The immunogenetics of multiple sclerosis.

The discoveries in the 1970s of strong associations between various diseases and certain human leukocyte antigen (HLA) factors were a revolution within genetic epidemiology in the last century by demonstrating for the first time how genetic markers can help unravel the genetics of disorders with complex genetic backgrounds. HLA controls immune response genes and HLA associations indicate the involvement of autoimmunity. Multiple sclerosis (MS) was one of the first conditions proven to be HLA associated involving primarily HLA class II factors. We review how HLA studies give fundamental information on the genetics of the susceptibility to MS, on the importance of linkage disequilibrium in association studies, and on the pathogenesis of MS. The HLA-DRB1*1501 molecule may explain about 50% of MS cases and its role in the pathogenesis is supported by studies of transgenic mice. Studies of polymorphic non-HLA genetic markers are discussed based on linkage studies and candidate gene approaches including complete genome scans. No other markers have so far rivaled the importance of HLA in the genetic susceptibility to MS. Recently, large international collaborations provided strong evidence for the involvement of polymorphism of two cytokine receptor genes in the pathogenesis of MS: the interleukin 7 receptor alpha chain gene (IL7RA) on chromosome 5p13 and the interleukin 2 receptor alpha chain gene (IL2RA (=CD25)) on chromosome 10p15. It is estimated that the C allele of a single nucleotide polymorphism, rs6897932, within the alternative spliced exon 6 of IL7RA is involved in about 30% of MS cases.

Graft rejection after hematopoietic cell transplantation with nonmyeloablative conditioning.

Graft rejection after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning is a rare but serious clinical problem. Graft rejection and salvage therapy in eight patients in a retrospective analysis of 124 consecutive patients is reported. The patients were conditioned with low-dose fludarabine and total body irradiation (TBI). The association of pretransplantation risk factors with rejection and the effect of chimerism and graft-versus-host disease on rejection were analyzed. Overall survival (OS) and progression free survival (PFS) were compared between patients with and without rejection. Retransplantation was performed with increased TBI conditioning for all patients, and with increased mycophenolate mofetil doses for recipients with HLA-identical sibling donors. No known pretransplantation risk factors were confirmed in this study. Rejection episodes were unevenly distributed over time. The storage temperature of the apheresis products was identified as a risk factor for rejection. Storage of the apheresis products at 5 degrees C diminished the risk of rejection. Low donor T cell chimerism at Day +14 significantly increased the risk of rejection. Seven patients were retransplanted. All but one engrafted successfully, but with decreased OS and PFS. Two patients received pentostatin infusion prior to donor lymphocyte infusions in unsuccessful attempts at reversing rejection. Storage temperature and donor chimerism had a significant effect on rejection. Following rejection, patients are at greater risk of dying from infections and progression/relapse of their malignancy. Retransplantation is feasible and well tolerated after HCT with nonmyeloablative conditioning and should be performed without delay in patients with imminent and manifest graft rejection.

C1q deficiency in an Inuit family: identification of a new class of C1q disease-causing mutations.

C1q deficiency is a rare condition associated with a systemic lupus erythematosus (SLE)-like syndrome and recurrent infections. Here we present the molecular basis behind C1q deficiency in three sisters of Inuit origin. Initial examination for complement deficiency showed no function of the classical complement activation pathway in the patients; the lectin and alternative pathways were intact. No C1q or low molecular weight C1q was detected in sera and no anti-C1q autoantibodies were found. Sequencing of the C1q genes revealed a novel missense mutation (Gly-Arg) in codon 217 of the B chain. All sisters were homozygous for the mutation: both parents were heterozygous. None of 100 healthy controls carried the mutation. Our findings define a third class of molecular mechanisms behind C1q deficiency, where missense mutations cause a lack of detectable C1q-antigen in serum.

Strong combined gene-environment effects in anti-cyclic citrullinated peptide-positive rheumatoid arthritis: a nationwide case-control study in Denmark.

