<p>Cytomegalovirus (CMV) is a widespread infection affecting 50-90% of the human population. A typical silent primary infection is followed by life-long persistence in the host under control by virus-specific CD8 (“killer”) and CD4 (“helper”) T cells. Although harmless in most people, CMV may cause disease and sequelae in patients with deficient cellular immunity, such as AIDS patients, recipients of organ transplants and children who have acquired the virus before birth. In this thesis we have characterized the cellular immunity to CMV in immunocompetent subjects, in patients receiving transplants and in infants.</p><p>In healthy individuals with latent CMV, the frequencies of CMV-specific CD8 T cells varied considerably between the donors. Within the same individual, the changes over time were usually small. In patients with primary, symptomatic CMV infection, the frequencies of CMV-specific CD8 T cells peaked within the first month after the appearance of symptoms. The frequencies then declined to levels similar to those in latently infected CMV carriers. The CD4 T-cell function followed the same pattern, but with lower peak values.</p><p>Immunosuppressed renal transplant patients with latent CMV had CMV-specific CD4 cell function similar to healthy controls. The frequencies of CMV-specific CD8 T cells were also comparable, but their function was impaired. When renal transplant recipients were investigated longitudinally, we found that their CMV-specific T cells decreased rapidly after transplantation. Whereas the frequencies and function of CD8 T cells rebounded within 3 months, CD4 T-cell recovery was impaired during the entire first year after transplantation.</p><p>Finally, the frequencies and function of CMV-specific T-cells were investigated in children with congenital and postnatal CMV. CMV-specific CD8 T cells could be detected in even the youngest children, suggesting that these cells can develop early in life. In contrast, CMV specific CD4 T cells were low or absent in the youngest children but increased slowly with age.</p>

Tc17 cells and the semi-invariant human mucosal associated invariant T (MAIT) cells are important CD8+ tissue-homing cell populations. Both are characterized by high expression of CD161 (++) and type-17 differentiation, yet their origins and relationships remain poorly defined. By transcriptional and functional analyses it is demonstrated that a pool of polyclonal, pre-committed type-17 CD161++CD8αβ+ T cells exists in cord blood, from which a prominent MAIT cell (TCR Vα7.2+/Vβ2 or 13.2) population emerges post-natally. During this expansion, CD8αα T-cells appear exclusively within CD161++CD8+/MAIT subset, sharing cytokine production (IL17, IL-22 and IFN-γ), chemokine-receptor expression (CCR2, CCR6 and CXCR6), TCR-usage and transcriptional profiles with their CD161++CD8αβ+ counterparts. These data demonstrate the origin and differentiation pathway of MAIT cells from a naïve type-17 pre-committed CD161++CD8+ T cell pool and the distinct phenotype and function of CD8αα cells in man. The CD161++CD8αβ and CD8αα T cell subsets are reduced in the peripheral circulation in chronic hepatitis B and C and are enriched in the liver in chronic hepatitis C. Their potential role in immunity to chronic viral hepatitis B and C is demonstrated by their expression of activation/exhaustion markers CD69, CD25, HLA-DR and PD-1. In addition a substantial distinct CD161-CD8β^low population is demonstrated in chronic hepatitis B, co-characterised by a CD28^low, HLA-DR^high phenotype and high expression of IFN-γ, with important implications for the development of immunotherapy and vaccination.

