Tumors are composed of multiple cell types besides the tumor cells themselves, including innate immune cells such as macrophages. Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). Here, they contribute to immunosuppression, enabling the establishment and persistence of solid tumors as well as metastatic dissemination. We have found that the pattern recognition scavenger receptor MARCO defines a subtype of suppressive TAMs and is linked to clinical outcome. An anti-MARCO monoclonal antibody was developed, which induces anti-tumor activity in breast and colon carcinoma, as well as in melanoma models through reprogramming TAM populations to a pro-inflammatory phenotype and increasing tumor immunogenicity. This anti-tumor activity is dependent on the inhibitory Fc-receptor, FcγRIIB, and also enhances the efficacy of checkpoint therapy. These results demonstrate that immunotherapies using antibodies designed to modify myeloid cells of the TME represent a promising mode of cancer treatment.

Marginal zone macrophages (MZM) are strategically located in the spleen, lining the marginal sinus where they sense inflammation and capture Ag from the circulation. One of the receptors expressed by MZM is scavenger receptor macrophage receptor with collagenous structure (MARCO), which has affinity for modified self-antigens. In this article, we show that engagement of MARCO on murine macrophages induces extracellular ATP and loss of CD21 and CD62L on marginal zone B cells. Engagement of MARCO also leads to reduction of Ag transport by marginal zone B cells and affects the subsequent immune response. This study highlights a novel function for MZM in regulating Ag transport and activation, and we suggest that MARCO-dependent ATP release regulates this through shedding of CD21 and CD62L. Because systemic lupus erythematosus patients were shown to acquire autoantibodies against MARCO, this highlights a mechanism that could affect a patient’s ability to combat infections.

Macrophages exist as innate immune subsets that exhibit phenotypic heterogeneity and functional plasticity. Their phenotypes are dictated by inputs from the tissue microenvironment. G-protein–coupled receptors are essential in transducing signals from the microenvironment, and heterotrimeric Gα signaling links these receptors to downstream effectors. Several Gαi-coupled G-protein–coupled receptors have been implicated in macrophage polarization. In this study, we use genetically modified mice to investigate the role of Gαi2 on inflammasome activity and macrophage polarization. We report that Gαi2 in murine bone marrow–derived macrophages (BMDMs) regulates IL-1β release after activation of the NLRP3, AIM2, and NLRC4 inflammasomes. We show this regulation stems from the biased polarity of Gαi2 deficient (Gnai2−/−) and RGS-insensitive Gαi2 (Gnai2G184S/G184S) BMDMs. We determined that although Gnai2G184S/G184S BMDMs (excess Gαi2 signaling) have a tendency toward classically activated proinflammatory (M1) phenotype, Gnai2−/− BMDMs (Gαi2 deficient) are biased toward alternatively activated anti-inflammatory (M2) phenotype. Finally, we find that Gαi2-deficient macrophages have increased Akt activation and IFN-β production but defects in ERK1/2 and STAT3 activation after LPS stimulation. Gαi2-deficient macrophages also exhibit increased STAT6 activation after IL-4 stimulation. In summary, our data indicates that excess Gαi2 signaling promotes an M1 macrophage phenotype, whereas Gαi2 signaling deficiency promotes an M2 phenotype. Understanding Gαi2-mediated effects on macrophage polarization may bring to light insights regarding disease pathogenesis and the reprogramming of macrophages for the development of novel therapeutics.

NKT cells comprise a rare, but important subset of T cells which account for ~0.2% of the total circulating T cell population. NKT cells are known to have anti-tumor functions and rapidly produce high levels of cytokines following activation. Several clinical trials have sought to exploit the effector functions of NKT cells. While some studies have shown promise, NKT cells are approximately 50% lower in cancer patients compared to healthy donors of the same age and gender, thus limiting their therapeutic efficacy. These studies indicate that baseline levels of activation should be assessed before initiating an NKT cell based immunotherapeutic strategy, thus the goal of this study was to develop a sensitive method to rapidly assess NKT cell function. We utilized artificial antigen presenting cells in combination with qPCR in order to determine NKT cell function in peripheral blood mononuclear cells from healthy donors and breast cancer patients. We found that NKT cell activation can be detected by qPCR, but not by ELISA, in healthy donors as well as in breast cancer patients following four hour stimulation. This method utilizing CD1d-expressing aAPC will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies.

