Immune microenvironment modulation unmasks therapeutic benefit of radiotherapy and checkpoint inhibition.

BACKGROUND: Immune checkpoint inhibitors (ICIs) for solid tumors, including those targeting programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), have shown impressive clinical efficacy, however, most patients do not achieve durable responses. One major therapeutic obstacle is the immunosuppressive tumor immune microenvironment (TIME). Thus, we hypothesized that a strategy combining tumor-directed radiation with TIME immunomodulation could improve ICI response rates in established solid tumors. METHODS: Using a syngeneic mouse model of human papillomavirus (HPV)-associated head and neck cancer, mEER, we developed a maximally effective regimen combining PD-1 and CTLA-4 inhibition, tumor-directed radiation, and two existing immunomodulatory drugs: cyclophosphamide (CTX) and a small-molecule inducible nitric oxide synthase (iNOS) inhibitor, L-n6-(1-iminoethyl)-lysine (L-NIL). We compared the effects of the various combinations of this regimen on tumor growth, overall survival, establishment of immunologic memory, and immunologic changes with flow cytometry and quantitative multiplex immunofluorescence. RESULTS: We found PD-1 and CTLA-4 blockade, and radiotherapy alone or in combination, incapable of clearing established tumors or reversing the unfavorable balance of effector to suppressor cells in the TIME. However, modulation of the TIME with cyclophosphamide (CTX) and L-NIL in combination with dual checkpoint inhibition and radiation led to rejection of over 70% of established mEER tumors and doubled median survival in the B16 melanoma model. Anti-tumor activity was CD8+ T cell-dependent and led to development of immunologic memory against tumor-associated HPV antigens. Immune profiling revealed that CTX/L-NIL induced remodeling of myeloid cell populations in the TIME and tumor-draining lymph node and drove subsequent activation and intratumoral infiltration of CD8+ effector T cells. CONCLUSIONS: Overall, this study demonstrates that modulation of the immunosuppressive TIME is required to unlock the benefits of ICIs and radiotherapy to induce immunologic rejection of treatment-refractory established solid tumors.

Anti-tumor immunity induced by ectopic expression of viral antigens is transient and limited by immune escape.

Immunotherapeutic treatments in head and neck cancer clinical trials include cancer vaccines targeting foreign viral antigens or mutational neoantigens derived from cancer-expressed proteins. Anti-tumor immune responses place cancer cells under selective pressure to lose or downregulate target antigens; therefore, vaccination against virus- or host- "driver" oncogenes are proposed as a strategy to overcome immune escape. Herein, we demonstrate the impact of immunogenic viral antigens on anti-tumor response and immune editing in MOC2-E6E7, a syngeneic murine oral cancer cell line expressing HPV-16 E6 and E7 oncoproteins. Using orthotopic syngeneic models, we observed in vivo tumor growth kinetics of MOC2-E6E7 is delayed in immunocompetent mice compared to parental MOC2 tumors. In contrast, tumor growth remained similar in Rag1-/- mice lacking adaptive immunity. MOC2-E6E7 tumors demonstrated an "inflamed" or immune-activated tumor microenvironment and greater infiltration of CD8+ T cells compared to MOC2. By real-time PCR, we detected downregulation of E6 and E7 genes in MOC2-E6E7 tumors only in immunocompetent mice, suggesting the loss of ectopic viral antigen expression due to immune editing. We then assessed the efficacy of a biomaterials-based mesoporous silica rod (MSR) cancer vaccine targeting HPV-16 E7 in our model. Vaccination induced robust infiltration of antigen-specific CD8+ T cells, which led to tumor growth delay and modestly prolonged survival in MOC2-E6E7 tumors. Increased efficacy was seen in a separate head and neck cancer tumor model, mEER, which obligately expresses E7 antigen. Collectively, our data highlight the need for both immunogenicity and 'driver' status of target antigens to be considered in cancer vaccine design.

Tumor microenvironment modulation enhances immunologic benefit of chemoradiotherapy.

BACKGROUND: Chemoradiotherapy (CRT) remains one of the most common cancer treatment modalities, and recent data suggest that CRT is maximally effective when there is generation of an anti-tumoral immune response. However, CRT has also been shown to promote immunosuppressive mechanisms which must be blocked or reversed to maximize its immune stimulating effects. METHODS: Therefore, using a preclinical model of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), we developed a clinically relevant therapy combining CRT and two existing immunomodulatory drugs: cyclophosphamide (CTX) and the small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). In this model, we treated the syngeneic HPV-HNSCC mEER tumor-bearing mice with fractionated (10 fractions of 3 Gy) tumor-directed radiation and weekly cisplatin administration. We compared the immune responses induced by CRT and those induced by combinatory treatment (CRT + CTX/L-NIL) with flow cytometry, quantitative multiplex immunofluorescence and by profiling immune-related gene expression changes. RESULTS: We show that combination treatment favorably remodels the tumor myeloid immune microenvironment including an increase in anti-tumor immune cell types (inflammatory monocytes and M1-like macrophages) and a decrease in immunosuppressive granulocytic myeloid-derived suppressor cells (MDSCs). Intratumoral T cell infiltration and tumor antigen specificity of T cells were also improved, including a 31.8-fold increase in the CD8+ T cell/ regulatory T cell ratio and a significant increase in tumor antigen-specific CD8+ T cells compared to CRT alone. CTX/LNIL immunomodulation was also shown to significantly improve CRT efficacy, leading to rejection of 21% established tumors in a CD8-dependent manner. CONCLUSIONS: Overall, these data show that modulation of the tumor immune microenvironment with CTX/L-NIL enhances susceptibility of treatment-refractory tumors to CRT. The combination of tumor immune microenvironment modulation with CRT constitutes a translationally relevant approach to enhance CRT efficacy through enhanced immune activation.

