Alloantibody Responses After Renal Transplant Failure Can Be Better Predicted by Donor–Recipient HLA Amino Acid Sequence and Physicochemical Disparities Than Conventional HLA Matching

We have assessed whether HLA immunogenicity as defined by differences in donor–recipient HLA amino‐acid sequence (amino‐acid mismatch score, AMS; and eplet mismatch score, EpMS) and physicochemical properties (electrostatic mismatch score, EMS) enables prediction of allosensitization to HLA, and also prediction of the risk of an individual donor–recipient HLA mismatch to induce donor‐specific antibody (DSA). HLA antibody screening was undertaken using single‐antigen beads in 131 kidney transplant recipients returning to the transplant waiting list following first graft failure. The effect of AMS, EpMS, and EMS on the development of allosensitization (calculated reaction frequency [cRF]) and DSA was determined. Multivariate analyses, adjusting for time on the waiting list, maintenance on immunosuppression after transplant failure, and graft nephrectomy, showed that AMS (odds ratio [OR]: 1.44 per 10 units, 95% CI: 1.02–2.10, p = 0.04) and EMS (OR: 1.27 per 10 units, 95% CI: 1.02–1.62, p = 0.04) were independently associated with the risk of developing sensitization to HLA (cRF > 15%). AMS, EpMS, and EMS were independently associated with the development of HLA‐DR and HLA‐DQ DSA, but only EMS correlated with the risk of HLA‐A and ‐B DSA development. Differences in donor–recipient HLA amino‐acid sequence and physicochemical properties enable better assessment of the risk of HLA‐specific sensitization than conventional HLA matching.     

Mast cells contribute to the induction of ocular mucosal alloimmunity

Beyond their historical role as the effector cells in allergic disorders, mast cells have been implicated in regulating both innate and adaptive immune responses. Possessing considerable functional plasticity, mast cells are abundant at mucosal surfaces, where the host and external environments interface. The purpose of this study was to evaluate the contribution of mast cells to allograft rejection at the ocular surface. Using a well‐characterized murine model of corneal transplantation, we report that mast cells promote allosensitization. Our data show mast cell frequencies and activation are increased following transplantation. We demonstrate that mast cell inhibition (a) limits the infiltration of inflammatory cells and APC maturation at the graft site; (b) reduces allosensitization and the generation of Th1 cells in draining lymphoid tissues; (c) decreases graft infiltration of alloimmune‐inflammatory cells; and (d) prolongs allograft survival. Our data demonstrate a novel function of mast cells in promoting allosensitization at the ocular surface.     

Shared alloimmune responses against blood and transplant donors result in adverse clinical outcomes following blood transfusion post–renal transplantation

De novo HLA donor‐specific antibodies (DSA) following transplantation are associated with alloimmune injury and allograft failure. Blood transfusions are allogeneic, and when given posttransplant (PTBT) they may independently increase the risk of HLA antibody development. This study aims to analyze the development of HLA transfusion‐specific antibodies (TSA) to blood donors of transfusions given posttransplant and examine the impact on clinical outcomes. A total of 244 blood donors of transfusions received by 86 transplant patients (46 who developed a DSA post transfusion and 40 who remained DSA negative) were HLA typed. De novo TSA developed against 150/244 (61.5%) blood donors. In 70/150 (46.7%) cases the TSA was of shared HLA antibody specificity with a DSA response in the recipient (DSA+ = TSA+). This occurred when there was a greater overall HLA match between the blood and transplant donor. DSA+ = TSA+ patients had increased risk of allograft failure (P = .0025) and AMR (P = .02) compared with the DSA+ ≠ TSA+ patients. To conclude, PTBT may elicit de novo HLA antibodies. Enhanced HLA matching between the blood and transplant donor is more likely to result in a DSA and TSA of shared antibody specificities. Transfusion avoidance or the use of HLA matched or selected blood may reduce this risk and improve outcomes.     

