Induction of Long-Lasting Regulatory B Lymphocytes by Modified Immune Cells in Kidney Transplant Recipients.

BACKGROUND: We recently demonstrated that donor-derived modified immune cells (MICs)-PBMCs that acquire immunosuppressive properties after a brief treatment-induced specific immunosuppression against the allogeneic donor when administered before kidney transplantation. We found up to a 68-fold increase in CD19 + CD24 hi CD38 hi transitional B lymphocytes compared with transplanted controls. METHODS: Ten patients from a phase 1 clinical trial who had received MIC infusions before kidney transplantation were followed to post-transplant day 1080. RESULTS: Patients treated with MICs had a favorable clinical course, showing no donor-specific human leukocyte antigen antibodies or acute rejections. The four patients who had received the highest dose of MICs 7 days before surgery and were on reduced immunosuppressive therapy showed an absence of in vitro lymphocyte reactivity against stimulatory donor blood cells, whereas reactivity against third party cells was preserved. In these patients, numbers of transitional B lymphocytes were 75-fold and seven-fold higher than in 12 long-term survivors on minimal immunosuppression and four operationally tolerant patients, respectively ( P <0.001 for both). In addition, we found significantly higher numbers of other regulatory B lymphocyte subsets and a gene expression signature suggestive of operational tolerance in three of four patients. In MIC-treated patients, in vitro lymphocyte reactivity against donor blood cells was restored after B lymphocyte depletion, suggesting a direct pathophysiologic role of regulatory B lymphocytes in donor-specific unresponsiveness. CONCLUSIONS: These results indicate that donor-specific immunosuppression after MIC infusion is long-lasting and associated with a striking increase in regulatory B lymphocytes. Donor-derived MICs appear to be an immunoregulatory cell population that when administered to recipients before transplantation, may exert a beneficial effect on kidney transplants. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: MIC Cell Therapy for Individualized Immunosuppression in Living Donor Kidney Transplant Recipients (TOL-1), NCT02560220.

Endothelial precursor cell cross-match using Tie-2-enriched spleen cells.

BACKGROUND: Non-HLA antibodies against human endothelial progenitor cells (EPC) in pre-transplant recipient serum can have a deleterious influence on the graft. EPC enriched from peripheral blood have been commonly used for EPC cross-matching. In the present study, we describe cross-matches using EPC enriched from fresh or frozen-thawed spleen cell preparations, thereby widening the sample source for deceased-donor cross-matching and retrospective studies. METHODS: EPC cross-matches were performed retrospectively using spleen cells and the flow cytometric XM-ONE cross-match test kit. RESULTS: Healthy controls (n = 28) showed no IgG antibodies against EPC. When sera of 11 random dialysis patients were studied, 2 patients (18%) exhibited IgG EPC antibodies. When pre-transplant sera of 20 kidney graft recipients with good long-term graft outcome (serum creatinine 1.0 ± 0.2 mg/dL measured 2463 ± 324 days post-transplant) were investigated using frozen-thawed and then separated Tie-2-enriched spleen cells of the original transplant donor, 3 patients (15%) had pre-transplant IgG EPC antibodies. When pre-transplant sera of 5 patients with intra-operative graft loss were studied employing the original donor spleen cells, 4 (80%) patients showed IgG EPC antibodies. CONCLUSIONS: Cross-matches with spleen cell-derived EPC using the XM-ONE assay are technically possible. Our very preliminary experience suggests clinical relevance.

Clinical relevance of preformed IgG and IgM antibodies against donor endothelial progenitor cells in recipients of living donor kidney grafts.

BACKGROUND: Literature reports suggest that non-HLA-antibodies against human endothelial progenitor cells (EPC) can be detected in pre-transplant recipient serum and that EPC antibodies can have a deleterious influence on the graft. METHODS: We investigated 71 renal transplant recipients from living donors for a possible influence of pre-transplant donor-specific IgG and/or IgM recipient antibodies against EPC of the donor using the flow cytometric XM-ONE cross-match. RESULTS: Eight of the 71 patients developed acute biopsy-proven rejection. Two of these patients showed IgM antibodies against EPC prior to transplantation while the other six patients had neither IgG nor IgM EPC antibodies. Conversely, pre-transplant IgG or IgM antibodies against EPC were detected in 19 patients without acute rejection (3 × both IgG and IgM, 1 × IgG and 15 × IgM). The remaining 44 patients had neither EPC antibodies nor experienced rejection. Comparing serum creatinine levels at one month and one yr post-transplant within and among the three patient groups revealed that serum creatinine levels were similar in patients with or without EPC antibodies (p > 0.05). CONCLUSION: In this series of 71 recipients with living donor kidneys, pre-transplant EPC antibodies detected with the XM-ONE test kit were neither associated with acute rejection nor with graft function at one month or one yr.

