Dissertations / Theses on the topic 'Immunology; Immunosuppression; Viral infection'

The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (T_H) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system.

To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65_495-503 peptide, a recombinant adenovirus coding for pp65, in vitro transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8⁺ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4⁺ T_H cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and T_H cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells ex vivo from virtually all stem cell donors for adoptive T cell transfer.

I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8⁺ T cells even when starting from undetectable frequencies (<0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8⁺ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer.

Natural killer (NK) cells are lymphocytes of the innate immune response with well-demonstrated activities against viral infections and tumors. Because of these abilities, we sought to glean insights into the mechanisms of NK cell activation so that they may be applied toward the design of new therapies.

NK cells are particularly critical for the control of poxviral infections. Vaccinia virus (VV) is the most-studied member of the poxviral family. It is robustly immunogenic and functions as the live vaccine responsible for the successful elimination of smallpox. VV infection provides a useful model for studying NK cell activation: NK cells play an important role in its clearance and the virus efficiently activates NK cells and recruits them to the site of infection. We had previously used this model to identify Toll-like receptor (TLR)-dependent and -independent mechanisms of NK cell activation to VV. One method of TLR-independent activation to VV requires the activation receptor NKG2D, which recognizes host ligands expressed upon viral infection by accessory cells such as dendritic cells (DCs) and macrophages.

In the first aim of this thesis, we sought to determine how the ligands for the NKG2D activation receptor become upregulated in the context of VV infection. Specifically, we asked whether interleukin-18 (IL-18), known to play a role in the innate immune response, could boost the expression of NKG2D ligands on DCs in response to viral infection. Using an in vivo infection model with IL-18R-deficient mice, our results confirmed an important role for IL-18 in NK cell activation to VV and viral control. We then made use of an NK-DC co-culture to show that IL-18 signaling on DCs, in addition to NK cells, is necessary to achieve efficient NK cell activation to viral infection. We further demonstrated in a cell-transfer experiment that cell-extrinsic IL-18 signaling is critical for NK cell activation in vivo. DC ablation via a mouse model designed to specifically ablate CD11c+ cells showed that DCs are also required for NK cell activation to VV in vivo. We finally showed how IL-18 can act on DCs in vivo and in vitro to boost the expression of Rae-1, an NKG2D ligand. Collectively, our data uncover a novel mechanism whereby NK cells become activated by IL-18 control of NKG2D ligand expression on DCs.

In the second aim of this project, we detailed how IL-18 signaling results in the upregulation of the NKG2D ligand Rae-1. Using an in vitro macrophage model, we showed how recombinant IL-18 was sufficient to upregulate Rae-1 expression. We compared IL-18 control of Rae-1 expression to LPS, a TLR ligand that also signals through the common adaptor MyD88 to govern Rae-1 expression. Using chemical inhibitors to cell signaling molecules, we then identified the importance of MyD88 signaling through PI3K. We then revealed that glycogen synthase kinase 3 (GSK-3) can act as a negative regulator of Rae-1 expression downstream of IL-18/TLR signaling. Specifically, we have shown that during inflammatory signaling, PI3K (acting downstream of MyD88) can inhibit GSK-3 to relieve its tonic suppression of Rae-1 expression and upregulate the NKG2D ligand. Finally, we showed that PI3K and GSK-3 signaling are also important to Rae-1 expression on DCs - the accessory cell where IL-18 signals to control Rae-1 expression to boost NK cell activation against VV.

In its entirety, this work seeks to address how NK cells become activated in the context of VV infection in order to identify new ways NK cells may be harnessed therapeutically.

