Benefits of BCG-induced metabolic switch from oxidative phosphorylation to aerobic glycolysis in autoimmune and nervous system diseases.

The most commonly used vaccine worldwide, bacillus Calmette-Guerin (BCG), appears to have the ability to restore blood sugar control in humans with early-onset but long-duration type 1 diabetes when a repeat vaccination strategy is used. This is a process that may be driven by a metabolic switch from overactive oxidative phosphorylation to accelerated aerobic glycolysis and a reset of the immune system. BCG is a live, attenuated strain of Mycobacteria bovis, a cousin of M. tuberculosis. Humans and Mycobacteria, which are found in the environment and in warm-blooded hosts, share a long coevolutionary history. In recent times, humans have had fewer exposures to these and other microorganisms that historically helped shape the immune response. By 're-introducing' an attenuated form of Mycobacteria via BCG vaccination, humans might benefit from an immunological perspective, a concept supported by a growing body of data in autoimmunity and robust data on the nonspecific immune effects of BCG related to protection from diverse infections and early mortality. New findings of immune and metabolic defects in type 1 diabetes that can be corrected with repeat BCG vaccination suggest that this therapeutic strategy may be applicable in other diseases with inadequate aerobic glycolysis, including Parkinson's disease, dementia, depression and other disorders affecting the nervous system.

Low levels of C-peptide have clinical significance for established Type 1 diabetes.

AIM: To determine whether the low C-peptide levels (< 50 pmol/l) produced by the pancreas for decades after onset of Type 1 diabetes have clinical significance. METHODS: We evaluated fasting C-peptide levels, duration of disease and age of onset in a large cross-sectional series (n = 1272) of people with Type 1 diabetes. We then expanded the scope of the study to include the relationship between C-peptide and HbA1c control (n = 1273), as well as diabetic complications (n = 324) and presence of hypoglycaemia (n = 323). The full range of C-peptide levels was also compared with 1,5-Anhydroglucitol, a glucose responsive marker. RESULTS: C-peptide levels declined for decades after diagnosis, and the rate of decline was significantly related to age of onset (P < 0.0001), after adjusting for disease duration. C-peptide levels > 10 pmol/l were associated with protection from complications (e.g. nephropathy, neuropathy, foot ulcers and retinopathy; P = 0.03). Low C-peptide levels were associated with poor metabolic control measured by HbA1c (P < 0.0001). Severe hypoglycaemia was associated with the lowest C-peptide levels compared with mild (P = 0.049) or moderate (P = 0.04) hypoglycaemia. All levels of measurable C-peptide were responsive to acute fluctuations in blood glucose levels as assessed by 1,5-Anhydroglucitol (P < 0.0001). CONCLUSIONS: Low C-peptide levels have clinical significance and appear helpful in characterizing groups at-risk for faster C-peptide decline, complications, poorer metabolic control and severe hypoglycaemia. Low C-peptide levels may be a biomarker for characterizing at-risk patients with Type 1 diabetes.

Murine pancreatic beta-cells express H-2K and H-2D but not Ia antigens.

Direct microcytotoxicity testing and absorption analyses were employed to determine whether H-2K, H-2D, and Ia antigens are present on murine islet of Langerhans cells. Products of the H-2K and H-2D loci were found on beta-cells from three different mouse strains, but I-region (Ia) antigens were not detected. The absence of Ia gene products from islet cells may be of importance in explaining the survival of islet allografts across major histocompatibility barriers.

Prevention of xenograft rejection by masking donor HLA class I antigens.

Destruction of target cells by cytotoxic T lymphocytes requires the presence of HLA (human lymphocyte antigen) class I antigens on the target cells for adhesion as well as for triggering of the antigen-specific T cell receptor. Rejection of xenogeneic human pancreatic islets and liver was circumvented by masking, before transplantation, donor antigens with F(ab')2 antibody fragments to HLA class I or tissue-specific epitopes. This strategy eliminated the need for recipient immunosuppression and allowed islet xenograft survival beyond 200 days, as demonstrated functionally by C peptide secretion as well as by histology. These in vivo observations are consistent with the importance of donor HLA class I in eliciting graft rejection and have potential applicability to the successful transplantation of other HLA class I-bearing donor tissues.

Prevention of allograft rejection by immunization with donor blood depleted of Ia-bearing cells.

Strain-specific unresponsiveness was induced in adult mice by immunizing them with donor blood treated with antiserum to Ia (I region-associated antigens) prior to the transplantation of islets of Langerhans. This regimen alone produced greater than 100-day survival of islet allografts transplanted across a major histocompatibility barrier.