Risk Stratification Before Living Donor Kidney Transplantation in Patients With Preformed Donor-specific Antibodies by Different Crossmatch Methods.

Background: Preformed donor-specific HLA antibodies (DSA) are a well-known risk factor in kidney transplantation. There is still considerable debate, however, about the optimal risk stratification among patients with preformed DSA. Additionally, data on the prognostic value of different crossmatch assays in DSA-positive patients are scarce. Methods: DSA-positive living kidney transplant recipients were selected from a multicenter study examining 4233 consecutive renal transplants. An additional 7 patients from 2 further centers were included. Flow cytometric crossmatches (FXM), Luminex-based crossmatches, and virtual crossmatches based on C1q- and C3d-binding antibodies (C1qXM and C3dXM) were performed retrospectively using pretransplant sera and lymphocytes isolated from fresh samples. These samples were obtained from 44 donor and recipient pairs from 12 centers. Clinical outcome data and the control group without DSA were compiled from the previous study and were supplemented by data on 10-y death-censored graft survival (10yGS). Results: Between 19% (C3dXM) and 46% (FXM) of crossmatches were positive. Crossmatch-positive patients showed high incidences of antibody-mediated rejection (AMR) within 6 mo (up to 60% in B-cell FXM+ patients). The incidence of AMR in crossmatch-negative patients ranged between 5% (FXM-) and 13% (C1qXM-). 10yGS was significantly impaired in patients with positive T-cell FXM and total FXM compared with both patients without DSA and those with DSA with negative FXM. Conclusions: Especially FXM are useful for risk stratification, as the outcome of DSA-positive, FXM-negative patients is similar to that of DSA-negative patients, whereas FXM-positive patients have both more AMR and decreased 10yGS. Because of their lower sensitivity, the significance of Luminex-based crossmatches, C1qXM, and C3dXM would have to be examined in patients with stronger DSA.

Five-year follow-up of a phase I trial of donor-derived modified immune cell infusion in kidney transplantation.

Background: The administration of modified immune cells (MIC) before kidney transplantation led to specific immunosuppression against the allogeneic donor and a significant increase in regulatory B lymphocytes. We wondered how this approach affected the continued clinical course of these patients. Methods: Ten patients from a phase I clinical trial who had received MIC infusions prior to kidney transplantation were retrospectively compared to 15 matched standard-risk recipients. Follow-up was until year five after surgery. Results: The 10 MIC patients had an excellent clinical course with stable kidney graft function, no donor-specific human leukocyte antigen antibodies (DSA) or acute rejections, and no opportunistic infections. In comparison, a retrospectively matched control group receiving standard immunosuppressive therapy had a higher frequency of DSA (log rank P = 0.046) and more opportunistic infections (log rank P = 0.033). Importantly, MIC patients, and in particular the four patients who had received the highest cell number 7 days before surgery and received low immunosuppression during follow-up, continued to show a lack of anti-donor T lymphocyte reactivity in vitro and high CD19+CD24hiCD38hi transitional and CD19+CD24hiCD27+ memory B lymphocytes until year five after surgery. Conclusions: MIC infusions together with reduced conventional immunosuppression were associated with good graft function during five years of follow-up, no de novo DSA development and no opportunistic infections. In the future, MIC infusions might contribute to graft protection while reducing the side effects of immunosuppressive therapy. However, this approach needs further validation in direct comparison with prospective controls. Trial registration: https://clinicaltrials.gov/, identifier NCT02560220 (for the TOL-1 Study). EudraCT Number: 2014-002086-30.

Living donor kidney transplantation in crossmatch-positive patients enabled by peritransplant immunoadsorption and anti-CD20 therapy.

