The chemokine receptor CXCR3 and its agonists CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC are involved in a variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. CXCL11 has also been reported to bind to an additional receptor, namely CXCR7, which also interacts with CXCL12. Two alternatively spliced variants of the human CXCR3 receptor have been described, namely CXCR3-B and CXCR3-alt. The human CXCR3-B has been found to bind CXCL9, CXCL10, CXCL11 as well as an additional agonist CXCL4/PF4. In contrast, CXCR3-alt only binds CXCL11. This work demonstrates that CXCL4 like the original CXCR3 agonists is capable of inducing biochemical signalling, namely intra-cellular calcium elevation, and activation of p44/p42 MAPK and PI3K/Akt pathways in activated human T lymphocytes. Phosphorylation of p44/p42 MAPK and Akt was inhibited by pertussis toxin, suggesting coupling to Gi protein. In contrast CXCR3 antagonists blocked CXCR3 agonists but not CXCL4-mediated responses. Surprisingly, stimulation of T cells with CXCL4 failed to elicit migratory responses of these cells and did not lead to loss of surface CXCR3 expression. Collectively our evidence shows that although CXCL4 is coupled to downstream biochemical machinery, its function in T cells is distinct from the function of CXCR3 agonists. The work presented in this thesis also indicates that despite considerably lower surface expression in comparison to the full length CXCR3, CXCR3-B and CXCR3-alt transduce biochemical signals in response to CXCL11 in transfected cells. According to previous reports the role of CXCR7 in signalling and chemotaxis in T cells could not be detected. In T cells and transfected cells system CXCR7 was localised at the plasma membrane and was efficiently internalized in response to CXCL11 and CXCL12. Studies of the involvement of methylation in T cell chemotaxis suggest that this modification may be required in this process as it was partially inhibited by methylation inhibitor- MTA. Moreover T cell co-stimulation caused increased levels of arginine mono-methylated proteins suggesting the importance of methylation in T lymphocyte signalling.

The respiratory system forms a major mucosal interface with the external environment. Consequently, the respiratory tract is constantly exposed to inhaled foreign antigens, commensal microorganisms, and potential pathogens. The respiratory system has evolved a complex regulatory network designed to prevent unnecessary inflammation to harmless antigens and to limit immune-mediated damage to the fragile lung epithelium in response to infection. The lung maintains a default anti-inflammatory state that is coordinated by the respiratory epithelium, alveolar macrophages, dendritic cells, and regulatory Foxp3+ CD4 T cells (Tregs). It is likely that all of these cells influence the development of pathogen-specific T cell responses in the lung. Following infection with a respiratory virus, virus-specific CD8 T cells in the lung are inhibited in their ability to produce cytokines. Current studies suggest that this functional inactivation occurs following infection with respiratory viruses within the Paramyxoviridae family. The data presented here demonstrate that suppression of effector functions of virus-specific CD8 T cells in the lungs occurs following infection with several unrelated respiratory viruses. These results indicate that the functional inhibition of virus-specific T cell responses is not restricted to infection with viruses from the Paramyxoviridae family. Furthermore, I show data indicating that the functional inactivation of virus-specific CD8 T cells in the lungs occurs in the absence of infection. I also demonstrate for the first time that the lung environment also regulates the effector functions of virus-specific CD4 T cells. Inhibition of cytokine production by pulmonary T cells is reversible as stimulation with exogenous peptide-pulsed antigen-presenting cells rescues IFN-gamma production. The inhibition of IFN-gamma production by virus-specific T cells occurs in other organs such as the kidney. These data suggest that regulation of T cell cytokine production by peripheral tissues may serve as an important mechanism to prevent immunopathology and preserve normal tissue function. Foxp3+ Tregs have been shown to inhibit conventional effector T cell responses in a large number of chronic infection models. However, their role during acute infections remains unclear. Examination of Foxp3+ Tregs during RSV infection showed that Tregs are rapidly recruited into the lungs and acquire an activated phenotype. Depletion of Foxp3+ Tregs prior to RSV infection revealed that Tregs facilitate the early recruitment of RSV-specific CD8 T cells from the draining lymph nodes to the lung and later limit the overall magnitude of the virus-specific CD8 T cell response. Depletion of Tregs increased TNF-αa production by RSV-specific CD8 T cells and enhanced T-cell-mediated immunopathology. These data demonstrate that Foxp3+ Tregs play a major role in regulating CD8 T cell responses to respiratory virus infections. Collectively, the data presented here demonstrate that CD8 T cell responses to respiratory pathogens are tightly regulated within the lung environment.

