Intratumoral dendritic cells in the anti-tumor immune response

Immunotherapy has become a popular approach to the treatment of cancer and is well known in the clinical management of cancer because of the exciting recent results from immunotherapy targeting the costimulatory blockade.¹ Another hot topic in this field is the combination of cancer immunotherapy and chemotherapy, and interestingly, T-cell immune response to the immunogenic cell death (ICD) induced by chemotherapeutic drugs is important to the therapeutic efficacy of certain types of chemotherapy. Therefore, the questions arising are what are the initiator and activator of the anti-tumor immune response in a tumor-bearing host receiving chemotherapy or combination therapy? Is there any possibility that effective antigen-presenting cells (APCs) exist in the immunosuppressive tumor microenvironment that process the tumor antigens released from the ICD of tumor cells and then initiate a tumor-specific T-cell response? Currently, it is well accepted that the tumor microenvironment, which consists of tumor cells, tumor-associated fibroblasts, vascular endothelial cells and immunosuppressive (or at least functionally impaired) immune cells, such as myeloid-derived suppressor cells, tumor-associated macrophages (TAMs), regulatory T cells and regulatory dendritic cells (DCs),² is immunosuppressive and tumor-promoting. Thus, the current cancer immunotherapies mainly aim to reverse this tumor immunosuppression as well as trigger an effective anti-tumor immune response, enhancing the cytotoxic ability of T cells to reject tumors.³ Therefore, further identification of the immune cell types in the tumor microenvironment, with immune-tolerating or immune-activating potential, will provide new opportunities for the design of cancer immunotherapeutics. More recently, intratumoral CD103⁺ DCs have been identified as active APCs in the tumor microenvironment that process antigens and are crucial to the induction of an anti-tumoral T-cell response,^4,5 thus challenging the concept that immune cells are all immunosuppressive or functionally impaired in the tumor microenvironment. These new findings will inspire immunologists to rethink the interactions between the host immune system and tumors and develop new approaches to cancer immunotherapy.

In the last few decades, the tumor microenvironment has been shown to be capable of inactivating various components of the immune system responsible for tumor clearance.⁶ This microenvironment becomes even more intricate and variable due to the tumor-promoting polarization of immune cell differentiation and function. For example, macrophages in tumors can enhance tumor cell proliferation, invasion and metastasis; stimulate angiogenesis; and inhibit T cell-mediated anti-tumor immune responses, thus promoting tumor progression.⁷ Although much work has been done to identify the molecular and cellular mechanisms underlying the anti-tumor immune response and immunosuppression in the tumor microenvironment, there are still some questions that remain to be investigated. For example, why do the APCs within tumors typically fail to maintain the cytotoxic T cell (CTL) function of killing the tumor? Are there any strongly stimulatory APCs within the tumor microenvironment to induce an anti-tumor immune response? To address these concerns, immunologists have been investigating the cell types and functions of APCs in the tumor microenvironment. As the most potent professional APCs, DCs have a superior ability to activate naive T cells and play a pivotal role in the induction and maintenance of anti-tumor responses.⁸ The stimulatory versus inhibitory function of DCs depends on their cell subset and maturation status. Regulatory DCs in the tumor microenvironment and systemic organs have been identified to mediate suppression of T-cell responses against tumors.^9,10 By contrast, marginating DCs of the tumor microenvironment can cross-present tumor antigens and stably engage tumor-specific T cells.¹¹ Expression of the neonatal Fc receptor in DCs can mediate protective immunity against colorectal cancer.¹² Moreover, DCs in tumor-associated tertiary lymphoid structures signal a Th1 cytotoxic immune contexture and promote a protective immune response mediated by T cells against cancer.¹³ Therefore, DCs represent a special population of cells that display different phenotypes and activities at the tumor site as well as exhibit differential pro-tumorigenic and anti-tumorigenic functions.

