Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLA-DR alleles by T lymphocytes of recent onset IDDM patients

Glutamic acid decarboxylase (GAD) has been defined as a major target antigen in insulin-dependent diabetes mellitus (IDDM). To identify the molecular ligands triggering a T cell response to GAD, a panel of human GAD65-specific T lymphocyte lines was generated from peripheral blood of three recent onset IDDM patients. All lines derived from a patient expressing the high-risk-conferring HLA-DR*0301/ *0401 haplotypes recognized a single epitope localized between amino acid positions 270 and 283 of GAD65, a stretch that is located in close proximity to the homology region shared with Coxsackie virus P2-C protein. All lines with this specificity were restricted to the DRA, B1*0401 product of the DR4 haplotype. Analysis of the GAD-specific T cell response in a second patient homozygous for DR4 haplotypes demonstrated that the same DRA, B1*0401 allele selected T cells specific for a different determinant. The T cell response profile in a third patient showed that DR*1501/ *1601-encoding haplotypes could present at least three different epitopes to GAD65-specific T lymphocytes. One of these epitopes was presented by a DR allele associated with the resistance-conferring DRB1*1501 haplotype. GAD-specific T cell lines could not be isolated from HLA class II-matched normal individuals. Our data reveal that (a) the T cell response to GAD65 is quite heterogenous in recent onset IDDM patients; (b) HLA-DR, not DQ, seems to be the principal restriction element used by T cells present at the onset of the disease; and (c) T cells responding to epitopes containing identical sequences to Coxsackie virus P2-C protein were not detected.     

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ alphabeta T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.     

Characteristics of the innervation of the head and neck

The association of HLA-DRB1 and DQB1 genes with IDDM in Koreans was assessed using 115 IDDM patients and 140 nondiabetic controls. DQB1*0201 is the only DQB1 allele positively associated with IDDM while DQB*0602, *0601 and *0301 are negatively associated. Three DRB1 alleles (DRB1*0301, DRB1*0407 and DRB1*0901) are positively associated while four DR allele groups (DRB1*15, DRB1*12, DRB1*10 and DRB1*14) are negatively associated. However, Haplotype analyses indicated that DQB1*0302, DRB1*0405 and DRB1*0401 may confer susceptibility because the DRB1*0405-DQB*0302 and DRB1*0401-DQB1*0302 haplotypes are positively associated with the disease. The lack of association in Koreans with the DQB1*0302 allele, which appears predisposing in studies of non-Orientals, is due to its strong linkage disequilibrium (LD) with the protective DRB1*0403 and *0406 alleles, while the lack of association with DRB1*0405 is because of its strong LD with the protective DQB1*0401 allele. Nine DR/DQ genotypes confer significantly increased risk to IDDM. Seven of the nine genotypes (DR3/4s, DR1/4s, DR4s/13, DR4s/8, DR4s/7, DR9/13 and DR3/9) were also found to be at high risk to IDDM in other populations, while the two others (DR1/9 and DR9/9) are only found in Koreans. Surprisingly, DR4/4 homozygotes are not associated with high risk to IDDM in Koreans. This observation can be explained by the high frequency of protective DR4 subtypes and the protective DQ alleles (0301 and 0401) associated with the susceptible DR4 alleles. Our analyses indicate that the counterbalancing act between susceptible DRB1 and protective DQB1, and vice versa, that has already been observed in Chinese and Japanese, is the major factor responsible for the low incidence of diabetes in Koreans.     

Insulin-dependent diabetes mellitus in Santiago, Chile: the role of immunogenetic and environmental factors

