Paul Brookes

Loss of Direct and Maintenance of Indirect Alloresponses in Renal Allograft Recipients: Implications for the Pathogenesis of Chronic Allograft Nephropathy

Chronic allograft nephropathy (CAN) is the principal cause of late renal allograft failure. This complex process is multifactorial in origin, and there is good evidence for immune-mediated effects. The immune contribution to this process is directed by CD4+ T cells, which can be activated by either direct or indirect pathways of allorecognition. For the first time, these pathways have been simultaneously compared in a cohort of 22 longstanding renal allograft recipients (13 with good function and nine with CAN). CD4+ T cells from all patients reveal donor-specific hyporesponsiveness by the direct pathway according to proliferation or the secretion of the cytokines IL-2, IL-5, and IFN-γ. Donor-specific cytotoxic T cell responses were also attenuated. In contrast, the frequencies of indirectly alloreactive cells were maintained, patients with CAN having significantly higher frequencies of CD4+ T cells indirectly activated by allogeneic peptides when compared with controls with good allograft function. An extensive search for alloantibodies has revealed significant titers in only a minority of patients, both with and without CAN. In summary, this study demonstrates widespread donor-specific hyporesponsiveness in directly activated CD4+ T cells derived from longstanding recipients of renal allografts, whether they have CAN or not. However, patients with CAN have significantly higher frequencies of CD4+ T cells activated by donor Ags in an indirect manner, a phenomenon resembling split tolerance. These findings provide an insight into the pathogenesis of CAN and also have implications for the development of a clinical tolerance assay.

De novo DQ donor-specific antibodies are associated with a significant risk of antibody-mediated rejection and transplant glomerulopathy.

Background The importance of human leukocyte antigen (HLA) matching in renal transplantation is well recognized, with HLA-DR compatibility having the greatest influence. De novo DQ donor-specific antibodies (DSAbs) are the predominant HLA class II DSAb after transplantation. The aim of this study was to establish the incidence and outcomes after the development of DQ DSAbs along with the impact of class II HLA mismatch on their development. Methods We retrospectively analyzed 505 patients who received a renal-alone transplant between 2005 and 2010. We excluded patients who received an ABO- and HLA-incompatible allograft, which we defined as those with a positive crossmatch or preformed DSAbs detected by single-antigen beads only. Results Of 505 patients, 92 (18.2%) developed DSAbs, with 50 (54.3%) of these 92 patients having DQ DSAbs. Patients who developed DQ DSAbs were at significant risk for antibody-mediated rejection, transplant glomerulopathy, and allograft loss (P<0.0001). Of 505 patients, 108 (21.4%) were matched at both the DR and DQ loci, 284 (56.2%) were mismatched at both loci, 38 (7.5%) were matched at DR alone, and 75 (14.9%) were matched at DQ alone. Patients mismatched at both DR and DQ were at risk for developing class II DSAbs when compared with those mismatched at either DR or DQ alone, P=0.001, and were at risk for antibody-mediated rejection, P=0.001. Conclusions DQ DSAbs are associated with inferior allograft outcomes. This study shows the importance of establishing the DQ match before transplantation to define immunologic risk.

The role of the allograft in the induction of donor-specific T cell hyporesponsiveness.

Background. With adequate immunosuppression the majority of renal allografts are accepted, despite the exceptional vigour of the T cell alloimmune response. Previous work from this laboratory has demonstrated that this is accompanied by significant reductions in the precursor frequencies of anti-donor T cells. We have also shown that parenchymal cells are tolerogenic in vitro. We propose that the reduction in T cell frequencies may be due to the interaction between circulating T cells and potentially tolerogenic graft parenchymal cells. Primed/memory T cells (CD45RO+) are the only subset capable of reaching the allograft and therefore we would predict that T cell hyporesponsiveness would develop predominantly in the CD45RO+ subset due to their trafficking properties. Methods. Frequencies of IL-2 secreting CD45RA+ and CD45RO+ CD4+ T cells in response to donor and third party stimulator cells were estimated in a series of renal transplant recipients, both before and after transplantation. Results. There were highly significant reductions in the frequencies of donor-specific CD4+CD45RO+ T cells, when adjusted to control for the generalised effects of immunosuppression. There were no significant alterations in the frequencies of donor-specific CD4+CD45RA+ T cells. Conclusions. In renal transplant recipients, donor-specific CD4+ T cell hyporesponsiveness occurs predominantly in CD4+ CD45RO+ T cells which is the subset capable of trafficking through the graft.

CYTOTOXIC T LYMPHOCYTE PRECURSOR FREQUENCY ANALYSES IN BONE MARROW TRANSPLANTATION WITH VOLUNTEER UNRELATED DONORS VALUE IN DONOR SELECTION

Between May 1989 and February 1994, we performed 48 volunteer unrelated donor BMTs for first chronic phase chronic myeloid leukemia using in vivo T cell depletion for acute graft-versus-host disease (aGvHD) prophylaxis. In 40 cases, adequate material was available to measure the frequency of antirecipient MHC cytotoxic T lymphocyte precursor (CTLp) cells in the blood of potential donors. This supplemented standard serological typing, one-dimensional isoelectric focusing for class I proteins, and allogenotyping for DR and DQ alleles using DNA RFLP analysis in the donor selection process. All recipients were conditioned with cyclophosphamide 120 mg/kg, TBI 1320 cGy, and intravenous Campath 1G. GvHD prophylaxis consisted of CsA, short-course methotrexate, and intravenous Campath 1G. Minimum follow-up in all surviving recipients was 100 days. The development of aGvHD and the probability of leukemia-free survival were compared between the high frequency group (CTLp>l in 100,000) (n=15) and the low frequency group (CTLp<l in 100,000) (n=25). There was a trend for increasing grade of aGvHD, which was statistically significant in the high frequency group when compared with the low frequency group (P=0.003). Both a high frequency of CTLp (relative risk [RR] = 9.0, P=0.016) and HLA mismatch (RR=6.7, P=0.023) were predictors of severe aGvHD (grade III or IV). Multivariate analysis showed that CTLp group (RR=3.4, P=0.015) and CMV status (RR=3.9, P=0.008) were predictors of leukemia-free survival. Further investigation showed an interaction between the two, such that CMV seropositive recipients in the high frequency group had a relative risk of 9.4 (P=0.0001) of treatment failure (death or relapse) when compared with other combinations. We conclude that with our present GvHD prophylaxis regimen, CTLp frequency analysis predicts post-BMT outcome and is a valuable aid in donor selection.

Comparison of helper and cytotoxic antirecipient T cell frequencies in unrelated bone marrow transplantation

Donor/recipient histocompatibility antigen differences initiate acute graft-versus-host disease (GVHD) after bone marrow transplantation. Frequency analysis, using limiting dilution techniques, of functionally defined (helper or cytotoxic) antirecipient T lymphocyte precursors in the peripheral blood of the donor has been shown to be an accurate predictor for the development of moderate-to-severe acute GVHD. Here, we describe a sensitive assay for measuring alloreactive helper (IL-2-producing) T lymphocyte precursor (HTLp) frequencies, and compare the ability of this assay and the cytotoxic T lymphocyte precursor (CTLp) assay to detect HLA- class II and class I differences and to predict clinical outcome in a cohort of unrelated donor/recipient BMT pairs. Twenty-two pairs underwent unrelated donor BMT. Patients with high (>1:100x103) HTLp or CTLp frequencies had a higher incidence of moderate-to-severe (grades II-IV) acute GVHD (80% and 100%, respectively) than pairs with low (<1:100x103) frequencies (40% and 57%, respectively). Ten (45%) patients have died, but all patients with both a low HTLp and low CTLp frequency remain alive. The HTLp and CTLp assays provided similar predictive information for outcome. Given that the HTLp assay is more rapid and less labor intensive, it offers an additional or alternative functional method for donor selection in unrelated donor BMT.