Linkage of faulty major histocompatibility complex class I to autoimmune diabetes.

Pancreatic islet cells are the targets of an autoimmune response in type I diabetes. In the nonobese diabetic (NOD) mouse model of autoimmune diabetes, expression of major histocompatibility complex (MHC) class I proteins was inversely correlated with diabetes; in this mouse a mutation in the MHC class II-linked gene for the putative MHC class I peptide transporter was also present. Mice deficient in MHC class I expression because they do not produce beta 2-microglobulin also developed late onset autoimmune diabetes. In cells from humans with type I diabetes expression of MHC class I was decreased; subsets of prediabetics categorized as most likely to become hyperglycemic also had low MHC class I. T cell responses to self antigens are faulty in diabetics. In sets of genetically identical twins that are discordant for diabetes, the defect appeared to reside with the antigen presenting cell. Thus, a lack of surface MHC class I protein is associated with autoimmune diabetes; the concomitant defect in antigen presentation may impair the development of self tolerance, which could result in autoimmune disease.

Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic antibodies in advanced Sézary syndrome.

Sézary syndrome (SS) is a rare form of cutaneous T-cell lymphoma often refractory to treatment. SS is defined as adenopathy, erythroderma with high numbers of atypical T cells. This offers an opportunity for new interventions and perhaps antibody-based therapeutic by virtue of its high expression of the TNFR2 oncogene on the tumor cells and on T-regulatory cells (Tregs). Potent human-directed TNFR2 antagonistic antibodies have been created that preferentially target the TNFR2 oncogene and tumor-infiltrating TNFR2+ Tregs. Here we test the therapeutic potential of TNFR2 antagonists on freshly isolated lymphocytes from patients with Stage IVA SS and from healthy controls. SS patients were on a variety of end-stage multi-drug therapies. Baseline burden Treg/T effector (Teff) ratios and the responsiveness of tumor and infiltrating Tregs to TNFR2 antibody killing was studied. We show dose-escalating concentrations of a dominant TNFR2 antagonistic antibody killed TNFR2+ SS tumor cells and thus restored CD26- subpopulations of lymphocyte cell numbers to normal. The abundant TNFR2+ Tregs of SS subjects are also killed with TNFR2 antagonism. Beneficial and rapid expansion of Teff was observed. The combination of Treg inhibition and Teff expansion brought the high Treg/Teff ratio to normal. Our findings suggest a marked responsiveness of SS tumor cells and Tregs, to targeting with TNFR2 antagonistic antibodies. These results show TNFR2 antibodies are potent and efficacious in vitro.

Reversal of established autoimmune diabetes by restoration of endogenous beta cell function.

In NOD (nonobese diabetic) mice, a model of autoimmune diabetes, various immunomodulatory interventions prevent progression to diabetes. However, after hyperglycemia is established, such interventions rarely alter the course of disease or allow sustained engraftment of islet transplants. A proteasome defect in lymphoid cells of NOD mice impairs the presentation of self antigens and increases the susceptibility of these cells to TNF-alpha-induced apoptosis. Here, we examine the hypothesis that induction of TNF-alpha expression combined with reeducation of newly emerging T cells with self antigens can interrupt autoimmunity. Hyperglycemic NOD mice were treated with CFA to induce TNF-alpha expression and were exposed to functional complexes of MHC class I molecules and antigenic peptides either by repeated injection of MHC class I matched splenocytes or by transplantation of islets from nonautoimmune donors. Hyperglycemia was controlled in animals injected with splenocytes by administration of insulin or, more effectively, by implantation of encapsulated islets. These interventions reversed the established beta cell-directed autoimmunity and restored endogenous pancreatic islet function to such an extent that normoglycemia was maintained in up to 75% of animals after discontinuation of treatment and removal of islet transplants. A therapy aimed at the selective elimination of autoreactive cells and the reeducation of T cells, when combined with control of glycemia, is thus able to effect an apparent cure of established type 1 diabetes in the NOD mouse.

Defective major histocompatibility complex class I expression on lymphoid cells in autoimmunity.

