Gram-positive bacterial infections have risen over recent years and current antibiotic treatments are not always sufficient to control these infections. Specifically, antibiotics target bacteria themselves, but not the bacterially secreted proteins that contribute to bacterial pathogenesis and host tissue damage (i.e. virulence factors). These virulence factors may linger after bacteria are eradicated, making their interaction with the host important to understand for the development of novel therapeutics to supplement antibiotics. One class of virulence factors studied in our laboratory is a large pore-forming toxin family known as the cholesterol-dependent cytolysins (CDC). These exotoxins are secreted by over twenty species of gram-positive bacteria and have been shown to contribute to the virulence of the bacteria that secrete them. We are interested in exploring the pathways initiated by CDC in host innate immune cells such as macrophages and dendritic cells. These cells would be expected to first encounter CDC after bacterial infection and therefore, pathways initiated in these cells by CDC could be targeted for the benefit of the host. We have characterized the mechanism of mature IL-1beta secretion induced by CDC tetanolysin O (TLO) from LPS-primed murine bone marrow-derived macrophages (BMDM). This process is dependent on TLO dose and relies on the caspase-1-containing NLRP3 inflammasome as well as associated signaling pathways, which include ion fluxes and iPLA2 and cathepsin B activities. Furthermore, TLO induces different cell death programs in BMDM that are dependent on TLO dose. High TLO doses induce conventional necrotic cell death while low TLO doses cause NLRP3 inflammasome-dependent and cathepsin B-dependent necrotic cell death that is characterized by lactate dehydrogenase (LDH) and high mobility group box 1 (HMGB1) release. Both IL-1beta and HMGB1 are pro-inflammatory cytokines that contribute to inflammation and may be useful therapeutic targets, in addition to the inflammasome. Finally, susceptibility to CDC-induced cell killing varies based on cell type. In order to determine pathways that might explain these differences, we created a variant murine dendritic cell line resistant to pore formation. Though this cell line has been characterized to some degree, future studies will be needed to pinpoint the pathways responsible for the phenotype observed.

There is a growing body of evidence indicating that the nervous and immune systems cross-talk during inflammatory and immune responses. Secretion of pro-inflammatory neuropeptides from the tachykinin family, including substance P (SP) and hemokinin-1 (HK-1), favors cellular immunity by binding the neurokinin 1 receptor (NK1R) to promote immune cell survival and activation. Dendritic cells (DCs) are essential for the stimulation of cellular immunity; however, the ability of pro-inflammatory tachykinins to affect the immune-stimulatory function of DCs remains elusive. Since DCs home strategically to peripheral and lymphoid tissues where tachykinins are secreted, we hypothesized that signaling via the NK1R enhances DC longevity and their T cell-stimulatory function, including the induction of type-1 CD4+ T cell helper (Th1) and CD8+ cytotoxic T cell (CTL/Tc1) responses. Using DCs generated from murine bone marrow precursors (BMDCs), I describe that BMDCs express functional NK1R, and agonistic signaling via the receptor rescues BMDCs from apoptosis. The immunological relevance of these findings were validated in vivo, as I demonstrate that adoptive transfer of NK1R-signaled BMDCs loaded with antigen (Ag) migrate efficiently to tissue-draining lymph nodes (DLNs) where they survive longer and induce superior type-1 DTH responses compared to adoptive transfer of control Ag-loaded BMDCs. Secondly, I investigated the mechanisms by which NK1R-signaled BMDCs favor cellular immunity, including their ability to generate Th1 and CTL/Tc1 responses. I show that agonistic signaling via the NK1R promotes the maturation of BMDCs and inhibits their secretion of IL-10, and adoptive transfer of NK1R-signaled BMDCs elicits enhanced Ag-specific Th1 and CTL/Tc1 responses. The individual roles of adoptively transferred NK1R-signaled BMDCs and endogenous DCs were further addressed by comparing the development of type-1 immunity in wild-type, IL-12 knockout (IL-12p35-/-) and Diphtheria Toxin Recetor (DTR) transgenic (inducible depletion of CD11c+ DCs) mice. With these models, I demonstrate that generation of robust Ag-specific Th1 and CTL/Tc1 responses requires secretion of IL-12p70 by endogenous DCs and inhibition of IL-10 production by transferred BMDCs. Collectively, our data strongly suggest that adoptive transfer of NK1R-signaled BMDCs promotes enhanced type-1 immunity by mechanisms involving both exogenous and endogenous DC populations.

