Michael A. Smith

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

BACKGROUNDnBronchiolitis 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.nnnMETHODSnOf 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.nnnRESULTSnUnivariate 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.nnnCONCLUSIONSnThese 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.

BACKGROUNDnDevelopment 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.nnnMETHODSnSera 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.nnnRESULTSnWhen 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.nnnCONCLUSIONSnOur 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.

Effect of development of antibodies to hla and cytomegalovirus mismatch on lung transplantation survival and development of bronchiolitis obliterans syndrome

OBJECTIVEnA retrospective analysis was performed to examine the role of HLA antibodies and cytomegalovirus mismatch on the development of bronchiolitis obliterans syndrome and survival after lung transplantation.nnnMETHODSnOf 339 consecutive lung transplantations performed over a 102-month interval, 301 patients survived at least 3 months. There was a minimum follow-up period of 13 months. Bronchiolitis obliterans syndrome was defined as a decline in forced expiratory volume in 1 second less than 80% of posttransplantation baseline and/or histologic presence of obliterative bronchiolitis and was defined as occurring early if documented within 3 years of transplantation. Variables analyzed included preoperative donor and recipient cytomegalovirus status and the development of antibodies to human leukocyte antigens after transplantation. Microcytotoxicity was used to determine the presence of antibodies to human leukocyte antigens. Variables were subjected to Kaplan-Meier analysis to determine their impact on freedom from bronchiolitis obliterans syndrome and survival.nnnRESULTSnThe development of antibodies to human leukocyte antigens after transplantation correlated significantly with bronchiolitis obliterans syndrome (P = .02). The development of antibodies to human leukocyte antigens did not affect survival (P = .33) unless they were detected within 2 years of transplantation (P = .04). There was greater frequency of early bronchiolitis obliterans syndrome in cytomegalovirus seronegative patients who received allografts from seropositive donors compared with all other combinations (P = .02). There was also a trend toward worse survival of cytomegalovirus seronegative patients who received allografts from seropositive donors (P = .13).nnnCONCLUSIONnThese data suggest that bronchiolitis obliterans syndrome is the result of an immune-mediated process in which HLA antibodies and cytomegalovirus may play a significant role.

Indirect recognition of donor HLA class I peptides in lung transplant recipients with bronchiolitis obliterans syndrome.

BACKGROUNDnThe presentation of donor MHC class II-derived peptides by host antigen-presenting cells in the context of self-MHC class II molecules has been suggested as a mechanism for the chronic rejection of kidney and heart allografts. The aim of this study was to determine whether indirect allorecognition of HLA class I-derived peptides occurred in lung transplant (LTx) recipients and whether it correlated with the development of bronchiolitis obliterans syndrome (BOS).nnnMETHODSnPeripheral blood mononuclear cells from LTx recipients were cultured with synthetic peptides corresponding to the hypervariable regions of the mismatched HLA class I antigens of the donor. Proliferation and precursor frequency (PF) of allopeptide reactive T cells were determined by the incorporation of [3H]thymidine into DNA and limiting dilution analysis.nnnRESULTSnPeripheral blood leukocytes of LTx recipients with BOS mismatched for HLA class I molecules showed a proliferative response three- to fourfold higher than those observed in mismatched recipients without BOS and in normal control individuals (P<0.001). Similarly, the PF of allopeptide-reactive T cell was 3- to 24-fold higher in recipients with BOS compared with recipients without BOS (P<0.05) as well as normal control individuals (P<0.03). The T cell PF to donor-specific allopeptides, as well as irrelevant allopeptides, was not significantly different in LTx recipients without BOS and normal control individuals.nnnCONCLUSIONSnThese data suggest that T cells from LTx recipients are sensitized to mismatched HLA class I antigens. The sensitization was significantly higher in LTx recipients with BOS compared with LTx recipients without BOS. Strategies to block T-cell responses generated by indirect allorecognition after lung transplantation may provide a means for the prevention or treatment of BOS in LTx recipients.

