Chronic rejection of human lung transplants remains an enigmatic and clinically intractable problem more than 2 decades after its first description (2
). Early studies of human lung allografts showed an elevated IgG2/IgG1 ratio in bronchoalveolar lavage fluid of patients with acute rejection (22
). However, instead of Abs to polymorphic HLA proteins, such as those commonly found in serum during lung transplant acute rejection (5
), analysis of the IgG2 Abs in bronchoalveolar lavage revealed a dominant specificity for an ECM protein, the minor col(V) (D.S. Wilkes, unpublished observations). Under normal circumstances, col(V) is largely confined to the interior of mature col(I):col(V) heterotypic fibrils; acid treatment or limited protease digestion is required to expose col(V) epitopes (16
). The ability of col(V) to become antigenic for a Th-dependent B cell response in lung transplants suggested both disruption of normal ECM structure and neoantigen creation. After extensive studies in rat indicated a critical role for col(V) autoimmunity in lung allograft tolerance and rejection (reviewed in ref. 24
), human translational studies were initiated. Our present finding of a high incidence of CD4-dependent TV-DTH responses to col(V), but not to col(IV) or col(II), in PBMCs from lung transplant recipients and the previous finding of outgrowth of col(V)-specific CD4+
T cell lines from long-term cultures of patient PBMCs (25
) confirm that col(V) becomes immunogenic after human lung transplantation. However, to our knowledge, evidence for a direct association of col(V)-specific cell-mediated immunity with lung transplant chronic rejection has been lacking until now.
The introduction of the TV-DTH assay (26
) when our prospective clinical study of BOS began was fortuitous, because it detected cell-mediated immune responses dependent on either Th1 (IFN-γ) or Th17 (IL-17) and monocyte (TNF-α, IL-1β) products (Figure ). It was also quite sensitive. The Th17 subset of CD4+
T memory cells is in low frequency in human peripheral blood relative to the Th1 (IFN-γ–producing) subset and typically produces 10- to 100-fold lower levels of cytokine after polyclonal stimulation (28
). We have thus far been unsuccessful in detecting col(V)-induced IL-17 protein or mRNA in cultures of lung transplant patient PBMCs and have relied on the much stronger monokine signal as an in vitro correlate of the col(V)-specific TV-DTH response (Figure ). Taking into account the low frequency of Th17 memory cells, and the fact that all patients were taking multiple immunosuppressive drugs, the detection of col(V)-specific cell-mediated immune responses in the majority of lung transplant recipients is quite remarkable. The critical role of both CD14+
monocytes and CD4+
T cells suggests an explanation: that immunosuppressive regimens currently used in lung transplantation may fail to prevent de novo Th17-dependent immune responses (29
). An interesting exception may be the macrolide antibiotic azithromycin, currently being used to treat established BOS; its unique ability to block downstream effects of IL-17 on lung tissue may account for its efficacy (30
). The phenomenon of monocyte–CD4+
T cell interactions leading to monokine production closely parallels findings in rheumatoid arthritis (31
), a disease that is resistant to calcineurin inhibitors but often responsive to treatment with TNF-α antagonists (32
). The correlation of col(V)-specific autoimmunity with BOS in our prospective study is also consistent with the ability of anti–TNF-α and anti–IL-1β Abs to block airway obliteration in the mouse tracheal allograft model (33
) and with the prominent role of monokines and monocyte recruitment in the alloantigen-independent phase of classical chronic rejection (34
Antibodies to HLA class I or class II antigens were detected in 14 of 52 (27%) patients tested, but did not prove to be a significant risk factor for BOS (Table ). In contrast, other published studies using ELISA and flow cytometry techniques have found a significant correlation between anti-HLA Abs in general, or donor-specific HLA Abs in particular, and subsequent BOS development (5
). Sample size may be one limiting factor in our study: for example, neither acute rejection nor HLA-DR mismatch were significant risk factors for BOS in the study subgroup (n
= 54), but both became significant in analysis of the cohort of 281 lung transplant recipients (Table ).
