Crossmatch Assay Research Articles

Simultaneous bone marrow and tissue transplantation in rats treated with a nonmyeloablative conditioning regimen promote tolerance to composite tissue allografts (141.18)

Abstract Approaches to safely induce graft acceptance through bone marrow chimerism and limited immunosuppression are currently being pursued. Here we evaluated the effect of nonmyeloablative conditioning on the establishment of tolerance and mixed chimerism in recipients of bone marrow transplantation (BMT) and reconstructive tissue allotransplantation (RTA). Twenty Wistar-Firth rats were treated with a short course of immunosuppressive therapy (anti-ab-TCR mAb, tacrolimus, and anti-lymphocyte serum). One day prior to BMT, rats received total body irradiation followed by transplantation of heterotopic osteomyocutaneous flaps taken from hind limbs of ACI rats. Following BMT, rats were monitored for rejection and mixed chimerism (>2% donor cells). Fifteen rats rejected their RTA, and five rats accepted the graft. Mixed chimerism was detected in peripheral blood at one month after RTA, but chimerism was lost in all transplant recipients by four months. After 1.5 years post-RTA, the number of donor cells in spleen, mesenteric lymph node, recipient bone marrow and thymus was <2%. To determine the tolerance mechanism, ten rats were given donor skin grafts at one year post-RTA and examined for anti-donor antibodies by flow cross-match assay. Acceptors of RTA were tolerant of donor skin and no anti-donor antibodies were detectable, which points to a deletional mechanism. These studies suggest that nonmyeloablative conditioning can establish long-term tolerance and indefinite allograft survival in rats. (Research supported by a grant from the US Department of Defense).

Comparison of Antibody Monitoring System with Flow Cytometric Crossmatch Test in Renal Transplant Recipients with High Panel-Reactive Antibody

Background/Aims: The antibody monitoring system (AMS) is a recently developed enzyme-linked immunosorbent assay (ELISA) crossmatch assay to detect donor-specific anti-HLA immunoglobulin G antibodies (DS-HLA Abs). This study was conducted to compare the AMS with the flow cytometric crossmatch (FCXM) test in renal transplant recipients with high panel-reactive antibody (PRA). Methods: Thirty-two sera were obtained from 10 patients with panel reactivity above 50%. When anti-HLA Ab was detected by ELISA PRA and the matched donor had the corresponding HLA antigen, it was considered to indicate DS-HLA Ab. The results of the AMS assay and FCXM were compared with the DS-HLA Abs. Results: Twenty-three (71.9%) sera were positive for DS-HLA Abs by ELISA PRA. The AMS assay showed that the number of compatible sera with DS-HLA Abs was 27 (84.4%), and it was significantly concordant (κ = 0.649, p < 0.0001). For FCXM, the number of compatible sera with DS-HLA Abs was 26 (81.3%), and it was also significantly concordant (κ = 0.614, p < 0.0001). There was a significant degree of concordance between the AMS assay and FCXM in detection of DS-HLA Abs (κ = 0.452, p = 0.010). Conclusion: The AMS assay is comparable to FCXM in detecting DS-HLA Abs in high PRA recipients.

Sensitization to Minor Histocompatibility Antigens Poses a Barrier in Transplantation and Can Be Prevented by CD154:CD40 Co-Stimulatory Blockade.

