Bolivar Wagers, Sara2022-11-142022-11-142022-08https://hdl.handle.net/11299/243074University of Minnesota Ph.D. dissertation. August 2022. Major: Microbiology, Immunology and Cancer Biology. Advisors: Bruce Blazar, Bryce Binstadt. 1 computer file (PDF); ix, 175 pages.Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be a curative option for many hematological disorders. Unfortunately, allo-HSCT efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality after transplant. Current GVHD prophylaxis leaves 30-70% of patients at risk of GVHD resulting in susceptibility to infections, relapse and secondary malignancies. Thus, novel therapies for GVHD suppression are warranted. Regulatory T-cells (Tregs) have shown efficacy in preclinical and clinical studies at preventing GVHD, but clinical use is limited by variable suppression at high doses. Here, we demonstrated two strategies that can be used to augment Treg cell therapy efficacy in an effort to reduce GVHD incidence and severity. First, we generated antigen specific Tregs via retroviral transduction with a construct to express an anti-human CD19 chimeric antigen receptor (hCAR19). hCAR19 Tregs were infused into lethally irradiated hCD19 hemizygous transgenic (hCD19TGTg/0) recipients following allo-HSCT. We found that recipients receiving hCAR19 Tregs as compared to control transduced Tregs had a significant decrease in acute GVHD (aGVHD) lethality and severity. Interestingly, we found that the graft-versus-tumor (GVT) response using hCD19 TBL12 lymphoma cells was not only preserved but was potentiated in hCD19TGTg/0 mice receiving hCAR19 Tregs. This effect was achieved as hCAR19 Tregs were shown to kill syngeneic hCD19+ but not hCD19- murine TBL12luc cells in vitro in a perforin-dependent, granzyme B-independent manner. With cytolytic potential, we evaluated whether hCAR19 Tregs had the potential for induction of cytokine release storm (CRS) as is seen with CD8 CAR T-cells. Cyclophosphamide treated hCD19TGTg/0 mice given hCAR19 cytotoxic T- lymphocytes experienced rapid lethality due to systemic toxicity, whereas hCAR19 Tregs avoided this severe complication. CAR19 Tregs are a novel and effective strategy to suppress GVHD while not only maintaining but also potentiating GVT responses. Secondly, we aimed to expand Tregs in vivo using an orthogonal IL-2Rβ (oIL2Rβ) that would selectively interact with its orthogonal IL-2 (oIL-2) cytokine and not with its wildtype counterpart. We used a murine MHC-disparate GVHD model to test whether the orthogonal system would preferentially drive donor Treg expansion and allow for GVHD suppression using lower numbers of Tregs. We found that recipient mice injected with oIL2Rβ Tregs and injected with oIL2 compared to PBS had enhanced GVHD survival with an associated significant Treg expansion and Tconventional (Tcon) suppression. Importantly, enhanced GVHD suppression did not limit the GVT response when tested against two distinct lymphoma and leukemia cell lines. These data support the novel use of this orthogonal system to enhance Treg cell therapies in the allo-HSCT setting. Overall, these studies demonstrated the mechanistic underpinning of two novel Treg strategies for GVHD suppression with GVT maintenance.enHASH(0x3fa7eb8)Thesis or Dissertation