The etiologic agent of Chagas disease is Trypanosoma cruzi. Patent parasitemia leads to parasite spread throughout the host during acute phase disease. Parasitemia concomitant with polyclonal lymphocyte activation has been reported and is thought to contribute to parasite evasion of host immunity and subsequent parasite persistence, which leads to chronic disease. In the present studies, polyclonal B cell activation was evaluated in relatively susceptible Balb/c versus resistant C57Bl/6 mouse models. Hypergammaglobulinemia and B cell activation in susceptible mice was associated with a large number of antibody secreting cells (ASC) without appreciable parasite-specific ASC. In contrast, in resistant mice, B cell activation and expansion was associated with generation of parasite-specific humoral immunity. These data indicate that the outcome of B cell activation during early T. cruzi experimental infection varies according to host susceptibility. T. cruzi encodes several proteins with mitogenic capacities that are thought to contribute to dysfunctional polyclonal B cell activation in susceptible mice. One recently identified T. cruzi mitogen is a proline racemase (TcPRAC). Characterization of B cell activation by recombinant protein in this study demonstrates that TcPRAC induced polyclonal B cell activation, evident by proliferation, antibody secretion, IL-10 production, and B cell surface phenotype. MZ B cells were more responsive to T-cell independent TcPRAC stimulation than were follicular mature (FM) B cells. These data provide the first comprehensive characterization of B cell activation by TcPRAC. During experimental T. cruzi infection, TcPRAC-specific IgG remained undetectable. Conversely, intradermal genetic immunization via gene-gun (GG) induced antigen-specific immunogenic responses, generating TcPRAC-specific high-titer IgG, bone marrow plasma cells, and memory B cells. TcPRAC-specific IgG bound mitogenic rTcPRAC, decreasing subsequent B cell activation. GG immunization with TcPRAC DNA was non-mitogenic and did not effect generation of specific IgG to another T. cruzi antigen, complement regulatory protein (CRP). These data demonstrate the utility of genetic immunization for the conversion of a protein mitogen into an effective immunogen. Furthermore, co-immunization of TcPRAC with another T. cruzi antigen indicated the usefulness of this approach for multivalent vaccine development.

Herpes Stromal Keratitis (HSK) is one of the leading causes of infectious blindness in the developed world. HSK is characterized by corneal damage and scarring that results from a Th1 cytokine-mediated immunopathology. It is triggered by reactivation of HSV-1 from latency in sensory neurons of the trigeminal ganglion (TG) and its subsequent transport to the eye through axons that innervate the cornea. My thesis work explored key components of both the HSV-specific CD8+ T cell response that maintains HSV-1 latency in sensory neurons, and the opposing roles of the immune system in controlling HSV-1 replication in the cornea and promoting the immunopathology that results in corneal scarring and blindness. We hypothesized and provide strong supporting evidence that CD4+ T cells act as a double-edged sword, on the one hand providing critical help to HSV-specific CD8+ T cells that enables them to control viral latency through avoidance of exhaustion within the infected TG. On the other hand, we employed HSV-specific CD4+ T cell clones to explore their role in orchestrating the immunopathology associated with HSK. We also explored the Ying and Yang of dendritic cell involvement in HSK. While our previous studies suggested a role for DC in promoting HSK, we now employ a unique localized DC depletion model to establish that first responding DCs are critical in mobilizing both the innate and adaptive immune response that clears virus from the cornea. Finally, we employed newly available recombinant mouse strains lacking IL-12p40, p35, or both p40 and p35 to explore the involvement of the IL-12 cytokine family in HSK. The heterodimeric cytokines IL-12 (p40/p35) and IL-23 (40/p19) are thought to regulate HSK by promoting production of the requisite TH1 cytokine IFN-. However, we find that HSK development does not require IL-12p40 and is thus independent of IL-12 and IL-23. Instead, HSK late progression actually requires a previously unrecognized IL-12p40-independent, proinflammatory function of IL-12p35.