The magnitude of estimated risk of BOS associated with col(V)-specific cell-mediated immunity was 3- to 5-fold for BOS-1; this risk increased to 5- to 10-fold for BOS-2, the severe form of lung chronic rejection. Besides col(V) TV-DTH response, certain other risk factors such as HLA-DR mismatch (Table and ref. 35
) and the appearance of lymphocytic bronchitis/bronchiolitis on surveillance biopsy (5
) have also been associated with a higher risk for BOS-2 versus BOS-1. HLA-DR mismatch tends to prevent establishment of immune regulation to allografts (36
); thus it may also predispose to an imbalance between self-reactive T regulatory and T effector cells that could promote a breakdown of col(V)-specific tolerance (25
). Lymphocytic bronchitis itself could be an indicator of an immune attack on areas of col(V) exposure (18
This possibility is supported by the ability, demonstrated here, (a) to generate an OB-like lesion in a rat lung isograft by adoptive transfer of LN cells from col(V)-immunized donors and (b) to detect col(V) antigen in the rat OB lung under nondenaturing conditions (Figure ). The latter finding is significant because col(V) epitopes cannot be readily detected by immunohistochemistry in normal tissues unless these tissues are first denatured to disrupt col(I) and/or col(V) fibrils, thereby exposing col(V) epitopes (16
). Col(V) transcripts are among the most highly upregulated of all matrix proteins in recently transplanted allografts (37
). Once the initial graft injury has healed, there may be lingering deposits of exposed col(V) within airway parenchyma and perivascular interstitium (21
). These deposits can serve as an antigen source for uptake and presentation of col(V) peptides, for example by monocytes and macrophages, to col(V)-specific Th17 effector cells that reach the graft long after the initial repair process has subsided. Furthermore, gastroesophageal reflux disorder (38
) and community-acquired respiratory viruses (39
) may promote BOS by causing acid- or collagenase-mediated exposure of col(V) from a normally sequestered position in the interior of mature heterotypic col(I) and/or col(V) fibrils (16
). In particular, during ECM repair, normally rare col(V) homotrimers, composed of 3 α1(V) chains, can occur. These would be excluded from heterotypic col(I)/col(V) fibrils and thus expose α1(V) epitopes (40
), which include the immunodominant antigens for CD4+
T cells detected by TV-DTH (Supplemental Figure 1B).
Persistent col(V) reactivity in the PBMCs without BOS development (for example, patients L16, L41, and L24; Figure C) may indicate a failure of monocytes and col(V)-specific CD4+
T cells to mobilize from the blood to the graft. Chemokine receptors specifically expressed by human Th17 cells have recently been identified (28
). Their ligands, when expressed by the target organ, may be particularly important in the mobilization process. In addition, collagen-specific activating receptors such as discoidin domain receptor 1 on monocytes and fibroblasts (41
) may synergize with signaling via IL-17 receptors, propagating lung fibroproliferative lesions.
We acknowledge limitations to our study. First, we relied upon a single measure of cell-mediated immunity, the TV-DTH assay, to prospectively monitor lung transplant recipients over a 7-yr period. This test requires crosstalk between human cytokines and cells in the SCID mouse footpad leading to chemokine-driven recruitment of a granulocyte-rich mouse leukocyte infiltrate (ref. 27
and L.D. Haynes, E. Jankowska-Gan, W. Burlingham, and J. Torrealba, unpublished observations). While in vitro analysis of cytokine release from antigen-stimulated PBMCs tends to validate the TV-DTH assay for type 1 and type 17 responses, we have not evaluated other types of human T cell–dependent responses, such as type 2 reactions, that may also be important in OB and/or BOS (42
). Second, there were gaps in the follow-up of some patients, while others were sampled more regularly; importantly, the average frequency of sampling was not different among low, intermediate, and high col(V) responders. Third, PBMCs from 1 of 4 col(V) TV-DTH+
patients produced IFN-γ in response to col(V) challenge in vitro; this finding suggests heterogeneity in the types of CD4+
T cells responding to this autoantigen and merits caution in the conclusion that all such cells are Th17 type.
In summary, prospective monitoring of human lung transplant patients revealed a critical role of col(V)-specific cellular immunity in the progression of OB, while confirming previous studies that indicate a role for HLA-specific immunity and acute rejection in the initiation of this process. Rat studies confirmed that col(V)-immune lymphoid cells caused OB lesions in a lung isograft but not in native lung of a syngeneic recipient. Analysis of patient responses suggests that an interplay between Th17 cells and monocytes is critically important in this allograft-induced autoimmune response. Recent data implicating IL-1β as the critical monokine driving differentiation of human Th17 cells (43
) is entirely in line with our findings and suggests an amplification loop whereby col(V) immunoreactivity begets more autoimmune effector T cells. The immunobiology of col(V)-reactive CD4+
effector and regulatory T cells, the biochemistry of exposure of the α1 chain of col(V) in the human lung transplant, and the mechanism of human Th17 cell–dependent monocyte activation and pulmonary fibrosis all warrant further investigation. Current clinical practice with respect to prophylaxis of BOS is clearly inadequate and may benefit substantially from consideration of the autoimmune component of this disease.