the T‐cell repertoire early in the disease. T‐cell activities to MBP and the MBP amino acid sequences 63–88, 110–128 and 148–165 were examined by short‐term cultures of mononuclear cells from cerebrospinal fluid (CSF) and blood in the presence of these antigens, and subsequent detection and counting of antigen‐specific T cells that responded by interferon‐gamma (IFN‐γ) secretion. Most patients with MS and ON had MBP and MBP peptide‐reactive T cells in CSF, amounting to mean values of between about 1 per 2000 and 1 per 7000 CSF cells and without immunodominance for any of the peptides. Numbers were 10‐fold to 100‐fold lower in the patients′ blood. Values were similar in ON and MS, and no evidence was obtained for a more restricted T‐cell repertoire in ON. The MBP peptide‐recognizing T‐cell repertoire was different in CSF than in blood in individual patients with ON and MS, thereby giving further evidence for an autonomy of the autoimmune T‐cell response in the CSF compartment. No relations were observed between numbers of autoreactive T cells and presence of oligoclonal IgG bands in CSF or abnormalities on magnetic resonance imaging of the brain in ON or clinical variables of MS. The high numbers of MBP and MBP peptide‐reactive T cells could play a role in the pathogenesis of ON via secretion of effector molecules, one of them being IFN‐γ, as well as in the transfer of ON to MS.

Autoreactive T and B cell responses to myelin antigens after diagnostic sural nerve biopsy

To study whether nervous tissue trauma provokes myelin antigen autoreactive T and B cell responses in humans we examined consecutive blood samples from 7 patients with polyneuropathy undergoing diagnostic sural nerve biopsy and 8 control patients undergoing other types of minor surgery. The antigen-specific T cells were assessed by enumerating cells secreting interferon-gamma (IFN-gamma) in response to the myelin components P0, P2, myelin basic protein (MBP) and myelin associated glycoprotein (MAG), and to 4 selected MBP peptides. B cell mediated immunity was assessed by counting numbers of cells secreting antibodies directed against the myelin proteins. On day 7 after biopsy, there were 3-10-fold increased numbers of T and B cells reactive with P0, P2, MBP and MAG in blood of polyneuropathy patients compared to controls, while levels of cells recognizing purified protein derivate or responding to phytohemagglutinin (PHA) did not differ significantly. Comparison of prebiopsy levels on day 0 with post-biopsy levels on day 7 in the polyneuropathy patients revealed a significant increase in T cells recognizing P0, P2 and MAG, and in B cells secreting IgG antibodies against P0 and P2. On day 14 after nerve biopsy these differences were no longer seen. We suggest that in patients with polyneuropathy, sural nerve biopsy with the ensuing wallerian degeneration and myelin breakdown causes transiently increased levels of circulating myelin autoreactive T and B cells. It remains to be determined if this has a physiological role in nerve trauma responses and/or affects the clinicopathological course of the peripheral neuropathy.

Lyme neuroborreliosis: evidence for persistent up-regulation of Borrelia burgdorferi-reactive cells secreting interferon-gamma.

The T‐cell response to the aetiologic pathogen Borrelia (B.) burgdorferi in patients with Lyme neuroborreliosis (LN) and in control patients with other neurological diseases was examined by enumerating B. burgdorferi‐reactive T cells secreting interferon‐γ (IFN‐γ) with an ELIspot assay. LN patients had elevated numbers of B. burgdorferi‐reactive IFN‐γ secreting cells in blood and approximately 20‐fold enriched in the cerebrospinal fluid (CSF). A positive correlation existed in CSF between B. burgdorferi‐reactive IFN‐γ secreting cells and B cells secreting anti‐B. burgdorferi IgG antibodies. The up‐regulation of antigen‐specific IFN‐γ secreting cells persisted in peripheral blood up to at least 9 months and in the CSF for at least 4 months after termination of treatment with antibiotics, when the patients were mostly free from clinical signs and symptoms due to LN. How IFN‐γ interplays with other cytokines and influences the pathogenesis of LN remains to be studied.

Immunopharmacologic modulation of experimental allergic encephalomyelitis: low-dose cyclosporin-A treatment causes disease relapse and increased systemic T and B cell-mediated myelin-directed autoimmunity.