CD4 and CD8 T cells play a vital role in mediating the clearance of viral pathogens following infection. Mice deficient- or depleted of their CD4 and/or CD8 T cells exhibit a diminished ability to control viral replication following infection and in some cases develop a persistent viral infection. CD8 T cells upregulate cytotoxic effector molecules such as granzyme B, Fas and TNF-related apoptosis-inducing ligand (TRAIL) that them to directly kill virus-infected cells. Following a systemic virus infection the CD8 T cell response is primed within secondary lymphoid organs, such as the spleen and lymph nodes (LNs). Although, it has been shown that the LNs are important for the generation of optimal CD8 T cell responses following systemic viral infections, the relative role of the spleen versus the LN in priming the CD8 T cell response is unknown. Studies in this thesis demonstrate that LNs, but not the spleen, are critical for the optimal generation of a CD8 T cell response following a systemic intraperitoneal (i.p.) lymphocytic choriomeningitis virus (LCMV) infection. Using adoptively transferred naïve LCMV-specific CD8 T cells, we demonstrate that the mediastinal LN (MedLN) serves as the initial draining LN and is responsible for priming the majority of the virus-specific CD8 T cell response following an i.p. LCMV infection. Moreover, the draining MedLN exhibits an increased frequency of CD62L- effector memory (TEM) CD8 T cells for up to 8 weeks following viral clearance. I demonstrate that the increased frequency of CD62L- TEM CD8 T cells is not due to residual viral antigen. Furthermore, a similar increase in CD62L- TEM CD8 T cells is found in the ipsilateral popliteal LN following a footpad LCMV infection. I demonstrate that the increased frequency of CD62L- TEM CD8 T cells in the draining LN is due to increased recruitment. CD4 T cells promote the generation of both effector and memory CD8 T cells either indirectly through their CD40-CD40L-dependent maturation of dendritic cells or through the production of cytokines such as IL-2 and IFN-γ that directly interact with CD8 T cells. CD4 T cells are also critical for the generation of germinal center B cells and promote the differentiation of activated B cells into memory B cells and plasma B cells. However, CD4 T cells often recognize epitopes derived from a broad array of pathogen-encoded proteins, making it difficult to accurately quantify the magnitude of virus-specific CD4 T cell responses. Therefore, I evaluated a large panel of activation and/or memory markers to determine a combination that could be used to reliably identify antigen-specific CD4 T cells following viral infection. I show that the integrins CD11a and CD49d are upregulated in an antigen-dependent manner on virus-specific CD4 T cells following LCMV infection. Furthermore, memory LCMV-specific CD4 T cells retain their CD11ahiCD49d+ expression pattern. Using CD11a and CD49d as surrogate makers for antigen-specific CD4 T cells, I show that approximately 50% of the CD4 T cells following LCMV infection are virus-specific, indicating that the virus-specific CD4 T cell response is substantially larger than previously recognized. Furthermore, I demonstrate that CD11a and CD49d can be used to accurately track newly-activated CD4 T cells following a heterologous virus challenge. In addition to LCMV, respiratory syncytial virus (RSV)-specific CD4 T cells are CD11ahiCD49d+. The two previously identified RSV CD4 T cell epitopes only account for ~3% of the CD11ahiCD49d+ CD4 T cell population during the peak of RSV infection, indicating that additional RSV-derived epitopes remain to be identified. Therefore, I used an overlapping peptide library spanning each of the RSV-derived proteins to identify novel RSV-specific CD4 and CD8 T cell epitopes. Using this approach, I identified 5 novel RSV-derived CD4 T cell epitopes and 4 novel CD8 T cell epitopes. Furthermore, I demonstrate that stimulation of CD4 T cells with 17-mer peptides results in over a 2-fold increase in the frequency of responding CD4 T cells as compared to stimulation with the commonly used 15-mer peptides. Collectively, the data shown here provides new insight into where and how the CD8 T cell response is initiated following a systemic virus infection, as well as provide a novel approach to track the endogenous CD4 T cell response following viral infections.

