In this dissertation, the roles of endocytosis and phagocytosis pathways in a variety of clinically relevant scenarios were examined. These scenarios include antibody-mediated internalization of cell surface proteins, titanium wear-particle uptake in failed joint replacements, and polymeric microparticle uptake and immune responses for drug delivery or adjuvant use. The use of antibodies specific for cell surface proteins has become a popular method to deliver therapeutics to target cells. As such, it is imperative to fully understand the ability of antibodies to mediate internalization and endosomal trafficking of the surface protein that it recognizes, so that drug delivery can be optimized. By comparing the internalization and endosomal localization of two different antibody-bound proteins, the transferrin receptor (TfR) and rabies G, we have found that there is a specific antibody-mediated internalization pathway that occurs when an antibody binds to a cell surface protein. Interestingly, the internalization pathway induced by antibody binding is different than that seen with recycling receptor internalization after ligand binding. This may have broad implications for the future development of antibody-based therapeutics. Joint replacement failure is a major clinical problem. Studies have indicated that a large amount of metal and polyethylene wear debris is found in the synovial membrane and tissue surrounding failed replacements. Through examination of the immune response following uptake of titanium particles, our results suggest that titanium wear-particle induced inflammation and subsequent joint replacement failure may be due to activation of the NLRP3 inflammasome, leading to increased IL-1ß secretion and IL-1 associated signaling. These findings introduce IL-1 as a target for potential therapeutics for patients exhibiting significant inflammation. Polymeric microparticles have been widely used in a variety of therapeutic applications, including drug delivery and vaccine adjuvants. It is essential to understand the ability of such particles to either activate or inhibit an immune response following uptake. Through comparison of particles with varying surface morphology, we have determined that particles with regions of high surface curvature (budding) are more immunogenic than particles with low surface curvature (spherical). Budding particles were more rapidly phagocytosed and induced higher levels of the inflammasome-associated cytokine, IL-1ß, when exposed to mouse macrophages. Additionally, budding particles induced a more rapid neutrophil response in vivo, when compared to spherical particles. These findings have broad implications for the development of future targeting vehicles for delivery of vaccines, drugs, proteins, and siRNA therapeutics.

Immunologic self-tolerance is maintained by both central and peripheral mechanisms. Furthermore, regulation of mature lymphocyte responses is governed by inhibitory as well as stimulatory signals. TCR recognition of cognate peptide bound to MHC molecules provides the initial stimulus leading to T lymphocyte activation and determines the antigen specificity of any subsequent response. However, lymphocytes must discriminate between foreign and self antigens presented by self-MHC molecules to maintain self tolerance and avoid pathological autoimmunity. Consequently, TCR ligation alone is reported to result in abortive activation, T cell anergy, apoptosis, and tolerance. Under normal physiological conditions, costimulatory signals modify lymphocyte responsiveness to TCR ligation to prevent autoimmunity while enabling robust responses to foreign antigen. Members of the CD28/B7 superfamily provide the critical secondary signals essential for normal immune cell function. CD28 is an essential positive costimulatory molecule with critical functions in thymic development, lineage commitment, and regulation of peripheral lymphocyte responses to antigenic stimuli. CD28 ligation by APC-expressed B7 molecules alters proximal signaling events subsequent to MHC/TCR interactions, and initiates unique signaling pathways that alter mRNA stability and gene transcription. Furthermore, CD28 signaling is required for regulatory T cell development and function. Thus, CD28 has a central role in both potentiating lymphocyte activation mediated by TCR engagement and regulating peripheral tolerance. In contrast, Ctla-4 mediates an inhibitory signal upon binding B7 molecules on an antigen-presenting cell. Its importance in governing lymphocyte responses is manifested in the fatal lymphoproliferative disorder seen in Ctla-4-/- mice. The lymphocyte proliferation is polyclonal, antigen and CD28 dependent, and arises from defects in peripheral CD4+T cell regulation. The high percentage of peripheral T lymphocytes expressing activation markers is accompanied by lymphocyte infiltration into numerous non-lymphoid tissues