Although current immune-checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of ...high-dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor-infiltrating cells by single-cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d, and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.
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•High-dimensional analyses of successful ICT in tumor-bearing mice•ICT induces changes in intratumoral myeloid and lymphoid cells•Tumor-associated monocytes/macrophages display complex cytokine-driven phenotypes•Different cytokines act on tumor-infiltrating monocytes to drive macrophage polarization
Comprehensive changes in the tumor microenvironment during successful immune-checkpoint therapy are profiled, implicating a key role for polarization of infiltrating macrophages in the anti-tumor immune milieu.
Cancer immunoediting is the process whereby the immune system suppresses neoplastic growth and shapes tumor immunogenicity. We previously reported that type I interferon (IFN-α/β) plays a central ...role in this process and that hematopoietic cells represent critical targets of type I IFN's actions. However, the specific cells affected by IFN-α/β and the functional processes that type I IFN induces remain undefined. Herein, we show that type I IFN is required to initiate the antitumor response and that its actions are temporally distinct from IFN-γ during cancer immunoediting. Using mixed bone marrow chimeric mice, we demonstrate that type I IFN sensitivity selectively within the innate immune compartment is essential for tumor-specific T cell priming and tumor elimination. We further show that mice lacking IFNAR1 (IFN-α/β receptor 1) in dendritic cells (DCs; Itgax-Cre(+)Ifnar1(f/f) mice) cannot reject highly immunogenic tumor cells and that CD8α(+) DCs from these mice display defects in antigen cross-presentation to CD8(+) T cells. In contrast, mice depleted of NK cells or mice that lack IFNAR1 in granulocytes and macrophage populations reject these tumors normally. Thus, DCs and specifically CD8α(+) DCs are functionally relevant targets of endogenous type I IFN during lymphocyte-mediated tumor rejection.
The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting
. Immunotherapies such as those that target immune checkpoint ...molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed
. Although the role of tumour neoantigen-specific CD8
T cells in tumour rejection is well established
, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8
and CD4
T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4
T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.
The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell ...immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.
Significance
Immune checkpoint therapy (ICT) has led to durable responses in a subset of cancer patients. Generally, patients who respond to ICT bear tumors with high mutational burden. Radiation is ...used for treatment of many types of cancers and has been shown to induce new mutations in treated tumor cells and to synergistically facilitate ICT. However, these latter actions have largely been explained by radiation-induced tumor cell death and/or effects on the host. Herein, we show that noncurative irradiation induces mutations in tumor cells lacking neoantigens and that these de novo-generated neoantigens function as targets for CD8
+
T cells, resulting in increased immunogenicity of nonimmunogenic tumor cells. This study thus identifies an additional mechanism that explains synergy between immunotherapy and radiation.
Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use Kras
G12D
x p53
−/−
sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8
+
T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.
Cancer immunoediting, the process by which the immune system controls tumour outgrowth and shapes tumour immunogenicity, is comprised of three phases: elimination, equilibrium and escape. Although ...many immune components that participate in this process are known, its underlying mechanisms remain poorly defined. A central tenet of cancer immunoediting is that T-cell recognition of tumour antigens drives the immunological destruction or sculpting of a developing cancer. However, our current understanding of tumour antigens comes largely from analyses of cancers that develop in immunocompetent hosts and thus may have already been edited. Little is known about the antigens expressed in nascent tumour cells, whether they are sufficient to induce protective antitumour immune responses or whether their expression is modulated by the immune system. Here, using massively parallel sequencing, we characterize expressed mutations in highly immunogenic methylcholanthrene-induced sarcomas derived from immunodeficient Rag2(-/-) mice that phenotypically resemble nascent primary tumour cells. Using class I prediction algorithms, we identify mutant spectrin-β2 as a potential rejection antigen of the d42m1 sarcoma and validate this prediction by conventional antigen expression cloning and detection. We also demonstrate that cancer immunoediting of d42m1 occurs via a T-cell-dependent immunoselection process that promotes outgrowth of pre-existing tumour cell clones lacking highly antigenic mutant spectrin-β2 and other potential strong antigens. These results demonstrate that the strong immunogenicity of an unedited tumour can be ascribed to expression of highly antigenic mutant proteins and show that outgrowth of tumour cells that lack these strong antigens via a T-cell-dependent immunoselection process represents one mechanism of cancer immunoediting.
Estrogen receptor alpha-positive (ERα+) luminal tumors are the most frequent subtype of breast cancer. Stat1−/− mice develop mammary tumors that closely recapitulate the biological characteristics of ...this cancer subtype. To identify transforming events that contribute to tumorigenesis, we performed whole genome sequencing of Stat1−/− primary mammary tumors and matched normal tissues. This investigation identified somatic truncating mutations affecting the prolactin receptor (PRLR) in all tumor and no normal samples. Targeted sequencing confirmed the presence of these mutations in precancerous lesions, indicating that this is an early event in tumorigenesis. Functional evaluation of these heterozygous mutations in Stat1−/− mouse embryonic fibroblasts showed that co-expression of truncated and wild-type PRLR led to aberrant STAT3 and STAT5 activation downstream of the receptor, cellular transformation in vitro, and tumor formation in vivo. In conclusion, truncating mutations of PRLR promote tumor growth in a model of human ERα+ breast cancer and warrant further investigation.
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•Mice lacking Stat1 develop mammary tumors that model human ERα+ breast cancer•DNA sequencing finds a Prlr truncating mutation hotspot in 100% of Stat1−/− tumors•At least 77.8% of DCIS examined also contain PRLR truncating mutations•Co-expression of truncated and full-length PRLR promote tumor growth in MEFs
Griffith et al. report on whole genome sequencing of mammary tumors that spontaneously develop in mice lacking Stat1. They find a 100% recurrent hotspot of mutations resulting in truncation of Prlr. Co-expression of truncated and full-length Prlr in mouse embryonic fibroblasts is shown to promote tumor growth.
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