OBJECTIVE: To study the role of shared epitope (SE) susceptibility genes, alone and in combination with tobacco smoking and other environmental risk factors, for risk of subtypes of rheumatoid arthritis (RA) defined by the presence or absence of serum antibodies against cyclic citrullinated peptides (CCPs). METHODS: To address these issues, a nationwide case-control study was conducted in Denmark during 2002-2004, comprising incident cases of RA or patients with recently diagnosed RA (309 seropositive and 136 seronegative for IgG antibodies against CCP) and 533 sex- and age-matched population controls. Associations were evaluated by logistic regression analyses, in which odds ratios (ORs) served as measures of relative risk. RESULTS: Compared with individuals without SE susceptibility genes, SE homozygotes had an elevated risk of anti-CCP-positive RA (OR 17.8, 95% confidence interval [95% CI] 10.8-29.4) but not anti-CCP-negative RA (OR 1.07, 95% CI 0.53-2.18). Strong combined gene-environment effects were observed, with markedly increased risks of anti-CCP-positive RA in SE homozygotes who were heavy smokers (OR 52.6, 95% CI 18.0-154), heavy coffee drinkers (OR 53.3, 95% CI 15.5-183), or oral contraceptive users (OR 44.6, 95% CI 15.2-131) compared with SE noncarriers who were not exposed to these environmental risk factors. CONCLUSION: Persons who are homozygous for SE susceptibility genes, notably those who are also exposed to environmental risk factors, have a markedly and selectively increased risk of anti-CCP-positive RA. A distinction between anti-CCP-positive RA and anti-CCP-negative RA seems warranted, because these RA subtypes most likely represent etiologically distinct disease entities.

Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins.

Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients. The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth-promoting effect depends on direct cell-cell contact and soluble factors such as interleukin-2. In conclusion, we demonstrate that SE triggers a bidirectional cross talk between nonmalignant T cells and malignant CTCL cells that promotes growth of the malignant cells. This represents a novel mechanism by which infections with SE-producing bacteria may contribute to pathogenesis of CTCL.

Cytokine gene expression in peripheral blood mononuclear cells and alloreactivity in hematopoietic cell transplantation with nonmyeloablative conditioning.

Cytokines are thought to play an important role in the pathophysiology of graft-versus-host disease (GVHD). To study the relationship between cytokines and GVHD, we obtained peripheral blood mononuclear cells (MNCs) from 21 patients with hematologic malignancies and their HLA-identical sibling donors before and sequentially after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning. The MNCs were cultured for 72 hours either alone or in mixed lymphocyte cultures with irradiated MNCs of recipient, donor, or HLA-mismatched third-party origin. The gene expression of interleukin (IL)-2, IL-4, IL-10, IL-18, tumor necrosis factor alpha, and transforming growth factor beta in each culture was then measured by real-time quantitative reverse transcriptase-polymerase chain reaction. The composition of the responder MNCs differed between patients and donors and changed after HCT, with a possible influence on the results. Early after transplantation (day +14), the IL-10 messenger RNA (mRNA) level in response to recipient or donor antigens was higher in patients who did not develop clinically significant acute GVHD when compared with the level in patients who subsequently developed acute GVHD grades II to IV (P = .005 and P = .004, respectively). The IL-10 mRNA level on day +14 was highly correlated with the pretransplantation mRNA level of the recipient MNCs but not with the level of the donor MNCs; this suggests that the IL-10 mRNA detected on day +14 originated from responder cells of recipient origin. A higher IL-10 mRNA level was found in MNCs obtained before transplantation from recipients whose disease progressed or relapsed after the transplantation when compared with the level in patients whose disease did not progress or relapse (P = .03). In conclusion, a high IL-10 gene expression in the recipient MNCs may be related to a reduced incidence of acute GVHD grades II to IV and a reduced graft-versus-tumor effect after HCT with nonmyeloablative conditioning.

A functional polymorphism in the Eta-1 promoter is associated with allele specific binding to the transcription factor Sp1 and elevated gene expression.

Early T lymphocyte activator 1 (Eta-1), also known as Osteopontin, is a cytokine produced by macrophages and T lymphocytes. It is involved in the regulation of IL-12 and IL-10 expression in macrophages and stimulates the polarization of T cells to the Th1 subset. Three promoter polymorphisms of the human Eta-1 gene, -443T/C, -156delG/G, -66T/G, were investigated for possible influence on gene expression. Electrophoretic mobility shift assays (EMSA) with nuclear extract from the human myeloid leukaemia premonocyte cell line, THP-1, revealed sequence specific binding of the transcription factor Sp1 to the -66T allele but not the -66G allele, and haplotype -443C/-156G/-66T showed a marked increase in promoter activity of a luciferase reporter gene. Thus, a substitution of the T-base with G at position -66 in the Eta-1 promoter modulates the promoter activity of the Eta-1 gene, which might influence the Th1 versus Th2 balance. These observations are discussed in relation to a recently reported related observation on the same gene, and it is argued that discrepancies between reporter gene assays in the two studies may be due to the use of different cell lines and may reflect requirements for different transcription factors in cells involved in immune responses compared with other cells.

