D. Phelan

HLA-A locus mismatches and development of antibodies to HLA after lung transplantation correlate with the development of bronchiolitis obliterans syndrome

BACKGROUND Bronchiolitis obliterans syndrome (BOS) is the most common cause of morbidity and mortality after lung transplantation (LT). A retrospective analysis of clinical and immunologic variables were done to identify those that might predict the development of BOS. METHODS Of 112 LT performed over a 42-month interval, 94 survived at least 3 months and form the basis of this analysis. There was a minimum of 21 months follow-up. BOS was defined on the basis of declining spirometry (FEV1 <80% of baseline) and/or the presence of histologic obliterative bronchiolitis. All variables analyzed were subjected first to a univariate analysis; those variables appearing to carry significance were then subjected to a multivariate logistic regression analysis. RESULTS Univariate analysis revealed the following to be predictors of the development of BOS: age (the probability of developing BOS declined with advancing age); donor/recipient HLA-A locus mismatch, with actuarial freedom from BOS being significantly greater with no A-locus mismatches versus cases with one or two mismatches (P=0.031); and development of anti-HLA antibodies after transplantation (P=0.006 vs. recipients without detectable antibodies). In multivariate analysis, only HLA locus mismatch and development of anti-HLA antibodies were significant independent predictors of the development of BOS. The remaining clinical variables (gender, type of LT, indication for LT, graft ischemic time, use of cardiopulmonary bypass, cytomegalovirus) and immunologic variables (crossmatch, frequent early acute rejection) did not correlate with the development of BOS. CONCLUSIONS These data suggest that BOS is the result of an immune process, that differences at the HLA-A locus may play an important role in this process, and antibody-mediated injury may play a role in BOS.

Development of ELISA-detected anti-HLA antibodies precedes the development of bronchiolitis obliterans syndrome and correlates with progressive decline in pulmonary function after lung transplantation.

BACKGROUND Development of anti-HLA antibodies after lung transplantation (LT) is thought to play an important role in the etiology of bronchiolitis obliterans syndrome (BOS). However, a cause-effect relationship between anti-HLA antibodies and BOS has not been established. This study was conducted to determine the temporal relationship between the development of anti-HLA antibodies and BOS after LT, and to determine the antigenic specificity of the antibodies developed in BOS patients. METHODS Sera from 15 BOS+ LT patients and 12 BOS- LT patients were obtained before LT and collected again at 6, 12, 24, 36, and 48 months after LT. Anti-HLA antibodies were detected by the PRA-STAT ELISA system and by complement-dependent cytotoxicity assays. Anti-HLA reactivity was further characterized by flow cytometry and absorption/elution with human platelets. RESULTS When analyzed by ELISA, 10 of 15 BOS+ patients developed anti-HLA antibodies, whereas 0 of 12 BOS- patients developed anti-HLA antibodies (P<0.001). When analyzed by complement-dependent cytotoxicity, only 2 of 15 BOS+ patients developed anti-HLA antibodies and 1 of 12 BOS- patients developed anti-HLA antibodies (P = 0.99). There was a significant difference of 20.1 months between the time of anti-HLA antibody detection and the time of BOS diagnosis (P = 0.005). A progressive decrease in pulmonary function correlated with a progressive increase in the anti-HLA reactivity 36 months after LT. The anti-HLA reactivity was directed to one of the donor HLA class I antigens and to other unrelated HLA class I antigens. No anti-HLA reactivity was found against HLA class II molecules. CONCLUSIONS Our study indicates that anti-HLA class I antibodies play an important role in the pathogenesis of BOS and that monitoring of anti-HLA class I antibody development by a highly sensitive assay such as the PRA-STAT ELISA after LT can provide an early identification of an important subset of LT patients with an increased risk of developing BOS.

Alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts

BACKGROUND Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality after lung transplantation (LTx). We sought to better understand the relationship between alloimmune responses and autoimmunity and, subsequently, how autoimmunity leads to chronic rejection. METHODS We analyzed the development of donor-specific antibodies (Abs) in LTx by flow PRA and the development of Abs to K-α1 tubulin (K-α1T) and collagen V (ColV) by ELISA. The frequency of K-α1T- and ColV-specific T cells that secrete IFN-γ, IL-17 and IL-10 in LTx recipients was measured by ELISPOT. RESULTS In a retrospective analysis of 42 LTx recipients, we demonstrated a strong correlation between development of donor-specific anti-HLA Abs, Abs to self-antigens and BOS (p < 0.05). To test the hypothesis that alloimmunity is related to an immune response to self-antigens, we analyzed 103 LTx patients prospectively for the development of donor-specific Abs (DSA) and Abs to self-antigens. A total of 42.7% of recipients developed DSA and 30.1% developed Abs to K-α1T and ColV. Development of DSA preceded development of Abs to self-antigens. BOS(+) patients had higher frequency of T cells secreting IL-17 (p < 0.01) and IFN-γ (p < 0.05) with decreased IL-10 (p < 0.05) when compared with BOS(-) patients. CONCLUSIONS Based on these results we propose that alloimmune responses to donor HLA can induce autoimmune responses to airway epithelial self-antigens, characterized by activation of the IL-17 pathway. These immune responses to self-antigens along with alloimmunity contribute to the pathogenesis of BOS. Strategies to prevent development of autoimmunity may be play a key role in preventing the development of chronic rejection.

Characterization of immune responses to cardiac self- antigens myosin and vimentin in human cardiac allograft recipients with antibody-mediated rejection and cardiac allograft vasculopathy

BACKGROUND Herein we study the role of donor-specific antibodies (DSA) to mismatched human leukocyte antigen (HLA) and antibodies (Abs) to the cardiac self-antigens myosin (MYO) and vimentin (VIM) in the pathogenesis of acute antibody-mediated rejection (AMR) in the early post-transplant period (EP, <12 months) and cardiac allograft vasculopathy (CAV) in the late post-transplant period (LP, >12 months) after heart transplantation (HTx). METHODS One hundred forty-eight HTx recipients (65 in EP, 83 in LP) were enrolled in the study. Development of DSA was determined by Luminex. Circulating Abs against MYO and VIM in sera were measured using enzyme-linked immunoassay (ELISA). Frequency of CD4+ T-helper cells (CD4+ Th) secreting interferon (IFN)-γ, interleukin (IL)-17, IL-10 or IL-5 specific to either MYO or VIM were analyzed in vitro using ELISpot assays. RESULTS AMR patients were more likely DSA positive (AMR-: 15%; AMR+: 70%; p = 0.03) and demonstrated increased Abs to MYO (AMR-: 144 ± 115 μg/ml; AMR+: 285 ± 70 μg/ml; p = 0.033) and VIM (AMR-: 37 ± 19 μg/ml; AMR+: 103 ± 43 μg/ml; p = 0.014). AMR patients demonstrated increased IL-5 CD4+ Th cells specific to MYO (5.2 ± 0.9 fold, p = 0.003) and VIM (7.3 ± 2.9-fold, p = 0.004) and decreased IL-10 CD4+ Th cells specific to MYO (2.2 ± 0.4-fold, p = 0.009) and VIM (1.7 ± 0.2-fold, p = 0.03). CAV patients were more likely DSA positive (CAV-): 25%; CAV+: 79%; p = 0.03) and demonstrated increased Abs to MYO (CAV-: 191 ± 120 μg/ml; CAV+: 550 ± 98 μg/ml; p = 0.025) and VIM (CAV-: 55 ± 25 μg/ml; CAV+: 255 ± 49 μg/ml; p = 0.001). CAV patients demonstrated increased IL-17 CD4+ Th cells specific to MYO (10.5 ± 7.3-fold, p = 0.002) and VIM (7.0 ± 3.9-fold, p = 0.003). CONCLUSIONS The presence of DSA in AMR and CAV is significantly associated with development of Abs to MYO and VIM in post-HTx patients. Induction of high CD4+ Th cells specific to cardiac self-antigens that secrete predominantly IL-5 and IL-17 plays a significant role in the development of Abs to self-antigens leading to AMR and CAV, respectively.

Pre-exposure to sub-saturating concentrations of HLA class I antibodies confers resistance to endothelial cells against antibody complement-mediated lysis by regulating Bad through the phosphatidylinositol 3-kinase/Akt pathway.