Natural killer (NK) cells are non-specific killer cells of the innate immune system that eliminate target cells based on discrimination between self and non-self. Activation is carefully regulated through integration of signals received through both activating and inhibitory receptors. During the course of a herpes simplex virus type-1 (HSV-1) infection, NK cells can influence host susceptibility to infection with severe infections occurring in individuals with genetic defects in the NK cell response. In response to HSV infection, NK cells are recruited to the inflammatory tissue where ensuing reciprocal interactions with accessory cells and proinflammatory cytokines induce NK cell activation, cytolytic activity, and cytokine production, contributing to innate immune response and ultimately influencing the adaptive immune response. The objective of this study was to elucidate the multiple roles of NK cells during the numerous steps in anti-HSV immune induction. Accordingly, we have demonstrated that NK cells are novel helpers that assist and influence an anti-HSV immune response via the secretion of cytokines that enhance HSV-specific CD8+ T cell effector function and cytokine production. Taken together, data from this study presented the critical importance of NK cells in mounting an essential and efficient anti-HSV immunity. The key findings of our study were: 1. In the absence of NK cells, dendritic cells have decreased capacity to prime HSV-specific T cells. 2. HSV infected NK cells can be directly activated via toll-like receptor (TLR) in a MyD88-dependent mechanism; however, interaction with HSV infected dendritic cells yields optimal NK cell activation and function (CD69 and IFNγ). 3. TRAIL-expressing NK cells eliminate antigen-bearing immature dermal DCs (CD11c+CD8α-DR5+), that migrate to draining lymphoid organs, to facilitate antigen transfer to lymphoid resident CD8α+ DC for T cell cross priming. 4. 'Helpless' CD8+ T cell function, generated in the absence of CD4+ T cells, can be partially restored to wild-type levels by NK cell supplementation. 5. Treatment of NK cells with anti-CD69 antibody results in a heightened NK activated state and augments the adaptive immune response, without increasing NK cell numbers. These findings may contribute to the potential revelation of avenues to manipulate NK cells for anti-viral therapies.

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.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) with characteristic multifocal lesions or ‘ plaques’ of demyelination mainly in the white matter of the brain ( involving cerebral cortex, cerebellar, brain stem and spinal cord). These MS plaques vary in size and shape, and are composed of infiltrates of lymphocytes and macrophages - which contain myelin debris. CD8 T cells are more prevalent in CNS lesions and display oligoclonal expansion. However, their role in disease remains unclear with studies showing both protective and pathogenic roles for myelin-specific CD8 T cells in the experimental autoimmune encephalomyelitis (EAE) model. Our studies have demonstrated a disease-suppressive function for CNS-specific CD8 T cells in a model where the antigen is exogenously administered in vivo and used for in vitro CD8 activation. My studies focus on probing the nature of the CD8 response elicited by endogenously presented myelin antigens in vivo utilizing a novel approach, infection with Listeria monocytogenes (LM) encoding for myelin proteolipid protein peptide PLP178-191 (LM-PLP). I show that LM-PLP infection preferentially induces PLP-specific CD8, but not CD4, T cell responses. Despite this, infection does not result in autoimmunity. In fact, routinely induced EAE is significantly ameliorated in LM-PLP-infected mice, compared to controls. Disease suppression is dependent on the presence of CD8 T cells, and the effector molecules IFN-g and perforin. CNS T cell infiltration and inflammatory responses are reduced in LM-PLP-protected mice, and CD4 T cells from LM-PLP-protected mice are less inflammatory than those from controls. Importantly, infection with LM-PLP ameliorates already established disease. My studies indicate that myelin-specific CD8 T cells induced by endogenous presentation of antigen attenuate CNS autoimmunity in multiple mouse models of EAE, implicating the potential of this approach as a novel immunotherapeutic strategy.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells identified in mice as Gr-1+CD11b+ cells with the ability to inhibit T cell function. MDSC are emerging as important regulators of T cell mediated immune responses. Current paradigm suggests that despite heterogeneity, all Gr-1+CD11b+ cells are suppressive when exposed to inflammatory stimuli. In vitro evaluation shows MDSC from multiple tissue sites have suppressive activity, and in vivo inhibition of MDSCenhances T cell function. However, the relative capacity of MDSC present at localized inflammatory sites or in peripheral tissues to suppress T cell responses in vivo has not been directly evaluated. Using a tissue specific acute inflammatory prostatitis model, we demonstrate that MDSC inhibition of CD8+ T-cell proliferation is restricted to the inflammatory site.Further, MDSC from inflammatory sites possess immediate capacity to inhibit T-cell function, whereas those isolated from peripheral tissues (spleens and liver) were not suppressive without activation of iNOS by exposure to IFN-_.Using two mouse models of prostate cancer, we extend these findings to thetumor micro-environment. During a chronic inflammatory response induced by tumorgrowth, we show Gr-1+CD11b+ cells from the tumor site possess immediate capacity toregulate effector T cell function whereas those from the spleen do not. In both tumormodels and in our prostatitis model, long term culture of activated T cells with splenicGr-1+CD11b+ cells converted precursor cells into functional MDSC during standard in vitro suppression assays. These data highlight the importance of MDSC in the prostate, and demonstrate the function of MDSC during a localized inflammatory response isrestricted to the site of an ongoing immune responseGrowing evidence suggests that prostatitis associated with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is mediated in part by the loss of T cell and B cell tolerance to prostate antigens. Clinical data demonstrates the presence of T cell proliferative responses to prostate auto-antigens in CP/CPPS patients. However, the mechanisms leading to this loss of tolerance are not clearly understood, largely because of a lack of available animal models. We report the development of a new mouse model for the study of chronic prostate inflammation (CPI), the Prostate Ovalbumin Expressing Transgenic-3 (POET-3) model. Adoptive transfer of antigen specific OT-I T cells induces CPI characterized by infiltration of exogenous (OT-I) and endogenous T cells into the prostate persisting as long as 45 days after transfer. In vitro and in vivo data demonstrate inflammation induced loss of T cell tolerance to prostate auto-antigens. Auto-antibody responses to prostate antigens were detected in POET-3 mice after induction of CPI. These data have important therapeutic implications for treatment of CPI.

