Faculty Bibliography

The immune system recognizes protein antigens expressed in transformed cells evidenced by accumulation of antigen-specific T cells in tumor and tumor draining lymph nodes. However, despite demonstrable immune response, cancers grow progressively suggesting that priming of antitumor immunity is insufficiently vigorous or that antitumor immunity is suppressed, or both. Compared to virus infection, antitumor T cells are low abundance that likely contributes to tumor escape and enhancement of priming is a long-sought goal of experimental vaccination therapy. Furthermore, patient treatment with antigen-specific T cells can in some cases overcome deficient priming and cause tumor regression supporting the notion that low numbers of T cells permits tumor outgrowth. However, tumor-induced suppression of antitumor immune response is now recognized as a significant factor contributing to cancer growth and reversal of the inhibitory influences within the tumor microenvironment is a major research objective. Multiple cell types and factors can inhibit T cell functions in tumors and may be grouped in two general classes: T cell intrinsic and T cell extrinsic. T cell intrinsic factors are exemplified by T cell expression of cell surface inhibitory signaling receptors that, after contact with cells expressing a cognate ligand, inactivate proximal T Cell Receptor-mediated signal transduction therein rendering T cells dysfunctional. T cell extrinsic factors are more diverse in nature and are produced by tumors and various non-tumor cells in the tumor microenvironment including: regulatory T cells, B cells, myeloid-derived cells, cancer associated fibroblasts, pericytes, mast cells, or vascular endothelia. These include proteins secreted by tumor or stromal cells (some which may be retained in the extracellular matrix), highly reactive soluble oxygen and nitrogen species, cytokines, chemokines, gangliosides, and toxic metabolites. These factors may restrict T cell entrance into the tumor parenchyma, cause inactivation of effector phase T cell functions, or induce T cell apoptosis ultimately causing diminished cancer elimination. Here, we review the contributions of inhibitory factors to tumor T cell dysfunction leading to tumor escape.

T cells play a critical role in host defense against viruses, intra- and extracellular microbes, and tumors. Because foreign antigen is presented amongst a vast majority of self-antigens, T cells have evolved the unique ability to discriminate "self" from "non-self" with high sensitivity and selectivity, enabling the elimination of foreign pathogens while largely avoiding self-reactivity. However, tissue-specific autoimmunity and tolerance to or eradication of cancer does not fit neatly into the self/non-self paradigm because the T cell responses in these situations are not directed to an exogenous pathogen, but rather most often to non-mutated self-proteins. Therefore, an important question is how the immune system establishes suitable thresholds that allow positively selected T cells to interact with selfligands in the periphery without causing overt activation. One hypothesis to explain how a T cell distinguishes among different types of self-ligands is the kinetic proof-reading theory, which relates signaling efficacy to the lifetime of the TCR (T cell receptor)-pMHC (peptide-major histocompatibility complex) interaction. More recently, T cell maturation associated signaling feedback pathways have also been hypothesized to play a role in T cell discrimination of between self-ligands. We are taking a variety of biophysical and cellular imaging approaches to determine how specific thresholds for T cell recognition of self-antigens are set. Our recent results [1] indicate that antitumor activity and autoimmunity are coupled and have a similar kinetic threshold; reducing autoimmunity cannot be accomplished without sacrificing efficacy of tumor killing. Therefore, an "optimal TCR affinity range" that leads to optimal tumor regression and minimal autoimmunity is elusive and treatment strategies focusing on increasing TCR affinities to a supraphysiological level has most likely little therapeutic benefit. Therefore, other approaches are needed to improve the balance between anti-tumor responses and autoimmunity. Our strategy to overcome this issue includes novel methods for careful biophysical engineering of tumor-specific TCRs to carefully balance tumorreactivity and autoimmunity. Furthermore, our recent preliminary data show that TCR-proximal signaling differs significantly between effector memory and central memory T cells due to differential constitutive activity and localization of signaling molecules. Understanding how activation signaling contributes to differences in memory T cell subset sensitivity may provide insight into how T cells can be manipulated to achieve optimal anti-tumor sensitivity. This could lead to adjuvants that target and enhance antigenspecific T cell anti-tumor efficacy. Together may lead to development of cancer immunotherapy approaches with improved outcomes

BACKGROUND & AIMS: Immune cells of the liver must be able to recognize and react to pathogens yet remain tolerant to food molecules and other nonpathogens. Dendritic cells (DCs) are believed to contribute to hepatic tolerance. Lipids have been implicated in dysfunction of DCs in cancer. Therefore, we investigated whether high lipid content in liver DCs affects induction of tolerance. METHODS: Mouse and human hepatic nonparenchymal cells were isolated by mechanical and enzymatic digestion. DCs were purified by fluorescence-activated cell sorting or with immunomagnetic beads. DC lipid content was assessed by flow cytometry, immune fluorescence, and electron microscopy and by measuring intracellular component lipids. DC activation was determined from surface phenotype and cytokine profile. DC function was assessed in T-cell, natural killer (NK) cell, and NKT cell coculture assays as well as in vivo. RESULTS: We observed 2 distinct populations of hepatic DCs in mice and humans based on their lipid content and expression of markers associated with adipogenesis and lipid metabolism. This lipid-based dichotomy in DCs was unique to the liver and specific to DCs compared with other hepatic immune cells. However, rather than mediate tolerance, the liver DC population with high concentrations of lipid was immunogenic in multiple models; they activated T cells, NK cells, and NKT cells. Conversely, liver DCs with low levels of lipid induced regulatory T cells, anergy to cancer, and oral tolerance. The immunogenicity of lipid-rich liver DCs required their secretion of tumor necrosis factor alpha and was directly related to their high lipid content; blocking DC synthesis of fatty acids or inhibiting adipogenesis (by reducing endoplasmic reticular stress) reduced DC immunogenicity. CONCLUSIONS: Human and mouse hepatic DCs are composed of distinct populations that contain different concentrations of lipid, which regulates immunogenic versus tolerogenic responses in the liver.

Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-beta. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1(low). We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease.

The transition of chronic pancreatic fibroinflammatory disease to neoplasia is a primary example of the paradigm linking inflammation to carcinogenesis. However, the cellular and molecular mediators bridging these entities are not well understood. Because TLR4 ligation can exacerbate pancreatic inflammation, we postulated that TLR4 activation drives pancreatic carcinogenesis. In this study, we show that lipopolysaccharide accelerates pancreatic tumorigenesis, whereas TLR4 inhibition is protective. Furthermore, blockade of the MyD88-independent TRIF pathway is protective against pancreatic cancer, whereas blockade of the MyD88-dependent pathway surprisingly exacerbates pancreatic inflammation and malignant progression. The protumorigenic and fibroinflammatory effects of MyD88 inhibition are mediated by dendritic cells (DCs), which induce pancreatic antigen-restricted Th2-deviated CD4(+) T cells and promote the transition from pancreatitis to carcinoma. Our data implicate a primary role for DCs in pancreatic carcinogenesis and illustrate divergent pathways in which blockade of TLR4 signaling via TRIF is protective against pancreatic cancer and, conversely, MyD88 inhibition exacerbates pancreatic inflammation and neoplastic transformation by augmenting the DC-Th2 axis.

CD8(+) tumor infiltrating T cells (TIL) lack effector-phase functions due to defective proximal TCR-mediated signaling previously shown to result from inactivation of p56(lck) kinase. We identify a novel interacting partner for p56(lck) in nonlytic TIL, Protocadherin-18 ('pcdh18'), and show that pcdh18 is transcribed upon in vitro or in vivo activation of all CD8(+) central memory T cells (CD44(+)CD62L(hi)CD127(+)) coincident with conversion into effector memory cells (CD44(+)CD62L(lo)CD127(+)). Expression of pcdh18 in primary CD8(+) effector cells induces the phenotype of nonlytic TIL: defective proximal TCR signaling, cytokine secretion, and cytolysis, and enhanced AICD. pcdh18 contains a motif (centered at Y842) shared with src kinases (QGQYQP) that is required for the inhibitory phenotype. Thus, pcdh18 is a novel activation marker of CD8(+) memory T cells that can function as an inhibitory signaling receptor and restrict the effector phase.

Myeloid-Derived Suppressor Cells (MDSC) are immature myeloid cells that are potent inhibitors of immune cell function and which accumulate under conditions of inflammation, especially cancer. MDSC are suggested to promote the growth of cancer by both enhancement of tumor angiogenesis and metastasis and also inhibition of antitumor immune responses. The presence of deficient and/or defective antitumor adaptive and innate immune responses, coincident with accumulation of MDSC in lymphoid organs and tumor parenchyma, supports the notion of a causal relationship. The potent ability of MDSC to inhibit several components and phases of immune response highlights the likelihood that targeting the inhibitory functions of MDSC may maximize the therapeutic potential of antitumor immunotherapy. In order to guide the rational development of immunotherapeutic strategies that incorporate inhibition of MDSC activity and enzymatic functions, thorough understanding of the role of MDSC in antitumor immune responses is required. In this manuscript we review the multifaceted inhibitory functions of MDSC and consider the role of MDSC-induced inhibition of antitumor T cell effector phase. Support for this research is from NIH R01 CA108573.

BACKGROUND & AIMS: The cellular mediators of acute pancreatitis are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis. METHODS: Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier method. RESULTS: Numbers of major histocompatibility complex II(+)CD11c(+) DCs increased 100-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes. Intrapancreatic DCs acquired a distinct immune phenotype in mice with acute pancreatitis; they expressed higher levels of major histocompatibility complex II and CD86 and increased production of interleukin-6, membrane cofactor protein-1, and tumor necrosis factor-alpha. However, rather than inducing an organ-destructive inflammatory process, DCs were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DCs and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DCs died from acinar cell death within 4 days. Depletion of DCs from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DCs did not require infiltrating neutrophils, activation of nuclear factor-kappaB, or signaling by mitogen-activated protein kinase or tumor necrosis factor-alpha. CONCLUSIONS: DCs are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress

The presence in cancer tissue of Ag-specific, activated tumor infiltrating CD8(+) T cells proves that tumors express Ags capable of eliciting immune response. Therefore, in general, tumor escape from immune-mediated clearance is not attributable to immunological ignorance. However, tumor-infiltrating lymphocytes are defective in effector phase function, demonstrating tumor-induced immune suppression that likely underlies tumor escape. Since exocytosis of lytic granules is dependent upon TCR-mediated signal transduction, it is a reasonable contention that tumors may induce defective signal transduction in tumor infiltrating T cells. In this review, we consider the biochemical basis for antitumor T cell dysfunction, focusing on the role of inhibitory signaling receptors in restricting TCR-mediated signaling in tumor-infiltrating lymphocytes