Cytotoxic T-lymphocytes are important for the control of viral replication during HIV infection, however the magnitude and breadth of HIV-specific CD8+ T-cell response does not correlate well. The purpose for this study was the examination of the HLA-B*2705-specific CD8+ T-cell response to the KRWIILGLNK (KK10) epitope as a definitive model of immune control over HIV replication. The breadth of the T-cell receptor (TCR) repertoire was determined for an association between the qualitative nature of this response and immune escape and therefore, disease progression. Methodology was developed and validated for TCR repertoire analysis in formaldehyde fixed antigen-specific CD8+ T-cells. The TCR repertoire for the KK10-specific CD8+ T-cell response was defined in cross-section and longitudinally for 6 HLA-B*2705+ patients. Comparison was made to cognate HLA-A*0201 CMV NV9 and HLA-B*2705 EBV RL9-specific CD8+ T-cell populations using the Simpson??s diversity index and the Morisita-Horn similarity index for standardized repertoire analysis. HLA-B*2705 KK10-specific TCR repertoire was not found to be a determinant of control. Greater clonotype variation was found within CMV-specific CD8+ T-cell populations, suggesting an association with reactivation of CMV and disease state. An association was found between KK10-specific population diversity and the prevalence of cognate KK10 epitope in vivo. Cross-reactivity observed for dominant KK10-specific clonotypes suggested that avidity of CD8+ T-cells was important for in vivo survival. Phenotype and function was tested through multiparameter analysis of HIV and CMV-specific CD8+ T-cells. Increased frequency of CD127 (IL-7R) and Bcl-2 expression within dominant populations was suggestive of selective advantage. Division of dominant and sub-dominant CMV-specific CD8+ T-cell populations into ??early?? and ??late?? differentiation phenotypes indicated virus-specific mechanisms of clonotype turn over. No simple association of TCR expression was found for HIV and CMV-specific CD8+ T-cells with published examples of definitive TCR bias. Over-represented TCR ??-chain families of patients were found in association with public clonotypes. Convergent recombination of TCR genes was demonstrated as a mechanism for the prevalence of shared clonotypes. Standardized assessment of T-cell repertoire successfully identified mechanisms of antigen-specific CD8+ T-cell recruitment. A substantial increase in sample numbers is required before this methodology can be used to accurately demonstrate the importance of TCR repertoire usage in the control of human viral infection.

A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

Thymocyte development is a complex process that requires precise regulation of differentiation and proliferation. Basic helix-loop-helix (bHLH) transcription factors have been shown to be crucial for proper T cell development. HEB and E2A are structurally and functionally related E proteins of the bHLH family. These proteins directly regulate the expression of a number of genes essential for lymphocyte development in a lineage- and stage-specific manner. Abrogation or compromise of their function results in the manifestation of B and T cell developmental defects. Genetic and biochemical studies have provided evidence of a significant degree of functional redundancy among E proteins. The existence of compensational abilities among different E proteins has hampered the investigation and elucidation of E protein function. As such, single gene knockouts demonstrate only limited defects in lymphocyte development. Double E2A-HEB knockouts that could eliminate E protein redundancy are embryonic lethal. In addition, conventional gene knockouts are not well-suited for discerning between intrinsic and extrinsic defects caused by E protein disruption. To eliminate functional compensation and to test the T cell intrinsic roles of E proteins during thymocyte development, we developed a conditional HEB-E2A double knockout. Specifically, we employed a loxP/Lck-Cre recombinase system to drive E protein deletion during early thymocyte development. Using this approach, we were able to reveal overlapping roles for HEB and E2A in thymocyte development that had been obscured in previous single gene knockout studies. We find that simultaneous deletion of HEB and E2A results in a severe block in thymocyte development at the DN to DP stage transition. This developmental block is accompanied by a dramatic decrease in total thymic cellularity, an increase in apoptosis, and a reduction of pTα expression. These developmentally arrested thymocytes exhibit increased proliferation in vivo and dramatic expansion ex vivo in response to IL-7 signaling. Our findings suggest that E2A and HEB are not only critical for the regulation of T cell differentiation but are also necessary to retain developing thymocytes in cell cycle arrest prior to pre-TCR expression. Together, these results imply that E proteins are required to coordinate thymocyte differentiation and proliferation. / Dissertation