Transfer of HLA‐Specific Allosensitization From a Highly Sensitized Deceased Organ Donor to the Recipients of Each Kidney

We report for the first time the adoptive transfer of donor HLA‐specific allosensitization in two recipients following kidney transplantation from a highly sensitized donor. Kidneys from a donation after circulatory death donor were transplanted into two nontransfused, HLA‐specific antibody negative males receiving their first transplant. Antibody screening 7 days after transplant showed high level de novo IgG HLA class I‐ and class II‐specific antibodies in both recipients, with largely overlapping antibody profiles but no antibodies to donor HLA. The unusually rapid appearance of de novo alloantibodies in immunosuppressed nonsensitized recipients and absence of donor HLA‐specific antibody prompted testing of stored donor serum that revealed high antibody levels with specificities very similar to those seen in both recipients, but in addition the presence of strong antibodies to each recipient HLA. Alloantibody levels gradually declined but were still detectable at 3 months. These findings suggest that alloreactive passenger B cells/plasma cells within the kidneys of highly sensitized donors may give rise to rapid development of posttransplant de novo HLA‐specific alloantibodies. While the clinical significance of this phenomenon is uncertain it provides one explanation for the appearance of de novo HLA‐specific antibodies directed against third party but not donor HLA.     

Impact of blood transfusion on renal transplantation.

The relationship between transfusion of different preparations of blood, sensitization to HLA antigens and survival of subsequent kidney transplants was investigated in 90 consecutive recipients. HLA lymphocytotoxins in transplant candidates precluded or greatly delayed receipt of an allograft (p less than 0.0005). Furthermore, only 17% of such sensitized recipients had functioning grafts one year after transplantation compared to 57% survival for nonsensitized recipients (p less than .02). A small number of nonsensitized patients who were never transfused had surprisingly poor one year graft survival (25%). If frozen blood is used for transfusion rather than whole/packed RBC, the incidence of patient sensitization can be markedly reduced without subsequent compromise in transplant survival (51%). It is concluded that as a consequence of avoiding HLA sensitization by transfusion of frozen blood (processed by agglomeration), the period of hemodialysis required for potential graft recipients will be shortened and an increased proportion of potential recipients will be successfully treated by transplantation.     

Donor‐targeted serotherapy as a rescue therapy for steroid‐resistant acute GVHD after HLA‐mismatched kidney transplantation

Acute graft‐versus‐host disease (GVHD) is a rare but frequently lethal complication after solid organ transplantation. GVHD occurs in unduly immunocompromised hosts but requires the escalation of immunosuppression, which does not discriminate between host and donor cells. In contrast, donor‐targeted therapy would ideally mitigate graft‐versus‐host reactivity while sparing recipient immune functions. We report two children with end‐stage renal disease and severe primary immune deficiency (Schimke syndrome) who developed severe steroid‐resistant acute GVHD along with full and sustained donor T cell chimerism after isolated kidney transplantation. Facing a therapeutic dead end, we used a novel strategy based on the adoptive transfer of anti‐HLA donor‐specific antibodies (DSAs) through the transfusion of highly selected plasma. After approval by the appropriate regulatory authority, an urgent nationwide search was launched among more than 3800 registered blood donors with known anti‐HLA sensitization. Adoptively transferred DSAs bound to and selectively depleted circulating donor T cells. The administration of DSA‐rich plasma was well tolerated and notably did not induce antibody‐mediated rejection of the renal allografts. Acute GVHD symptoms promptly resolved in one child. This report provides a proof of concept for a highly targeted novel therapeutic approach for solid organ transplantation–associated GVHD.     

Immunological transformations in the recipient of grafted allogeneic human bone

Twenty-one patients received allogeneic human bone grafts following deep freezing according to various orthopaedic indications. The HLA antigens of all donors and recipients had been determined preoperatively, and grafting was performed without any respect to the HLA match. The immunological follow-up of the recipients was managed by two different methods: MLC (mixed lymphocyte culture) and MAILA (monoclonal antibody-specific immobilisation of lymphocyte antigens). No immunosuppression was performed. The follow-up lasted up to 6 years. Allogeneic grafting of human cancellous bone induces specific immunological reactions in the recipient. The consequences of these observations are: (1) allogeneic bone grafting may induce second-set reactions following subsequent blood transfusion, tissue grafting or organ transplantation; (2) transplantation of fresh, perfused, vascularised allogeneic bone or joint may become a therapeutic approach in the near future. Then the employment of standard immunosuppressive protocols will be mandatory in order to fight acute rejection of the graft.The project Human Bone Transplantation is supported by the Karl -Wilder-Stiftung Munich, a grant of the German Life Insurances.     

Non-HLA T-cell reactivity during the first year after HLA-identical living-related kidney transplantation

Abstract: Background: It has been reported that donor‐reactive T‐cell responses may decrease during the first year after HLA‐mismatched organ transplantation. We wondered whether donor‐reactive T‐cell responses directed to minor histocompatibility antigens (mHAgs) or other non‐HLA antigens also decrease after HLA‐identical living‐related (LR) kidney transplantation. Methods: We studied donor‐reactive T‐cell responses by IFN‐γ and granzyme B (GrB) Elispot assays in 15 HLA‐identical LR kidney transplant recipients before, six months and one yr after transplantation. Third‐party reactivity was used as control. Patient and donor peripheral blood mononuclear cells were typed for 11 known mHAgs. Results: During the study period, 60% and 36% of the patients demonstrated donor‐reactive IFN‐γ and GrB producing cells (pc), respectively. The number of donor‐reactive IFN‐γ and GrB pc was significantly lower than the number of third‐party reactive IFN‐γ and GrB pc. After transplantation, donor‐reactivity and third‐party reactivity were comparable to pre‐transplant values. No relation was found in mHAg mismatches between donor and recipient and donor‐reactive T‐cell response. Conclusions: Donor‐reactivity could be detected before and after HLA‐identical LR kidney transplantation, but was not related with the number of mHAg mismatches, and did not decrease after transplantation.     

Reassessment of the clinical impact of preformed donor‐specific anti‐HLA‐Cw antibodies in kidney transplantation

Anti‐denatured HLA‐Cw antibodies are highly prevalent, whereas anti‐native HLA‐Cw antibodies seem to lead to random flow cytometry crossmatch results. We aimed to reassess crossmatch prediction for anti‐HLA‐Cw using 2 types of single antigen flow beads (classical beads and beads with diminished expression of denatured HLA), and to compare the pathogenicity of preformed anti‐denatured and anti‐native HLA‐Cw antibodies in kidney transplantation. We performed 135 crossmatches with sera reacting against donor HLA‐Cw (classical beads fluorescence ≥500); only 20.6% were positive. Forty‐three (31.6%) were anti‐denatured HLA antibodies (beads with diminished expression of denatured HLA fluorescence <300); all were crossmatch negative. The correlation between classical beads fluorescence and the crossmatch ratio was low (ρ = 0.178), and slightly higher with beads with diminished expression of denatured HLA (ρ = 0.289). We studied 52 kidney recipients with preformed anti‐HLA‐Cw donor‐specific antibodies. Those with anti‐native HLA antibodies experienced more acute and chronic antibody‐mediated rejections (P = .006 and .03, respectively), and displayed a lower graft survival (P = .04). Patients with anti‐native HLA‐Cw antibodies more frequently had previous sensitizing events (P < .000001) or plausibility of their antibody profile according to known anti‐native HLA‐Cw eplets (P = .0001). Anti‐native but not anti‐denatured HLA‐Cw antibodies are deleterious, which underscores the need for reagents with diminished expression of denatured HLA.     

Immunoadsorption of sensitized kidney transplant candidates immediately prior to surgery

Patients with anti-human leucocyte antigen (HLA) antibodies from previous transplantation, blood transfusion are highly sensitized and at risk to hyperacute renal graft loss. As these antibodies are identified to be of pathogenic importance, an effective removal may allow successful transplantation. Six 'high risk patients' [panel-reactive antibodies (PRA) >30% or retransplanted patients with an acutely rejected first graft within 6 months from surgery] were treated by protein A immunoadsorption (IA) immediately prior to transplantation. We treated the calculated plasma volume one to three times prior to surgery: mean 4600 mL (range 2100-10 200 mL). After transplantation we repeated the sessions according to antibody (Ab) recurrence, graft function and signs of rejection. The panel reactive Ab were reduced from mean 65% pre-IA (range 35-85) to lowest 15% (range 0-55). After the course they reappeared to 30% (range 0-90). Five of the six patients had no clinical signs of vascular rejection. At a follow-up of mean 54 months (+/-14) four grafts still function with a mean serum creatinine of 172 micromol/L (+/-57). Protein A IA is a safe and effective adjunct in the treatment of highly sensitized patients awaiting renal transplantation. The treatment immediately prior to operation can prevent hyperacute rejection and increases the graft survival in these patients.     

Assessing the utilization of high‐resolution 2‐field HLA typing in solid organ transplantation

HLA typing in solid organ transplantation (SOT) is necessary for determining HLA‐matching status between donor‐recipient pairs and assessing patients’ anti‐HLA antibody profiles. Histocompatibility has traditionally been evaluated based on serologically defined HLA antigens. The evolution of HLA typing and antibody identification technologies, however, has revealed many limitations with using serologic equivalents for assessing compatibility in SOT. The significant improvements to HLA typing introduced by next‐generation sequencing (NGS) require an assessment of the impact of this technology on SOT. We have assessed the role of high‐resolution 2‐field HLA typing (HR‐2F) in SOT by retrospectively evaluating NGS‐typed pre‐ and post‐SOT cases. HR‐2F typing was highly instructive or necessary in 41% (156/385) of the cases. Several pre‐ and posttransplant scenarios were identified as being better served by HR‐2F typing. Five different categories are presented with specific case examples. The experience of another center (Temple University Hospital) is also included, whereby 21% of the cases required HR‐2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti‐HLA antibody issues.     

Development of clinical immunosuppression for organ transplantation

The progress in the understanding of allograft rejection since the first modern kidney transplantation is enormous. The concept of the histocompatibility complex (HLA system) was born and the loci for the related genes are now identified. The actual structure of HLA antigens and the molecules (lymphokines) released by them are being understood. A population of lymphocytes (suppressor cells) which reduces the host immune response to tissue allografts has also been identified. With advanced understanding, ideas and methods for immunosuppression have been developed. Hyperacute rejection due to presensitization (secondary to preformed HLA antibody) ought to be avoided or attenuated, if it were to happen. The significance of previous blood transfusion or multiple pregnancies were clarified in this regard. The tests to determine such immunological reactivity were devised. Steroids, azathioprine and cyclosporine which are presently in use for immunosuppression were reviewed as to their actions, effects and side-effects. Total lymphoid irradiation presently appears as a potential effective immunosuppressive procedure and is currently being tried in certain transplant centers. The superiority of monoclonal antibodies against polyclonal antilymphocyte antibodies has been confirmed, although the latter also has various useful actions. Finally, the need and possible means to facilitate donor specific unresponsiveness are mentioned in perspectives for the future management of clinical organ transplantation.     