Deficiency of somatic hypermutation of immunoglobulin G transcripts is a better predictor of severe respiratory tract infections than lack of memory B cells in common variable immunodeficiency.

Defects of memory B cells and of somatic hypermutation (SHM) are involved in the pathogenesis of common variable immunodeficiency (CVID). Here we report for the first time a systematic study of the relationship between memory B cell deficiency and SHM abnormalities in CVID, and relate these variables to prediagnostic infections. Isotype switched Vh3-23 transcripts were undetectable or low in 30% (IgG) and 63% (IgA) of the patients, but never in controls (P < 0.001). When measurable, the SHM fraction of transcripts was significantly lower in patients (IgM: median 32% vs. 56% (P = 0.0002); IgG: 72% vs. 87% (P = 0.0002); IgA: 81% vs. 88% (P = 0.04)). The concentration of switched (CD19+/CD27+/IgG+) and unswitched (CD19+/CD27+/IgM+/IgD+) memory cells was reduced in 75% and 58% of the patients, respectively. Patients with reduced concentrations of switched memory B cells had normal or low SHM, and only the IgG SHM fraction correlated with prediagnostic incidence of severe respiratory tract infections (P = 0.004).

Evaluation and automation of hematopoietic chimerism analysis based on real-time quantitative polymerase chain reaction.

Abstract Chimerism analysis is an essential tool in the follow-up of patients after allogeneic stem cell transplantation. High-resolution methods for chimerism analysis based on real-time quantitative polymerase chain reaction (RQ-PCR) with a detection limit of 0.1% marker-specific cells are especially valuable in the detection of patient-derived subpopulations for the monitoring of minimal residual disease. Using artificial chimeric mixtures of genotypically different cells, we optimized and evaluated the intrasample variation, accuracy, and detection limit of chimerism analysis based on RQ-PCR of short insertion and deletion polymorphisms. Furthermore, automated setup by robot was evaluated. The results were accurate, with acceptable intrasample variation at and above 0.1% marker-specific cells. The sensitivity was mainly limited by background values. Chimerism results based on RQ-PCR were similar to results based on PCR of short tandem repeats when samples from recipients of transplants with nonmyeloablative conditioning were analyzed. Furthermore, automated setup was feasible in a time-, labor-, and reagent-conserving manner.

Molecular characterization of the circulating anti-HIV-1 gp120-specific B cell repertoire using antibody phage display libraries generated from pre-selected HIV-1 gp120 binding PBLs.

Human bone marrow is a major repository for maturated antibody-secreting plasma cells, which produce the majority of the antibodies found in serum, making it an attractive source for generating human immune antibody libraries. Unfortunately, bone marrow is not always readily available and, although human immune libraries can be generated from circulating B cells, the low frequency of antigen-specific B cells in the circulation yield few monoclonal antibodies of interest. We used a pre-selection strategy to enrich for antigen-specific B cells prior to library generation, and applied this approach to evaluate, at a molecular level, the nature of the human anti-HIV-1 gp120 repertoire encoded by circulating B cells. IgG antibody phage display libraries were generated from HIV-1 seropositive individuals using either affinity-selected anti-gp120 IgG-bearing circulating B cells, predominantly exhibiting memory/activated B cell phenotype, or unselected PBMCs. These libraries were selected against HIV-1 gp120, resulting in isolation of a panel of gp120-specific antibodies. Whereas only 2 gp120-specific antibodies were retrieved from the non-pre-selected HIV-1 library, 9 gp120-specific antibodies were retrieved from the 10-fold smaller library generated from the pre-selected B cells, demonstrating the feasibility of the approach. The anti-gp120 antibodies derived from the circulating B cells of HIV-1 donors generally resembles those from bone marrow plasma cells with respect to epitope specificity, affinity and neutralization ability. They exhibit high affinity for gp120, are directed against a variety of epitopes, but rarely exhibit the ability to neutralize HIV-1.

Deficiency of somatic hypermutation of the antibody light chain is associated with increased frequency of severe respiratory tract infection in common variable immunodeficiency.

