A Novel Mechanism for Immune Regulation After Human Lung Transplantation
Thalachallour Mohanakumar, Monal Sharma, Ranjithkumar Ravichandran, Sandhya Bansal, *Michael A Smith, *Ross Bremner
St. Joseph's Hospital & Medical Center, Phoenix, AZ
Background: Human lung transplantation (LTx) is a therapeutic option for patients with end-stage lung diseases, but long-term survival after LTx is limited due to chronic rejection, clinically diagnosed as bronchiolitis obliterans syndrome (BOS). The mechanisms leading to BOS remain unknown; however, allo- and autoimmune responses play a role in its pathogenesis. Risk factors associated with BOS are primary graft dysfunction (PGD), respiratory viral infection (RVI), acute rejection (AR), and development of antibodies to mismatched donor HLA (DSA). We sought to define a common denominator in LTx recipients (LTxRs) diagnosed with PGD, RVI, AR, or DSA that triggers immune responses and ultimately leads to BOS. We demonstrate that exosomes (ie, nanovesicles of 40-100 nm, released from the transplanted organ) critically affect the pathogenesis of chronic rejection.
Methods: Blood was collected from patients who underwent LTx at our institution during the study period. LTxRs with PGD, RVI requiring treatment, AR, DSA, and stable LTxRs were included. Serum samples were collected and exosomes were isolated by ultracentrifugation; purity was confirmed by sucrose density cushion and by size (40-100 nm) using electron microscopy. Exosomes were analyzed with immunoblot with specific antibodies to test for lung self-antigens (ie, K-alpha 1Tubulin [Kα1T], Collagen V [Col V]), costimulatory molecules (ie, CD80, CD86), transcription factors (ie, NFKβ, HIFα, CIITA), and 20S proteasome.
Results: Eighty nine patients underwent LTx at our institution in 2017. For this analysis we selected 6 LTx diagnosed with PGD grade 3, 5 LTx diagnosed with PGD grade 0, 15 LTx with RVI requiring intervention, 10 LTx with AR, 10 with de novo DSA, 5 without DSA, and 10 stable LTx. Serum samples collected from LTxRs with PGD grade 3, RVI requiring intervention, AR, and DSA had circulating exosomes that contained the lung self-antigens Kα1T and Col V, indicating that the circulating exosomes indeed originated from the transplanted organ. In contrast, exosomes isolated from stable LTxRs or those with PGD grade 0, viral infections not requiring intervention, or without detectable de novo DSA contained no lung self-antigens. In addition, exosomes isolated from LTxRs with PGD grade 3, RVI, AR, or DSA also contained immune-activating costimulatory molecules (ie, CD80, CD86), transcription factors (ie, NFKβ, CIITA), 20S proteasome, and stress proteins EIFα and PERK (Table 1). Furthermore, our preliminary findings suggest that patients with PGD or RVI whose lung self-antigens persist for a month are at increased risk for BOS.
Conclusions: Stress on the transplanted lungs after PGD grade 3, RVI, AR, or de novo DSA induces exosomes, which contain lung self-antigens and immune-activating molecules. Our preliminary findings show that if stress persists, it can cause continued release of circulatory exosomes, perpetuating immune responses and resulting in increased risk for the development of BOS after LTx.
Table 1. Exosome phenotype and expression of the lung self-antigens Kα1T and Col V.
|Col V Expression|
|PGD Grade 0||5||0||0|
|PGD Grade 3||6||1.7||1.3||Kα1T: 0.065, Col V: 0.048|
|DSA Positive||10||3||1.5||Kα1T: 0.065, Col V: 0.048|
|Acute Rejection||10||2||Col V: 0.002|
|Viral Infection||15||3.0||5.0||Kα1T: 0.045, Col V: 0.024|
* pValues: PGD3 versus PGD0, DSA+ versus DSA-, viral and AR versus stable.
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