Successful isolated intestinal transplantation in sensitized recipients with the use of virtual crossmatching.

We evaluated virtual crossmatching (VXM) for organ allocation and immunologic risk reduction in sensitized isolated intestinal transplantation recipients. All isolated intestine transplants performed at our institution from 2008 to 2011 were included in this study. Allograft allocation in sensitized recipients was based on the results of a VXM, in which the donor-specific antibody (DSA) was prospectively evaluated with the use of single-antigen assays. A total of 42 isolated intestine transplants (13 pediatric and 29 adult) were performed during this time period, with a median follow-up of 20 months (6-40 months). A sensitized (PRA ≥ 20%) group (n = 15) was compared to a control (PRA < 20%) group (n = 27) to evaluate the efficacy of VXM. With the use of VXM, 80% (12/15) of the sensitized patients were transplanted with a negative or weakly positive flow-cytometry crossmatch and 86.7% (13/15) with zero or only low-titer (≤ 1:16) DSA. Outcomes were comparable between sensitized and control recipients, including 1-year freedom from rejection (53.3% and 66.7% respectively, p = 0.367), 1-year patient survival (73.3% and 88.9% respectively, p = 0.197) and 1-year graft survival (66.7% and 85.2% respectively, p = 0.167). In conclusion, a VXM strategy to optimize organ allocation enables sensitized patients to successfully undergo isolated intestinal transplantation with acceptable short-term outcomes.

Investigation of the putative immunodominant T cell epitopes in coeliac disease.

BACKGROUND: Coeliac disease (CD) is an enteropathy mediated by gluten specific T cells which secrete interferon gamma (IFN-gamma) when stimulated by gluten peptides presented by HLA-DQ2 or DQ8 molecules. Residues 62-75 of alpha(2) gliadin have been proposed as the immunodominant epitope in the majority of CD patients. Deamidation by tissue transglutaminase (tTG) of the glutamine (Q) at position 65 to glutamic acid (E) is essential for T cell stimulation. AIMS: To investigate the antigenicity of this peptide and to establish whether its T cell activating properties can be downregulated by the formation of altered peptide ligands. PATIENTS: Individuals with known CD. METHODS: Peptide G4 corresponding to alpha(2) gliadin residues 62-75, Q-E65 and analogues, substituting each amino acid, except E65, in turn for alanine residues, were synthesised. Small intestinal biopsies were obtained from patients. Biopsies were cultured overnight with a peptic/tryptic digest of gliadin (PTG). Lymphocytes were cultured and restimulated with tTG treated PTG. A T cell line was cloned and clones tested for stimulation and IFN-gamma production in response to G4 and its analogues. RESULTS: Some high activity clones were isolated with, for example, a stimulation index (SI) of 15 to G4 and secreting 327 pg/ml of IFN-gamma. Substitution of amino acids at several positions abolished or downregulated stimulation and IFN-gamma production. CONCLUSIONS: Peptide G4 is highly immunogenic. Certain amino acid substitutions in peptide G4 abolish T cell reactivity while others are partial agonists which may have potential in immunomodulation in this condition.

Coeliac disease: follow-up linkage study provides further support for existence of a susceptibility locus on chromosome 11p11.

Susceptibility to coeliac disease has a strong genetic component. The HLA associations have been well described but it is clear that other genes outside this region must also be involved in disease development. Two previous genome-wide linkage studies using the affected sib pair method produced conflicting results. Our own family based linkage study of 16 highly informative pedigrees identified 17 possibly linked regions, each of which produced a result significant at p & 0.05 or less. We have now investigated these 17 regions in a larger set of pedigrees using more finely spaced markers. Fifty multiply affected families were studied, comprising the 16 pedigrees from the original genome screen plus 34 new highly informative pedigrees. A total of 128 microsatellite markers were genotyped with an average spacing between markers of 5 cM. Two-point and three-point linkage analysis using classical and model free methods identified five potential susceptibility loci with heterogeneity lod scores > 2.0, at 6p12, 11p11, 17q12, 18q23 and 22q13.3. The most significant was a heterogeneity lod of 2.6 at D11S914 on chromosome 11p11. This marker maps to a position implicated in one of the two previous genome scans and taken together these results provide strong support for the existence of a susceptibility locus in this region.

A genome-wide family-based linkage study of coeliac disease.

The susceptibility to develop coeliac disease (CD) has a strong genetic component, which is not entirely explained by HLA associations. Two previous genome wide linkage studies have been performed to identify additional loci outside this region. These studies both used a sib-pair design and produced conflicting results. Our aim is to identify non-MHC genetic loci contributing to coeliac disease using a family based linkage study. We performed a genome wide search in 16 highly informative multiply affected pedigrees using 400 microsatellite markers with an average spacing of 10 cM. Linkage analysis was performed using lod score and model free methods. We identified two new potential susceptibility loci with lod scores of 1.9, at 10q23.1, and 16q23.3. Significant, but lower lod scores were found for 6q14 (1.2), 11p11 (1.5), and 19q13.4 (0.9), areas implicated in a previous genome wide study. Lod scores of 0.9 were obtained for both D78507, which lies 1 cM from the gammaT-cell receptor gene, and for D2S364, which lies 12 cM from the CTLA4 gene.

Family linkage study of the T-cell receptor genes in coeliac disease.

BACKGROUND: The susceptibility to coeliac disease is genetically determined by possession of certain HLA DQ alleles, together with a one or more non-HLA genes. The central role of the T-cell receptor in disease pathogenesis makes the T-cell receptor genes strong candidates as disease susceptibility genes, and previous studies had provided equivocal ambiguous results. METHODS: A pedigree based genetic linkage study was used to determine if any of the T-cell receptor genes have a role in the genetic aetiology of coeliac disease. Intragenic microsatellite markers were used to study T-cell receptor alpha, beta, and delta, while gamma was studied using two flanking microsatellites D7S484 and D7S629. RESULTS: Conventional linkage analysis was performed using the MLINK computer package. Model-free linkage analysis was performed using MFLINK. No evidence of linkage between coeliac disease and the T-cell receptor genes was found in these pedigrees. CONCLUSIONS: Mutations in the T-cell receptor genes are not implicated in the genetic aetiology of coeliac disease.

Gluten-induced nitric oxide and pro-inflammatory cytokine release by cultured coeliac small intestinal biopsies.

OBJECTIVES: To determine whether there was increased nitric oxide (NO) production from coeliac small intestinal biopsies cultured in vitro with gluten and whether the inhibition of NO production could prevent gluten-induced enterotoxicity. The relationship between NO production with the pro-inflammatory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was evaluated. DESIGN: Small intestinal biopsies from ten patients with treated coeliac disease and six controls were studied. METHODS: Small intestinal biopsies were taken from each patient and set up in culture with Frazer's fraction III (FFIII), a peptic/tryptic digest of gluten, FFIII plus L-NMMA and L-NMMA alone, culture medium alone and ovalbumin which served as a control protein. The biopsies were cultured for 20 h at 37 degrees C. The supernatants were then collected and analysed for nitrite using the Greiss reaction; cytokine levels were determined using ELISA kits. Enterocyte height was determined by microscopy using a calibrated eyepiece graticule and cryostat sections of the cultured biopsies. RESULTS: Coeliac biopsies cultured with FFIII produced significantly greater nitrite compared to culture medium alone (P< 0.05) and this could be blocked with L-NMMA (P< 0.01). A reduction in enterocyte height was seen in coeliac biopsies cultured with FFIII compared to culture medium alone (P < 0.01) and this was ameliorated but not completely blocked when FFIII was cultured with L-NMMA. These changes were not seen in the controls. There was a significant reduction in IL-1beta levels in the supernatant of coeliac biopsies cultured with FFIII compared to culture medium alone (P< 0.05), but when cultured with FFIII and L-NMMA there was a significant increase in IL-1beta levels (P< 0.05). An increase in IFN-gamma levels was also seen when coeliac biopsies were cultured with FFIII and L-NMMA (P< 0.05). This pattern was not seen with TNF-alpha. CONCLUSIONS: Increased levels of NO can be found when coeliac biopsies are cultured with gluten in an in vitro small intestinal culture system, and NO may play a role in the observed enterotoxicity as the inhibition of NO production ameliorates the enterocyte damage. The results suggest that NO is involved in the regulation of pro-inflammatory cytokines, particularly IL-1beta. This is likely to be one of many pathways leading to the observed mucosal pathology in coeliac disease and demonstrates the close interactions between them.

The detection and localization of inducible nitric oxide synthase production in the small intestine of patients with coeliac disease.

OBJECTIVES: To investigate whether there are increased numbers of inducible nitric oxide synthase (iNOS) containing cells in the small intestine of patients with coeliac disease and the localization of nitric oxide synthase production. DESIGN: Small intestinal biopsy specimens from patients with coeliac disease (11 untreated, 10 treated) and nine disease controls were studied. METHODS: Histochemical staining of sections for NADPH-diaphorase activity was performed, which gives an indication of NOS activity. iNOS protein was detected with immunohistochemistry and iNOS mRNA expression was detected using in situ hybridization with an oligonucleotide probe cocktail for iNOS. Cell phenotype was detected using monoclonal antibodies to CD3 (T-lymphocytes) and CD45 (total inflammatory cell infiltrate). RESULTS: There was significantly greater NADPH diaphorase staining in the lamina propria of patients with untreated coeliac disease (P < 0.005). The same pattern was found for immunohistochemical and in situ hybridization methods of staining for iNOS in each of the patient groups (P < 0.005) but no epithelial staining was seen with any method. The pattern of iNOS staining in the lamina propria appeared in a similar distribution to that of the inflammatory cell infiltrate. At least 80% of the significantly increased total inflammatory cell infiltrate (CD45) in the lamina propria of patients with untreated coeliac disease was lymphocytic (CD3) whilst the iNOS staining cells made up less than 15% of the total inflammatory cell infiltrate. CONCLUSIONS: There is a significant increase in the number of NOS staining cells of the inducible isoform in the lamina propria of patients with untreated coeliac disease. The lamina propria and not the epithelium is the site of iNOS production in coeliac disease. It appears that inflammatory cells other than T-lymphocytes are likely to be the cellular sources of iNOS production within the lamina propria. This is the first study to demonstrate increased numbers of iNOS producing cells in the small intestine of patients with untreated coeliac disease and suggests a role for nitric oxide in the pathogenesis of the histological changes seen in coeliac disease although it may be a non-specific inflammatory response to immune activation by gluten in susceptible individuals.

Binding of gluten-derived peptides to the HLA-DQ2 (alpha1*0501, beta1*0201) molecule, assessed in a cellular assay.

The nature of the immunopathogenic relationship underlying the very strong association of coeliac disease (CD) to the HLA-DQ (A1*0501, B1*0201) genotype is not known, but probably relates to binding of gluten-derived epitopes to the HLA-DQ (alpha1*0501, beta1*0201) heterodimer (DQ2). These epitopes have not yet been defined. In this study we have tested the binding of various gluten-derived peptides to DQ2 in a cellular assay using Epstein-Barr virus (EBV)-transformed B lymphocytes and murine fibroblast transfectants. One of these peptides (peptide A), which has previously been shown to exacerbate the CD lesion in vitro and in vivo, was found to bind to DQ2, albeit only moderately, lending further credence to its possible role in the pathogenesis of CD. The nature of peptide A's binding to DQ2 was explored with truncated and conservative point substituted analogues and compared with the published DQ2 binding motif, the results of which explain the observed level of binding.

Measurement of gluten using a monoclonal antibody to a coeliac toxic peptide of A-gliadin.

BACKGROUND: Future European Community regulations will require a sensitive and specific assay for measurement of coeliac toxic gluten proteins in foods marketed as gluten-free. To avoid spurious cross reactions with non-toxic proteins, specific antibodies and target antigens are required. A synthetic 19 amino acid peptide of A gliadin has been shown to cause deterioration in the morphology of small intestinal biopsy specimens of coeliac patients in remission. AIMS: To develop an assay for detection of gluten in foods, based on measurement of a known toxic peptide. METHODS: A monoclonal antibody raised against the toxic A gliadin peptide, with a polyclonal anti-unfractionated gliadin capture antibody, was used to develop a double sandwich enzyme linked immunosorbent assay (ELISA) for the measurement of gluten in foods. RESULTS: Standard curves for gliadin and for rye, barley, and oat prolamins were produced. The sensitivity of the assay was 4 ng/ml of gliadin, 500 ng/ml for rye prolamins, and 1000 ng/ml for oat and barley prolamins. The assay could detect gluten in cooked foods, although at reduced sensitivity. Prolamins from coeliac non-toxic rice, maize, millet, and sorghum did not cross react in the assay. A variety of commercially available gluten-free foods were analysed; small quantities of gluten were detected in some products. CONCLUSION: The assay may form the basis of a sensitive method for measurement of gluten in foods for consumption by patients with coeliac disease.

Detection and characterisation of anti-endomysial antibody in coeliac disease using human umbilical cord.

