Many groups are working to improve the results of clinical allogeneic islet transplantation in a primate model. However, few studies have focused on the optimal islet dose for achieving normal glycemia without exogenous insulin after transplantation in primate models or on the relationship between rejection and islet amyloid polypeptide (IAPP) expression. We evaluated the dose (10,000, 20,000, and > 25,000 islet equivalents (IEQ)/kg) needed to achieve normal glycemia without exogenous insulin after transplantation using eleven cynomolgus monkeys, and we analyzed the characteristics exhibited in the islets after transplantation. 10,000 IEQ/kg (N = 2) failed to control blood glucose level, despite injection with the highest dose of exogenous insulin, and 20,000 IEQ/kg group (N = 5) achieved unstable control, with a high insulin requirement. However, 25,000 IEQ/kg (N = 4) achieved normal glycemia without exogenous insulin and maintained it for more than 60 days. Immunohistochemistry results from staining islets found in liver biopsies indicated that as the number of transplanted islets decreased, the amount of IAPP accumulation within the islets increased, which accelerated CD3⁺ T cell infiltration. In conclusion, the optimal transplantation dose for achieving a normal glycemia without exogenous insulin in our cynomolgus monkey model was > 25,000 IEQ/kg, and the accumulation of IAPP early after transplantation, which depends on the transplanted islet dose, can be considered one factor in rejection.

Recent reports on donor-specific antibodies documented an overwhelming frequency of antibodies to one specific locus - human leukocyte antigen DQ (HLA-DQ). This article provides a short summary of clinical observations, a historic perspective to account for the late recognition of the role of HLA-DQ antibodies as well as potential explanations.

The basic understanding of the complexity of HLA-DQ molecules (antigens and antibodies) existed already 3-4 decades ago. However, only more recent advancements in molecular techniques as well as solid phase platforms, that allow for testing antibody specificities against individual HLA targets, provided state-of-the-art tools that are also amenable to mass applications. Thus, the significance of the polymorphic nature of both polypeptide chains of the DQ molecule, DQα and DQβ, is only now re-emerging.

HLA-DQ antibodies are real, relevant, and abundant. In order to achieve a clinically useful understanding of this phenomenon, HLA-DQ antigens and antibodies should be viewed at the level of the physiologic structure, as it appears on the cell surface, namely, one unit composed as DQαβ. Preliminary data demonstrated that such an approach is likely to lead to more equitable calculation of calculated panel reactive antibody, improving the accuracy of virtual crossmatch prediction, and increasing the likelihood of finding a compatible donor for the very highly sensitized patients.

This report describes a detailed analysis how donor-specific HLA class II epitope mismatching affects antibody reactivity patterns in 75 solid organ transplant recipients with an in situ allograft and who were considered for retransplantation. Sera were tested for antibodies in a sensitive antigen-binding assay (Luminex) with single class II alleles. Their reactivity was analyzed with HLAMatchmaker, a structural matching algorithm that considers so-called eplets to define epitopes recognized by antibodies. Only 24% of the patients showed donor-specific anti-DRB1 antibodies and there was a significant correlation with a low number of mismatched DRB1 eplets. This low detection rate of anti-DRB1 antibodies may also be due to allograft absorption. In contrast, antibodies to DRB3/4/5 mismatches were more common. Especially, 83% of the DRB4 (DR53) mismatches resulted in detectable antibodies against an eplet uniquely found on DR53 antigens. Donor-specific DQB mismatches led to detectable anti-DQB antibodies with a frequency of 87%. Their specificity correlated with eplets uniquely found on DQ1-4. The incidence of antibodies induced by 2-digit DQA mismatches was 64% and several eplets appeared to play a dominant role. These findings suggest that both alpha and beta chains of HLA-DQ heterodimers have immunogenic epitopes that can elicit specific antibodies. About one-third of the sera had anti-DP antibodies; they reacted primarily with two DPB eplets and an allelic pair of DPA eplets. These data demonstrate that HLA class II reactive sera display distinct specificity patterns associated with structurally defined epitopes on different HLA-D alleles.

We have successfully performed heart transplantation despite the most unfavourable risk factors for graft and patient survival: the presence of a high level of antibodies (Abs) against the donor's human leukocyte antigens (HLA) class I/II and blood group A1 antigens. The present study concerns post-transplant follow-up and characterization of donor reactive antibodies (DRA).

