Decline in Club Cell Secretory Proteins, Exosomes Induction and Immune Responses to Lung Self-antigens, Kα1 Tubulin and Collagen V, Leading to Chronic Rejection After Human Lung Transplantation

Abstract

Background: Chronic lung allograft dysfunction (CLAD), is a major hurdle for long-term lung allograft survival after lung transplant and roughly 50% of lung transplant recipients (LTxRs) develop CLAD within 5 years. The mechanisms of CLAD development remain unknown. Donor-specific immune responses to HLA and lung self-antigens (SAgs) are vital to the pathogenesis of CLAD. Reduction in Club cell secretory protein (CCSP) has been reported in bronchoalveolar lavage (BAL) fluid samples from LTxRs with bronchiolitis obliterans syndrome (BOS). CCSP levels in BAL fluid and development of antibodies to lung SAgs in plasma were determined by ELISA. Cytokines in BAL fluid were analyzed by 30-plex Luminex panel. Exosomes from BAL fluid or plasma were analyzed for SAgs, natural killer (NK) cells markers, and cytotoxic molecules.

Results: We demonstrate that LTxRs with BOS have lower CCSP levels up to 9 months before BOS diagnosis. LTxRs with antibodies to SAgs 1-year posttransplant also developed DSA (43%) and had lower CCSP. BOS with lower CCSP also induced Interleukin-8 and reduced vascular endothelial growth factor. Exosomes from BOS contained increased SAgs, NK cells markers, and cytotoxic molecules.

Conclusions: We conclude lower CCSP leads to inflammation, pro-inflammatory cytokine production, immune responses to HLA and SAgs, and induction of exosomes. For the first time, we demonstrate that CCSP loss results in exosome release from NK cells capable of stimulating innate and adaptive immunity posttransplant. This increases the risk of BOS, suggesting a role of NK cell exosomes in CLAD development.

Figure 1:

(A) Lung transplant recipients with bronchiolitis obliterans syndrome (BOS) had low levels of club cell secretory protein (CCSP) in bronchoalveolar lavage fluid samples both at the time of BOS diagnosis and (B) 7 to 9 months before BOS diagnosis.

Figure 2:

Lung transplant recipients with bronchiolitis obliterans syndrome (BOS) (n=5) with antibodies to self-antigens (SAgs) Collagen V and K-alpha 1 tubulin had low levels of club cell secretory protein (CCSP) in bronchoalveolar lavage fluid samples compared to stable (n=24).

Figure 3:

(A) Lung transplant recipients (LTxRs) with low levels of club cell secretory protein (CCSP) developed antibodies (Abs) to lung self-antigens (SAgs) within 1 year of transplant, compared to LTxRs without Abs. (B) LTxRs with low CCSP levels developed de novo donor-specific anti-HLA (DSA), along with Abs to lung SAgs within 1 year of LTx. (C) Abs to SAgs (Collagen V and K-alpha 1 tubulin), if persistent, lower CCSP levels in bronchoalveolar lavage fluid samples.

Figure 4:

(A) Interleukin-8 (IL8) was upregulated and (B) vascular endothelial growth factor (VEGF) was downregulated in bronchoalveolar lavage fluid samples from lung transplant recipients with bronchiolitis obliterans syndrome (BOS; n=18).

Figure 5:

MT1H, GRANZYME A, and NKG7 were upregulated in bronchoalveolar lavage fluid samples from lung transplant recipients with bronchiolitis obliterans syndrome.

Figure 6:

(A) Exosome size, verified by Nanosight, was less than 200nm. (B) Exosomes from bronchoalveolar lavage fluid samples from lung transplant recipients (LTxRs) with bronchiolitis obliterans syndrome (BOS) contained increased levels of self-antigens (CIITA, 20S proteasome, NFκB, CD56, NKG2D, FasL and perforin). (C) Densitometry analysis of self-antigens (CIITA, 20S proteasome, NFκB). (D) Representative densitometry analysis of natural killer (NK) cells related and cytotoxic molecules in LTxRs with BOS (n=2) vs stable LTxRs (n=2), demonstrating increased levels of CD56, NKG2D, FasL, and perforin.

Figure 7:

(A) Exosomes isolated from plasma samples of healthy volunteers (HV), stable lunt transplant recipients (LTxRs), and LTxRs with bronchiolitis obliterans syndrome (BOS) demonstrate increased levels of CD56, NKG2D, FasL, and perforin in LTxRs with BOS. (B) Densitometry analysis of natural killer (NK) cells related and cytotoxic molecules optical density in LTxRs with BOS (n=14) compared with stable LTxRs (n=14).

Figure 8:

(A) Cytotoxicity assay demonstrated increased toxicity of club cell line incubated with plasma of BOS compared with stable LTxRs. (B) Immunofluorescence studies demonstrated BOS LTxRs plasma had increased level of IgG Abs bound to club cells compared to stable LtxRs.

Figure 9:

Proposed role of club cell secretory proteins (CCSPs) regulating natural killer (NK) cells and NK-cell-derived exosomes. Decline of CCSPs and NK cell activation, thereby inducing pro-inflammatory cytokines and oxidative stress and circulating exosomes with lung SAgs and NK-cell-related protein. This induction in turn leads to de novo development of Abs to donor HLA and/or non-HLA lung SAgs, before ultimately manifesting into CLAD.