Introduction: Manipulation of the immune system to eliminate cancer, known as cancer immunotherapy, is an emerging field that has shown impressive clinical success and promise. Adoptive transfer of T cells engineered for tumor reactivity is an avenue of therapy for patients with previously untreatable disease. Our lab has developed a novel chimeric receptor, called a T cell antigen coupler (TAC), which redirects T cell cytotoxicity towards a tumor target. Although considerably effective, this receptor does not provide T cell costimulation necessary for optimal anti-tumor effectiveness. Methods: We explored two methods to deliver costimulation to TAC-engineered T cells. First, we designed a receptor to be utilized in conjunction with the TAC in a dual receptor system. This chimeric costimulatory receptor (CCR) was generated by fusion of the T cell TIGIT and CD28 receptors. In our second approach, we investigated direct incorporation of costimulatory domains into the TAC design. To do so, we substituted in regions from the CD28 or 4-1BB costimulatory receptors. Results: Three TIGIT/CD28 chimeras were successfully generated. Of these, two were well surface-expressed on primary human T cells. Despite testing of these receptors in several biological assays, we were unable to confirm functionality of these receptors in transmitting CD28 signals. We next generated the 4-1BB and CD28TAC variants. The 4-1BBTAC was poorly surface-expressed M.Sc Thesis – Arya Afsahi McMaster University – Medical Sciences iv and was difficult to introduce into T cells at high efficiency. The CD28TAC-variant was virtually absent from the T cell surface membrane. Further analysis indicated that the CD28TAC was retained in the endoplasmic reticulum (ER) and the 4-1BBTAC was produced at an extremely low amount. Conclusions: Our investigation into delivery of costimulation through a novel CCR or TAC receptor was inconclusive. We recommend several optimizations to both receptor design and experimental analysis to further elucidate the potential of these receptors. / Thesis / Master of Science (MSc)

<p>T cells and B cells each express unique antigen receptors used to identify, eliminate, and remember pathogens. These receptors are generated through a process known as V(D)J recombination, in which T cell receptor and B cell receptor gene loci undergo genomic recombination. Interestingly, recombination at certain genes is regulated so that a single in-frame rearrangement is present on only one allele per cell. This phenomenon, termed allelic exclusion, requires two steps. First, recombination can occur only on one allele at a time. In the second step, additional recombination must be prevented. Though the mechanism of the second step is well-understood, the first step remains poorly understood.</p><p>The first step of recombination necessitates that alleles rearrange one at a time. This could be achieved either through inefficient recombination or by halting further recombination in the presence of recombination. To separate these mechanisms, we analyzed recombination in nuclei unable to complete recombination. We found that rearrangement events accumulated at antigen receptor loci, suggesting that the presence of recombination does not stop additional rearrangements and asynchronous recombination likely results from inefficient recombination at both alleles.</p><p>Association with repressive subnuclear compartments has been proposed to reduce the recombination efficiency of allelically excluded antigen receptor loci. Of the alleleically excluded loci, <italic>Tcrb</italic> alleles are uniquely regulated during development. Other allelically excluded alleles are positioned at the transcriptionally-repressive nuclear periphery prior to recombination, and relocate to the nuclear interior at the stage in which they recombine. However <italic>Tcrb</italic> alleles remain highly associated with the nuclear periphery during rearrangement. Here we provide evidence that this peripheral subnuclear positioning of <italic>Tcrb</italic> alleles does suppress recombination. We go on to suggest that peripheral localization mediates the first step of allelic exclusion.</p><p>In search of the mechanism by which recombination is suppressed on peripheral <italic>Tcrb</italic> alleles, we investigated the subnuclear localization of a recombinase protein. Two recombinase proteins are required for recombination, one of which is recruited to actively transcribing (and more centrally located) DNA. Here we demonstrate that one recombinase protein is unable to localize to peripheral <italic>Tcrb</italic> alleles, potentially serving as the mechanism by which recombination is suppressed on peripheral alleles.</p> / Dissertation