Living donor kidney transplantation in crossmatch-positive patients is a challenge that requires specific measures. Ten patients with positive crossmatch results (n = 9) or negative crossmatch results but strong donor-specific antibodies (DSA; n = 1) were desensitized using immunoadsorption (IA) and anti-CD20 antibody induction. IA was continued after transplantation and accompanied by HLA antibody monitoring and protocol biopsies. After a median of 10 IA treatments, all patients were desensitized successfully and transplanted. Median levels of mean fluorescence intensity (MFI) of Luminex-DSA before desensitization were 6203 and decreased after desensitization and immediately before transplantation to 891. Patients received a median of seven post-transplant IA treatments. At last visit, after a median follow-up of 19 months, 9 of 10 patients had a functioning allograft and a median Luminex-DSA of 149 MFI; serum creatinine was 1.6 mg/dl, and protein to creatinine ratio 0.1. Reversible acute antibody-mediated rejection was diagnosed in three patients. One allograft was lost after the second post-transplant year in a patient with catastrophic antiphospholipid syndrome. We describe a treatment algorithm for desensitization of living donor kidney transplant recipients that allows the rapid elimination of DSA with a low rate of side effects and results in good graft outcome.

Human Leucocyte Antigens as Prognostic Markers in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: The induction and regulation of immune responses depend on human leucocyte antigen (HLA) molecules that present peptides derived from mutated neoantigens or tumor-associated antigens to cytotoxic T cells. The natural variation of HLA molecules might differ between tumor patients and the normal population. Thus, there might be associations between the frequencies of HLA alleles and the survival of tumor patients. METHODS: This issue was studied in a cohort of 84 patients with head and neck squamous cell carcinomas (HNSCCs) of different localizations. The cohort was followed up for more than 10 years. HLA-A/B/C CTS-PCR-SSP typing at 1 field level from blood samples was performed, and the results were correlated with survival. RESULTS: HLA-A*02 was the most prevalent allele in our cohort and was present in 51.1% of patients. The HLA-A*25 and HLA-C*06 alleles exhibited a significantly higher frequency in cancer patients than in the normal population of 174 blood and kidney donors (p = 0.02 and p = 0.01, respectively, Fisher's exact test). For HLA-C*04, a negative impact on overall survival in univariate analysis (p = 0.045) and a negative, but statistically insignificant effect on survival toward poorer survival in multivariate analysis (HR: 1.82; 95% CI: 0.99-3.34, p = 0.053) were observed. In addition, HLA-A*02 was also beneficial for overall survival and progression-free survival in multivariate analysis (HR 0.54; 95% CI: 0.31-0.92; p = 0.023). CONCLUSION: HLA-A*02 allele expression might not only predict better survival but might also indicate superior tumor antigen presentation and, thus, help to select patients who could benefit from T-cell-dependent immunotherapies.

Individualised immunosuppression with intravenously administered donor-derived modified immune cells compared with standard of care in living donor kidney transplantation (TOL-2 Study): protocol for a multicentre, open-label, phase II, randomised controlled trial.

INTRODUCTION: Donor-derived modified immune cells (MIC) induced long-term specific immunosuppression against the allogeneic donor in preclinical models of transplantation. In a phase I clinical trial (TOL-1 Study), MIC treatment resulted in a cellular phenotype that was directly and indirectly suppressive to the recipient's immune system allowing for reduction of conventional immunosuppressive therapy. Here, we describe a protocol for a randomised controlled, multicentre phase-IIb clinical trial of individualised immunosuppression with intravenously administered donor MIC compared with standard-of-care (SoC) in living donor kidney transplantation (TOL-2 Study). METHODS AND ANALYSIS: Sixty-three living donor kidney transplant recipients from six German transplant centres are randomised 2:1 to treatment with MIC (MIC group, N=42) or no treatment with MIC (control arm, N=21). MIC are manufactured from donor peripheral blood mononuclear cells under Good Manufacturing Practice conditions. The primary objective of this trial is to determine the efficacy of MIC treatment together with reduced conventional immunosuppressive therapy in terms of achieving an operational tolerance-like phenotype compared with SoC 12 months after MIC administration. Key secondary endpoints are the number of patient-relevant infections as well as a composite of biopsy-proven acute rejection, graft loss, graft dysfunction or death. Immunosuppressive therapy of MIC-treated patients is reduced during follow-up under an extended immunological monitoring including human leucocyte antigen-antibody testing, and determination of lymphocyte subsets, for example, regulatory B lymphocytes (Breg) and antidonor T cell response. A Data Safety Monitoring Board has been established to allow an independent assessment of safety and efficacy. ETHICS AND DISSEMINATION: Ethical approval has been provided by the Ethics Committee of the Medical Faculty of the University of Heidelberg, Heidelberg, Germany (AFmu-580/2021, 17 March 2022) and from the Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Institute, Langen, Germany (Vorlage-Nr. 4586/02, 21 March 2022). Written informed consent will be obtained from all patients and respective donors prior to enrolment in the study. The results from the TOL-2 Study will be published in peer-reviewed medical journals and will be presented at symposia and scientific meetings. TRIAL REGISTRATION NUMBER: NCT05365672.

