Aviva Aloush

De Novo Production of K-α1 Tubulin-Specific Antibodies: Role in Chronic Lung Allograft Rejection

Lung transplantation is the treatment option for a variety of end-stage pulmonary diseases. Posttransplant development of Abs against donor HLA and non-HLA Ags have been associated with acute and chronic rejection of transplanted organs. Development of bronchiolitis obliterans syndrome (BOS) following lung transplantation has been correlated with de novo production of anti-donor-HLA Abs. However, only a portion of the patients with BOS demonstrate detectable anti-donor-HLA Abs. Airway epithelium is considered as a major target for lung allograft rejection. In this study we demonstrate that many BOS+ patients (12 of 36) develop Abs reactive to epithelial cell Ag that are distinct from HLA. Furthermore, de novo production of antiepithelial cell Ab precedes clinical onset of BOS. N-terminal sequencing and blastx analysis as well as blocking with K-α1 tubulin-specific Ab identified the epithelial Ag as K-α1 tubulin. Binding of the de novo-produced anti-K-α1 tubulin Abs to the airway epithelial cells resulted in the increased expression of transcription factors (TCF5 and c-Myc), leading to increased expression of fibrogenic growth factors, activation of cell cycle signaling, and fibroproliferation, the central events in immunopathogenesis of BOS following human lung transplantation.

Anti-human leukocyte antigen antibodies and preemptive antibody-directed therapy after lung transplantation

BACKGROUND Because the development of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) after lung transplantation has been associated with acute and chronic rejection, we implemented a clinical protocol to screen all transplant recipients for DSA and preemptively treat those who developed DSA with rituximab and intravenous immune globulin (IVIG), or IVIG alone. METHODS We conducted a prospective observational study of this protocol and used the LABScreen Single Antigen assay to detect DSA after transplantation. We compared the incidence of acute rejection, lymphocytic bronchiolitis, and bronchiolitis obliterans syndrome (BOS) between those who developed DSA and those who did not using Cox proportional hazards models. We used the Kaplan-Meier method to compare freedom from BOS and survival between those who had persistent DSA and those who had successful depletion of DSA. RESULTS Among 116 recipients screened, DSA developed in 65 during the study period. Those who developed DSA and received antibody-directed therapy had a similar incidence of acute rejection, lymphocytic bronchiolitis, and BOS as those who did not develop DSA. Furthermore, recipients who had successful depletion of DSA had greater freedom from BOS and better survival than those who had persistent DSA. Finally, those treated for DSA had a similar incidence of infectious complications as those who did not develop DSA. CONCLUSIONS The development of DSA is surprisingly common after lung transplantation. Antibody-directed therapy may reduce the risk of rejection associated with DSA, but a randomized controlled trial is necessary to critically evaluate the efficacy of this treatment protocol.

The significance of a single episode of minimal acute rejection after lung transplantation.

Background. Bronchiolitis obliterans syndrome (BOS) remains the leading obstacle to better long-term outcomes after lung transplantation. Acute rejection has been identified as the primary risk factor for BOS, but the impact of minimal acute rejection, especially a solitary episode, has usually been discounted as clinically insignificant. Methods. We performed a retrospective cohort study of 259 adult lung transplant recipients to determine the risk of BOS associated with a single episode of A1 rejection, without recurrence or subsequent progression to a higher grade. The cohort was divided into 3 groups based on the severity of acute rejection (none, single episode of A1, and single episode of A2). We determined the risks of BOS stages 1, 2, 3, and death for each group using univariate and multivariate Cox regression analyses. Results. A solitary episode of A1 rejection was a significant risk factor for BOS stages 1 and 2, but not stage 3 or death, in the univariate analysis. Multivariate Cox regression models confirmed that the risk of BOS attributable to a single episode of A1 rejection was independent of other potential risk factors, such as community acquired respiratory viral infections, number of HLA mismatches, and cytomegalovirus pneumonitis. Likewise, univariate and multivariate analyses demonstrated that a single episode of A2 rejection was a significant risk factor for all stages of BOS but not death. Conclusions. A single episode of minimal acute rejection without recurrence or subsequent progression to a higher grade is a significant predictor of BOS independent of other risk factors.

