Collagen type-V is a danger signal associated with primary graft dysfunction in lung transplantation

Primary graft dysfunction (PGD) is the leading cause of early mortality after lung transplantation. Anti-collagen type-V (col(V)) immunity has been observed in animal models of ischemia-reperfusion injury (IRI) and in PGD. We hypothesized that collagen type-V is an innate danger signal contributing to PGD pathogenesis. Methods: Anti-col(V) antibody production was detected by flow cytometric assay following cultures of murine CD19+ splenic cells with col(V). Responding murine B cells were phenotyped using surface markers. RNA-Seq analysis was performed on murine CD19+ cells. Levels of anti-col(V) antibodies were measured in 188 recipients from the Lung Transplant Outcomes Group (LTOG) after transplantation. Results: Col(V) induced rapid production of anti-col(V) antibodies from murine CD19+ B cells. Subtype analysis demonstrated innate B-1 B cells bound col(V). Col(V) induced a specific transcriptional signature in CD19+ B cells with similarities to, yet distinct from, B cell receptor (BCR) stimulation. Rapid de novo production of anti-col(V) Abs was associated with an increased incidence of clinical PGD after lung transplant. Conclusions: This study demonstrated that col(V) is an rapidly recognized by B cells and has specific transcriptional signature. In lung transplants recipients the rapid seroconversion to anti-col(V) Ab is linked to increased risk of grade 3 PGD. Overall design: Three biological replicate harvest of C57BL/10 male mouse spleens were used in 7 different experimental conditions. CD19+ cells (1×106 ml) were then resuspended in 6 well plates in a total volume of 4 ml, and incubated for 3 h. Conditions included media (RPMI 1640 (Invitrogen) containing 10% heat-inactivated FBS (HyClone) and 1% penicillin/streptomycin, 1% glutamine (Invitrogen)), media plus 5mM HOAc, or media plus 5mM HOAc with either 200 µg/ml col(I), 200 µg/ml col(V), 10 µg/ml LPS (E. coli 0111:B4 (Sigma L4391), or 10 µg/ml goat anti-mouse F(ab')2 anti-IgM (Jackson ImmunoResearch 115-006-075). Purified col(I) and col(V) were soluble in 50mM or 100mM HOAC, respectively, so the culture media had 20mM HEPES pH 7.5 added to restore the pH to neutrality.