David S. Wilkes

IL-17–dependent cellular immunity to collagen type V predisposes to obliterative bronchiolitis in human lung transplants

Bronchiolitis obliterans syndrome (BOS), a process of fibro-obliterative occlusion of the small airways in the transplanted lung, is the most common cause of lung transplant failure. We tested the role of cell-mediated immunity to collagen type V [col(V)] in this process. PBMC responses to col(II) and col(V) were monitored prospectively over a 7-year period. PBMCs from lung transplant recipients, but not from healthy controls or col(IV)-reactive Goodpastures syndrome patients after renal transplant, were frequently col(V) reactive. Col(V)-specific responses were dependent on both CD4+ T cells and monocytes and required both IL-17 and the monokines TNF-alpha and IL-1beta. Strong col(V)-specific responses were associated with substantially increased incidence and severity of BOS. Incidences of acute rejection, HLA-DR mismatched transplants, and induction of HLA-specific antibodies in the transplant recipient were not as strongly associated with a risk of BOS. These data suggest that while alloimmunity initiates lung transplant rejection, de novo autoimmunity mediated by col(V)-specific Th17 cells and monocyte/macrophage accessory cells ultimately causes progressive airway obliteration.

Efficacy of infliximab in extrapulmonary sarcoidosis: results from a randomised trial

The aim of the present study was to investigate the efficacy of infliximab for the treatment of extrapulmonary sarcoidosis. A prospective, randomised, double-blind, placebo-controlled trial was conducted, with infliximab at 3 and 5 mg·kg−1 body weight administered over 24 weeks. Extrapulmonary organ severity was determined by a novel severity tool (extrapulmonary physician organ severity tool; ePOST) with an adjustment for the number of organs involved (ePOSTadj). In total, 138 patients enrolled in the trial of infliximab versus placebo for the treatment of chronic corticosteroid-dependent pulmonary sarcoidosis. The baseline severity of extrapulmonary organ involvement, as measured by ePOST, was similar across treatment groups. After 24 weeks of drug-therapy study, the change from baseline to week 24 in ePOST was greater for the combined infliximab group compared with the placebo group. After adjustment for the number of extrapulmonary organs involved, the improvement in ePOSTadj observed in the combined infliximab group was also greater than that observed in placebo-treated patients, after 24 weeks of therapy. The improvements in ePOST and ePOSTadj were not maintained during a subsequent 24-week washout period. Infliximab may be beneficial compared with placebo in the treatment of extrapulmonary sarcoidosis in patients already receiving corticosteroids, as assessed by the severity tool described in the present study.

Evidence for Immune Responses to a Self-Antigen in Lung Transplantation: Role of Type V Collagen-Specific T Cells in the Pathogenesis of Lung Allograft Rejection

We have reported that lung allograft rejection involves an immune response to a native protein in the lung, type V collagen (col(V)), and that col(V)-induced oral tolerance prevented acute and chronic rejection. In support of these findings col(V) fragments were detected in allografts during rejection, but not in normal lungs. The purpose of the current study was to isolate and characterize col(V)-specific allograft-infiltrating T cells and to determine their contribution to the rejection response in vivo. Two col(V)-specific T cell lines, LT1 and LT3, were isolated from F344 (RT1lv1) rat lung allografts during rejection that occurred after transplantation into WKY (RT1l) recipients. Both cell lines, but not normal lung lymphocytes, proliferated in response to col(V). Neither LT1 nor LT3 proliferated in response to alloantigens. LT1 and LT3 were CD4+CD25− and produced IFN-γ in response to col(V). Compared with normal CD4+ T cells, both cell lines expressed a limited V-β TCR repertoire. Each cell strongly expressed V-β 9 and 16, but differed in expression of other V-βs. Adoptive transfer of each cell line did not induce pathology in lungs of normal WKY rats. In contrast, adoptive transfer of LT1, but not LT3, caused marked peribronchiolar and perivascular inflammation in isograft (WKY) lungs and abrogated col(V)-induced oral tolerance to allograft (F344) lungs. Collectively, these data show that lung allograft rejection involves both allo- and autoimmune responses, and graft destruction that occurs during the rejection response may expose allograft-infiltrating T cells to potentially antigenic epitopes in col(V).

An international ISHLT/ATS/ERS clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome

Bronchiolitis obliterans syndrome (BOS) is a major complication of lung transplantation that is associated with poor survival. The International Society for Heart and Lung Transplantation, American Thoracic Society, and European Respiratory Society convened a committee of international experts to describe and/or provide recommendations for 1) the definition of BOS, 2) the risk factors for developing BOS, 3) the diagnosis of BOS, and 4) the management and prevention of BOS. A pragmatic evidence synthesis was performed to identify all unique citations related to BOS published from 1980 through to March, 2013. The expert committee discussed the available research evidence upon which the updated definition of BOS, identified risk factors and recommendations are based. The committee followed the GRADE (Grading of Recommendation, Assessment, Development and Evaluation) approach to develop specific clinical recommendations. The term BOS should be used to describe a delayed allograft dysfunction with persistent decline in forced expiratory volume in 1 s that is not caused by other known and potentially reversible causes of post-transplant loss of lung function. The committee formulated specific recommendations about the use of systemic corticosteroids, cyclosporine, tacrolimus, azithromycin and about re-transplantation in patients with suspected and confirmed BOS. The diagnosis of BOS requires the careful exclusion of other post-transplant complications that can cause delayed lung allograft dysfunction, and several risk factors have been identified that have a significant association with the onset of BOS. Currently available therapies have not been proven to result in significant benefit in the prevention or treatment of BOS. Adequately designed and executed randomised controlled trials that properly measure and report all patient-important outcomes are needed to identify optimal therapies for established BOS and effective strategies for its prevention. Diagnosis of BOS requires careful exclusion of other complications that can cause delayed lung allograft dysfunction http://ow.ly/AZmbr

Anti-Type V Collagen Lymphocytes that Express IL-17 and IL-23 Induce Rejection Pathology in Fresh and Well-Healed Lung Transplants

Immunity to collagen V [col(V)] contributes to lung ‘rejection.’ We hypothesized that ischemia reperfusion injury (IRI) associated with lung transplantation unmasks antigenic col(V) such that fresh and well‐healed lung grafts have differential susceptibility to anti‐col(V)‐mediated injury; and expression of the autoimmune cytokines, IL‐17 and IL‐23, are associated with this process. Adoptive transfer of col(V)‐reactive lymphocytes to WKY rats induced grade 2 rejection in fresh isografts, but induced worse pathology (grade 3) when transferred to isograft recipients 30 days post‐transplantation. Immunhistochemistry detected col(V) in fresh and well‐healed isografts but not native lungs. Hen egg lysozyme‐reactive lymphocytes (HEL, control) did not induce lung disease in any group. Col(V), but not HEL, immunization induced transcripts for IL‐17 and IL‐23 (p19) in the cells utilized for adoptive transfer. Transcripts for IL‐17 were upregulated in fresh, but not well‐healed isografts after transfer of col(V)‐reactive cells. These data show that IRI predisposes to anti‐col(V)‐mediated pathology; col(V)‐reactive lymphocytes express IL‐17 and IL‐23; and anti‐col(V)‐mediated lung disease is associated with local expression of IL‐17. Finally, because of similar histologic patterns, the pathology of clinical rejection may reflect the activity of autoimmunity to col(V) and/or alloimmunity.

Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation.

Obliterative bronchiolitis (OB) is the key impediment to the long‐term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining OB immunopathogenesis. In the current study, lungs from C57BL/10 H‐2b mice that are MHC compatible, but minor histocompatability antigen incompatible, were transplanted into C57BL/6 mice. Histological features and cytokine profiles of OB were assessed. Moderate rejection (grade A3) developed by day 14, with evidence of OB at that time point. At 21 days, OB was present in 55% of grafts and moderate to severe rejection (grade A3‐A4) was present in all mice. At 28 days, OB was present in 44% of mice and severe rejection (grade A4) was present in all. IL‐17A, but not IL‐17F, splenic mRNA transcripts and serum protein levels were increased only in mice that developed OB, whereas IL‐10 transcripts and protein were increased only in non‐OB mice. Neutralizing IL‐17 prevented OB, down regulated acute rejection, and upregulated systemic IL‐10. Collectively, these data show that transplantation of minor histoincompatible lungs from C57BL/10 mice into C57BL/6 mice results in a highly reproducible preclinical model of OB. In addition, these data indicate that neutralizing IL‐17A or augmenting IL‐10 could be therapeutic interventions to prevent OB.

Prevention of bronchiolitis obliterans in rat lung allografts by type V collagen-induced oral tolerance.

Background. We have reported that feeding type V collagen (col(V)) to lung allograft recipients induces immune tolerance that prevents acute lung allograft rejection. Repeated acute rejection is a risk factor for or associated with chronic rejection, known as bronchiolitis obliterans (BO), the leading cause of death in lung allograft recipients. The current study examines if col(V)-induced oral tolerance prevents BO. Methods. WKY rats (RT1l) were fed either col(V) or diluent before orthotopic transplantation of F344 (RT1lvl) lung allografts. No rats received any immunosuppression. At 10 weeks posttransplantation the time to onset of BO, delayed type hypersensitivity (DTH) responses to donor antigens, and col(V) were examined. In addition, proliferative responses of recipient T lymphocytes to donor antigens, and ability of recipient antigen presenting cells to present alloantigens in lung allografts were evaluated. Results. The data show that recipient rats have sustained DTH responses to donor antigens and col(V). T lymphocytes from col(V)-fed lung allograft recipients were unable to proliferate in response to donor antigens, but feeding col(V) had no effect on the presentation of donor alloantigens by recipient antigen presenting cells. All diluent fed rats developed BO, but only mild acute rejection (grade 2) was present in all rats fed col(V). Transforming growth factor (TGF)-&bgr; production was up-regulated systemically in col(V)-fed, but not diluent fed, lung allograft recipients, and neutralizing TGF-36 recovered the DTH response to donor antigens in col(V)-fed rats. Conclusions. Collectively these data show that col(V)-induces oral tolerance that prevents BO, and that tolerance may be mediated by systemic production of TGF-36.

