Paolo Serafini

Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells

Active suppression of tumor-specific T lymphocytes can limit the efficacy of immune surveillance and immunotherapy. While tumor-recruited CD11b+ myeloid cells are known mediators of tumor-associated immune dysfunction, the true nature of these suppressive cells and the fine biochemical pathways governing their immunosuppressive activity remain elusive. Here we describe a population of circulating CD11b+IL-4 receptor alpha+ (CD11b+IL-4Ralpha+), inflammatory-type monocytes that is elicited by growing tumors and activated by IFN-gamma released from T lymphocytes. CD11b+IL-4Ralpha+ cells produced IL-13 and IFN-gamma and integrated the downstream signals of these cytokines to trigger the molecular pathways suppressing antigen-activated CD8+ T lymphocytes. Analogous immunosuppressive circuits were active in CD11b+ cells present within the tumor microenvironment. These suppressor cells challenge the current idea that tumor-conditioned immunosuppressive monocytes/macrophages are alternatively activated. Moreover, our data show how the inflammatory response elicited by tumors had detrimental effects on the adaptive immune system and suggest novel approaches for the treatment of tumor-induced immune dysfunctions.

Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function

Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, tadalafil, and vardenafil) are agents currently in clinical use for nonmalignant conditions. We report the use of PDE5 inhibitors as modulators of the antitumor immune response. In several mouse tumor models, PDE5 inhibition reverses tumor-induced immunosuppressive mechanisms and enables a measurable antitumor immune response to be generated that substantially delays tumor progression. In particular, sildenafil, down-regulates arginase 1 and nitric oxide synthase–2 expression, thereby reducing the suppressive machinery of CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs) recruited by growing tumors. By removing these tumor escape mechanisms, sildenafil enhances intratumoral T cell infiltration and activation, reduces tumor outgrowth, and improves the antitumor efficacy of adoptive T cell therapy. Sildenafil also restores in vitro T cell proliferation of peripheral blood mononuclear cells from multiple myeloma and head and neck cancer patients. In light of the recent data that enzymes mediating MDSC-dependent immunosuppression in mice are active also in humans, these findings demonstrate a potentially novel use of PDE5 inhibitors as adjuncts to tumor-specific immune therapy.

Myeloid Suppressor Lines Inhibit T Cell Responses by an NO-Dependent Mechanism

CD11b+Gr-1+ myeloid suppressor cells (MSC) accumulate in lymphoid organs under conditions of intense immune stress where they inhibit T and B cell function. We recently described the generation of immortalized MSC lines that provide a homogeneous source of suppressor cells for dissecting the mechanism of suppression. In this study we show that the MSC lines potently block in vitro proliferation of T cells stimulated with either mitogen or antigenic peptide, with as few as 3% of MSC cells causing complete suppression. Inhibition of mitogenic and peptide-specific responses is not associated with a loss in IL-2 production or inability to up-modulate the early activation markers, CD69 and CD25, but results in direct impairment of the three IL-2R signaling pathways, as demonstrated by the lack of Janus kinase 3, STAT5, extracellular signal-regulated kinase, and Akt phosphorylation in response to IL-2. Suppression is mediated by and requires NO, which is secreted by MSC in response to signals from activated T cells, including IFN-γ and a contact-dependent stimulus. Experiments with inducible NO synthase knockout mice demonstrated that the inhibition of T cell proliferation by CD11b+Gr-1+ cells in the spleens of immunosuppressed mice is also dependent upon NO, indicating that the MSC lines accurately represent their normal counterparts. The distinctive capacity of MSC to generate suppressive signals when encountering activated T cells defines a specialized subset of myeloid cells that most likely serve a regulatory function during times of heightened immune activity.

