Pathways and mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a novel, multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naïve, central and effector memory, and terminal effector subsets is contingent on both differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is dependent on CD4+ or CD8+ T cell lineage, subset differentiation and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting new pathways for human T cell fate determination and homeostasis. Memory T cells can remain in tissues as non-circulating, resident memory populations, which provide optimal protection against infection at barrier surfaces. Lung-resident memory T cells (TRM) mediate in situ protection to respiratory pathogens, though mechanisms for their maintenance and retention are unknown. Through whole transcriptome profiling, we identify a cohesive network of genes enriched in lung CD4+ TRM, including Itgad (CD11d), Cd69, and IFN-associated responders. We find that upregulation of CD11d enhances CD69 expression through type I IFN signaling downstream of homotypic cell adhesion, and is required for optimal T cell differentiation and lung retention. Moreover, blockade of IFNαR1 reduces CD11d expression and retention of influenza-generated lung TRM, suggesting that CD11d-dependent type I IFN signaling promotes TRM establishment. Our results implicate CD11d and type I IFN in retaining lung CD4+ TRM cells, and identify potential targets for modulating tissue immunity.

The study of B and T cell receptor repertoires from high throughput sequencing is a recent development that allows for unprecedented resolution and quantification of the adaptive immune response. The immense diversity and long tailed distribution of these repertoires has up until now limited such studies to expanded clonal signatures or to analysis of imprecise signals with limited dynamic range collected by techniques such as radioactive and fluorescent labeling. This thesis presents a number of quantitative methods to characterize the repertoire and examine the questions of sequence diversity and inter-repertoire divergence of T cell repertoires. These approaches attempt to accurately parametrize the inherent distribution of T cell clones drawing from statistical tools derived from ecological literature and information theory. The methods presented are applied to T cell analyses of various tissue compartments of the human body, including peripheral blood mononucleocytes, thymic tissues, spleen, inguinal lymph nodes, lung lymph nodes and the brain. A number of applications are explored with strong implications for translational use in medicine. Novel insights are made into the mechanism of maintenance and compartmentalization of na{\"i}ve T cells from human donors of many different ages. Diversity and divergence of the tumor infiltrating sequence repertoire is measured in low grade gliomas and glioblastomas from cancer patients, and potential sequence based biomarkers are assessed for studying glioma phenotype progression. A careful investigation of the immune response to allogeneic stimulus reveals the effect of HLA on sequence sharing and diversity of the alloresponse, and quantifies for the first time using sequence data the fraction of T cells in a repertoire that are alloreactive. The use of repertoire sequencing and mathematical models within immunology is a new and emerging concept within the rapidly expanding field of systems immunology and will undoubtedly have a profound impact on the future of immunology research. It is hoped that the tools presented in this thesis will give insight into how to quantitatively explore the breadth and depth of the T cell receptor repertoire, and provide future directions for TCR repertoire analysis.