Anti-type V collagen humoral immunity in lung transplant primary graft dysfunction

Primary graft dysfunction (PGD) is a major complication following lung transplantation. We reported that anti-type V collagen (col(V)) T cell immunity was strongly associated with PGD. However, the role of preformed anti-col(V) Abs and their potential target in PGD are unknown. Col(V) immune serum, purified IgG or B cells from col(V) immune rats were transferred to WKY rat lung isograft recipients followed by assessments of lung pathology, cytokines, and PaO2/FiO2, an index of lung dysfunction in PGD. Immune serum, purified IgG, and B cells all induced pathology consistent with PGD within 4 days posttransfer; up-regulated IFN-γ, TNF-α, and IL-1β locally; and induced significant reductions in PaO2/FiO2. Depleting anti-col(V) Abs before transfer demonstrated that IgG2c was a major subtype mediating injury. Confocal microscopy revealed strong apical col(V) expression on lung epithelial, but not endothelial cells; which was consistent with the ability of col(V) immune serum to induce complement-dependent cytotoxicity only in the epithelial cells. Examination of plasma from patients with or without PGD revealed that higher levels of preformed anti-col(V) Abs were strongly associated with PGD development. This study demonstrates a major role for anti-col(V) humoral immunity in PGD, and identifies the airway epithelium as a target in PGD.

Th-17, Monokines, Collagen Type V, and Primary Graft Dysfunction in Lung Transplantation

RATIONALE The pathogenesis of primary graft dysfunction (PGD), a serious complication of lung transplantation, is poorly understood. Human studies and rodent models have shown that collagen type V (col[V]), stimulates IL-17-dependent cellular immunity after lung transplantation. OBJECTIVES To determine whether patients with end-stage lung disease develop pretransplant col(V)-specific cellular immunity, and if so, the impact of this response on PGD. METHODS Trans-vivo delayed-type hypersensitivity (TV-DTH) assays were used to evaluate memory T-cell responses to col(V) in 55 patients awaiting lung transplantation. Pa(O(2))/Fi(O(2)) index data were used to assess PGD. Univariate risk factor analysis was performed to identify variables associated with PGD. Rats immunized with col(V) or irrelevant antigen underwent lung isografting to determine if prior anti-col(V) immunity triggers PGD in the absence of alloreactivity. MEASUREMENTS AND MAIN RESULTS We found that 58.8% (10/17) of patients with idiopathic pulmonary fibrosis, and 15.8% (6/38) of patients without idiopathic pulmonary fibrosis tested while on the wait list for a lung transplant were col(V) DTH positive. Col(V) reactivity was CD4(+) T-cell and monocyte mediated, and dependent on IL-17, IL-1beta, and tumor necrosis factor (TNF)-alpha. Pa(O(2))/Fi(O(2)) indices were impaired significantly 6-72 hours after transplantation in col(V)-reactive versus nonreactive patients. Univariate risk factor analysis identified only preoperative TV-DTH to col(V) and ischemic time as predictors of PGD. Finally, in a rat lung isograft model, col(V) sensitization resulted in significantly lower Pa(O(2))/Fi(O(2)), increased local TNF-alpha and IL-1beta production, and a moderate-to-severe bronchiolitis/vasculitis when compared with control isografts. CONCLUSIONS The data suggest that activation of innate immunity by col(V)-specific Th-17 memory cells represents a novel pathway to PGD after lung transplantation.

Differential Expression of Smad7 Transcripts Identifies the CD4+CD45RChigh Regulatory T Cells That Mediate Type V Collagen-Induced Tolerance to Lung Allografts

Regulatory T cells (Tregs) induced by oral tolerance may suppress immunity by production of TGF-β that could also enhance Treg activity. However, all cells that are phenotypically Tregs in rats (CD4+CD45RChigh-RChigh) may not have regulatory function. Because Smad7 expression in T cells is associated with inflammation and autoimmunity, then lack of Smad7 may identify those cells that function as Tregs. We reported that feeding type V collagen (col(V)) to WKY rats (RT1l) induces oral tolerance to lung allografts (F344-RT1lvl) by T cells that produce TGF-β. The purpose of the current study was to identify the Tregs that mediate col(V)-induced tolerance, and determine Smad7 expression in these cells. RChigh cells from tolerant rats were unresponsive to allogeneic stimulation and abrogated rejection after adoptive transfer. In contrast, CD4+CD45RClow (RClow) cells from tolerant rats and RChigh or RClow cells from normal rats or untreated allograft recipients proliferated vigorously in response to donor Ags, and did not suppress rejection after adoptive transfer. TGF-β enhanced proliferation in response to col(V) presented to tolerant RChigh, but not other cells. In contrast to other cells, only RChigh cells from tolerant rats did not express Smad7. Collectively, these data show that the Tregs that mediate col(V)-induced tolerance to lung allografts do not express SMAD7 and, therefore, are permissive to TGF-β-mediated signaling.