Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia, and despite recent introduction of targeted therapies, remains incurable. An important hallmark of CLL is severe immune deficiency, including the failure to mount effective anti-tumour immune responses. This can partly be explained by insufficient antigen presentation, but also by the existence of complex CLL-induced T-cell defects. Based on the cancer immuno-editing hypothesis that the immune system not only protects a host against tumour formation but can also be compromised to actively provide a pro-tumour microenvironment, modulating cancer-induced T-cell defects could restore the full anti-tumour response and result in more durable clinical responses. The immune checkpoint molecules PD-1 (expressed on activated immune effector cells) and PD-L1 (expressed on antigen-presenting and microenvironmental cells including tumour cells) have emerged as important mediators of T-cell suppression. Several studies suggest that PD-L1/PD-1 inhibitory signalling in CLL might be overcome by the immune modulatory drug lenalidomide. Furthermore, directly targeting PDL-1/PD-1 interactions produces significant responses in solid cancers. However, similar studies are notably absent in CLL, and the effect of PDL-1/PD-1 blockade on restoring cancer-induced immune dysfunction is not understood. Transgenic Eμ-TCL1 mice have been extensively validated as an adequate preclinical model of aggressive human CLL, and our group showed their suitability to mirror T-cell defects observed in human CLL. Using the Eμ-TCL1 model, this dissertation project substantially extends our previous characterization of CLL-induced T-cell dysfunction and evaluates the functional impact of PD-L1/PD-1 inhibitory signalling both in parallel with disease development and in different microenvironments. The findings to be described here demonstrate that developing CLL is associated with specific T-cell subset alterations, phenotypic changes, and functional defects that are very similar in peripheral blood and secondary lymphoid organs. In addition to PD-L1, PD-L2 is identified as a potential mediator of inhibitory signalling in CLL. CD8+ T cells in leukaemic mice are characterised as a functionally heterogeneous population, in which subsets of cells are able to exert effector functions despite PD-1 expression. In vivo lenalidomide treatment repairs selected phenotypic alterations and immune synapse formation, and a PD-L1 IgG blocking antibody effectively controls disease and reverses global T-cell defects even in cells expressing PD-1. In sum, this work provides a strong rationale to explore PD-L1/PD-1 targeting in CLL clinical trials, potentially in combination with novel agents.

Malaria affects 200 million people annually, resulting in 584,000 - 1,238,000 deaths. The majority of these deaths occur in children, less than 5 years of age, in sub-Saharan Africa and are due to cerebral malaria (CM), a neuropathology induced primarily by the species Plasmodium (P.) falciparum. The pathogenesis of CM remains poorly understood and the mechanisms involved in acquired protection against the syndrome in malaria-endemic regions are undefined. Utilising the well characterised P. berghei ANKA experimental infection model of cerebral malaria (ECM), results presented in this thesis show that the development of ECM is associated with the accumulation and arrest of pathogenic CD8+ T cells within the perivascular spaces of the brain. Accumulation of activated CD8+ T cells, without arrest, was observed in the perivascular spaces of the brains of mice infected with the non-ECM causing P. berghei NK65 strain. These data show that the behaviour of intracerebral CD8+ T cells specifies their pathogenic function during malaria infection. The development of ECM was associated with extensive disruption to the BBB, which developed in the absence of extensive CD8+ T cell-dependent endothelial cell apoptosis. We modified the ECM model, establishing an infection-drug cure strategy, to investigate the immunological basis of parasite exposure-induced resistance to ECM development. Three rounds of infection-drug cure promoted resistance to ECM, which was associated with reduced intracerebral expression of genes involved in defence response, regulation of apoptosis, chemotaxis, CTL activity, antigen processing and presentation and cell adhesion, compared with ECM susceptible mice. Additionally, CD8+ T cell activation was suppressed in exposure-induced resistant mice and was associated with the antibody dependent expansion of a splenic plasmacytoid DC population, with a regulatory phenotype. The infection-induced protection against ECM was critically dependent upon secreted antibody production. A long standing problem in studying the immune response to malaria infection has been the inability to track parasite-specific CD4+ T cell responses. To address this, we generated and validated new transgenic P. berghei parasites expressing the model antigen, ovalbumin (OVA), either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). We found that cellular location and expression level of the antigen influence the induction and magnitude of parasite-specific T-cell responses. These parasites thus provide knowledge on the factors that influence the recognition of parasite antigens by the immune system and represent useful tools to study the development and function of antigen-specific T-cell responses during malaria infection. The results in this thesis improve our understanding of the events that lead to the development of CM, and the host immune responses that develop following parasite exposure to protect against it. The results should contribute towards the rational development of adjunctive therapies and effective vaccines for human CM.