Memory T cells are critical for maintaining lifelong immunity by protecting against reinfection with previously encountered pathogens. In recent years, a subset of memory T cells termed tissue-resident memory T cells (TRM) has emerged as the primary mediator of protection at many tissue sites. Numerous studies in mice have demonstrated that TRM accelerate pathogen clearance compared with other subsets of memory T cells. The defining characteristic of TRM is that they are retained within tissues and do not circulate in the blood. The lack of TRM in blood has proved to be a barrier for investigating the role of TRM in healthy humans. As a result, there are many outstanding questions about TRM biology in humans, including which phenotypic markers identify TRM, if TRM represent a unique memory subset, as well as defining transcriptional and functional characteristics of this subset. Through a unique collaboration with the local organ procurement agency, we obtained samples from >15 tissue sites from healthy organ donors of all ages. We found that the surface marker CD69 was expressed by memory CD4+ and CD8+ T cells in multiple tissues including spleen and other lymphoid tissues, lung, and intestines, but not in blood, suggesting that this marker may identify TRM in human tissues. We identify a core transcriptional signature that distinguishes CD69+ memory T cells in tissues from CD69- memory T cells in tissues and blood with key homologies to the transcriptional profile of TRM in mice, suggesting that CD69 expression identifies TRM in humans. We show that human TRM have a distinct profile of adhesion and migration markers, and a unique dual functional capacity encompassing effector cytokine production but also the upregulation of inhibitory markers and the ability to produce IL-10 upon stimulation. These results suggest unique adaptations for TRM to maintain long-term residence within tissues and carry out pathogen clearance. We found substantial heterogeneity within human TRM in lymphoid and mucosal tissue sites, including a substantial fraction (40-60%) of TRM in various human tissues with the ability to efflux fluorescent dyes. These efflux(+) TRM had phenotypic and transcriptional characteristics associated with quiescence, including expression of immunomodulatory markers, reduced expression of exhaustion markers, and reduced turnover at steady state. Upon TCR stimulation, efflux(+) TRM produced lower levels of proinflammatory cytokines and cytotoxic molecules but had a superior ability to proliferate compared with efflux(-) TRM. However, efflux(+) also had an enhanced capacity for IL-17 production along with transcriptional features of IL-17 signaling following stimulation. Overall, these studies establish universal properties of human TRM and hint at the function of distinct TRM subsets in mediating tissue immunity.

T cells are key agents in the adaptive immune response, responsible for robust and selective protection of the body against foreign pathogens. T cells are activated through their interaction with antigen-presenting cells (APCs) via a dynamic cell-cell interface called the immune synapse (IS). Numerous studies in recent years have shown that T cell activation is a mechanoresponsive process. Modulation of substrate rigidity and topology are emerging as powerful tools for controlling T cell activation. However, the majority of systems used to investigate the IS have used substrates that lack the rigidities and topographical complexities inherent in the physiological T cell - APC interface. Circumventing these limitations, elastomer micropillar arrays can be fabricated with physiologically-relevant rigidities and provide a topographically-deformable activating substrate. In this thesis, we examine the mechanisms behind T cell mechanosensing in order to gain a more complete understanding of T cell activation. More specifically, we take advantage of micropillar substrate properties to examine the IS in both 2D and 3D, seeking new insights into how the structural and mechanical features of the IS modulate T cell activity. We first investigate the traditional paradigm of T cell force generation at the 2D IS by seeking to characterize the temporal relationship between TCR signaling and force generation. We find that in both mouse naive and preactivated CD4+ T cells, TCR signaling is robust, dynamic, and localized to the pillar features. However, no temporal correlation is found between signaling and force generation. A potential reason for this lack of correlation is recent research showing that the physiological IS is a 3D interface that is topographically dynamic. This phenomenon complicates our interpretation of the 2D IS, as our micropillar system is protrusion-inducing substrate. In order to investigate the implications of topographical cues, we then characterize T cell activation in the 3D IS with respect to force generation and cytoskeletal development over time. We demonstrate that preactivated CD4+ T cells exhibit a dynamic and robust penetration into micropillar arrays. In the 3D IS, actin polymerization is again not correlated with force generation, but we find that microtubules (MTs) have a critical role in 3D T cell mechanosensing. Namely, MT architecture is correlated with the spatial distribution of force generation in the 3D IS, the centralization of microtubule-organizing center (MTOC) to the 3D IS is a mechanosensitive process that is modulated by surface rigidity, and while MT polymerization is not necessary for force generation, it is critical for maintaining synaptic integrity over time. Together, this work reveals important aspects of the underlying dynamics of the T cell cytoskeleton in IS formation and maintenance. The conclusions will help advance the concept of mechanobiology in immunology, which may in turn be leveraged towards the development of biomaterials that enhance T cell manufacturing in adoptive cell therapy.