Anaplastic Large Cell Lymphoma (ALCL) is a peripheral T cell lymphoma divided into three distinct entities: ALCL, Anaplastic Lymphoma Kinase (ALK)+, ALCL ALK- and cutaneous ALCL. In the majority of ALCL, ALK+, ALK is expressed as the result of a chromosomal translocation generating Nucleophosmin 1(NPM)-ALK, which is considered the main driver. ALCL have an unusual immunophenotype; they rarely express a T cell receptor (TCR), but are often positive for CD4 and produce cytotoxic proteins such as perforin and Granzyme B, but in the absence of CD8, questioning the origin and pathogenesis of this malignancy. Expression of NPM-ALK in mice from the T-cell specific CD4 promoter gives rise to thymic lymphomas not modelling human ALCL suggesting that other events and/or expression of NPM-ALK at a defined stage of T cell ontogeny is required for peripheral T cell lymphoma development. Indeed, back-crossing the CD4/NPM-ALK line onto a RAG competent, MHC class I restricted ovalbumin-specific TCR, OTI transgenic line (CD4/NPM-ALK/OTI) permits peripheral lymphoma development mimicking human ALCL (but CD4/NPM-ALK/OTII mice still develop thymic lymphoma); tumours contain cells histopathologically identical to ALCL hallmark cells. Interestingly, peripheral tumours developing in this model also lack cell surface expression of the OTI TCR in fitting with observations of a lack of TCR expression on human ALCL. It follows that stimulation of T cells in vivo by infection with MHV-ova prevents lymphomagenesis suggesting that the TCR is detrimental to tumour growth. Indeed, strong stimulation via the TCR of NPM-ALK-expressing primary T cells in vitro, impedes cell proliferation but cell growth is favoured when a weaker stimulus is employed. Overall, data presented in this thesis identifies a potential mechanism of lymphomagenesis accounting for the unusual immunophenotype of ALCL and an explanation as to why cells lack a TCR and associated proximal signaling.

No description available.

CD4+ and CD8+ T cells are critical components of the adaptive arm of immune responses. During viral infection, CD8+ T cells utilize their cytotoxic function to kill infected cells and clear the infection. In addition, CD4+ T cells differentiate into either T helper 1 (Th1) or T follicular helper (Tfh) cells, which provide essential help to enhance the efficacy of other response immune cells, including macrophages, CD8+ T cells, and B cells. The transcription factor, T cell factor-1 (TCF1), and its homologue, Lymphoid enhancer-binding factor-1 (LEF1), have critical roles in the development, differentiation, and persistence of both CD4+ and CD8+ T cells. However, the influence of TCF1 and LEF1 on Th1 and Tfh differentiation remains to be examined. Furthermore, due to alternative promoter usage, TCF1 and LEF1 are expressed as both long and short isoforms. The distinct roles of the long and short isoforms of TCF1 in the context of CD4+ and CD8+ T cell responses have not been defined. My studies utilized multiple novel mouse strains to examine the roles of TCF1 and LEF1 in Tfh and Th1 differentiation during viral infection, and the unique requirements of TCF1 long isoforms in CD4+ and CD8+ T cell responses. Specifically, my initial studies characterized a new TCF1 reporter construct (referred to as p45GFP reporter) and used this reporter to address the specific contributions of TCF1 long isoforms to the CD8+ T cell response. Previous studies have abrogated all TCF1 isoforms and shown that in the absence of TCF1, the memory CD8+ T cell population is dramatically impaired and exhibits defective persistence over time. Here, I showed that TCF1 short isoforms are sufficient for the generation of memory CD8+ T cells, however TCF1 long isoforms are important for the maturation of memory CD8+ T cells. Another critical component of pathogen clearance and long-term protection is a productive humoral response, which is optimized by the B cell help provided by Tfh cells. Using the p45GFP reporter, I showed that TCF1 is specifically retained in Tfh cells, but downregulated in Th1 cells. I utilized a huCd2-Cre system to conditionally delete TCF1 and LEF1 in mature T cells. In response to viral infection, TCF1 and LEF1 double-deficient mice showed normal Th1 responses, but severely defective Tfh differentiation and a concomitant impaired B cell response. I further demonstrated that TCF1 promotes Tfh differentiation by directly regulating many Tfh-associated genes. Furthermore, I used the p45GFP reporter to I identified distinct, but critical, roles for both long and short isoforms of TCF1 in driving Tfh differentiation and repressing differentiation toward Th1 or germinal center Tfh cells. Finally, while TCF1 is known to be critical in the formation of memory CD8+ T cells, its impact on memory CD4+ T cell generation has not been assessed. Once again utilizing the p45GFP reporter, my studies identified an important role for TCF1 long isoforms in the survival of both Th1 and Tfh cells through contraction. In the absence of TCF1 long isoforms, the memory CD4+ T cell population is severely reduced. Taken together, my work has demonstrated critical roles for TCF1 during both effector and memory phases of the CD4+ T cell response to viral infection. In summary, TCF1 is crucial for CD4+ T cells to effectively differentiate and provide important help to B cells during viral infection. Moreover, my studies have identified critical and unique roles for long and short isoforms of TCF1. Finally, TCF1 is necessary for optimal formation of memory CD4+ and CD8+ T cells, and thus is an essential component in achieving protective immunological memory after viral infection.

