Characteristics of graft-infiltrating lymphocytes after human heart transplantation HLA mismatches and the cellular immune response within the transplanted heart

The influence of HLA mismatches between donor and recipient on the phenotypes, function, and specificity of T-lymphocyte cultures derived from endomyocardial biopsies was studied in 118 heart transplant recipients. In case of HLA-DR mismatches, the majority of the EMB-derived cultures were dominated by CD4⁺ T cells while, in patients with HLA-A and -B mismatches but without DR mismatches, CD8⁺ T cells comprised the predominant T-cell subset. Cytotoxicity against donor antigens was observed in 75% of the cultures. A significantly (p < 0.005) lower proportion of the cultures showed cytotoxicity against HLA-A antigens (36%) when compared with HLA-B (53%) or HLA-DR (49%). An HLA-A2 mismatch elicited a cytotoxic response that was comparable to that found against HLA-B and -DR antigens: 62% of the cultures from HLA-A2 mismatched donor-recipient combinations was reactive against A2. A higher number of A, B, or DR mismatches resulted in a higher number of cytotoxic cultures directed against these antigens. A higher number of HLA-B and -DR mismatches was associated with a lower freedom from rejection. Our data indicate that, despite the use of adequate immunosuppressive therapy, the degree of HLA matching plays a crucial role in the immune response against a transplanted heart, resulting in a significant effect on freedom from rejection.     

Naturally processed HLA-DR bound peptides of fibroblast cell lines as facultative antigen-presenting cells

Beside professional APC, fibroblasts and epithelial cells may gain antigen presenting capacity after induction of MHC-class II antigens with γ-interferon. Fibroblast cell lines derived from normal kidney biopsies were investigated for HLA-class II bound peptide after stimulation with γ-interferon and compared with naturally processed peptides bound to similar HLA-class II alleles on EBV-transformed B cell lines. Three major naturally processed peptides acid extracted from immunoaffinity-purified HLA-DR molecules were identified by mass spectrometry and Edman microsequencing. One exogenous peptide of the HLA-DRB1^*07x/HLA-DRB1^*1303 cell line originated from AA 80-97 of invariant chain, and two peptides of the HLA-DRB1^*0101/HLA-DRB1^*0801 cell line were derived from the endogenous transferrin-receptor (AA215-232) and the bovine Apolipoprotein B-100 (AA 1942-1956) which both possessed the characteristic anchor at relative positions 1, 6 and 9 for HLA-DRB1^*0101. Corresponding synthetic peptides showed high binding to HLA-DR1 or HLA-DR7 in a high-performance size exclusion chromatography binding assay. Thus professional and facultative APC do not differ in dominant HLA-DR associated peptides.     

A peptide from the known HLA class I restricted melanoma-specific tumor antigen GP100 is presented by HLA-DR in the melanoma cell line FM3

A number of HLA-class I restricted melanoma tumor antigens such as MAGE-1, MAGE-3, MART-1/Melan-A or gp100 have been characterized but there is thus far no direct proof if peptides derived from these antigens are also presented in vivo by HLA-class II molecules. To examine this we cultured, up to 1 × 10¹⁰ cells of the constitutively class II and CD80 expressing melanoma cell line FM3 (DR B1^*0401, DR B1^*15x, DQ B1^*0301, DQ B1^*0602) that has been shown to be able to serve as antigen presenting cells, lysed the cells, isolated the HLA-DR and HLA-DQ by immunoaffinity chromatography and released the MHC-bound self peptides by a shift to pH 2. The peptide pool was separated by HPLC using a C18 reversed phase column. The peaks were collected and tested for purity by matrix assisted laser desorption spectrometry (MALDI). Homogeneous peaks were sequenced by Edman degradation, inhomogeneous peaks were further purified by rechromatography before submitting to the sequencer.

In this way we were able to find amongst other peptides a HLA-DR-bound 16mer peptide derived from the melanocyte specific protein gp100 with the sequence WNRQLYPEWTEAQRLD. To prove the specific binding of this peptide to one of the two DR alleles we synthesized the peptide and submitted it to our in vitro peptide binding/competition assay using gel filtration, isolated HLA-DR alleles and a fluorescently labeled peptide. This is the first reported HLA-DR-restricted epitope of a known tumor-associated antigen in melanoma.     

An invariant chain peptide different from the clip region is a dominant self peptide of HLA-DP1

In contrast to HLA-DR and a few HLA-DQ alleles there is little information about HLA-DP associated peptides due to the relative low expression of HLA-DP on antigen presenting cells.