Pre-transplant treatment consisted of mycophenolate mofetil (MMF), prednisolone, tacrolimus, intravenous immunoglobulin (IVIG), rituximab, protein-A immunoadsorption (PAIA) and per-operative plasma exchange. A standard triple-drug immunosuppressive protocol was used post-operatively. Abs were analyzed by the complement dependent cytotoxicity (CDC) test against donor and panel B/T cells and by flow cytometry (FlowPRA tests detecting isolated HLA class I/II antigens). Abs against the donor's erythrocytes were analyzed using a standard direct agglutination test for immunoglobulin M (IgM) Abs and a Bio-Rad AHG gel card test detecting IgG Abs and C3d.

Pre-transplant treatment reduced Ab titers against the donor's lymphocytes from 128 to 16 and against the donor's blood group A1 antigen from 256 to 0. The patient was emergently transplanted with a heart from a blood group incompatible donor (A1 secretor to O). No hyperacute rejection was seen. DRA were present against all mismatched HLA class I and class II antigens at the time of transplantation; two of these DRA Abs disappeared within the first year post-transplant (anti-B62 and anti-DR4), one showed weakened reactivity (anti-A24) and one is still strongly reactive (anti-DQ3). The donor-specific CDC cross-match is still positive (titers 2 to 8). The level of panel reactive antibodies (PRA) remained unchanged from 6 months on post-transplant. Rising anti-A1 blood group Abs preceded the second rejection and were adsorbed by two blood group specific immunoadsorptions (Glycosorb)-ABO) and remained at a low level. IgM anti-A1 blood group Abs disappeared at 1 yr post-transplant and IgG Abs are still reactive with blood group A1 erythrocytes but at low titers (1 to 2).

The patient is clinically well 2 years after heart transplantation despite the constant persistence of donor reactive IgG Abs against blood group A1 and HLA-DQ antigens. The reactivity of DRA against other mismatched HLA antigens disappeared or weakened during the follow-up period.

The relevance of anti-HLA class II antibodies for kidney graft survival is still controversial. In part, this can be attributed to difficulties to detect and differentiate anti-HLA class II antibodies. Anti-HLA class II IgG antibody screening was performed by enzyme-linked immunosorbent assay. Subsequently, all anti-HLA class II-positive sera were subjected to the determination and specification using color-coded microspheres coated with purified HLA antigens. In a cohort of 934 patients awaiting kidney transplantation, 41 sera (4.4%) were positive for IgG anti-HLA class II antibodies. The presence was confirmed in 90.2% sera by retesting. Subsequently, all anti-HLA class II-positive patients (n = 27) who in the past had undergone a kidney transplantation with an HLA-DR and/or -DQ-mismatched graft were selected. In 25 of 27 sera (92.6%), the alloantibody specificities corresponded to the known previous transplant mismatches on a broad antigen level. In 20 of 27 sera (74.1%), anticlass I antibodies were detected as well. Anti-HLA-DP antibodies were seen in 24 of the 27 sera of this cohort. In the majority of the cases, the reactivities with different DPB1 alleles could be explained by involvement of a single, specific DPB1 epitope. Donor-specific anti-HLA-DR and -DQ antibodies were seen in the majority of cases with graft failure following HLA class II alloantigen exposure in prior kidney transplantations. In addition, HLA-DP may serve as a transplantation antigen in kidney transplantation, leading to a humoral response.

A renal transplant is the best possible treatment for patients with terminal renal failure. Advances in the development of techniques of screening of pre-formed antibodies have contributed to a notable improvement in the results obtained with allogenic transplants.

The aim of the present work is to study the nature, class, isotype and specificity of antibodies detected in patients awaiting renal transplantation at Complejo Hospitalario Universitario Juan Canalejo, as well as their relation with the level of anti-human leucocyte antigen (HLA) sensitization.

In all patient groups, there was a predominance of IgG. The distribution of anti-HLA antibody class showed that the most frequent pattern corresponded to a mixture of class I and class II antibodies for all groups. The study of specificity of anti-HLA antibody showed that of the patients with at least one previous transplant, 72% developed specific anti-HLA antibodies against some of the incompatible antigens of the donor, 12% against HLA antigens not related with the phenotype of the donor and in 16% it was not possible to determine their specificity. Most patients developed antibodies against antigens of locus B, probably because of that the number of incompatibilities contributed by the donors is also greater for locus B.

The exhaustive study of sera of patients on a waiting list for transplantation with respect to the nature, isotype, class and specificity of the antibody is important since it is possible that different antibodies can bring about, in the transplant, events that will have different consequences for the survival of the graft.

