F. Carl Grumet

Complex HLA-DR and -DQ Interactions Confer Risk of Narcolepsy- Cataplexy in Three Ethnic Groups

Human narcolepsy-cataplexy, a sleep disorder associated with a centrally mediated hypocretin (orexin) deficiency, is tightly associated with HLA-DQB1*0602. Few studies have investigated the influence that additional HLA class II alleles have on susceptibility to this disease. In this work, 1,087 control subjects and 420 narcoleptic subjects with cataplexy, from three ethnic groups, were HLA typed, and the effects of HLA-DRB1, -DQA1, and -DQB1 were analyzed. As reported elsewhere, almost all narcoleptic subjects were positive for both HLA-DQA1*0102 and -DQB1*0602. A strong predisposing effect was observed in DQB1*0602 homozygotes, across all ethnic groups. Relative risks for narcolepsy were next calculated for heterozygous DQB1*0602/other HLA class II allelic combinations. Nine HLA class II alleles carried in trans with DQB1*0602 were found to influence disease predisposition. Significantly higher relative risks were observed for heterozygote combinations including DQB1*0301, DQA1*06, DRB1*04, DRB1*08, DRB1*11, and DRB1*12. Three alleles-DQB1*0601, DQB1*0501, and DQA1*01 (non-DQA1*0102)-were found to be protective. The genetic contribution of HLA-DQ to narcolepsy susceptibility was also estimated by use of lambda statistics. Results indicate that complex HLA-DR and -DQ interactions contribute to the genetic predisposition to human narcolepsy but that additional susceptibility loci are also most likely involved. Together with the recent hypocretin discoveries, these findings are consistent with an immunologically mediated destruction of hypocretin-containing cells in human narcolepsy-cataplexy.

Prevention of transfusion-acquired cytomegalovirus infections in newborn infants

Transfusion-acquired cytomegalovirus occurred in 13.5% of 74 infants of seronegative mothers who were exposed to one or more blood donors who had a CMV indirect hemagglutination titer of 1:8 or higher. None of 90 infants of seronegative mothers exposed only to donors with CMV IHA titers of less than 1:8 became infected. Ten of 41 (24%) infants of seronegative mothers who received more than 50 ml of packed red blood cells and who were exposed to at least one seropositive donor became infected. None of 23 infants of seronegative mothers who received this amount of blood but who were exposed only to seronegative donors became infected. Fatal or serious symptoms developed in 50% of the infected infants of seronegative mothers and in none of the 32 infected infants of seropositive mothers. Acquired CMV infections occurred in 15% of infants of, seropositive mothers who were exposed to the red blood cells of seropositive donors and in 17.6% of infants of seropositve mothers exposed only to seronegative donors. Use of seronegative donors reduced the prevalence of excretion of CMV among hospitalized infants who were 4 weeks of age or older from 12.5 to 1.8% and eliminated acquired CMV infections in infants of seronegative mothers.

Narcolepsy and immunity

Narcolepsy is a neurological disorder known to be associated with human leukocyte antigen (HLA)-DQB1*0602 in humans. In a canine model, the disorder is also genetically linked to a gene of high homology with the human mu-switch-like immunoglobulin (Ig) gene (current LOD score 13.6 at 0% recombination). Since association with HLA or other immune function polymorphic genes (T cell receptor of Ig, mainly) is a hallmark of most autoimmune diseases, it is proposed that autoimmunity may also play a role in the development of narcolepsy. Arguments for and against this hypothesis are reviewed. It is shown that both on the basis of the most recent molecular studies, and because of some of its clinical features, narcolepsy may be an autoimmune disorder. However, neither systemic nor central nervous system (CNS) evidence of any autoimmune abnormality have ever been found. To reconcile this discrepancy, it is suggested that the pathological immune process involved in narcolepsy could be difficult to detect because it is restricted to a very small region of the brain or targets a low abundance neuroeffector. Alternatively, it is possible that a more fundamental relationship is involved between sleep generation and immune regulation. The pathophysiology of narcolepsy may then involve new CNS-immune mechanisms that may shed new light on the sleep process itself.