Starting from 50×10⁹ cells of the EBV-transformed B cell line WT 100 (DPA1^* 0201 DPB1^*0101), we have been able to isolate 300 ug of solubilized HLA-DP from the intracellular fraction by affinity chromatography on a B7/21 (anti HLA-DP) column. Peptides were then eluted by acidification and separated by reversed phase chromatography on a micro bore HPLC. Nine different peptides with a length of 15-46 amino acids were identified by Edman degradation. Five of these peptides were derived from HLA-B or HLA-DR molecules, one from a ribosomal protein and one peptide that could not be identified. Most interestingly, we found two peptides with an identical core region from the class II associated invariant chain which is different from the CLIP region.

Using our standardized HPSEC binding/competition assay with fluorescent labeled self peptides, we will present data for the agretopic conditions for peptide association, pH optimum and kinetic measurements.

As a main topic we will discuss the specific properties of our new dominant li self peptide in the contex of the well known CLIP peptide.     

The HLA-DR phenotype of the responder is predictive of humoral response against HLA class I antigens

Recent studies suggest that the immunogenicity of an human leukocyte antigen (HLA) incompatibility should be considered in the context of the HLA phenotype of the recipient. The HLA-DR phenotype of the responder is thought to be predictive for the strength of the alloimmune response. In order to analyze the humoral response against HLA class I antigens in the context of the HLA-DR phenotype of the responder, we selected all HLA-DR homozygous Dutch patients that were present on the Eurotransplant waiting list between 1967 and 2000 (n=1,317 patients). By logistic regression it was determined whether antibody production against a specific HLA class I antigen is associated with a particular HLA-DR antigen in the patient. Furthermore, it was analyzed whether a patient, expressing a particular HLA-DR antigen, preferentially produces antibodies against particular HLA class I antigens. The results demonstrate that patients, homozygous for a certain HLA-DR antigen, cannot be considered high or low responders when analyzing the antibody response in terms of panel reactive antibody (PRA) value. However, a correlation can be found between the HLA-DR phenotype of the patient and the specific antibody response against HLA class I antigens. For example, antibodies against HLA-A10, -A11, -A19, and -B35 are produced more frequently by HLA-DR6 positive individuals, whereas antibodies against HLA-A3, -B5, -B7, -B8, and -B12 are produced more frequently by HLA-DR4 positive individuals. These data confirm that the HLA-DR phenotype of the responder plays a determinative role in the immunogenicity of mismatched HLA antigens. The results indicate that selection of HLA class I mismatches of the donor in the context of the HLA-DR phenotype of the responder might reduce the incidence of humoral graft rejection and minimize the sensitization grade of retransplant candidates.     

HLA-A, B and DR antigens in patients with gonadal dysgenesis

HLA (human leukocyte antigens) antigens A, B, and DR were determined in a series of 50 patients with gonadal dysgenesis (GD), separated into different groups according to karyotype. There were no significant differences in frequency of HLA antigen types between GD patients and the population control. When frequencies of the HLA antigens in the various GD patient groups by karyotype were compared, only one significant difference was found: HLA-A3 was more common among GD patients with isochromosome X than among GD patients with karyotype 45, X (p<0.001, corr. p<0.008). Although GD patients have a higher expectancy for development of autoimmune disorders, and in our 50 patients thyroglobulin and/or microsomal antibodies were detected in 20 (i.e., 40%), we failed to find any increased frequency of specific HLA antigen types known to be associated with juvenile autoimmune thyroiditis.     

HLA-C allele frequencies and linkage disequilibrium in 604 random Caucasian donors by high resolution PCR-SSP.

Sequence-specific PCR (PCR-SSP) methods have been successfully used to type HLA-C to a medium resolution level through the use of group-specific amplifications. We have developed high resolution PCR-SSP utilising 48 allele and group-specific PCR reactions to define HLA-C to the allelic level in order to find out the true level of polymorphism in a random Caucasian population and hence define the linkage disequilibrium between HLA-A, HLA-B and HLA-C.