There is an increased rate of graft failure associated with nonmyeloablative conditioning approaches for hematopoietic stem cell (HSC) transplantation, often resulting in subsequent sensitization to donor alloantigens. Recipient sensitization is among the most critical of problems currently facing clinical transplantation, resulting in hyperacute rejection of transplanted HSC and/or organ grafts by existing donor-specific antibodies (Ab) against major histocompatibility (MHC) antigens in secondary transplants. The humoral immune response to minor histocompatibility antigens (Mi-HAg) has not been fully evaluated in BMC transplantation to date. In this study, we explored the role of allosensitization to Mi-HAg in BMC transplant rejection using a mouse model. AKR (H-2k) mice were sensitized with donor skin grafts from MHC-matched, but minor antigen-disparate B10.BR (H-2k) mice. Significantly higher levels of Abs against donor were generated as detected at 4 weeks after skin grafting by flow cytometry cross match assay (mean fluorescence intensity (MFI) 231.3 ± 151.1; P < 0.0001) compared with the Ab levels in sera of naïve AKR mice (MFI 4.2 ± 2.0) . The MFI of Ab titer to anti-Mi-HAg was similar to that in mice after rejecting skin grafts with MHC disparity (MFI: 231.3 ± 139.0) or MHC plus Mi-HAg disparity (MFI: 301.1 ± 141.1). IgG subclasses of IgG1, IgG2a, and IgG2b were detected in all tested AKR mice with high levels of anti–donor total IgG. Surprisingly, the Abs generated to minor antigens were not donor-specific as they also bound to splenocytes from B6 (H-2b), BALB/c (H-2d), C3H (H-2k) and B10.D2 (H-2d) mice. In subsequent HSC transplantation performed 5–7 weeks after skin grafting, only 20% of B10.BR skin sensitized AKR mice engrafted after ablative conditioning. In contrast, all naïve AKR control mice engrafted but none did in mice sensitized to MHC alloantigens. These findings correlated with in vivo cytotoxicity assays performed at the same time point. Mi-HAg sensitized AKR mice eliminated CFSE labeled donor splenocytes with a significantly faster rate than naïve AKR but significantly slower rate than the mice sensitized to MHC alloantigen. We previously reported that blockade of signaling via the CD40/CD154 co-stimulatory pathway with anti-CD154 induces B cell tolerance and abrogates donor-specific Ab generation in a MHC plus minor antigen mismatched mouse model. Here, we therefore examined whether CD154 blockade during exposure to minor antigens would also prevent sensitization to minor antigens. The production of anti-donor-Mi-HAg antibodies was totally prevented in mice treated with anti-CD154 mAb (MFI 6.1±3.48l P=0.33) compared with the Ab in naïve mice (3.9±0.25), suggesting a dominant role of the CD154:CD40 pathway in B-cell responses to Mi-HAg. Moreover, anti-CD154 treatment promoted bone marrow engraftment to 100% in recipients previously exposed to donor Mi-HAg, suggesting that antibody and not T cells play a dominant role in sensitization to minor antigens. These data demonstrate that sensitization to minor antigens poses a significant barrier in transplantation, but with less intensity than sensitization to MHC antigens, and can be prevented by anti-CD154 mAb. Our findings may identify a novel and clinically relevant approach in the development of conditioning approaches to enhance engraftment but prevent sensitization in the event of graft failure.

Costimulatory Blockade Enhances Engraftment and Prevents Anti-Donor Antibody Generation in Nonmyeloablative Conditioned Prediabetic NOD Mice

Chimerism induces tolerance to organ and tissue grafts. Although the toxicity of conditioning has been significantly reduced with nonmyeloablative approaches, it would be beneficial to further reduce the intensity of conditioning in organ transplant recipients. Studies in normal mice and autoimmune NOD mice have revealed that the primary role for conditioning is to control host-versus-graft alloreactivity, allowing for immune-based conditioning to establish chimerism. Recent insights into the mechanism of interaction and crossregulation between innate and adaptive immune responses have led to the development of new approaches for immune-based conditioning. We previously demonstrated that preconditioning of diabetes-prone NOD mice with anti-CD8 monoclonal antibody (mAb) combined with anti-CD154 mAb allowed chimerism to be established with 550 cGy total body irradiation (TBI). Here we investigated whether combined costimulatory blockade can improve the nonmyeloablative regimen and enhance bone marrow chimerism in autoimmune recipients. Prediabetic NOD (H-2d) mice were treated with anti-CD8 antibody (day -3). After 500 cGy TBI (day 0), recipient NOD mice were transplanted with 30 × 106 B10.BR (H-2k) bone marrow cells (day 0) and received anti-CD154, anti-OX40L, and anti- inducible co-stimulatory molecule (ICOS) mAbs intraperitoneally (IP) (day 0, 1, 2, 3). Chimerism and multilineage analysis were quantitated by flow cytometry. Chimeric animals produced multilineage donor chimerism. While 18 prediabetic NOD mice demonstrated an average PB chimerism of ~37% at 1 month and 61% engraftment, 15 of 18 mice lost PB chimerism by 6 months. NOD mice were then transplanted with B10.BR donor skin grafts 1 month post BM transplant. Flow cytometry cross-match assays were performed to detect anti-donor antibody (Ab) in the sera collected 1 month post skin grafting. 44% of chimeric NOD mice (n=9) accepted their skin grafts for over 90 days, while 100% of non-chimeric animals (n=4) rejected their grafts within 60 days. NOD mice transplanted with B10.BR skin grafts alone rejected their grafts within 2 weeks and generated significantly higher levels of donor-specific Abs (mean fluorescence intensity: 251.2 ± 61.5, P<0.0001, n=6) compared to chimeric NOD (4.5 ± 0.6, P<0.0005, n=17) or non-chimeric NOD (12.6 ± 2.4, P<0.0005, n=10). Both chimeric NOD and non-chimeric NOD mice transplanted with 3rd party B6 skin grafts rejected their grafts within 30 days. These results suggest that rejection of skin grafts in chimeric and nonchimeric animals is not associated with anti-donor antibody generation in preconditioned animals. Studies are currently underway to determine if impaired generation of effector/memory T cells (CD44high/CD62Llow/−) is responsible for enhanced engraftment and to determine the potential mechanisms that promote tolerance in prediabetic NOD mice after loss of PB chimerism.