and results in death by 3-4 weeks. While still controversial, Ctla-4 signaling has been reported to be essential for induction of peripheral T lymphocyte tolerance in vivo and in some model systems is proposed to regulate both T lymphocyte anergy induction and the immune suppressive effects of some regulatory T cells in the prevention of autoimmunity. Signaling pathways activated by TCR ligation and CD28 costimulation have been extensively characterized. In contrast, the mechanisms mediating Ctla-4 maintenance of tolerance remain largely unknown. Ctla-4 gene expression is tightly controlled during T cell development and activation, and its intracellular localization and expression on the cell surface is regulated by numerous pathways and intermediates. While a tailless Ctla-4 mutant is capable of inhibiting T cell activation, recent studies have shown that a ligand independent form of Ctla-4 is also capable of providing an inhibitory signal to T lymphocytes. In conjunction with the strictly controlled expression kinetics and the perfect amino acid homology between the intracellular domains of mouse and human Ctla-4, this data suggests that Ctla-4 may participate in the modulation or initiation of intracellular signaling pathways. Positive and negative costimulatory receptors on the T cell modify lymphocyte responses by altering both quantitative and qualitative aspects of the lymphocyte response including threshold of activation, cytokine secretion, and memory responses. Positive costimulation augments T cell responses, in part, by downregulating the expression of genes that actively maintain the quiescent phenotype. This study was initiated to determine the role of Ctla-4 ligation in modifying the global gene expression profile of stimulated T cells and to determine if the Ctla-4 mediated maintenance of T cell tolerance was achieved, in part, by altering the transcription of quiescence genes necessary for the prevention of T cell activation subsequent to TCR and CD28 stimulation. Previous studies investigating the influence of Ctla-4 ligation on transcriptional profiles of activated lymphocytes detected only quantitative alterations in the transcriptional regulation initiated by CD28 signaling. In contrast, our data suggests that quantitative effects of Ctla-4 ligation that differentially influence pathways acting downstream of stimulatory receptors results in a stable and qualitatively unique phenotype detectable at the level of the transcriptome. Thus, the cumulative effect of Ctla-4 signaling is unique and not constrained to reversing alterations in expression initiated by CD28. In addition, Ctla-4 ligation can be shown to influence T lymphocyte responsiveness and the resulting global expression profile within 4 hours after stimulation and prior to detectable Ctla-4 surface expression. In a subpopulation of T cells, TCR stimulation activates pathways that result in commitment to activation with 2-6 hours. In contrast, CD28 signaling must be maintained for 12-16 hours to ensure maximal responses at the population level. The period of sensitivity to Ctla-4 inhibition of activation is more constrained and does not extend beyond 12 hours. Together, these data support a potential role for Ctla-4 in modification of the early transcriptional response and may explain various alterations in phenotype resulting from Ctla-4 ligation that have been reported in secondary responses. Identification of genes involved in lymphocyte activation, maintenance of selftolerance, and attenuation of immune responses opens the door to therapeutic manipulation of the pathways implicated. CD28 costimulation results in general amplification of TCR-initiated transcriptional responses, and specifically alters the expression profile of a subset of genes. In contrast, Ctla-4 ligation directly and specifically alters the expression of a select group of genes when ligated, and results in minimal suppression of the global CD28-mediated costimulatory transcriptional response. Ctla-4 regulated genes comprise a heterogeneous family, but include known quiescence factors, transcriptional regulators, and various determinants of cell cycle progression and senescence. The role of Ctla-4 in maintaining self-tolerance indicates that targeted manipulation of these gene products presents a novel therapeutic opportunity, and suggests that the mechanisms involved in Ctla-4-mediated maintenance of peripheral T cell tolerance and regulation of immune responsiveness is more nuanced than previously thought. In addition, this study provides the most comprehensive description of global gene expression during primary lymphocyte activation yet available. The integration of statistical and bioinfomatics analyses with large scale data mining tools identifies genes not previously characterized in lymphocytes and can direct future work by predicting potentially interacting gene products and pathways.