Humoral graft-specific alloreactivity was investigated in 110 renal transplant (RTx) recipients (group A) starting immediately postTx and in 32 RTx candidates sensitized against a failed graft (group B) using an enzyme-linked immunosorbent assay (ELISA) assay. All patients received a human leukocyte antigen (HLA) class I and II incompatible graft. Donor-specific (DS) antibodies were detected in 11 of 110 (90.9%) group-A patients, predominately during the first 6 months postTx. In all 11 cases, only HLA class II antibodies were detected. Ten of 11 antibody-positive patients received an HLA-DR, HLA-DQ incompatible graft, and all patients had HLA-DQ DS antibodies, either alone (n=8) or with HLA-DR antibodies (n=2). HLA-DQ antibodies were also detected in 80.9% of group-B patients. The presence of HLA-DQ DS antibodies in the early postTx period does not identify patients with rejection or deterioration of graft function. Whether these patients are at high risk for graft loss remains to be clarified.

The predictive power of a positive B-cell crossmatch remains controversial due to the presence of cofactors, such as sensitization and human leukocyte antigen (HLA) mismatch levels. UNOS OPTN/Scientific Registry data were analyzed on 9031 cadaveric kidney graft recipients who were B-cell crossmatched during 1994 and 1995 for graft outcome. This 2-year time period was chosen so that most US transplant recipients in this study would have had a similar regimen of immunosuppression consisting of prednisone, Sandimmune, and azathioprine The two patient groups that were analyzed were B-pos (n = 336) and B-neg (n = 8,695). All T-cell crossmatches were negative. Data analyzed included donor-recipient demographics, sensitization levels, B-cell crossmatch techniques, histocompatibility mismatching, graft rejection incidence, early graft loss, cause of graft failure, and statistical analyses (univariate and multivariate) in primary and repeat graft recipients. Significant factors in both crossmatch groups included pretransplant transfusions, peak and most recent class I PRA levels, a previous kidney graft, histocompatibility mismatching at HLA-A plus -B, urine in first 24 h, and rejection incidence between discharge and 6 months post-transplantation. Class II antibody specificities and panel reactive antibody (PRA) levels were not available from the UNOS database. Fifty-seven percent of 15,896 (1994-1995) transplant recipients (n 9031) were B-cell crossmatched, and 336 of 9031 recipients (3.7%) were transplanted with a B-pos crossmatch. Sixteen percent of B-pos recipients experienced early graft loss (< 6 months) compared with 11% of B-neg recipients (p < 0.001). Both primary and repeat grafts with B-pos crossmatches experienced an increase in rejection incidence (p = 0.023) and early graft loss (p < 0.001). In the sensitized (PRA > 10%) recipient subset (n = 2,789), both primary (n = 93) and regraft (n = 52) recipients with B-pos crossmatches had a higher incidence of early graft loss at 3 months, p < 0.001 and p = 0.016, respectively. HLA-DR mismatch levels in both patient groups were not different (p = 0.109). There was a 68% increase in the odds of 3-month graft loss in B-pos versus B-neg recipients (multivariate logistic regression analysis p = 0.054, 95% confidence interval 0.99-2.85). In conclusion, a B-pos crossmatch in primary and regraft recipients, including a sensitized subset, is predictive of inferior kidney graft outcome.

HLA-specific humoral immunity, as a result of recipient allosensitization, induces hyperacute rejection of allogenic kidney grafts. Cross-match tests are performed to avoid this complication. However, current techniques do not allow determination of HLA-specificity of donor-reactive antibodies in the acute cadaver-donor situation. New methods are described and discussed in this report as well as the alloantibody specificities that are of clinical importance. Alloantibodies not only mediate hyperacute rejection but may also participate in the acute rejection of organ grafts. Clinical associations between early immunological complications, such as acute rejection, in heart, liver and kidney allografted patients and pre-transplantation humoral alloimmunity emphasize the need for proper determination of donor-specific humoral immunity prior to transplantation.

Disadvantages inherent to complement-dependent cytotoxicity cross-match (CDC XM) methods are the requirements for complement and viable target cells, detection of antibodies (Abs) against non-HLA antigens, and subjective scoring. Cross-Stat (SangStat Medical Corp., Menlo Park, CA), a recently developed enzyme-linked immunosorbent assay XM procedure for the detection of IgG anti-donor HLA Abs, is theoretically devoid of these flaws.

We compared results of Cross-Stat and our standard anti-human globulin (AHG)-enhanced CDC XM procedure on 524 sera from 230 transplant candidates, which were evaluated against 51 cadaveric donors.