OBJECTIVES: To verify the effectiveness of human umbilical cord (HUC) in the detection of anti-endomysial antibodies (AEA) in coeliac disease and to characterize further these antibodies by studying tissue adsorption characteristics and antibody inhibition studies. METHODS: AEA were detected on HUC and primate oesophagus in a blind study, using sera from 46 patients with untreated coeliac disease and 108 controls. Tissue adsorption studies were performed using homogenized tissue from rodent liver, HUC, primate oesophagus and human liver. Sera were adsorbed with each of these homogenates and antibody was detected using HUC, primate oesophagus and rat kidney. In the inhibition experiments AEA was detected on HUC, and inhibition of binding was attempted by pre-incubating the sections with antibodies against collagen types I, III and IV. RESULTS: The sensitivity of AEA was 91% when detected on HUC, 89% when detected on primate oesophagus (93% and 91%, respectively, after exclusion of 1 patient with IgA deficiency). Specificity was 100% for both assays. Tissue adsorption studies showed identical results for AEA detected on both HUC or primate oesophagus, whereas antireticulin antibody was adsorbed only by rodent tissue. Blocking of the HUC with anticollagen antibodies did not prevent binding of AEA. CONCLUSIONS: HUC is an effective substrate for the detection of AEA and may be superior to primate oesophagus. The antibody detected by HUC shows identical tissue adsorption specificities to that detected on primate oesophagus.

Analysis of interleukin-4 and interleukin-10 and their association with the lymphocytic infiltrate in the small intestine of patients with coeliac disease.

BACKGROUND: Concentrations of pro-inflammatory cytokines are raised in the small intestine of patients with coeliac disease after ingestion of gluten but there are equivalent data on interleukin-4 (IL-4) and interleukin-10 (IL-10) producing cells. These cytokines are known to exert important regulatory effects on pro-inflammatory cytokine production from lymphocytes and macrophages. AIMS: To investigate whether there is a primary deficiency of IL-4 and IL-10 producing cells and their site of production in the small intestine of patients with coeliac disease in relation to the changes in inflammatory cell infiltrate. PATIENTS: Jejunal biopsy specimens from patients with coeliac disease (11 untreated, 10 treated) and nine disease controls were studied. METHODS: Immunohistochemical staining of sections for IL-4 and IL-10 cytokines and the cell phenotypic markers CD3 (T lymphocytes) and CD45 (total inflammatory cell infiltrate) was carried out using monoclonal antibodies. Expression of IL-4 and IL-10 messenger RNA was detected by in situ hybridisation with oligonucleotide probe cocktails for each cytokine. RESULTS: IL-4 and IL-10 mRNA and protein were detected in the lamina propria of treated and untreated coeliac patients and disease controls but not in the epithelium. A significant increase in the number of CD45 (p < 0.005) and CD3 (p < 0.05) positive cells was found in the lamina propria of patients with untreated coeliac disease compared with treated coeliac patients and disease controls but there were no differences in IL-4 or IL-10 between these groups with either method. CONCLUSIONS: There is no primary deficiency of IL-4 and IL-10 producing cells in the small intestine of patients with coeliac disease. Detectable concentrations of IL-4 and IL-10 were found in control patients which suggests that these cytokines are involved in normal mucosal immunoregulation. The increased number of T lymphocytes but not IL-4 or IL-10 producing cells in the lamina propria of patients with untreated than in those with treated disease suggests not only that the lamina propria is the major mucosal compartment for cytokine production but that newly recruited mucosal T lymphocytes are directed to a predominant Th1 and not a Th2 cytokine response in coeliac patients on a diet containing gluten.

Diverse usage of human T-cell receptor gene segments in HLA-DR1 allospecific T-cell clones.

T-cell recognition of alloantigen involves both the MHC molecule and its associated peptide ligand. To understand the relationship between the specificity of alloantigen recognition and the structure of TCR molecules, we have investigated TCR gene utilization by sequencing TCR genes from well-defined allospecific T-lymphocyte clones. Alloreactive TLC consisted of a panel of clones primed to recognize DR1-related alloantigens. Our sequencing results revealed extensively diverse, but nonrandom, usage of TCR AV and BV gene segments and essentially no conservation in CDR3 or junctional sequences. Such observations are consistent with allospecific TCR that interact with MHC molecules on a generic level while recognizing specific peptides. They also reduce potential enthusiasm for anti-TCR therapy in allograft rejection.

The relative importance of individual DR binding motif positions as defined by peptide anchor analysis of influenza hemagglutinin peptide 306-318 and human myelin basic protein peptide 152-165 binding to several DR molecules: definition of a common extended DR binding motif.

Definition of peptide binding motifs for DR molecules has proven difficult as the peptides that bind to a DR molecule have shown extensive variability at putative motif positions. Recent studies suggest that specific peptide anchor residues (motif positions) and specific DR residues can differ in importance for peptide binding to a DR molecule. To assess further the relevance of individual peptide anchor residues, the binding of serial alanine-substituted analogs of influenza virus hemagglutinin (HA) 306-318 and human myelin basic protein (MBP) 152-165 to a panel of transfected wild-type DR molecules was examined. This analysis included DR molecules from a wide range of allelic families and, unlike most earlier studies, multiple members of single DR allelic families. The data show that different peptide residues serve as critical anchors for binding to different DR molecules. For example, MBP binding to DR(alpha, beta 1*0303) required peptide residues F154 (i), R159 (i + 5) and R162 (i + 8). In contrast, MBP binding to DR(alpha, beta 1*0102) required peptide residues I153 (i) and L156 (i + 3). More importantly, the combination of critical anchor residues in HA and MBP differed for binding to a single DR molecule [e.g. V309 (i) for HA and I153 (i) and L156 (i + 3) for MBP binding to DR(alpha, beta 1*0102)]. Although the location of the binding pocket in each DR molecule compared to the DR (alpha, beta 1 *0101) crystal is expected to be similar and suggests a common extended DR binding motif, the present results suggest that the relative importance of individual peptide anchor residues and of the corresponding DR binding pockets will differ for each DR/peptide complex.

Different requirements of ICAM-1/LFA-1 adhesion in allorecognition and self-restricted antigen recognition by class II-specific T cell clones.

We have analyzed the influence of non-antigen-specific interactions between ICAM-1 and LFA-1 in target recognition by allospecific and antigen-specific T cells at the clonal level, using human and mouse fibroblasts transfected with HLA-DR1 or DR2 with or without co-expression of ICAM-1, as antigen-presenting cells. The results show a great heterogeneity in the requirements for ICAM-1/LFA-1 interactions for antigen-specific and alloreactive T cell responses and this requirement may depend on the avidity of any particular interaction. The data also show that for most alloreactive clones, ICAM-1/LFA-1 adhesion is not sufficient to facilitate efficient T cell recognition of its target molecule. HLA class II recognition by a large proportion of the DR1- and DR2-specific alloreactive clones studied was different for class II molecules expressed on murine or human fibroblasts compared to human lymphoid cells, and was independent of ICAM-1 expression on the stimulator cells. The inability of some T cell clones to recognize HLA-class II expressed on non-lymphoid cells suggests the absence of specific epitopes and could be due to the lack of the relevant peptides, either because they are derived from species-specific proteins or to differences in processing of endogenous antigen in the transfected stimulator cells.

HLA-A*8001 is a member of a newly discovered ancient family of HLA-A alleles.

A member of a new HLA-A locus family, HLA-A*8001, has been characterized from 3 African-American individuals expressing a unique HLA-A serologic specificity. The HLA-A*8001 sequence is most closely related to alleles of the HLA-A1/3/11 family, although it also contains residues characteristic of the HLA-A2/28, -A9, -A10, and -A19 families. More importantly, the HLA-A*8001 sequence contains four unique nonsynonymous (amino acid replacing) nucleotide substitutions absent from all other primate A locus alleles. In addition, five other nucleotide substitutions, four nonsynonymous and one synonymous (silent), observed only in non-human primate A locus alleles were found. Neighbor-joining tree analysis of HLA-A*8001 supports the notion that the HLA-A*8001 allele is a member of a new HLA-A locus family which was derived from the ancestral A3 lineage but diverged early in the evolution of the HLA-A locus.

Nonrandom T cell receptor usage in the allorecognition of HLA-DR1 microvariation.

