Alan B. Frey

Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population expanded in cancer and other chronic inflammatory conditions. Here the authors identify the challenges and propose a set of minimal reporting guidelines for mouse and human MDSC.

Protein zero of peripheral nerve myelin: Biosynthesis, membrane insertion, and evidence for homotypic interaction

Protein zero (P0), an integral membrane glycoprotein synthesized by Schwann cells, is the major glycoprotein of peripheral nerve myelin. The predicted disposition of P0 with respect to the membrane bilayer postulates the existence of extracellular and intracellular domains, that mediate compaction of the myelin lamellae. We used in vitro translations programmed with sciatic nerve mRNA and cells transfected with a P0 cDNA construct to study the biosynthesis and topology of P0 in the bilayer. The behavior of P0 at the cell surface, when expressed under physiological conditions, was also examined. We have verified the topological predictions of an earlier model, derived from analysis of a P0 cDNA, and provide evidence that the extracellular domain of P0 mediates homotypically cell-cell interactions in the transfectants.

Type I IFN Modulates Innate and Specific Antiviral Immunity

IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-γ was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication.

In liver fibrosis, dendritic cells govern hepatic inflammation in mice via TNF-α

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.

Distinct Requirements for IFNs and STAT1 in NK Cell Function

NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNα receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1−/− mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1−/− bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-γ were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1−/− mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-α and IFN-γ are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity.

CD8+ Tumor-Infiltrating T Cells Are Deficient in Perforin-Mediated Cytolytic Activity Due to Defective Microtubule-Organizing Center Mobilization and Lytic Granule Exocytosis

Tumor-infiltrating lymphocytes (TIL) are well known to be functionally impaired typified by the inability to lyse cognate tumor cells in vitro. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8+ TIL are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cytokine mRNAs, and contain perforin and granzyme B proteins which are packaged into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, but not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rapid; mitogen-activated protein/extracellular signal-related kinase kinase, extracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein kinase C are activated; and IL-2 and IFN-γ is secreted. However, on conjugate formation between nonlytic TIL and cognate tumor cells in vitro, the microtubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis of preformed lytic granules and accounting for defective TIL lytic function. Recovery of TCR-mediated, activation-dependent MTOC mobilization and lytic activity requires proteasome function, implying the existence of an inhibitor of MTOC mobilization. Our findings show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction.

Tumor-Infiltrating Macrophages Induce Apoptosis in Activated CD8 + T Cells by a Mechanism Requiring Cell Contact and Mediated by Both the Cell-Associated Form of TNF and Nitric Oxide

We have investigated the ability of different cells present in murine tumors to induce apoptosis of activated CD8+ T cells in vitro. Tumor cells do not induce apoptosis of T cells; however, macrophages that infiltrate tumors are potent inducers of apoptosis. Tumor macrophages express cell surface-associated TNF, TNF type I (CD120a) and II (CD120b) receptors, and, upon contact with T cells which induces release of IFN-γ from T cells, secrete nitric oxide. Killing of T cells in vitro is blocked by Abs to IFN-γ, TNF, CD120a, or CD120b, or N-methyl-l-arginine. In concert with that finding, tumor macrophages isolated from either TNF type I or type II receptor −/− mice are not proapoptotic and do not produce nitric oxide upon contact with activated T cells. Control macrophages do not express TNF receptors or release nitric oxide. Tumor cells or tumor-derived macrophages do not express FasL, and blocking Abs to either Fas or FasL have no effect on macrophage-mediated T cell killing. These results demonstrate that macrophages which infiltrate tumors are highly proapoptotic and may be responsible for elimination of activated antitumor T cells within the tumor bed.

MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells

MyD88 blockade exaggerates the ability of dendritic cells to promote the transition from chronic pancreatitis to pancreatic cancer.

Signaling defects in anti-tumor T cells

Summary: 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.

Distinct populations of metastases‐enabling myeloid cells expand in the liver of mice harboring invasive and preinvasive intra‐abdominal tumor

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.