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Clonally related B cells infiltrate the brain, meninges and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients, but the mechanisms driving the B-cell response and shaping the immunoglobulin repertoires remain unclear. Here, we used single-cell full-length RNA-seq and B-cell receptor reconstruction to simultaneously assess the phenotypes, isotypes, constant region polymorphisms, and the paired heavy- and light-chain repertoires in intrathecal B-lineage cells. We detected extensive clonal connections between the memory B cell and antibody-secreting cell (ASC) compartments and observed clonally related cells of different isotypes, including IgM/IgG1, IgG1/IgA1, IgG1/IgG2, and IgM/IgA1. There was a strong dominance of the G1m1 allotype constant region polymorphisms in ASCs, but not in memory B cells. Tightly linked to the G1m1 allotype, we found a preferential pairing of theIGHV4gene family with the κ variable(IGKV)1gene family. These results link IgG constant region polymorphisms to stereotyped B-cell responses in MS, indicating that the intrathecal B-cell response in these patients could be directed against structurally similar epitopes. The data also suggest that the dominance of the G1m1 allotype in ASCs may occur as a result of biased differentiation of intrathecal memory B cells.

We have previously shown that plasmablasts of the G1m1 allotype of IgG1 are selectively enriched in the cerebrospinal fluid of G1m1/G1m3 heterozygous patients with multiple sclerosis, whereas both allotypes are equally used in neuroborreliosis. Here, we demonstrate a strong preference for the G1m1 allotype in the intrathecal humoral immune responses against measles, rubella, and varicella zoster virus in G1m1/G1m3 heterozygous multiple sclerosis patients. Conversely, intrathecally synthesized varicella zoster virus‐specific IgG1 in varicella zoster virus meningoencephalitis comprised both allotypes. This implies that G1m1 B cells are selected to the central nervous system of multiple sclerosis patients regardless of specificity and suggests that an antigen‐independent mechanism could drive the intrathecal humoral immune response. publishedVersion

The mechanisms driving the intrathecal synthesis of IgG in multiple sclerosis (MS) are unknown. We combined high-throughput sequencing of transcribed immunoglobulin heavy-chain variable (IGHV) genes and mass spectrometry to chart the diversity and compartmentalization of IgG-producing B cells in the cerebrospinal fluid (CSF) of MS patients and controls with other neuroinflammatory diseases. In both groups, a few clones dominated the intrathecal IGHV transcriptome. In most MS patients and some controls, dominant transcripts matched the CSF IgG. The IGHV transcripts in CSF of MS patients frequently carried IGHV4 genes and had more replacement mutations compared to controls. In both groups, dominant IGHV transcripts were identified within clusters of clonally related B cells that had identical or related IGHV transcripts in the blood. These findings suggest more pronounced affinity maturation, but an equal degree of diversity and compartmentalization of the intrathecal B-cell response in MS compared to other neuroinflammatory diseases.

Background B cells may contribute to the immunopathogenesis of multiple sclerosis (MS). Dimethyl fumarate (DMF) has recently been shown to reduce the frequency of memory B cells in blood, but it is not known whether the drug influences the cellular composition in the cerebrospinal fluid (CSF). Methods A cross-sectional study examining the cellular composition in blood and cerebrospinal fluid (CSF) from 10 patients treated with DMF and 18 patients receiving other disease modifying drugs or no treatment. Results Patients treated with DMF had reduced proportions of memory B cells in blood compared to other MS patients ( p = 0.0007), and the reduction correlated with treatment duration ( r s = −0.75, p = 0.021). In the CSF, the absolute number of mononuclear cells were significantly lower in DMF-treated patients compared to the other patients ( p = 0.023), and there was a disproportionate decrease of plasmablasts ( p = 0.031). Conclusion The results of this exploratory study support a B-cell mediated mechanism of action for DMF in both blood and CSF.

Epstein-Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS). Here, we used high-throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV-reactivity of the T-cell receptor (TCR) repertoires in MS. TCR-β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T-cell clones, represented by TCR-β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR-β libraries generated from peripheral blood T cells reactive against autologous EBV-transformed B cells were highly enriched for public EBV-specific sequences and were used to quantify EBV-reactive TCR-β sequences in CSF. TCR-β sequences of EBV-reactive CD8+ T cells, including several public EBV-specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV-reactive CD4+ T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T-cell response in MS. The presented strategy links TCR sequence to intrathecal T-cell specificity, demonstrating enrichment of EBV-reactive CD8+ T cells in MS.

