Purpose Immune responses against lung self-antigens (SAg) [Kα1tubulin (Kα1T) and Collagen V (ColV)] are critical in the pathogenesis of chronic rejection in human lung transplantation (LT). Our goal was to determine the mechanisms by which antibodies (Abs) to SAgs lead to rejection in co-stimulatory blockade induced tolerant murine LT. Methods and Materials LT was done from Balb/c to Bl/6 and tolerance was induced with MR1 (250μg) and CTLA4-Ig (200μg) on post-LT days 1 and 2. Abs to Kα1T or ColV or both (200μg/dose, i/p) or isotype control were given on days -10, -2, 0 & weekly following LT. Lungs were analyzed by H&E, trichrome and immunostains. Kα1T and ColV specific IFN-γ, IL-10, IL-4 and IL-17 cells were enumerated by ELISpot. Abs to allo-MHC was determined by FACS. Cytokine and growth factors in lung were done by RTPCR. Results Allo-LT with co-stimulatory blockade did not show evidence of rejection for >100 days. However, administration of Abs to Kα1T or ColV or both resulted in loss of tolerance and rejection (cellular infiltration, airway remodeling) by day 45-60. Isotype controls did not demonstrate rejection pathology. Abs to SAgs in tolerant mice led to Infiltration of CD4 and CD8 T cells, B-cells and neutrophils, increase in SAg specific T-cells secreting IFNγ (p=0.002), IL-4 (p=0.047) and IL-17 (p=0.001) and decrease in IL-10 (p=0.03). Abs to either SAgs resulted in immune responses to both the SAgs and donor specific Abs to allo-MHC (MFI>2000 against Balb/c - H2kd), demonstrating epitope spreading to other SAgs and linked allo-MHC. Upregulation of IL-6 (7.1 fold), IL-1β (17 fold), IFN-γ(4.8 fold), IL-17 (10.1 fold), VEGF (2.9 fold), TGF-β (3.8 fold) was also noted in the lung tissue. Conclusions Administration of Abs to lung Sags resulted in epitope spreading of immune responses not only to Sags but also to the linked allo-MHC leading to loss of tolerance induced by co-stimulatory blockade.