There was a significant correlation between AHG-CDC IgG XM and Cross-Stat results (P<0.001). For false negative sera, repeat AHG-CDC IgG XMs were still positive after platelet absorption, indicating that the Abs present were either non-HLA Abs or anti-HLA class II. Flow cytometry testing of false positive sera usually (42/62) substantiated Cross-Stat results, indicating that the discrepancy with AHG-CDC IgG XM is caused by greater sensitivity of Cross-Stat. Relative to the AHG-CDC XM, the sensitivity of Cross-Stat was 100%, the specificity was 93%, the positive predictive value was 73%, and the negative predictive value was 100%. A technical shortcoming of the Cross-Stat assay is that the frequency of indeterminate samples in the assays was 15%. Among 49 Cross-Stat negative vs. 13 Cross-Stat positive primary cadaveric renal allograft recipients (all AHG-CDC IgG-XM negative), there was no statistical difference in overall graft survival.

Given the important theoretical advantages of enzyme-linked immunosorbent assay-based XM methods over the CDC XM, however, further testing of the clinical relevance of the Cross-Stat is warranted.

Before an organ transplant is performed, donor-recipient compatibility must be established by a crossmatch in much the same way as it is done for blood transfusions. The target antigens in organ transplantation, however, are HLA rather than blood group molecules, and the target cells are lymphocytes instead of red cells. If antidonor antibodies are detected, it is important to know whether they are IgG or IgM, whether they recognize T and/or B cells, and whether the antibody reactivity is weak or strong. These test requirements are better met by flow cytometry than by the standard cytotoxicity technique. A growing body of evidence now indicates that flow cytometry can provide more sensitive and timely crossmatch information than cytotoxicity assays to decide whether or not a transplant should be done. Flow cytometry crossmatch (FCXM) is a new and evolving technique that has already been found to be extremely useful in the clinical transplantation setting, even though significant questions yet remain about the precision and reliability of using flow cytometry to quantify alloantibodies and about the limits of normal reactivity in the assay. This article reviews important technical details of the FCXM and its interpretation and clinical application in transplantation medicine.

Sera from 505 patients awaiting renal transplantation with known panel-reactive cytotoxic antibody (PRA) status were tested for HLA-DP antibodies of the IgG class by means of the monoclonal antibody immobilization of leukocyte antigens (MAILA) technique. The overall incidence of HLA-DP antibodies was 7.3%. A positive HLA-DP antibody status correlated only weakly (r = 0.23) with a positive cytotoxic antibody status. After retrospective analysis, patients with HLA-DP antibodies prior to retransplantation revealed a significantly (P < 0.025) higher graft function rate than HLA-DP-negative patients. One patient was found to possess IgG HLA-DP autoantibodies prior to transplantation; thus far, his graft has been functioning for more than 2 years.

There is no doubt that the interpretation of a positive lymphocytotoxic crossmatch test has changed over the past 10-15 years. It is now generally accepted that the original dogma put forward in the mid-1960s "that a renal transplant must not be performed in the presence of a positive lymphocytotoxic crossmatch" is no longer tenable, and many positive crossmatch transplants have already been successfully carried out. However, the precise conditions under which such a transplant can be performed are still not fully understood. Some factors which should be considered when deciding whether or not to transplant in the presence of a positive crossmatch are: (1) the specificity of the antibody, and this can be HLA class I, class II or non-HLA; (2) the time interval between the last positive crossmatch serum and transplantation; and (3) the immunoglobulin class of the antibody, either IgG or IgM.

Parental disparity for trophoblast-lymphocyte crossreactive (TLX) antigens may promote successful pregnancy. A TLX antigen system has been defined on peripheral blood lymphocytes by heteroantisera. More recently, we have reported additional activity against antigens on B lymphocytes alone termed trophoblast-B lymphocyte crossreactive (TBX) antigens. In the present study we have investigated ten TLX sera in order to determine if their target antigens are linked to the human leucocyte antigen (HLA) gene complex. The sera showed no selective activity when tested against target B lymphocytes from ten normal donors. Cytotoxic activity of TLX antisera against peripheral blood lymphocytes from six normal donors was not reduced when the class I HLA antigens of the target cells were blocked with a monoclonal antibody (PA 2.6). Similarly the cytotoxic activity of both TBX antisera against B lymphocytes from six normal donors was not decreased when class II HLA antigens were blocked by a monoclonal antibody (FMC 4). Within a family the cytotoxic activity of the TLX antisera was absorbed equally by lymphocytes from siblings who shared neither HLA haplotype. Antibody content in TLX and TBX antisera is not directed toward the classically defined HLA class I or class II antigens and is not linked to the HLA gene complex.