Murine infection by the Lyme disease spirochete, B. burgdorferi, results in the generation of pathogen-specific antibody that can provide protection against Lyme disease, but the cells involved in this response are poorly characterized. T cells are not required for generating a protective antibody response to B. burgdorferi infection, but their exact role in providing protection against tissue colonization had not been previously determined. We found that TCRβxδ;-/- mice were susceptible to high pathogen loads and decreased antibody titers, but inhibition of CD40L-dependent interactions resulted in partial protection suggesting that a portion of the help provided by T cells was not dependent on CD40L-CD40 interactions between T and B cells. RAG1-/- mice reconstituted with either un-fractionated or B1-enriched peritoneal cells from previously infected mice generated B. burgdorferi-specific antibody, and upon spirochetal challenge suffered significantly lower levels of pathogen load in the joint and heart. Peritoneal cells from previously infected TCRβxδ-/- mice or B2-enriched or B1-purified peritoneal cells conferred little to only moderate protection, suggesting T cells play an important role in protection against spirochetal infection the joint. Consistent with this, T cells from previously infected donor mice, when transferred with B1 or B2 cells into RAG1-/- mice, generated increased antibody titers and were capable of diminishing bacterial burden in the joint and heart. A previously identified class of protective antibody is directed against the spirochetal surface lipoprotein DbpA, and we found that DbpA is a prominent protein antigen recognized by RAG1-/- mice reconstituted with B1-enriched peritoneal cells. Additionally, we found that mice reconstituted with B1 cells also make antibody directed towards the spirochetal glycolipid antigen, BbGL-IIc, which is recognized by Vα14iNKT cells. Consistent with the idea that T cells are important in providing protection against spirochetal infection, RAG1-/- mice reconstituted with B1 and T cells generated a more robust response against DbpA and BbGL-IIc. These results support the hypothesis that T cells act with B1 cells in a CD40L-independent manner to promote the production of antibodies that play an important role in protection of the joint from spirochetal infection.

Erythropoiesis maintains stable tissue oxygenation in the basal state, while accelerating red cell production in anemia, blood loss or high altitude. The principal regulator of erythropoiesis is the hormone erythropoietin (Epo). In response to hypoxic stress, Epo can increase a 1000-fold, driving erythropoietic rate by up to 10-fold. It’s been suggested that survival pathways activated by the Epo receptor (EpoR) underlie its regulation of erythropoietic rate. A number of apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here I assessed the roles of three survival pathways activated by EpoR in erythroblasts in-vivo: the suppression of cell-surface Fas and FasL, the suppression of the pro-apoptotic regulator Bim, and the induction of the anti-apoptotic regulator Bcl-xL. I used the novel CD71/Ter119 flow-cytometric method of identifying erythroblast maturation stages in vivo to measure these apoptotic pathways in fetal liver and adult erythropoietic tissues. I found that these pathways differ markedly in their regulation of erythropoietic rate. Using mouse genetic models, I found that apoptosis mediated by interaction between erythroblasts that co-express cell-surface Fas and FasL plays a key autoregulatory role in stabilizing the size of the erythroblast pool in the basal state. Further, mice mutant for Fas or FasL showed a delayed erythropoietic response to hypoxia or high Epo. This suggests that Fas and FasL accelerate the stress response by providing an apoptotic ‘cell reserve’ that can be rescued by Epo in stress. I also examined the in-vivo behavior of two cell-intrinsic apoptotic regulators, Bcl-xL and Bim, previously unexamined in stress. The induction of Bcl-xL was rapid but transient, whilst the suppression of Bim was slower but persistent. My data suggest that Bcl-xL is a key mediator of EpoR’s anti-apoptotic signal very early in the stress response, before Bim and Fas are suppressed. Bcl-xL adaptation to high Epo occurs through inhibition of Stat5 activation, and resets it for the next acute stress. My findings suggest that in vivo, Epo regulates erythropoietic rate through erythroblast apoptosis, and that various apoptotic regulators play distinct and unique roles in this process. My work provides new molecular insights into erythropoiesis that are relevant to cytokine biology and to clinical approaches of disease treatment.