Reduced levels of somatic hypermutation (SHM) have recently been described in IgG-switched immunoglobulin genes in a minority of patients with common variable immunodeficiency (CVID), demonstrating a disruption of the normal linkage between isotype switch and SHM. To see if, irrespective of isotype, there is a tendency to use unmutated immunoglobulin genes in CVID, we studied SHM in kappa light-chain transcripts using a VkappaA27-specific restriction enzyme-based hot-spot mutation assay (IgkappaREHMA). Hot-spot mutations were found in 48% (median; reference interval, 28%-62%) of transcripts from 53 healthy controls. Values were significantly lower in 31 patients (median, 7.5%; range, 0%-73%; P < .0000001) of whom 24 (77%) had levels below the reference interval. Low levels of SHM correlated with increased frequency of severe respiratory tract infection (SRTI; P < .005), but not with diarrhea (P = .8). Mannose-binding lectin (MBL) deficiency also correlated with SRTI score (P = .009). However, the correlation of SHM and SRTI was also seen when only patients with normal MBL genotypes were analyzed (n = 18, P = .006). A slight decline of mutated fractions over years was noted (P = .01). This suggests that most patients with CVID fail to recruit affinity-maturated B cells, adding a qualitative deficiency to the quantitative deficiency characterizing these patients.

Haematopoietic stem cell transplantation with non-myeloablative conditioning in the outpatient setting: results, complications and admission requirements in a single institution.

Thirty patients with haematological malignancies received peripheral blood stem cells from human leucocyte antigen (HLA)-identical sibling donors after non-myeloablative conditioning with fludarabine and total body irradiation. Twenty-seven patients received the transplant as an outpatient procedure. All patients engrafted. The probability of acute graft-versus-host disease (GVHD) grades II-IV and extensive chronic GVHD was 57% and 80%, respectively. Patients alive on day +365 experienced a median of 44 d (range 4-151) of hospitalization during the first year. In the entire cohort, GVHD accounted for 22%, infections for 18%, thrombotic thrombocytopenic purpura (TTP) for 16% and engraftment syndrome for 14% of the time in hospital. The 1-year risk of TTP was 26%. Acute GVHD was a risk factor for the development of TTP (P = 0.008). With a median follow-up of 602 d, the 2-year estimates for overall survival, progression-free survival, non-relapse mortality and relapse related mortality were 68%, 43%, 22% and 13%, respectively. This transplantation regimen is feasible and induces long-term remissions in heavily pretreated patients. The procedure can be performed in the outpatient setting, but complications could result in a substantial number of admissions during the first year.

Concordance for disease course and age of onset in Scandinavian multiple sclerosis coaffected sib pairs.

BACKGROUND: Investigation of coaffected sib pairs is one method to determine the genetic influence on the clinical presentation of many complex diseases, such as multiple sclerosis (MS). Investigation of the clinical concordance in coaffected sib pairs may be a prerequisite to identify genes that modify the clinical outcome. The aim of this study was to investigate a possible genetic influence on selected demographic and clinical variables among familial Scandinavian MS cases. MATERIAL AND METHODS: We identified 136 Caucasian Scandinavian families with MS coaffected sib pairs from Denmark, Norway and Sweden. Cohen's kappa coefficient and the intraclass correlation coefficient were used to assess concordances in sib pairs. Furthermore, clinical features and HLA-DR2 carrier status were compared among the probands of sib pairs. RESULTS: We found significant concordance of the disease course (kappa = 0.28, P < 0.001) and adjusted age of onset (r = 0.23, P = 0.028). Among probands of sib pairs, HLA-DR2 carrier patients had a younger age of onset (P = 0.024). CONCLUSION: Analyses of Scandinavian coaffected sib pairs suggest that disease course and age of onset are partly under genetic control. Furthermore, HLA-DR2 in probands of sib pairs suggests importance for age of onset.

Crystal structure of HLA-DQ0602 that protects against type 1 diabetes and confers strong susceptibility to narcolepsy.

The MHC class II molecule DQ0602 confers strong susceptibility to narcolepsy but dominant protection against type 1 diabetes. The crystal structure of DQ0602 reveals the molecular features underlying these contrasting genetic properties. Structural comparisons to homologous DQ molecules with differential disease associations highlight a previously unrecognized interplay between the volume of the P6 pocket and the specificity of the P9 pocket, which implies that presentation of an expanded peptide repertoire is critical for dominant protection against type 1 diabetes. In narcolepsy, the volume of the P4 pocket appears central to the susceptibility, suggesting that the presentation of a specific peptide population plays a major role.

Knocking out IL-6 by vaccination.