Therapies with immunosuppressive drugs in autoimmune experimental diseases often down‐regulate disease but sometimes may lead to paradoxical disease exacerbation. To elucidate possible mechanisms behind such phenomena the effects were studied of mitoxantrone (Mx) and cyclosporin A (CsA) given at high and low doses on clinical course, and on autoreactive T‐ and B‐cell responses in actively induced experimental allergic encephalomyelitis (EAE) in Lewis rats. Treatment with mx and high dose CsA abrogated EAE and decreased dramatically the measured immune responses compared to vehicle‐treated control EAE rats. Low‐dose CsA treatment caused a disease relapse 20–30 days post immunization (p.i.). This relapse was accompanied by increased numbers of cells spontaneously producing IFN‐γ in the CNS and regional lymph nodes. Furthermore, anti‐myelin and anti‐MBP secreting cells were increased as were numbers of primed T cells that produced IFN‐γ in response to myelin antigens. It was concluded that these aspects of the myelin autoreactive immune response correlated well with clinical disease and are useful in evaluating immunotherapeutic intervention. Low‐dose CsA treatment may interfere with systemic down‐regulatory mechanisms acting on both T‐ and B‐cell myelin‐directcd autoimmunity.

The T-Cell Repertoire in Myasthenia Gravis Involves Multiple Cholinergic Receptor Epitopes

Antibodies against the α‐subunit of the acetylcholine receptor (AChR) are found in most patients with myaslhenia gravis and are considered to contribute to the receptor damage which leads to the characteristic signs and symptoms of the disease. This B‐cell response is T‐cell driven. Elevated T‐cell reactivities to AChR and its α‐subunit have been described in myasthenia gravis, and AChR α‐subunit peptide reactive T‐cell lines and clones preferentially recognizing certain defined sequence segments have been reported, thereby disclosing the possibility of specific immunotherapy. We have defined the T‐cell repertoire to AChR, its α‐subunit and the synthetic peptide sequences 1OO‐117,113‐130,143‐163,161‐179,207‐225,221‐240, and 235‐255 of the α‐subunit in an immunospot assay which is based on secretion of interferon‐gamma (IFN‐γ) by individual memory T cells upon stimulation with specific antigen in short‐term cultures. Most patients with myasthenia gravis displayed T‐cell reactivities to 1 to 6 different peptides. The mean numbers of T cells recognizing individual peptides varied in the myasthenia gravis patients between 1 per 77,000 and 1 per 167,000 peripheral blood mononuclear cells. None of the seven peptides evaluated could be identified as an immunodominant T‐cell epitope, and any of them was found to dominate in individual patients. The numbers of T cells reacting with AChR and recombinant human AChR α‐subunit were slightly higher (mean numbers 1 per 26,000 and 1 per 50,000 mononuclear cells, respectively). Such cells, as well as AChR α‐subunit peptide reactive T cells, were also found in patients with other neurological diseases and in healthy subjects, but at lower frequencies and numbers. In myasthenia gravis, the elevated numbers of memory T cells recognizing multiple AChR α‐subunit peptides may be crucial for the development of the disease, and the IFN‐γ released by such T cells might be important for its perpetuation.

Myasthenia gravis: T and B cell reactivities to the β-bungarotoxin binding protein presynaptic membrane receptor

Antibodies against acetylcholine receptor (AChR) can be detected in most patients with myasthenia gravis (MG) and are considered to be involved in the immunopathogenesis of this disease. AChR are isolated from crude receptor preparations by binding to alpha-bungarotoxin (alpha-BuTx). Patients with MG have also antibodies against a second protein tentatively named presynaptic membrane receptor (PsmR), which has been isolated from crude receptor utilizing beta-bungarotoxin (beta-BuTx). PsmR could represent another antigen besides AChR relevant for the development of MG. We have now evaluated the T cell reactivity to PsmR in MG and controls by analysing the frequencies of cells which in response to PsmR in short-term cultures secreted interferon-gamma (IFN-gamma). The B cell response to PsmR was analysed in parallel by counting cells secreting anti-PsmR antibodies. Most patients with MG had PsmR reactive T cell in blood with a median number of about 1 per 44,000 mononuclear cells. Cells secreting anti-PsmR antibodies belonging to the IgG and IgA isotypes, less frequently of the IgM isotype were detected in most MG patients. A positive correlation was found between T cells reactive with PsmR and anti-PsmR IgG antibody secreting cells. PsmR reactive T and B cells were also detected in control patients, but at much lower numbers. Our results indicate that MG is accompanied by T as well as B cell responses to PsmR, in addition to the previously recognized responses to AChR. It remains to be shown whether these PsmR reactivities are of pathogenetic importance in MG.