Sensitivity, specificity and clinical relevance of different cross-matching assays in deceased-donor renal transplantation

To assess the significance of antibodies detected by complement-dependent cytotoxicity (CDC), solid phase (SPA) and flow cytometry (FC) assays we compared their predictive value in 354 consecutive cases of deceased-donor kidney transplantation. Pre-transplantation screening of anti-HLA class I and class II antibodies was performed by CDC and SPA. The direct crossmatch between recipients' sera and donors' T and B cells was performed by CDC followed by FC and SPA (“virtual cross-match”). The past history of antibodies displayed by the recipient was not considered a contraindication for transplantation even when it showed DSA. A side-by-side comparison of the correlation between graft loss, history of DSA and cross-match results indicated that sensitivity was 5%, 16% and 17% while specificity was 99%, 93% and 86% in CDC, SPA, FC crossmatches respectively. There was no significant difference between the 3 year survival of primary and secondary kidney allografts. We conclude that screening and cross-matching the sera by CDC provides reliable results and optimizes the patient's chances to receive a transplant. SPA and FC, however, are of great importance for identifying patients which require close monitoring by biopsy and serology for early diagnosis and treatment of acute antibody mediated rejection (AAMR).

CD154:CD40 Co-Stimulatory Blockade: A Specific Approach To Prevent Sensitization to Donor Alloantigens.

Introduction: Recipient sensitization is one of the most critical problems facing clinical transplantation. Patients with sickle cell disease are presensitized to HLA-alloantigens from chronic transfusion therapy. These allosensitized recipients experience a higher failure rate in engraftment and require more intense conditioning. A method to prevent sensitization would have a significant impact on transplant outcomes in these recipients. We have identified that B-cell responses and humoral immunity are the dominant factor in sensitization to MHC alloantigens. T-cell-dependent B-cell responses require physical cell:cell contacts that drive the different steps of B-cell differentiation to memory B-cells or to Ab-producing cells. Several signal pathways have been identified that are important for initiation of B-cell differentiation, including costimulation through the CD28/B7 or TNF/TNFR families. Here, we examined whether blocking the costimulatory interaction between T- and B-cells with anti-CD154, anti-OX40L, or anti-ICOS during exposure to alloantigen would prevent allosensitization.Methods: B6 (H-2b) mice received allogeneic BALB/c (H-2d) skin grafts on day 0 and were treated with anti-CD154 (day 0 and day +3), anti-OX40L (day 0 to +4), or anti-ICOS (day +4, 6, 8 and 10). Anti-donor specific antibodies were measured by flow cytometry cross-match assay 4 weeks after skin grafting and reported as mean fluorescence intensity (MFI).Results: Recipients treated with anti-CD154, anti-OX40L, or anti-ICOS rejected BALB/c skin grafts with a time course similar to normal B6 controls (12.7±2.4 days). The anti-donor antibody titer in mice treated with anti-CD154 (5.9±3.9) was slightly, but not significantly (P>0.05) higher than for naïve mice (3.8±0.6). Levels of allospecific Ab were markedly higher in mice treated with mAb anti-ICOS (123.6±38.1; P<0.001) and anti-OX40L (80.6±23.3; P<0.0001) compared with naïve controls or anti-CD154 treated animals. The Ab level in mice treated with mAb anti-ICOS was similar to sensitized controls (skin grafting only without mAb treatment; MFI: 132.6±38.5; P=0.69). The anti-donor Ab in mice treated with mAb anti-OX40L was significantly lower (P=0.02) compared to sensitized controls, but the difference was much more significant when compared with naïve controls. To evaluate sensitization in vivo, mice were conditioned with 950 cGy and transplanted with 15×106 of BALB/c BM cells 5–7 weeks after skin grafting. As expected, engraftment did not occur in sensitized control mice (no Ab treatment). Engraftment also did not occur in mice treated with anti-ICOS. Only 16.7% of mice engrafted with anti-OX40L treatment. In contrast, 75.0% of mice engrafted with anti-CD154 treatment.Conclusion: Blocking molecular interactions between CD40/CD154 abrogated the generation of antibody against donor antigens. The CD154 blockade effect on Ab production was specific and not a general feature of co-stimulatory blockade, as blockade of ICOS or OX40L did not completely inhibit Ab generation. These data suggest that the engagement of B-cell molecule CD40 and its ligand CD154 plays a central role in T-cell-dependent B-cell responses. These findings could have a significant impact on management of sensitized recipients in the clinic. [Display omitted]

