PROJECT SUMMARY / ABSTRACT The current half-life of a lung allograft is unacceptably short at just 6.7 years, and all recipients encounter significant complications, both from allograft rejection and from immunosuppression-associated toxicities. Immune tolerance provides the ultimate solution to these issues, with the promise of life-long graft acceptance without chronic immunosuppression, and, or critical importance, with maintenance of protective immunity. While tolerance to kidneys has been achieved in non-human primates (NHPs) and humans with transient mixed chimerism, the same protocols have thus far failed for lungs. Importantly, however, we have recently successfully induced durable (ie stable for the life of the transplant) mixed-chimerism and tolerance during NHP lung transplantation, representing a major advance. However, this first success employed non-clinically available agents and conferred a high risk of post- transplant lymphoproliferative disorder (PTLD). These data suggest that durable chimerism-based lung transplant tolerance is possible, but that improved strategies are needed, to reset recipient immunity towards tolerance, preserve protective immunity, and enable rapid clinical translation. To address these goals, Project 2 focuses on 3 areas: 1) Developing safe, targeted bone marrow conditioning regimens for chimerism-induction; (2) Discovering Treg-supportive immunomodulation strategies; and (3) Engineering optimal CD4+/FoxP3+ regulatory T cell (Treg)- based cellular therapies, to enhance and stabilize chimerism-based immune tolerance. We do so through the following two Specific Aims: Aim 1: CD45-antibody-drug-conjugate (ADC)-based conditioning and targeted immunomodulation: the new era of mixed-chimerism induction for clinical translation. Aim 1 encompasses 3 Objectives: (1) To test the hypothesis that a CD45-ADC can successfully create marrow and immune space. (2) To test the hypothesis that Treg- supportive, tolerogenic immunomodulation, through anti-OX40L, CD137-ADC, or anti- CD154, will successfully induce durable chimerism across MHC barriers and maintain protective immunity. (3) To test the hypothesis that CD45-ADC- based chimerism and targeted immune modulation can induce lung allograft tolerance by establishing a Treg- supportive systemic and intragraft environment. Aim 2: Engineering an optimal Treg cellular therapy to augment chimerism-based tolerance induction. Aim 2 encompasses 3 Objectives designed to optimize Treg safety/efficacy for chimerism- and tolerance-induction. (1) To test the hypothesis that OX40L- or CD83-CAR-Tregs will be more suppressive than unmodified Treg, and will enhance control of inflammatory signaling, inhibit alloreactivity, and stabilize chimerism. (2) To test the hypothesis that Tregs that are base-edited to enforce FOXP3 expression and be resistant to calcineurin inhibitors, sirolimus, and/or steroids, will be maximally persistent and stable after infusion. (3) To test the hypothesis that these Tregs will optimize chimerism- based lung allograft tolerance, by reinforcing a Treg- supportive systemic and intragraft environment. Together, these Aims are designed to develop innovative approaches for durable chimerism-based lung transplant tolerance.

PROJECT NARRATIVE The current half-life of a lung allograft is unacceptably short, and all recipients encounter significant complications, both from allograft rejection and from immunosuppression-associated toxicities. Given these issues, the ultimate goal of lung transplant is immune tolerance-induction, which promises life-long graft acceptance without chronic immunosuppression, and with maintenance of protective immunity against viruses and other infections. The proposed research is highly relevant to public health because it aims to improve clinical outcomes for patients that are undergoing lung transplantation, by discovering new strategies to induce immune tolerance.