Abrogation of IFN-γ Signaling May not Worsen Sensitivity to PD-1/PD-L1 Blockade.

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-ß 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-ß 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.

Experimental Combined Immunotherapy of Tumours with Major Histocompatibility Complex Class I Downregulation.

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of a significant anti-tumor response required a combination of DNA vaccination with the administration of an adjuvant, either the synthetic oligodeoxynucleotide ODN1826, carrying immunostimulatory CpG motifs, or α-galactosylceramide (α-GalCer). The most profound anti-tumor effect was achieved when these adjuvants were applied in a mix with a one-week delay relative to DNA immunization. Combined immunotherapy induced tumor infiltration with various subsets of immune cells contributing to tumor regression, of which cluster of differentiation (CD) 8⁺ T cells were the predominant subpopulation. In contrast, the numbers of tumor-associated macrophages (TAMs) were not markedly increased after immunotherapy but in vivo and in vitro results showed that they could be repolarized to an anti-tumor M1 phenotype. A blockade of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) immune checkpoint had a negligible effect on anti-tumor immunity and TAMs repolarization. Our results demonstrate a benefit of combined immunotherapy comprising the activation of both adaptive and innate immunity in the treatment of tumors with reduced MHC-I expression.

CD80 Expression on Tumor Cells Alters Tumor Microenvironment and Efficacy of Cancer Immunotherapy by CTLA-4 Blockade.

Cluster of differentiation (CD) 80 is mainly expressed in immune cells but can also be found in several types of cancer cells. This molecule may either activate or inhibit immune reactions. Here, we determined the immunosuppressive role of CD80 in the tumor microenvironment by CRISPR/Cas9-mediated deactivation of the corresponding gene in the mouse oncogenic TC-1 cell line. The tumor cells with deactivated CD80 (TC-1/dCD80-1) were more immunogenic than parental cells and induced tumors that gained sensitivity to cytotoxic T-lymphocyte antigen 4 (CTLA-4) blockade, as compared with the TC-1 cells. In vivo depletion experiments showed that the deactivation of CD80 switched the pro-tumorigenic effect of macrophages observed in TC-1-induced tumors into an anti-tumorigenic effect in TC-1/dCD80-1 tumors and induced the pro-tumorigenic activity of CD4+ cells. Moreover, the frequency of lymphoid and myeloid cells and the CTLA-4 expression by T helper (Th)17 cells were increased in TC-1/dCD80-1- compared with that in the TC-1-induced tumors. CTLA-4 blockade downregulated the frequencies of most immune cell types and upregulated the frequency of M2 macrophages in the TC-1 tumors, while it increased the frequency of lymphoid cells in TC-1/dCD80-1-induced tumors. Furthermore, the anti-CTLA-4 therapy enhanced the frequency of CD8+ T cells as well as CD4+ T cells, especially for a Th1 subset. Regulatory T cells (Treg) formed the most abundant CD4+ T cell subset in untreated tumors. The anti-CTLA-4 treatment downregulated the frequency of Treg cells with limited immunosuppressive potential in the TC-1 tumors, whereas it enriched this type of Treg cells and decreased the Treg cells with high immunosuppressive potential in TC-1/dCD80-1-induced tumors. The immunosuppressive role of tumor-cell-expressed CD80 should be considered in research into biomarkers for the prediction of cancer patients' sensitivity to immune checkpoint inhibitors and for the development of a tumor-cell-specific CD80 blockade.

Establishment and characterization of a mouse tumor cell line with irreversible downregulation of MHC class I molecules.

In the majority of human tumors, downregulation of major histocompatibility complex class I (MHC­I) expression contributes to the escape from the host immune system and resistance to immunotherapy. Relevant animal models are therefore needed to enhance the efficacy of cancer immunotherapy. As loss of ß­2 microglobulin expression results in irreversible downregulation of surface MHC­I molecules in various human tumors, the ß­2 microglobulin gene (B2m) was deactivated in a mouse oncogenic TC­1 cell line and a TC­1/dB2m cell line that was negative for surface MHC­I expression was derived. Following stimulation with interferon γ, MHC­I heavy chains, particularly the H­2Db molecules, were found to be expressed at low levels on the cell surface, but without ß­2 microglobulin. B2m deactivation in TC­1/dB2m cells led to reduced proliferation and tumor growth. These cells were insensitive to DNA vaccination and only weakly responsive to combined immunotherapy with a DNA vaccine and the ODN1826 adjuvant. In vivo depletion demonstrated that NK1.1+ cells were involved in both reduced tumor growth and an antitumor effect of immunotherapy. The number of immune cells infiltrating TC­1/dB2m­induced tumors was comparable with that in tumors developing from TC­1/A9 cells characterized by reversible MHC­I downregulation. However, the composition of the cell infiltrate was different and, most importantly, infiltration with immune cells was not increased in TC­1/dB2m tumors after immunotherapy. Therefore, the TC­1/dB2m cell line represents a clinically relevant tumor model that may be used for enhancement of cancer immunotherapy.

The effect of clinically relevant doses of immunosuppressive drugs on human mesenchymal stem cells.

Immunosuppressive drugs are used to suppress graft rejection after transplantation and for the treatment of various diseases. The main limitations of their use in clinical settings are severe side effects, therefore alternative approaches are desirable. In this respect, mesenchymal stem cells (MSCs) possess a regenerative and immunomodulatory capacity that has generated considerable interest for their use in cell-based therapy. Currently, MSCs are tested in many clinical trials, including the treatment of diseases which require simultaneous immunosuppressive treatment. Since the molecular targets of immunosuppressive drugs are also present in MSCs, we investigated whether immunosuppressive drugs interact with the activity of MSCs. Human MSCs isolated from the bone marrow (BM) or adipose tissue (AT) were cultured in the presence of clinical doses of five widely used immunosuppressive drugs (cyclosporine A, mycophenolate mofetil, rapamycin, prednisone and dexamethasone), and the influence of these drugs on several factors related to the immunosuppressive properties of MSCs, including the expression of immunomodulatory enzymes, various growth factors, cytokines, chemokines, adhesion molecules and proapoptotic ligands, was assessed. Glucocorticoids, especially dexamethasone, showed the most prominent effects on both types of MSCs and suppressed the expression of the majority of the factors that were tested. A significant increase of hepatocyte growth factor production in AT-MSCs and of indoleamine 2,3-dioxygenase expression in both types of MSCs were the only exceptions. In conclusion, clinically relevant doses of inhibitors of calcineurin, mTOR and IMPDH and glucocorticoids interfere with MSC functions, but do not restrain their immunosuppressive properties. These findings should be taken into account before preparing immunosuppressive strategies combining the use of immunosuppressive drugs and MSCs.