Potent anti-retroviral therapy has transformed HIV infection from an acute to a chronic disease. Consequently, diseases previously not prevalent in HIV+ persons have emerged. For example, HIV-infected persons are at increased risk for developing COPD. Pneumocystis (Pc), a fungal opportunistic pathogen, has been associated with HIV and COPD. Pc colonization- the presence of Pc in subjects without clinical symptoms of Pneumocystis pneumonia- is increased in COPD patients. Furthermore, HIV+ individuals are at elevated risk for both Pc colonization and emphysema. Together, these observations suggest that COPD in HIV+ individuals involves Pc colonization. We used a simian/human immunodeficiency virus (SHIV) model of HIV infection to study pulmonary effects of Pc colonization. SHIV-infected/Pc-colonized monkeys developed obstructive pulmonary disease characterized by increased emphysematous tissue and bronchial-associated lymphoid tissue. Elevated Th2 cytokines and pro-inflammatory mediators in bronchoalveolar lavage fluid coincided with Pc colonization and pulmonary function decline. These results indicate that Pc colonization may be a risk factor for development of HIV-associated COPD. Gene expression profiles in the lung tissue of these animals evaluated by microarray analysis revealed differential expression of 243 genes in the obstructed SHIV/Pc monkeys compared to SHIV-only monkeys with normal lung function. Potentially relevant differentially expressed genes included genes involved in inflammation, protease/antiprotease balance, redox balance and tissue homeostasis, thus indentifying factors and pathways involved in early development of SHIV-associated COPD and revealing several novel, possible therapeutic targets. In a second cohort of animals, airway obstruction development associated with Pc colonization was recapitulated. To directly correlate pulmonary function decline with presence of Pc, a subset of the Pc-colonized monkeys was treated with the anti-Pc drug, TMP-SMX, after significant airway obstruction had occurred. No further pulmonary function decline was observed in either the treated or untreated animals up to a year after initiating TMP-SMX treatment. These results indicate that Pc-associated induction of airway obstruction takes place early after onset of colonization followed by an extended period of containment of the effects of Pc. These results demonstrate a key role for Pc in the early development of SHIV-associated COPD. Furthermore, they reveal multiple potential mediators of Pc-induced airway obstruction.

According to the American Cancer Society, over 1.5 million new cancer cases will be diagnosed this year, a figure that is expected to rise with the aging population. Chemotherapy and radiation are the current gold standards for cancer treatment, but these therapies are marginally effective, toxic, and serve to diminish the quality of life for cancer patients. Immunotherapy represents an attractive alternative to these traditional treatment regimens. Despite overwhelming evidence that the immune system is capable of recognizing and eliminating tumors, both spontaneously and in response to immune-based therapy, such protection is abrogated in the face of compensatory immunosuppressive events characteristic of progressive disease. Thus, a major goal of novel immune-based therapies is the coordinate silencing of regulatory circuits and amplification of protective T cell function. While immune modulating reagents that trigger the T cell costimulatory molecules OX40 and GITR are currently being evaluated in early-phase clinical trials, little pre-clinical information is available regarding the efficacy and mechanism(s) of action for these agents in the setting of advanced, well-established disease. To further characterize the molecular, cellular, and treatment-associated consequences of OX40 and GITR engagement, novel agonistic reagents directed against murine OX40 and GITR (ligand-Fc fusion proteins) were recently constructed and characterized in vitro. We now show that the growth of well-established, day 17 sarcomas is significantly inhibited or ablated by a short course of either treatment, with OX40L-Fc demonstrating superior anti-tumor efficacy over GITRL-Fc at comparable dosing. Both treatments were capable of eliminating regulatory T cells within tumors, inducing profound proliferation of T effector cells in the tumor-draining lymph node, and promoting the recruitment of these expanded effector cells to the tumor microenvironment. However, OX40L-Fc therapy mediated additional, T cell-independent effects, including the activation of tumor-localized dendritic and endothelial cell subsets. These changes rendered the tumor microenvironment more immunogenic and permissive to the infiltration of treatment-induced, protective immune cells. The pleiotropic anti-tumor effects demonstrated in this model by OX40L-Fc, and to a lesser extent GITRL-Fc, strongly supports the further translation of such modalities into human clinical trials, either as single agents or in the context of combinational immunotherapy.