Virus infection is sensed by the innate immune system through germline encoded pattern recognition receptors (PRRs). Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) serve as PRRs that recognize different viral components. Microbial nucleic acids such as Ribonucleic acid (RNA) are important virus-derived pathogen-associated molecular patterns (PAMPs) to be recognized by PRRs. Virus recognition may occur at multiple stages of the viral life cycle. Replication intermediates such as single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) are detected by the RNA-sensing PRRs that initiate innate and adaptive immune responses. Triggering of the innate immune system is a critical event that can shape the adaptive immune response to virus infection. Better vaccination strategies that lead to improved T-cell and antibody responses are needed for protection against pathogens. We sought to delineate the RNA-sensing PRR pathways that are activated during infection with an RNA virus, the signaling mediators involved and the influence on subsequent virus-specific adaptive immune responses. To analyze the role of RNA-sensing PRRs in T-cell immune responses in vitro, we performed direct co-stimulation experiments on CD4+ T-cells of high purity. We utilized synthetic RNA-like immune response modifiers (IRMs) R-848 (MyD88-dependent) and poly I:C (MyD88-independent) as RNA PAMPs to determine the direct effects of RNA-sensing PRR activation on CD4+ T-cells. RNA PAMPs can act directly on CD4+ T-cells and modulate their function and phenotype. Maximal direct co-stimulatory effects were observed in CD4+ T-cells cultured with poly I:C compared to R-848. The cytoplasmic dsRNA-dependent protein kinase R (PKR) was also involved in poly I:C-mediated signaling in CD4+ T-cells. We found differences in the RNA-sensing PRRs activated by R-848 between mouse and human CD4+ T-cells. We observed minimal direct co-stimulatory effects by R-848 in mouse CD4+ T-cells. In contrast, augmentation of Th1 responses by R-848 was observed in human CD4+ T-cells. TLR8 activation in human CD4+ T-cells may explain the observed differences. We next explored the signaling pathways activated by RNA PAMPs in conventional dendritic cells (cDCs) and CD4+ T-cells that drive Th1 CD4 T-cell responses in isolated cDC/CD4 T-cell interactions. Allogeneic cDCs and CD4+ T-cells of high purity were cultured together with R-848 and poly I:C in MHC congenic mixed leukocyte reactions (MLRs). R-848 and poly I:C stimulation of type I IFN production and signaling was essential but not sufficient for driving CD4+ Th1 responses. The early production of IL-1α and IL-1β was equally critical. To analyze the role of RNA-sensing PRRs in T-cell immune responses in vivo, we utilized a mouse model of heterosubtypic influenza A virus (IAV) infections. Using MyD88-/-, TLR7-/- and IL-1-deficient mice, we explored the role of MyD88-signaling in the generation of heterosubtypic memory CD4+ T-cell, CD8+ T-cell and antibody responses. We found that MyD88 signaling played an important role in anti-IAV spleen and lung CD4+ T-cell, spleen CD8+ T-cell and Th1 antibody immune responses. Anti-IAV lung heterosubtypic CD8+ T-cell responses were not dependent on MyD88 signaling. Our in vitro and in vivo results show the pivotal role of RNA-sensing PRR pathway activation in T-cell immune responses. Understanding the complexity of the PRR pathways involved during viral infections and defining the subsequent immune response would have important implications for the generation of more effective vaccine strategies.