In multiple sclerosis (MS), B cells are trafficking across the blood-brain barrier, but it is not known how this relates to the synthesis of oligoclonal IgG. We used quantitative mass spectrometry of oligoclonal bands and high-throughput sequencing of immunoglobulin heavy-chain variable transcripts to study the longitudinal B cell response in the cerebrospinal fluid (CSF) and blood of two MS patients. Twenty of 22 (91%) and 25 of 28 (89%) of oligoclonal band peptides persisted in samples collected 18 months apart, in spite of a dynamic exchange across the blood-CSF barrier of B lineage cells connecting to oligoclonal IgG.

Immunoglobulin gamma (IgG) heavy chain genes are associated with susceptibility to multiple sclerosis (MS) and IgG levels in the cerebrospinal fluid (CSF). However, how these variants are implicated in disease mechanisms remains unknown. Here, we show that proliferating plasmablasts expressing the G1m1 allotype of IgG1 are selectively enriched in CSF of G1m1/G1m3 heterozygous MS patients, whereas plasmablasts expressing either G1m1 or G1m3 are evenly distributed in blood. Moreover, there was a preferential intrathecal synthesis of oligoclonal IgG1 of the G1m1 allotype in heterozygous patients, whereas controls with Lyme neuroborreliosis displayed oligoclonal IgG1 of both allotypes. This points to a disease‐specific mechanism involved in B‐cell establishment within the central nervous system in MS. publishedVersion

Epidemiological data suggest that the Epstein-Barr virus (EBV) is associated with several autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. However, it is not clear whether EBV plays a role in the pathogenesis of these diseases, and if so, by which mechanisms the virus may contribute. In this review, we discuss possible viral and immunological mechanisms that might explain associations between EBV and autoimmune diseases and whether these associations represent causes or effects of inflammation and autoimmunity.

Background: Intrathecal synthesis of oligoclonal IgG antibodies against measles virus (MeV), varicella zoster virus (VZV) and herpes simplex virus type-1 (HSV-1) is a characteristic feature multiple sclerosis (MS). Methods: We have used isoelectric focusing-immunoblot to define the clonal patterns of IgG and of IgG antibodies to MeV, VZV and HSV-1 in supernatants of in vitro cultures of peripheral blood lymphocytes (PBL) and cerebrospinal fluid (CSF) cells and in sera and CSF from three patients with MS and three patients with clinically isolated syndromes (CIS) suspective of demyelinating disease. Results: In vitro synthesis of IgG by PBL was not detected in any patient. In contrast, in vitro synthesis by CSF cells of oligoclonal IgG and oligoclonal IgG antibodies to one or two of the three viruses tested was observed in all six patients. The clonal patterns of the in vitro synthesized IgG and virus specific IgG differed to varying extent from those synthesized intrathecally in vivo. However, in each patient, the in vitro and in vivo intrathecally produced antibodies displayed specificity for the same viruses. The addition of B cell activating factor (BAFF) had no effect on the amounts or clonal patterns of either total IgG or virus-specific IgG produced by CSF cells in vitro. Conclusion: Virus specific B cells capable of spontaneous IgG synthesis are clonally expanded in the CSF of patients with MS. The B-cell repertoire in CSF samples is only partially representative of the intrathecal B-cell repertoire.

Most patients with stiff person syndrome (SPS) display intrathecal synthesis of oligoclonal and high-avidity IgG against the 65 kDa isoform of glutamic acid decarboxylase (GAD65 IgG), but little is known about the mechanisms driving this immune response. We hypothesized that GAD65-specific T cells accumulating in the central nervous system drive the intrathecal GAD65 IgG production. Accordingly, we were able to clone HLA-DR or DP restricted GAD65-specific T cells from the cerebrospinal fluid (CSF) of all three patients with, but not in one patient without substantial intrathecal production of GAD65 IgG. The CSF T cells recognized four GAD65 epitopes, which were unique to each patient. In two patients, identical or closely related GAD65-specific CSF T cell clones were expanded in vivo . In contrast to the findings in CSF, only one GAD65-specific T cell clone could be raised from the blood of one single patient. Cysteine in amino acid position 474, which is important for enzymatic function of GAD65, was critical for recognition of GAD65 474–484 by HLA-DP restricted CSF T cells. We conclude that GAD65-specific T cells and clonally expanded GAD65-specific B cells coexist intrathecally, where they may collaborate in the synthesis of GAD65 IgG.