The role of HLA class II alleles in the genetic susceptibility to develop insulin-dependent diabetes mellitus (IDDM) was examined by means of PCR and oligospecific probes in 63 IDDM children and 74 controls subjects. In diabetic patients we found a significant increase in the alleles frequency DR3, DR4, DQB1*0302 and DQA1*0301 compared to the control group, where the most prevalent alleles were DR2, DR14 (DRB1*1402), DQA1*0101 and DQA1*0201. All the risk genotypes in the diabetic group were similar than in other caucasian groups: DR3/DR4-DQB1*0201/0302-DQA1*0301/0501 and DR4/DR4-DQB1*0302/0302-DQA1*0301/0301. The homozygote character no asp57 conferred an absolute risk (AR) of 3.87 and the marker Arg52 an AR of 5.78/100.000 bab year. The homozygosis for both markers (no Asp57 + Arg52) had an AR of 7.56/100.000 bab year. Regarding environmental factors associated with IDDM, our population under study showed a low prevalence of infectious agents (mainly mumps and rubella, specifically associated with IDDM) and a high prevalence of effective breast-feeding (over 3 months). These factors could be exercising a protector role in the development of IDDM. The factors that appear to be important in the low incidence of IDDM in Santiago de Chile are: the low prevalence of infectious agents related to IDDM, the high percentage of breast-feeding children in the population, the reduced frequency of susceptible molecules as DR3, DQB1*0201 (compared to other caucasian groups) and the presence of protective genotypes related to DR13 and DR14 observed in the non diabetic children.     

Associations of GAD65- and IA-2- autoantibodies with genetic risk markers in new-onset IDDM patients and their siblings. The Belgian Diabetes Registry

OBJECTIVE: To investigate the association of GAD (65-kDa) autoantibodies (GAD65-Abs) and IA-2 autoantibodies (IA-2-Abs) with human leukocyte antigen (HLA)-DQ and insulin gene (INS) risk markers in patients with recent-onset IDDM and their siblings. RESEARCH DESIGN AND METHODS: Blood was sampled from 608 recent-onset IDDM patients and 480 siblings, aged 0-39 years and consecutively recruited by the Belgian Diabetes Registry, to determine GAD65- and IA-2-Ab (radiobinding assay), HLA-DQ- (allele-specific oligonucleotyping), and INS-genotypes (restriction fragment length polymorphism analysis; siblings, n = 439). RESULTS: At the onset of IDDM, GAD65-Abs were preferentially associated with two populations at genetic risk but only in the 20- to 39-year age-group: 1) their prevalence was higher in carriers of DQA1*0301-DQB1*0302 (88 vs. 73% in non[DQA1*0301-DQB1*0302], P = 0.001), and 2) an association was found in patients lacking this haplotype but carrying DQA1*0501-DQB1*0201, together with INS I/I (87 vs. 54% vs. non[INS I/I], P = 0.003). Siblings of IDDM patients also presented the association of GAD65-Abs with DQA1*0301-DQB1*0302 (13 vs. 2% non[DQA1*0301-DQB1*0302], P < 0.001), while associations with the second genetic risk group could not yet be assessed. At the onset of IDDM, IA-2-Ab prevalence was higher in carriers of DQA1*0301-DQB1*0302 (69 vs. 39% non[DQA1*0301-DQB1*0302], P < 0.001) but not of DQA1*0501-DQB1*0201 or INS I/I. This association was present in both the 0- to 19- and the 20- to 39-year age-groups. It was also found in siblings of IDDM patients (4 vs. 0% non[DQA1*0301-DQB1*0302], P < 0.001). CONCLUSIONS: Both GAD65- and IA-2-Abs exhibit higher prevalences in presence of HLA-DQ- and/or INS-genetic risk markers. Their respective associations differ with age at clinical onset, suggesting a possible usefulness in the identification of subgroups in this heterogeneous disease.     

Effects of residues of deltamethrin in cattle faeces on the development and survival of three species of dung-breeding insect

We report on the role of HLA-DQA1 and DQB1 alleles in determining susceptibility to insulin-dependent diabetes mellitus (IDDM) in Hong Kong Chinese and investigate whether these alleles affect the age of onset of the disease. We studied 76 unrelated Chinese patients and 250 controls. There was no apparent predisposing effect of non-aspartic acid residues at position 57 of the DQ beta chain (Asp57-) but there was an excess of homozygous genotypes containing arginine at position 52 of the DQ alpha chain (Arg52+). This excess was mainly attributable to the genotype DQA1*0301/DQA1*05011 in early-onset disease. There was a significant excess of heterodimers of DQ alpha and DQ beta carrying Arg52+ and Asp57- in both early-onset and late-onset disease, but the excess in early-onset disease was mainly attributable to a single heterodimer formed by DQA1*05011 and DQB1*0201. Of three DQA1/DQB1 genotypes containing a double dose of Arg52+ and Asp57-, only one had a strong association with both early-onset and late-onset disease. We show that early-onset IDDM and late-onset IDDM in Chinese may be separated on the basis of their associated DQA1 and DQB1 genotypes and we conclude that previously reported associations of IDDM with Arg52+ and Asp57- residues in Chinese are secondary to specific combinations of DQA1 and DQB1 alleles. We also show that DRB1 molecules play a distinct role in determining susceptibility to early-onset IDDM but the greatest effect is exerted by specific DR/DQ genotypic combinations.     