Memory CD8 T cells offer increased protection to immune hosts by rapidly eliminating pathogen-infected cells during re-infection. Generating and sustaining a protective memory CD8 T cell response is considered a hallmark of adaptive immunity. Extensive research has been devoted to understanding the parameters affecting memory CD8 T cell generation after infection or immunization in order to design the most effective vaccines. An accepted notion in the field is that increased protection from re-infection is afforded by the generation of a large number of memory CD8 T cells. Consecutive prime-boost immunization strategies that elicit secondary responses are often used to increase the absolute numbers of memory CD8 T cells. While parameters affecting the generation of primary memory CD8 T cells are well known, the factors influencing the development of re-stimulated secondary CD8 T cell responses remain understudied. Here, I addressed the mechanisms involved in the generation and development of secondary memory CD8 T cells. I found that the time at which primary memory CD8 T cells enter into an immune response during re-infection impacts their fate and differentiation into secondary memory CD8 T cells. Late-entry of primary memory CD8 T cells into an immune response (relative to the initiation of infection) not only facilitates expression of transcription factors associated with memory formation in secondary effector CD8 T cells, but also influences the ability of secondary memory CD8 T cells to localize within the lymph nodes, produce interleukin-2 cytokine (IL-2), and undergo robust antigen-driven proliferation. The timing of stimulation of primary memory CD8 T cells also impacts the duration of expression of the high-affinity IL-2 receptor (CD25) on secondary effector CD8 T cells and their sensitivity to IL-2 signaling. Importantly, by blocking or enhancing IL-2 signaling in developing secondary CD8 T cells, I verify the role of IL-2 in controlling the differentiation of secondary CD8 T cell responses. The data I present herein suggest that the process of primary memory-to-secondary memory CD8 T cell differentiation is not fixed and can be manipulated, a notion with implications in the design of future prime-boost vaccination approaches. Although vaccines are designed and intended to benefit a range of individuals, at times the efficacy of a vaccination regime depends on the overall health status of a host. Thus, in another portion of my thesis work I explored the extent to which obesity compromises the differentiation and maintenance of protective memory CD8 T cell responses. I found that diet-induced obesity did not impact the maintenance of pre-existing memory CD8 T cells, including their acquisition of a long-term memory phenotype (i.e., CD27hi, CD62Lhi, KLRG1low) and function (i.e., cytokine production, antigen-driven secondary expansion, and memory CD8 T cell-mediated protection). Additionally, diet-induced obesity did not influence the differentiation and maintenance of newly evoked memory CD8 T cell responses, in inbred and outbred hosts, that were generated in response to different types of systemic (LCMV, L. monocytogenes) and/or localized (influenza virus) infections. Interestingly, I found that the rate of naïve-to-memory CD8 T cell differentiation after a peptide-coated dendritic cell immunization was similar in lean and obese hosts. This suggests that obesity-associated inflammation is unlike pathogen- or adjuvant-induced inflammation, and does not influence the development of an endogenous memory CD8 T cell response. My studies reveal that the obese environment does not influence the development or maintenance of memory CD8 T cell responses that are either primed before or after obesity is established. This is a surprising notion with implications for future studies aiming to elucidate the role of obesity in susceptibility to infection and vaccine efficacy. Collectively, the data presented here further the understanding of memory CD8 T cell responses in contexts of health and disease.

