Gerald Krystal

Although the importance of immunomodulatory myeloid cells in both normal physiology and carcinogenesis is well established, many questions remain regarding the specific roles and regulation of these cells. In this thesis, we explore the immunosuppressive features of macrophages [Mφs] and elucidate the mechanisms by which they suppress T cell proliferation/activation, the factors that regulate their suppressive properties, the relative potency of macrophage suppression compared to other myeloid cells, such as myeloid-derived suppressor cells (MDSCs), and the role these cells play in promoting tumor growth and metastasis. We demonstrate herein that in response to interferon (IFN)-beta, which is secreted by activated T cells, resident macrophages from non-tumor-bearing mice acquire immunosuppressive properties that are mediated by nitric oxide (NO). Moreover, our data reveal a novel role for Toll-like receptor (TLR)-induced IFN-beta in regulating the immunosuppressive properties of macrophages. We also demonstrate for the first time that in vitro culture conditions profoundly affect the immunosuppressive functions of MDSCs. Specifically, we show that serum antagonizes the suppressive abilities of MDSCs from 4T1 tumor-bearing mice and that the major serum protein albumin mediates these effects, in part by reducing reactive oxygen species (ROS) production from MDSCs. These findings have important implications, since the accurate detection and quantification of immunosuppression is critical for both the identification and functional analysis of tumor-induced MDSCs. We also explore the phenotypic and functional heterogeneity of tumor-induced myeloid cells and compare the immunosuppressive functions of different populations isolated from normal and tumor-bearing mice. We show that tumors that induce the accumulation of myeloid cells also enhance the suppressive functions of these cells. In addition, we demonstrate that, in vitro, tumor-induced macrophages are significantly more potent immune suppressors than tumor-induced MDSCs on a per cell basis, and suppress T cell responses via distinct mechanisms. Finally, we present data showing that treating metastatic mammary tumor-bearing mice with all-trans-retinoic acid (ATRA) decreases MDSCs, increases macrophages, and enhances metastatic growth. Taken together, these findings advance our understanding of the factors that regulate myeloid cell functions in normal and neoplastic tissues and may lead to improved immunotherapies to treat human disease.

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The tumor microenvironment encompasses all of the factors and accessory cells which interact with a tumor and, more often than not, are co-opted by tumor-secreted factors to help the cancer grow. In this thesis, we examined elements of the tumor microenvironment, specifically the cancer-promoting M2 macrophages (MΦs) and tumor glucose supply and metabolism. In the vast majority of advanced cancer patients and tumor-bearing animals, their tumors contain MΦs that are profoundly skewed to a cancer-promoting M2 phenotype, which often correlates with a poor prognosis. As well, these same tumor tissues are more dependent on high glucose levels for energy and survival than most normal tissues. Although MΦ phenotype and tumor cell glucose metabolism are quite disparate fields of study, we employed similar strategies to uncover what regulates them in order to explore mechanisms to reduce tumor growth. In our M2 MΦ studies, we assessed the phenotypic plasticity of mature IL-4-induced M2 MΦs and found their phenotype (i.e., cell-surface markers, cytokine secretion, and T cell stimulatory properties) could be fully reversed with IL-4 withdrawal and subsequent IFN-γ priming, demonstrating that M2 MΦs, indeed, can be reprogrammed even if they are phenotypically polarized. As well, we uncovered a circuit of M2-MΦ generation which may be relevant in vivo in the M2-skewed SHIP-/- mouse model. This circuit involves the sensitization of MΦs and MΦ progenitors, via IgG and TGF-β-containing mouse plasma, to low levels of constitutive IL-4 uniquely secreted by SHIP-/- basophils. In a similar vein, and based on the Warburg effect, which describes the propensity of cancer cells to consume glucose via glycolysis rather than oxidative phosphorylation (OXPHOS), we designed low carbohydrate (CHO) diets to limit the glucose supply to tumors. We found that mice fed our low CHO diets had lowered blood glucose, insulin, and lactate, and this correlated with a slower growth rate of implanted tumors, and a lower cancer incidence in a spontaneous mouse mammary carcinoma model. Furthermore, our diets worked additively with known anti-cancer agents (i.e.,Temsirolimus, Celebrex) to slow tumor growth.

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SHIP (SH2-containing inositol 5'-phosphatase) is a hematopoietic restricted enzyme responsible for the hydrolysis of the phosphatidylinositol 3-kinase-generated second messenger PI-3,4,5-P₃ to PI-3,4-P₂ and, thereby, negatively regulates cell survival, proliferation and differentiation. Herein, we demonstrate a role for SHIP in the differentiation and function of dendritic cells (DCs). We found that SHIP restrains in vitro generation and survival of bone marrow derived DCs cultured with granulocyte macrophage colony stimulating factor (GM-CSF) or fms-like tyrosine kinase 3 ligand (Flt3L). These results are consistent with the in vivo finding that SHIP-deficient mice have increased numbers of splenic DCs. We provide evidence that Ship-/- GM-CSF-derived DCs (GM-DCs) have impaired ability to activate T cells – a defect associated with deficient DC maturation and interleukin-12 (IL-12) production in response to Toll-like receptor (TLR) agonists. Reduced antigen (Ag)-specific T cell activation was associated with defective TH1 cell induction in vitro and in vivo. SHIP’s role is more restricted in Flt3L-derived DCs (FL-DCs) since the functional abnormalities of Ship-/- FL-DCs, leading to reduced DC maturation and Ag-specific T cell proliferation, are limited to MyD88-independent TLR activation pathways. Thus, we conclude that the function of SHIP in DC biology depends on the derivation context and the nature of the activating pathogen.Next, we evaluated the role of Ship-/- DCs in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis previously thought to primarily depend on a TH1-mediated immune response. We found that SHIP-deficient mice develop EAE despite reduced DC-induced T cell activation. Notwithstanding evidence of a disease suppressive environment, we show evidence that Ship-/- mice have robust clinical symptoms after EAE induction resulting from enhanced production of Ag-specific IgMs in vivo.In addition to T cell activation, under certain conditions DCs may suppress T cell proliferation using a variety of mechanisms. We show Ship-/- GM-DCs, despite expressing high levels of the T cell suppressing enzyme arginase 1, do not have enhanced suppressive ability. Intriguingly, in contrast to WT GM-DCs, we found that Ship-/- GM-DCs do not use interferon gamma-induced nitric oxide production to suppress T cell proliferation but rather an alternative contact-dependent suppressive mechanism.

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