Allografts transplanted across HLA‐sensitization results in an antibody‐mediated rejection known as hyperacute rejection. Depleting anti‐graft antibodies from the recipient by plasmapheresis prior to transplantation can prevent this rejection. We developed an in vitro model using polyclonal HLA class I antibodies obtained from highly sensitized patients awaiting transplantation,and analyzed their ability to provide signals following binding to human aortic endothelial cells (EC). Using this model, we show that EC undergo caspase 3‐dependent cell death by apoptosis upon exposure to saturating concentrations of HLA class I antibodies and complement accompanied by loss of Akt activation and phosphorylation of Bad. In contrast, exposure of EC to sub‐saturating concentrations of HLA class I antibodies conferred resistance towards antibody/complement‐mediated lysis termed accommodation. Accommodated EC exhibited reduction in the expression of the adhesion molecules ICAM‐1 and VCAM‐1 and a significant increase in the expression of anti‐apoptotic genes Bcl‐xL, Bcl‐2 and heme oxygenase‐1. Further, induction of phosphatidylinositol 3‐kinase (PI3K) and Akt activities thatfacilitate the phosphorylation of Bad were also noted. In conclusion, exposure of sub‐saturating concentrations of HLA class I antibodies results in the induction of PI3K/Akt pathway that confers resistance to endothelial cells against antibody/complement‐mediated cell death.

A significant role for histocompatibility in human islet transplantation

Background. In recent years, transplantation of islets and pancreas has become a viable option for patients debilitated with type I diabetes. The success of islet transplantation has been attributed to the ability to isolate high quality islets for transplantation and capacity to maintain the recipients immunosuppressive levels within a specific target range following transplantation. The purpose of this study was to determine the role of pretransplant sensitization to human leukocyte antigen (HLA) in islet transplantation. Methods. We retrospectively analyzed seven patients that were transplanted with islets under the auspices of the Juvenile Diabetes Research Foundation and Islet Cell Resource Center/National Institutes of Health. Humoral sensitization towards donor antigens both prior to and following islet transplantation was detected by FLOW panel reactive antibodies (PRA) and donor-specific cellular sensitization was detected by performing enzyme-linked immunospot assay analysis for cytokines interferon-γ and interleukin-2. Results. Our analysis demonstrates that humoral and cellular sensitization to histocompatibility antigens prior to and after islet transplantation are associated with the failure of transplanted islets Conclusion. Patient selection based on sensitization to donor HLA may be one of the factors crucial for the success of islet transplant. Further, in some patients, rejection of islets can be associated with sensitization to mismatched donor histocompatibility antigens.

Living donor renal transplantation in the presence of donor-specific human leukocyte antigen antibody detected by solid-phase assay

Presensitization of donor human leukocyte antigens (HLA) demonstrated through a positive crossmatch is detrimental to allograft function and best avoided through donor exclusion. The clinical significance of alloantibody detectable by sensitive solid-phase assay is not completely defined and is the focus of this study. Pretransplant sera from 64 consecutive living-donor renal transplant recipients were screened by enzyme-linked immunosorbent assay (ELISA) and Luminex assays. Results were analyzed for correlation with clinical outcome. Luminex proved more sensitive than ELISA for alloantibody detection, with three identifiable patterns. Twenty-eight patients were antibody negative, 24 had non-donor-specific antibody (non-DSA), and 12 had donor-specific antibody (DSA). The highest number of rejections (n = 4) and graft losses (n = 6) occurred in the antibody-negative group. The non-DSA group had two graft losses, as did the DSA group. The two graft losses in the DSA group were caused by recurrent focal segmental glomerulosclerosis (FSGS) at 35 months and death with a functioning graft at 32 months. Overall, there were no cases of antibody-mediated rejection and allograft function to 4 years was comparable among all three groups. Under our standard immunosuppression protocol and crossmatch criteria for histocompatibility, alloantibody detectable by Luminex was not detrimental to successful living-donor transplantation.

A Role for Antibodies to Human Leukocyte Antigens, Collagen-V, and K-α1-Tubulin in Antibody-Mediated Rejection and Cardiac Allograft Vasculopathy

