HLA-A2 reactive antibodies in a patient who types as HLA-A2: The importance of high resolution typing and epitope-based antibody analysis.

This report describes a case of a highly sensitized patient who had serum antibodies reacting with HLA-A2 but whose phenotype included HLA-A2. The determination of HLA mismatch acceptability at the antigen level was problematic, but high-resolution HLA typing information and epitope-based antibody specificity analysis based on the nonself-self paradigm of HLA epitope immunogenicity have provided a solution. This case supports the concept that HLA typing at the allele level offers a better approach to identifying suitable donors for sensitized patients.

Promoter-region alleles of the TNF-alpha and IL-10 genes have no effect on pretransplant alloantibody production.

BACKGROUND: The presence of high levels of alloantibodies are known to be a risk factor in renal graft outcome. Expression level polymorphisms in cytokine genes are also thought to have an effect on allograft outcome, but the studies examining this have been inconsistent. This may be due to center-specific differences in immunosuppressive protocols. Therefore, we studied the effects of these polymorphisms on pretransplant class I alloantibody production in nonexogenously immunosuppressed candidates. METHODS: Tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) gene polymorphisms were assayed genotypically by PCR-SSP on 177 renal transplant candidates. Candidates with a peak goat antihuman immunoglobulin-enhanced T-cell panel reactive antibody (PRA) of >or=10% were considered to be positive for alloantibody (32% of 177 total). RESULTS: Previous transplants, transfusions, or pregnancies were all associated with alloantibody production, but TNF-alpha and IL-10 phenotypes were not. High levels of alloantibody production (peak PRA >50%) were also not effected by cytokine phenotype. CONCLUSIONS: These data suggest that differences in TNF-alpha and IL-10 phenotype do not effect a patient's likelihood of becoming sensitized by transfusions, pregnancies, and prior transplants.

The association of lymphocytotoxic antibodies with corneal allograft rejection in high risk patients. The Collaborative Corneal Transplantation Studies Research Group.

The Collaborative Corneal Transplantation Studies are a pair of multicenter prospective clinical trials evaluating the effectiveness of histocompatibility matching in high risk keratoplasty patients. The antigen matching study (AMS) evaluated HLA matching in patients without circulating lymphocytotoxic antibody to HLA antigens and the cross-match study (CS) evaluated the effect of using cross-match-negative donors in patients with identified circulating lymphocytotoxic antibodies to HLA antigens. Sera from 510 patients considered for enrollment in the studies were screened preoperatively for the presence of anti-class I lymphocytotoxic antibodies (LA). The 42 patients (8%) found to have detectable LA entered the CS. The 468 patients found not to have detectable LA preoperatively entered the AMS. Fifteen of the 37 transplanted CS patients were found to have donor-specific anti-class I antibody (before or after surgery). These patients were also screened for anti-class II LA and 25 had anti-class II panel reactive antibody > or = 5%. Forty-nine of the 419 transplanted AMS patients (12%) were found to have produced anti-class I LA after surgery, and in 19 patients, antibody specificities were those of donor HLA antigens. There was a significant association between the number of mismatched class I antigens and the number of donor-specific LA produced. The production of LA by AMS patients was significantly associated with reaction episodes; eighty-two percent of patients (40 of 49) with LA had reaction, compared with 63% of patients (230 of 365) without LA (P = 0.02). Likewise, production of donor-specific LA was significantly associated with immune-mediated graft failure (P = 0.025). For CS patients, there was no correlation between the production of donor-specific anti-class I or nonspecific anti-class II antibodies and graft outcome. However, the CS patients had poorer graft survival than did AMS patients at 3 years (57% vs. 66%, P = 0.01). These data demonstrate that LA, especially directed against donor class I HLA antigens following corneal transplantation in high risk patients, are associated with immune graft rejection and can be an indicator of allograft rejection.

TNF-alpha, IL-6, IFN-gamma, and IL-10 gene expression polymorphisms and the IL-4 receptor alpha-chain variant Q576R: effects on renal allograft outcome.

BACKGROUND: There has been much interest recently in the effects of various cytokine gene expression polymorphisms on graft outcome. However, the results of these investigations reveal the outcomes to be organ-specific and center-specific. We sought to confirm and add to some of the earlier findings by studying the impact of tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6) polymorphisms and the interleukin-4 (IL-4) receptor alpha-chain variant on posttransplant renal allograft outcome. METHOD: TNF-alpha, IL-6, IFN-gamma, and IL-10 gene promoter region polymorphisms were assayed genotypically by PCR-SSP on 120 patients transplanted at the Albany Medical Center. These patients were also typed for the IL-4 receptor alpha-chain variant Q576R. RESULTS: Producers of high levels of the proinflammatory cytokine TNF-alpha were found to be at increased risk for acute rejection episodes if the allograft was mismatched for the molecular products of the class II region of the human major histocompatibility complex (HLA-DR). Expression level polymorphisms of the IL-6, IFN-gamma, and IL-10 genes were not associated with acute rejection episodes, nor was the IL-4 receptor alpha-chain variant Q576R. CONCLUSIONS: These data would suggest that the production of high levels of the cytokine TNF-alpha is especially detrimental to graft survival when the recipient's T-helper lymphocytes are being activated by mismatched donor HLA-class II antigens. Typing all potential kidney recipients for TNF-alpha, and providing well-matched organs for high producers of this cytokines, may be expected to increase rejection-free graft survival in these patients.

Origin of pleural cells after lung transplantation: from donor or recipient?

We evaluated the change in the percentage of cells of donor origin in pleural fluid of 13 consecutive patients who underwent lung transplantation. Pleural fluid was sampled 2, 4, and 8 days after lung transplantation. DNA, which was extracted from the blood of donors and recipients and from the pleural fluid, was amplified using a polymerase chain reaction technique. The reaction products were electrophoresed, and bands indicating amplified human leukocyte antigen (HLA)-DR alleles were quantified by determining the area under the curve (AUC) by a densitometric analysis. HLA-DR alleles, which were present only in recipient cells (recipient allele), were analyzed and compared to HLA-DR alleles that were present only in donor cells (donor allele). A dilution study was first performed to provide a standard curve relating the percentage of donor and recipient cells in a mixture to their AUC. The AUC of the recipient alleles did not change significantly over the first 8 postoperative days. The AUC of the donor alleles was less on postoperative days 4 and 8 than on day 2 (p<0.05). The donor allele AUC on day 8 was <20% of the shared allele AUC, corresponding to <1% of all cells by the dilution study. We conclude that donor cells are rapidly cleared from the pleural space after lung transplantation, with <1% of cells of donor origin by postoperative day 8.

Rapid method for the analysis of peripheral chimerism in suspected graft-versus-host disease after liver transplantation.

The effects of microchimerism and possible tolerance have been well studied in orthotopic liver transplantation. In some patients, greater levels of donor cells persist in the periphery. These cells were characterized and their effects on clinical outcome were studied. Peripheral blood was obtained from patients at various times posttransplantation. HLA class II typing was performed by the polymerase chain reaction-sequence-specific primer method on unfractionated blood and lymphocyte subpopulations. Relative levels of amplification of donor and recipient alleles were compared. All patients studied had a low degree of chimerism that was most apparent in the CD8(+)T/natural killer (NK) cell population. One patient with persistently high levels of donor alleles in his CD8(+)T/NK cell population was diagnosed with severe graft-versus-host disease (GVHD) and died of opportunistic infections. Another patient with biopsy-proven GVHD was chimeric in several cell populations. On resolution of her symptoms, donor alleles were reduced to levels undetectable by this assay. These results suggest that persistently elevated levels of donor CD8(+)T/NK cells in the periphery may indicate GVHD in liver transplant recipients. This technique aids in rapid diagnosis, which facilitates appropriate treatment and thus may improve clinical outcome.

Anti-Qa-2-induced T cell activation. The parameters of activation, the definition of mitogenic and nonmitogenic antibodies, and the differential effects on CD4+ vs CD8+ T cells.

The MHC Ag Qa-2 is a glycolipid anchored class I molecule expressed at high levels on all peripheral T lymphocytes. In this study we found that anti-Qa-2 antibodies could stimulate the proliferation of murine T cells in vitro. Anti-Qa-2-induced proliferation required secondary cross-linking with anti-Ig antibody and the presence of PMA. Only Qa-2+ strains could be induced to proliferate by anti-Qa-2 antibody, but under the conditions employed, anti-CD3 could induce proliferation in Qa-2+ and Qa-2-strains. Interestingly, only anti-Qa-2 reagents directed against the alpha 3 domain of the Qa-2 class I molecule were effective in inducing proliferation. Furthermore, unlike purified CD4+ cells, purified CD8+ cells were unable to be stimulated by the anti-Qa-2 antibodies. These results lead to the inclusion of Qa-2 in a group of physiologically relevant, glycolipid-anchored, cell-surface molecules, mobilization of which can generate signals that initiate the proliferation of T cells. Such molecules may play a secondary role in cellular activation after the primary engagement of the TCR.

Signals delivered via the Qa-2 molecule can synergize with limiting anti-CD3-induced signals to cause T lymphocyte activation.