Lymphocytes from patients with insulin-dependent diabetes mellitus (IDDM), a chronic autoimmune disease, have recently been shown to have decreased surface expression of MHC class I antigens. Since IDDM and other autoimmune diseases share a strong genetic association with MHC class II genes, which may in turn be linked to genes that affect MHC class I expression, we studied other autoimmune diseases to determine whether MHC class I expression is abnormal. Fresh PBLs were isolated from patients with IDDM, Hashimoto's thyroiditis, Graves' disease, systemic lupus erythematosis, rheumatoid arthritis, and Sjogren's syndrome. Nondiabetic and non-insulin-dependent diabetes mellitus patients served as controls. MHC class I expression was measured with a conformationally dependent monoclonal antibody, W6/32. Freshly prepared PBLs from the autoimmune diseases studied and the corresponding fresh EBV-transformed B cell lines had decreased MHC class I expression compared with PBLs from normal volunteers and non-insulin-dependent (nonautoimmune) diabetic patients. Only 3 of more than 180 donors without IDDM or other clinically recognized autoimmune disease had persistently decreased MHC class I expression; one patient was treated with immunosuppressive drugs, and subsequent screening of the other two patients revealed high titers of autoantibodies, revealing clinically occult autoimmunity. Patients with nonautoimmune inflammation (osteomyelitis or tuberculosis) had normal MHC class I expression. Autoimmune diseases are characterized by decreased expression of MHC class I on lymphocytes. MHC class I expression may be necessary for self-tolerance, and abnormalities in such expression may lead to autoimmunity.

Lymphocyte transfusions prevent diabetes in the Bio-Breeding/Worcester rat.

The Bio-Breeding/Worcester (BB/W) rat develops spontaneous autoimmune diabetes similar to human insulin-dependent diabetes mellitus. Transfusions of whole blood from the nondiabetic W-line of BB/W rats prevent the syndrome in diabetes-prone recipients. We report three experiments designed to determine which blood component is protective. In all experiments, diabetes-prone BB/W rats 23 to 35 d of age were given four or six weekly intravenous injections. In the first experiment, animals received either saline or transfusions of erythrocytes, white blood cells, or plasma from W-line donors. Diabetes occurred in 7/22 (32%) erythrocyte, 2/27 (7%) white cell, 14/24 (58%) plasma, and 15/27 (56%) saline recipients (P less than 0.001). At 120 d of age, peripheral blood was obtained from nondiabetic rats. Fluorescence-activated cell sorter analysis of OX 19 tagged leucocytes revealed 35% T lymphocytes in white cell recipients (n = 13), compared with 9% in saline recipients (n = 7; P less than 0.001). Responsiveness to concanavalin A was also increased in the white cell group, whereas the frequency of both insulitis and thyroiditis was decreased. In the second experiment, 1/19 (5%) rats transfused with W-line spleen cells developed diabetes, as contrasted with 12/18 (67%) recipients of diabetes-prone spleen cells and 19/31 (61%) noninjected controls (P less than 0.001). In the third experiment, diabetes-prone rats received either W-line blood treated with a cytotoxic anti-T lymphocyte antibody plus complement, untreated blood, or saline. Diabetes occurred in 8/20 (40%), 1/20 (5%), and 13/19 (68%) rats in each group, respectively (P less than 0.001). We conclude that transfusions of W-line T lymphocytes prevent diabetes in the BB/W rat.

NOD mice are defective in proteasome production and activation of NF-kappaB.

The nonobese diabetic (NOD) mouse is an animal model of human type I diabetes with a strong genetic component that maps to the major histocompatibility complex (MHC) of the genome. We have identified in NOD lymphocytes a specific proteasome defect that results from the lack of the LMP2 subunit. The pronounced proteasome defect results in defective production and activation of the transcription factor NF-kappaB, which plays an important role in immune and inflammatory responses as well as in preventing apoptosis induced by tumor necrosis factor alpha. The defect in proteasome function in NOD mouse splenocytes was evident from impaired NF-kappaB subunit p50 and p52 generation by proteolytic processing and impaired degradation of the NF-kappaB-inhibitory protein IkappaBalpha. An obligatory role of MHC-linked proteasome subunits in transcription factor processing and activation has been established in a spontaneous-disease model and mutant cells similarly lacking the MHC-encoded subunit. These data suggest that NOD proteasome dysfunction is due to a tissue- and developmental-stage-specific defect in expression of the MHC-linked Lmp2 gene, resulting in altered transcription factor NF-kappaB activity, and that this defect contributes to pathogenesis in NOD mice. These observations are consistent with the diverse symptomatology of type I diabetes and demonstrate clear sex-, tissue-, and age-specific differences in the expression of this error which parallel the initiation and disease course of insulin-dependent (type I) diabetes mellitus.

T-lymphocyte changes linked to autoantibodies. Association of insulin autoantibodies with CD4+CD45R+ lymphocyte subpopulation in prediabetic subjects.