67-P: Rituximab does not interfere in a solid phase donor-specific crossmatch (DSA) assay

67-P: Rituximab does not interfere in a solid phase donor-specific crossmatch (DSA) assay

Immunoglobulin Class (IgG, IgM) Determination by Dithiothreitol in Sensitized Kidney Transplant Candidates

Immunoglobulin class plays an important role in the histocompatibility crossmatch test to predict hyperacute rejection in kidney transplantation. The existing data indicates that immunoglobulin (Ig) M antibodies, particularly when they are autoantibodies, are not deleterious to the renal allograft. We used the reducing agent dithiotreitol (DTT) to inactivate IgM but not IgG in the crossmatch assay to help sensitized patients have the chance for successful transplantation. In this descriptive study, 57 candidates for kidney transplantation with final positive crossmatches who had a history of panel-reactive antibody (PRA) greater than 30% were selected. Two of 57 patients had systemic lupus erythematous (SLE). The sera of patients were treated by DTT and then measured for cytotoxicity against donor lymphocytes and a panel of 12 cells using the complement-dependent cytotoxicity (CDC) method. Autocrossmatch was also performed to differentiate autoantibodies and alloantibodies by the CDC method. Of the 57 patients, six subjects (10.53%) had IgM and 51 patients (89.47%) IgG in their serum against donor lymphocytes. Also against panel cells, 39 of 57 patients (68.43%) had IgG, three patients (5.26%) had IgM, and 15 patients (26.31%) had both IgG and IgM antibodies. Autolymphocytotoxic antibodies were detected in 1.75% patients (1 of 57) who had SLE. According to our results, 5.26% of the patients who were IgM-positive and IgG-negative for both crossmatch and PRA assays may experience successful kidney transplantation.

Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure.

The detection of donor-specific anti-HLA antibodies by standard procedures such as complement-dependent cytotoxicity assay (CDC) or flow cytometric (FACS) analysis is limited by its low sensitivity and the quality of the donor cells. Therefore, an ELISA-based technique was employed using solid phase-immobilized monoclonal antibodies to capture HLA class I or class II molecules of the donor, respectively. In this HLA class I and class II antibody monitoring system (AMS) the donor-specific anti-HLA antibodies from the sera of recipients bind to the HLA molecules of the donor which have been immobilized by monoclonal antibodies (mAb) recognizing non-polymorphic epitopes. Upon binding of donor-specific anti-HLA antibodies they are recognized by secondary enzyme-conjugated anti-human immunoglobulin (Ig) antibodies. A newly established modification of the standard protocol allows the differentiation between bound antibodies of the IgG and IgM isotype. Furthermore, this assay was adapted for investigating small amounts of solid tissue of donors from whom no other cells (e.g. from blood) were available. We here provide an overview of the classical crossmatch methods with their advantages and limits. In addition, the design of the novel AMS-ELISA is described in terms of quality and sensitivity of the approach using exemplary cases of different application. The selected cases show that the AMS-ELISA represents a valuable tool for the post-transplantation monitoring of donor-specific anti-HLA antibodies during reaction crisis, after transfusion reactions and in particular cases of tissue transplantations lacking single cells.