Qa-2 is a glycolipid anchored, MHC encoded class I molecule expressed at high levels on all murine peripheral T lymphocytes. Anti-Qa-2 antibodies have previously been found to stimulate T cells to proliferate in the presence of crosslinking antibody and PMA. We have examined the effect of anti-Qa-2 antibodies on T cells stimulated with a suboptimal concentration of immobilized anti-CD3. When anti-Qa-2 antibodies were co-immobilized with limiting anti-CD3, in the absence of PMA, a clear augmentation of T cell proliferation was seen. Interestingly, the co-stimulatory anti-Qa-2 antibodies could be directed against epitopes mapped to either the alpha 3 or the alpha 1/alpha 2 Qa-2 domains. As was the case with activation induced by soluble/crosslinked anti-Qa-2 antibodies plus PMA, CD8+ T cells were less able to be costimulated with anti-Qa-2 antibodies than CD4+ cells. Surprisingly, Ca2+ mobilization was only seen when two anti-Qa-2 antibodies reactive to separate structural domains were co-crosslinked on the surface of Indo-1 loaded T cells with a suboptimal concentration of anti-CD3. Collectively these results raise questions regarding the mechanism of Qa-2 mediated signaling and its potential role in T cell activation.

Sperm protein 17 (Sp17) in multiple myeloma: opportunity for myeloma-specific donor T cell infusion to enhance graft-versus-myeloma effect without increasing graft-versus-host disease risk.

We recently found that sperm protein 17 (Sp17), a spermatozoa-restricted protein, is aberrantly expressed on the tumor cells in patients with multiple myeloma (MM). It may therefore be possible to generate donor-derived Sp17-specific CTL for administration following allogeneic stem cell transplant to augment graft-versus-myeloma (GVM) effect without inducing a global GVHD. To assess this approach, we have produced recombinant Sp17 protein and used Sp17 protein-pulsed dendritic cells to generate HLA class I-restricted Sp17-specific CTL from a previously unimmunized healthy donor. These CTL were able to lyse autologous Epstein-Barr virus-transformed lymphoblastoid cells in a Sp17-dependent manner. Target lysis was HLA-A1 and HLA-B27 restricted. Cytotoxicity could be blocked by antibodies against monomorphic HLA class I, HLA-A1 and HLA-B27 molecules but not HLA class II molecules. Most importantly, the CTL lysed HLA class I-matched Sp17-positive tumor cells, suggesting that Sp17 is processed and presented in association with the HLA class I molecules in Sp17-positive tumor cells in a concentration and configuration that could be recognized by recombinant protein-primed CTL. Analysis by flow cytometry of the CTL indicated that they were predominantly CD8 in phenotype and they produced IFN-gamma and very little IL-4. Our results suggest the potential for the generation and administration of donor-derived Sp17-specific CTL to augment GVM without inducing GVHD following allogeneic stem cell transplant for MM.

Association of HLA-DR and -DQ alleles with idiopathic achalasia.

BACKGROUND & AIMS: Idiopathic achalasia is a motility disorder of the esophagus characterized by incomplete relaxation of the lower esophageal sphincter and a loss of normal peristaltic activity in the body of the esophagus. The loss of inhibitory neurons in the distal esophagus, as well as abnormalities in the vagus nerve, dorsal motor nucleus of the vagus nerve, and autonomic nervous system, have been described in achalasia. Although the underlying cause of idiopathic achalasia is unknown, the diffuse neuronal effects found suggest a possible viral or neurodegenerative mechanism. By use of serological methods, a significant association between the HLA-DQ1 phenotype and idiopathic achalasia has been found, suggesting a possible immunogenetic mechanism. To further define immunogenetics in the pathogenesis of idiopathic achalasia, we performed tissue typing in patients with achalasia to determine their specific HLA phenotypes. METHODS: We prospectively studied 32 patients (23 white and 9 black) with idiopathic achalasia. Peripheral blood was collected, and HLA-DR and -DQ typing by polymerase chain reaction with sequence-specific primers was performed. Results were compared with those from 268 racially matched local controls. RESULTS: Idiopathic achalasia and the broad HLA-DQ1 allele were not significantly associated in either population, although a trend was found in white subjects (odds ratio [OR], 2.16; chi2 = 5.36, P corrected [Pc] = 0.0824). Further subtyping in white subjects revealed a significant association between idiopathic achalasia and the DQB1*0602 allele (OR, 3.10; chi2 = 7.32, Pc = 0.0408). A strong trend was also found with the DRB1*15 allele (OR, 2.83; chi2 = 8.11, Pc = 0.0572). In the black population, there was no association between idiopathic achalasia and DQB1*0602 or DRB1*15, but a trend was found with DRB1*12 (OR, 6. 19; chi2 = 5.19, P = 0.0227 uncorrected, Pc = 0.295). CONCLUSIONS: Idiopathic achalasia is associated with HLA alleles in a race-specific manner. These results support an immunogenetic mechanism in the pathogenesis of idiopathic achalasia.