The onset of insulin-dependent (type I) diabetes is predictable before hyperglycemia by the presence of islet cell autoantibodies (ICAs) and competitive insulin autoantibodies (CIAAs). CIAA+ICA+ first-degree relatives of individuals with type I diabetes have increased numbers of CD4 cells bearing the CD45R antigen and reciprocal depressions of the CD4 cells bearing the CD29 determinant. In addition, depressed CD4/CD8 ratios are present. In this study, we investigated the correlation between autoantibody levels and T-lymphocyte changes in the prediabetic state. The data demonstrate a clear linear relationship between rising CIAA levels, a marker of disease rate, and rising elevations in the CD4+CD45R+/CD4+CD29+ ratio in 37 CIAA+ICA+ and CIAA+ICA- relatives (r = 0.93). In marked contrast, the degree of CD4/CD8 depression found in individuals with prediabetes or long-term diabetes failed to correlate with either CIAA (r = 0.32) or ICA (r = 0.29) levels. The investigation of T-lymphocyte changes in siblings of individuals with type I diabetes with different stable autoantibody patterns (CIAAs and/or ICAs), and thus varying risks for diabetes, revealed differences in the prediabetic groups. Fifteen CIAA+ICA- relatives with high CIAA levels (greater than 80 nU/ml) had high CD4+CD45R+/CD4+CD29+ ratios (P = 0.03) and depressed CD4/CD8 ratios (P = 0.008). In contrast, CIAA+ICA- relatives with low CIAA levels (39-80 nU/ml), and thus low risk of diabetes, had no alteration in their CD4/CD8 ratio (P = 0.75) or CD4+CD45R+/CD4+CD29+ ratio (P = 0.33). Nineteen CIAA-ICA+ siblings with a predicted intermediate risk for diabetes showed heterogeneity in the presence of T-lymphocyte abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Faulty major histocompatibility complex class II I-E expression is associated with autoimmunity in diverse strains of mice. Autoantibodies, insulitis, and sialadenitis.

Many regions and loci of the murine genome contribute to the pancreatic and salivary gland autoimmunity observed in the diabetic NOD mouse. Examination of the major histocompatibility complex region of the NOD mouse has revealed a defect in the expression of the major histocompatibility complex class II gene, I-E. To determine the isolated role of faulty I-E expression in abnormal self-recognition, we examined six commonly used inbred strains of mice on diverse genetic backgrounds that also do not express I-E, i.e., C57BL/10, SJL, ACA, DBA/1, NOD, and 129. Autoimmunity was assessed by the presence of inflammatory cell infiltrates (0,+/-,+,++, , +) within and among the pancreatic islets and salivary glands, and autoantibodies to self determinants. At 6 mo of age, inflammatory infiltrates in the pancreas (0, 3 mice; +/-, 3 mice; +, 7 mice; ++, 6 mice; , 1 mouse; +, 5 mice) and/or salivary glands (0, 0 mice; +/-, 3 mice; +, 1 mouse; ++, 4 mice; , 6 mice; +, 10 mice) were detected as well as autoantibodies in all 24 mice of all I-E- mouse strains on diverse genetic backgrounds. This indicates that defective expression of this single locus, in isolation, is sufficient for the spontaneous development of autoreactivity. In contrast, the simultaneous examination of 19 I-E+ mice on five commonly used inbred strains of mice (BALB/c, C67/KsJ, B10.BR, B10.A [2R], and B10.A [5R]) demonstrated no signs of humoral or cellular autoimmunity with target gland destruction or lymphocytic invasion. Our data suggest that many commonly used inbred strains of mice represent models of autoimmunity attributable to this single defective gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal T-lymphocyte subsets in type I diabetes.