A Memory B Cell Crossmatch Assay for Quantification of Donor‐Specific Memory B Cells in the Peripheral Blood of HLA‐Immunized Individuals

Humoral responses against mismatched donor HLA are routinely measured as serum HLA antibodies, which are mainly produced by bone marrow–residing plasma cells. Individuals with a history of alloimmunization but lacking serum antibodies may harbor circulating dormant memory B cells, which may rapidly become plasma cells on antigen reencounter. Currently available methods to detect HLA‐specific memory B cells are scarce and insufficient in quantifying the complete donor‐specific memory B cell response due to their dependence on synthetic HLA molecules. We present a highly sensitive and specific tool for quantifying donor‐specific memory B cells in peripheral blood of individuals using cell lysates covering the complete HLA class I and class II repertoire of an individual. Using this enzyme‐linked immunospot (ELISpot) assay, we found a median frequency of 31 HLA class I and 89 HLA class II–specific memory B cells per million IgG‐producing cells directed at paternal HLA in peripheral blood samples from women (n = 22) with a history of pregnancy, using cell lysates from spouses. The donor‐specific memory B cell ELISpot can be used in HLA diagnostic laboratories as a cross‐match assay to quantify donor‐specific memory B cells in patients with a history of sensitizing events.     

Biologic mechanisms and clinical consequences of pregnancy alloimmunization

Alloimmunization occurs during pregnancy when tissue antigens derived from the fetus and/or placenta prime maternal immune cells to divide and differentiate. For many women, the result of pregnancy alloimmunization is the formation of anti‐HLA antibody that can endure for decades and preclude transplantation by limiting donor compatibility. Pregnancy alloimmunization may also generate memory B cells that can rapidly produce anti‐HLA antibody after transplantation as well as pathogenic memory T cells, which pose a threat to graft survival. However, emerging data suggest that pregnancy also programs the differentiation of anergic, dysfunctional, and regulatory T cell populations, which may not mediate accelerated graft rejection. Hence, some of the immune mechanisms responsible for maternal immunologic tolerance of the fetus may persist into postpartum life and affect the response to an allograft. This review discusses these emerging data as well as the persistent knowledge gaps that affect women at multiple stages of their transplant care.     

Influence of HLA matching and blood transfusion on renal allograft survival.

One hundred and seven consecutive cadaver kidney transplants have been followed for up to 6 years. The beneficial effect of HLA matching, shown in previous studies, has been confirmed. The 2-year failure rate from rejection was 29% for grafts with less than two incompatibilities, in comparison with a figure of 52% where there were two or more incompatibilities. In contrast to some reports, the presence of HLA antibodies did not have an adverse effect on the survival of first grafts. Patients not transfused prior to transplantation had a much higher 1-year graft failure rate (72%) than those given either frozen-thawed red cells (29%) or whole blood (23%). This apparently beneficial effect of blood transfusion was no greater in patients transfused with more than five units compared with those given less than five units. We believe that blood transfusion has an important influence on the outcome of renal transplantation.     

Allosensitization after transplant failure: the role of graft nephrectomy and immunosuppression – a retrospective study

There are conflicting data about the role of transplant nephrectomy and immunosuppression withdrawal on the development of allosensitization and the impact on re‐transplantation. We divided 109 first graft recipients into two groups according to whether they underwent nephrectomy (NX+, n = 61) or their graft was left in situ (NX?, n = 48). Sera were assessed for HLA‐A/B/Cw/DR/DQ antibodies at the time of NX/transplant failure and after 3, 6, 12, 24 months. The NX+ group showed a higher rate of donor specific antibody (DSA) and non‐DSA human leukocyte antigen (HLA) antibody production at all the time points. Multivariable analysis showed that nephrectomy was a strong, independent risk factor for the development of DSAs after 12 and 24 months (P = 0.005 and 0.008). In the NX? group, low tacrolimus levels correlated with DSA formation (AUC 0.817, P = 0.002; best cut‐off level 2.9 ng/ml). Analysis with a standardized pool of UK donors showed a more difficult grade of HLA matchability following nephrectomy compared with the NX? group. Nephrectomy is followed by the long‐term production of DSA and non‐DSA HLA antibodies and negatively impacts on the chances of finding a HLA‐compatible kidney. Tacrolimus levels ≥3 ng/ml are protective against the development of allosensitization and could facilitate re‐transplantation in the NX? group.     

