Tumor-associated macrophages (TAMs) plentifully infiltrate the tumor microenvironment (TME), but their role in anti-tumor immunity is controversial. Depending on the acquired polarization, they can either support tumor growth or participate in the elimination of neoplastic cells. In this study, we analyzed the TME by RNA-seq and flow cytometry and examined TAMs after ex vivo activation. Tumors with normal and either reversibly or irreversibly decreased expression of major histocompatibility complex class I (MHC-I) molecules were induced with TC-1, TC-1/A9, and TC-1/dB2m cells, respectively. We found that combined immunotherapy (IT), composed of DNA immunization and the CpG oligodeoxynucleotide (ODN) ODN1826, evoked immune reactions in the TME of TC-1- and TC-1/A9-induced tumors, while the TME of TC-1/dB2m tumors was mostly immunologically unresponsive. TAMs infiltrated both tumor types with MHC-I downregulation, but only TAMs from TC-1/A9 tumors acquired the M1 phenotype upon IT and were cytotoxic in in vitro assay. The anti-tumor effect of combined IT was markedly enhanced by a blockade of the colony-stimulating factor-1 receptor (CSF-1R), but only against TC-1/A9 tumors. Overall, TAMs from tumors with irreversible MHC-I downregulation were resistant to the stimulation of cytotoxic activity. These data suggest the dissimilarity of TAMs from different tumor types, which should be considered when utilizing TAMs in cancer IT.

Programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockade is a promising therapy for various cancer types, but most patients are still resistant. Therefore, a larger number of predictive biomarkers is necessary. In this study, we assessed whether a loss-of-function mutation of the interferon (IFN)-γ receptor 1 (IFNGR1) in tumor cells can interfere with anti-PD-L1 therapy. For this purpose, we used the mouse oncogenic TC-1 cell line expressing PD-L1 and major histocompatibility complex class I (MHC-I) molecules and its TC-1/A9 clone with reversibly downregulated PD-L1 and MHC-I expression. Using the CRISPR/Cas9 system, we generated cells with deactivated IFNGR1 (TC-1/dIfngr1 and TC-1/A9/dIfngr1). In tumors, IFNGR1 deactivation did not lead to PD-L1 or MHC-I reduction on tumor cells. From potential inducers, mainly IFN-α and IFN-β enhanced PD-L1 and MHC-I expression on TC-1/dIfngr1 and TC-1/A9/dIfngr1 cells in vitro. Neutralization of the IFN-α/IFN-β receptor confirmed the effect of these cytokines in vivo. Combined immunotherapy with PD-L1 blockade and DNA vaccination showed that IFNGR1 deactivation did not reduce tumor sensitivity to anti-PD-L1. Thus, the impairment of IFN-γ signaling may not be sufficient for PD-L1 and MHC-I reduction on tumor cells and resistance to PD-L1 blockade, and thus should not be used as a single predictive marker for anti-PD-1/PD-L1 cancer therapy.

BackgroundDepletion of tumor-associated macrophages (TAMs), which are regarded as M2, pro-tumor cells, is one of the strategies for cancer treatment. However, repolarization of TAMs to the M1 anti-tumor phenotype could constitute an immunotherapeutic alternative for tumors with defective major histocompatibility complex class I (MHC-I), where the anti-tumor effect of cytotoxic CD8+ T cells could be limited.Materials and MethodsIn this study, we characterized TAMs from mouse tumor models of human papillomavirus 16-associated tumors, characterized by either reversibly (TC-1/A9) or irreversibly (TC-1/dB2m) downregulated MHC-I expression. Tumors were treated with DNA immunization against the papillomaviral E7 oncoprotein combined with intraperitoneal injection of the synthetic oligodeoxynucleotide ODN1826, a Toll-like receptor 9 agonist. TAMs were characterized ex vivo by flow cytometry. In vitro, F4/80+ TAMs from naïve tumors were stimulated to M1 or M2 phenotype and co-cultures with TC-1/A9 or TC-1/dB2m cells were established. The cytotoxic effect of polarized TAMs was investigated, and the role of nitric oxide (NO) and tumor necrosis factor (TNF)-α was examined. Finally, interleukin (IL)-10, IL-12 and TNF-α concentrations were determined by ELISA in the culture media from polarized TAMs.ResultsWe demonstrated that TAMs infiltrated both tumor types and this effect was moderately enhanced after combined immunotherapy. Increase in MHC-II molecules, broadly regarded as an M1 marker, was observed solely in TAMs from treated TC-1/A9 tumors. In contrast, TAMs from TC-1/dB2m tumors expressed high MHC-II levels, regardless of the treatment. Therefore, the new CD38+/Egr2+ classification1 was applied and showed to be a better descriptive parameter for M1/M2 TAMs, respectively, because the number of Egr2+ TAMs decreased in both tumor types after combined immunotherapy. While CD38+ TAMs were significantly increased after treatment of TC-1/A9 tumors, they did not increase substantially in TC-1/dB2m tumors. In vitro, co-cultures with tumor cells resulted in increase of NO production by M1 TAMs. However, NO and TNF-α contributed to the cytotoxic effect only in TAMs from TC-1/A9 tumor. Finally, in vitro polarized M1 TAMs were able to produce TNF-α and IL-10 but not IL-12.ConclusionsOur results showed different effects of immunostimulation on cytotoxicity of TAMs from tumors with distinct MHC-I expression. While TAMs from TC-1/A9 tumors acquired M1 phenotype and became cytotoxic, TAMs from TC-1/dB2m tumors were more resistant to repolarization. This project was supported by grants GA19–00816S provided by the Czech Science Foundation and LQ1604 provided by the Ministry of Education, Youth and Sports of the Czech Republic.ReferenceJablonski KA, Amici SA, Webb LM, Ruiz-Rosado JdD, Popovich PG, Partida-Sanchez S, Arellano M. Novel Markers to Delineate Murine M1 and M2 Macrophages. PLoS ONE 2015; 10(12); 1–25.Disclosure InformationA. Piataková: None. I. Poláková: None. M. Šmahel: None.