Infection with human gastrointestinal (GI) parasites, such as Trichuris trichiura, affects more than billion people worldwide, causing significant morbidity and health problems especially in poverty-stricken developing countries. Despite extensive research, the mechanisms of induction and regulation of effector immune responses against these parasites are incompletely understood, which hinders the development of anti-parasite therapies. Infection with GI parasite is usually chronic suggesting that parasites are capable of modulating immune responses of their host to prevent expulsion. However, mechanisms by which parasites control host immunity to allow infection are still ill-defined. The aim of this PhD study was to characterise the role of different immunoregulatory mechanisms in immunity to GI parasite infection, with a focus on dendritic cells (DCs), regulatory T cells (Tregs) and the regulatory cytokine transforming growth factor β (TGF-β).Here we showed for the first time that loss of TGF-β-activating integrin alphavβ8 specifically on DCs resulted in protection from chronic infection with Trichuris muris, a mouse model of T. trichiura infection in man. Accelerated expulsion was immune-mediated and although increased levels of protective Th2 cytokines were observed very early during infection, elevated levels of non-protective Th1 cytokines were also detected. Partial depletion of CD4+ or FcεRI+ cells had no effect on the observed phenotype. Since deletion of alphavβ8 on DCs results in decreased numbers of Tregs in the gut, we tested whether depletion of Tregs using a mouse model that allows conditional ablation of Foxp3+ Tregs (DEREG mice) would alter infection development. Although transient Treg depletion at the beginning of infection had no major effect on expulsion kinetics, we observed a tendency for enhanced Th2 responses in DEREG mice. Moreover, even though DC-mediated TGF-β activation via alphavβ8 integrin was essential for T. muris infection development, transient depletion of DCs had no effect on the induction of Th2 responses or parasite expulsion. These data indicate a novel role for the TGF-β-activating integrin alphavβ8 and DCs in regulating effector immune responses during T. muris infection and may contribute to the development of new anti-parasite therapies.

CD46 is a ubiquitously expressed transmembrane protein in humans with a role in immune homeostasis. Originally identified as a complement regulator, CD46 has since been regarded a receptor for several pathogens and most recently, described as a T cell costimulatory molecule. Its coligation with CD3 and consequent cleavage from the T cell surface serves as a costimulatory stimulus for T cell activation. In addition, in the presence of IL-2, CD46 induces Tr1 cell differentiation which is characterised by low IFN-γ and high IL-10 secretion. CD3/CD46-induced Tr1 differentiation is defective in patients with MS, rheumatoid arthritis and asthma, highlighting the need to investigate the mechanisms involved in the regulation of the CD46 pathway. CD46 is a highly glycosylated protein with three N-glycosylation sites in the short consensus repeats and multiple O-glycosylation sites in the STP region. Previous data from the lab have shown that CD3 activation causes a change in CD46 glycosylation. Herein, I convey that this change is more pronounced in memory than naive CD4+ T cells and is mainly due to changes in CD46 O-glycosylation. Furthermore, these changes are required for the T cell responses triggered by CD46 costimulation including T cell activation and Tr1 differentiation. Interestingly, CD46 is recruited to the immunological synapse formed between a T cell and an antigen presenting cell and I illustrate that the STP region is needed for this also. These data suggest that the glycosylation status of CD46 regulates its function. In MS, vitamin D deficiency is considered to be a significant risk factor and many patients take vitamin D supplement to help manage their condition. Herein, I report that treatment of healthy and MS CD4+ T cells with vitamin D does not prevent T cell activation but it decreases adhesion molecule expression. Moreover, vitamin D supplementation in MS enhances CD46 cleavage. Therefore, vitamin D also plays a role in the regulation of the