Given that Streptococcus pneumoniae can cause life-threatening pulmonary and systemic infection, an apparent paradox is that the bacterium resides, usually harmlessly, in the nasopharynx of many people. Humoral immunity is thought to be the primary defense against serious pneumococcal infection, and we hypothesized that nasopharyngeal colonization of mice results in the generation of an antibody response that provides long-term protection against lung infection. We found that survival of of C57L/6 mice after intranasal inoculation with wild-type serotype 4 strain TIGR4 pneumococci required B cells but not T cells, suggesting that nasopharyngeal colonization elicited a protective humoral immune response. In fact, intranasal inoculation resulted in detectable pneumococcal-specific antibody responses, and protected mice against a subsequent high-dose S. pneumoniae pulmonary challenge. B cells were required for this response, and transfer of immune sera from i.n. colonized mice, or monoclonal antibodies against phosphorylcholine, a common surface antigen of S. pneumoniae, was sufficient to confer protection. IgA, which is thought to participate in mucosal immunity, contributed to but was not absolutely required for protection from pulmonary challenge. Protection induced by i.n. colonization lasted at least ten weeks. Although it was partially dependent on T cells, depletion of CD4+ T cells at the time of challenge did not alter protection, suggesting that T cells did not provide essential help in activation of conventional memory cells. Peritoneal B1b cells and radiation-resistant, long-lived antibody secreting cells have previously been shown to secrete anti-pneumococcal antibodies and mediate protection against systemic infection following immunization with killed bacteria or capsular polysaccharide [1, 2]. We found that peritoneal cells were not sufficient for colonization-induced protection, but sub-lethally irradiated mice largely survived pulmonary challenge. Thus, our results are consistent with the hypothesis that nasopharyngeal colonization, a common occurrence in humans, is capable of eliciting extended protection against invasive pneumococcal disease by generating long-lived antibody-secreting cells.

Inhibiting BCR-ABL kinase activity with tyrosine kinase inhibitors (TKIs) has been the frontline therapy for CML. Resistance to TKIs frequently occurs, but the mechanisms remain elusive. First, to uncover survival pathways involved in TKI resistance in CML, I conducted a genome-wide RNAi screen in human CML cells to identify genes governing cellular sensitivity to the first generation TKI called IM (Gleevec). I identified genes converging on and activating the MEK/ERK pathway through transcriptional up-regulation of PRKCH. Combining IM with a MEK inhibitor synergistically kills TKI-resistant CML cells and CML stem cells. Next, I performed single cell RNA-seq to compare expression profiles of CML stem cells and hematopoietic stem cells isolated from the same patient. Among the genes that are preferentially expressed in CML stem cells is PIM2, which encodes a pro-survival serine-threonine kinase that phosphorylates and inhibits the pro-apoptotic protein BAD. Inhibiting PIM2 function sensitizes CML stem cells to IM-induced apoptosis and prevents disease relapse in a CML mouse model. Last, I devised a CRISPR-Cas9 based strategy to perform insertional mutagenesis at a defined genomic location in murine hematopoietic Ba/F3 cells. As proof of principle, we showed its capability to perform unbiased, saturated point mutagenesis in a 9 amino acid region of BCR-ABL encompassing the socalled “gatekeeper” residue, an important determinant of TKI binding. We found that the ranking order of mutations from the screen correlated well with their prevalence in IM-resistant CML patients. Overall, my findings reveal novel resistance mechanisms in CML and provide alternative therapeutic strategies.

