*Purpose: Bronchiolitis obliterans syndrome (BOS) remains a major cause of death after lung transplantation. Unfortunately, we lack effective treatments for BOS as the mechanisms by which BOS develops remain poorly understood. Through a retrospective analysis of BOS risk factors using our lung transplant single nucleotide polymorphism database we noted that human lung recipient carriers of a T300A mutation in ATG161, a highly common allelic variant in the autophagy related protein, developed significantly earlier BOS relative to non-carriers. T300A is known to be proteolytically unstable and result in deficiency of ATG16L1 in monocyte-derived antigen presenting cells (Mo-APCs). To gain insight into how loss of ATG16L1 in Mo-APCs is associated with BOS, we generated a mouse model with deletion of ATG16L1 restricted to Mo-APCs and a knock-in T300A mouse. To study these mechanisms, we analyzed the effects of ATG16L1 deficiency in Mo-APCs in a mouse lung transplant model of BOS.

*Methods: LysMCreATG16L1^loxp/loxp (ATG16L1^Δ/Δ) and control ATG16L1^loxp/loxp recipients received major MHC-mismatched FVB lungs, with immunosuppression for tolerance. After induced epithelial injury, allografts were assessed for obliterative airway lesions (OB) at day 16. The ability of ATG16L1^Δ/Δ and T300A Mo-APCs to stimulate T cell alloimmune responses was examined by T cell proliferation assays with CFSE. We measured IL1β levels by mRNA and ELISA. Intragraft Mo-APCs were analyzed for bulk RNA sequencing, then by FACS for mitochondrial dysfunction by measuring mitochondrial mass and ROS. Mitophagic flux was visualized by confocal using Mt-kiema mitophagy reporter mice. Mitochondrial fitness and glycolysis flux were examined by Seahorse and tritiated glucose, and the glycolytic transcription factor HIF1a was measured by immunoblotting.

*Results: In line with human data, murine ATG16L1^Δ/Δ recipients also showed early OB. Cultured ATG16L1^Δ/Δ Mo-APCs showed an increased ability to stimulate T cell proliferative responses and higher IL1β, suggesting enhanced alloimmunity. ATG16L1-deficient Mo-APCs also showed evidence of mitochondrial dysfunction with increased mass, ROS and hyperpolarization and attenuated mitophagic flux. RNASeq revealed loss of subunits encoding for mitochondria electron complexes. In line with this, we noted a shift from decreased maximal mitochondrial respiratory capacity to increased glycolysis along with increased HIF1a expression.

*Conclusions: Mitochondrial dysfunction and metabolic adaptation in ATG16L1-deficient Mo-APCs may drive alloimmunity and BOS pathogenesis in lung transplant recipients that carry a T300A mutation.

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