We previously reported an association between the SH2D2A gene encoding TSAd and multiple sclerosis (MS). Here a total of 2128 Nordic MS patients and 2004 controls were genotyped for the SH2D2A promoter GA repeat polymorphism and rs926103 encoding a serine to asparagine substitution at amino acid position 52 in TSAd. The GA(16)-rs926103()A haplotype was associated with MS in Norwegians (OR 1.4, P=0.04). A similar trend was observed among Danes. In the independent Norwegian, Danish and Swedish sample sets the GA(16) allele showed a combined OR of 1.13, P=0.05. Thus, the present study shows that the SH2D2A gene may contribute to susceptibility to MS.

T cells and B cells are crucial in the initiation and maintenance of multiple sclerosis (MS), and the activation of these cells is believed to be mediated through specific recognition of antigens by the T‐ and B‐cell receptors. The antigen receptors are highly polymorphic due to recombination (T‐ and B‐cell receptors) and mutation (B‐cell receptors) of the encoding genes, which can therefore be used as fingerprints to track individual T‐ and B‐cell clones. Such studies can shed light on mechanisms driving the immune responses and provide new insights into the pathogenesis. Here, we summarize studies that have explored the T‐ and B‐cell receptor repertoires using earlier methodological approaches, and we focus on how high‐throughput sequencing has provided new knowledge by surveying the immune repertoires in MS in even greater detail and with unprecedented depth.

The highly diversified variable regions of immunoglobulin (Ig) molecules contain immunogenic determinants denoted idiotopes. We have previously reported that T cells from multiple sclerosis (MS) patients recognize IgG from autologous cerebrospinal fluid (CSF), and mapped a T-cell epitope to an IgG idiotope. To test the ability of CSF IgG molecules to elicit a broad polyclonal T-cell response in MS, we have analysed T-cell responses in the blood and CSF against idiotope peptides spanning complementarity determining region (CDR) 3 and somatic mutations within the variable regions of monoclonal CSF IgG. Consistent with a diversified idiotope-specific T-cell repertoire, CD4+ T cells from both patients recognized several idiotope peptides presented by HLA-DR molecules. Mutations were critical for T-cell recognition, as T cells specific for a mutated CDR1 peptide did not recognize corresponding germline-encoded peptides. One T-cell clone recognized both an idiotope peptide and the B-cell clone expressing this idiotope, compatible with endogenous processing and presentation of this idiotope by B cells. These results suggest that mutated CSF IgG from MS patients carry several T-cell epitopes, which could mediate intrathecal IgG production and inflammation in MS through idiotope-driven T–B-cell collaboration.

Chromosome region 2q33 encodes several regulators of the immune system, among these the CD28, CTLA4, and ICOS molecules. Involvement of these genes in multiple sclerosis (MS) is not yet clear. We investigated six microsatellites and three SNPs in a relatively large and clinically well characterised Norwegian MS cohort. No associations were observed for any of the markers analysed in 575 MS patients and 551 controls. Associations were neither found when stratifying the material for the HLA-DRB1*1501, DQB1*0602 haplotype, gender, age at onset, disease course nor familial aggregation. In conclusion, this study could not confirm association with the CD28/CTLA4/ICOS gene region.

The association between multiple sclerosis and Epstein-Barr virus infection could involve Epstein-Barr virus-specific T cells, provided that these T cells get access to the intrathecal compartment. We report that CD4+ T cells from the cerebrospinal fluid of six out of six multiple sclerosis patients, and four out of six patients with other neurological diseases, recognized autologous B cells transformed with Epstein-Barr virus. The cerebrospinal fluid T-cell responses were predominantly HLA-DR restricted. These T cells did not recognize B cells activated through stimulation of CD40 or the inducible autoantigen alphaB crystalline. These findings support that the immunological response to Epstein-Barr virus could contribute to the pathogenesis of multiple sclerosis.