Human influenza is a contagious respiratory disease resulting in substantial morbidity and mortality worldwide. With the recent cases of avian influenza infections in humans and the heightened concern for an influenza pandemic arising from these infections, it is essential to understand host responses that would confer protective immunity to influenza. The cell-mediated immune responses to influenza virus play an important role during influenza infection. To analyze the specificity and diversity of memory T-cell responses, we performed a genome-wide screening of T cell epitopes to influenza A virus in healthy adult donors. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-(IFN-γ) enzyme-linked immunosorbent spot assays (ELISPOT) using peripheral blood mononuclear cells (PBMCs) from four healthy adult donors. We found that among 11 influenza viral proteins, hemagglutinin (HA) and matrix protein 1 (M1) had more T-cell epitopes than other viral proteins. The donors were not previously exposed to H5N1 subtype, but we detected H5 HA T cell responses in two of the four donors. To confirm that HA is a major target of T cell responses we also analyzed H1 and H3 HA-specific T-cell responses using PBMC of additional 30 adult donors. Fifteen out of thirty donors gave a positive response to H3 HA peptides, whereas five of thirty donors gave a positive response to H1 HA peptides. Because we detected T cell responses to the H5 HA peptides in donors without prior exposure to H5N1 subtype, we asked if cross-reactive T cells to H5 HA peptides can be attributed to a prior exposure to H2N2 subtype, the closest HA to the H5 based on their phylogeny. We compared younger donors who have no prior exposure to H2N2 subtype and older donors who were likely to be exposed to H2N2 subtype, and both groups responded H2N2 peptides at similar level, suggesting that memory T cells cross-reactive to H5 HA peptides can be generated by prior exposure to the H1N1 and H3N2 subtypes, and the exposure to H2N2 subtype is not necessary. We subsequently identified a CD4+ T cell epitope that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of HA of influenza A and the HA of the influenza B virus. A CD4+ T cell line specific to this epitope recognizes target cells infected with various influenza A viruses including seasonal H1N1 and H3N2, a reassortant H2N1, the 2009 pandemic H1N1, H5N1 and influenza B virus in cytotoxicity assays and intracellular cytokine staining assays. Individuals who have the HLA-DRB1*09 allele have ex vivo IFN-γ responses to this epitope peptide in ELISPOT. Although natural infection or standard vaccination may not induce strong T and B cell responses to this very conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4+ T cells which are cross-reactive to both influenza A and B viruses.

The generation of memory T cells following primary exposure to a pathogen is a critical feature of the vertebrate immune system which has evolved as a protective mechanism in order to defend the host against repeated assaults by the patnogen. Memory T cells are long-lived, undergo rapid proliferation upon re-activation, mediate a robust secondary response and clear the pathogen much more efficiently. These aspects have made the generation of memory T cells an attractive goal for the production of both prophylactic and therapeutic vaccines. However, the degeneracy of the T cell receptor, whereby a given T cell recognizes more than one epitope, allows the T cell to be modulated by epitope variants which could be self-ligands, ligands related to the original epitope but altered in sequence, or completely unrelated epitopes. Experiments in both mice and humans show that such cross-reactive stimulation of memory T cells results in complete, partial, or no activation of T cells, and in some cases, even alters the functional identity of the T cell (for example, T helper 1 cells start secreting IL-4, IL-5 and become part of a T helper 2 response). In the context of secondary infection of immune organisms with pathogens containing mutated or related T cell epitopes, such alterations at the cellular level translate into drastic changes in the overall clinical outcome of the infection. Thus, the presence of cross-reactive T cells in the memory population implies that the protective or pathologic nature of the secondary immune response is a consequence of the host's infection history. Although several murine models of heterologous infection resulting in altered pathological outcome have been studied, the exact immune correlates of protection versus immunopathology are still unclear. This thesis addresses this issue in dengue virus infections in humans. Dengue fever (DF) and Dengue Hemorrhagic Fever (DHF) are two disease manifestations caused by infections of humans by the dengue viruses. These are a group of 4 serologically distinct flaviviruses (D1-4) which often co-circulate among endemic populations. While primary infection with any of the four serotypes can result in the more severe clinical disease characterized by DHF, epidemiological data from several outbreaks show that 80% - 90% of DHF cases occur among individuals with secondary infection. This implies that prior immunity to dengue is actually a risk factor for developing severe disease. In these DHF cases, there are increased numbers of CD69+ CD8+ T cells in circulation, with increases observed in the frequency of epitope-specific T cells, and the serum levels of several T cell produced cytokines, chemokines, and immune receptors are highly elevated. Since the four serotypes share 65% - 75% amino acid sequence homology, the possibility that unconserved T cell epitope sequences stimulated cross-reactive responses was borne out in in vitroexaminations. In these studies, peripheral blood mononuclear cells (PBMC) and cloned T cells from both vaccinated and infected donors contained large populations of memory T cells that were cross-reactive for heterologous viral serotypes in proliferation and CTL assays. These data suggest that the severity of disease seen in DHF patients can be attributed to an immunopathologic secondary response during heterologous infection, and highlight a role for serotype cross-reactive T cells in this process. This thesis addresses the hypothesis that the recognition of the natural variants of dengue virus T cell epitopes by serotype cross-reactive CD8+ T cells of a dengue-immune donor results in an altered secondary response profile, with the changes reflected in both the quantitative and qualitative nature of the response. In order to compare the functional profile of the secondary response of dengue-immune PBMC re-activated with heterologous serotypes, we focused on a panel of 4 donors who were vaccinated with live attenuated monovalent vaccines corresponding to D1, D2, or D4 serotypes. We screened a panel of peptides predicted to bind to HLA-A*0201 for cytokine responses and identified 4 novel epitopes that were highly immunogenic in all four donors. Direct ex vivo stimulation of donor PBMC with the heterologous sequences of these epitopes also showed sizeable serotype cross-reactive T cell populations. CFSE- and intracellular staining for cytokines and chemokines showed that these cross-reactive T cells not only expanded but also produced IFNγ, TNFα, and MIP-1β. Multi-parameter staining revealed functionally diverse populations comprised of single cytokine (IFNγ+, TNFα+, MIP-1β+, double cytokine (IFNγ+TNFα+, IFNγ+MIP-1β+, TNFα+MIP-1β+, and triple cytokine (IFNγ+TNFα+MIP-1β+ secreting sub-sets. Stimulation with the epitope variants altered the magnitude of the overall response as well as the relative sizes of these sub-sets. The patterns of responses revealed the effects of epitope immunogenicity, infection history and donor-specific variability. All 4 donors showed the highest cytokine response to a -single epitope (NS4b 2353). The same two peptide variants (D2 NS4a 2148 and D3 NS4b 2343) induced the highest response in all 4 donors regardless of the serotype of primary dengue infection. Interestingly, the epitope variants which showed the highest immunogenecity in our donors corresponded to the D2 and D3 serotypes which have been documented as being more virulent as well as a viral risk factor for DHF. In one donor, the response to all peptide variants was dominated by the same cytokine sub-sets. These data suggested that the dengue-immune memory T cell repertoire was functionally diverse and underwent alterations in size after secondary stimulation. Therefore, we also investigated the effect of epitope variants on dengue-specific CD8+T cell clones isolated from vaccinated and infected donors in order to determine if epitope variants induced altered functional outcomes at the clonal level. The epitope variants functioned either as strong agonists (particularly the D2 and D3 sequences), partial agonists, or null ligands. Some variants were able to induce cytolysis but not other effector functions at low concentrations. The variant ligands also influenced the hierarchy of cytokine responses within each clone. The third part of this thesis focused on the characterization of the frequency and phenotypic profile of epitope-specific CD8+ T cells in patients with DHF and DF at different times in the disease course in order to better understand the kinetics of the response and delineate any differences between the immune profile of severe vs. moderate disease. Tetramer staining for a previously identified HLA-B*07 restricted epitope was combined with staining for activation markers (CD69, CD38, HLA-DR), homing receptors (CCR7, CD62L), and programmed death receptor 1 (PD-1). The DHF subjects had early T cell activation with higher frequencies of tetramer+CD69+ cells as compared to DF subjects, in whom T cell frequencies peaked around the time of defervescence. While each subject had a unique phenotypic profile of tetramer+ cells, there was a difference between DF and DHF subjects in terms of CCR 7 expression; all subjects expressed low levels of CCR7 during acute illness but only the DHF subjects did not show upregulation of CCR7 on tetramer+ cells during convalescence. These data suggest that there is a sustained alteration in memory phenotype in those who recovered from severe dengue disease. A majority of the tetramer+cells also expressed PD-1 during acute illness but not during convalescence. Double-staining with variant tetramers allowed us to directly visualize serotype cross-reactivity of the epitope-specific population, and showed that secondary stimulation did induce the expansion of cells with low avidity for that secondary serotype and higher avidity to the variant. Furthermore, the ratios of these sub-sets changed during the course of the response. Taken together, these studies suggest that the immune response to heterologous secondary dengue infection is mediated by a heterogeneous population of serotype-cross reactive T cells that have different functional avidities to epitope variants and is influenced by the serotype of the secondary infection as well as the prior infection history of the individual. The preferential expansion of clones which secrete IFNγ but not inflammatory MIP-1β or TNFα or a repertoire characterized by a higher ratio of cytolytic to cytokine producing clones could limit immune mediated damage while efficiently clearing the virus. This information will be useful in the design of vaccine strategies aimed at inducing protective cross-reactive responses against all 4 dengue serotypes while preventing immunopathological outcomes following secondary infection.