TGF-ß1 programmed myeloid-derived suppressor cells (MDSC) acquire immune-stimulating and tumor killing activity capable of rejecting established tumors in combination with radiotherapy.

Cancer-induced myeloid-derived suppressor cells (MDSC) play an important role in tumor immune evasion. MDSC programming or polarization has been proposed as a strategy for leveraging the developmental plasticity of myeloid cells to reverse MDSC immune suppressive functions, or cause them to acquire anti-tumor activity. While MDSC derived ex vivo from murine bone marrow precursor cells with tumor-conditioned medium efficiently suppressed T cell proliferation, MDSC derived from conditioned medium in presence of TGF-ß1 (TGFß-MDSC) acquired a novel immune-stimulatory phenotype, losing the ability to inhibit T cell proliferation and acquiring enhanced antigen-presenting capability. Altered immune function was associated with SMAD-2 dependent upregulation of maturation and costimulatory molecules, and downregulation of inducible nitric oxide synthase (iNOS), an effector mechanism of immunosuppression. TGFß-MDSC also upregulated FAS-ligand expression, leading to FAS-dependent killing of murine human papillomavirus (HPV)-associated head and neck cancer cells and tumor spheroids in vitro and anti-tumor activity in vivo. Radiation upregulated FAS expression on tumor cells, and the combination of radiotherapy and intratumoral injection of TGFß-MDSC strongly enhanced class I expression on tumor cells and induction of HPV E7 tetramer-positive CD8 + T cells, leading to clearance of established tumors and long-term survival. TGFß-MDSC derived from human PBMC with tumor conditioned medium also lost immunosuppressive function and acquired tumor-killing activity. Thus, TGFß1 mediated programming of nascent MDSC leads to a potent anti-tumor phenotype potentially suitable for adoptive immunotherapy.

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Cyclophosphamide treatment regulates the balance of functional/exhausted tumor-specific CD8+ T cells.

An important question is how chemotherapy may (re-)activate tumor-specific immunity. In this study, we provide a phenotypic, functional and genomic analysis of tumor-specific CD8+ T cells in tumor (P815)-bearing mice, treated or not with cyclophosphamide. Our data show that chemotherapy favors the development of effector-type lymphocytes in tumor bed, characterized by higher KLRG-1 expression, lower PD-1 expression and increased cytotoxicity. This suggests re-engagement of T lymphocytes into the effector program. IFN-I appears involved in this remodeling. Our findings provide some insight into how cyclophosphamide regulates the amplitude and quality of tumor-specific immune responses.

Chemotherapy and immunotherapy: A close interplay to fight cancer?

In theory, the immunotherapy of cancer should induce the selective destruction of cancer cells and a long-term specific protection, based on the specificity and memory of immunity. This contrasts with the collateral damages of conventional therapies and their toxic effects on host tissues. However, recent data suggest that chemotherapy may potentiate ongoing immune responses, through homeostatic mechanisms. Massive tumor death, empty "immune" niches and selected cytokines may act as a danger signal, alerting the immune system and amplifying pre-existing antitumor reactivity.

[The interplay of immunotherapy and chemotherapy, a novel approach]. / L'immunothérapie au service de la chimiothérapie, de nouvelles avancées.

In the last decade, a growing body of evidence has highlighted the major role of cancer immunosurveillance. The immune system can recognize tumor cells and keep them under check for long period of time, but is impeded by escape mechanisms induced by the tumor itself. Interestingly, the efficacy of chemotherapy has been shown to depend on the immune response, which in turn is potentiated by chemical agents, creating a positive feedback loop leading to long term tumor resistance. In this review, we summarize some mechanisms underlying the synergy between chemo- and immuno-therapy.

Cyclophosphamide treatment induces rejection of established P815 mastocytoma by enhancing CD4 priming and intratumoral infiltration of P1E/H-2K(d) -specific CD8+ T cells.

There is increasing evidence that the effect of chemotherapy on tumor growth is not cell autonomous but relies on the immune system. The objective of this study was therefore to decipher the cellular and molecular mechanisms underlying the role of innate and adaptive immunity in chemotherapy-induced tumor rejection. Treatment of DBA/2 mice bearing P815 mastocytoma with cyclophosphamide induced rejection and long-term protection in a CD4- and CD8-dependent manner. A population of inflammatory-type dendritic cells was dramatically expanded in the lymph nodes of mice that rejected the tumor and correlated with CD4-dependent infiltration, in tumor bed, of tumor-specific CD8+ T lymphocytes. Our data point to a major role of CD4+ T cells in inducing chemokine expression in the tumor, provoking migration of tumor-specific CXCR3+ CD8+ T lymphocytes. Importantly, the analysis of CD8+ T cells specific to P1A/H-2L(d) and P1E/H-2K(d) revealed that cyclophosphamide altered the P815-specific CD8 T repertoire by amplifying the response specific to the mutated P1E antigen.