CD46 pathway and it would be interesting to investigate whether vitamin D affects CD46 glycosylation. During my MSc, I showed that a recombinant protein derived from adenovirus serotype 35 (which naturally binds CD46) known as Ad35K++ controls the CD46 pathway in CD4+ T cells. Lymphoma cells treated with Ad35K++ in combination with monoclonal antibody therapy rituximab have demonstrated increased sensitivity to rituximab and prove that virus-derived recombinant proteins that target CD46 have therapeutic potential. Considering the key role of CD46 as a T cell costimulatory molecule, I have investigated the effects of Ad35K++ on the CD46 pathway following its use in vivo and confirm CD46 is still cleaved from the cell surface and the cells still become activated. Overall these results provide insight into the mechanisms involved in the regulation of the CD46 pathway and highlight how it can be manipulated for therapeutic use.

A balanced immune response is required to mediate clearance of a virus infection without immune-mediated disease. CD4 and CD8 T cells are capable of both exerting antiviral effector functions and regulating the immune response. The regulatory T cell (Treg) subset of CD4 T cells helps to modulate immune activation and inflammation. During respiratory syncytial virus (RSV) infection in mice, conventional CD4 T-cell-mediated cytokine production has been shown to contribute to immune-mediated pathology. I demonstrate that Tregs are critical to control immunopathology during RSV infection. This was demonstrated through diphtheria toxin (DT)-mediated Treg elimination in a mouse strain expressing the DT receptor (DTR) under the control of the Foxp3 promoter. However, these mice were unable to maintain extended Treg depletion limiting the effectiveness of this model. In addition, DT-treated wild-type (WT) mice were found to be a necessary control for adverse DT-induced disease. In humans, I have shown that activated Tregs are reduced in the peripheral blood of RSV-infected infants compared to controls. RSV-infected infants also exhibited an increased proinflammatory cytokine response in nasal aspirates. However, the alarmin cytokine IL-33, which has been shown to mediate Treg homeostasis, was the only cytokine that exhibited reduced protein levels in RSV-infected infants compared to controls. Thus, severe RSV infection in infants may be due to lack of proper Treg-mediated immune regulation. Similar to RSV, regulation of the T cell response during chronic viral infection with lymphocytic choriomeningitis virus (LCMV) is vital to prevent immune-mediated pathology. During LCMV and human chronic viral infections, CD4 and CD8 T cells exhibit T cell exhaustion where they lose the ability to exert effector functions. However, a functional CD4 and CD8 T cell response is required for viral clearance. During human chronic viral infection, an association between increased CD4 and CD8 T cell function and enhanced viral control has been identified that can be influenced by genetic factors. I aimed to identify the contribution of the host genetic factors that contribute to enhanced CD8 T cell function and viral control using the LCMV model. I found that increasing the major histocompatibility complex (MHC) diversity resulted in enhanced viral control in both a C57BL and BALB genetic background. Thus, induction of a broader T cell response was associated with enhanced viral control. However, mice expressing a heterozygous MHC on the C57BL background also exhibited mortality following chronic viral infection. Both CD4 and CD8 T cells were shown to contribute to this mortality and exhibited reduced T cell exhaustion during LCMV infection in these mice. Heterozygous MHC expression on the C57BL mouse background was also associated with an increased T helper (Th)-1 skewed CD4 T cell response compared to mice on the BALB background. Furthermore, CD4 T-cell-mediated IFN-γ production contributed to both CD8 T cell effector activity and mortality during chronic LCMV infection. Thus, both T cell epitope diversity and host genetics contribute to LCMV-induced mortality. Collectively, my data highlight both the need for effective immune-meditated viral control and regulation of T-cell-mediated pathology during both acute and chronic viral infections.