Mechanisms for human memory T cell differentiation and maintenance have predominantly been inferred from studies of peripheral blood, though the majority of T cells reside in lymphoid and non-lymphoid sites. Studies in mice have shown that memory T cells in non-lymphoid sites provide superior protection to pathogens compared to those in blood, defining a subset known as tissue-resident memory T cells (TRM), with emerging roles in lymphoid sites. There are many key unknown aspects of TRM biology in human tissues including if TRM have superior functional abilities, the mechanisms for maintenance of TRM in lymphoid and non-lymphoid sites, and the relatedness of tissue and blood localized T cell subsets. Through a collaboration with the local organ procurement agency, we obtained samples from >15 tissue sites from healthy organ donors of all ages. We analyzed CD8+ T cells in diverse sites and found the majority of TRM cells in lymph nodes (LNs) display an increased proliferative capacity, increased expression of TCF-1, and decreased turnover compared to TRM and effector memory (TEM) cells in other sites including blood, bone marrow (BM), spleen and lung. Further, we identified that exposure to type 1 interferons results in increased downregulation of TCF-1 expression during cell divisions driven by T cell receptor (TCR) stimulation. We investigated the relatedness of CD4+ and CD8+ T cell subsets, including central memory (TCM), effector memory (TEM), TRM, and terminal effectors (TEMRA) by sequencing TCR rearrangements. From diversity analysis of TCR repertoires we found that effector and memory subsets are maintained in a hierarchy from most to least diverse (TCM > TEM and TRM > TEMRA) that is largely conserved across tissues and CD4+ and CD8+ T cell lineages. Overlap analysis revealed the low and high relatedness of TCM and TEMRA cells respectively and this was highly conserved across tissues; in contrast, we found the relatedness of TEM and TRM was more dynamic across tissues. Together, these findings have implications for immune monitoring and modulation, highlighting that lymph nodes may function as reservoirs for long-lived memory T cells with high functional capacity; additionally, we identify cell extrinsic signals that regulate tissue-specific maintenance of T cell memory in lymph node sites.