FAMILIAL PATTERNS OF NARCOLEPSY

Familial patterns of narcolepsy were investigated in a clinic population of 334 unrelated narcoleptic patients. 40% of probands had at least 1 family member with an isolated daytime sleepiness complaint and 6% had a positive family history of narcolepsy. Multicase families were rare; only two families were found with 3 or more affected relatives. Family members often shared the same HLA-DR2 haplotype as the proband but did not have narcolepsy. However, the risk of disease for first-degree relatives was six to eighteen times greater than that for unrelated individuals. Although most patients were HLA-DR2+, 2 new HLA-DR2- individuals were found. The data predict that as many as 9% of unrelated North-American white patients with narcolepsy will be DR2-. Analysis of these and other data indicates that although strongly associated with disease, the HLA-DR2 haplotype is neither sufficient nor necessary for the development of narcolepsy.

HLA-class II genes modify outcome of Toxoplasma gondii infection

Associations between Human Leukocyte Antigen (HLA) (i.e. human major histocompatibility complex [MHC]) genes and susceptibility to infections and inflammatory processes have been described, but causal relationships have not been proven. We characterized effects of HLA-DQ alleles on outcome of congenital toxoplasma infection and found that among Caucasians, the DQ3 gene frequency was significantly higher in infected infants with hydrocephalus (0.783) than infected infants without hydrocephalus (0.444) or published normal controls (0.487). We then developed a novel animal model to definitively determine the effect of these HLA DQ molecules on the severity of toxoplasmosis. Human MHC-Class II transgenes reduced parasite burden and necrosis in brains of mice infected with Toxoplasma gondii. Consistent with the observed association between DQ3 and hydrocephalus in human infants, in the murine model the DQ3(DQ8; DQB1*0302) gene protected less than DQ1 (DQ6; DQB1*0601). Our findings definitively prove a cause and effect relationship between human MHC genes and resistance to infection, provide novel means to characterise human immune responses that are protective or pathogenic in infections, and are important for vaccine development.

Monoclonal antibody (B27M2) subdividing HLA-B27

Abstract Because HLA-B27 shows strong association with ankylosing spondylitis, it was of interest to produce murine monoclonal antibodies to study this antigen in detail. The first such anti-B27 antibody, anti-B27M1, reacted with 100% of B27 + cells and cross-reacted with homozygous B7 cells. In the present report a second monoclonal antibody, anti-B27M2, is described which subdivides HLA-B27 into two variants. Among healthy B27 + individuals, 87% were B27M1 + and B27M2 + , and 13% were B27M1 + but B27M2 + by standard lymphocytotoxicity assays. The specificity of B27M2 antibody for the HLA-B27 molecule was confirmed with blocking studies using F(ab′) 2 fragments of HLA alloantibodies. Both the B27M2 + and B27M2 variants of HLA-B27 bred true in family studies. Unlike B27M1, B27M2 antibody did not react with B7 but did react with the rare Bw47 allele. For antibody binding studies Epstein-Barr virus-transformed B lymphoblastoid cell lines were derived from normal donors KCA (by lymphocytotoxicity shown to be B27 + , B27M2 + . VC (B27 + , B27M1 + , B27M2 − ), and WH (B27 − , B27M1 − , B27M2 − ). Each cell line bound equivalent amounts of W6/32 (monoclonal anti-HLA-ABC): KCA and VC bound similar amounts of anti-B27M1, but only KCA bound substantial anti-B27M2 antibody. These data are consistent with a model in which all B27 antigens possess a B27M1 epitope: whereas most, but not all, possess an additional and distinct epitope, B27M2. Although the relation of these genetic variants to disease susceptibility remains to be determined, the availability of epitope-specific monoclonal antibodies should help to refine our understanding of the structure and function of HLA molecules.

MONOCLONAL ANTI-HLA-B27 ANTIBODY (B27M1): PRODUCTION AND LACK OF DETECTABLE TYPING DIFFERENCE BETWEEN PATIENTS WITH ANKYLOSING SPONDYLITIS, REITER'S SYNDROME, AND NORMAL CONTROLS

Abstract A monoclonal murine anti-HLA-B27 antibody (designated anti-B27M 1 ) has been produced by the technique of somatic cell hybridisation. When tested by standard lymphocytotoxicity against panels of normal controls and patients with spondyloarthropathies, anti-B27M 1 showed as good a correlation with the HLA-B27 alloantigen as did conventional typing alloantisera (r= 0·9). No differences were detected between the typings of patients and of controls, suggesting that the B27 alloantigens of each group share a common epitope and that this specificity may not be divisible into normal and disease-associated variants. These results show that specified hybridoma antibodies of reagent quality can be produced and applied to HLA typing

Donor-specific antibody against denatured HLA-A1: Clinically nonsignificant?