Sublobar resection is equivalent to lobectomy for clinical stage 1A lung cancer in solid nodules

OBJECTIVESnA single randomized trial established lobectomy as the standard of care for the surgical treatment of early-stage non-small cell lung cancer. Recent advances in imaging/staging modalities and detection of smaller tumors have once again rekindled interest in sublobar resection for early-stage disease. The objective of this study was to compare lung cancer survival in patients with non-small cell lung cancer with a diameter of 30 mm or less with clinical stage 1 disease who underwent lobectomy or sublobar resection.nnnMETHODSnWe identified 347 patients diagnosed with lung cancer who underwent lobectomy (n = 294) or sublobar resection (n = 53) for non-small cell lung cancer manifesting as a solid nodule in the International Early Lung Cancer Action Program from 1993 to 2011. Differences in the distribution of the presurgical covariates between sublobar resection and lobectomy were assessed using unadjusted P values determined by logistic regression analysis. Propensity scoring was performed using the same covariates. Differences in the distribution of the same covariates between sublobar resection and lobectomy were assessed using adjusted P values determined by logistic regression analysis with adjustment for the propensity scores. Lung cancer-specific survival was determined by the Kaplan-Meier method. Cox survival regression analysis was used to compare sublobar resection with lobectomy, adjusted for the propensity scores, surgical, and pathology findings, when adjusted and stratified by propensity quintiles.nnnRESULTSnAmong 347 patients, 10-year Kaplan-Meier for 53 patients treated by sublobar resection compared with 294 patients treated by lobectomy was 85% (95% confidence interval, 80-91) versus 86% (confidence interval, 75-96) (P = .86). Cox survival analysis showed no significant difference between sublobar resection and lobectomy when adjusted for propensity scores or when using propensity quintiles (P = .62 and P = .79, respectively). For those with cancers 20 mm or less in diameter, the 10-year rates were 88% (95% confidence interval, 82-93) versus 84% (95% confidence interval, 73-96) (P = .45), and Cox survival analysis showed no significant difference between sublobar resection and lobectomy using either approach (P = .42 and P = .52, respectively).nnnCONCLUSIONSnSublobar resection and lobectomy have equivalent survival for patients with clinical stage IA non-small cell lung cancer in the context of computed tomography screening for lung cancer.

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.

Increased expression of inflammatory cytokines and adhesion molecules by alveolar macrophages of human lung allograft recipients with acute rejection: decline with resolution of rejection

BACKGROUNDnAlveolar macrophages (AM) are the major population in bronchoalveolar lavage (BAL) cells; we assessed their role in human lung allograft recipients by correlating the expression of adhesion molecules and inflammatory cytokines with clinical outcome of allograft.nnnMETHODSnWe obtained BAL samples from patients and enriched them for AM in plastic petri dish for 2 hours at 37 degrees C in 5% CO(2). Expression of intercellular adhesion molecule-1 (ICAM-1, CD54), platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31), and CD11c was assessed by flow cytometry using monoclonal antibodies. We assessed cytokine profile using Multi-Probe RNase protection assay.nnnRESULTSnAlveolar macrophages that express CD11c, CD31 and CD54 were increased in patients with either rejection or infection compared with those without rejection and infection. The difference in the percentage of AM expressing CD11c and CD31 between the rejection group and patients without rejection and infection group was statistically significant (CD11c, p < 0.01; CD31, p < 0.03). Interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1Ra), and IL-6 expression was higher in the rejection group than in patients without rejection. Five out of 9 patients in the rejection group expressed high levels of IL-15 and tumor necrosis factor-alpha compared with patients without rejection and infection. The increased number of AM expressing adhesion molecules and elevated expression of cytokines observed during acute rejection declined to basal levels after successful treatment and resolution of rejection. This study demonstrates that lung allograft rejection is associated with increased expression of adhesion molecules and inflammatory cytokines by AM, which could facilitate mononuclear cell adhesion and extravasation contributing to the allograft injury in lung transplant recipients.

Indirect recognition and antibody production against a single mismatched HLA-A2-transgenic molecule precede the development of obliterative airway disease in murine heterotopic tracheal allografts