Pancreas Transplantation Utilizing Thymoglobulin, Sirolimus, and Cyclosporine

This study aims to determine the impact of thymoglobulin-sirolimus-cyclosporine immunosuppression on the alloimune response of pancreas-kidney transplant recipients. Thirty-six pancreas transplant recipients received an induction protocol of thymoglobulin, sirolimus, reduced-dose cyclosporine, and corticosteroids. Ten recipients were also enrolled in a study to measure immune responsiveness. Flow PRA determined HLA antibody, donor-specific flow cytometry crossmatching (FCXM), T-cell subset, and suppressor cell assays were performed during the first posttransplant year. One-year patient, kidney, and pancreas graft survivals were 97%, 94%, and 92%, respectively. There was one death and three graft losses. There were no acute rejection episodes. Recipients in the immune-monitoring study (n=10) displayed>80% depression of CD3, CD4, and CD8 (+) cell counts up to 3 months posttransplant. At transplantation 9/10 patients displayed<10% class I and no class II HLA antibody. By 3 months, 7/10 monitored recipients showed a transient elevation in class I HLA antibodies, including 2 patients who expressed>80% Flow PRA. One patient was pretransplant FCXM positive, whereas by 3 months posttransplant 2/10 patients demonstrated a positive FCXM. There were no clinical consequences of the presence of HLA antibody or the positive FCXMs. By 6 months, 7/9 patients demonstrated immunoregulatory suppressor cells. The absence of acute rejection events was likely due to inhibition of donor-specific immunity by the immunosuppressive regimen. Seventy percent of patients demonstrated an early, non-donor-directed HLA antibody response that had no adverse effect on graft function and 78% of the monitored patients displayed immunoregulatory cells probably contributing to the successful outcomes.

CD154:CD40 Co-Stimulatory Blockade: A Novel Approach To Prevent Sensitization to Donor Alloantigens.

Introduction: Recipient sensitization is one of the most critical problems facing clinical transplantation. Allosensitized recipients often rapidly reject vascularized solid organ grafts as a result of preformed anti-donor antibody. Similarly, bone marrow transplantation for sickle cell disease and thalassemia is limited by sensitization from transfusion. A method to prevent sensitization would have a significant impact on transplant outcomes. Until recently, T cells were believed to be the primary effector cell in the induction of adaptive immune responses. We recently found that humoral immunity provides a dominant barrier in allosensitization to MHC antigens. B cell activation occurs through T-cell-dependent responses via signaling from the co-stimulatory molecule CD154 (on T cells) to its ligand CD40 (on B cells). Here, we examined whether blocking the costimulatory interaction between T and B cells during exposure to alloantigen would prevent allosensitization.Materials and Methods: Mice deficient for CD154 molecule (CD154−/ −, H-2b), α β-TCR+ T cells (TCRβ −/ −, H-2b); or wild type B6 (H-2b) mice received allogeneic BALB/c (H-2d) skin grafts (SG) on day 0. Some B6 mice were also treated with anti-CD154 (day0 and day+3) and/or anti-α β-TCR mAb (day-3) peritransplant. Antibodies were detected by flow cytometry cross-match (FCM) assay and reported as mean fluorescence intensity (MFI).Results: CD154−/ − mice rejected primary BALB/c SG with a time course similar to normal B6 controls (12.4 ± 2.1 vs. 12.7 ± 2.4 days). TCRβ −/ − mice accepted SG permanently (>120 days). Notably, anti-donor antibody was not generated in either the CD154−/ − or TCRβ −/ − mice (MFI: 4.1 ± 0.1 and 4.2 ± 0.4) after SG compared with Ab in naïve serum (3.0±0.2). Sensitized B6 mice had significantly higher antibody titers (106.8 ± 35.1) 4 weeks after SG rejection. A second SG transplanted 5 to 7 weeks after the first graft was rejected at an accelerated rate (9.0 ± 0.8 days, P < 0.05) in the CD154−/ − mice, but no anti-donor MHC antibody was produced. Second grafts placed on TCRβ −/ − mice were accepted, as were the primary SG. In normal B6 recipients pretreated with anti-CD154 or anti-α β-TCR alone, SG survival was not significantly prolonged. The Ab titers were only slightly higher in mice treated with anti-CD154 (5.9±3.4; P>0.05) than in naïve mice, and significantly higher in mice treated with mAb anti-α β-TCR (45.1±25.6; P=0.03). The combined treatment with both mAbs resulted in complete abrogation of Ab production (4.2±0.9) and 70% of skin grafts survived >100 days. Germinal center formation, reflective of B cell activation, was completely disrupted in mice treated with anti-CD154 alone or combined with anti-α β-TCR.Conclusion: These results suggest that the CD40/CD154 co-stimulatory pathway is critically important in B cell activation to generate alloantibody. Notably, blocking molecular interactions between CD40/CD154 abrogated the generation of antibody and blocked germinal center formation, inducing B cell tolerance. The additional removal of recipient T cells in the context of co-stimulatory blockade resulted in the induction of T as well as B cell tolerance. These findings are the first demonstration that sensitization can be prevented through blockade of co-stimulatory interactions in the generation of adaptive immune responses and could have a significant impact on management of sensitized recipients in the clinic.

Graft Survival and Immune Regulation of Pancreas Allograft Recipients Induced With Thymoglobulin, Sirolimus, and Cyclosporine

Background Our objective was to determine the impact of thymoglobulin–sirolimus–cyclosporine immunosuppression on the alloimune response of pancreas–kidney transplant recipients. Methods Thirty-six pancreas transplant recipients received an induction protocol of thymoglobulin, sirolimus, reduced-dose cyclosporine, and corticosteroids. A subset of 10 recipients were also enrolled in a study to measure immune responsiveness. Flow PRA–determined HLA antibody, donor-specific flow cytometry crossmatching (FCXM), T-cell subset, and suppressor cell assays were performed at various timepoints during the first posttransplant year. Results One-year patient, kidney, and pancreas survivals were 97%, 94%, and 92%, respectively. There was 1 death due to sepsis, and 1 kidney and 2 pancreas graft losses. There were no acute rejection episodes. Recipients in the immune-monitoring study displayed depression of CD3, CD4, and CD8 counts (<80%) until 3 months posttransplant. At transplantation, 9 of 10 patients displayed <10% class I HLA antibody. By 3 months, 7 of 10 showed a transient elevation in class I HLA antibodies, with 2 patients expressing >80% flow PRA. At transplant 1 patient was FCXM-positive, whereas, by 3 months posttransplant, 2 of 10 patients demonstrated a positive FCXM. There were no clinical consequences to either the presence of HLA antibody or the positive FCXMs. By 6 months, 7 of 9 patients expressed immunoregulatory suppressor cell activity. Conclusions The absence of acute rejection events was likely due to inhibition of donor-specific immunity. Seventy percent of patients demonstrated an early non–donor-directed HLA antibody response that had no adverse effect on graft function and 78% of the patients displayed immunoregulatory suppressor cell function, probably contributing to the successful clinical outcome.

HISTOCOMPATIBILITY TESTING PREDICTS ACUTE RENAL ALLOGRAFT REJECTION RISK IN EARLY CORTICOSTEROID CESSATION (ECS) REGIMENS

O140 Aims: Little data exists regarding the ability of standard histocompatibility testing to predict acute rejection (AR) in kidney transplant (Tx) recipients receiving corticosteroid (CS)-free immunosuppression (IS). Moreover, no data exists regarding the ability of newer techniques (flow cytometric PRA testing with Class I and II MHC beads) to predict AR risk. The purpose of the present study was to evaluate old and new histocompatibility techniques in predicting AR risk in kidney Tx recipients who undergo early (<7 day) CS withdrawal (WD). Methods: 234 patients (pts) were entered in 6 IRB-approved early CSWD regimens. Histocompatibility testing included: serologic PRA, flow cytometic PRA testing by Class I and Class II MHC beads, and B cell crossmatching. The flow B cell crossmatch assay was optimized by pronase treatment of cells. Influence of individual tests was examined using logistic regression analysis. Results: Results are presented in Table I.FigureLogistic Regression Analysis Cadaveric and LURD Tx recipients were at signficantly higher risk of AR. Accordingly, HLA AB and DR locus mismatching was associated with increased AR risk, with DR mismatching conferring a greater risk. Serologic, but not flow cytometic PRA analysis predicted AR risk, with current PRA testing the stronger predictor (1.7% increase in AR risk for each 1% increase in PRA). Finally, pronase-optimized flow cytometric B cell crossmatch also predicted AR risk. Conclusions: 1)Histocompatibility testing can predict AR risk, 2) HLA AB and DR mismatching are associated with increased AR risk, 3) serologic PRA, but not flow cytometric PRA testing predicted AR risk, 4) current PRA provides a stronger predictor or AR risk than peak PRA, and 5) pronase-optimized flow cytometric B cell crossmatching may provide an additional means for identifying recipients for high risk of AR. These data support the use of histocompatibility testing as a means for predicting AR risk in renal transplant pts who receive early CSWD.

Presence of Gal-alpha1,3Gal epitope on xenogeneic lines: implications for cellular gene therapy based on the encapsulation technology.

Exposure to human serum induces the lysis of xenogeneic cells through natural antibodies and complement activation. The carbohydrate Galactose-alpha1,3-Galactose (Gal-alpha1,3-Gal) epitope, has been shown to be the principal antigenic determinant on target cells. This reaction is, therefore, particularly important for xenogeneic cell-based therapy. As a first step toward the evaluation of the impact of this phenomenon for encapsulated xenogeneic cells, we have evaluated the presence of the Gal-alpha1,3Gal epitope on two cell lines currently being used for the systemic delivery of protein in the periphery or the treatment of neurodegenerative diseases. In the second part of the study, we have tested and compared human serum and cerebrospinal fluid (CSF) for the presence of xenoreactive natural antibodies (XNAs) and their potential impact on the survival of xenogeneic cells. Fluorescence-activated cell sorting analysis indicated that baby hamster kidney (BHK) cells expressed low levels of the alpha-Gal epitope, whereas mouse myoblast C2C12 cells were extensively stained with the specific IB4-lectin. There was a direct correlation between serum killing and the level of Gal-alpha1,3-Gal epitope expression on these cells. Importantly, we showed that CSF did not lyse BHK and C2C12 cells as determined by cytotoxic crossmatch assays. The reaction was specific as the addition of soluble Gal-alpha1,3-Gal sugar to human serum effectively reduced cell killing, and the overproduction of alpha-1,3-galactosyltransferase in BHK cells significantly increased inactivation by human serum. To interfere with this antibody-antigen reaction and develop cell lines particularly suitable for cell-based therapy, we either selected C2C12 clones expressing low levels of Gal-alpha1,3-Gal or high levels of alpha-1,2-fucosyltransferase. These cells were found to be resistant to complement-mediated cytolysis. These strategies may, therefore, protect encapsulated xenogeneic cells transplanted in the periphery or the central nervous system even in an unlikely event of a blood-brain barrier breakage and the post-transplantation development of an antibody response.

Flow cytometry in human leukocyte antigen testing

Stem cell transplantation is a recognized treatment for many hematologic malignancies. Human leukocyte antigen (HLA) testing is a key assessment in the evaluation of potential recipients and selection of the appropriate stem cell donor. Over the past several years, technological advances have moved HLA testing from a serologic art form to a sophisticated and high-resolution molecular-based procedure. HLA typing is now performed by DNA-based methods, while flow cytometry has significantly improved our ability to detect HLA antibodies. Although DNA-based typing methods have advanced our understanding of HLA polymorphisms, HLA antibodies still play a role in both stem cell and solid organ transplantation and can lead to failed engraftment or allograft graft rejection, respectively. Flow cytometric crossmatching and HLA antigen-specific microparticle assays represent the new HLA serology. Together, these new technologies are impacting patient outcomes and are expanding our understanding of the immune response to HLA alloantigens.

Impact of donor-specific antibodies on chronic rejection occurrence and graft loss in renal transplantation: posttransplant analysis using flow cytometric techniques.

Improvements in immunosuppressive therapy have greatly reduced acute rejection (ARj) episodes, ensuring better short-term graft outcome, but have not modified long-term survival in renal transplantation. It is now well accepted that chronic rejection (CRj) can be determined by both immune and/or nonimmune mechanisms. The aim of this study was to evaluate the importance of the posttransplant humoral immune response towards mismatched HLA graft antigens in CRj occurrence and graft outcome. Serum samples from 120 nonpresensitized renal transplant recipients were prospectively screened for 1 year after surgery by means of flow cytometry cross-match (FCXM) and FlowPRA beads (microbeads coated with purified HLA class I and class II antigens) assays. All transplants were followed-up for 2 years or until graft removal. FCXM monitoring identified donor-specific antibodies (DS-Abs) in 29 (24.2%) of 120 transplanted patients. Correlation with clinical data highlighted a higher incidence of ARj in DS-Abs-positive patients compared to negative patients (62% vs. 13%, P<0.00001). Furthermore, graft failure occurred more frequently among FCXM-positive patients than among negative patients (34% vs. 1%, P<0.00001). The deleterious effect of DS-Abs on graft function was confirmed by serum creatinine levels 2 years after transplantation. These were in fact higher in subjects producing DS-Abs than in subjects with only ARj (mean creatinine: 2.5+/-1.3 mg/dL vs.1.7+/-0.5 mg/dL, P=0.04). FlowPRA analysis of DS-Ab HLA specificity highlighted the presence of anti-HLA class I antibodies in 85% of FCXM-positive patients, who also presented with a higher incidence of HLA-B mismatches than FCXM-negative patients (1.23+/-0.66 vs. 0.92+/-0.59, P=0.02). Flow cytometric techniques are precious tools for investigating the activation of the humoral response against HLA antigens of the graft in renal transplantation. DS-Abs production has a worse impact on organ function and survival than ARj episodes. These findings represent further proof of the threat posed by DS-Abs on long-term graft function and draw attention to the need for a specific immunosuppressive therapy aimed at counteracting the different kinds of immune activation toward graft.

Human aortic valve allografts elicit a donor-specific immune response

Objective: The nature and magnitude of the immunologic response to implantation of human cryopreserved aortic valve allografts was investigated. Methods: Twenty aortic valve allograft recipients were investigated for donor-specific antibody and T-cell–mediated responses with serial flow cytometric and microlymphocytotoxic crossmatch assays and one-way mixed lymphocyte cultures. Results: Donor-specific immunoglobulin G antibodies to class I and II human leukocyte antigens were first detected in the serum of all aortic valve allograft recipients at 30 days after implantation and persisted in substantial amounts in all but one of the recipients at day 365. Recipient T-cell alloreactivity toward donor lymphocytes was significantly increased at day 30 compared with levels before and 10 days after operation. Conclusions: Cryopreserved aortic valve allografts elicit a substantial allogeneic response in recipients. This alloreactivity may contribute to the observed morphologic changes in aortic valve allografts and eventual long-term deterioration of allograft function. (J T HORAC C ARDIOVASC S URG 1996;112:1260-7)

Experimental hyperacute rejection in pancreas allotransplants.

A model of sensitization by intraperitoneal lymph node inoculation was developed to test the hypothesis that hyperacute rejection (HAR) could occur in sensitized recipients of vascularized pancreas allografts. Ten pairs of outbred mongrel dogs that were lymphocytotoxic cross-match assay negative were inoculated with homogenized lymph nodes on either three or four occasions at fortnightly intervals before renal transplantation. A renal allograft from the same donor was used to test the HAR response and to further enhance sensitization by rejection of a vascularized organ. Pancreas transplants were performed 2 weeks later, with biopsies of the graft and blood samples taken at 0, 10, 20, and 30 min and then at 30-min intervals until the grafts were no longer viable. All renal and pancreas grafts were rejected in a classical hyperacute pattern. Within 4 min of revascularization of the pancreas, central lobular hemorrhage and vascular congestion appeared, followed by general edema. Histology demonstrated parallel changes of edema, vascular congestion, necrosis, hemorrhage, and leukocytic infiltrate, which all preceded graft infarction. A sharp decline in both arterial and venous white blood cell count and platelets occurred within 10 min of revascularization with initial sequestration and subsequent release of platelets from the graft (P=0.02). In summary, HAR of the allografted pancreas can be observed by the surgeon within minutes of revascularization, with predictable macroscopic and microscopic changes. This study supports the use of routine lymphocytotoxic cross-match tests for all recipients of pancreas transplants and implies that particular care is warranted in regraft pancreas allograft recipients.

CLINICAL APPLICATION OF FLOW CYTOMETRY IN LIVE RELATED DONOR RENAL TRANSPLANTATION

To monitor the activity of the humoral component in graft dysfunction following renal transplantation using live related donors, flowcytometric cross-match procedure was adopted. Antidonor antibodies were detected in the sera of both pre- and post-transplant patients using conventional serological cytotoxicity cross-match and flowcytometric cross-match assays. In the 52 pretransplant samples no significant differences were observed in flowcytometric and cytotoxicity tests except in 2 secondary transplant cases which were negative by cytotoxicity test. However, in post-transplant samples, floweytometry was found to be a more objective and useful test than cytotoxicity testing in distinguishing 6 out of 7 mild acute-graft-rejection episodes. Both tests were found to be negative in alt 5 cases of cyclosporin-A nephrotoxicity, 7 cases of acute tubular necrosis and 10 out of 11 cases of chronic rejection.

Detection of antilineage specific leucocyte antibodies by a quantitative immunocytometry method in sera from candidates for renal allografts

A sensitive and quantitative flow cytometry method for detecting antileucocyte antibodies was developed to study retrospectively samples from candidates using frozen donor cells and frozen recipient sera. This immunofluorescence method was used to compare levels of reactivities of serum antibodies before and after donor specific blood transfusion treatment and after renal transplantation. The results demonstrate that the flow cytometry crossmatch is a sensitive and accurate method which should be used prospectively before precluding transplantation in the presence of a positive B cell standard crossmatch. Antibodies detected by flow cytometry before transplantation could be responsible for an early acute rejection episode. Finally, combination of flow cytometry crossmatch and standard crossmatch assays might thus be useful before precluding transplantation in living related donors.