Squamous cell carcinoma of the head and neck (SCCHN) cells can escape recognition and lysis by tumor antigen (TA)-specific cytotoxic T lymphocytes (CTL) by downregulation of antigen processing machinery (APM) components, such as the transporter associated with antigen processing (TAP)-1/2 heterodimer. APM component upregulation by interferon gamma (IFN-g) restores SCCHN cell susceptibility to lysis by CTL, but the mechanism underlying TAP1/2 downregulation in SCCHN cells is not known. Because IFN-g activates signal transducer and activator of transcription (STAT)-1, we investigated phosphorylated (p)-STAT1 as a mediator of low basal TAP1/2 expression in SCCHN cells. SCCHN cells were found to express basal total STAT1 but low to undetectable levels of pSTAT1. The association of increased pSTAT1 levels and APM components likely reflects a cause-effect relationship, since STAT1 knockdown significantly reduced both IFN-g-mediated APM component expression and TA-specific CTL recognition of IFN-g treated SCCHN cells. On the other hand, since oncogenic pSTAT3 is overexpressed in SCCHN cells and was found to heterodimerize with pSTAT1, we also tested whether pSTAT3 and STAT1:STAT3 heterodimers inhibited IFN-g-induced APM component expression. First, STAT3 activation or depletion did not affect basal or IFN-g induced expression of pSTAT1 and APM components, or recognition of SCCHN cells by TA-specific CTL. Second, STAT1:STAT3 heterodimers did not interfere with IFN-g induced STAT1 binding to the TAP1 promoter or APM component protein expression. These findings demonstrate that APM component downregulation is regulated primarily by an IFN-g-pSTAT1-mediated signaling pathway, independent of STAT3 in SCCHN cells. Interestingly, treatment of SCCHN cells with a broad phosphatase inhibitor, sodium orthovanadate, increased pSTAT1 levels, suggesting that a phosphatase might be responsible for maintaining low basal pSTAT1 and APM component levels, as a mechanism for CTL escape by tumor cells. Indeed, immunohistochemical analyses of 14 SCCHN tumors and paired adjacent normal mucosa demonstrated that src homology-2 domain-containing phosphatase (SHP2) was significantly upregulated in the tumor tissue compared to surrounding mucosa. Moreover, SHP2 specific knockdown using siRNA resulted in significant upregulation of pSTAT1, APM components, and HLA class I in SCCHN cells. Furthermore, SHP2 depletion restored the recognition of SCCHN cells by TA-specific CTL, and induced secretion of Regulation upon Activation, Normal T cell Expressed, and presumably Secreted (RANTES) and IFN-g-inducible protein 10 (IP10). These novel findings identify SHP2 as an important molecular regulator contributing to low basal pSTAT1 levels and APM-mediated immune escape in SCCHN cells, and provide a potential inhibitory strategy for enhancing the clinical activity of T cell-based immunotherapy.

Dendritic cells (DCs) are integral to differentiation of T helper cells into Th1, Th2 and Th17 subsets. We have dissected two novel pathways in DCs that specifically regulate CD4 T cell responses. The first is the role of the c-Kit-Phosphatidyl inositol 3 kinase (PI3 kinase)-interleukin-6 (IL-6) axis and the second that of vascular endothelial growth factor (VEGF). IL-6 plays a central role in regulating CD4 T cell immune responses by limiting a Th1 response and promoting Th2 and Th17 responses. We investigate pathways in DCs that promote IL-6 production and show that the allergen house dust mite or the mucosal adjuvant cholera toxin but not Th1-inducing adjuvant, CpG oligodeoxynucleotide (ODN) promote cell surface expression of c-kit and its ligand, stem cell factor (SCF), in DCs. This dual upregulation of c-kit and SCF results in sustained PI3-kinase signaling promoting IL-6 secretion. Intranasal administration of antigen into c-kit mutant mice or neutralization of IL-6 blunted Th2 and Th17 but promoted Th1 responses in lung-draining lymph nodes. DCs lacking functional c-kit elicit diminished allergic airway inflammation when adoptively transferred into mice. Expression of the Notch ligand, Jagged-2, which has been associated with Th2 differentiation, was reduced in DCs from c-kit mutant mice. DCs generated from mice expressing a catalytically inactive form of the p110Ô (p110D910A) subunit of PI3-kinase secrete lower levels of IL-6 upon stimulation with CT. These results collectively highlight the importance of the c-kit-PI3-kinase-IL-6 signaling axis in DCs in regulating T cell responses. We also investigated mechanisms underlying the production of VEGF, which has been recently shown to be a Th2-skewing cytokine and to promote allergic asthma. We found that CT-stimulated DCs secrete high levels of VEGF while LPS induces minimal VEGF production. Activation of iNOS, NF-ÛB and PI3 kinase enhanced production of VEGF in DCs whereas IL-12, a Th1-skewing cytokine, inhibited VEGF production. This mechanism highlights a critical but previously unknown role for DC-derived VEGF. Taken together, these findings broaden our understanding of diverse mechanisms in DCs that enable T cell polarization and offer novel targets for therapeutic interventions.