Vaccinia virus (VV) is the most thoroughly studied member of the Poxviridae family and the vaccine used to achieve the only successful eradication of a human disease. Over the years, it has proven itself as a useful tool for the study of antiviral immunity, vaccine development, and potentially cancer immunotherapy. VV is capable of eliciting a robust immune response; however the mechanisms by which VV accomplishes this task remain unknown. The overall goal of this thesis project is to determine how VV activates the innate immune system, and how this activation contributes to viral clearance in vivo. We determined that VV or VV-DNA activated the TLR8-MyD88 pathway in plasmacytoid dendritic cells (pDC), resulting in the production of type I interferons (IFN). We also demonstrated that TLR8-mediated production of type I IFN by pDC was crucial to efficient VV control and clearance in vivo. Moreover, we identified the polyA- and polyT-rich sequences in VV-DNA was the possible motif recognize by TLR8. Type I IFN, known for ability to establish the "antiviral state", are also critical mediators of NK cell activation. In the setting of VV infection, we demonstrated that direct action of type I IFN on NK cells, but not accessory cells such as DC, was necessary for NK cell activation in vivo. We further demonstrated that type I IFN-dependent activation of NK cells was required for optimal VV clearance in vivo. Given the importance of NK cells in anti-VV innate immunity, we next examined what role the TLR2-MyD88 pathway, critical for activation of cDC, played in the activation of NK cells. NK cells from TLR2-/- or MyD88-/- mice displayed a reduction in activation and cytolytic function, and this defect was independent of pro-inflammatory cytokine signaling. We were able to demonstrate that direct TLR2 signaling on NK cells was required for their optimal activation and function in response to VV infection. Moreover, we were able to demonstrate that TLR2-MyD88 signaling resulted in the activation of the PI3K-ERK pathway, which was necessary for NK cell cytotoxicity. In addition, we identified the NKG2D pathway as critical for efficient NK cell activation and function in response to VV infection, independent of the TLR2 pathway. Both the NKG2D and TLR2 pathways were crucial for optimal VV clearance and control in vivo. Collectively, this project illuminates the roles and mechanisms of the innate immune system in the control of VV in vivo.

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell-dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help-dependent, it remains controversial when the "help" is needed. The goal of this thesis project is to determine requirement and mechanisms of CD4 help during the CD8 response to vaccinia viral (VV) infection.

The first aim of this project was to determine when CD4 T cell help is required during the CD8 response to VV infection. Using both CD4-deficient mice and mice with transient depletion of CD4 T cells, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to VV infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to VV infection and may help to design effective vaccine strategies.

Given that CD4 T cell help is critical for the survival of activated CD8 T cells during both the primary and memory recall responses, it is still unclear how CD4 T cell help promotes CD8 T cell survival. The second aim of this project was to determine the mechanism of CD4 help for the survival of activated CD8 cells. We first showed that CD4 help in vitro was mediated by IL-21, a cytokine produced predominantly by activated CD4 T cells. We then demonstrated direct action of IL-21 on CD8 T cells was critical for the VV-specific CD8 T cell response in vivo. This intrinsic IL-21 signaling was essential for the survival of activated CD8 T cells and the generation of long-lived memory cells. We further revealed that IL-21 promoted CD8 T cell survival in a mechanism dependent on activation of the STAT1 and STAT3 pathways and subsequent upregulation of the pro-survival molecules Bcl-2 and Bcl-xL. Collectively, these results identify a critical role for CD4-derived IL-21 signaling in CD8 T cell responses to acute VV infection in vivo and may help design effective vaccine strategies in situations where CD4 cells are not fully functional.

A fraction of HIV-1 patients are able to successfully control the virus and avoid developing AIDS. It has become increasingly clear that variations in the immune response during the initial days of acute infection including the period of peak viral replication determine long term differences in disease outcomes. While the precise factor(s) necessary and sufficient for protection from AIDS is as yet unidentified, a number of factors have been correlated with protection from AIDS. Among these are the presence of a strong proliferative and multifunctional T-cell response as well as the HLA allele status of a patient. Therefore the goal of this thesis project was to 1) broadly identify the major contributors to the proliferative and multifunctional T-cell response during acute infection with HIV, 2) examine the durability of these responses and 3) elucidate the gene regulation pathway(s) by which HLA allele status determines disease outcomes.

