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Item Restricted The Role of Gamma Delta T Cells in Haematopoietic Stem Cell Transplantation and Malignancies(Saudi Digital Library, 2025) Alagrafi, Faisal Sultan; Michael, Uhlin; Mikael, Sundin; supervisor, Main; Thomas, PoiretAllogeneic haematopoietic stem cell transplantation (aHCT) is a potentially curative immunotherapeutic approach for several lethal hematological disorders. However, morbidity and mortality rates remain high after aHCT due to complications such as relapse, graft-versus-host disease (GVHD), and infections. Successful aHCT requires rapid and effective immune reconstitution, particularly within the T-cell compartment, to protect against opportunistic infections and to eliminate residual tumor cells without aggravating GVHD during the immunocompromised period following transplantation. Early reconstitution of γδ T cells plays a substantial role in immune surveillance post-aHCT via mediating anti-infection and anticancer immunity, correlating with favorable clinical outcomes with less GVHD incidence. These unique features have attracted increasing attention towards harnessing γδ T cells as effector cells for cancer immunotherapy. This thesis presents three papers to bring further knowledge on the role of γδ T cells in the dimension of aHCT and their potential use in hematological malignancy therapy. Study I aimed to investigate long-term homeostatic steady-state γδ T cell reconstitution after aHCT, focusing on its associations with previous clinical outcomes. We performed an in-depth analysis of γδ T cell phenotypes, TCR-Y repertoire, and functional responses upon stimulation in 20 recipient/donor pairs using multiparametric flow cytometry and next-generation sequencing of the TCR-y chain. Results showed a comparable phenotypic profile between recipients and donors. Upon PMA/lonomycin stimulation, recipient γδ T cells secreted high levels of cytokines. Furthermore, the TRG repertoire in recipients was almost completely restored, with no significant differences in diversity, clonality, or gene segment usage compared to donors. However, we found an association between overrepresented donor-derived clonotypes and elevated HLA-DR expression in Vδ1 T cells with increased severity of chronic GVHD in some recipients. Study II focused on augmenting the antileukemic activity of expanded γδ T cells by CD34/CD3 bispecific T-cell engager (BTE) in vitro. We demonstrated that the CD34/CD3 BTE effectively activates and redirects γδ T cells (effector cells) against CD34-expressing leukemia cell lines (target cells), as evidenced by their specific cytotoxicity in a dose-dependent manner and high cytokine release. Furthermore, CD34/CD3 BTE induced γδ T cell-mediated killing of primary CD34+ AML blasts. In the presence of CD34/CD3, γδ T cells showed superior antileukemic activity compared to conventional aß T cells, while demonstrating no cytotoxic effects against CD34+ normal cells. In Study III, we aimed to further enhance the antileukemic activity of expanded yo T cells by sensitizing leukemia cells with thymoquinone (TQ) treatment. TQ is a phytochemical compound featuring epigenetic activity and immunomodulatory properties with growing evidence as a potent anticancer candidate. We observed that γδ T cells exhibited rapid and increased cytotoxicity when co-cultured with pre-treated leukemia cell lines with TQ compared to vehicle control and untreated conditions. This enhanced cytotoxicity could be attributed to the upregulated expression of NKG2D and DNAM-1 ligands on leukemia cells after TQ treatment. We also showed that TQ pretreatment in leukemia cell lines supports the condition of the γδ T cells-based CD34/CD3 approach. Altogether, this thesis investigated that long-term γδ T cell reconstitution reaches a homeostatic state with a normalized repertoire. Elevated HLA-DR expression on Vδ1 T cells in cGVHD recipients could be a potential therapeutic target, warranting further investigation. It also highlights the potential use of expanded γδ T cells in hematological malignancy therapy by targeting CD34 and sensitizing leukemia cell lines through thymoquinone for CD34/CD3 BTE treatment11 0