Type 1 diabetes (T1D), a disease characterized by the autoimmune-mediated destruction of the insulin-secreting beta cells of the pancreas, affects approximately 1% of the US population, with an incidence that is increasing at a rate of 3% per year. Beta cell killing is accomplished through various immune-mediated mechanisms, with the production of reactive oxygen species (ROS) contributing to both inflammation and cell death. While previous reports suggested that antioxidant scavenging protected beta cells against ROS-mediated damage, islet-specific over-expression of antioxidants was not fully protective. As systemic elevation in free radical defenses had a positive impact on islet survival, I hypothesize that control of oxidative stress at the level of the immune system will regulate proinflammatory responses. Genetic studies using the ALR mouse have provided strong support for my hypothesis. ALR-derived diabetes resistance and reduced oxidative burst from neutrophils and macrophages, as well as elevated Superoxide Dismutase 1 (SOD1) activity all map to the Suppressor of superoxide production (Susp) locus on Chr. 3. NAPDH oxidase (NOX) function from ALR cells could be rescued with inhibition of SOD1, demonstrating that dissipation was modifying immune effector function. Elevated SOD1 activity was associated with increased dimer stability, suggesting a post-translational modification is enhancing dimerization. Introduction of Susp into the NOD background was highly protective against T1D. This resistance is linked to the loss of T lymphocyte diabetogenic potential. The loss of T cell ROS in T1D protection was confirmed using NOX-deficient NOD mice. T cell lineage commitment and proinflammatory cytokine synthesis were dependent on ROS signaling. Macrophages and T cells from NOD-Ncf1m1J mice exhibited a skewed cytokine response, with increased synthesis of IL-17 and IL-10, as opposed to the predominant IFN-γ production typically observed from NOD lymphocytes. Genome-wide analyses were performed to fine map Susp in order to define the mechanism leading to altered SOD1 activity. Positional cloning experiments mapped Susp between D3Mit180 (34.4 Mbp) and D3Mit223 (34.8 Mbp) on Chr. 3. This mapping defines a novel candidate region involved in the regulation of SOD1 activity and dimerization stability, resulting in reduced superoxide release via NADPH oxidase activity.

MUC1 glycoprotein is overexpressed on the surface of a variety of epithelial tumors and has been under investigation as a target for immunotherapy. A number of cytotoxic lymphocyte clones were generated in our laboratory from breast and pancreatic cancer patients that recognized MUC1 on the surface of tumor cells in a TCR-mediated MHC-unrestricted manner. The purpose of this study was to test the feasibility, efficacy and safety of using MHC-unrestricted MUC1-specific T cell receptor (TCR) gene transfer as a tool for cancer immunotherapy. The TCR £ and £] chains were cloned from one MHC-unrestricted MUC1-specific CTL clone (MA). Various configurations of chimeric TCRs were constructed and were expressed on the surface of a variety of cell lines in vitro. The TCR-deficient T cell line, Jurkat JRT3.5, transfected with the TCR £ and £] chains from MA CTL clone fluxed calcium in response to stimulation by a MUC1+ pancreatic human tumor, HPAF. BWZ murine thymoma cells transfected with a single-chain TCR (scTCR) consisting of the TCR extracellular domain and the CD3 ê signaling domain) were triggered to secrete IL-2 in response to stimulation with different MUC1+ tumor cells. The tumor recognition and rejection functions of this scTCR were tested in vivo when SCID mice were reconstituted with bone marrow (BM) cells transduced with scTCR-MFG retroviral supernatant and challenged with HPAF tumor cells. Tumor growth in mice reconstituted with scTCR-transduced BM cells was significantly slower (P<0.05) than that seen in the control group. Tumor sections from TCR-reconstituted mice were infiltrated by neutrophils and macrophages, and to lesser extent, by NK cells. FACS analyses showed that BM cells transduced with scTCR-MFG could differentiate in vivo into multiple immune lineages including T cells, B cells, granulocytes, monocytes and NK cells that express the scTCR. The scTCR was expressed on higher percentages of cells of the innate immune system when compared to T and B cells. Human MUC1 transgenic (Tg.) mice reconstituted with BM cells transduced with this MUC1-specific TCR did not show any signs of autoimmunity, abnormal cellular infiltration or destruction of MUC1-expressing tissues. Transduction of BM with tumor-specific TCR represents a potentially efficacious gene therapy/immunotherapy approach. MUC1-specific MHC-unrestricted TCR will make this treatment applicable to all cancer patients with MUC1+ tumors, regardless of their HLA type.