Background. We determined the role of donor-specific antibodies (DSA) and antibodies (Abs) to self-antigens, collagen-V (Col-V), and K-&agr;1-Tubulin (KAT) in pathogenesis of acute antibody-mediated rejection (AMR) and cardiac allograft vasculopathy (CAV) after human heart transplantation (HTx). Methods. One hundred thirty-seven HTx recipients, with 60 early period (≤12 months) and 77 late period (>12 months), were enrolled in this study. Circulating DSA was determined using LUMINEX. Abs against Col-I, II, IV, V, and KAT were measured using ELISA. Frequency of CD4+T helper cells (CD4+Th) secreting interferon (IFN)-&ggr;, interleukin (IL)-5, -10, or -17 specific to self-antigens were determined using Enzyme Linked Immunosorbent Spot assay. Results. A significant association between AMR and DSA was demonstrated. Development of DSA in AMR patients correlated well with the development of auto-Abs to Col-V (AMR[+]: 383±72 &mgr;g/mL, AMR[−]: 172±49 &mgr;g/mL, P=0.033) and KAT (AMR[+]: 252±49 &mgr;g/mL, AMR[−]: 61±21 &mgr;g/mL, P=0.014). Patients who developed AMR demonstrated increased frequencies of CD4+Th secreting IFN-&ggr; and IL-5 with reduction in IL-10 specific for Col-V/KAT. Patients diagnosed with CAV also developed DSA and auto-Abs to Col-V (CAV[+]: 835±142 &mgr;g/mL, CAV[−]: 242±68 &mgr;g/mL, P=0.025) and KAT (CAV[+]: 768±206 &mgr;g/mL, CAV[−]: 196±72 &mgr;g/mL, P=0.001) with increased frequencies of CD4+Th secreting IL-17 with reduction in IL-10 specific for Col-V/KAT. Conclusions. Development of Abs to human leukocyte antigens and self-antigens are associated with increases in CD4+Th secreting IFN-&ggr; and IL-5 in AMR and IL-17 in CAV, with reduction in CD4+Th secreting IL-10 in both AMR and CAV.

Donor-specific antibodies to human leukocyte antigens are associated with and precede antibodies to major histocompatibility complex class I–related chain A in antibody-mediated rejection and cardiac allograft vasculopathy after human cardiac transplantation

Humoral immune responses to mismatched donor human leukocyte antigen (HLA) and major histocompatibility complex (MHC) class I-related chain A (MICA) have been reported to contribute to immunopathogenesis of antibody-mediated rejection (AMR) in the early period and cardiac allograft vasculopathy (CAV) in the late period after cardiac transplantation (HTx). The goal of this study is to define the roles of donor-specific antibodies (DSA) and anti-MICA in AMR and CAV. A total of 95 post-HTx recipients were enrolled; 43 patients in the early period (≤ 12 months post-HTx) and 52 patients in the late period (>12 months post-HTx). Development of DSA and anti-MICA were serially monitored using Luminex. Development of DSA (AMR+: n = 6/8.75%, AMR-: n = 4/35.11%, p = 0.009) and anti-MICA (AMR+: n = 5/8.63%, AMR-: n = 4/35.11%, p = 0.002) was significantly associated with AMR. AMR+DSA+ patients demonstrated increased anti-MICA levels compared with AMR+DSA- patients (p=0.01). Serial monitoring revealed DSA (2.7 ± 1.4 months) preceded development of anti-MICA (6.5 ± 2.1 months) in recipients diagnosed with AMR at 8.3 ± 2.5 months post-HTx. Development of DSA (CAV+: n = 8/12.67%, CAV-: n = 5/40.13%, p = 0.004) and anti-MICA (CAV+: n = 9/12.75%, CAV-: n = 5/40.13%, p = 0.001) was significantly associated with CAV. CAV+DSA+ patients demonstrated increased anti-MICA levels compared with CAV+DSA- patients (p = 0.01). Antibodies to HLA are associated with and precede development of anti-MICA in AMR and CAV. Therefore, DSA and anti-MICA can be used as noninvasive markers for monitoring AMR and CAV.

Probable antibody-mediated failure of two sequential abo-compatible hepatic allografts in a single recipient

Two sequential ABO-compatible orthotopic liver allografts failed, despite excellent initial posttransplant function, in a patient with preformed donor-specific alloantibodies. There was no evidence of cell-mediated rejection. Retrospective crossmatching of recipient serum, obtained immediately prior to the first transplant, revealed the presence of lymphocytotoxic antibodies directed against donor class I HLA B17, at a titer of greater than 1:32,768. Similarly, lymphocytotoxic antibodies directed against the second donors class I HLA A2 phenotype were detected on retrospective crossmatching utilizing both the three-wash Amos technique (TWA-CDC), and the anti-human immunoglobulin augmented technique (AHG-CDC), at a titer of greater than 1:32,768. Anti-class I specific alloantibodies were eluted from both failed liver grafts at titers of 1:256. The hepatic necrosis in zones 3 and 2 that were observed on histologic examination, and the profound refractory consumptive thrombocytopenia subsequent to each transplant may have been the result of antibody-mediated rejection by preformed lymphocytotoxic antibodies. Despite the livers remarkable capacity to withstand antibody-mediated injury, primary humoral rejection following ABO compatible liver transplantation may occur if extremely high titers of performed allospecific lymphocytotoxic antibodies are present.