In the last decade, six CAR T cell therapies against hematological malignancies have been approved for commercial manufacturing and several clinical trials are underway. This has led to extensive preclinical research focused on optimizing individual manufacturing steps of adoptive T cell therapeutics. Ex vivo expansion of T cells is one of the crucial manufacturing steps, as is necessary to obtain clinically required cell numbers for infusion. However, ex vivo expansion is also a complex step as it involves multiple different variables including culture medium, serum and cytokine supplementation, activation reagent and mode of genetic modification. Consequently, our understanding of changes in T cells during ex vivo expansion and the impact of expansion conditions on the final product; and thus the outcome of the therapy remain mostly elusive. Therefore, this project was designed to understand the changes in T cells at different stages of ex vivo expansion compared to freshly isolated T cells with a focus on understanding the ongoing transcriptional changes. The T cells were isolated from healthy blood donor buffy coats using FABian®-Cell Isolation System based on Fab-TACS technology. T cells were cultured for 7 days in X-VIVO 15 media (supplemented with 5% human serum and 50 IU/ml IL-2) with activation for initial 3 days using anti-CD3/CD28 TranAct. The T cell kinetics during ex vivo expansion was characterized based on cell activation, differentiation and proliferation. For whole transcriptome sequencing, Total RNA was harvested from 6 different time points, freshly isolated cells (0 hr), and cells cultured for 4, 12, 24, 72 and 168 hr (7 days). The RNA sequencing libraries were prepared using “Illumina TruSeq Stranded Total RNA library prep' workflow and whole transcriptome sequencing was performed on Illumina Novaseq 6000. Further, changes in T cell trafficking capabilities, cell size and cell cycle progression were studied in freshly isolated T cells and cultured cells. The changes in T cell trafficking was studied by analyzing the changes in VLA4 mediated T cell adhesion to VCAM1 coated surface under increasing shear stress. The cell size and volume of freshly isolated T cells and cultured cells were analyzed using multisizer instrument. Additionally, an in vitro model was developed to simulate the behavior of cultured T cells upon re-infusion into the blood and changes in cell cycle was analyzed. The components of in vitro reconstituted blood model were pooled human AB serum, erythrocyte concentrates and cultured T cells. The absolute lymphocyte count in buffy coats and total number of T cells isolated per buffy coat were in range compared to cell isolation and enrichment through standard leukapheresis. Thus suggesting that healthy donor-derived buffy coats and enrichment of T cells using Fab-TACS technology can be a suitable starting material and cell enrichment device respectively. The T cell growth kinetics was analyzed based on surface expression of specific markers, which also closely resembled their gene expression. The T cell kinetics observed during ex vivo expansion was similar to T cell kinetics observed in several preclinical CAR T cell expansion studies. The T cell proliferation in terms of increase in cell numbers and gene ontology (GO) terms related to DNA replication and cell division were significantly enriched only after 3 days of ex vivo expansion. The final cell numbers after 7 days of ex vivo expansion were approx. 1.0E+9 T cells, which was well above the clinically required infusion dosage of currently approved CAR T cell therapies. Taken together, the ex vivo expansion protocol followed in this study generates T cells in range required for clinical infusion dose and the growth kinetics of T cells observed were in line with the commercial expansion protocol. Hierarchical clustering of genes based on their expression over time identified 29 different gene-clusters which followed the pattern of mono-, bi- and triphasic modulation. The gene-clusters 11 and 18 were significantly enriched with T cell immune function related GO terms. The GO analysis of differentially expressed genes identified enrichment several bioprocesses, signaling pathways and T cell immune functions including commonly known activation, differentiation and proliferation. The ex vivo expansion of T cells was associated with early (i.e. upto 24 hr time point) enrichment of several GO terms associated with cytokine production such as IL-1, IL-2, IL-5, IL-6, IL-10, IL-13, IL-17, TNF and IFN-γ. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) and Mitogen-activated protein kinase (MAPK) signaling cascades related GO terms were enriched as a result of autocrine signaling mediated by cytokines released during ex vivo expansion. These data demonstrate that cytokine release in T cells is activated during ex vivo manufacturing, and should be considered during future optimization. The gene expression analysis of commonly known exhaustion markers revealed two different patterns of expression. The CTLA4, TIGIT, TBX21 and BATF was upregulated at early time points. Whereas, the expression of TIM3, LAG3 and CX3CR1 was upregulated at later time points. The early expression of exhaustion markers can be attributed to immune check point function to prevent over-activation, and later expression of exhaustion markers may contribute to inhibitory function. In vitro investigation of ex vivo expanded T cells exhibited stronger VLA4 mediated adhesion to VCAM1 coated surface compared to freshly isolated cells under increasing shear stress. This was in contrast to the downregulation of alpha 4/beta 1 integrin gene expression during ex vivo expansion. The cell size analysis revealed cultured T cells were larger in terms of both size and volume at the end of 7-day culture period with doubled cell volume compared to freshly isolated T cells. These results taken together, suggest that increased adhesion capacity and increased cell size, after T cell expansion may be associated with accumulation of T cells in lungs upon infusion. The freshly isolated T cells that closely represent the T cells circulating in peripheral blood were arrested in G0/G1 phase. However, during ex vivo expansion T cells entered cell cycle, and T cells were found to be predominantly in S+G2/M phase on day 3, 5 and 7. Surprisingly, the cultured T cells were still in cell cycle even after 48 h of incubation in reconstituted blood in vitro. This suggests that a prolonged resting phase of ex vivo expanded T cells for more 48 hr before infusion into the patients can be advantageous in minimizing the risks associated with T cell therapy. In conclusion, this study has revealed a number of novel insights into transcriptional regulation and signaling processes occurring during culture expansion. In the study, the different patterns of transcriptional regulation and enrichment of various associated bio-processes and signaling pathways during ex vivo expansion were explored. In addition, an in-depth analysis of genes related to T cell activation and differentiation, adhesion and migration, and exhaustion markers was performed. The protein-protein interaction analysis and transcriptional factor enrichment analysis provide valuable data for further in silico investigations of transcriptional changes in T cells during ex vivo expansion. Additionally, this study provides a comprehensive overview of long non-coding RNAs at different stages of ex vivo expansion of T cells, thus providing a resource for novel understanding of impact of lncRNAs on T cells during ex vivo expansion for adoptive T cell therapies. The complete data of 48 transcriptomes derived from 8 donors over 6 time points is reposited (GEO: GSE250311) to a publicly available database and will allow exploration for future studies which aim at the characterization of alterations in expanded T cells for therapy, and optimization of conditions for their future use in patients.