On T-cell recognition of nickel as a hapten

T-cells recognize antigens as peptides associated with self-molecules encoded by genes of the HLA region. In patients with contact allergy to nickel, T-cells that are specific for non-peptide haptens have been described. Previously, we have isolated HLA class II-restricted nickel-specific T-cell clones from patients with nickel sensitivity. In this paper, data on the fine specificity of a nickel-specific HLA-DR4-restricted clone have been reevaluated. Genomic tissue typing employing polymerase chain reaction and sequence-specific primers were used. Nickel was presented to the T-cell clone by all three subtypes of HLA-DR4 included in our panel. Two different DRB4*0404-positive cells presented nickel, whereas only 3 of the 7 DRB1*0401-positive and one of the 3 DRB1*0408-positive cells restimulated the T-cell clone. These findings are compatible with the notion that nickel interacts with endogenous peptides in the antigen-presenting groove of the HLA molecule, thereby changing these peptides' antigenicity rather than their ability to bind to the HLA molecule. Variations of the endogenous peptide in the antigen-presenting groove as well as differences of the HLA molecules give the DR4 specificity of the nickel-specific clone MCE2.     

Contribution of DRB1*04 variants to predisposition to or protection from insulin dependent diabetes mellitus is independent of dq

Certain DQ alpha/beta dimeric molecules have been shown to play a major role in determining susceptibility or resistance to IDDM. Whether or not predisposition associated with DR4 haplotypes is exclusively due to linkage to DQB1*0302 and DQA1*0301 alleles is still a controversial issue. A modifying effect of certain DRB1*04 subtypes on the susceptibility encoded by DQ alleles is possible, since not all DRB1*04-DQB1*0302 haplotypes are associated with the disease. The distribution of DRB1*04 subtypes was analysed in 240 DR4-positive Caucasian IDDM patients and 110 DR4-positive healthy controls using allele-specific hybridization after genomic amplification. Although an important contribution to IDDM predisposition was encoded by the DQB1*0302 allele which was found in the majority of patients (94.2% vs 64.7% in controls, Odd's ratio OR = 8.8, P < 0.0001), differences between DRB1*04 variants persisted after the effect of the DQB1 locus was removed by matching patients and controls for DQB1*0302. Thus, the DRB1*0402 allele conferred a strong IDDM-predisposing effect (OR = 3.1, P < 0.02) which was highly significant in the absence of DR3 on the second haplotype (OR = 5.6, P < 0.0001) but was not visible among DR3/4 heterozygote individuals. Conversely, the DRB1*0404 allele conferred a strong protective effect (OR = 0.26, P < 0.0001) which was dominant even in the presence of the associated high risk DR3 haplotype (OR = 0.21, P < 0.03). These data indicate that DQ molecules are not the sole contributors to the DR4-associated IDDM predisposition, and that peculiar DR4 subtypes play a significant role in susceptibility to or protection from the disease. DRB1*0402 differs from DRB1*0404 by only two acidic residues at positions 70 and 71 within the peptide binding groove, instead of amide and basic amino acids. This might induce changes of peptide binding specificity that correlate with the genetic linkage of IDDM predisposition.     