Stable engraftment of allogeneic T lymphocytes in a patient with severe combined immunodeficiency following a transfusion with unirradiated blood. Specific recognition of host histocompatibility antigens and engraftment failure of a bone marrow transplant HLA-identical to the host

We have analyzed the immunocompetence of blood donor-derived, allogeneic T cells that had engrafted in a patient with severe combined immunodeficiency following transfusion with unirradiated blood. The blood donor T cells multiply and persist in the patients' bone marrow and peripheral blood over several months without leading to a graft-versus-host disease. The allogeneic T cells can be expanded in vitro and restimulated by cells expressing host HLA-DR antigens. The presence of blood donor-derived T cells prevented engraftment of a bone marrow graft from an HLA-identical sibling; in contrast, engraftment and immunological reconstitution by bone marrow donor-derived cells was seen following elimination of the allogeneic T cells in vivo. We conclude that the allogeneic T cells sensitized against host histocompatibility antigens were responsible for bone marrow graft failure, even though this sensitization did not lead to a graft-versus-host disease.     

Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection

Regulatory T cell (Treg) therapy using recipient‐derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor‐MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA‐A2‐specific CARs were engineered: one comprising a CD28‐CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA‐A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA‐A2‐expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune‐mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation.     

Human leukocyte antigens antibodies after lung transplantation: Primary results of the HALT study

Donor‐specific antibodies (DSA) to mismatched human leukocyte antigens (HLA) are associated with worse outcomes after lung transplantation. To determine the incidence and characteristics of DSA early after lung transplantation, we conducted a prospective multicenter observational study that used standardized treatment and testing protocols. Among 119 transplant recipients, 43 (36%) developed DSA: 6 (14%) developed DSA only to class I HLA, 23 (53%) developed DSA only to class II HLA, and 14 (33%) developed DSA to both class I and class II HLA. The median DSA mean fluorescence intensity (MFI) was 3197. We identified a significant association between the Lung Allocation Score and the development of DSA (HR = 1.02, 95% CI: 1.001‐1.03, P = .047) and a significant association between DSA with an MFI ≥ 3000 and acute cellular rejection (ACR) grade ≥ A2 (HR = 2.11, 95% CI: 1.04‐4.27, P = .039). However, we did not detect an association between DSA and survival. We conclude that DSA occur frequently early after lung transplantation, and most target class II HLA. DSA with an MFI ≥ 3000 have a significant association with ACR. Extended follow‐up is necessary to determine the impact of DSA on other important outcomes.     

Drug‐induced alloreactivity: A new paradigm for allorecognition

Abacavir administration is associated with drug‐induced hypersensitivity reactions in HIV+ individuals expressing the HLA‐B*57:01 allele. However, the immunological effects of abacavir administration in an HLA‐B57 mismatched transplantation setting have not been studied. We hypothesized that abacavir exposure could induce de novo HLA‐B57‐specific allorecognition. HIV‐specific CD8 T cell clones were generated from HIV+ individuals, using single cell sorting based on HIV peptide/HLA tetramer staining. The T cell clones were assayed for alloreactivity against a panel of single HLA‐expressing cell lines, in the presence or absence of abacavir. Cytokine assay, CD137 upregulation, and cytotoxicity were used as readout. Abacavir exposure can induce de novo HLA‐B57 allorecognition by HIV‐specific T cells. A HIV Gag RK9/HLA‐A3‐specific T cell did exhibit interferon‐γ production, CD137 upregulation, and cytolytic effector function against allogeneic HLA‐B57, but only in the presence of abacavir. Allorecognition was specific to the virus specificity, HLA restriction, and T cell receptor TRBV use of the T cell. We provide proof‐of‐principle evidence that administration of a drug could induce specific allorecognition of mismatched HLA molecules in the transplant setting. We suggest that HIV‐seropositive recipients of an HLA‐B57 mismatched graft should not receive abacavir until further studies are completed.