The Human Immunodeficiency Virus (HIV) is able to infect CD4+ T cells as well as macrophages. Macrophage-tropism has been linked to determinants in the envelope of HIV. These determinants allow envelopes to exploit low levels of CD4 for infection. Macrophages are an important reservoir of virus, especially during chronic infection, and are likely responsible for the bulk of virus produced after CD4+T cells have declined. Viral factors that may impact the ability to infect macrophages are worth studying because this cell type is so important in infection. It was previously reported that the macrophage-tropic primary isolate AD8 was vpu-independent. The molecular clone YU-2, derived from brain tissue without culture, was also reported to be macrophage-tropic despite having a mutation in the vpu start codon. It was therefore possible that vpu-independent envelopes could evolve in vivo. To examine this possibility, I constructed chimeras containing wild type or defective vpu start codons, and gp160 sequences from AD8, YU-2 or SF162 (a vpu-dependent control). I also used full length AD8 and YU-2 with wild type or defective vpu start codons. I infected macrophages with equal amounts of virus, and measured viral output over two weeks. Viruses with defective vpu start codons were released to lower levels compared to their wild type vpucounterparts. In contrast to previous reports, the AD8 envelope is not vpu-independent for replication in macrophages. The YU-2 envelope is also not vpu-independent. Macrophage-tropic envelopes from late stages of infection can be sensitive to antibodies that bind the CD4 binding site on gp120, implying that macrophage-tropic envelopes have more exposed CD4 binding sites. Neutralizing antibodies may act as modulators of macrophage-tropism over the course of infection. Using chimeras containing gp120 sequences derived from the PBMC of four HIV+patients, I examined the capacity for envelopes to infect macrophages. Three patients (MM1, 4, and 8) had macrophage-tropic envelopes before and after developing autologous neutralizing antibodies. Three patients (MM1, 4, and 23) developed heterologous antibodies against IIIB, an easily neutralized T-cell line adapted strain of HIV-1. This data indicates that macrophage-tropism in these patients is not modulated by the presence of neutralizing antibodies. The macrophage-tropism of envelopes tends to segregate depending on the tissue origin of the virus. Envelopes from two separate tissues from the same patient exhibit very different infectivity characteristics. The B33 envelope, from brain tissue, is very infectious and is macrophage-tropic, while the LN40 envelope, from lymph node tissue, is weakly infectious and is not macrophage-tropic. Replacing the entire gp41 of LN40 with that of B33 restores some infectivity to LN40. The cytoplasmic domain of gp41 contains many motifs important for assembly and infectivity. To examine which motifs are responsible for the weak infectivity of LN40, I made chimeras of gp41, as well as point mutations in gp41. The LN40 chimera containing the entire gp41 of B33 restored the most infectivity. Point mutations in the palmitoylation site, Pr55gagbinding region, and dileucine motif at the C-terminus also restored infectivity when combined. Determinants in the gp41 cytoplasmic domain are responsible for the weak infectivity of LN40; however, it is possible that there are contributing determinants in gp120, such as the ability to use low levels of CD4. Here, I examined how changes in the vpu and env genes of HIV-1 can impact infectivity, especially infectivity of macrophages. Changes that adversely impact the virus’ ability to infect macrophages may also impact the overall course of disease. However, the data here show that retaining the ability to infect, and replicate in, macrophages give HIV an advantage. I speculate that retaining the ability to infect macrophages gives the virus a reservoir for later in disease, when CD4+ T cells have been depleted, as well as way of avoiding neutralizing antibodies. This work further defines the importance of macrophages in HIV-1 infectivity and disease.