Pre-transplant screening of a woman with end-stage renal disease (ESRD) showed no anti-human leukocyte antigen (HLA) alloantibodies by anti-human globulin-complement-dependent cytotoxicity (AHG-CDC; class I) or enzyme-linked immunosorbent assay (class II). Following a negative AHG-CDC crossmatch, an HLA*01:01+ deceased donor (DD) kidney was transplanted in September 2005. Subsequent screening of pre-transplant serum by LABScreen Single Antigen (SA) array showed strong reactivity versus A*01:01. Despite that reactivity, at 5 years post-transplant, the patient has a serum creatinine of 1.6 mg/dl and has never experienced humoral or cellular rejection. Retrospective flow-cytometric crossmatch of pre- and post-transplant sera versus DD cells was negative. Rescreening of multiple pre- and post-transplant sera revealed anti-A1 reactivity persisting from the first through the last samples tested. The patients anti-A1 was almost two fold more reactive with denatured A*01:01 FlowPRA SA beads after denaturation with acid treatment (pH 2.7) than with untreated beads. Parallel results were observed with pH 2.7 treated versus untreated A1+ T cells in FXM. These data highlight the difficulty in interpreting screening results obtained using bead arrays, because of antibodies that appear to recognize denatured but not native class I HLA antigens. We suggest that such bead-positive, flow cytometric crossmatch negative antibodies are not associated with humoral rejection, may not necessarily be detrimental to a graft, and deserve further evaluation before becoming a barrier to transplantation.

H-Y antibody development associates with acute rejection in female patients with male kidney transplants.

Background. Human minor histocompatibility antigens (mHA) and clinically relevant immune responses to them have not been well defined in organ transplantation. We hypothesized that women with male kidney transplants would develop antibodies against H-Y, the mHA encoded on the Y-chromosome, in association with graft rejection. Methods. We tested sera from 118 consecutive transplant recipients with kidney biopsies. Antibodies that specifically recognized the recombinant H-Y antigens RPS4Y1 or DDX3Y were detected by IgG enzyme-linked immunosorbent assay and western blotting. Immunogenic epitopes were further identified using overlapping H-Y antigen peptides for both the H-Y proteins. Results. In the 26 female recipients of male kidneys, H-Y antibody development posttransplant (1) was more frequent (46%) than in other gender combinations (P<0.001), (2) showed strong correlation with acute rejection (P=0.00048), (3) correlated with plasma cell infiltrates in biopsied kidneys (P=0.04), and (4) did not correlate with C4d deposition or donor-specific anti-human leukocyte antigen (HLA) antibodies. Of the two H-Y antigens, RPS4Y1 was more frequently recognized (P=0.005). Conclusion. This first demonstration of a strong association between H-Y antibody development and acute rejection in kidney transplant recipients shows that in solid organ allografts, humoral immune responses against well defined mHA have clear clinical correlates, can be easily monitored, and warrant study for possible effects on long-term graft function.

Soluble form of an HLA-B7 class I antigen specifically suppresses humoral alloimmunization

A soluble HLA-B7 molecule, designated sB7 and generated by genetically engineering the B7 gene to remove the transmembrane and cytoplasmic domains, was tested as a tolerogen. Supernatants from cultures of C1R cells transfected with the gene for sB7 were harvested and concentrated, as were control supernatants. From days -17 to -1, C57Bl/6 mice were pretreated with a total of 11 intraperitoneal doses of 1.0 microgram each of sB7 or appropriate control supernatant, and then were challenged intraperitoneally on each of days 0, 7, and 14 with 10(6) C1R-B7 cells (expressing surface HLA-B7). Antibody kinetics revealed (1) anti-B7 was not induced after sB7 pretreatment; (2) the anti-B7 response of sB7-pretreated mice was marginal and of apparent low avidity compared with the brisk anti-B7 response of control mice; (3) none of the mice made antibody to a control HLA antigen, A24; (4) all mice made strong antibody responses to the non-B7 surface antigens of C1R; (5) free sB7 did not appear in the blood of the treated mice; and (6) all mice appeared to be generally healthy. These data show soluble B7 antigen is not immunogenic and appears to specifically block humoral immune response to cell membrane-bound HLA-B7 in a nontoxic manner.