T cell effector functions depend on focused secretion. This is accomplished by secretory vesicle (SV) clustering around the microtubule organizing center (MTOC) and MTOC translocation to the specialized site of cell-cell contact - the immunological synapse (IS). The dynein molecular motor has been implicated in both processes. To investigate the roles of dynein and dynein-associated proteins we used Jurkat cells expressing fluorescent CTLA-4 for SV tracking and molecular traps targeting dynein subunits to show that dynein intermediate chain (DIC) and the light chain LC8 are needed for both SV clustering and MTOC translocation. We also found that immunostaining with different anti-DIC antibodies labeled different pools of dynein at the IS in activated Jurkat cells. To discern how dynein separately accomplishes both MTOC and SV activities we cloned DIC cDNAs from Jurkat cell mRNA and obtained two isoforms, DIC2B and DIC2C. However, both isoforms were concentrated around the MTOC and formed a ring-like structure at the IS. We also saw little difference in dynein-binding proteins that co-immunoprecipitated with each isoform. We then investigated the roles of the dynactin component p150Glued and Lis1 protein in MTOC translocation and SV clustering. Surprisingly, p150Glued was concentrated around the MTOC but was not present at the IS. SVs marked by CTLA-4 showed clustering defects while MTOC translocation was not significantly affected in p150Glued siRNA knockdown cells. On the other hand, Lis1 immunostaining labeled a ring at the IS where it mimicked the distribution of the dynein ring thought to be involved in MTOC translocation. MTOC translocation was potently blocked in Lis1 siRNA knockdown cells but dynein recruitment was only slightly disrupted and there was no visible effect on actin localization at the IS. Overexpression of Lis1 or expression of Lis1 deletion mutants interfered with MTOC translocation and interfered with dynein recruitment, while actin was still localized at the IS. However, studies of calcium flux in response to T cell receptor (TcR) stimulation showed that these mutant-expressing cells had deficiencies in cell signaling from the TcR. These results suggest that MTOC translocation and SV clustering are mediated by dynein but likely involve different dynein-binding proteins. / text

The use of both multipotent progenitors and fully differentiated cells has been demonstrated to be effective for cell-based immunotherapy. The goal of this thesis was to establish an in vitro hematopoietic differentiation system to generate hematopoietic progenitor cells (HPCs) and functional T cells from pluripotent stem cells. Generation of progenitor T cells by co-culturing stem cells on Notch ligand-expressing OP9 stromal cells (OP9-DL1) had been successfully employed previously. However, further differentiation of these cells in vitro into mature, antigen-specific, functional T cells, without retroviral transduction of T cell receptors (TcRs), had not been achieved. In the thymic niche, differentiation of T cells to a state of antigen specificity is controlled by the interaction of their developing TcRs with the Major Histocompatibility Complex (MHC) on thymic stromal cells. We hypothesized that, by providing exogenous antigen-specific MHC/TcR signals, stem and progenitor cells could be engineered into functional effector T cells specific for the same antigen. In Chapter 3 and 4, we demonstrate that both thymus-derived double positive (DP: CD4+CD8+) immature T cells and mouse Embryonic Stem (ES) cells can be efficiently differentiated into antigen-specific CD8+ T cells using either MHC tetramers or peptide-loaded stromal cells. DP cells, following MHC/TcR signaling, retained elevated RAG1 levels, suggesting continuing TcR gene rearrangement. Both DP and ES cell-derived CD8+ T cells showed significant Cytotoxic T Lymphocyte (CTL) activity against antigen-loaded target cells, indicating that these cells are functional. This directed differentiation strategy could provide an efficient method for generating functional, antigen-specific CTLs from stem cells for potential use in adoptive T cell therapies. The use of ES cells in the clinic has been hindered by the unavailability of patient-specific ES cells and the ethical issues surrounding the use of human embryos. Induced pluripotent stem (iPS) cells offer great hope to regenerative medicine as their use can circumvent both the patient-specific and ethical issues associated with ES cells. In Chapter 5, we have developed a feeder cell-free suspension culture system supplemented with OP9-DL1 secretary factors to efficiently generated HPCs from iPS and ES cells. The differentiation potential of these HPCs was demonstrated by generation of DCs in the presence of GM-CSF and IL-3. The DCs express the activation molecules, CD86 and CD80 in response to LPS stimulation and are able to stimulate T cell proliferation in a mixed lymphocyte reaction. We employed extensive quantitative RT-PCR analysis to identify a number of differentially expressed genes in HPCs generated from the feeder-free culture. / text