604 consecutive samples from random individuals consisting of 552 cadaver donors and 52 random blood donors were typed by high resolution PCR-SSP. The PCR conditions and parameters are described in Tissue Antigens 95, vol. 46 pg 355. Cw^*0101, ^*0201, ^*0302, ^*0402/3, ^*0703, ^*0801/3, ^*1201,^* 1301, ^*1401,^* 1403 ^*1501/3/4 were not detected in our population although we have detected Cw^*0302, ^*1403, ^*0801 and ^*1504 in other populations. Cw7 is the most common Caucasian HLA-C antigen with frequencies of 30, 28 and 3.8% for Cw^*0701,^* 0702 and ^*0704 respectively. Cw^*0701 is in linkage disequilibrium with B8, B49 and B18, Cw^*0702 is associated with B7 and B39 whilst Cw^*0704 is associated with B44 and B^*1518. Complete frequencies, linkage disequilibria, statistics and full information on the primer mixes will be given.     

Increased Frequency of HLA-DR/v3 in Systemic Lupus Erythematosus

The distribution of HLA antigens was studied in 40 patients suffering from systemic lupus erythematosus. For loci A and B, increased frequencies of A1, B8 and the presence of only one B antigen were found. Nevertheless, in relation to the number of antigens tested, this increase was not significant. For the DRw locus, DRw3 was significantly increased (P < 0.001, after correction for the 39 antigens tested). However, the patients with DRw3 did not show any correlation with a specific clinical picture or the presence in their serum of lymphocytotoxic antibodies or autoantibodies against T and B lymphocytes.     

Application of Mouse Antibodies to Somatic Antigen for Detection of Salmonella enteritidis by Competitive ELISA

Competitive ELISA estimation based on application of polyclonal mouse antibodies to somatic antigen O:9, 12 was developed. The optimization of the protocol is reported. The optimal concentration of immobilized somatic antigen O:9, 12 was found to be 4.9 ×10⁴ cells ml^-1; optimal concentration of mouse IgG was 6.25 μg ml^-1; and the optimal concentration of peroxidase labelled antibody to mouse IgG was 8 μg ml^-1. The tested antibody exhibited neither cross reactions with chosen strains (serotypes) of salmonellas group 04 (B), 07 (C₁), 08 (C₂-C₃), nor with members of Enterobacteriaceae: Escherichia coli, Klebsiella pneumonia, Citrobacter freundii and non-fermenting bacterium Pseudomonas fluorescens. Application of chemiluminiscent substrates increased the sensitivity of S. enteritidis detection up to three times. Competitive ELISA tested on model samples produced results comparable with standard cultivation techniques for Salmonella spp.     

Frequency of HLA-DPB1 alleles, including a novel DPB1 sequence, in the Northern Ireland population

HLA-DPB1 allele frequencies in 150 unrelated normal individuals from Northern Ireland were determined using oligonucleotide typing methods. HLA-DPB1^*0401 was the most common allele in the population possessed by 75.3% of subjects, followed by DPB1^*0201 (20.7%). In addition to these alleles, only HLA-DPB1^*0402, -DPB1^*0301, and -DPB1^*0501 were present in subjects at frequencies greater than 10%. The results in this study are in broad agreement with other Caucasoid studies, but there is regional and ethnic variation in HLA-DP allele frequencies. Three DPB1 alleles were found to be in linkage disequilibrium with HLA-DR antigens determined by RFLP, namely, DPB1^*0101 with DRw17 (Dw24 associated) REFLP, DPB1^*0501 with DRw13-Dw19 RFLP, and DPB1^*1901 with DRw13-Dw18 (Dw25 associated) RFLP. One individual revealed a novel DPB1 pattern of probe reactivity, which following DNA sequencing was found to be HLA-DPB1^*2001. To assess the system used and to compare consistency of results between laboratories, 62 cell lines were oligotyped for HLA-DP. The results revealed the system described here to be extremely accurate and showed excellent aggreement of HLA-DP typing results for cell lines between laboratories.     

Monoclonal antibodies against serogroup B salmonellae: production, characterisation and use in a sandwich ELISA

Ten monoclonal antibodies (MAbs) against serogroup B salmonellae were obtained after immunisation of BALB/c mice with outer membrane proteins (OMPs) from Salmonella enterica serovar Typhimurium. Affinity constants, measured by enzyme-linked immunosorbent assay (ELISA), ranged from 8×0⁵ to 6×0⁷ l mol^-1. Additivity ELISA demonstrated that most MAbs recognise different epitopes. ELISA determined the antigen specificity of the MAbs. The MAbs were more reactive with live Salmonella Typhimurium than with heat-treated cells. Two MAbs were used to develop a sandwich ELISA for rapid detection of Salmonella Typhimurium in experimentally contaminated meats. Its detection limit was 10⁵ CFU ml^-1 and it was able to detect 1-10 CFU in post-enrichment broth from 25 g of beef and chicken meat samples. The sandwich ELISA developed was shown to be specific and sensitive for the detection of Salmonella Typhimurium in meat, and produced results comparable to the culture methods in 57 h.     