BACKGROUNDnPrevious studies have implicated the allogeneic immune response in the development of obliterative bronchiolitis after lung transplantation. However, the progression of specific pathogenic events leading to this form of chronic allograft dysfunction have not been well characterized. We used a murine tracheal transplantation model in which a single mismatched HLA-A2-transgenic molecule is indirectly recognized by the recipient CD4(+) T cells to show that obliterative airway disease (OAD) that developed in these allografts was preceded by indirect recognition of the HLA-A2 molecule and subsequent development of anti-HLA-A2 antibodies.nnnMETHODSnTracheas from HLA-A2(+) C57BL/6 mice were heterotopically transplanted into C57BL/6 mice. Allograft histopathology as well as anti-HLA-A2 T-cell proliferative responses and anti-HLA-A2 antibody development were determined at days 5, 10, 20, and 28 after transplantation.nnnRESULTSnAll of the HLA-A2(+) tracheal allografts transplanted into C57BL/6 recipients demonstrated complete development of OAD by day 20. Spleen cells from the mice that underwent transplantation demonstrated significant proliferation against HLA-A2(+) cells by day 5. Indirect recognition of HLA-A2-derived peptides by spleen cells from allograft recipients was also higher on days 5 and 10 as compared with irrelevant peptides derived from HLA-A1, HLA-A3, and HLA-B44. Allograft recipients showed detectable levels of anti-HLA-A2 antibodies by day 5 and full development of anti-HLA-A2 antibodies by day 20.nnnCONCLUSIONnThese results show that sensitization of CD4+ T cells against the mismatched HLA-A2 alloantigen precedes the development of anti-HLA antibodies as well as OAD, suggesting an important role for alloreactive CD4(+) T-cell activation and alloantibody development in the immunopathogenesis of OAD.

Temporal relationship between the development of anti-HLA antibodies and the development of bronchiolitis obliterans syndrome after lung transplantation.

HE MAJOR CAUSE of late morbidity and mortality after lung transplantation (LT) is the development of bronchiolitis obliterans syndrome (BOS), which is believed to represent chronic allograft rejection. The etiology and pathogenesis of this late-onset chronic lung allograft dysfunction are not yet known. However, a growing body of evidence suggests that BOS is caused by an immunologically mediated injury directed against endothelial and epithelial cells in the airways of the lung allograft. Previous studies have established an association between development of BOS and several predisposing factors, including HLA class I mismatch, and development of cytotoxic antiHLA antibodies (Ab) post-LT. 1‐ 4 In addition, recent studies have suggested an important role for noncytotoxic Abs directed against HLA class I antigens in the development of chronic rejection. 5,6 These in vitro studies show that antiHLA class I Abs stimulated the proliferation of endothelial cells (EC) and smooth muscle cells (SMC). In vivo, noncytotoxic anti-HLA Abs may contribute to the development of chronic rejection by binding to HLA class I antigens in the allograft and stimulate the proliferation of different cell types including EC and SMC. A cause‐ effect relationship has not been established between development of anti-HLA Abs and development of BOS. In addition, a role for noncytotoxic anti-HLA Abs in the development of BOS after LT has not been assessed. This study was conducted to determine if a temporal relationship exists between the development of cytotoxic as well as noncytotoxic anti-HLA Abs and the development of BOS after LT.

Balancing curability and unnecessary surgery in the context of computed tomography screening for lung cancer

OBJECTIVEnSurgical management is a critical component of computed tomography (CT) screening for lung cancer. We report the results for US sites in a large ongoing screening program, the International Early Lung Cancer Action Program (I-ELCAP).nnnMETHODSnWe identified all patients who underwent surgical resection. We compared the results before (1993-2005) and after (2006-2011) termination of the National Lung Screening Trial to identify emerging trends.nnnRESULTSnAmong 31,646 baseline and 37,861 annual repeat CT screenings, 492 patients underwent surgical resection; 437 (89%) were diagnosed with lung cancer; 396 (91%) had clinical stage I disease. In the 54 (11%) patients with nonmalignant disease, resection was sublobar in 48 and lobectomy in 6. The estimated cure rate based on the 15-year Kaplan-Meier survival for all 428 patients (excluding 9 typical carcinoids) with lung cancer was 84% (95% confidence interval [CI], 80%-88%) and 88% (95% CI, 83%-92%) for clinical stage I disease resected within 1xa0month of diagnosis. Video-assisted thoracoscopic surgery and sublobar resection increased significantly, from 10% to 34% (Pxa0<xa0.0001) and 22% to 34% (Pxa0=xa0.01) respectively; there were no significant differences in the percentage of malignant diagnoses (90% vs 87%, Pxa0=xa0.36), clinical stage I (92% vs 89%, Pxa0=xa0.33), pathologic stage I (85% vs 82%, Pxa0=xa0.44), tumor size (Pxa0=xa0.61), or cell type (Pxa0=xa0.81).nnnCONCLUSIONSnThe frequency and extent of surgery for nonmalignant disease can be minimized in a CT screening program and provide a high cure rate for those diagnosed with lung cancer and undergoing surgical resection.