Two reports in the newest issue of Cancer Cell described the intratumoral CD103⁺ DC population as a remarkable CTL stimulator that plays an important role in the induction of anti-tumor immune responses.^4,5 Dr Krummel and his colleagues utilized a broad range of tumors to analyze the distinct composition of the myeloid tumor microenvironment and attempt to understand the lineage relationships amongst these populations and how each cell type influences anti-tumor T-cell responses and outcome. By using flow cytometry, the authors identified rare tumoral DC subsets among abundant macrophages, which were delineated into two populations (CD11b⁺ DC1 and CD103⁺ DC2) based on their differential expression of CD11b and CD103. Then, they found that CD103⁺ DC2s express CD135 (Flt3), CD117 (cKit) and CD26, whereas TAM populations do not. When determining the cytokines driving differentiation into these DC populations, this group demonstrated that GMCSF expression by the tumor dramatically increased the proportion of CD11b⁺ DC1s, whereas the rare CD103⁺ DC2s are programmed by FLT3L at the tumor site. Gene profile analysis of the DC populations identified DC-defining transcription factors such as interferon regulatory factor 8 (IRF8; specific for CD103⁺ DC2s alone), zinc finger and BTB domain-containing protein 46 (Zbtb46) and basic leucine zipper transcription factor ATF-like 3 (Batf3) as the genes most distinctly expressed by CD103⁺ DC2s. Knockdown experiments confirmed that the development of CD103⁺ DC2s is uniquely dependent upon IRF8, Zbtb46 and Batf3. Thus, the authors concluded that CD103⁺ DC2s represent a distinct lineage of APCs independent of the highly abundant TAMs and CD11b⁺ DC1s in the tumor microenvironment.

Importantly, CD103⁺ DC2s have been functionally shown to exhibit unique antigen processing and presentation capabilities and are superior stimulators of naive and activated CD8⁺ T cells. Two-photon intravital imaging and live tumor slice imaging revealed that CD11b⁺ DC1s and CD103⁺ DC2s are sparse near tumor margins and can interact with T cells when present there. Moreover, the authors showed that CD103⁺ DC2s are capable of competing for T-cell occupancy using an ex vivo assay. The intratumoral CD103⁺ DC2s, although rare in vivo, were found to be required for the efficient suppression of tumor growth by adoptive T-cell immunotherapy. To extend the physiological relevance and clinical significance, the group took advantage of TCGA data (The Cancer Genome Atlas- Data Portal[https://tcga-data.nci.nih.gov/tcga/]) that quantifies relative gene expression from numerous human cancer types with matched outcome data. After selecting high level transcripts from the RNAseq data on CD103⁺ DC2 cells as their molecular signatures, they analyzed associations of the molecular signatures (human homologs of mouse genes) with the prognosis and outcome of the cancer patients. The group demonstrated that the molecular signatures of intratumoral CD103⁺ DC2 abundance was predictive of good prognosis and outcome across human cancers, profoundly so in breast cancer, head-neck squamous cell carcinoma and lung adenocarcinoma. Taken together, these results show that intratumoral CD103⁺ DC2s are the stimulatory APCs in the induction of anti-tumor T-cell immune responses.

In another article, Dr Coussens's group demonstrated that intratumoral DCs promote the anti-tumor CTL response by producing IL-12 when they tried to identify the molecular mechanisms by which macrophages suppress CD8⁺ T-cell responses to chemotherapy in mammary carcinoma models. Using a series of methods, Dr Coussens found that macrophages are the primary source of IL-10 in tumors and that the improved response to chemotherapy by blockade of the IL-10 receptor is CD8⁺ T cell-dependent. The intratumoral DCs express higher levels of IL-10R and the highest levels of Il12b mRNA relative to other cells in the tumor. Moreover, IL-12p40 protein was detectable only by intracellular flow cytometry in a small population of tumor-associated CD103⁺ DCs. IL-12 is known to enhance CD8⁺ T-cell proliferation and effector function. Therefore, the authors investigated a possible role for TAM-derived IL-10 in suppressing IL-12 production by DCs and thereby influencing CD8⁺ T-cell responses during chemotherapy. Experiments in breast cancer-bearing mouse models and TCGA data in human breast cancers have confirmed that IL-12 is functionally significant with regard to enhanced CD8-dependent anti-tumor responses. Taken together, these results indicate a critical role of intratumoral CD103⁺ DCs and their IL-12 in determining anti-tumor immune responses to combination therapy. Suppressing intratumoral DC production of IL-12 by TAM-derived IL-10 can limit CTL responses to chemotherapy in mammary carcinomas. These new findings not only enrich our understanding of intratumoral DC immunobiology, but also reveal possible risk stratification biomarkers and provide new opportunities for prognostic as well as therapeutic approaches across multiple types of cancer.

