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

Abstract

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 Goodpasture's 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.

Figures

Figure 1. CD4+ T cell–dependent, col(V)-specific cell-mediated immunity in lung transplant (Tx) recipients.

(A) TV-DTH responses by PBMCs obtained from normal healthy controls (n = 6), renal transplant recipients with GPS at 2–10 yr after transplant (n = 5), or lung transplant recipients at 0.5–3.5 yr after transplant (n = 15). Lung primary disease types represented are chronic obstructive pulmonary disease (n = 7), cystic fibrosis (n = 3), idiopathic pulmonary fibrosis (n = 2), α-1 antitrypsin deficiency (n = 2), and other (n = 1). All had stable graft function on standard immunosuppression at time of testing. TV-DTH responses to EBV and TT/DT were determined separately and averaged to yield a positive control swelling response (Recall) for each subject. Responses to 5 μg col(II), col(IV), or col(V) were averaged from duplicate tests and are shown as individual data points. Horizontal bars denote group means. **P < 0.001 among treatment groups in response to a specific collagen, Wilcoxon rank-sum test. (B) TV-DTH responses (mean ± SEM) to TT/DT and col(V) in col(V)-reactive patients L52 and L84 in the presence of isotype control, anti–IFN-γ, anti–TNF-α, anti–IL-1β, or anti–IL-17 Abs. *P < 0.05 versus IgG, Student’s t test. (C) TV-DTH responses (mean ± SEM) to TT/DT and col(V) by sham-depleted whole PBMCs (10 × 106) or subset-depleted PBMCs (8 × 106) of patients L84 (α-1 antitrypsin deficiency) and L52 and L16 (chronic obstructive pulmonary disease). NT, not tested. *P < 0.05 versus whole PBMC, Student’s t test.

Figure 2. In vitro cytokine responses to col(V) indicate a key role for monocytes.

(A) PBMCs from controls (n = 6) and lung transplant recipients (n = 4; patients L84, L52, L16, and L143) were stimulated with TT or col(V). Release of IFN-γ, TNF-α, or IL-1β was measured in culture supernatants after 20 h. Release of TNF-α and IL-1β was significantly higher for lung transplant recipients compared with controls. *P = 0.03 versus respective control, Wilcoxon rank-sum test. (B) TNF-α responses of patient L84 to col(V) in sham-depleted PBMCs or PBMCs depleted of the indicated subpopulations after 20 h culture, or in the presence of media alone, anti–IL-17, or anti–IFN-γ at the indicated concentrations. *P < 0.05 versus PBMCs or media alone as appropriate, Student’s t test. (C) Most intracellular TNF-α is derived from monocytes. PBMCs from patient L84 were cultured with col(V) or col(II) for 5 h. TNF-α+ cells are shown for CD14+ monocytes and CD3+ T cells.

Figure 3. Lung transplant recipients develop anti-col(V) TV-DTH responsiveness prior to onset of severe BOS.

Patient timelines (n = 54) were grouped by maximum post-transplant TV-DTH response to col(V) as assessed by net footpad swelling, either low (A, DTH <25 × 10–4 in.; n = 22), intermediate (B, DTH 25 × 10–4 in.; n = 8) or strongly positive (C, DTH >25 × 10–4 in.; n = 24). Each dot represents a separate blood sample/test result. Patient timelines were sorted by follow-up time. The outcomes as of December 31, 2005, include time points of BOS-1, BOS-2, and death or retransplant.

Figure 4. An alloantigen-independent form of OB is induced by transfer of col(V)-immune rat LN cells.

LN cells from WKY rats immunized with HEL or col(V) were adoptively transferred to naive WKY rats 1 d prior to transplantation of a WKY lung isografts. Top: H&E staining of both native lungs and isografted lungs (30 d after transplant) from animals transferred with LN cells from HEL-immunized animals and col(V)-immunized animals. Bottom: Trichrome staining and col(V) immunostaining of native and isograft lungs from animals that received LN cells from col(V)-immunized animals was performed to determine tissue exposure of col(V). H&E staining revealed lymphocytic infiltrates (Li), an open bronchus (B), an occluded vessel (OV), and an occluded bronchiole (OB). Arrow on occluded bronchiole shows residual airway. Original magnification, ×200 (top); ×400 (bottom).