CD4⁺ T cells are a critical component of the adaptive immune compartment. Each T cell expresses a clonotypic T cell receptor (TCR) that must discriminate between self and foreign peptides presented in major histocompatibility molecules (pMHC) on the surface of antigen presenting cells to direct T cell fate decisions. Information regarding TCR-pMHC interactions must then be transmitted to the TCR-associated CD3 signaling modules, which contain ITAMs that serve as signaling substrates for Src kinases. The Src kinase, Lck, is recruited to the pMHC-bound TCR-CD3 complex via association with the CD4 coreceptor that binds MHCII. It is therefore through the coordinated interactions within the TCR-CD3-pMHC-CD4 macro-complex that productive TCR signaling can occur to inform T cell activation and fate decisions. While much is known regarding the structure of the individual subunits that make up the TCR-CD3-pMHC-CD4 macro-complex, there is little information regarding how these components come together to initiate TCR signaling and determine functional outcomes. Here, we have interrogated how interaction of these individual components leads to productive T cell activation. Specifically, we interrogated the nature of TCR-MHC interactions and provide evidence that there is intrinsic specificity of the TCR for MHCII. We have also built mouse models to determine the role of TCR-CD3 interactions and TCR dimerization in the transmission of information from the TCR to the CD3 subunits following TCR-pMHC engagement. Finally, we show that both the CD4 transmembrane and extracellular domains contribute to T cell activation in vitro. Overall, this work provides insight into how the constituents of the TCR-CD3-pMHC-CD4 macro-complex interact to initiate T cell fate and function.

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