Regulatory T cells (TRegs) are stable markers of immune functioning, acting to suppress inflammation. TRegs are important during implantation and early pregnancy where they suppress immune-mediated rejection of the embryo. Given the role of TRegs in the maintenance of pregnancy, their depletion can be associated with obstetric complications. Through the completion of two studies, this thesis seeks to identify the role of TRegs in two forms of perinatal pathology: depression and arterial thickening. The first study examines whether decreased TReg levels during pregnancy are associated with an increase in depressive symptoms, and if this relationship is mediated by maternal stress. We predicted that the TReg-depression relationship would be unique to pregnancy, and not occur in the postpartum. In the second study we assessed if decreased TRegs were inversely correlated with carotid arterial thickness. TReg samples were obtained from women between 24 and 32 weeks gestation (N=16), and at 12 weeks postpartum (N=19). Depression was assessed using the Edinburgh Perinatal Depression Scale (EPDS) and the Mongomery-Asberg Depression Rating Scale (MADRS) , and stress with the Perceived Stress Scale (PSS). TRegs were measured using flow cytometry. In the first study, we showed that lower TRegs were associated with increased levels of depression in pregnancy, and that this association was mediated by perceived stress. In the postpartum period, TRegs were not associated with changes in mood. In the second study, we found no relationship between TRegs and carotid arterial thickness. Our results suggest that TReg changes in pregnancy may be associated with maternal mood in pregnancy, but not in the postpartum period. Despite the fact that we failed to find a correlation between TRegs and carotid arterial thickness during pregnancy, our limited sample size leads us to recommend that the presence of an inverse correlation between these two markers not be ruled out, but suggest that these links be further examined using a larger sample and more precise imaging. Together, these two studies may provide very early insights into the role of TRegs in perinatal mood disorders and cardiovascular health and highlight the need for further research. / Thesis / Master of Science (MSc)

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a low platelet count (less than 100 x 10^9 platelets/L) and an increased risk of bleeding. ITP is difficult to diagnose and manage due to the deficiencies in our understanding of the pathophysiological mechanisms leading to thrombocytopenia. Anti-platelet autoantibodies are believed to be the primary mechanism of thrombocytopenia in ITP. In this thesis, I demonstrate that autoantibodies can only be detected in half of all ITP patients; therefore, other mechanisms should be investigated. CD8+ T cells have been implicated as a mechanism of disease in ITP, but platelet-specific CD8+ T cells have yet to be identified. I have characterized CD8+ T cells in ITP patients and found that platelet-specific CD8+ T cells can be detected in ITP patients. These platelet-specific CD8+ T cells can also be detected in healthy individuals, so they are not specific to ITP. However, regulatory defects were observed in ITP patients and CD8+ T cell activity was elevated in ITP patients relative to healthy individuals and thrombocytopenic non-ITP patients. Investigating whether platelet-specific CD8+ T cells can actively participate in platelet destruction and underproduction will be an essential step towards better understanding the role of CD8+ T cells as a disease mechanism in ITP, which will lead to improvements in the management of ITP. / Thesis / Doctor of Philosophy (PhD) / Platelets are small blood cells that are involved in minimizing blood loss at the site of a wound by forming a plug. In a disease called immune thrombocytopenia (ITP), patients have a low platelet count, which can result in bleeding. The bleeding symptoms of ITP decrease the quality of life for ITP patients and can be life-threatening in rare cases. It is believed that ITP is caused by proteins produced by the immune system called antibodies. I found that the antibodies that cause ITP can only be detected in half of all ITP patients. Therefore, there are probably additional causes of ITP. It is suspected that CD8+ T cells might cause ITP in some patients. CD8+ T cells are part of the immune system and they typically destroy other cells that are cancerous or infected by viruses. CD8+ T cells might also destroy healthy cells, like platelets. My goal was to characterize CD8+ T cells in order to determine their role in ITP. I found that CD8+ T cells from ITP patients can target platelets, and that healthy people have these CD8+ T cells as well. In regard to CD8+ T cells that target platelets, the difference between ITP patients and healthy people appears to be related to immune system regulation and CD8+ T cell activity. In the future, we should focus on understanding how platelet-specific CD8+ T cells can cause a low platelet count in order to improve the clinical management of ITP.

