SRP230631 PRJNA590495 GSE140681 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. GSE140681 pubmed 31325493