Role of donor and recipient antigen-presenting cells in priming and maintaining t cells with indirect allospecificity

BACKGROUND It has been suggested that the sensitization of recipient T lymphocytes against peptides derived from allogeneic major histocompatibility complex (MHC) antigens in the context of self-MHC molecules may contribute to the pathogenesis of chronic allograft rejection. The purpose of this study was to quantitate and characterize the indirect alloresponse in renal transplantation. METHODS An HLA-A2-negative patient whose A2-positive kidney transplant failed as a result of chronic rejection was selected for this study. T-cell clones were raised using a cocktail of peptides corresponding to polymorphic regions of the A2 sequence and studied by measuring their proliferation using [3H]thymidine incorporation. The presence in vivo of HLA-A2-specific T cells was assessed using limiting dilution analysis. RESULTS T-cell clones were specific for a single peptide of HLA-A2, residues 92-120, and restricted by HLA-DRB1*1502. The frequency of interleukin-2-secreting T cells specific for this A2 peptide was 1:86,000, only 2-fold lower than that measured against the recall antigen tetanus toxoid. Capitalizing on the similarity of the donor and recipient DR15 alleles (DRB1*1501 and 1502), the question was addressed as to how these T cells had been primed in vivo. Although the large majority of clones responded to A2 synthetic peptide presented by both DR15 alleles, only 3 of 10 clones responded to cells co-expressing DRB1*1501 and A2. CONCLUSION These data suggest that antigen presentation by recipient APCs is responsible for maintaining T cells with indirect allospecificity in vivo and that, in the context of partial DR matching, indirect presentation by the parenchymal cells of the graft may serve to induce tolerance in T cells with indirect allospecificity.

Preformed complement-activating low-level donor-specific antibody predicts early antibody-mediated rejection in renal allografts.

Background. Donor-specific anti-HLA antibodies (DSA) are a major cause of alloimmune injury. Transplant recipients with negative complement-dependent cytotoxic crossmatch (CDC-XM) and donor cell-based flow cytometric crossmatch (flow-XM) but low level DSA (i.e., by Luminex) have worse outcomes compared with nonsensitized patients. The aim of this study was to establish whether complement-activating ability in this low-level DSA, present before transplantation, as determined by this technique is important in dictating pathogenicity. Methods. We retrospectively studied 52 patients with preformed DSA detected by single-antigen flow cytometric fluorescent beads (SAFBs). Patients were transplanted using a steroid-sparing regimen consisting of alemtuzumab induction, 1 week of corticosteroids and tacrolimus monotherapy.Fifteen (29%) of 52 patients experienced antibody-mediated rejection (AMR), whereas 37 (71%) patients did not. There were no demographic differences between patients with AMR and those without. Pretransplant sera were retested using a modified (SAFB) assay, which detects the presence of the complement fragment C4d as a result of DSA-induced complement activation. Results. C4d+DSA were detected in 10 (19%) of 52 patients. Biopsy-proven AMR occurred in 7 (70%) of the 10 patients with C4d+DSA and in 8 (19%) of 42 patients with C4d-DSA. AMR-free survival was worse in patients with C4d+DSA (P<0.001). Conclusions. The ability of preformed, low-level, DSA to trigger C4d fixation in vitro in patients with negative conventional crossmatch tests is predictive for AMR. C4d SAFB is potentially a powerful tool for risk stratification prior to transplantation and may allow identification of unacceptable donor antigens, or patients who may require enhanced immunosuppression.

Kidney Transplantation With Minimized Maintenance: Alemtuzumab Induction With Tacrolimus Monotherapy—an Open Label, Randomized Trial