We report the first two genome-wide screens for linkage disequilibrium between putative multiple sclerosis (MS) susceptibility genes and genetic markers performed in the genetically homogenous Scandinavian population, using 6000 microsatellite markers and DNA pools of approximately 200 MS cases and 200 controls in each screen. Usable data were achieved from the same 3331 markers in both screens. Nine markers from eight genomic regions (1p33, 3q13, 6p21, 6q14, 7p22, 9p21, 9q21 and Xq22) were identified as potentially associated with MS in both screens.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of unknown etiology. Nitric oxide (NO) is a free radical that participates in a variety of biological processes. It is an important mediator in the immune response. Several studies indicate involvement of NO in the pathogenesis of MS. We studied five markers within the three NO synthase genes with regards to susceptibility and disease course in 156 affected sib-pairs and in 96 “benign” and 96 “severe” definite MS patients and 148 controls. We found no significant association or evidence for linkage in our data sets.

The T cell specific adapter protein (TSAd) encoded by the SH2D2A gene is involved in the control of T cell activation. The gene is located in the 1q21 region, which has been implicated in susceptibility to experimental allergic encephalomyelitis in the mouse. We therefore evaluated whether a polymorphic GA repeat (GA(13)-GA(33)) within the promoter region of the SH2D2A gene shows association to multiple sclerosis (MS). The frequency of the short alleles GA(13-16) was increased among 313 Norwegian MS patients compared to 277 healthy controls (0.332 vs 0.249, OR 1.5, Pc = 0.03). Transmission disequilibrium analysis in 146 Scandinavian families with at least two affected sibs showed increased transmission of GA(16) to MS patients. No linkage or association of MS to four genetic markers flanking the SH2D2A gene was observed. After activation of naive CD4(+) T cells, T cells homozygous for MS associated short alleles displayed lower level of TSAd ex vivo than T cells carrying at least one long allele, which were not associated to MS. Since the SH2D2A protein modulates T cell activation, this may be a mechanism for how short SH2D2A alleles confer susceptibility to develop MS.

The HLA-DR2, DQ6 (i.e., HLA-DRB1*1501, DQA1*0102, DQB1*0602) haplotype contributes to the risk of developing multiple sclerosis (MS) in Caucasoids of Northern European heritage. A correlation between the clinical expression of MS and the presence of HLA-DR2, DQ6 has, however, not convincingly been shown. In this study conventional bivariate analysis and logistic regression analysis were used to study the relationship between HLA-DR2, DQ6 and four disease variables in a cohort of 286 Norwegian MS patients from the Oslo area. Logistic regression analysis showed that HLA-DR2, DQ6 was significantly more frequent among female than male patients ( P =0.0251), and was negatively correlated with age at diagnosis regardless of sex ( P =0.0254). No significant correlation was observed between HLA-DR2, DQ6 and type of disease (relapsing–remitting versus primary chronic progressive MS) or presence/absence of oligoclonal bands in the cerebrospinal fluid.

Background. A single-center study of 655 nonsensitized recipients of primary cadaveric kidney grafts is presented. Results. Graft survival in serologically HLA-DR 1-10 antigen-matched grafts to nonsensitized recipients at 1 year was 90%, compared with 82% (P=0.004) and 73% (P=0.001) in one and two DR antigen-mismatched grafts. The corresponding figures at 5 years were 76%, 62%, and 56%, respectively. Matching for the DR antigens 11-14, or for some DR alleles only detectable by genomic typing, further improved graft survival, but the differences did not reach statistical significance. Matching also for the serologically defined HLA-A and -B antigens did not significantly further improve overall graft survival, but some effects for grafts surviving at least 1 year were observed. Among recipients of grafts mismatched for zero, one, or two HLA-DR antigens, acute rejection episodes were experienced in 48%, 64% (P

The pathogenesis of multiple sclerosis (MS) may involve intrathecal Epstein–Barr virus nuclear antigen-1 (EBNA-1) specific T cells susceptible to modulation by vitamin D. We established EBNA-1 reactive T cell lines from the cerebrospinal fluid (CSF) and blood of three MS patients and cloned EBNA-1 specific CD4+ T cells from two of these. T cell clones from CSF and blood displayed Th1 or Th17 phenotypes and were restricted by HLA-DR molecules, in one patient encoded by the DRB1*0403 or DRB1*1501 haplotypes. 1,25-dihydroxyvitamin D inhibited proliferation and suppressed secretion of IFN-γ and IL-17, irrespective of T cell origin and HLA restriction.

The susceptibility to develop celiac disease (CD) seems to be primarily associated to a particular HLA-DQ α/s heterodimer encoded by the DQA1∗0501 and DOB1∗0201 alleles, in cis position on the DR3-DQ2 haplotype or in trans position by DR5-DQ7/DR7-DQ2 heterozygotes. However, exceptional patients exist who are neither DR3 nor DR5/DR7, particularly among Southern European populations. We therefore examined the DRB1, DQA1, and DQB1 alleles of 13 Spanish CD patients who were serologically typed to be neither DR3 nor DR5/DR7. Five patients were found to carry the DQA1∗0501 and DQB1∗0201 alleles either in cis or in trans position, three of them had previously been serologically mistyped. However, two of these patients carried DQA1∗0501 and DQB1∗0201 on haplotypes other than DR3 or DR5 in combination with DR7. One of the latter patients carried an unusual DR4-DQ2 haplotype, while another had an unusual DR8-DQ2 haplotype. Four of the remaining eight patients carried DR4-DQ8 haplotypes. Taken together, our findings provide further evidence that the DQ α/s heterodimer encoded by the DQA1∗0501 and the DQB1∗0201 alleles confers the primary HLA-associated susceptibility to develop CD. However, our studies also corroborate that a second (and “weaker”) HLA-associated CD susceptibility gene may be present on some DR4-carrying haplotypes.

B cells present idiotopes (Id) from their B cell receptor to Id-specific CD4 + T cells. Chronic Id-driven T-B cell collaboration can cause autoimmune disease in mice. We propose that Id-driven T-B cell collaboration mediates the development of multiple sclerosis by perpetuating immune responses initiated against infectious agents. During germinal centre reactions, B cells express a multitude of mutated Ids. While most mutations lead to decreased affinity and deletion of the B cell, some B cells could be rescued by Id-specific T cells. Such Id-connected T-B cell pairs might initiate inflammatory foci in the central nervous system. This model may explain the intrathecal synthesis of low-avidity IgG against viruses, and the synthesis of oligoclonal IgG with unknown specificity in multiple sclerosis.

Background - The immune system may attack the brain and cause inflammatory disorders like multiple sclerosis (MS). On the other hand, the immune system may protect and support neurons. Methods -There are two obstacles to study this paradox in humans. First, the target antigens in many human central nervous system (CNS) disorders are unknown. Second, it is often difficult to separate pathogenic from protective events, as well as primary from secondary phenomena. Idiopathic stiff person syndrome (SPS) circumvents the first obstacle, because most patients secrete antibodies against glutamic acid decarboxylase (GAD) 65. The immune response against glatiramer acetate (GA) may circumvent the second obstacle. Migration of activated T helper cells to the intrathecal compartment could be a common denominator in GA treatment and SPS. Results - We here discuss recent results on T cells in MS and SPS, showing that GAD65-specific and GA-reactive lymphocytes in the cerebrospinal fluid are not a simple reflection of those in blood. Conclusion - The rules and mechanisms governing T cell selection and maintenance in the CNS may provide a key to the understanding of protective and detrimental aspects of CNS immunity.

Background and purpose: Persistent intrathecal production of IgG autoantibodies against glutamic acid decarboxylase 65 (GAD65 IgG) and oligoclonal IgG of undetermined specificity has been reported in stiff person syndrome (SPS). Methods: To chart the avidity and clonal patterns of GAD65 IgG, we performed scatchard plot of binding characteristics and isoelectric focusing-immunoblot of cerebrospinal fluid (CSF) and serum from five SPS patients. Results: Oligoclonal GAD65 IgG bands, predominantly restricted to the IgG1 subclass, were detected in CSF and serum in all patients. The distribution of GAD65-specific IgG bands in serum and CSF revealed intrathecal synthesis of oligoclonal GAD65 IgG in all five patients, whilst radioimmunoassay demonstrated intrathecal synthesis of GAD65 IgG in four. The binding avidity of GAD65 IgG from CSF was more than 10 times higher than in serum in two of the patients but did not differ substantially in the remaining three. These differences were not related to symptom severity. The pattern of oligoclonal GAD65 IgG bands in CSF and serum in three patients examined remained unchanged for up to 7 years after symptom debut. Conclusion: This study confirms the persistent systemic and intrathecal production of GAD65-specific IgG in SPS, and further shows that this immune response is oligoclonal and mediated by a stable population of affinity maturated B cell clones.

During the last few years, the concept of multiple sclerosis (MS) as a pure inflammatory disease mediated by myelin reactive T cells has been challenged. Neither the specificity nor the mechanisms triggering or perpetuating the immune response are understood. Genetic studies have so far not identified therapeutic targets outside the HLA complex, but epidemiological and immunological studies have suggested putative pathogenetic factors which may be important in therapy or prevention, including the Epstein-Barr virus and vitamin D. Advances in the treatment of MS have been reached by manipulating the immune response where the pathogenesis of MS intersects experimental autoimmune encephalomyelitis, most recently by blocking T-cell migration through the blood-brain barrier. Antigen-specific approaches are effective in experimental models driven by a focused immune response against defined autoantigens, but MS may not fit into this concept. Novel candidate autoantigens which are not constitutively expressed in the brain, such as protein alpha-B crystallin or IgG V-region idiotopes, as well as evidence of pathogenetic heterogeneity and complexity, suggest that treating MS by tolerizing the immune system against an universal MS antigen may be a fata morgana. Further characterization of MS subtypes may lead to individualized treatment. However, shared immunological features, such as intrathecal production of oligoclonal IgG, suggest that potential therapeutic targets may be shared by most MS patients.

Epstein-Barr virus-specific CD4+ T cells could be involved in the pathogenesis of multiple sclerosis, provided they can gain entry to the intrathecal compartment. The authors have previously demonstrated that cerebrospinal fluid T cells from multiple sclerosis patients recognize autologous Epstein-Barr virus-transformed B cells. They now report that CD4+ T cells specific for the Epstein-Barr virus DNA polymerase peptide EBV 627-641 were present in the cerebrospinal fluid from one of two multiple sclerosis patients, and that a high proportion of these CD4+ T cells cross-recognized an immunodominant myelin basic protein peptide, MBP 85-99. In the observed patient, the proportion of EBV 627-641-specific CD4+ T cells seemed to exceed 1/10,000 in cerebrospinal fluid, compared to approximately 1/100,000 in blood. These findings prove that Epstein-Barr-virus specific CD4+ T cells can gain access to the intrathecal compartment, and suggest that Epstein-Barr virus-specific CD4+ T cells could target myelin basic protein in the central nervous system.

MS is associated with genes in the HLA complex. We have previously proposed that the primary HLA-associated susceptibility may be conferred by particular DQ alleles. Furthermore, we have previously found that there is no association of MS to DP alleles. This study shows that MS is not associated to alleles of the TAP2 locus, which is located close to DQ on its centromeric side. This observation provides further support to the notion that HLA-associated susceptibility to MS maps telomeric to the TAP2 locus.

Celiac disease (CD) is a malabsorptive disorder of the small intestine characterized by villous atrophy, hyperplastic crypts and T cell infiltration in the epithelium and in the lamina propria [1]. The disease is caused by an abnormal immune response to gluten, probably initiated by the activation of T cells in the intestinal mucosa to gluten-derived peptides. CD is strongly associated to specific genes in the HLA complex and is a unique model for studies of type 1 diabetes and other HLA associated diseases since (a) the primary HLA associations have been established, (b) the disease-inducing agent (gluten) is known and (c) gluten-specific T cells from the target organ, the intestinal mucosa, are accessible from biopsies for in vitro studies.

Celiac disease (CD) is most probably an immunological disease, precipitated in susceptible individuals by ingestion of wheat gliadin and related proteins from other cereals. The disease shows a strong HLA association predominantly to the cis- or trans-encoded HLA-DQ(alpha1*0501, beta1*02) (i.e., DQ2) heterodimer. T cell recognition of gliadin peptides presented by DQ2 in the intestinal mucosa is central in the immunopathogenesis of CD. Here we describe a study where overlapping peptides from the N-terminal region of alpha-gliadin have been tested in biochemical assays for binding to affinity-purified DQ2 and DR3 (i.e., DR(alpha, beta1*0301)) molecules. The peptides were also tested for binding to DQ2 in a functional binding assay, where binding was measured as the capacity to inhibit the stimulation of a gliadin-specific, DQ2-restricted T lymphocyte clone RNnTalpha33. In both assay systems the overlapping gliadin peptides were found to bind with weak or intermediate affinity to DQ2. No or only very weak binding was found to DR3 in the biochemical binding assay. Overall, the results question the role of these peptides in the T-cell-mediated immunopathogenesis of CD. The in vitro assays described here provide new methods for the screening of potentially toxic peptides.

Objective. To examine the distribution of HLA class II alleles in clinically distinct juvenile rheumatoid arthritis (JRA) subsets. Methods. We typed 298 patients and 181 controls for HLA–DRB1, DQA1, DQB1, and DPB1 alleles using polymerase chain reaction and oligonucleotide probe techniques. Results. Each JRA subset was characterized by a distinct distribution of HLA class II alleles. For the persistently pauciarticular and rheumatoid factornegative polyarticular JRA subsets, certain combinations of DRB1 and DPB1 alleles were characteristic. In patients without antinuclear antibodies and chronic iridocyclitis, there was an increase of DRB1*0101/02 and DQA1*0101. Conclusion. Findings of HLA typing support clinical subdivisions of the disease and suggest the existence of a novel DRB1*0101/02 and DQA1*0101 associated disease subset.

Japanese MS patients and controls were examined for the distribution of HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 alleles using in vitro amplification of genomic DNA and probing with sequence-specific oligonucleotides. No significant difference in frequency of the examined alleles was observed among the two groups. This is in contrast to Norwegian MS patients, where an association to a combination of certain DQA1 and DQB1 alleles has previously been demonstrated.

Genomic typing of in vitro amplified DNA with sequence-specific oligonucleotide (SSO) probes was performed for DRB1, DQA1, DQB1, DPA1 and DPB1 alleles in 54 random Norwegian rheumatoid arthritis (RA) patients and 181 healthy controls. DRB1 alleles encoding the serological specificity DR4 were found in 80% of the patients, compared to 34% of the controls (relative risk = 7.9, p less than 0.0001). All DR4-positive RA patients carried either DRB1*0401 (Dw4), 0404 (Dw14), or 0405 (Dw15), while no patients were found to carry DRB1*0402 (Dw10) or 0403 (Dw13). The frequency of the DRB1*0101 allele encoding DR1 was not increased, even among DR4-negative RA patients, and we were unable to detect any sharing of other class II alleles among DR4-negative patients. No contribution of any DQA1, DQB1, DPA1 or DPB1 alleles to RA susceptibility could be detected. The results suggest that in the Norwegian population RA is primarily associated with a shared sequence at residues 67-74 of the DR beta 1 chain, but only when this sequence is expressed on DR4 molecules.

Of 61 Norwegian multiple sclerosis patients tested, 59, i.e., 97%, were positive for at least one of the HLA specificities DR2, DR4, or DRw6. Typing with sequence-specific oligonucleotide probes revealed that the same 59 patients carried DR2-, DR4-, or DRw6-associated HLA-DQB1 genes which encode shared polymorphic amino acid sequences in the membrane-distal part of their HLA-DQB chains. This shared DQB polymorphism may explain previously reported DR associations and could thus be the primary HLA association in MS.

An imprint electroimmunofixation method (IEIF) was used to characterize antibodies to eight viral antigens (measles, mumps, rubella, herpes simplex type 1, varicella-zoster, vaccinia, cytomegalovirus, adenovirus) and four bacterial antigens (beta-hemolytic streptococcus, Hemophilus influenzae type B, Escherichia coli, enterococcus) in serum and cerebrospinal fluid (CSF) of 12 patients with multiple sclerosis (MS). Twelve patients matched for age and sex sex served as controls. Evidence for intrathecal synthesis of oligoclonal antibodies to one or more antigens was found in all 12 MS patients and in 1 of the controls. In the MS group, antibodies to viruses with neurotropic properties were more frequently associated with local synthesis than antibodies to other viruses and bacteria. The types and number of locally synthesized antibodies showed no correlation with disease duration and severity. The antibodies were not associated with oligoclonal CSF IgG and appear to account for only a minor fraction of the locally synthesized CSF IgG in MS.

Here we summarize some of our own most recent studies on the moloecular basis of the HLA associations in insulin-dependent diabetes mellitus, multiple sclerosis and coeliac disease. Secondly, the functional importance for T cell activation of some of these disease-associated HLA polymorphisms will be treated

Oligoclonal IgG was present in the cerebrospinal fluid (CSF) of each of three patients with neurosyphilis studied. Conventional serological tests disclosed reduced serum/CSF ratios of antibodies to Treponema pallidum (TP) in each patient, consistent with intrathecal production of treponemal antibodies. Antibody analyses of electrofocused specimens by an immunofixation technique disclosed intrathecal production of oligoclonal TP antibodies in all patients. Treponemal antibody light chains showed a close correlation with the light chains of the oligoclonal CSF IgG. Absorption of the CSF with TP caused removal of the oligoclonal IgG. The results provide strong evidence that the oligoclonal CSF IgG in neurosyphilis represents TP antibodies, reflecting a specific immune response in the central nervous system to the infectious agent.