Due to the growing burden of malignancy and chronic infections, manipulating CD8+ T cell responses for clinical use has become an important goal for immunologists. CD8+ T cells have the unique capacity to recognize and kill tumor cells and intracellular pathogens. Thus far, failed or only minimally effective T cell vaccines against chronic pathogen infections and tumors have highlighted basic knowledge gaps for eliciting memory CD8+ T cell protection. Defining the immunological mechanisms that determine protective capacity and longevity in T cells will be critical to both therapeutic and prohylactic vaccine efficacy. My studies focus on strategies to manipulate effector and memory CD8+ T cell responses, including their mechanisms of action. Specifically, I show that dendritic cell (DC) immunization coupled with relatively early (days 1-3) or late (days 4-6) administration of enhanced IL-2 signals drive either effector or memory programs. DC + IL-2c administered 4-6 days post-DC transfer is shown to enhance Ag-specific effector CD8+ T cell responses; this approach is further explored in the context of a cancer immunotherapy, demonstrating effective control of tumor burden in multiple murine models of cancer. Temporal alterations of IL-2 signaling from day 4-6 to day 1-3 post-DC immunization is shown to increase memory potential and memory CD8+ T cell numbers long-term. Additional studies reveal CTLA-4-mediated down-regulation of B7-ligands on DCs after IL-2c treatment, demonstrating that weaker or more transient signaling through the CD28-B7 axis may favor memory CD8+ T cell programs. My work contributes valuable concepts in memory CD8 T cell generation to develop T cell vaccines that are both safe and predictable.

Les lymphocytes T régulateurs Foxp3+ jouent un rôle crucial dans l'établissement de la tolérance au soi, le contrôle des réponses inflammatoires et le maintien de l'homéostasie du système immunitaire. La compréhension des mécanismes moléculaires impliqués dans la fonction de ces cellules représente un défi important. Chez le rat, la déficience en Themis1, une nouvelle molécule de la signalisation du TCR, associée à un locus de 117kb d'origine BN induit un défaut fonctionnel des Tregs et le développement spontané d'une maladie inflammatoire des intestins. Au sein de ce locus, le rat BN présente deux polymorphismes non-synonymes, un au niveau du gène C3 et un au niveau du gène Vav1 (R63W). Ce dernier est un candidat potentiel du fait du rôle joué par Vav1 dans l'activation des lymphocytes T et de sa régulation par Themis1. Dans ce travail de thèse, j'ai étudié l'effet de la déficience en Themis1 associé au polymorphisme R63W de Vav1 chez la souris de fond génétique C57BL/6 sur les fonctions des lymphocytes T régulateurs. J'ai montré que la déficience en Themis1 associé au polymorphisme R63W induit un défaut fonctionnel des Tregs in vitro et in vivo dans un modèle de colite. Ce défaut est associé à une production accrue de cytokines pro-inflammatoires par ces Tregs. J'ai également mis en évidence que l'association de ces deux mutations induit une sensibilité accrue à la colite induite par le DSS. Au niveau moléculaire, j'ai mis en evidence que ce défaut fonctionnel est associé à une réduction de la signalisation du TCR impliquant les vois Erk et NF-ĸB. De plus, j'ai montré que l'inhibition d'une phosphatase de la signalisation du TCR, SHP-1, permet de restaurer les fonctions suppressives des Tregs Vav1R63W-Themis1-/-. Cette étude souligne l'importance de l'intégrité du hub de la signalisation impliquant Vav1, Themis1 et SHP-1 dans la plasticité des lymphocytes T régulateurs et dans le maintien de leur fonction suppressive. Ainsi ce hub de signalisation représente une cible thérapeutique pour augmenter les fonctions des Tregs dans le cadre des maladies inflammatoires ou réduire leurs fonctions suppressives pour favoriser les réponses immunes anti-tumorales dans le cadre du cancer. / Regulatory T cells (Treg) are of paramount importance for restraining excessive immune responses and their manipulation holds enormous therapeutic potential. Our recent results using a congenic rat model suggested that the integrity of Vav1/Themis1 T-cell receptor signaling hub plays a crucial role in Treg suppressive function. Indeed, Themis1 deficiency in BN, but not in LEW rats, led to the development of inflammatory bowel disease (IBD), linked to a defect in Treg suppressive function. Genetic studies revealed that this phenotype depended on a 117 Kb genomic locus, containing the R63W polymorphism on Vav1 that impacted its expression and functions. To test the importance of the Vav1/Themis1 TCR signaling hub in Treg function, we generated Themis1-T-/- mice expressing conditionally Themis1 in thymocytes, but not in peripheral T cells. In contrast to regular germline Themis1 knockout mice, these mice were not lymphopenic and exhibited normal proportions of CD4+ T cells in the thymus and in peripheral lymphoid organs. Next, Themis1-T-/- mice were crossed with Vav1R63W mice to assess the impact of these combined mutations on Treg suppressive functions. Using in vitro approaches, together with in vivo analyses of IBD, we showed that suppressive activity of Treg was impaired in Themis1-deficient mice harboring the mutated Vav1; this defect is linked to higher production of IL-17 and IFNg. Functional studies showed that Themis1-deficient associated with the mutated Vav1 induced a defect in Erk and P65 phosphorylation after TCR engagement. Interestingly, the inhibition of the SHP-1 phosphatase restore the functional defect of Tregs. Together, these data showed that Themis1, Vav1 and SHP-1 cooperate in the signaling hub to regulate the suppressive function of regulatory T cells. Thus, this signaling hub represents a therapeutic target to enhance the suppressive functions of Tregs in the context of autoimmune and inflammatory diseases or to decrease their functions to favor anti-tumoral immune responses.

Infection with Plasmodium species leads to nearly 400,000 deaths a year despite widespread use of mosquito bed nets, insecticides, and anti-malarial drugs. To date, there is not a licensed vaccine capable of providing complete protection from Plasmodium infection to vaccinees. Whole parasite vaccination of humans and rodents can achieve complete protection in vaccines, but the dose of sporozoites, number of administrations, and production concerns in generating these types of vaccines will likely prevent these approaches from achieving worldwide use. However, the protective immunological responses against Plasmodium parasites engendered by these vaccination approaches can be studied and aid in the development of advanced subunit vaccines against Plasmodium. Using rodent models of malaria to elucidate the features of protective immunity engendered by whole parasite vaccination, it has been repeatedly shown that CD8 T cell responses directed against liver-stage parasite antigens can provide complete protection with some contribution by CD4 T cells and antibody responses depending on the model system studied. However, the quantatitive and qualitative requirements for CD8 T cell immunity against Plasmodium remains largely undefined. To enhance our understanding of how to generate protective immunity against Plasmodium, I have utilized rodent models of malaria to study the superior protection afforded from single-dose vaccination with virulent sporozoites administered under prophylatic chloroquine-cover, referred to as chemoprophylaxis sporozoites (CPS) vaccination, compared to the well-studied approach of administering radiation-attenuated Plasmodium sporozoites (RAS). RAS vaccination has long been considered the “gold standard” in vaccination due the ability of RAS vaccination to engender complete protection following sporozoite challenge of vaccinated humans and rodents. However, CPS vaccination is arguably a superior vaccination approach since it can achieve protection through less vaccine administrations relative to RAS vaccination, but the immunological basis of this enhanced CPS vaccine-induced immune response was unclear. In my study, I utilized a stringent host/parasite model to find that C57Bl/6 mice administered CPS vaccination with P. yoelii sporozoites elicit substantially higher parasite-specific CD8 T cell responses than RAS vaccination, but CPS-induced CD8 T cells were not necessary for protection following liver-stage sporozoite or blood-stage parasite challenge. CPS vaccination resulted in a low grade, transient parasitemia shortly following cessation of chloroquine treatment, which lead to the generation of potent antibody responses to blood-stage parasites; this blood-stage parasite-specific antibody response correlated with sterilizing protection in sporozoite challenged CPS-vaccinated mice. Therefore, my data provide a mechanistic basis for enhanced protective immunity elicited by single-dose CPS vaccination in a rodent model that is independent of CD8 T cells. The other portion of my work examines how CD8 T cell specificity impacts protective capacity against Plasmodium. I show that robust CD8 T cell responses of similar phenotype are mounted following prime-boost immunization against three novel Plasmodium berghei protein-derived epitopes in addition to a previously described protective, immunodominant epitope. I show that only CD8 T cells specific to sporozoite