Functional Fc Gamma Receptor Gene Polymorphisms and Long-Term Kidney Allograft Survival.

The functional Fc gamma receptor (FcγR) IIIA polymorphism FCGR3A-V/F158 was earlier suggested to determine the potential of donor-specific HLA antibodies to trigger microcirculation inflammation, a key lesion of antibody-mediated renal allograft rejection. Associations with long-term transplant outcomes, however, have not been evaluated to date. To clarify the impact of FCGR3A-V/F158 polymorphism on kidney transplant survival, we genotyped a cohort of 1,940 recipient/donor pairs. Analyzing 10-year death-censored allograft survival, we found no significant differences in relation to FCGR3A-V/F158. There was also no independent survival effect in a multivariable Cox model. Similarly, functional polymorphisms in two other activating FcγR, FCGR2A-H/R131 (FcγRIIA) and FCGR3B-NA1/NA2 (FcγRIIIB), were not associated with outcome. There were also no significant survival differences among patient subgroups at increased risk of rejection-related injury, such as pre-sensitized recipients (> 0% panel reactivity; n = 438) or recipients treated for rejection within the first year after transplantation (n = 229). Our study results suggest that the earlier shown association of FcγR polymorphism with microcirculation inflammation may not be strong enough to exert a meaningful effect on graft survival.

Analysis of de novo donor-specific HLA-DPB1 antibodies in kidney transplantation.

HLA matching and avoidance of unacceptable mismatches are important aspects in the selection of donors for solid organ transplantation. The impact of HLA-DPB1 incompatibility on the outcomes of kidney transplantation is not fully understood. We investigated a potential effect of mismatching for HLA-DPB1 at allele, eplet, or Terasaki epitope (TerEp) level on the formation of de novo donor-specific antibodies (dnDSA) and also asked whether polymorphisms associated with HLA-DPB1 expression level may influence dnDSA induction. Furthermore, we analyzed the correlation between graft survival and HLA-DPB1 mismatches defined by different approaches. A cohort of 366 patients who received a kidney transplant at the Heidelberg University Hospital, Germany, with availability of pre- and post-transplant HLA antibody results by single antigen testing as well as of donor and recipient HLA-DPB1 high-resolution typing were analyzed retrospectively. Susceptibility to increased HLA-DPB1 expression was predicted by the linked dimorphism rs9277534 A/G of the HLA-DPB1 gene. Neither HLA-DPB1 mismatches at allele, eplet or TerEp level nor exposure to donor's high HLA-DPB1 expression were significantly associated with the risk of developing dnDSA against HLA-DPB1. However, HLA-DPB1 eplet and TerEp mismatches had a significant negative impact on graft survival (p < 0.001 and p = 0.003, respectively). Matching for HLA-DPB1 at epitope instead of allele level appears to have potential to improve graft survival in kidney transplantation.

Pre-transplant HLA Antibodies and Delayed Graft Function in the Current Era of Kidney Transplantation.