Multiple epitopes of HLA-DRB1*0411 are recognized by T-cell clones originated from individuals carrying other DR4 subtypes

HLA polymorphism dictates the binding and recognition of specific peptides, leading to variations in individual immune responses and may contribute to autoimmune disorders and outcome in organ transplantation. We have studied the molecular basis for the cellular recognition of DRB1*0411 in individuals carrying other sequence-related DR4-alleles by characterization of T-cell clones (TLC). A set of 166 TLC were raised by priming cells from DRB1*0401,0402 and DRB1*0405,0901 individuals and 52 of them recognized DRB1*0411. Five distinct patterns of T-cell allorecognition were found: DRB1*0411 alone, DRB1*0411 and 0405, DRB1*0411 and 0406, DRB1*0411 and 0407 and DRB1*0411, 0406 and 0407, depending on responder phenotypes and epitopes recognized by their T cells. A stretch of 30 amino acids on DRB1*0411 from positions 57 to 86 behaves as a functional domain and residues S57, R71, E74 and V86 seem to be crucial in forming immunogenic determinants recognized by these TLC. The knowledge of shared amino acid residues between closely related DR4 alleles, which show similar patterns of recognition by T cells could also be useful in the selection of prospective donors for clinical transplantation of solid organs or bone marrow.     

Taking the pith out of reality: a reflexive methodology for psychiatric nursing research

HLA-DQ genes are the main inherited factors predisposing to IDDM. This gene region harbors long terminal repeat (DQ LTR) elements of the human endogenous retrovirus HER V-K, which we analyzed for a possible association with disease. We first investigated whether LTR segregate with DQ alleles in families. Members (n = 110) of 29 families with at least one diabetic child, unrelated patients with IDDM (n = 159), and healthy controls (n = 173) were analyzed. Genomic DNA was amplified for DQ LTR3 by a nested primer approach as well as for DQA1 and DQB1 second exons, to assign DQA1 and DQB1 alleles. DQ LTR segregated in 24 families along with DQ alleles. Of the 29 families, 20 index patients were positive for DQ LTR. The DQ LTR was in all patients on the haplotype carrying the DQA1 *0301 and DQB1 *0302 alleles. A majority of patients had DQ LTR (62%) compared with controls (38%) (p < 1.3 x 10(-5)), even after matching for the high-risk alleles DQA1 *0501, DQB1 *0201-DQA1 *0301, and DQB1 *0302 (79% of patients and 48% of controls; p < 0.02). Subtyping for DRB1 *04 alleles in all DQB1 *0302+ individuals showed 56% DRB1 *0401, DQB1 *0302 [LTR' patients vs. 29% controls with the same haplotype (p < 0.002)]. In conclusion, these data demonstrate the segregation of DQ LTR with DQA1, DQB1 alleles on HLA haplotypes. Furthermore their presence on DRB1 *0401-, DQA1 *0301-, and DQB1 *0302-positive haplotypes suggest that they contribute to DQ-related susceptibility for IDDM.     

Persistent GAD 65 antibodies in longstanding IDDM are not associated with residual beta-cell function, neuropathy or HLA-DR status

Persistent humoral autoimmunity to the enzyme glutamic acid decarboxylase (GAD) has been described in a substantial proportion of patients with insulin-dependent diabetes mellitus (IDDM) of long duration. The source of the stimulus for this autoimmune reactivity is still unknown. Because the GAD 65 isoform is mainly expressed in pancreatic beta-cells and in the nervous system we investigated in the present study of the largest number of well characterized patients with longstanding IDDM (n = 105; median duration: 21 years; range: 10-46 years) the presence of autoantibodies to GAD 65 and their relationship to a residual C-peptide response or peripheral and autonomic neuropathy. Additionally we studied the HLA-DR status relative to GAD 65 antibodies in 86 out of the 105 individuals. One hundred healthy control subjects and 100 recent onset IDDM patients were also studied for GAD 65 antibodies. GAD 65 antibodies were detected in a radioligand-binding-assay with recombinant human GAD 65 and were present in 32% of the long-term diabetic patients, 82% of the recent onset IDDM patients and in 3% of the healthy control subjects. A preserved C-peptide response to i.v. glucagon (Hendriksen criteria) was observed in 23% of the long-term IDDM patients. Autonomic neuropathy and peripheral neuropathy was identified using criteria based on both symptoms and formal testing giving a frequency of 67% vs 79%. The HLA specific DR 4/X was observed in 47% and HLA-DR 3/X in 22% of the long-term IDDM patients. Patients who were heterozygous for DR3/DR4 were found in 23% of the cases. GAD 65 antibodies were significantly less frequent in the long-term IDDM patients compared to recent onset IDDM (p < 0.001), and diabetes duration showed a significant negative correlation with GAD 65 antibody index levels (r = 0.22, p < 0.01). Interestingly, GAD 65 antibodies were not significantly correlated either with residual beta-cell function or neuropathy and no particular HLA-DR status was associated with persistent GAD 65 antibodies. In conclusion neither residual beta-cell function nor diabetic neuropathy or a certain HLA-DR specificity are exclusively associated with persistent autoimmunity directed to GAD 65 in longstanding IDDM. The stimulus for the persistent humoral immune response and its significance for the disease process and its complications remain to be established.     

Prevention strategies for occupational low back pain

Ethnic comparisons are extremely important and useful for studying the HLA component involved in insulin-dependent diabetes mellitus (IDDM) predisposition. To date there have been only a few reports on the association of HLA loci and IDDM in Chinese. We report here a study on DQA1*Arg52, DQB1*nonAsp57, and DRB1*04 in IDDM children and control adults among Han Chinese living in Taiwan. One hundred and fourteen unrelated children (62 boys) with IDDM were studied. Their ages at diagnosis were between 0.3 and 15.0 years (6.8 +/- 3.6 years). The control population consisted of 120 randomly selected normal adults. DQA1*Arg52(+/+), DQB1*nonAsp57(+/+), and DRB1*04(+/-) were associated with IDDM (RR = 11.50, 2.21, and 2.82; p = 1.11 x 10(-15), 2.84 x 10(-3), and 1.98 x 10(-4), respectively). DQA1*Arg52, DQB1*nonAsp57, and DRB1*04 conferred risks for IDDM (RR = 12.79, 7.11, and 2.83; pc = 8.22 x 10(-4), 5.35 x 10(-3), and 5.68 x 10(-4), respectively). Combinations of DQA1*Arg52 and DRB1*04 conferred the highest risk for IDDM (RR = 19.64, pc = 5.4 x 10(-5)). DQA1*Arg52 was associated with IDDM in subjects with DQB1*nonAsp57+ (RR = 14.87, pc = 2.41 x 10(-4)) and DQB1*nonAsp57 was also associated with IDDM in subjects with DQA1*Arg52+ (RR = 8.41, pc = 1.54 x 10(-3)), suggesting that DQA1*Arg52 and DQB1*nonAsp57 are interacting. This study demonstrates that DQA1*Arg52, DQB1*nonAsp57, and DRB1*04 confer susceptibility for IDDM to Chinese children. A combination of DQA1*Arg52 and DRB1*04 confers the highest risk and it is suggested that a susceptibility gene might be situated between DQA1*Arg52 and DRB1*04 or both are synergistic. There is an interaction between DQA1*Arg52 and DQB1*nonAsp57 and homozygosity for DQA1*Arg52/DQB1*nonAsp57, which encodes four susceptibility DQ heterodimers, confers a high risk.     

Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4(+) human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4(+) T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1-20. DR3.Ab0 mice, immunized with bacillus Calmette-Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1-20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette-Guérin but not to p1-20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171-200, 311-340, and 411-440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51-70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1-20 and p51-70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.     

Identification of mimicry peptides based on sequential motifs of epitopes derived from 65-kDa glutamic acid decarboxylase

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease with a predominantly non-hereditary etiology that results in a destruction of pancreatic beta cells by autoaggressive T lymphocytes. Neither the mechanism of initial stimulation of these T cells nor the nature of the environmental factors implicated in the disease have so far been identified. However, both issues are taken into account by the hypothesis of initial T cell activation by viral or bacterial mimicry peptides with sequence similarities to pancreatic self antigens. We determined sequential epitope motifs to search for mimicry peptides stimulating T cell lines specific for two epitopes derived from the IDDM autoantigen 65-kDa glutamic acid decarboxylase (GAD65). These were GAD65 (88-99), presented by HLA-DRB1*0101, and GAD65 (248-257), presented by HLA-DRB5*0101. T cell stimulation by peptides with substitutions in HLA anchor or T cell contact positions was analyzed to establish degenerate epitope motifs for database searching. Out of 28 tested candidate mimicry peptides derived from bacterial, viral and human proteins, 3 stimulated T cell lines and a T cell clone specific for epitope GAD65 (248-257). Our results demonstrate that mono- and polyclonal GAD65-specific T cells from IDDM patients can be stimulated by viral and bacterial peptides with little apparent sequence homology with autoantigenic epitopes. Moreover, in a synopsis with related published studies, our findings suggest that simple degenerate search motifs comprising principal T cell contacts plus HLA class II binding motifs may suffice to identify most mimicry peptides.     