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Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. Immune system dysregulation, excessive deposition of collagen and microvascular damage in the skin and multiple internal organs are the main pathologic characteristics of the disease. Little is known about the mechanisms that are responsible for the pathogenesis of SSc. However, evidence has been accumulated demonstrating that T cells play a key role in the initiation and propagation of the disease. Previous studies in our laboratory have identified the presence of high proportions of identical β–chains TCR transcripts, demonstrating the presence of clonal expansion of T cells in skin biopsies from patients with SSc of recent onset. These T cells have undergone proliferation and clonal expansion in response to as yet unidentified antigen(s). The hypothesis that has been tested in this study is whether clonally expanded T cells in skin biopsies of patients with SSc of recent onset recognize self or non–self (possibly viral) putative SSc antigens, including DNA topoisomerase I, cytomegalovirus (CMV) and parvovirus. With the objective to identify the antigens recognized by clonally expanded T cells in skin biopsies of patients with SSc, we examined the presence of α– and β–chain TCR transcripts. Amplification of α–chain TCR transcripts by the non–palindromic adaptor PCR (NPA–PCR)/Vα specific PCR followed by cloning and sequencing revealed the presence of several clonally expanded α–chain TCR transcripts in skin biopsies from four patients with SSc and peripheral blood from one of these patients. Additionally, several clonally expanded β–chain TCR transcripts were identified in skin biopsies from all three of these patients with SSc examined, after NPA–PCR/Vβ specific amplification followed by cloning and sequencing. To identify the antigens recognized by these in vivo clonally expanded α– and β–chain TCR clones, full length α– and β– chain TCR transcripts containing the identified CDR3 regions from the clonally expanded TCR clones from the patients SSc–21 and SSc–22 were constructed. Pairs of clonally expanded, full length α– and β–chain TCR transcripts and appropriate controls were expressed in mutant TCR negative cells of the Jurkat T cell line (J.RT3–T3.5) by using a retroviral gene transfer and expression system. Each clonally expanded α–chain TCR transcript was combined with each clonally expanded β–chain TCR transcript from the same patient, generating T cells lines containing all pairing combinations of the clonally expanded TCR transcripts for each SSc patient. A total of 52 T cell lines were generated, including 10 control T cell lines. The surface expression of the TCR complex on these T cell lines was verified by flow cytometric analysis using antibodies against the α/β TCR and CD3epsilon. We employed an intracellular calcium mobilization assay to examine whether the Jurkat T cell lines transduced with the clonally expanded TCR transcripts from skin biopsies from patients with SSc (SSc–21 and SSc–22) recognize putative SSc antigens or their peptides presented by autologous EBV–transformed B cell lines. The putative SSc antigens that were tested are the self–antigen, DNA topoisomerase I and the viral antigens, cytomegalovirus and parvovirus which have been previously suggested to be involved in the pathogenesis of SSc. Significant intracellular calcium mobilization was observed in response to 3 DNA topoisomerase I and 2 CMV peptides by 5 T cell lines transduced with clonally expanded α– and β–chain TCR transcripts from patients SSc–21 and SSc–22. / Microbiology and Immunology

BACKGROUND: Patients with relapsed or refractory large B-cell lymphoma (LBCL) who have relapsed after at least 2 lines of therapy had a poor prognosis before the introduction of chimeric antigen receptor (CAR) T-cell therapy. The FDA approved three CD19 CAR T-cell products, axicabtagene ciloleucel (axi-cel), tisagenlecleucel, and lisocabtagene maraleucel (liso-cel), based on the results of pivotal phase 2 clinical trials. High response rates and long-term remissions in these multiply relapsed patients led to randomized trials as a second-line therapy against the current standard of care for primary refractory and early relapsing LBCL. Axi-cel and liso-cel are now approved as second-line treatments for patients with relapsed or refractory large B-cell lymphoma based on these trials, while tisagenlecleucel failed to improve upon second-line standard of care. This has led to greater axi-cel and liso-cel usage as compared with tisagenlecleucel. Clinical trials and real-world trials show a higher toxicity profile for axi-cel as compared to liso-cel with similar efficacy outcomes, leading to selection of liso-cel for older patients with more medical comorbidities. However, axi-cel manufacturing is faster and more reliable making it a preferred choice for rapidly progressive lymphomas. No direct comparison has been made between the two in order to