surface-expressed protein-derived epitopes, but not the intracellular protein-derived epitopes, are efficiently recognized by sporozoite-infected hepatocytes in vitro. These results suggest that antigenic targets must be efficiently presented by infected hepatocytes for CD8 T cells to eliminate liver-stage Plasmodium infection and proteins expressed on the surface of sporozoites may be good target antigens for protective CD8 T cells. Collectively, my work highlights the ability to generate protective CD8 T cell independent and dependent immunity against Plasmodium infections, whether achieved through potent blood-stage-specific antibody responses, or via numerically large monospecific CD8 T cell responses that target parasite antigens that are efficiently presented during liver-stage infection. These studies are relevant in understanding how to efficiency engender protective immunity against Plasmodium, and could aid in the advancement of subunit vaccination approaches that generate immunity through the priming of responses from multiple arms of the immune response, targeting both the liver- and blood-stages of Plasmodium.

The proper regulation of inducible costimulator (ICOS) and its ligand (ICOSL) have been shown to be essential for maintaining immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant antibody production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that ADAM10 is the primary physiological sheddase of ICOSL in both mouse and human. Using an in vivo system in which ADAM10 is deleted only on B cells (ADAM10B-/-), elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wildtype (WT) mice, interaction of ICOSL/ICOS results in ADAM10 induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper (TFH) development and Th2 polarization, seen in a house dust mite exposure model. In addition, enhanced Th1 and Th1 immune responses are seen in experimental allergic encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and at least partially rescues both TFH numbers and the abnormal antibody production previously reported in these mice. Overall, we propose a novel regulation of the ICOS:ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL as well as indirectly regulating ICOS, thus controlling ICOS:ICOSL-dependent responses. Additionally, we report a specific role for the metalloprotease ADAM10 on B cells in regulating both ICOSL and ICOS in a mouse model of increased humoral immunity using mir146a-/- mice and a model of lymphoproliferative disease using the well characterized lpr model. B6lpr mice lacking ADAM10 on B cells (A10Blpr) have decreased nodal proliferation and T cell accumulation compared to control B6lpr mice. Additionally, A10Blpr mice have a drastic reduction in autoimmune anti-dsDNA antibody production. In line with this, we found a significant reduction in follicular helper T cells (TFH) and germinal center (GC) B cells in these mice. We also show that lymphoproliferation in this model is closely tied to elevated ICOS levels and decreased ICOSL levels. Overall, our data not only shows a role of B cell ADAM10 in controlling autoimmunity, but also increases our understanding of the regulation of ICOS and ICOSL in the context of autoimmunity. Additionally, we found that ADAM17 is important for marginal zone (MZ) B cell development as well as responses to T-independent type 2 (TI2) immunizations. Mice which lack ADAM17 on B cells (A17B) have decreased MZ B cell numbers but have increased levels of antigen specific antibodies in response to TI2 Immunizations. ADAM17 also regulates the level of several surface molecules on plasma cells and MZ B cells necessary for their function and survival. We also show a role for ADAM17 in ILC2 responsiveness to IL-33. In vivo, mice that lack ADAM17 specifically on ILC2s (ADAM17ILC2-/-) exhibit decreased ILC2 expansion in response to intranasal IL-33 as well as Nippostrongylus brasiliensis (Nb) infection. However, ADAM17ILC2-/- mice have normal ILC2 numbers in a naïve state, suggesting this defect in ILC2 function is limited to cell activation. In vitro, ADAM17 inhibited ILC2s have an increased level of apoptosis and less IL-13 production in response to IL-33 compared to vehicle treated ILC2s. The defect in cytokine production following ADAM17 inhibition is not observed in response to IL-25 stimulation, suggesting this defect is limited to IL-33 stimulation Mechanistically, ADAM17 inhibition in ILC2s specifically causes a defect in IL-33 mediated ERK activation, potentially explaining the defective survival and IL-13 production following ADAM17 inhibition in these cells. Additionally, ADAM17 regulates the level of surface IL1R2 which may affect IL-33 signaling in ILC2s.