Type I (insulin-dependent) diabetes mellitus is a slow autoimmune disease associated with the selective destruction of beta-cells in the islets of Langerhans. Recent studies in humans indicate that autoantibodies to insulin and islets of Langerhans appear years before overt diabetes and identify a normoglycemic prediabetic state. To determine whether type I diabetes mellitus represents a generalized immunologic disorder, we studied the phenotypic characteristics of peripheral blood lymphocytes from all stages, i.e., prediabetic, new-onset diabetic, and long-term diabetic patients, with the anti-2H4 monoclonal antibody CD45R, which defines a human suppressor-inducer subset, and the anti-4B4 monoclonal antibody CDw29, which defines a human helper-inducer subset of peripheral blood lymphocytes. All 22 prediabetic patients had elevated T4+2H4+ (suppressor-inducer) cells and reciprocal depressed T4+4B4+ (helper-inducer) cells compared with healthy age-matched control subjects. In addition, the T4/T8 ratio in prediabetic patients was decreased compared with the age-matched control subjects. The abnormal T4+2H4+ and T4+4B4+ subsets were resolved in 20 new-onset and 15 long-term diabetic patients. Family studies showed that the changes in the 2H4 and 4B4 antigens were not part of an inherited polymorphic determinant, because these markers were normal in unaffected siblings and parents. Ten prediabetic patients were restudied greater than 1 yr after the original analysis and showed the persistence of the observed changes. This abnormal increase in suppressor-inducer cells and decrease in helper-inducer cells among islet and insulin antibody-positive prediabetic patients may be of help in understanding diabetes pathogenesis and may also be an early noninvasive screening tool for the detection of the prediabetic state.

Transplantation without immunosuppression.

Exciting new findings have been reported in the past few years that indicate that islets can be transplanted successfully across major histocompatibility barriers without the continuous use of immunosuppressive agents by techniques designed to eliminate passenger leukocytes. In vitro culture of donor islets either at a low temperature (24 degrees C) with a single injection of antilymphocyte serum or culture of megaislets in the presence of 95% O2 before transplantation permitted the successful transplantation of islet allografts and xenografts (rat to mouse). Definitive evidence in support of the passenger leukocyte concept has been obtained recently. Mouse islet cells have been shown to express the class I antigens of the H-2 complex, but do not express Ia antigens. Treatment of fresh mouse islets with specific anti-Ia sera and complement completely prevented rejection of islets transplanted across a major histocompatibility barrier. These findings indicate that Ia-positive cells (possibly dendritic cells) are primarily responsible for the initiation of immune rejection of the transplants. This is of particular importance with respect to the eventual transplantation of islets into human diabetics since it may be feasible to utilize antisera to HLA-DR antigens for pretreatment of islets before transplantation. In addition, these new developments may also be applicable to the transplantation of other organs such as the parathyroid, heart, kidney, liver, and skin.

New monoclonal antibody diagnostic reagents for type I diabetes: differential lymphocyte surface antigen expression related to disease.

New cellular-based reagents are needed to diagnose type I diabetes as well as to monitor the outcomes of clinical trials at early time points. Four new monoclonal antibodies (mAbs) have been shown to demonstrate reduced binding to lymphocytes from identical twins with long-term type I diabetes relative to that observed with lymphocytes from their twin partners without diabetes or from control subjects. Biochemical analysis revealed mAb 3G12EG recognized an unidentified 45-kDa protein, whereas mAb 2E8F1 and 5B6E11 did not appear to precipitate specific proteins as detected by SDS-PAGE. Electrophoresis under reducing and nonreducing conditions and peptide mapping revealed that mAb 8F410 recognizes a novel dimeric form of HLA class I molecule. Predictions from crystallography studies suggested previously this class I dimer as the optimal activation of a single CD8 T-cell. In B-cells from both normal and diabetic individuals, the class I dimer was minimally associated with beta2-microglobulin rapidly formed in the endoplasmic reticulum. These new reagents appear to be able to identify new lymphocyte surface phenotypes associated with diabetes expression in both fresh blood samples and Epstein-Barr virus-established cell lines.

Elimination of self-peptide major histocompatibility complex class I reactivity in NOD and beta 2-microglobulin-negative mice.

Nonobese diabetic (NOD) mice and beta 2-microglobulin-gene-ablated mice (beta 2M -/-) show impaired presentation of major histocompatibility complex (MHC) class I and self-peptides, structures now recognized as critical for T-cell education to endogenous peptides. The naturally occurring NOD class I presentation abnormality appears to be attributable to, in part, a quantitative defect in the production of Tap-1 mRNA; Tap-1 with Tap-2 normally functions as a transporter for stable self-peptide and class I assembly. This study attempts to reverse NOD and beta 2-M -/- mouse autoreactivity by introduced or reestablished syngeneic class I presentation. Introduction of MHC class I and self-peptides on syngeneic MHC class I-matched cells specifically prevented diabetes in NOD mice and eliminated in vitro class I-directed T-cell autoreactivity in NOD and beta 2M -/- mice. Reestablishment of endogenous class I and self-peptide presentation in NOD mice was achieved with two well-described cures for the NOD mouse, complete Freund's adjuvant and mouse hepatitis virus. Both treatments induced Tap-1 mRNA, reestablished class I presentation of endogenous antigens, and eliminated in vitro and in vivo T-cell autoreactivity of self-peptides in the class I groove. These results substantiate a therapeutic role of self-peptide complexed with class I for T-cell education and suggest that some well-described NOD treatments may work, in part, through reestablishment of tolerance through class I and self-peptide.

