TAM-like Macrophages Research Articles

Adenosine-generating ovarian cancer cells attract myeloid cells which differentiate into adenosine-generating tumor associated macrophages – a self-amplifying, CD39- and CD73-dependent mechanism for tumor immune escape

BackgroundOvarian cancer (OvCA) tissues show abundant expression of the ectonucleotidases CD39 and CD73 which generate immunomodulatory adenosine, thereby inhibiting cytotoxic lymphocytes. Little, however, is known about the effect of adenosine...

Determination of Optimal Target Antigen and Immune Modulatory Approaches to Improve and Standardize Epstein-Barr Virus-Specific Adoptive T Cell Therapy

Epstein-Barr Virus (EBV) is a ubiquitous gamma herpesvirus that infects lymphocytes and epithelial cells, establishing lifelong latency. Immunosuppression is associated with a transition from a quiescent, latent EBV infection to either a lytic or active latent life cycle, which involves transformation of memory B cells to rapidly growing lymphoblastoid cell lines (LCLs). Due to the immunogenicity of EBV viral proteins, cells expressing most latent or lytic peptides are eradicated through immune surveillance mechanisms. EBV-associated lymphoproliferative disorders (LPDs) arise in immunocompromised individuals and are facilitated by the expression of the same viral proteins that are expressed in the immune tolerant environment. Furthermore, EBV-LPDs acquire various mechanisms to suppress immune function, such as expression of the programmed death ligand (PD-L1), CTLA-4, amino acid depletion, and propagation of suppressive immune cell subsets, including tumor-associated macrophages (TAMs) that attenuate T cell receptor signaling and cytokine production. These immunosuppressive mechanisms increase the risk of acquiring EBV viremia and can facilitate the development of LPDs. In other work reported by our group (Patton et al, ASH abstract 2014), a TAM-like population has recently been discovered to spontaneously expand in peripheral blood mononuclear cell (PBMC)/LCL co-cultures. These TAM-like macrophages are capable of potent cytotoxicity against EBV-specific T cells, thus novel strategies to counteract this negative regulatory network are needed.One effective strategy developed to reestablish EBV-specific immunity is adoptive T cell therapy. However, the optimal combination of target antigens required to protect against EBV-LPD is not well characterized. It remains plausible that expansion of cytotoxic and/or helper T cell populations specific for multiple EBV antigens will improve the efficiency of the T cell response. The suppression of negative immune regulators may further enhance the effectiveness of the adoptive therapies to provide optimal control of viremia and effectively treat EBV-driven malignancies.Here, we generated EBV-specific T cell preparations by culturing PBMCs supplemented with interleukins 4 and 7 (IL4, IL7) in the presence of latent (EBNA1, EBNA3, LMP1, LMP2) or lytic (BZLF1, BRLF, BMLF1, BMRF1) PepMixes (complete protein-spanning pools of overlapping peptides). Cells were separated into two groups, a test and expansion group, to determine optimal ex vivo expansion. Activity of T cells was measured by direct flow-based cytotoxicity against LCL targets and interferon gamma (IFNγ) production via Enzyme-Linked Immuno Spot (ELISpot).Preliminary ELISpot assays from two donors in the test group showed that BZLF1-specific T cells produced as much as 34-fold more IFNγ in comparison to other EBV antigens (standardized to a negative control). T cells produced in culture with a peptivator PepMix, a collection of 13 latent and lytic peptide pools, on average exhibited the second highest levels of IFNγ release, expressing as much as 1.4-fold more IFNγ than the next highest antigen-specific response, depending on the donor. Additionally, LMP1 produced as much as 24-fold higher levels of IFNγ. Therefore, given the heterogeneous EBV viral gene expression profiles and variation of immune dominance across human leukocyte antigen (HLA) types, use of polyclonal immunogen-specific T cells may be more effective in improving survival and long-term protection from EBV-driven complications via improved memory T cell surveillance. Additional ex vivo work is currently underway examining combinations of lytic and latent antigens based on immunodominance analysis across HLA types. Finally, use of immune modulatory mechanisms to address newly discovered TAM-like populations contributing to reduced immune effector function against EBV-LPD are currently being evaluated. We are testing strategies to promote PD-L1 and CTLA-4 blockade, inhibition of amino acid depletion, and down regulation of regulatory T cells, to allow for enhanced expansion of antigen-specific T cell populations. Confirmation of these hypotheses will be directed toward developing streamlined methods for efficient, rapid, and personalized T cell preparation that can be standardized for translation into clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Identifying and Targeting Cytotoxic Tumor-Associated Macrophages in Epstein-Barr Virus-Driven Lymphoproliferative Disease

Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with the development of a range of malignant B-cell lymphoproliferative diseases that are associated with immune suppression, aggressive clinical courses, and poor outcomes. Current treatment options typically lead to further immune suppression, increasing the risk of EBV reactivation and other potentially lethal opportunistic infections. These complications make it essential to identify novel therapeutic approaches targeting mechanisms of immune evasion while promoting tumor immune surveillance.Immune evasion of tumors is supported by aberrant ligand and cytokine expression within the microenvironment, and tumor-associated macrophages (TAM) play an important role in inhibiting anti-tumor immune responses. In particular, TAMs are known to suppress T cell activation, and increased TAM density and ratio to cytotoxic T lymphocytes (CTL) in the tumor microenvironment are negative prognostic indicators.Here we demonstrate the use of an in vitro co-culture (CoCx) system in which EBV-transformed lymphoblastoid cell lines (LCL) are cultured with autologous peripheral blood mononuclear cells (PBMC). Media from these CoCx collected after 48 hrs., showed elevated levels of the monocyte/macrophage associated cytokines and chemokines MCP, IL-8, IP-10, MIG, GRO and MMP-9, which were absent in media from LCL or PBMC cultures. Furthermore, LCL+PBMC CoCx showed a notable outgrowth of a CD14+ monocyte/macrophage population. To analyze the direct effect of these secreted factors, conditioned media was collected from cultures of PBMC, LCL or CoCx and added to purified autologous monocytes. MTS assays at 7 days showed pronounced proliferation of monocytes in CoCx conditioned media relative to monocytes in PBMC or LCL conditioned media. Microscopy indicated that monocytes cultured with CoCx conditioned media formed non-adherent, proliferating colonies, whereas monocytes incubated in unconditioned, PBMC conditioned, or LCL conditioned media remained adherent. Depletion of CD8+ lymphocytes (but not CD4+, CD19+, CD56+ or CD14+ subsets) from CoCx led to a decrease in proliferation of monocytes, indicating a role of CTL in this phenomenon. This monocyte-derived cell population retained monocyte markers CD14 and CD11b and showed pronounced increases in both canonical M1 and M2 macrophage markers (HLA-DR and CD163, respectively), consistent with a TAM phenotype. Furthermore, the T cell inhibitory molecule PD-L1 was prominently expressed on these cells. This phenotype was confirmed to be the same as for the CD14+ cells that expanded from PBMC cultured with LCL. The functional activity of these macrophages on T cells was assessed by incubating autologous T cells alone or with macrophages derived from CoCx conditioned media for 24 hrs. Quantitative flow cytometric analysis showed a moderate decrease in CD4+ T helper cells and CD8+ CTL in the CoCx, compared to T cells cultured alone. However, if T cells were first activated (anti-CD3/CD28), the total numbers of viable CD3+ cells in the CoCx dropped 10-fold compared with activated T cells cultured alone. We previously reported that the eIF4A-specific translation initiation inhibitor silvestrol elicits marked anti-tumor activity against EBV-driven lymphoma. This occurs both by direct cytotoxicity to EBV-transformed B cells and by facilitating the activation, proliferation and cytotoxic activity of anti-tumor immune subsets including EBV-specific CTL. Here, the addition of 10nM silvestrol into CoCx blocked the release of monocyte/macrophage-associated cytokines, and silvestrol-treated CoCx conditioned medium did not drive proliferation or colony expansion of TAM-like macrophages. This finding indicates that selective targeting of the eIF4F translation initiation complex, which includes eIF4A, may block TAM formation in the setting of EBV-driven lymphoma.In summary, we have identified an immune evasion mechanism by which exposure of normal PBMC to EBV+ lymphoma cells results in expansion of a TAM-like macrophage population with potent cytotoxic activity against T cells. Low-dose silvestrol treatment abrogates the outgrowth of this TAM-like population and allows expansion of EBV-specific CTL. This finding provides an entirely new approach to modulate the immune response in this challenging group of EBV-related diseases. DisclosuresNo relevant conflicts of interest to declare.

RNAi screen in apoptotic cancer cell-stimulated human macrophages reveals co-regulation of IL-6/IL-10 expression

Tumor-associated macrophages (TAM) are a major supportive component within neoplasms and are characterized by a plethora of functions that facilitate tumor outgrowth. Mechanisms of macrophage attraction and differentiation to a tumor-promoting phenotype, defined among others by distinct cytokine patterns such as pronounced interleukin (IL-10) production, are ill-defined. We aimed to identify signaling pathways that contribute to the generation of TAM-like macrophages using an adenoviral RNAi-based approach. Primary human monocyte-derived macrophages were stimulated with apoptotic tumor cell supernatants (ACM) to induce a TAM-like phenotype, characterized by secretion of IL-10, IL-6, IL-8 but repression of IL-12. For the high-throughput screen, macrophages were transduced with 8495 constructs of the adenoviral shRNA SilenceSelect® library of Galapagos BV, which aims at identifying druggable targets. We identified 96 genes involved in IL-10 production in response to ACM and observed a pronounced cluster of targets regulating both IL-10 and IL-6. Validation of five targets within the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors in human primary macrophages. Among those, interleukin 4 receptor-α and cannabinoid receptor 2 were confirmed as regulators of IL-10 and IL-6 secretion by ACM-stimulated macrophages. Our approach characterizes cellular functions of transfection-resistant, highly plastic and versatile cells and identifies novel targets involved in the generation of a TAM-like phenotype in human macrophages.