HLA linked immune response to S and pre-S2 gene products in hepatitis B vaccination

In order to detect a possible HLA linked genetic control of human immune responses to hepatitis B virus, forty healthy adult persons of the same age typed for HLA-A, -B and -DR antigens, were vaccinated against virus hepatitis B and sequentially tested for anti-HBs and anti-pre-S2 antibodies. They received three injections of Hevac-B Pasteur vaccine, the second 1 month and the third 3 months after the first. Following the third immunization, 38 individuals (95%) had a protective level of anti-HBs antibodies and 17 (42.3%) had a positive level of anti-pre-S2 antibodies. HLA-A11 antigen was significantly more frequent (pc = 0.007) among anti-HBs high responders than low responders. In addition, anti-HBs high responders were more frequently HLA-DR1, and less frequently HLA-DR4 and DR7 positive; corrected values, however, were not significant. Anti-pre-S2 high responders showed an apparent increase of HLA-B7, B14 or DR3 antigens, when compared to low responders (pc not significant).     

Down-Regulation of CD25 Expression on the Surface of Activated Tumor-Infiltrating Lymphocytes in Human Cervical Carcinoma

ABSTRACT: To investigate the activation status of tumor-infiltrating lymphocytes (TILs) within the tumor milieu of human cervical carcinoma, we quantitatively measured and compared the activation markers on lymphocyte subpopulations which infiltrating normal and neoplastic cervix. A total of 20 patients with stage IA to IIA cervical cancer (cancer group) and 10 women with normal cervix (control group) were enrolled in this study. Mononuclear cells were isolated from tissue specimens by mechanical dispersal technique and three-color flow cytometry was utilized for the quantification of activation markers on lymphocyte subsets. Compared with control group, lymphocytes isolated from cancer tissue consisted of higher proportions of B cells (7.23% ± 4.49% vs. 3.67% ± 3.19%, P = 0.016) and T cells (72.33% ± 8.70% vs. 53.15% ± 17.36%, P = 0.004), but an inverted CD4:CD8 ratio (0.74 ± 0.27 vs. 1.14 ± 0.28, P = 0.002) and decreased NK cells (7.53% ± 4.33% vs. 16.00% ± 11.82%, P = 0.035). Low expression of CD25, but not CD69 and HLA-DR was observed on both CD4⁺CD3⁺ and CD8⁺CD3⁺ T cells derived from cervical cancer (P < 0.0001). Further dual activation marker analysis demonstrated that the expression of CD25 was dissociated from CD69 and HLA-DR on the same TILs in cancer tissue (P < 0.001). TILs in the tumor microenvironment can be functionally inhibited and lose the ability of clonal proliferation due to depressed expression of CD25.     

CD8 is involved in both class I- and class II—induced proliferation of a cytolytic T-cell clone with dual specificity for HLA-B27 and HLA-DR2 antigens

A CD3⁺ CD4⁻ CD8⁺ cytolytic T-lymphocyte (CTL) clone, CTL 47, could be induced to proliferate in the presence of exogenous interleukin 2 by either HLA-B27.1⁺ or HLA-DR2⁺ cells. B27.1-induced proliferation was strongly and equally inhibited by an anti-B27 and by an anti-CD8 monoclonal antibody (MoAb). DR2-induced proliferation was inhibited by the same anti-CD8 MoAb less efficiently and with a different time course than anti-class II blocking, only being significant when the antibody was added ab initio or very early during the assay. These results indicate that CD8 is essential for class I—induced proliferation but that it also enhances class II—induced stimulation of this CTL clone. It is proposed that the necessary role of CD8 in class I—induced proliferation is related to its interaction with the same class I molecule bound by the T-cell receptor. The accessory role in class II—induced proliferation would be due to an additive effect on the avidity of cell adhesion, resulting from interaction of CD8 with the class I antigens on the stimulator cell, or perhaps to a regulatory role of CD8 as a transducer of early signals for T-cell activation.     

Strong association between an HLA-DR antigen and thyroid carcinoma

A group of 20 patients affected with thyroid carcinoma, all from Southern Italy, was typed for HLA-A,-B,-C and -DR antigens. Sixteen of them (80%) typed for DR1, while in the control group (120 unrelated healthy individuals from the same region) only 22 (18.3%) shared the same antigen ( x ²2 = 32.96). The data indicate a highly significant association between the HLA-DR1 gene and the thyroid carcinoma.     