Intratumoral DCs play a pivotal role in anti-tumor immune responses within the so-called ‘immunosuppressive' tumor microenvironment (Figure 1). The two above-mentioned studies identified the characteristics and function of CD103⁺ DCs, a specific subset of intratumoral DCs that robustly stimulate CTLs and activate immune responses even in immuno-evasive tumors. This challenges the previous concept that tumors contain only weak or suppressive myeloid lineages. These studies also provided the additional insight that key Batf3- and IRF8-dependent DCs play important roles in repriming the immune response within the tumor, which shifts the emphasis from the lymph nodes to the tumor for T-cell control. These new findings reveal possible prognostic biomarkers for cancer patients and provide new opportunities for developing strategies of cancer immunotherapy. However, a series of related issues are also raised. Is the CTL anti-tumor immune response controlled only by CD103⁺ DCs? What roles do other intratumoral DC subsets play in tumor microenvironments? What are the phenotypes and functions of DCs in other types of cancer models? How does one translate the basic findings from mouse models to clinical practice for cancer immunotherapy? These questions need further investigation in the future.

Figure 1.

Intratumoral dendritic cells in the anti-tumor immune response. Tumors can actively drive the generation of immunosuppressive or regulatory immune cell subtypes and can induce massive accumulation of tumor-promoting myeloid immune cells in the tumor microenvironment. The anti-tumor immune response induced by intratumoral DCs can be divided into several major steps, starting with the uptake of antigens released from the tumor cells undergoing ICD and ending with the killing of tumor cells. Antigen presentation to T cells will elicit a response depending on the type of DC maturation stimulus received and upon the interaction of T-cell costimulatory molecules with their surface receptors on DCs. CTL, cytotoxic T lymphocyte; DC, dendritic cell; ICD, immunogenic cell death; MDSC, myeloid-derived suppressor cell; TAM, tumor-associated macrophage; Treg, regulatory T cell.

Just as immunotherapy is moving to the forefront of cancer therapy, DC-based therapy is becoming increasingly important in cancer immunotherapy. To trigger an efficient systemic anti-tumor immune response via DCs, several key steps should be closely functionally connected, and their efficacies should be improved. For example, tumor antigens should be released upon tumor cell death, efficiently disposed and presented by DCs as damage-associated molecular patterns. To our knowledge, ICD involves changes in the composition of the cancer cell surface. Such signals operate on a series of receptors expressed by DCs to stimulate the presentation of tumor antigens to T cells, thus promoting a productive immune response to the tumor.^14,15 Therefore, it is necessary to find a suitable therapeutic method or clinically successful anticancer agents that can induce the hallmarks of ICD. Because of the importance and function of DCs in stimulating T-cell responses, they are often regarded as ‘nature's adjuvants', and a number of DC-based vaccines have been approved by the FDA for the treatment of several different cancers.¹⁶ It will be of considerable interest to develop more rational DC-based vaccines, even a whole new array of therapeutic options that include rewiring DC molecular pathways. However, there are also some studies reporting that the antigen-presenting function of DCs may be lost or inefficient in the tumor microenvironment, and they might be polarized into immunosuppressive/tolerogenic regulatory DCs, which limit the activity of effector T cells and supports tumor growth and progression.^17,18 These key properties of DCs render them as important targets for therapeutic interventions in cancer.

It will be interesting to understand how cancer cells alter DC physiology and how we can generate novel cancer immunotherapies based on the powerful properties of DCs. Our sufficient understanding of the functional heterogeneity and mechanisms of distinct intratumoral DC subsets, the lineage relationships amongst immune cells (such as TAMs, DCs, regulatory T cells and myeloid-derived suppressor cells) in tumor microenvironments and how each influences anti-tumor immune responses and outcomes will significantly accelerate the development of the next generation of cancer immunotherapy.