The increasing threat of epidemic and pandemic influenza underscore the need to better-understand the immune response to influenza virus infections and to better understand the factors that contribute to the clearance of virus without complications of immunopathology. A hallmark of the adaptive immune response to primary influenza virus infections is the induction of influenza-specific CD8+ T cell responses. These T cells target and kill influenza-infected epithelial cells in the airway, thereby clearing the virus and allowing recovery of the infected host. Recent reports demonstrated that CD8+ T cells express TNF-related apoptosis-inducing ligand (TRAIL) after influenza virus infection. While roles for perforin/granzyme and Fas:FasL interactions in clearing influenza virus infections had been established, little was known about the role of TRAIL in the CD8+ T cell responses to influenza virus infection. We hypothesized that influenza-specific CD8+ T cells would express TRAIL after influenza infection and could utilize TRAIL to induce the apoptosis of virally-infected cells. We discovered that CD8+ T cells do express TRAIL after influenza infection, and that this expression occurs in an influenza-specific fashion. Further, we demonstrated that these influenza-specific CD8+ T cells utilize this TRAIL to kill virally infected cells and protect the host from death, while T cells lacking TRAIL were unable to kill targets as efficiently and provided reduced protection. These data supported our hypothesis that CD8+ T cells utilize TRAIL to kill infected cells. Unexpectedly, when we increased the initial viral inoculum, the pulmonary cytotoxicity of T cells in TRAIL-/- mice was increased compared to those in TRAIL+/+ mice. Investigation of this phenomenon revealed that changes in cytotoxicity correlated not with changes in effector molecule expression on the T cells, but with increased recruitment of T cells to the lung. T cell recruitment to the lungs of TRAIL-/- mice was dependent on CCR5 and CXCR3, and likely the result of aberrant expression of MIG and MIP-1α in the lungs. Together, these data suggest that TRAIL expression contributes not only to T cell cytotoxicity, but also to the regulation of chemokine expression and associated cell recruitment after influenza virus infections. To confirm the relevance of our animal model to the study of human disease, we examined the potential role for TRAIL in the human immune response to infection. We determined that in vitro influenza infection stimulates upregulation of functional TRAIL on the surface of CD3+, CD14+, CD19+, and CD56+ PBMC populations. This expression was not caused by infection of the cells, but by interferon produced as a result of the infection. Infected (TRAIL-expressing) PBMCs killed influenza-infected lung epithelial cells, revealing that influenza infection sensitizes epithelial cells to TRAIL-induced apoptosis. Surprisingly, blocking TRAIL signaling, but not FasL signaling, was able to abrogate this killing of infected epithelial cells. Together, these data support a role for TRAIL in the human immune response to influenza virus infections. Considered as a whole, the data from these studies suggest an additional, previously-unappreciated mechanism by which CD8+ T cells can kill virally infected cells, TRAIL. They also suggest additional, previously-unappreciated roles for TRAIL in immune responses: in helping clear virally infected cells after infections and in helping control cytokine/chemokine expression, and thus the immune response, after virus infection.