Inappropriate expression of IL-6 plays a role in various inflammatory conditions, degenerative diseases, and cancers. Several model systems have been developed that can specifically block IL-6-receptor interactions. Here we present a simple and highly effective approach based on vaccination with a pool of specifically mutated IL-6 analogues to induce a neutralizing IL-6 antibody response in mice. Judged by the ability of the analogues to bind to heterologous anti-IL-6 antibodies and cellular IL-6 receptors the IL-6 analogues seemed to have a three-dimensional structure comparable to that of wild-type IL-6. Injection of them broke self-tolerance and induced an immune response to IL-6, presumably because of the amino acid differences between the analogues and wild-type IL-6. This resulted in a long-lasting anti-IL-6 antibody-mediated IL-6 deficiency that blocked experimentally induced IL-6-mediated pathology.

Bi-phasic effect of interferon (IFN)-alpha: IFN-alpha up- and down-regulates interleukin-4 signaling in human T cells.

Interferon (IFN)-alpha/beta is produced by virally infected cells and is believed to play an important role in early phases of the innate immune response. In addition, IFN-alpha/beta inhibits interleukin (IL)-4 signaling in B cells and monocytes, suggesting that IFN-alpha/beta (like IFN-gamma) is a Th1 cytokine. Here, we study cross-talk between IFN-alpha and IL-4 in human T cells. As expected, stimulation with IFN-alpha for 12-24 h inhibits IL-4 signaling. Surprisingly, however, IFN-alpha has the opposite effect on IL-4 signaling at earlier time points (up to 6 h). Thus, IFN-alpha enhances IL-4-mediated STAT6 activation in both CD4+ and CD8+ human T cells. The effect is specific because (i) another interferon, IFN-gamma, does not enhance IL-4-mediated STAT6 activation, (ii) IFN-alpha-mediated STAT1 and STAT2 activation is not modulated by IL-4, and (iii) activation of Janus kinases is not enhanced or prolonged by simultaneous stimulation with IFN-alpha and IL-4. Moreover, co-stimulation results in a selective increased STAT6/STAT2 association and an association between IFNAR/IL-4R components, suggesting that the IFNAR provides an additional STAT6 docking site via STAT2, leading to a more efficient dimerization/activation of STAT6 only. The co-stimulatory effect on STAT6 activation correlates with a cooperative increase in nuclear translocation, DNA binding, transcriptional activity, and mRNA expression of STAT6 target genes (IL-4Ralpha and IL-15Ralpha). In conclusion, we provide evidence that IFN-alpha both up- and down-regulates IL-4-mediated STAT6 signaling and thereby regulates the sensitivity to IL-4 in human T lymphocytes. Thus, our findings suggest that IFN-alpha has a complex regulatory role in adaptive immunity, which is different from the "classical" Th1 profile of IFN-gamma.

Two genome-wide linkage disequilibrium screens in Scandinavian multiple sclerosis patients.

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.

Immunogenetic heterogeneity in single-system and multisystem langerhans cell histiocytosis.

Langerhans cell histiocytosis is a rare disease with an unknown etiology and poorly understood pathogenesis. Immunologic, viral, and proliferative clonality causes have all been considered. To determine whether Langerhans cell histiocytosis and its two main subgroups, single-system and multisystem disease, are associated with HLA-A, -B, -Cw, or -DR alleles, a total of 84 patients <15 y of age at the time of diagnosis and of Nordic origin were analyzed, 82 for HLA class I and 76 for HLA class II. Stratification of the patients into two subgroups, single-system disease (skin only, and monostotic and polyostotic disease) and multisystem disease with or without organ dysfunction, showed that patients with single-system disease (17 of 45, 38%) more often (p = 0.00018 and, after correction, p = 0.029) had the phenotype HLA-DRB1*03 compared with patients with multisystem disease (1 of 31, 3%). In the patients with multisystem disease a nonsignificant reduction of the frequency of this phenotype was seen compared with controls (p = 0.02, uncorrected). In 14 of the patients with single-system disease, but none with multisystem disease, the deduced haplotype HLA-A*01, B*08, DRB1*03 was found. High-resolution typing, performed in nine patients, revealed that all had the HLA-A*0101, B*0801, DRB1*0301, DQB1*0201 alleles. Our findings suggest an immunogenetic heterogeneity in the two clinical entities of Langerhans cell histiocytosis and indicate that HLA-DRB1*03 may play a protective role against developing multisystem disease. Further studies to confirm these findings are desired.