Bayesian analysis of case control polygenic etiology studies with missing data.

Many genetic studies are based on analysing multiple DNA regions of cases and controls. Usually each is tested separately for association with disease. However, some diseases may require interacting polymorphisms at several regions, and most disease susceptibility is polygenic. In this paper, we develop new methods for determining combinations of polymorphisms that affect the risk of disease. For example, two different genes might produce normal proteins, but these proteins improperly function when they occur together. We consider a Bayesian approach to analyse studies where DNA data from cases and controls have been analysed for polymorphisms at multiple regions and a polygenic etiology is suspected. The method of Gibbs sampling is used to incorporate data from individuals who have not had every region analysed at the DNA sequence or amino acid level. The Gibbs sampling algorithm alternatively generates a sample from the posterior distribution of the sequence of combinations of polymorphisms in cases and controls and then uses this sample to impute the data that are missing. After convergence the algorithm is used to generate a sample from the posterior distribution for the probability of each combination in order to identify groups of polymorphisms that best discriminate cases from controls. We apply the methods to a genetic study of type I diabetes. The protein encoded by the TAP2 gene is important in T cell function, and thus may affect the development of autoimmune diseases such as insulin dependent diabetes mellitus (IDDM). We determine pairs of polymorphisms of genetic fragments in the coding regions of linked HLA genes that may impact the risk of IDDM.

Strategies for circumventing transplant rejection: modification of cells, tissues and organs.

New technologies may permit the therapeutic use of transplanted cells or whole organs to be extended to the treatment of a wider range of diseases and a greater number of individuals. At present, the replacement of failing organs or the treatment of disease by transplantation is restricted by the need for co-administration of toxic immunosuppressive drugs to prevent rejection, and the limited availability of donor organs. Recent advances, however, suggest that genetically engineered or immunologically modified donor organs or cells can eliminate the need for host immunosuppression and can allow organ and cell survival across species. Transplantation could thus become a widespread therapeutic approach in the next century.

Tap-1 and Tap-2 gene therapy selectively restores conformationally dependent HLA Class I expression in type I diabetic cells.

Genetic susceptibility to many autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM) is statistically linked to the HLA class II region of chromosome 6. However, a distinguishing feature of patients with HLA class II-linked autoimmune disease is an abnormally low density of conformationally correct, self-peptide filled HLA class I molecules on the lymphocyte cell surface. The transporters associated with antigen processing (Tap-1 and Tap-2) are essential for normal class I expression and presentation of intracellular peptides, and these genes are located within the HLA class II region. The aims of this project were to determine if Tap genes could be implicated in the defective class I expression associated with IDDM by using a novel Epstein-Barr virus (EBV)-mediated gene transfer system to introduce a cloned, normal Tap-2 or Tap-1 gene into B cell lines from normal and IDDM patients and analyzing the effect on conformationally dependent class I expression. The results show that Tap-2 gene transfer in B cells from 40% of randomly selected IDDM patients increased expression of conformationally correct, cell-surface class I molecules to levels comparable with similarly treated B cells from normal control individuals. B cells from another 40% of IDDM patients responded to Tap-1 gene transfer. These effects were specific because B cells from normal individuals did not respond to Tap-1 or Tap-2 gene transfer with increased class I expression, and B cells from IDDM patients responding to Tap-2 gene transfer did not respond to Tap-1 gene transfer and vice versa. Thus, these complementation studies identify distinct, non-overlapping subsets of IDDM patients whose class I defect in B cells can be reversed by Tap-1 or Tap-2 gene transfer. The increase in class I expression induced by Tap gene transfer is associated with a reduction in the number of peptide-empty class I molecules as demonstrated by the response to exogenous peptide loading. Furthermore, the increase in self-peptide filled class I molecules induced by Tap gene transfer into B cells from IDDM patients is associated with restored antigen presentation to autologous T cells. These studies conclude that Tap gene dysfunctions may contribute to the defect in class I phenotype and antigen presentation demonstrated by IDDM patients. Defective presentation of self-peptides by antigen presenting cells can lead to the failed T cell education and tolerance to self antigens evident in IDDM. These studies functionally identify HLA class II region genes that contribute to an immunologic defect in IDDM.