Faculty Bibliography

The normal liver is characterized by immunologic tolerance. Primary mediators of hepatic immune tolerance are liver sinusoidal endothelial cells (LSECs). LSECs block adaptive immunogenic responses to Ag and induce the generation of T regulatory cells. Hepatic fibrosis is characterized by both intense intrahepatic inflammation and altered hepatic immunity. We postulated that, in liver fibrosis, a reversal of LSEC function from tolerogenic to proinflammatory and immunogenic may contribute to both the heightened inflammatory milieu and altered intrahepatic immunity. We found that, after fibrotic liver injury from hepatotoxins, LSECs become highly proinflammatory and secrete an array of cytokines and chemokines. In addition, LSECs gain enhanced capacity to capture Ag and induce T cell proliferation. Similarly, unlike LSECs in normal livers, in fibrosis, LSECs do not veto dendritic cell priming of T cells. Furthermore, whereas in normal livers, LSECs are active in the generation of T regulatory cells, in hepatic fibrosis LSECs induce an immunogenic T cell phenotype capable of enhancing endogenous CTLs and generating potent de novo CTL responses. Moreover, depletion of LSECs from fibrotic liver cultures mitigates the proinflammatory milieu characteristic of hepatic fibrosis. Our findings offer a critical understanding of the role of LSECs in modulating intrahepatic immunity and inflammation in fibro-inflammatory liver disease

The liver is the most common site of adenocarcinoma metastases, even in patients who initially present with early disease. We postulated that immune-suppressive cells in the liver of tumor-bearing hosts inhibit anti-tumor T cells, thereby accelerating the growth of liver metastases. Using models of early preinvasive pancreatic neoplasia and advanced colorectal cancer, aims of this study were to determine immune phenotype, stimulus for recruitment, inhibitory effects, and tumor-enabling function of immune-suppressive cells in the liver of tumor-bearing hosts. We found that in mice with intra-abdominal malignancies, two distinct CD11b(+)Gr1(+) populations with divergent phenotypic and functional properties accumulate in the liver, becoming the dominant hepatic leukocytes. Their expansion is contingent on tumor expression of KC. These cells are distinct from CD11b(+)Gr1(+) populations in other tissues of tumor-bearing hosts in terms of cellular phenotype and cytokine and chemokine profile. Liver CD11b(+)Gr1(+) cells are highly suppressive of T cell activation, proliferation, and cytotoxicity and induce the development of Tregs. Moreover, liver myeloid-derived suppressor cells accelerate the development of hepatic metastases by inactivation of cytotoxic T cells. These findings may explain the propensity of patients with intra-abdominal cancers to develop liver metastases and suggest a promising target for experimental therapeutics

Hepatic fibrosis occurs during most chronic liver diseases and is driven by inflammatory responses to injured tissue. Because DCs are central to modulating liver immunity, we postulated that altered DC function contributes to immunologic changes in hepatic fibrosis and affects the pathologic inflammatory milieu within the fibrotic liver. Using mouse models, we determined the contribution of DCs to altered hepatic immunity in fibrosis and investigated the role of DCs in modulating the inflammatory environment within the fibrotic liver. We found that DC depletion completely abrogated the elevated levels of many inflammatory mediators that are produced in the fibrotic liver. DCs represented approximately 25% of the fibrotic hepatic leukocytes and showed an elevated CD11b+CD8- fraction, a lower B220+ plasmacytoid fraction, and increased expression of MHC II and CD40. Moreover, after liver injury, DCs gained a marked capacity to induce hepatic stellate cells, NK cells, and T cells to mediate inflammation, proliferation, and production of potent immune responses. The proinflammatory and immunogenic effects of fibrotic DCs were contingent on their production of TNF-alpha. Therefore, modulating DC function may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease

Mononuclear phagocytes (MPCs) at the tumor site can be divided into subclasses, including monocyte-lineage myeloid-derived suppressor cells (MDSCs) and the immunosuppressive tumor-infiltrating macrophages (TIMs). Cancer growth coincides with the expansion of MDSCs found in the blood, secondary lymphoid organs, and tumor tissue. These MDSCs are thought to mature into macrophages and to promote tumor development by a combination of growth-enhancing properties and suppression of local antitumor immunoresponses. As little is known about either subset of MPCs, we investigated MPCs infiltrating into murine adenocarcinoma MCA38 tumors. We found that these MPCs displayed immunosuppressive characteristics and a MDSC cell-surface phenotype. Over 70% of the MPCs were mature (F4/80(+)Ly6C(-)) macrophages, and the rest were immature (F480(+) Ly6C(+)) monocytes. MPC maturation was inhibited when the cells infiltrated a tumor variant expressing IL-2 and soluble TNF type II receptor (sTNFRII). In addition, the IL-2/sTNFRII MCA38 tumor microenvironment altered the MPC phenotype; these cells did not survive culturing in vitro as a result of Fas-mediated apoptosis and negligible M-CSFR expression. Furthermore, CD4(+) tumor-infiltrating lymphocytes (TILs) in wild-type tumors robustly expressed IL-13, IFN-gamma, and GM-CSF, and CD4(+) TILs in IL-2/sTNFRII-expressing tumors expressed little IL-13. These data suggest that immunotherapy-altered Th cell balance in the tumor microenvironment can affect the differentiation and maturation of MPCs in vivo. Furthermore, as neither the designation MDSC nor TIM can sufficiently describe the status of monocytes/macrophages in this tumor microenvironment, we believe these cells are best designated as MPCs

The immune response to cancer has been long recognized, including both innate and adaptive responses, showing that the immune system can recognize protein products of genetic and epigenetic changes in transformed cells. The accumulation of antigen-specific T cells within the tumor, the draining lymph node, and the circulation, either in newly diagnosed patients or resultant from experimental immunotherapy, proves that tumors produce antigens and that priming occurs. Unfortunately, just as obviously, tumors grow, implying that anti-tumor immune responses are either not sufficiently vigorous to eliminate the cancer or that anti-tumor immunity is suppressed. Both possibilities are supported by current data. In experimental animal models of cancer and also in patients, systemic immunity is usually not dramatically suppressed, because tumor-bearing animals and patients develop T-cell-dependent immune responses to microbes and to either model antigens or experimental cancer vaccines. However, inhibition of specific anti-tumor immunity is common, and several possible explanations of tolerance to tumor antigens or tumor-induced immunesuppression have been proposed. Inhibition of effective anti-tumor immunity results from the tumor or the host response to tumor growth, inhibiting the activation, differentiation, or function of anti-tumor immune cells. As a consequence, anti-tumor T cells cannot respond productively to developmental, targeting, or activation cues. While able to enhance the number and phenotype of anti-tumor T cells, the modest success of immunotherapy has shown the necessity to attempt to reverse tolerance in anti-tumor T cells, and the vanguard of experimental therapy now focuses on vaccination in combination with blockade of immunosuppressive mechanisms. This review discusses several potential mechanisms by which anti-tumor T cells may be inhibited in function

CD8(+) tumor-infiltrating lymphocytes (TIL) lack in vivo and in vitro lytic function due to a signaling deficit characterized by failure to flux calcium or activate tyrosine kinase activity upon contact with cognate tumor cells. Although CD3 zeta is phosphorylated by conjugation in vitro with cognate tumor cells, showing that TIL are triggered, PLC gamma-1, LAT, and ZAP70 are not activated and LFA-1 is not affinity-matured, and because p56(lck) is required for LFA-1 activation, this implies that the signaling blockade is very proximal. Here, we show that TIL signaling defects are transient, being reversed upon purification and brief culture in vitro, implying a fast-acting 'switch'. Biochemical analysis of purified nonlytic TIL shows that contact with tumor cells causes transient activation of p56(lck) ( approximately 10 s) which is rapidly inactivated. In contrast, tumor-induced activation of p56(lck) in lytic TIL is sustained coincident with downstream TCR signaling and lytic function. Shp-1 is robustly active in nonlytic TIL compared with lytic TIL, colocalizes with p56(lck) in nonlytic TIL, and inhibition of Shp-1 activity in lytic TIL in vitro blocks tumor-induced defective TIL cytolysis. Collectively, our data support the notion that contact of nonlytic TIL with tumor cells, and not with tumor-infiltrating myeloid-derived suppressor cells, causes activation of Shp-1 that rapidly dephosphorylates the p56(lck) activation motif (Y394), thus inhibiting effector phase functions