Optic neuritis and multiple sclerosis: the T cell repertoires to myelin proteins and MBP peptides change with time.

Autoreactive T cells recognizing myelin basic protein (MBP), proteolipid protein (PLP) and MBP peptides have been described in multiple sclerosis (MS) and optic neuritis (ON), but their role in disease pathogenesis, if any, is unknown. A consistency of the T cell repertoire over the course of MS and ON should facilitate the development of specific immunotherapies. We have examined the T cell responses to autoantigens in two consecutive blood specimens taken from patients with ON and MS, and in two consecutive CSF specimens obtained from ON patients. As read‐out numbers of T cells responding to antigen stimulation by the secretion of interferon‐γ were estimated. Pronounced differences in occurrence and numbers of T cells recognizing MBP, MBP peptides with the amino acid sequences 63–88, 110–128 and 148–165, and PLP were noticed in individual ON and MS patients over the course of disease. The MBP peptide among those three included, that was predominantly recognized by T cells in the individual patient, also varied over the course. The quantitative and qualitative changes of the myelin antigen‐specific T cell response in MS and in ON, the latter to a certain extent reflecting the situation in early MS, do not favor the future useful development of specific immunotherapies in these diseases.

Multiple sclerosis: occurrence of myelin basic protein peptide‐reactive T cells in healthy family members

Genetic factors influence the susceptibility to multiple sclerosis (MS). This disease is accompanied by augmented T cell responses to CNS myelin components such as myelin basic protein. To evaluate the familial occurrence of such T cell autoreactivity, we have studied 12 MS families including 37 healthy first‐degree relatives for occurrence of numbers of interferon‐gamma (IFN‐γ) secreting cells among blood mononuclear after culture in presence of myelin basic protein (MBP), eight synthetic MBP peptides and the control antigen acetylcholine receptor (AChR). There were no differences between MS patients and healthy family members regarding frequencies of autoreactive T cells recognizing MBP, the eight different MBP peptides or AChR. None of the MBP peptides predominated as T cell antigen among the MS patients or their unaffected family members. In some families the highest number of MBP peptide reactive T cells were found among unaffected family members. No correlation was observed between numbers of MBP or MBP peptide reactive T cells in various subjects and their HLA‐DR‐DQ phenotypes. In conclusion, this study has revealed the presence of MBP and MBP peptide reactive T cells of similar frequencies in MS patients and their healthy family members.

Cord blood contains cells secreting antibodies to nervous system components

Umbilical cord blood of newborns and peripheral blood or healthy adults were investigated by an immunospot assay for cells secreting IgG, IgA and IgM antibodies against myelin basic protein (MBP), proteolipid protein (PLP), myelin‐associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) which represent putative antigens for an autoimmune attack in multiple sclerosis (MS) and against acetylcholine receptor (AChR) which is considered an important autoantigen in myasthenia gravis. Cells secreting antibodies against one or more of these autoantigens were detected in 18 out of 24 newborns, and in eight out of 20 adults. Eight of the cord blood samples contained cells secreting antibodies of IgG, IgA and/or IgM isotypes to one antigen, five to two antigens, two to three antigens, two to four antigens, and one to five antigens. Most prominent were anti‐MBP IgG antibody secreting cells which were detected in 13 newborns at a mean number of 1/20000 cord blood cells, and in six adults at a mean number of l/105 peripheral blood cells. Anti‐AChR IgG antibody secreting cells were detected in four out of 12 newborns versus four out of 14 peripheral blood specimens, at mean values of 1/105 cells in both instances. Cells secreting autoantibodies of IgA and IgM isotypes were less frequent both in cord blood and peripheral blood. The occurrence of nervous tissue autoantibody secreting cells in newborns must be related to a possible primary role of such autoantibodies in MS and myasthenia gravis.