Background. Immunosuppressive regimens for kidney transplantation which reduce the long-term burden of immunosuppression are attractive, but little data are available to judge the safety and efficacy of the different strategies used. We tested the hypothesis that the simple, cheap, regimen of alemtuzumab induction combined with tacrolimus monotherapy maintenance provided equivalent outcomes to the more commonly used combination of interleukin-2 receptor monoclonal antibody induction with tacrolimus and mycophenolate mofetil combination maintenance, both regimens using steroid withdrawal after 7 days. Methods. One hundred twenty-three live or deceased donor renal transplant recipients were randomized 2:1 to receive alemtuzumab/tacrolimus or daclizumab/tacrolimus/mycophenolate. The primary endpoint was survival with a functioning graft at 1 year. Results. Both regimens produced equivalent, excellent outcomes with the primary outcome measure of 97.6% in the alemtuzumab arm and 95.1% in the daclizumab arm at 1 year (95% confidence interval of difference 6.9% to −1.7%) and at 2 years 92.6% and 95.1%. Rejection was less frequent in the alemtuzumab arm with 1- and 2-year rejection-free survival of 91.2% and 89.9% compared with 82.3% and 82.3% in the daclizumab arm. There were no significant differences in terms of the occurrence of opportunistic infections. Conclusion. Alemtuzumab induction with tacrolimus maintenance monotherapy and short-course steroid use provides a simple, safe, and effective immunosuppressive regimen for renal transplantation.

Antibody-mediated rejection after alemtuzumab induction: incidence, risk factors, and predictors of poor outcome.

Background. Antibody-mediated rejection (AMR) is associated with allograft loss. Identification of factors associated with poor outcome has not been extensively studied. Methods. We retrospectively studied 469 patients who received a negative crossmatch renal transplant with alemtuzumab induction. Forty-eight of 469 (10.2%) patients were treated for AMR. Thirty of 48 (62.5%) of the cases fulfilled the Banff criteria for definite AMR, whereas 18 of 48 (37.5%) were categorized as suspicious for AMR (tissue injury with C4d staining or donor-specific antibodies [DSAbs]). Sensitization, high human leukocyte antigen, and -DR mismatch were risk factors for the development of AMR (P=0.0016, 0.001, and 0.012, respectively). Results. Allograft survival was inferior in the AMR group (70.2%) compared with the nonrejector group (97.0%) (P<0.001). Forty-two of 48 (87.5%) of patients with acute AMR had DSAbs. Patients with CII DSAbs at the time of AMR, whether alone or in combination with CI DSAbs had the worst allograft survival (P=0.014). Both the mean cumulative and immunodominant mean fluorescence index were higher in those patients who subsequently lost their grafts (P<0.001). Patients with diffuse C4d staining had inferior allograft survival than those with focal C4d or no staining (P=0.02). There was no significant difference in survival by histological grade but a trend to inferior outcomes in those with vascular involvement (P=0.06). Those patients who met the full Banff criteria had worse survival than those with suspicion for AMR only (P=0.04). Conclusion. This study identifies patients at risk of graft failure from AMR. These patients may benefit from newer therapeutic strategies including the use of eculizumab or bortezomib.

Optimizing a limiting dilution culture system for quantifying the frequency of interleukin-2-producing alloreactive T helper lymphocytes.

BACKGROUND The development of sensitive, specific, and reproducible techniques to quantify T cells with direct allospecificity has potential applications in the selection of bone marrow donors and in the monitoring of the antidonor alloresponse in patients after organ transplantation. Such data may provide an objective basis for altering existing immunosuppression, monitoring novel antirejection therapies, and predicting long-term graft outcome. We have previously published a correlation between donor antirecipient T helper frequencies (HTLf) and the severity of acute graft-versus-host disease after bone marrow transplantation. Using the same assay protocol, we have described the development of donor-specific hyporesponsiveness in a proportion of renal transplant recipients. However, several imperfections existed in the protocols used in these studies. Cellular interactions within the stimulator and the responder cell populations, and back stimulation of T cells within the stimulator cell population, could give rise to extraneous interleukin-2 and alter the validity or estimation of derived recipient antidonor HTLf. METHODS Using peripheral blood mononuclear cells as the responding population and splenic mononuclear cells as the stimulating population, we have examined the possible effects of these cellular interactions on the results of limiting dilution analysis assays for HTLf measurement. RESULTS These interactions have the ability to alter the validity or estimation of HTLf. We show that by depleting the responder population of HLA class II+ cells and depleting T cells from the stimulating population, these interactions are effectively abrogated. CONCLUSIONS On the basis of the findings reported here, we describe an optimized HTLf assay which is sensitive, specific, and reproducible. This has obvious applications in the analysis of alloimmune responses in transplantation.