Lytic granule exocytosis is the major pathway used by CD8+ CTL to kill virally infected and tumor cells. Despite the obvious importance of this pathway in adaptive T cell immunity, the molecular identity of enzymes involved in the regulation of this process is poorly characterized. One signal known to be critical for the regulation of granule exocytosis-mediated cytotoxicity in CD8+ T cells is Ag receptor-induced activation of protein kinase C (PKC). However, it is not known which step of the process is regulated by PKC. In addition, it has not been determined to date which of the PKC family members is required for the regulation of lytic granule exocytosis. By combination of pharmacological inhibitors and use of mice with targeted gene deletions, we show that PKCdelta is required for granule exocytosis-mediated lytic function in mouse CD8+ T cells. Our studies demonstrate that PKCdelta is required for lytic granule exocytosis, but is dispensable for activation, cytokine production, and expression of cytolytic molecules in response to TCR stimulation. Importantly, defective lytic function in PKCdelta-deficient cytotoxic lymphocytes is reversed by ectopic expression of PKCdelta. Finally, we show that PKCdelta is not involved in target cell-induced reorientation of the microtubule-organizing center, but is required for the subsequent exocytosis step, i.e., lytic granule polarization. Thus, our studies identify PKCdelta as a novel and selective regulator of Ag receptor-induced lytic granule polarization in mouse CD8+ T cells

It is well known that tumor necrosis factor (TNF) can have both contrary and pleiotropic effects in anti-tumor immune response. In the present study, we prepared two different tumor cell-based immunotherapy models: MCA38 adenocarcinoma and GL261 glioma intracranial interleukin-2 (IL-2)-based. Each tumor was transfected to express IL-2 with or without expression of the soluble form of tumor necrosis factor receptor type II (sTNFRII). Although mice in which TNF is blocked survive longer than IL-2 alone (35.2 versus 26 days), the reverse was observed for GL261 glioma. The differential effect on tumor growth implies enhanced TNF sensitivity of GL261 compared to MCA38. This notion is supported by the observation that TNF induces apoptosis in GL261 but not MCA38 tumors. We further examined tumor infiltrating CD11b+F4/80+ macrophages (or tumor-associated macrophages: TAM) for TNF production in vivo and found that TAM express cell surface TNF implying a role in eliminating glioma cells mediated by the cell surface form of TNF

Inflammation resultant from tumor growth, infection with certain pathogens, or in some cases, trauma, can result in systemic release of cytokines, especially GM-CSF, that in turn stimulate the abundant production and activation of a population of immature myeloid cells, termed myeloid suppressor cells (MSCs), that have potent immunosuppressive functions. In this issue of the JCI, Gallina and colleagues have illuminated some complex issues concerning the development, activation, and function of MSCs (see the related article beginning on page 2777). They show that activation of MSCs is initiated in response to IFN-gamma, presumably produced in situ by antitumor T cells in the tumor microenvironment. After this triggering event, MSCs express 2 enzymes involved in l-arginine metabolism, Arginase I and iNOS, whose metabolic products include diffusible and highly reactive peroxynitrites, the ultimate biochemical mediators of T cell immune suppression. The multifaceted regulation of this complex suppressive effector system provides several potential therapeutic targets

CD8(+) tumor-infiltrating lymphocytes (TIL) are defective in cytolysis due to tumor-induced inhibition of proximal TCR-mediated signaling, a defect that is relieved upon purification and brief culture. We show in this study that frequency of conjugation in vitro of nonlytic TIL with tumor cells is low in comparison with their lytic counterparts, and the strength of interaction and duration of conjugation are also reduced. Previous reports show that p56(lck) activation is required for TCR-initiated LFA-1 avidity up-regulation, raising the question: is low LFA-1 avidity the basis of reduced TIL conjugation frequency? When stimulated with phorbol ester, nonlytic TIL bind purified ICAM-1 equivalently as lytic TIL, suggesting that LFA-1 can be activated if proximal TCR signaling is bypassed. However, when treated with phorbol ester, the conjugation frequency of nonlytic TIL does not increase. CD2 and CD8 also mediate T cell adhesion to cognate target cells and are both expressed at lower levels in nonlytic TIL in addition to being excluded from the immune synapse formed upon conjugation. Collectively, these results imply that adhesion defects in nonlytic TIL result from a combination of decreased cell surface levels of adhesion molecules, deficient LFA-1 activation, and the failure to recruit essential adhesion receptors to the membrane contact site formed with cognate target cells