Background: Tumor-associated macrophages (TAM) play an important role in tumor-promoting inflammation and have been associated with poor prognosis in many human cancers. Recently, we showed that class A scavenger receptor MARCO is a specific marker for the immune-suppressive tumor-promoting TAM subtype in different tumor models. Targeting MARCO by monoclonal antibodies (Ab) could reprogram the protumorigenic TAM towards a proinflammatory phenotype with antitumor activity, leading to decreased tumor growth and metastases formation in three different mouse tumor models. Aim(s): The current immunotherapies are estimated to treat less than 50% of all cancers; therefore, it is of high importance to develop new targets and combinatorial therapies. In this study, we aim to define the role of scavenger receptor MARCO on TAM in more detail and the mechanisms of how targeting MARCO by monoclonal Ab modulates macrophage phenotype and tumor microenvironment. Moreover, we are investigating how it can be used as a possible anticancer therapeutic approach used as single or combinatorial treatment. Results: Our recent data demonstrate that using anti-MARCO Ab in combination with other immune checkpoint inhibitors, such as anti-CTLA-4 Ab, further decreases the tumor volume by 40-50% in the B16 melanoma model. For example, different checkpoint Ab combined with anti-MARCO Ab shows a reduction from average tumor volume 500mm3 in single treatments to 250mm3 (p=0.0101) in the combinatorial treatment. Currently, we are investigating what happens in the tumor microenvironment on a cellular level after using the different combinatorial treatments. Moreover, we see increased ATP release of MARCO+ macrophages in vitro after engagement of MARCO as well as enhanced glycolysis in anti-MARCO Ab treated macrophages compared to the control group. Conclusion: Checkpoint inhibitors targeting T cells are already used in the clinics and the anti-MARCO Ab targeting TAM is a promising candidate to use in combination with checkpoint inhibitors to even further increase the survival rate of patients. The understanding of MARCO signaling and how this leads to a shift towards proinflammatory macrophages would not just benefit the field of basic macrophage biology but will also be an important step in translating the anti-MARCO Ab therapy from mice to humans as a new approach for cancer immunotherapy. Citation Format: Vanessa F. Boura, Sofia Tyystjärvi, Ganna Oliynyk, Marie Arsenian-Henriksson, Mikael C.I. Karlsson, Silke Sohn. Targeting scavenger receptors for immunotherapy of cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B09.

An important role of keratinocyte (KC) is to serve as a barrier against environmental stimuli including microbial infections. S. aureus acts as pathobiont on human skin often worsening atopic dermatitis, while normally colonizing in nasal cavity and gut. Therefore, to address how S. aureus behaves as pathobiont in skin through KC activation, we first examined the role of MyD88 signaling in KC. S. aureus were colonized epicutaneously in wild-type (WT), MyD88-/and K14-CreMyd88-/mice. WT mice exhibited severe skin inflammation 7 days after S. aureus colonization, whereas Myd88-/mice showed no skin inflammation. Importantly, K14-CreMyd88-/mice developed dramatically reduced skin inflammation, suggesting the importance of MyD88 in KC. IL-1 family cytokines, upstream mediators of MyD88, are reportedly expressed in KC and trigger skin inflammation. S. aureus-colonized Il1r-/mice showed moderately reduced skin inflammation. In addition, IL-36R-blocking antibody (Ab) treatment in Il1r-/mice further reduced the inflammation, indicating the importance of IL-1 and IL-36. We next examined IL-17 expressions in S. aureus-colonized skin. S. aureuscolonized K14-CreMyd88-/and IL-36R-blocking Ab-injected Il1r-/mice showed dramatically decreased IL-17 expressions compared to WT mice. Additionally, S. aureus-colonized Il17af-/mice showed significantly less skin inflammation. Taken together, IL-17 plays a pivotal role in the skin inflammation. Moreover, we discovered that phenol soluble modulin a (PSMa) from S. aureus induced IL-36 and IL-1 release from primary KC along with inducing cell death in vitro, and psma-deleted S. aureus (LACDpsma) did not induce the skin inflammation in vivo. In conclusion, we identified that PSMa as a key virulence factor in epicutaneous S. aureus-induced skin inflammation through IL-36 and IL-1 release from KC.

Tumor suppressor protein p53 is mutated in close to 50% of human tumors. Under steady state conditions, the two E3 ligases MDM2 and MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 has therefore been considered a therapeutic strategy. In this study, we tested p53-MDM2 protein-protein interaction inhibition using the small molecule AMG 232, which is currently being tested in phase 1b/2a clinical trials. In vitro, AMG 232 induced a significant, p53-dependent growth arrest in the mouse melanoma cell line B16-F10. Using mass spectrometry-based proteomics, we identified differential protein expression patterns following AMG 232 treatment of B16-F10 melanoma cells. In vivo, the growth of B16-F10 melanoma cells implanted in WT C57BL/6 mice was significantly reduced by AMG 232 treatment. Our data demonstrate that AMG 232 induces a p53-dependent tumor growth arrest in an immunocompetent mouse model, and we are currently testing whether AMG232 can synergize with checkpoint immunotherapy. Citation Format: Katrine Ingelshed, Danai Lianoudaki, Dilraj Lama, Silke Sohn, Nicolas Fritz, Long Jiang, Fredrik Wermeling, Mikael Karlsson, David P. Lane, Saikiran K. Sedimbi. Inhibition of p53-MDM2 protein interaction reduces tumor growth in a mouse melanoma model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A79.