Early posttransplant inflammation promotes the development of alloimmunity and chronic human lung allograft rejection

Background. Chronic human lung allograft rejection, represented by bronchiolitis obliterans syndrome (BOS), is the single most important factor that limits the long-term survival following lung transplantation (LT). However, the pathogenesis of BOS remains unclear. We hypothesized that the early posttransplant inflammation would promote the development of donor anti–human leukocyte antigen (HLA) alloimmunity and predispose to BOS. Methods. Serum levels of interleukin (IL)-1&bgr;, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, IP-10, MIG, MCP-1, MIP-1&agr;, MIP-1&bgr;, RANTES, tumor necrosis factor (TNF)-&agr;, interferon (IFN)-&agr;, IFN-&ggr;, granulocyte-macrophage colony-stimulating factor, IL-1R&agr;, and IL-2R were serially analyzed in 31 BOS+ and matched 31 BOS− patients using quantitative multiplex bead immunoassays. Donor-specific HLA class II cellular immunity was analyzed using enzyme-linked immunospot (ELISPOT) by testing recipient peripheral blood mononuclear cells against mismatched donor HLA-DR peptides. Anti-HLA class II antibodies were monitored using flow panel reactive antibodies. Results. There was early posttransplant elevation in basal serum levels of proinflammatory chemokines IP-10 and MCP-1 and Th1-cytokines IL-1&bgr;, IL-2, IL-12, and IL-15 in BOS+ patients, compared to BOS− and normal subjects. In addition, a threefold decline in IL-10 levels was found during BOS development. BOS+ patients revealed increased development of HLA class II alloantibodies and Th1-predominant donor-specific cellular immunity with high frequency of IFN-&ggr; and low IL-5 producing T-cells. Conclusion. Early posttransplant elevation of proinflammatory mediators is associated with alloimmunity and chronic human lung allograft rejection.

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.

Minimal acute rejection after lung transplantation: a risk for bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is a major cause of lung allograft dysfunction. Although previous studies have identified mild to severe rejection (grade ≥A2) as a risk factor for BOS, the role of minimal rejection (grade A1) remains unclear. To determine if A1 rejection by itself is a risk factor for BOS, we performed a retrospective cohort study on 228 adult lung transplant recipients over a 7‐year period. Cohorts were defined by their most severe rejection episode (none, A1 only, and ≥A2) and analyzed for the subsequent development and progression of BOS using univariate and multivariate time‐dependent Cox regression analysis. In the univariate model, the occurrence of isolated minimal rejection was a risk factor for all stages of BOS. Similarly, multivariate models that included HLA mismatch, cytomegalovirus pneumonitis, community acquired viral infection, underlying disease and type of transplant demonstrated that A1 rejection was a distinct risk factor for BOS. Furthermore, the associated risk with A1 rejection was slightly greater than the risk from ≥A2 and treatment of A1 rejection decreased the risk for subsequent BOS stage 1. We conclude that minimal rejection is associated with an increased risk for BOS development and progression that is comparable to A2 rejection.

Immunological Link Between Primary Graft Dysfunction and Chronic Lung Allograft Rejection

BACKGROUND Primary graft dysfunction (PGD) in the immediate post-lung transplant period strongly increases the risk of chronic rejection (broncholitis obliterans syndrome). Here, we hypothesized that PGD-induced inflammation augments alloimmunity, thereby predisposing to broncholitis obliterans syndrome. METHODS Primary graft dysfunction and broncholitis obliterans syndrome were diagnosed according to the established International Society for Heart and Lung Transplantation criteria. Anti-human leukocyte antigen (HLA) alloantibodies were analyzed using Flow-PRA. Donor HLA class II-specific T cells were analyzed using interferon (IFN)-gamma ELISPOT. Serum levels of 25 cytokines and chemokines were measured using LUMINEX. RESULTS Of the 127 subjects, 29 (22.8%) had no PGD (grade 0), 42 (33.2%) had PGD-1, 36 (28.3%) had PGD-2, and 20 (15.7%) had PGD-3. Patients with PGD grades 1 to 3 (PGD(1-3)) had elevated proinflammatory mediators MCP-1, IP-10, interleukin (IL)-1 beta, IL-2, IFN-gamma, and IL-12 in the sera during the early posttransplant period compared with patients with PGD grade 0 (PGD(0)). On serial analysis, PGD(1-3) patients revealed increased development of de novo anti-HLA-II (5 years: 52.2% versus PGD(0) 13.5%, p = 0.008). However, no difference was found in anti-HLA-I alloantibody development (PGD(1-3) patients 48% versus PGD(0) 39.6%, p = 0.6). Furthermore, PGD(1-3) patients had increased frequency of donor HLA class II-specific CD4(+) T cells [(91.4 +/- 19.37) x 10(-6) versus (23.6 +/- 15.93) x 10(-6), p = 0.003]. CONCLUSIONS Primary graft dysfunction induces proinflammatory cytokines that can upregulate donor HLA-II antigens on the allograft. Increased donor HLA-II expression along with PGD-induced allograft inflammation promotes the development of donor specific alloimmunity. This provides an important mechanistic link between early posttransplant lung allograft injury and reported association with broncholitis obliterans syndrome.

Antibodies to K-α 1 tubulin and collagen V are associated with chronic rejection after lung transplantation.

Bronchiolitis obliterans syndrome (BOS), the clinical correlate of chronic rejection after lung transplantation, is the leading obstacle to better long‐term outcomes. We previously instituted a clinical protocol to screen for donor‐specific human leukocyte antigen (HLA) antibodies (DSA) and a preemptive antibody‐directed therapy protocol consisting of rituximab and/or intravenous immune globulin. In this study, we retrospectively analyzed serum samples from lung transplant recipients (n = 108) for antibodies to self‐antigens (K‐α 1 tubulin and collagen V) before and after antibody‐directed therapy and correlated the results with the subsequent development of BOS. Seventy‐two of the 108 recipients developed antibodies to self‐antigens. There was a correlation between the development of antibodies to self‐antigens and DSA. Sixteen of the 54 patients who had antibodies to self‐antigens and were treated with antibody‐directed therapy cleared the antibodies, and they were significantly less likely to develop BOS than those who had persistent antibodies. Furthermore, those who cleared DSA after treatment but had persistent antibodies to self‐antigens were significantly more likely to develop BOS than those who cleared these antibodies. We conclude that antibodies to self‐antigens are an important risk factor for the development of BOS.

Activation of human airway epithelial cells by non-HLA antibodies developed after lung transplantation: a potential etiological factor for bronchiolitis obliterans syndrome.

Background. The main cause of morbidity and mortality after lung transplantation (LT) is bronchiolitis obliterans syndrome (BOS). Anti-HLA antibodies development after LT has been shown to play an important role in BOS pathogenesis. However, the nature of non-HLA antibodies developed after LT and their role in BOS pathogenesis have not been determined. Methods. Sera from 16 BOS+ patients and 11 BOS− patients were collected at 12, 24, 36, and 48 months after LT. Anti-HLA class I and class II antibodies were absorbed with pooled human platelets and pooled human lymphoblastoid cell lines, respectively. Then, the presence of non-HLA antibodies against several cell lines from different origin was determined by flow cytometric analysis. Antibody-positive samples were tested for induction of proliferation and growth factor production in two selected airway epithelial cell (AEC) lines. Results. Five of 16 BOS+ patients (31.2%) and 0 of 11 BOS- patients (0%) developed anti-AEC antibodies after LT (P =0.05). No reactivity against endothelial cells, lymphocytes, monocytes, or granulocytes was detected. Further analysis of two selected sera demonstrated the development of reactivity against a 60-kDa antigen expressed by 60% of AEC lines and only 12% of cell lines from other tissues. Antibody binding to this antigen induced intracellular Ca++ influx, tyrosine phosphorylation, proliferation, and up-regulation of transforming growth factor-&bgr; and heparin-binding epidermal growth factor mRNA transcription in AECs. Conclusions. These results indicate that anti-AEC antibodies may play a role in the immunopathogenesis of BOS in the absence of anti-HLA antibodies.

Pre-transplant antibodies to Kα1 tubulin and collagen-V in lung transplantation: Clinical correlations

BACKGROUND Immune responses to lung-associated self-antigens (SAgs) have been implicated in chronic lung allograft rejection. The goals of this study were to determine the prevalence of pre-existing antibodies (Abs) to the SAgs in pulmonary diseases and the association between pre-existing Abs to SAgs and the development of primary graft dysfunction (PGD), donor-specific antibodies (DSA), and chronic rejection. METHODS Pre- and post-transplant sera were analyzed from 317 lung transplant (LTx) recipients between 2000 and 2011 with diagnosis of chronic obstructive disease (n = 161), idiopathic pulmonary fibrosis (IPF; n = 50), cystic fibrosis (CF; n = 55), and others (n = 51). Samples were analyzed for Abs to SAgs by enzyme-linked immunosorbent assay, and DSA and cytokines by Luminex. The clinical diagnosis of PGD and bronchiolitis obliterans syndrome (BOS) was based on International Society for Heart and Lung Transplantation guidelines. RESULTS The overall prevalence of Abs to SAgs was 22.71%, including 18% in chronic obstructive pulmonary disease (p = 0.033), 34% in IPF (p = 0.0006), 29% in CF (p = 0.0023), and 19.6% in other diagnoses (p = 0.044). The incidence of PGD (88% vs 54%, p < 0.05), DSA (70% vs 45%, p < 0.01), and BOS (90% vs 38% (p < 0.001) after LTx was significantly higher in patients with pre-LTx Abs to SAgs than without. Pro-inflammatory cytokines (interleukin-1β, interleukin-17, and interferon-γ) were elevated in patients who had pre-LTx Abs to SAgs, along with a reduction in anti-inflammatory interleukin-10. CONCLUSIONS Patients with IPF and CF have the highest prevalence of Abs to SAgs. Patients with pre-existing Abs to SAgs are at increased risk for development of PGD, DSA, and BOS. Strategies to remove pre-existing Abs to SAgs should be considered to improve lung allograft outcome.