Varicella-zoster (VZ) virus meningoencephalitis was diagnosed in a 72-year-old man without other clinical signs of VZ infection, on the basis of intrathecal virus-specific IgG, IgA and IgM antibody responses demonstrated by imprint immunofixation (IIF) and by serological analyses of serum and CSF. The intrathecally produced antibodies displayed oligoclonal characteristics. The intrathecal production of VZ-IgG and -IgA antibodies persisted throughout the observation period of 20 months, while that of VZ-IgM antibodies was not detectable later than 3 months after onset. Part of the intrathecally produced VZ-IgG and -IgA antibody populations, but no IgM antibodies, were shown to cross-react with herpes simplex virus. Oligoclonal IgG bands were demonstrated in the CSF throughout the observation period. The bulk of the IgG bands was shown to represent VZ-specific antibodies.

Intrathecal production of oligoclonal mumps-specific IgG was demonstrated in nine out of 10 children with mumps meningitis by imprint immunofixation (IIF) of sera and cerebrospinal fluids (CSF) separated by agarose electropheresis and by thin-layer electrofocusing. Four of the patients had intrathecal mumps antibody synthesis demonstrable also by conventional serological tests. Oligoclonal CSF IgG was demonstrable by agarose electrophoresis in four of the patients. A dominance of λ over κ type oligoclonal Ig and mumps antibodies was observed in the CSF of three of these patients. The bulk of the oligoclonal CSF IgG was concluded to represent mumps-specific antibodies on the basis of the IIF as well as virus absorption analysis. Intrathecal production of oligoclonal IgG antibodies to one, two, or three other (measles, rubella, herpes simplex) viruses was demonstrated by IIF in four patients. These antibodies were not associated with the oligoclonal CSF IgG present in three of the patients. It is concluded that a specific intrathecal IgG antibody response is a common feature in children with mumps meningitis. This response sometimes reaches a magnitude that permits detection of oligoclonal IgG in the CSF. In some patients, the specific response appears to be associated with a non-specific activation of cells producing antibodies of other (unrelated) specificity.

The terminal complement complex (TCC) was determined in plasma and cerebrospinal fluid (CSF) from 208 neurological patients. Elevated CSF TCC levels were observed in higher frequencies in patients with infectious diseases (80%), radiculoneuritis (62%), multiple sclerosis (30%), and miscellaneous autoimmune diseases (27%) than in patients with miscellaneous non-inflammatory diseases (2–13%). The plasma level of TCC was significantly increased only in the infectious group. No positive correlation was observed between the plasma and the CSF TCC concentration in the whole patient population nor in subgroups divided according to blood-brain barrier function. Furthermore, the CSF TCC concentration did not correlate with the serum/CSF albumin ratio or with CSF total protein concentration when this was below 1.0 g/l. It is concluded that an elevated TCC concentration in CSF reflects intrathecal complement activation and that quantification of TCC in CSF may be a valuable supplement in the examination of neurological diseases.

Epstein-Barr virus (EBV) transformed B-cells from patients with multiple sclerosis (MS) and healthy controls were analysed for reactivity with the HLA-DQ-specific human-human hybridoma Ab TrB12 (anti-DQw6 + DQw8 + DQw9) by indirect immunofluorescence (IIF). Positive results were obtained with 34 out of 35 MS patients (97.1%) and 79 out of 106 controls (74.5%) (p less than 0.005, RR = 11.6). Thus, DQ molecules that express the TrB12 epitope may contribute to the susceptibility to develop MS.

We have studied 87 unrelated Caucasian insulin-dependent diabetes mellitus (IDDM) patients and 181 healthy controls by oligotyping for 20 DRB1, eight DQA1 and 13 DQB1 alleles, and established their DR-DQ haplotypes and DQ genotypes. An increase of DRB1 alleles encoding DR4 was found among IDDM patients, but the distribution of DR4 subtypes did not differ among DR4-positive IDDM patients and controls. The frequency of certain DRB1-DQA1-DQB1 haplotypes and DQA1-DQB1 genotypes was significantly increased among IDDM patients. Taken together, the data suggest that IDDM is primarily associated with several (at least five) different DQ alpha beta heterodimers.