Histocompatibility antigens in progressive systemic sclerosis (PSS; scleroderma)

Patients with progressive systemic sclerosis (PSS; scleroderma) were typed for the HLA-A, -B, and -DR antigens. No significant differences in the frequencies of any HLA-A or -B antigen were found. In the subgroup of patients with PSS and diffuse scleroderma (PSS-DS), the frequency of Bw35 was increased (0.30 vs 0.17 in controls;P<0.005, correctedP>0.2). Although patients with PSS-DS also had an increased frequency of DR1 antigen (0.27 vs 0.12 in local controls;P<0.005, correctedP<0.05), no association between Bw35 and DR1 antigens could be detected. We found no increase in the frequencies of the DR3 or DR5 antigens in patients with PSS. However, in a subset of PSS patients with pulmonary fibrosis, an increase in DR3 and a decrease in DR4 antigens (P<0.005) were observed. Serum antibodies to centromere occurred more frequently in DR1-positive than DR1-negative patients (0.46 vs 0.18;P<0.005). This study of a large number of patients with PSS failed to confirm previously reported associations of PSS with the HLA-B8/DR3 haplotype or HLA-DR5 antigen.     

Endogenous retroviral long terminal repeats of the HLA-DQ region are associated with susceptibility to insulin-dependent diabetes mellitus

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 HERV-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 × 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. Human Immunology 50, 103–110 (1996)     

Planned immunization of human volunteers with HLA-DR incompatible lymphocytes

Aiming at the production of anti HLA-DR test sera, eight healthy human volunteers were immunized by repeated intradermal injections of lymphocytes which were selected to be incompatible for one HLA-DR antigen, and matched as well as possible for HLA-A,-B,-C antigens. One out of 3 recipients immunized exclusively against HLA-DR produced lyrnphocytotoxic HLA-DR antibodies. The remaining 5 recipients were immunized against 1 or more HLA-A,-B,-C antigens in addition to one HLA-DR antigen. After 3 immunizations, 3 of these reacted with strong HLA-A or -B antibody production; however, only one showed a parallel anti HLA-DR antibody response detectable by complement dependent lymphocytotoxicity.
Testing of the recipient sera in the antibody dependent cell-mediated cytotoxicity (ADCC) assay revealed that 6 of the 8 recipients did react early to the immunizations with HLA specific antibody production. However, in spite of repeated booster injections it was not possible to obtain more than the above-mentioned 2 sera with HLA-DR antibodies strong enough to react in the lymphocytotoxicity microtechique.     

Homozygosity at the dopamine D3 receptor gene in schizophrenic patients

Dopamine receptors have long been implicated in the etiology of schizophrenia. It has been reported an association of schizophrenia with homozygosity at the dopamine D3 receptor gene locus. We have investigated the distribution of a D3 receptor gene polymorphism (BalI) in 107 schizophrenic Spanish patients and 100 healthy matched controls. No statistically significant differences between the patients and control group were detected with respect to either allele frequencies or genotype distribution. However, if not corrected for multiple testing, a correlation was found between homozygosity and early age of schizophrenia (χ² = 3.1, df = 1, P = 0.03) and between A1 allele frequency and disorganized and undifferentiated schizophrenia (χ² = 3.4, df = 1, P = 0.03; χ² = 2.7, df = 1, P = 0.05, respectively). These results suggest the possibility that D3 polymorphisms may be among the physiological factors underlying schizophrenia; though not the determining factor.     

Triggering of T cell-mediated immune responses by allogenic tumor cell vaccine in patients with oral cancer

We investigated the immunomodulatory activity of allogenic whole tumor cell vaccine in oral cancer patients in vitro by two-color flow cytometry. Vaccine treatment significantly increased the expression of CD69 and HLA-DR in CD3⁺ T-cell subsets. The frequency of Interferon-gamma and Interleukin (IL)-2 expressing CD4⁺/CD8⁺ T-cell subsets was significantly higher with a concomitant reduction in IL-4 and IL-10 expressing T-cells in the vaccine treated group as compared with the untreated controls. Vaccine treatment significantly increased T-cell receptor (TCR), Vβ3, Vβ5 and Vβ8 usage. The results indicate that the allogenic whole tumor cell vaccine is able to trigger T-cell mediated immunity in patients with intraoral squamous cell carcinoma.