optimally inform product selection. OBJECTIVE: We aimed to compare the toxicity profile and efficacy outcomes between two cohorts, one treated with axi-cel and the other with liso-cel, ideally well matched, during the same period of time. METHODS: We retroactively gathered patient data for patients treated between June 2021 to September 2022 with both products. We compared the cohorts for patient characteristics that are proven to affect the toxicity and efficacy in order to identify significant differences that could influence our results and to increase the likelihood that the two cohorts were well matched. We then assessed associated toxicities and long-term efficacy outcomes. RESULTS: The two cohorts were comparable for all patient and disease variables other than age (median age of 62 years old in axi-cel compared to 71 years old liso-cel [p < 0.001]). There was no significant difference between high-grade cytokine release syndrome (CRS) (3% vs 5% for axi-cel vs. liso-cel cohorts, respectively; p = 0.58), high-grade immune effector cell-associated neurotoxicity syndrome (ICANS) (18% [ASTCT] or 19% [CTCAE], 14% [ASTCT] or 12% [CTCAE] for axi-cel vs. liso-cel cohorts, respectively, p = 0.055). There were higher rates of any grade CRS with axi-cel, and duration of hospitalization was longer for axi-cel vs. liso-cel (10 vs. 14 days, respectively). Best overall response rates (ORR) (93% vs. 84% axi-cel vs. liso-cel, respectively) and complete response (CR) rates (71% vs. 56% axi-cel vs liso-cel, respectively) did not statistically differ between the two groups. 12-month overall survival (OS) (76% vs. 81% axi-cel vs. liso-cel, respectively) and progression free survival (PFS) (61% vs. 45% of patients axi-cel vs. liso-cel, respectively) did not statistically differ between the two groups (p =0.94, p =0.51 for OS and PFS, respectively). CONCLUSIONS: Our study showed both products are similar in their high-grade toxicity profile as well as their efficacy. While axi-cel has more any grade CRS and ICANS, the lack of significantly higher high-grade toxicities likely reflects better and more aggressive toxicity mitigation strategies when patients present with low grade side effects. As a result, axi-cel in our study was found to be less toxic than previously seen in past clinical trials as well as real-world studies. Many factors go into selection of a CAR T-cell product, ranging from product performance attributes like safety and efficacy, to product manufacturing qualities like turnaround time and fidelity of manufacturing. With equivalency with regards to product performance, manufacturing qualities may then be most important in guiding product selection for LBCL patients.

Glioblastoma is a deadly brain cancer with discouraging standard of care. New methods like convection enhanced delivery and chimeric antigen receptor T cells (CAR-T) are promising treatments that can be translated to glioblastoma. In this study, CAR-T cell flow through a hydrogel was explored in the context of in-vitro convection enhanced delivery. A culture method to create large spheroids mimicking tumors from preexisting glioblastoma stem cell lines was fabricated, a convection enhanced delivery system for in-vitro testing was designed, and characterization of the CAR-T cells using the in-vitro system took place. The spheroid culture method was successfully optimized to produce spheroids large enough to act as a sufficient tumor in little time, the in-vitro set-up successfully administered treatment, and CAR-T cells were found to increase their velocities through a medium as their injection velocity increased. It was discovered that the density of the spheroid plays a crucial role in treatment delivery, often times driving how treatment will move through the spheroid. This system can be used in the future studies to test the killing potential of CAR-T cells to a tumor in-vitro. / Master of Science / Glioblastoma is a deadly brain cancer with current treatments that are discouraging at best. New methods must be utilized to aid in patient recovery. Chimeric antigen receptor T-Cells (CAR-T) are a promising treatment that can be used in glioblastoma. In this study, CAR-T cell behavior is defined in the context of in-vitro convection enhanced delivery. A large spheroid, or sphere of cells, mimicking a tumor was created, a convection enhanced delivery system set-up for in-vitro testing was designed, and characterization of CAR-T cell behavior using the in-vitro system took place. The spheroids were successfully cultured to act as a sufficient tumor, the in-vitro set-up successfully administered treatment, and CAR-T cells were found to increase their velocities in a gel as their injection velocity increases. It was discovered that the density of the spheroid plays a crucial role in treatment delivery, often times driving how treatment will move through the spheroid. This system can be used in the future studies to test the killing potential of CAR-T cells to a tumor in-vitro.