In order to identify the major contributors to the proliferative and multifunctional T-cell response to HIV we utilized PBMC samples from a cohort of acutely infected HIV patients in the Duke and University of North Carolina infectious disease clinics. These samples were stimulated in vitro with peptides representing the HIV clade B consensus sequence and the T-cells were analyzed for proliferation and multifunctionality. Through this analysis we identified CD4+CD8+ (DP) T-cells as overrepresented within the proliferative response and the primary contributor to multifunctionality. Additionally, the acute multifunctional T-cell response was highly focused on the Nef, Rev, Tat, VPR and VPU sections of the HIV proteome. We also discovered similar response patterns among a cohort of HIV controllers recruited from the Duke infectious disease clinic. In fact, the frequency of multifunctional DP T-cells was inversely correlated with viral loads among the controller cohort.

Having identified DP T-cells as HIV responding cells of interest, we next examined their durability following the removal of widespread antigenic stimulation via administration of HAART. Utilizing longitudinal samples from the acute HIV cohort we again examined T-cell proliferation and multifunctionality at approximately 24 weeks and 104 weeks post infection among patients. This experiment demonstrated that among patients who initiated HAART during acute infection there was a significant reduction in the frequency of multifunctional DP T-cells at 24 and 104 weeks post infection compared to study entry. Meanwhile the proliferative DP T-cell response was maintained longitudinally. Additionally, these patients did not exhibit the previously described increase in frequency of multifunctional CD8 T-cells as infection progressed to the chronic phase. Although the majority of patients initiated HAART during the acute stage of infection, a minority delayed HAART initiation for various lengths up to and including study cessation. Among this group of patients the frequency of multifunctional DP T-cells was maintained longitudinally. Therefore, the early initiation of HAART reduces long term frequencies of multifunctional DP T-cells while delayed HAART initiation leads to a durable multifunctional DP T-cell response. Since HIV controllers with higher frequencies of multifunctional DP T-cells maintain lower viral loads, early HAART initiation may be detrimental to the development of immune cells capable of controlling the virus.

Finally, we examined the effect HLA alleles have on gene regulation during the initial interactions between HIV and the host immune system. This work employed 2 HIV negative patient cohorts. One cohort expressed HLA-B*35 which has previously been shown to correlate with rapid progression to AIDS following infection with HIV. The second cohort expressed HLA-B*57 which has been associated with long term non-progression following infection with HIV. PBMCs from each group were infected with HIV in vitro. Twenty-four hours after infection these cells were sorted into CD4+ T-cells, CD8+ T-cells and NK-cells. Following cell sorting, mRNA was isolated and interrogated for expression changes using whole genome microarrays. This analysis revealed HLA allele specific differences in the magnitude by which CD4+ T-cells, CD8+ T-cells and NK-cells activate the interferon response pathway following exposure to HIV.

In total, these findings provide insight into the cell types responsible for significant portions of the acute immune response to HIV and the mechanisms by which individuals protected from progression to AIDS differ from their peers.

The mammalian urinary bladder is a highly specialized organ that must be able to withstand considerable amounts of osmotic pressure at its mucosal surface, in addition to maintaining an impenetrable barrier against potential pathogens. The lower urinary tract's virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected with uropathogenic E.coli (UPEC) in combination with clinical observations that antibody responses following bladder infections are not detectable, suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells. This mast cell-dependent phenomenon involves localized production of IL-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of mast cells orchestrating the early innate immune responses in the bladder during infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of mast cell-maintained immune privilege.

Interestingly, though the bladder is not capable of initiating an effective adaptive immune response during bladder infections, we have generated data showing that it was possible to circumvent the immune limitations of the bladder to provoke a strong adaptive and protective immune response by vaccinating against UPEC at an alternate mucosal site. We reasoned that by immunizing the nasal regions of mice with a vaccine formulation comprising of FimH adhesin, a highly conserved adhesive moiety of type 1 fimbriae expressed on UPEC, and an effective mucosal adjuvant we would evoke protective immunity against UPEC infections. We found that a FimH vaccine coupled with either a mast cell activating adjuvant c48/80 or CpG oligodeoxynucleotide, a TLR9 agonist, evoked high levels of FimH specific IgG antibody in the serum and IgA in the urine of immunized mice. We also observed that following UPEC challenge, these FimH/adjuvant immunized mice exhibited significantly reduced bacterial load in the bladders compared to mice challenged with just FimH. These studies reveal that immunization of nasal regions with a FimH vaccine is an effective strategy to overcome the limitation in adaptive immunity observed in the bladder.

Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (TReg) are a cell lineage that develops in the thymus and exits to the periphery, where they represent 5-10% of the peripheral CD4+ T cell population. Phenotypically, they are characterized by the expression of the cell surface markers CD25, as known as the IL-2 receptor alpha chain, glucocorticoid-induced tumor necrosis factor receptor (GITR), and cytotoxic T-lymphocyte antigen-4 (CTLA-4), as well as forkhead box P3 (Foxp3), a transcription factor considered to be the most specific TReg marker. Functionally, TReg cells are defined by their ability to suppress the activation of multiple cell types including CD4+ and CD8+ T cells, B cells, natural killer (NK) cells, and dendritic cells (DCs). Suppression can be achieved by the production of immunosuppressive cytokines or direct cell-to-cell contact, with these mechanisms directly affecting suppressed cells or indirectly affecting them by modulating antigen presenting cells (APCs). The suppressive abilities of TReg cells are crucial in maintaining dominant tolerance--the active, trans-acting suppression of the immune system for the prevention of autoimmune diseases. In addition to preventing autoimmune diseases, studies have also demonstrated critical roles for TReg cells in down-modulating anti-tumor immunity, suppressing allergic diseases, such as asthma, and achieving transplant tolerance. Recent studies have also demonstrated roles for TReg cells during pathogen infection, which will be the focus of this thesis.

Studies examining TReg cells during infection have largely focused on chronic infection models. These studies have shown that TReg cells can affect responses to pathogens in various ways that can be beneficial or detrimental for either the host or the invading pathogen. In some infections, TReg cells downregulation effector responses, which can lead to pathogen persistence and, in some cases, concomitant immunity. TReg cell-mediated suppression can also reduce immunopathology at sites of infection, which can occur as a result of a vigorous anti-pathogen immune response.

In contrast to chronic infection, how TReg cells behave and function following acute infections remains largely unknown as, to date, very few studies have been conducted. Current work with acute infection models has indicated that TReg cells affect immune responses in some acute infection models, but not in all. Furthermore, the results of these studies have implicated that current approaches to examine TReg cells during acute infection by depleting the total TReg cell repertoire, as opposed to targeting pathogen-specific TReg cells, may not be ideal. Finally, it is unclear what happens to activated TReg cells following the resolution of infection.

Due to the lack of knowledge about the role of pathogen-specific TReg cells during acute infection, we sought to employ a different approach to address some of the outstanding questions in the field. Here, we utilized CD4+ non-TReg and TReg cells from T cell receptor (TCR) transgenic mice that recognize a pathogen-specific epitope found in three different models of acute viral infection: recombinant vaccinia virus, recombinant adenovirus, and influenza. Using this model system, we were able to track pathogen-specific TReg cells following acute viral infection to determine their kinetics during the course of infection, as well as their influence on CD4+ non-TReg cells during different times after infection. We also employed major histocompatibility complex (MHC) Class II tetramer technology to track the fate of endogenous pathogen-specific TReg cells following infection with influenza.

Using these models systems, we show that pathogen-specific TReg cells can be activated and expand upon acute viral infections in vivo. The activated TReg cells then contract to form a "memory" pool after resolution of the infection. These "memory" TReg cells expand rapidly upon a secondary challenge, secrete large amounts of IL-10, and suppress excessive immunopathology, which is elicited by the expansion of non-TReg cells, via an IL-10-dependent mechanism. The work presented in this thesis reveals a previously unknown "memory" TReg cell population that develops after acute viral infections and may help design effective strategies to circumvent excessive immunopathology.