Delayed graft function (DGF) occurs in a significant proportion of deceased donor kidney transplant recipients and was associated with graft injury and inferior clinical outcome. The aim of the present multi-center study was to identify the immunological and non-immunological predictors of DGF and to determine its influence on outcome in the presence and absence of human leukocyte antigen (HLA) antibodies. 1,724 patients who received a deceased donor kidney transplant during 2008-2017 and on whom a pre-transplant serum sample was available were studied. Graft survival during the first 3 post-transplant years was analyzed by multivariable Cox regression. Pre-transplant predictors of DGF and influence of DGF and pre-transplant HLA antibodies on biopsy-proven rejections in the first 3 post-transplant months were determined by multivariable logistic regression. Donor age ≥50 years, simultaneous pre-transplant presence of HLA class I and II antibodies, diabetes mellitus as cause of end-stage renal disease, cold ischemia time ≥18 h, and time on dialysis >5 years were associated with increased risk of DGF, while the risk was reduced if gender of donor or recipient was female or the reason for death of donor was trauma. DGF alone doubled the risk for graft loss, more due to impaired death-censored graft than patient survival. In DGF patients, the risk of death-censored graft loss increased further if HLA antibodies (hazard ratio HR=4.75, P < 0.001) or donor-specific HLA antibodies (DSA, HR=7.39, P < 0.001) were present pre-transplant. In the presence of HLA antibodies or DSA, the incidence of biopsy-proven rejections, including antibody-mediated rejections, increased significantly in patients with as well as without DGF. Recipients without DGF and without biopsy-proven rejections during the first 3 months had the highest fraction of patients with good kidney function at year 1, whereas patients with both DGF and rejection showed the lowest rate of good kidney function, especially when organs from ≥65-year-old donors were used. In this new era of transplantation, besides non-immunological factors, also the pre-transplant presence of HLA class I and II antibodies increase the risk of DGF. Measures to prevent the strong negative impact of DGF on outcome are necessary, especially during organ allocation for presensitized patients.

Relevance of donor-specific antibody monitoring after kidney transplantation: Findings from the Collaborative Transplant Study and the Heidelberg Transplant Center.

Monitoring of donor-specific HLA antibodies (DSA) has become part of the clinical routine in kidney transplantation. This paper gives a brief overview on data from the Collaborative Transplant Study (CTS) and the Heidelberg Transplant Center on the clinical relevance of post-transplant DSA monitoring in patients undergoing renal transplantation. The obtained findings underline the importance of DSA monitoring in the post-operative course in immunologically high-risk patients and patients with deterioration of graft function. Especially in patients with a pre-activated immune system, a gap in the immunosuppressive therapy appear to lead to persistence, reappearance or de novo occurrence of strong, complement-activating DSA, resulting in severe antibody-mediated rejection (AMR) and, without timely intervention, in AMR-related graft loss.

No association of factor V Leiden, prothrombin G20210A, and MTHFR C677T gene polymorphisms with kidney allograft survival: a multicenter study.

BACKGROUND: It has been proposed that inherited risk factors of venous thromboembolism, such as factor V G1691A (FV-Leiden), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T, might be associated with poorer survival rates of transplanted kidneys. On the basis of this hypothesis, we performed a multicenter study, involving recipients of primary and repeat kidney transplants, to investigate the potential effect of these three single nucleotide polymorphisms (SNP) on graft survival. METHODS: The study consisted of 676 first and 651 retransplant patients. Using the polymerase chain reaction-sequence specific primers method, we typed all patients for the three SNP and analyzed graft survival. RESULTS: We could not find a statistically significant association between graft survival and factor V Leiden or MTHFR C677T genotypes. A better 3-yr graft survival was found for first transplant recipients with the genotype prothrombin 20210 G/G as compared to those with the G/A genotype (P=0.031). However, Bonferroni correction for the three SNPs investigated in this series rendered the P value insignificant (P(corrected)=0.093). CONCLUSION: We did not find a statistically significant association of SNP factor V Leiden G1691A and MTHFR C677T with renal graft survival. Prothrombin G20210A resulted in a significant association that was not sustained after Bonferroni correction. This SNP might be an interesting candidate for future studies.

Donor-specific antibodies require preactivated immune system to harm renal transplant.

BACKGROUND: It is an unresolved issue why some kidney transplant recipients with pretransplant donor-specific HLA antibodies (DSA) show a high transplant failure rate, whereas in other patients DSA do not harm the graft. We investigated whether help from preactivated T-cells might be necessary for DSA to exert a deleterious effect. METHODS: The impact of pretransplant DSA and immune activation marker soluble CD30 (sCD30) on 3-year graft survival was analyzed in 385 presensitized kidney transplant recipients. FINDINGS: A deleterious influence of pretransplant DSA on graft survival was evident only in patients who were positive for the immune activation marker sCD30. In the absence of sCD30 positivity, 3-year graft survival was virtually identical in patients with or without DSA (83.1±3.9% and 84.3±2.8%, P=0.81). A strikingly lower 3-year graft survival rate of 62.1±6.4% was observed in patients who were both sCD30 and DSA positive (HR 2.92, P<0.001). Even in the presence of strong DSA with ≥5000 MFI, the 3-year graft survival rate was high if the recipients were sCD30 negative. INTERPRETATION: Pretransplant DSA have a significantly deleterious impact on graft survival only in the presence of high pretransplant levels of the activation marker sCD30.