Role of donor and recipient antigen-presenting cells in priming and maintaining T cells with indirect allospecificity

BACKGROUND: It has been suggested that the sensitization of recipient T lymphocytes against peptides derived from allogeneic major histocompatibility complex (MHC) antigens in the context of self-MHC molecules may contribute to the pathogenesis of chronic allograft rejection. The purpose of this study was to quantitate and characterize the indirect alloresponse in renal transplantation. METHODS: An HLA-A2-negative patient whose A2-positive kidney transplant failed as a result of chronic rejection was selected for this study. T-cell clones were raised using a cocktail of peptides corresponding to polymorphic regions of the A2 sequence and studied by measuring their proliferation using [3H]thymidine incorporation. The presence in vivo of HLA-A2-specific T cells was assessed using limiting dilution analysis. RESULTS: T-cell clones were specific for a single peptide of HLA-A2, residues 92-120, and restricted by HLA-DRB1*1502. The frequency of interleukin-2-secreting T cells specific for this A2 peptide was 1:86,000, only 2-fold lower than that measured against the recall antigen tetanus toxoid. Capitalizing on the similarity of the donor and recipient DR15 alleles (DRB1*1501 and 1502), the question was addressed as to how these T cells had been primed in vivo. Although the large majority of clones responded to A2 synthetic peptide presented by both DR15 alleles, only 3 of 10 clones responded to cells co-expressing DRB1*1501 and A2. CONCLUSION: These data suggest that antigen presentation by recipient APCs is responsible for maintaining T cells with indirect allospecificity in vivo and that, in the context of partial DR matching, indirect presentation by the parenchymal cells of the graft may serve to induce tolerance in T cells with indirect allospecificity.     

Cytoplasmic islet cell antibodies recognize distinct islet antigens in IDDM but not in stiff man syndrome

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals. Mouse-reactive ICAs, however, remained absent in 36% of the patients at diagnosis of IDDM.     

Recognition of HLA-DR1/DRB1*0101 molecules presenting HLA-A2 derived peptides by a human recombinant antibody, Fab-5 A1

MHC molecules present peptides in their binding groove to T-cell receptors inducing proliferation or cytotoxicity of alloreactive T cells. A previously generated human monoclonal antibody (mAb) UL-5 A1, recognizing a conformational epitope formed by HLA DR1/DRB1*0101 molecules and HLA-A2 derived peptides, demonstrates T-cell-like recognition of the peptide/MHC complex (PMC). To study the genes of the antigen binding region, the nucleotide sequences of the rearranged genes in the variable regions of UL-5 A1 were determined and the V-gene usage (VH3, V lambda 2) was identified by comparison with published germlines. The genes encoding heavy (Fd) and light (L) chains of UL-5 A1 were linked and expressed in a bacterial system. Specificity of the recombinant Fab-5 A1 was determined with HLA-typed LCLs by flow cytometric analysis. As demonstrated in competitive inhibition assays, UL-5 A1 and Fab-5 A1 recognize the same PMC epitope on HLA-A2+, -DR1/DRB1*0101+ typed LCLs. Additionally, mAb UL-5 A1 and Fab-5 A1 both recognize HLA-A2-, -DR1/DRB1*0101+ LCLs exogenously loaded with HLA-A2 peptides (105-117, 103-117). UL-5 A1-like antibodies against peptide/MHC complexes could prove valuable tools for research on T-cell recognition and MHC function.     