Myeloid derived suppressor cells promote cross-tolerance in B cell lymphoma by expanding regulatory T cells

Tumor-induced T-cell tolerance is a major mechanism that facilitates tumor progression and limits the efficacy of immune therapeutic interventions. Regulatory T cells (Treg) play a central role in the induction of tolerance to tumor antigens, yet the precise mechanisms regulating its induction in vivo remain to be elucidated. Using the A20 B-cell lymphoma model, here we identify myeloid-derived suppressor cells (MDSC) as the tolerogenic antigen presenting cells capable of antigen uptake and presentation to tumor-specific Tregs. MDSC-mediated Treg induction requires arginase but is transforming growth factor-beta independent. In vitro and in vivo inhibition of MDSC function, respectively, with NOHA or sildenafil abrogates Treg proliferation and tumor-induced tolerance in antigen-specific T cells. These findings establish a role for MDSCs in antigen-specific tolerance induction through preferential antigen uptake mediating the recruitment and expansion of Tregs. Furthermore, therapeutic interventions, such as in vivo phosphodiesterase 5-inhibition, which effectively abrogate the immunosuppressive role of MDSCs and reduce Treg numbers, may play a critical role in delaying and/or reversing tolerance induction.

L-arginine metabolism in myeloid cells controls T-lymphocyte functions

Although current attention has focused on regulatory T lymphocytes as suppressors of autoimmune responses, powerful immunosuppression is also mediated by a subset of myeloid cells that enter the lymphoid organs and peripheral tissues during times of immune stress. If these myeloid suppressor cells (MSCs) receive signals from activated T lymphocytes in the lymphoid organs, they block T-cell proliferation. MSCs use two enzymes involved in arginine metabolism to control T-cell responses: inducible nitric oxide synthase (NOS2), which generates nitric oxide (NO) and arginase 1 (Arg1), which depletes the milieu of arginine. Th1 cytokines induce NOS2, whereas Th2 cytokines upregulate Arg1. Induction of either enzyme alone results in a reversible block in T-cell proliferation. When both enzymes are induced together, peroxynitrites, generated by NOS2 under conditions of limiting arginine, cause activated T lymphocytes to undergo apoptosis. Thus, NOS2 and Arg1 might act separately or synergistically in vivo to control specific types of T-cell responses, and selective antagonists of these enzymes might prove beneficial in fighting diseases in which T-cell responses are inappropriately suppressed. This Opinion is the second in a series on the regulation of the immune system by metabolic pathways.

High-Dose Granulocyte-Macrophage Colony-Stimulating Factor-Producing Vaccines Impair the Immune Response through the Recruitment of Myeloid Suppressor Cells

Tumor vaccines have shown promise in early clinical trials. Among them, tumor cells genetically engineered to secrete biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF) can generate a systemic antitumor immune response. Although the minimal required GM-CSF dose produced by modified tumor cells to achieve a measurable antitumor effect is well known, no data examined whether an upper therapeutic limit may exist for this vaccination strategy. Because recent data demonstrate an immunosuppressive effect of GM-CSF produced by growing tumors, we thus sought to determine whether high GM-CSF doses administered in a vaccine formulation could impair antitumor immunity. Using a vaccine strategy involving a GM-CSF-producing bystander cell line (B78H1-GM) admixed with autologous tumor, we assessed the impact of varying doses of GM-CSF while maintaining a constant antigen dose. Our results defined a threshold above which a GM-CSF-based vaccine not only lost its efficacy, but more importantly for its clinical implications resulted in substantial immunosuppression in vivo. Above this threshold, GM-CSF induced Gr1+/CD11b+ myeloid suppressor cells that substantially impaired antigen-specific T-cell responses and adversely affected antitumor immune responses in vivo. The dual effects of GM-CSF are mediated by the systemic and not local concentration of this cytokine. Myeloid suppressor cell-induced immunosuppression is mediated by nitric oxide production via inducible nitric oxide synthase (iNOS) because the specific iNOS inhibitor, l-NMMA, restored antigen-specific T-cell responsiveness in vitro. Taken together, our data demonstrated the negative impact of supra-therapeutic vaccine doses of GM-CSF and underscored the importance of identifying these critical variables in an effort to increase the therapeutic efficacy of tumor vaccines.

IL-4-Induced Arginase 1 Suppresses Alloreactive T Cells in Tumor-Bearing Mice

We previously demonstrated that a specialized subset of immature myeloid cells migrate to lymphoid organs as a result of tumor growth or immune stress, where they suppress B and T cell responses to Ags. Although NO was required for suppression of mitogen activation of T cells by myeloid suppressor cells (MSC), it was not required for suppression of allogenic responses. In this study, we describe a novel mechanism used by MSC to block T cell proliferation and CTL generation in response to alloantigen, which is mediated by the enzyme arginase 1 (Arg1). We show that Arg1 increases superoxide production in myeloid cells through a pathway that likely utilizes the reductase domain of inducible NO synthase (iNOS), and that superoxide is required for Arg1-dependent suppression of T cell function. Arg1 is induced by IL-4 in freshly isolated MSC or cloned MSC lines, and is therefore up-regulated by activated Th2, but not Th1, cells. In contrast, iNOS is induced by IFN-γ and Th1 cells. Because Arg1 and iNOS share l-arginine as a common substrate, our results indicate that l-arginine metabolism in myeloid cells is a potential target for selective intervention in reversing myeloid-induced dysfunction in tumor-bearing hosts.

Derangement of immune responses by myeloid suppressor cells

In tumor-bearing mice and cancer patients, tumor progression is often associated with altered hematopoiesis leading to the accumulation of myeloid cells. Extensive studies in preclinical models indicate that these cells share the CD11b and the Gr-1 markers, possess a mixed mature-immature myeloid phenotype, and are responsible for the induction of T-cell dysfunctions, both tumor-specific and nonspecific. Moreover, CD11b+Gr-1+ myeloid cells are described under different unrelated situations associated with temporary impairment of the T-lymphocyte reactivity. This review examines recent findings on the nature, properties, and mechanisms of action of these myeloid suppressor cells (MSCs).

Tumor-induced immune dysfunctions caused by myeloid suppressor cells.

In the late 1970s, several findings suggested that accessory cells distinct from lymphocytes might suppress immune reactivity in tumor-bearing hosts. Studies in animal models and patients later confirmed that cells driven to act as dominant immune suppressors by growing cancers could subvert the immune system. These cells have also been termed natural suppressors, a functional definition connoting their ability to hamper various T- and B-lymphocyte responses without prior activation and independently from antigen and MHC restriction. These properties were attributed to distinct cell populations. The phenotypic discrepancies, together with the lack of antigen specificity, have generated serious restraints to research on tumor-induced suppression. Recent evidence indicates that suppressor cells are closely related to immature myeloid precursors and can be found in several situations that can exert adverse effects on the immunotherapy of cancer. The present review is an attempt to address the nature and properties of immature myeloid suppressors and their relationship to dendritic cells and macrophages, with the aim of clarifying the complex network of tumor-induced, negative regulators of the immune system.

Immortalized myeloid suppressor cells trigger apoptosis in antigen-activated T lymphocytes

We described a generalized suppression of CTL anamnestic responses that occurred in mice bearing large tumor nodules or immunized with powerful recombinant viral immunogens. Immune suppression entirely depended on GM-CSF-driven accumulation of CD11b+/Gr-1+ myeloid suppressor cells (MSC) in secondary lymphoid organs. To further investigate the nature and properties of MSC, we immortalized CD11b+/Gr-1+ cells isolated from the spleens of immunosuppressed mice, using a retrovirus encoding the v-myc and v-raf oncogenes. Immortalized cells expressed monocyte/macrophage markers (CD11b, F4/80, CD86, CD11c), but they differed from previously characterized macrophage lines in their capacities to inhibit T lymphocyte activation. Two MSC lines, MSC-1 and MSC-2, were selected based upon their abilities to inhibit Ag-specific proliferative and functional CTL responses. MSC-1 line was constitutively inhibitory, while suppressive functions of MSC-2 line were stimulated by exposure to the cytokine IL-4. Both MSC lines triggered the apoptotic cascade in Ag-activated T lymphocytes by a mechanism requiring cell-cell contact. Some well-known membrane molecules involved in the activation of apoptotic pathways (e.g., TNF-related apoptosis-inducing ligand, Fas ligand, TNF-α) were ruled out as candidate effectors for the suppression mechanism. The immortalized myeloid lines represent a novel, useful tool to shed light on the molecules involved in the differentiation of myeloid-related suppressors as well as in the inhibitory pathway they use to control T lymphocyte activation.