Analysis of KIR ligand incompatibility in human renal transplantation.

Natural killer cells express killer immunoglobulin-like receptors (KIR) that bind to MHC class I antigens. Lack of self-MHC on the target cell can cause NK-cell mediated killing. Here, we analyzed the effect of KIR ligand incompatiblity on renal allograft survival in humans. Kidney recipient/donor pairs were separated according to their HLA-Cw alleles and HLA-Bw4 specificity which are considered epitopes for KIR. A total of 2,757 renal transplants were examined. Graft survival rates were computed according to the Kaplan-Meier method. No effect of KIR ligand matching on graft survival was observed in cadaver kidney transplants. Our results indicate that KIR ligand matching cannot be recommended as a strategy for improving renal allograft survival.

Seven novel HLA alleles reflect different mechanisms involved in the evolution of HLA diversity: description of the new alleles and review of the literature.

The human leukocyte antigen (HLA) loci are among the most polymorphic genes in the human genome. The diversity of these genes is thought to be generated by different mechanisms including point mutation, gene conversion and crossing-over. During routine HLA typing, we discovered seven novel HLA alleles which were probably generated by different evolutionary mechanisms. HLA-B*41:21, HLA-DQB1*02:10 and HLA-DQA1*01:12 likely emerged from the common alleles of their groups by point mutations, all of which caused non-synonymous amino acid substitutions. In contrast, a deletion of one nucleotide leading to a frame shift with subsequent generation of a stop codon is responsible for the appearance of a null allele, HLA-A*01:123N. Whereas HLA-B*35:231 and HLA-B*53:31 were probably products of intralocus gene conversion between HLA-B alleles, HLA-C*07:294 presumably evolved by interlocus gene conversion between an HLA-C and an HLA-B allele. Our analysis of these novel alleles illustrates the different mechanisms which may have contributed to the evolution of HLA polymorphism.

Association of Kidney Graft Loss With De Novo Produced Donor-Specific and Non-Donor-Specific HLA Antibodies Detected by Single Antigen Testing.

BACKGROUND: The association of donor-specific HLA antibodies (DSA) with kidney graft failure has been addressed previously; however, the majority of studies were based on small numbers of patients with graft failure. METHODS: We investigated 83 patients with failed kidney transplants for a possible association of de novo development and persistence or loss of pre-existing DSA with graft failure. Single Antigen Bead assay-detected DSA and non-DSA antibodies were compared between patients with graft loss and matched controls with functioning grafts. RESULTS: The incidence of weak de novo DSA or non-DSA at a mean fluorescence intensity of 500 or higher was higher in the graft loss than in the nonrejector group (76% vs 40%, P < 0.001). Because of the low number of patients developing de novo DSA, the DSA results did not reach statistical significance (only 22% of patients with graft loss developed de novo DSA). However, at all cutoffs, there was a significantly higher rate of graft loss in patients with de novo non-DSA. The incidence of strong pretransplant DSA that persist after transplantation was higher in the graft loss group (10% vs 1%, P = 0.034). When C1q-binding ability in sera of rejectors and nonrejectors with posttransplant de novo or persistent DSA was compared, none of the nonrejectors demonstrated C1q positivity, whereas 43% of patients with graft loss showed C1q-positive antibodies, although not necessarily donor-specific (P < 0.001). CONCLUSIONS: Our data show that the posttransplant presence of persisting or de novo HLA antibodies, especially if C1q binding, is associated with graft loss, even if the antibodies are not specific for mismatched donor HLA.

Matrix analysis for the dissection of interactions of G-protein beta3 subunit C825T genotype, allograft function, and posttransplant hypertension in kidney transplantation.

BACKGROUND: Complex relationships between genes and environment and the resulting biological impact have been dissected predominantly by conventional association studies. A major limitation of such studies results from the fact that only bidirectional investigations of genes and clinical end-points are commonly performed. The authors, therefore, applied matrix analyses to account for interactions between genetic and environmental factors influencing kidney allograft function. METHODS: By using matrices of correlation coefficients we tested the genetic effect of a variant within the gene encoding the beta3-subunit of heterotrimeric G-proteins (Gbeta3-C825T polymorphism) on posttransplant hypertension and kidney allograft function. This strategy allowed the authors to account for the influence of additional well-established genetic, clinical, and environmental confounders. The authors studied 281 consecutive white kidney recipients recruited between 1988 and 1993. Correlation coefficients of indices of relative change (percent) of systolic blood pressure (BP) and creatinine clearance (CrCl) were used in correlation coefficient matrices to elucidate interactions of parametrical biological parameters with environmental and genetic risk factors. RESULTS: A significant relationship was found between decreasing CrCl and increasing systolic BP in only those recipients who carried the Gbeta3-825TT genotype and did not lose graft during the first 3 years (R2 = 0.25; P = 0.021). CONCLUSIONS: In transplant recipients who did not lose their graft during the first 3 years after transplantation, the Gbeta3-TT genotype contributed to accelerated loss of allograft function by exaggeration of posttransplant hypertension. This relationship could only be elucidated by means of matrix analyses that allow the detection of complex relations between clinical, genetic, and environmental factors.

HIV-Specific CD8(+) T Lymphocytes in Blood of Long-Term HIV-Infected Hemophilia Patients.

Hemophilia patients infected with human immunodeficiency virus (HIV) 30 years ago show increased proportions of activated CD8(+)DR(+) blood lymphocytes. We hypothesized that this might indicate a cellular immune response directed against HIV and might be the reason for long-term clinical stability of these patients. CD8(+) peripheral blood lymphocytes (PBL) reactive with six HIV and two cytomegalovirus (CMV) pentamers were determined in heparinized whole blood. Additional lymphocyte subsets as well as plasma cytokines and HIV-1 load were studied. Long-term HIV-infected hemophilia patients with (n=15) or without (n=33) currently detectable HIV-1 load in the plasma showed higher proportions of CD8(+) lymphocytes reactive with HIV (p<0.001) and CMV pentamers (p=0.010) than healthy individuals. The cellular anti-HIV response tended to be stronger and more polyclonal in patients during periods of viral replication than in patients with retroviral quiescence (p=0.077). Anti-HIV CD8(+) lymphocyte responses were strongest in patients with high counts of activated CD8(+)DR(+) T (r=0.353; p=0.014) and low CD19(+) B lymphocyte counts (r=-0.472; p=0.001). Patients with or without HIV-1 viral load showed normal Th1 and Th2 plasma cytokine levels and high plasma interleukin-6 (versus healthy controls, p=0.001) and tumor necrosis factor-α (p=0.020). Hemophilia patients who have been living with HIV for more than 30 years showed a polyclonal CD8(+) T-cell response against HIV and CMV. This cellular antiviral immune response was strongest during periods of HIV-1 replication and remained detectable during periods of HIV-1 quiescence. We hypothesize that the consistent cellular anti-HIV-1 response in combination with highly active antiretroviral therapy ensures stability and survival of these chronically HIV-1-infected hemophilia patients.

Deleterious impact of HLA-DRB1 allele mismatch in sensitized recipients of kidney retransplants.

BACKGROUND: Whether mismatches between donor and recipient of a kidney transplant at the HLA allele level can elicit an immune response strong enough to impact graft survival is not known. METHODS: We examined the influence of HLA-DRB1 allele level mismatch on graft survival based on high-resolution typing, utilizing blood samples and clinical data provided by the Collaborative Transplant Study. HLA-DRB1*04 was selected as a model for this investigation because it is the most common HLA-DRB1 allele group and consists of several alleles with relatively high frequencies, allowing for analysis of transplants matched at the antigen level but mismatched at the allele level. Nine hundred and ninety-six recipient/donor pairs were typed for HLA-DRB1 at high resolution. RESULTS: No effect of HLA-DRB1*04 allele mismatch was observed in first transplants. However, in retransplants, HLA-DRB1*04 allele mismatch was associated with significantly decreased graft survival, albeit only in sensitized (PRA>5%) patients (hazard ratio 3.98, P=0.014). CONCLUSION: Our finding reinforces the concept that HLA compatibility significantly influences the outcome of kidney transplants, in sensitized retransplant recipients even at the allele level.