Influenza is a significant cause of morbidity and mortality worldwide. Individuals with underlying immune conditions, including the very young, are particularly vulnerable. Infection elicits lasting antibody and T cell-mediated immune responses although antibody-mediated protection is limited due to the mutagenic nature of influenza viral surface antigens. T cell responses, in contrast, target conserved viral proteins and can protect from highly disparate strains. Compared to circulating memory, non-circulating, lung tissue-resident memory T cells (TRM) generated following influenza infection mediate enhanced viral clearance and protection following challenge. Thus, vaccination strategies promoting TRM may convey enhanced protection from disease compared to those relying on circulating responses. The factors governing TRM generation, however, are unclear and whether individuals most susceptible to infection, such as the very young, generate functional TRM is not known. This body of work investigates the nature of T cell responses and TRM establishment following influenza vaccination and infection in early life and adulthood. We have identified distinct capacities of commercially available inactivated influenza virus (IIV) and live-attenuated influenza virus (LAIV) vaccines to elicit protective responses with IIV inducing strain-specific neutralizing antibodies and LAIV generating lung-localized, virus-specific TRM capable of providing heterosubtypic protection upon viral challenge. We have further found that infants generate robust primary T cell responses following influenza infection or LAIV vaccination comparable to adults. However, mice infected or vaccinated in infancy fail to efficiently generate TRM and are less protected from subsequent infection in adulthood. We have identified enhanced expression of T-bet, known to promote effector differentiation while limiting memory T cell establishment, by primary infant effectors and further demonstrate that heterozygous infants expressing reduced T-bet generate lung TRM comparable to adults. Together, these findings have implications in influenza vaccine design, highlighting differing mechanisms of protection between IIV and LAIV, establishing TRM as a correlate of vaccine-mediated protection to influenza, as well as identifying cell-intrinsic dysregulation of a transcriptional pathway early in life necessary for effective lung TRM generation.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children. RSV induces variable disease severities in infected children. Severe cases of RSV-induced disease result in bronchiolitis, with a subset of children going onto develop long-term airway morbidities. The host antiviral T cell response is believed to contribute to the severity of pulmonary disease following acute RSV infection. However recent work has questioned the relative proportion of T cells that migrate into the lung tissue following a respiratory virus infection. Using in vivo intravascular antibody labeling, >80% of antigen-specific effector T cells were found to remain in the pulmonary vasculature following an intratracheal infection with the systemic viral pathogen lymphocytic choriomeningitis virus (LCMV). Therefore, I determined the proportion of RSV-specific CD4 T cells located within the lung tissue following infection. In contrast to recent reports with LCMV-specific CD8 T cells, I found approximately 85% of RSV-specific CD4 T cells were located within the lung tissue, indicating that the vast majority of virus-specific effector CD4 T cells are located within the lung tissue and not in the pulmonary vasculature following an acute RSV infection. Genetic variations can occur in the circulating RSV strains both within and between infectious seasons. Therefore, I questioned if different RSV strains could induce differential CD4 T cell responses. I demonstrate that RSV strains induce differential CD4 T helper responses, which are associated with the differential activation of the innate immune response. The RSV line 19 strain induced the early production of the pro-inflammatory cytokines IL-1Β and IL-6 resulting in an increased Th17 response as compared to the RSV strains A2 and 2-20. Blockade and/or neutralization of IL-1Β and IL-6 inhibited the ability of RSV line 19 to induce a Th17 response. These results demonstrate that RSV strains can differentially activate innate immunity that subsequently influences the type of adaptive immune response. This in part may contribute to differential RSV pathogenesis and the development of long-term airway morbidities observed in humans. IL-10 is a pleotropic cytokine able to suppress the adaptive immune response. Because the host adaptive immune response is believed to contribute to RSV-induced pulmonary disease, I evaluated the role of IL-10 in modulating the RSV-specific immune response. I found that IL-10 protein levels in the lung were increased following acute RSV infection with maximum production corresponding to the peak of the virus-specific T cell response. Multiple populations of CD4 T cells accounted for the majority of IL-10 produced in the lung including Foxp3+ Tregs, Foxp3- CD4 T cells that co-produce IFN-Γ, and Foxp3- CD4 T cells that do not co-produce IFN-Γ. Furthermore, RSV-induced disease severity was increased in both the absence of IL-10 and following IL-10 receptor blockade as compared to control mice. I also observed an increase in the magnitude of the RSV-induced CD8 and CD4 T cell response that correlated with increased disease severity following IL-10 receptor blockade. IL-10 receptor blockade during acute RSV infection altered CD4 T cell subset distribution, resulting in a significant increase in IL-17A-producing CD4 T cells and a concomitant decrease in Foxp3+ regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell-mediated pulmonary inflammation and injury. Overall, my data demonstrate that RSV-specific CD4 T cells migrate into the lung tissue with their differentiation influenced by the strain-specific activation of innate immune response. IL-10 is then produced by CD4 T cells to regulate the RSV-specific T cell responses and inhibit virus-induced immunopathology. My data indicate that there are multiple targets for immunotherapy for individuals with severe RSV-induced disease.

No description available.

Marsupials and eutherians are the two principal groups of modern mammals. Mammalian immunological studies, to date, have focused on eutherian systems with little or no comprehensive work having been carried out on marsupials. This project investigates the functional and developmental aspects of T-cell responses in the marsupial, Macropus eugenii (Tammar wallaby) in both adults and pouch young at various stages of development. Determination of the age at which the Tammar wallaby immune system becomes competent has been examined through the use of cellular and molecular studies carried out on developing pouch young tissue. The capacity for generating an immunological response in adult and pouch young marsupials has been studied by following cellular proliferation in response to mitogens or mixed lymphocyte culture (MLC). After examining adult responses to mitogens and allogenic lymphocytes, optimised conditions were then used to examine the development of responsiveness in pouch young. Several further tests were conducted and findings shown. The study has shown that the earliest age at which Macropus eugenii is capable of mounting a T-cell mediated immune response is between 5 to 13 days post-partum / Doctor of Philosophy (PhD)

"October 2001" Amendments (4 leaves) inserted inside back cover. Includes bibliographical references (leaves 215-266)