Microvariation within the DR1 Ag family has created two DR molecules which differ only at beta-chain residues 85 (Val/Ala) and 86 (Gly/Val). TCR utilized by human alloproliferative T lymphocyte clones which can distinguish between these microvariants have been characterized by cDNA sequencing. The alpha- and beta-chain cDNA utilize a diverse set of variable (V) gene segments although the same V segment may be used by different individuals suggesting that V segment usage by the alloreactive T lymphocyte clones is nonrandom. There appears to be no difference in the repertoire of V segments utilized by T lymphocytes that preferentially recognize specific DR1 allelic products (DR(alpha,beta 1*0101) or DR(alpha,beta 1*0102)) and T lymphocytes that recognize both DR1 molecules. In contrast, the junctional regions of both alpha- and beta-chains are diverse in length and sequence although some common elements can be observed among TCR which share V gene segments. Two TCR which share V alpha and V beta gene segments differ in fine specificity for specific DR1 allelic products implicating the junctional regions of alpha- and beta-chains in the recognition of differentially bound peptides and/or in recognition of DR beta-chain residues 85 and 86. The stimulation of many diverse TCR by the limited allelic variation between DR(alpha,beta 1*0101) and DR(alpha,beta 1*0102) molecules suggests that the effect of DR microvariation on human immune responsiveness may be substantial.

Peptide-induced nonresponsiveness of HLA-DP restricted human T cells reactive with Dermatophagoides spp. (house dust mite).

The activation of CD4+ T lymphocytes, which play a central role in allergic inflammation, depends on the recognition of allergen-derived peptides in association with major histocompatibility complex class II gene products. In this report we demonstrate, at a clonal level, that a component of the T-cell repertoire reactive with Dermatophagoides spp. (house dust mite) in atopic individuals, is restricted by HLA-DP class II molecules. This supports the recent results emerging from genetic epidemiologic studies that indicate positive associations between the HLA-DP phenotype and immune responsiveness to a variety of common allergens. Our findings also reveal that the T cells restricted by HLA-DP recognize a species-specific epitope located in the group I allergen of Dermatophagoides pteronyssinus (residues 101-119). Furthermore, we report that the pretreatment of the T cells restricted by HLA-DP with the Der p I peptide renders them nonresponsive to an immunogenic challenge with house dust mite allergen, and the loss of antigen-dependent proliferation is associated with downregulation of membrane expression of the T-cell antigen receptor. The ability to functionally inactivate T cells restricted by HLA-DP, as well as those that recognize allergen in association with HLA-DR class II molecules, suggests that desensitization with allergen-derived peptides may have therapeutic potential in the management of allergic diseases irrespective of their HLA class II association.

Efficient cDNA expression vectors for stable and transient expression of HLA-DR in transfected fibroblast and lymphoid cells.

cDNA expression vectors with several useful features were constructed. First, the long terminal repeat of Rous sarcoma virus was used as a promoter to obtain high levels of expression in various cells of human and mouse origin. Second, cis-linked expression units that confer resistance either to mycophenolic acid or the neomycin analog G418 were inserted to facitate the isolation of transfected cells expressing the cDNA of interest. Third, by replicating in simian COS cells, these vectors can be used for efficient transient expression. cDNA fragments encoding the DR alpha or DR beta chains of human class II major histocompatibility complex antigens were inserted into these vectors and high levels of cell surface HLA-DR antigen were obtained after cotransfection into mouse and human fibroblasts. These vectors were also successfully used to correct the inability of a class II-negative B cell line, derived from a patient with a congenital immunodeficiency, to present peptide antigen to DR-restricted T cells.

Polymorphic HLA-DR7 beta 1 chain residues that are involved in T cell allorecognition.

The contributions to allorecognition of polymorphic amino acids in the HLA-DR7 beta 1 chain were analyzed by using mutant DR7 beta 1 chains with single amino acid substitutions at position 4, 11, 13, 25, 30, 37, 57, 60, 67, 70, 71, 74, or 78. Transfectants expressing mutant DR7 molecules were used as stimulators for six DR7-alloreactive T cell clones. The majority of the substitutions had profound effects on the ability of the DR7 molecule to stimulate one or more T cell clones. Nine of the 13 substitutions completely abrogated recognition by at least one clone. The finding that each of the substitutions in the beta-strands in the floor of the peptide binding groove affected T cell allorecognition supports the model of allorecognition in which the complex of a self-peptide bound to a class II molecule is recognized by the TCR. Interestingly, the substitution at position 4, which is predicted to be located outside the peptide binding groove, decreased the ability of the DR7 molecule to stimulate some clones. Each of the DR7-alloreactive T cell clones had a unique reactivity pattern in response to the different mutant molecules, indicating that the TCR of each clone recognized the DR7 molecule differently. Surprisingly, many of the mutant DR7 molecules induced proliferation by one or more clones that was greater than 125% of the proliferation induced by the wild-type DR7 molecule. These data indicate that multiple polymorphic residues, predicted in the class II model to be located in both the beta-strands and alpha-helix of the DR7 beta 1 chain, contribute to allorecognition of the DR7 molecule.