Radioimmunoassay detects the frequent occurrence of autoantibodies to the Mr 65,000 isoform of glutamic acid decarboxylase in Japanese insulin-dependent diabetes

Glutamic acid decarboxylase antibodies (GAD65Ab) are common in new onset Caucasian insulin-dependent diabetic (IDDM) patients but it is unclear if this marker is also prevalent in patients of other ethnic backgrounds. We determined antibodies against human recombinant GAD in Japanese diabetic patients using a radioimmunoassay with competition between in vitro translated 35S-GAD65 and non-labelled recombinant human GAD65 (rhGAD65). GAD67 antibodies (GAD67Ab) were similarly analyzed but without antigen competition. In 73 Japanese diabetic patients, GAD65Ab were found in 11/16 (69%) of patients with short-duration (less than 5 yrs) IDDM, 6/23 (26%) with long-duration (5 or more yrs) IDDM and 10/20 (50%) with slowly progressive diabetes. High GAD65Ab levels were associated with concomitant autoimmune diseases (p = 0.021). GAD67Ab were found in 4/16 (25%) of patients with short-duration IDDM, 3/23 (13%) with long-duration IDDM and 2/20 (10%) with slowly progressive diabetes. In 14 non-insulin dependent diabetic (NIDDM) patients, GAD65Ab and GAD67Ab were not found (0/14) and 1/50 (2%) healthy controls were positive in either assay. Among the GAD67Ab-positive samples, 8/9 (88%) were also high level GAD65Ab positive, 7/9 (77%) were displaced by an excess of rhGAD65 and the antibody levels correlated (r2 = 0.573; p = 0.003). Our data are consistent with a strong association of GAD65Ab also in Japanese IDDM, and suggest that, when present, GAD67Ab are frequently directed to epitope(s) common to GAD65 and GAD67.     

A phase I/II study to evaluate the effect of fractionated hemibody irradiation in the treatment of osseous metastases--RTOG 88-22

Previous studies have indicated that certain alleles of HLA-DR and -DQ genes were strongly associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM), and the role of DQ molecule in IDDM has been suggested. To further clarify the association of DQ alleles with IDDM, we determined the nucleotide sequences of full-length cDNA from 13 DQA1 alleles and 14 DQB1 alleles. The sequencing analysis revealed sequence polymorphisms outside the hypervariable region of DQ genes. We then analyzed the DQA1 and DQB1 polymorphisms along with that of DRB genes in 86 B-lymphoblastoid cell lines (B-LCLs) from various ethnic groups and in healthy unrelated Japanese and Norwegian individuals. The allelic and haplotypic distributions in each population revealed the characteristic haplotypic formation in the HLA class II region. HLA genes in 139 Japanese and 100 Norwegian IDDM patients were analyzed. DQB1*0301 was negatively associated with IDDM in both ethnic groups, irrespective of associated DRB1 and DQA1 alleles. In DQB1*0302 positive populations, which represented a positive association with IDDM in both ethnic groups, DRB1*0401, *0404, *0802 haplotypes increased in the patients, whereas DRB1*0406 haplotype decreased. Considering about the hierarchy in DRB1 alleles with IDDM susceptibility (DRB1*0401>*0404>*0403 in Norwegian and DRB1*0802>*0403>*0406 in Japanese), the genetic predisposition to IDDM is suggested to be defined by the combination of DR-associated susceptibility and DQ-associated susceptibility and by the DQ-associated resistance which is a dominant genetic trait.     

Induction of autoimmune arthritis in HLA-DR4 (DRB1*0401) transgenic mice by immunization with human and bovine type II collagen

Although associations between the expression of particular HLA genes and the susceptibility to specific autoimmune diseases has been known for some time, the role that these HLA molecules play in the autoimmune response is unclear. Through the establishment of a chimeric HLA-DR/I-E transgene, we have examined the function of the rheumatoid arthritis (RA) susceptibility allele HLA-DR4 (DRB1*0401) in presenting antigenic peptides derived from the model Ag, type II collagen (CII), and in mediating an autoimmune response. As a transgene, the chimeric DR4 molecule conferred susceptibility to an autoimmune arthritis induced by immunization with human CII or bovine CII. These mice developed an inflammatory, autoimmune arthritis that was similar both histologically and in severity to that previously described for the collagen-induced arthritis model. The DR4-mediated autoimmune arthritis was accompanied by T cell and B cell responses to both the immunogen and the autoantigen, murine CII. The DR4-restricted T cell response to human CII was focused on an immunodominant determinant within CII263-270 and a minor determinant within CII286-300, the same CII determinants recently identified for yet another RA susceptibility allele, HLA-DR1 (DRB1*0101). Thus these data demonstrate that, like HLA-DR1, HLA-DR4 is capable of binding peptides derived from human CII and therefore probably plays a role in the autoimmune response to human CII observed in RA patients.