Infectious diseases remain amongst leading causes of death in people aged 65 years and older; therefore, much research is focused on determining the immune components that contribute to age-dependent increased susceptibility to, and increased mortality from, infections. CD8⁺ T cells are critical for clearing intracellular pathogens through production of cytokines and direct killing of infected cells. Age-dependent CD8⁺ T cell alterations have been described, including decreased numbers of naïve CD8⁺ T cell precursors and decreased numbers and function during infection. This dissertation explores the mechanisms contributing to these changes. First, we demonstrated that multiple mechanisms contribute to changes in the CD8⁺ T cell pool with age. CD8⁺ T cells from unimmunized T cell receptor transgenic (TCRTg) old mice undergo massive virtual memory (VM) conversion with age; both homeostatic proliferation and cross-reactivity may contribute to the generation and accumulation of VM cells with age. These VM cells exhibit an age-dependent replicative impairment to cognate antigen, which points to possible detrimental functional consequences due to changes in the overall T cell pool. Second, we evaluated the cell intrinsic contribution to the decreased old CD8⁺ T cell response. With in vitro stimulation, old CD8⁺ T cells exhibit decreased ability to enter into late cell divisions and decreased production of effector molecules. In addition, we found that old CD8⁺ T cells have decreased expression of the master transcription factor T-bet, which correlates to decreased effector function and terminal differentiation in vivo. Collectively, these results identify possible cell-intrinsic targets for improving CD8⁺ T cell immunity. Finally, we measured whether a Listera monocytogenes live vaccine model induces protective immune responses in old mice. We found that vaccination conferred little protection in old mice upon pathogen challenge. These results contrast with other vaccine models, which may allow for pinpointing both the vaccine and immune components required for generating strong protective immunity in the elderly. Collectively, this dissertation demonstrates that CD8⁺ T cell precursors, effector cells, and memory cells exhibit profound changes with, age and identifies both possible mechanisms contributing to these alterations as well as possible therapeutic/vaccine targets for improving immunity in the elderly.

The oncogene Ahi-1 was recently identified through provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in several leukemic cells lines, particularly in cutaneous T-cell lymphoma (CTCL) cell lines (Hut 78 and Hut 102). Hut 78 is derived from a patient with Sezary syndrome, a common leukemic variant of the human CTCL mycosis fungoides. Aberrant expression of AHI-1 mRNA and protein has been found in CD4⁺CD7⁻ leukemic Sezary cells from patients with Sezary syndrome. Moreover, stable suppression of AHI-1 using retroviral-mediated RNA interference in Hut 78 cells inhibits their transforming activity in vitro and in vivo. In an effort to identify genes involved in AHI-1-mediated leukemic transformation in CTCL, microarray analysis was performed to compare six RNA samples from AHI-1 suppressed Hut 78/sh4 cells to five samples from Hut 78 control cells. Limma and dChip analyses identified 218 and 95 differentially expressed genes, respectively, using a fold change criteria of > or < 2 and a p-value threshold of ≤ 0.01. After evaluation of both analyses, 21 genes were selected based upon interesting structural and functional information, specificity to hematopoietic cells or T-cells, and previous connections to cancer. Expression patterns of these 21 genes were validated by qRT-PCR with p-values < 0.05 ranging from 1.97 x 10⁻¹⁰ to 6.55 x 10⁻³, with the exception of BRDG1 at 5.88 x 10⁻². The observed up-regulation of both BIN1 and HCK in AHI-1 suppressed Hut 78/sh4 cells as compared to control cells further confirmed at the protein level. The tumor suppressor BIN1 is known to physically interact with c-MYC, which also exhibits differential protein expression in these cells. Characterization of BIN1 identified 4 isoforms all of which contain exon 10 and demonstrate alternative splicing of exons 12A and 13. Additionally, qRT-PCR results from primary Sezary samples indicate there is clinical significance in the expression changes detected for BIN1, HCK, REPS2, BRDG1, NKG7 and SPIB. These findings identify several new differentially expressed genes that may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells.