Andrés Jaramillo

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.

Anti-HLA class I antibody binding to airway epithelial cells induces production of fibrogenic growth factors and apoptotic cell death: a possible mechanism for bronchiolitis obliterans syndrome.

Development of anti-HLA class I antibodies is associated with bronchiolitis obliterans syndrome (BOS) after lung transplantation. BOS is characterized histologically by significant fibrosis and airway epithelial cell (AEC) apoptosis. Thus, this study was designed to determine whether anti-HLA class I antibodies can activate AECs to produce growth factors and to undergo apoptosis. KCC-266 AECs were activated with the W6/32 anti-HLA class I monoclonal antibody. Proliferation and apoptosis levels were determined after 24, 48, and 72 hours. The induction of fibroblast and bronchial smooth muscle cell proliferation by anti-HLA class I activated AECs was assessed in the presence of neutralizing antibodies against various growth factors. The anti-HLA class I induced AEC proliferation after 24 hours followed by significant induction of apoptosis after 48 hours. Anti-HLA class I activated AECs produced soluble growth factors that stimulated fibroblasts but not bronchial smooth muscle cells. The stimulation of fibroblast proliferation was inhibited by antibodies against platelet-derived growth factor, heparin-binding epidermal growth factor, insulin-like growth factor 1, and basic fibroblast growth factor. The results from this study suggest that anti-HLA class I alloantibodies may play an important role in the pathogenesis of BOS by inducing proliferation, growth factor production, and apoptotic cell death in AECs.

Indirect Allorecognition of Mismatched Donor HLA Class II Peptides in Lung Transplant Recipients with Bronchiolitis Obliterans Syndrome

A correlation between indirect allorecognition of mismatched donor HLA class I peptides and development of bronchiolitis obliterans syndrome (BOS) after lung transplantation has been previously observed. The aim of this study was to determine whether there was a correlation between indirect allorecognition of mismatched donor HLA class II peptides and development of BOS after lung transplantation. Peripheral blood mononuclear cells from nine BOS+ and nine BOS– lung transplant recipients were cultured with synthetic peptides corresponding to the β‐chain hypervariable region of a mismatched donor HLA‐DR molecule. Then, proliferative alloreactivity as well as frequency of alloreactive T cells were determined. In addition, the immunodominant epitopes from the donor HLA‐DR molecules were identified in selected patients. T cells from BOS+ patients showed a dose‐dependent proliferative alloreactivity against donor HLA‐DR peptides that was significantly higher than that observed in BOS– patients (p = 0.001). Similarly, the frequency of HLA‐DR alloreactive T cells was significantly higher in BOS+ patients than in BOS– patients (p = 0.001). This T‐cell alloreactivity was directed against a single immunodominant HLA‐DR peptide. These results suggest that indirect alloreactivity to donor HLA class II molecules may play a role in the pathogenesis of BOS after lung transplantation.

Anti-HLA antibody binding to hla class I molecules induces proliferation of airway epithelial cells: a potential mechanism for bronchiolitis obliterans syndrome.

OBJECTIVE Development of anti-HLA antibodies is associated with development of bronchiolitis obliterans syndrome after lung transplantation. We sought to determine the mechanism by which anti-HLA antibodies affect the development of bronchiolitis obliterans syndrome. We postulated that anti-HLA antibodies bind to the donor lung epithelium and stimulate phosphorylation and proliferation. METHODS The A549 lung epithelial carcinoma cell line was cultured in serum-deficient medium to produce static growth. Then the cells were treated with anti-HLA sera from lung transplant recipients, pooled anti-HLA serum from highly sensitized patients, or normal human serum. The cells were also treated with the W6/32 mouse anti-HLA class I monoclonal antibody or control mouse IgG. Tritiated thymidine uptake was determined at 24, 48, and 72 hours. In parallel experiments the cells were treated as described above, and the levels of tyrosine phosphorylation were determined by Western blot analysis. RESULTS Cells treated with anti-HLA serum or the W6/32 monoclonal antibody exhibited significantly greater proliferation and tyrosine phosphorylation of proteins of approximately 170, 130, 110, and 70 kd compared with cells treated with normal human serum or mouse IgG, respectively. CONCLUSIONS These data indicate that anti-HLA antibodies have the ability to stimulate airway epithelial cell proliferation and that they may play an important role in the development of bronchiolitis obliterans syndrome. Prevention of HLA sensitization and immunosuppression with agents capable of blocking indirect antigen presentation and the humoral immune response against the allograft may be pivotal in preventing the development of bronchiolitis obliterans syndrome after lung transplantation.

Immune mechanisms in the pathogenesis of bronchiolitis obliterans syndrome after lung transplantation

Abstract:  Lung transplantation is recognized as the only viable treatment option in a variety of end‐stage pulmonary diseases. However, the long‐term survival after lung transplantation is limited by the development of obliterative bronchiolitis, and its clinical correlate bronchiolitis obliterans syndrome (BOS), which is considered to represent chronic lung allograft rejection. Histopathologically, BOS is an inflammatory process that leads to fibrous scarring of the terminal and respiratory bronchioles and subsequent total occlusion of the airways. The specific etiology and pathogenesis of BOS are not well understood. The current premise is that BOS represents a common lesion in which different inflammatory insults such as ischemia‐reperfusion, rejection, and infection can lead to a similar histological and clinical outcome. However, the low incidence of BOS in non‐transplanted individuals and the observation that early development of BOS is predicted by the frequency and severity of acute rejection episodes indicate that alloimmune‐dependent mechanisms play a crucial role in the pathogenesis of BOS. The evidence presented in this review indicates that BOS is the result of humoral and cellular immune responses developed against major histocompatibility complex molecules expressed by airway epithelial cells of the lung allograft. This process is aggravated by alloimmune‐independent mechanisms such as ischemia‐reperfusion and infection. Currently, treatment of BOS is frequently unsuccessful. Therefore, a better understanding of the immunopathogenesis of BOS is of paramount importance toward improving long‐term patient and graft survival after lung transplantation.

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.

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.

Airway Epithelium is the Primary Target of Allograft Rejection in Murine Obliterative Airway Disease

Murine heterotopic tracheal allografts develop obliterative airway disease (OAD), a suitable model of chronic lung allograft rejection. This model, however, fails to account for the behavior of the allograft when adjacent to recipient airway tissues, particularly the epithelium. This study was performed to determine the immunologic role of the epithelium in development of OAD. BALB/c (H2d) tracheal allografts were transplanted orthotopically into C57BL/6 (H2b) mice and harvested 14–150 days post‐transplantation. The phenotype of the allograft epithelium after orthotopic transplantation was determined with immunofluorescent staining. Orthotopic BALB/c tracheal allografts harvested at 28 days were re‐transplanted heterotopically into BALB/c or C57BL/6 mice, harvested after 28 days, and assessed for OAD. Orthotopic allografts displayed mild cellular infiltration, no fibrosis and preserved epithelium at 28 days post‐transplant. The presence of recipient‐derived epithelium within the allograft was demonstrated with immunofluorescent staining at day 14. Significantly, BALB/c orthotopic allografts re‐transplanted heterotopically into BALB/c mice developed OAD by day 28, whereas BALB/c orthotopic allografts re‐transplanted heterotopically into C57BL/6 mice did not. Repopulation of orthotopic tracheal allografts with recipient‐derived epithelium confers a protective effect against OAD after heterotopic re‐transplantation. This indicates that the airway epithelium plays a crucial role in OAD development.

Identification of HLA-A3-restricted CD8+ T cell epitopes derived from mammaglobin-A, a tumor-associated antigen of human breast cancer.

Mammaglobin‐A is highly overexpressed in breast cancer cell lines and primary breast tumors. This pattern of expression is restricted to mammary epithelium and metastatic breast tumors. Thus, mammaglobin‐A‐specific T cell immune responses may provide an important approach for the design of breast cancer‐specific immunotherapy. The purpose of our study was to define the T cell‐mediated immune response to mammaglobin‐A. We determined that the frequency of mammaglobin‐A‐reactive CD8+ and CD4+ T cells in breast cancer patients is significantly higher than that observed in healthy female controls using limiting dilution analyses (p = 0.026 and p = 0.02, respectively). We identified 8 mammaglobin‐A‐derived 9‐mer peptides with the highest binding affinity for the HLA‐A3 molecule (Mam‐A3.1–8) using a computer‐assisted analysis of the mammaglobin‐A protein sequence. Subsequently, we determined that CD8+ T cells from breast cancer patients reacted to peptides Mam‐A3.1 (23–31, PLLENVISK), Mam‐A3.3 (2–10, KLLMVLMLA), Mam‐A3.4 (55–63, TTNAIDELK) and Mam‐A3.8 (58–66, AIDELKECF) using an IFN‐γ enzyme‐linked immunospot assay. A CD8+ T cell line generated in vitro against HLA‐A*0301‐transfected TAP‐deficient T2 cells loaded with these peptides showed significant cytotoxic activity against the Mam‐A3.1 peptide. This CD8+ T cell line showed a significant HLA‐A3‐restricted cytotoxic activity against mammaglobin‐A‐positive but not mammaglobin‐A‐negative breast cancer cells. In summary, our study identified four HLA‐A3‐restricted mammaglobin‐A‐derived epitopes naturally expressed by breast cancer cells, indicating the immunotherapeutic potential of this novel antigen for the treatment and prevention of breast cancer.

PROLONGED COLD-PRESERVATION OF NERVE ALLOGRAFTS

The goal of this study was to determine the effect of varying durations of cold‐preservation on the immunogenicity of nerve allografts and their subsequent ability to facilitate neuroregeneration across a short nerve gap. Allografts preserved for 1, 4, and 7 weeks were compared to untreated allografts and isografts. There was a shift from an interferon‐γ–producing cellular response (untreated allografts) to an absence of response (7‐week cold‐preserved allografts and isografts). There were no detectable alloantibodies by flow cytometry. Histomorphometry distal to the graft showed robust regeneration in the isograft and 7‐week cold‐preserved groups when compared to the untreated allograft group. Increasing duration of cold‐preservation diminished the cellular immune response. This cold‐preservation does not preclude subsequent nerve regeneration across a short nerve graft. Prolonged cold‐preservation of nerve allograft tissue could serve as a means to produce unlimited graft material for use in peripheral nerve reconstruction. Muscle Nerve, 2005