Cytomegalovirus (CMV) is a widespread infection affecting 50-90% of the human population. A typical silent primary infection is followed by life-long persistence in the host under control by virus-specific CD8 (“killer”) and CD4 (“helper”) T cells. Although harmless in most people, CMV may cause disease and sequelae in patients with deficient cellular immunity, such as AIDS patients, recipients of organ transplants and children who have acquired the virus before birth. In this thesis we have characterized the cellular immunity to CMV in immunocompetent subjects, in patients receiving transplants and in infants. In healthy individuals with latent CMV, the frequencies of CMV-specific CD8 T cells varied considerably between the donors. Within the same individual, the changes over time were usually small. In patients with primary, symptomatic CMV infection, the frequencies of CMV-specific CD8 T cells peaked within the first month after the appearance of symptoms. The frequencies then declined to levels similar to those in latently infected CMV carriers. The CD4 T-cell function followed the same pattern, but with lower peak values. Immunosuppressed renal transplant patients with latent CMV had CMV-specific CD4 cell function similar to healthy controls. The frequencies of CMV-specific CD8 T cells were also comparable, but their function was impaired. When renal transplant recipients were investigated longitudinally, we found that their CMV-specific T cells decreased rapidly after transplantation. Whereas the frequencies and function of CD8 T cells rebounded within 3 months, CD4 T-cell recovery was impaired during the entire first year after transplantation. Finally, the frequencies and function of CMV-specific T-cells were investigated in children with congenital and postnatal CMV. CMV-specific CD8 T cells could be detected in even the youngest children, suggesting that these cells can develop early in life. In contrast, CMV specific CD4 T cells were low or absent in the youngest children but increased slowly with age.

Cytomegalovirus (CMV) is a β-herpesvirus that, after primary infection, establishes a life-long persistence in the human host. Up to 90% of humans are infected with CMV, that is kept under control by CMV-specific CD8+ and CD4+ T cells. In patients with an impaired cellular immunity, however, CMV infections can be life-threatening. Thus, it is vital to identify risk factors and target high-risk patients. In this thesis we have evaluated low-dose valacyclovir prophylaxis in renal transplant patients and studied CMV-specific T cell immunity in healthy and renal transplant patients. In renal transplant patients, the CMV serostatus of both the recipient (R) and the donor (D) has a major impact on the risk of developing CMV disease. In the high-risk D+/R- population, &gt;50% are likely to develop CMV disease in the absence of prophylaxis and/or pre-emptive therapy. We have used low-dose valacyclovir prophylaxis for high-risk renal transplant patients, and graft and patient survival up to 5 years after transplantation was comparable to data reported for other prophylactic protocols. The incidence of CMV disease and graft rejection during the first year after transplantation was also comparable to that achieved with other protocols, and without the adverse effects reported for other therapies. In the D+/R+ population, with a 15-35% risk of developing CMV disease, it is important to identify those individuals that are subject to a higher risk because of risk factors other than CMV serostatus. We therefore measured several immunologic parameters in renal transplant patients and in immunocompetent individuals with latent and primary CMV infection. In patients with a primary symptomatic CMV infection, CMV-specific CD8+ T cells peaked within a month after onset of symptoms but declined rapidly. In renal transplant patients, we found that the reduction in IFNγ-producing CMV-specific CD4+ T cells at 2 months post-transplantation may predict high-grade CMV DNAemia.

Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein.