Daniel M. Andrews

Primary Tumor Hypoxia Recruits CD11b+/Ly6Cmed/Ly6G+ Immune Suppressor Cells and Compromises NK Cell Cytotoxicity in the Premetastatic Niche

Hypoxia within a tumor acts as a strong selective pressure that promotes angiogenesis, invasion, and metastatic spread. In this study, we used immune competent bone marrow chimeric mice and syngeneic orthotopic mammary cancer models to show that hypoxia in the primary tumor promotes premetastatic niche formation in secondary organs. Injection of mice with cell-free conditioned medium derived from hypoxic mammary tumor cells resulted in increased bone marrow-derived cell infiltration into the lung in the absence of a primary tumor and led to increased metastatic burden in mammary and melanoma experimental metastasis models. By characterizing the composition of infiltrating bone marrow-derived cells, we identified CD11b+/Ly6Cmed/Ly6G+ myeloid and CD3-/NK1.1+ immune cell lineages as key constituents of the premetastatic niche. Furthermore, the cytotoxicity of natural killer (NK) cells was significantly decreased, resulting in a reduced antitumor response that allowed metastasis formation in secondary organs to a similar extent as ablation of NK cells. In contrast, metastatic burden was decreased when active NK cells were present in premetastatic lungs. Together, our findings suggest that primary tumor hypoxia provides cytokines and growth factors capable of creating a premetastatic niche through recruitment of CD11b+/Ly6Cmed/Ly6G+ myeloid cells and a reduction in the cytotoxic effector functions of NK cell populations.

DNAM-1/CD155 interactions promote cytokine and NK cell-mediated suppression of poorly immunogenic melanoma metastases.

A role for NK cells in therapeutic intervention for hematologic malignancies, such as acute myeloid leukemia and multiple myeloma, and nonhematologic malignancies, such as melanoma, is becoming more apparent. DNAM-1 is an NK cell receptor whose importance in facilitating activation signals received by NK cells in natural and cytokine-driven responses to tumor metastases in vivo is poorly explored. In this study, we used matched tumor lines expressing a variety of relevant ligands, neutralizing monoclonal Abs, and DNAM-1 gene-targeted mice to determine the relative importance of DNAM-1–ligand interactions in controlling tumor metastases. Our results demonstrate that NK cells require DNAM-1 for natural or cytokine (IL-2, IL-12, or IL-21) suppression of tumor metastases or their variants expressing CD70 or CD80. In contrast, DNAM-1 was dispensable when tumor cells were targets of Ab-dependent cellular cytotoxicity or presented ligands for NKG2D. CD155 appeared to be a key ligand recognized by DNAM-1 in NK cell-mediated suppression of metastases, and DNAM-1-mediated suppression coincided with perforin activity. Overall, these data implied a general role for DNAM-1-CD155 interactions in NK cell-mediated killing of tumors, even in the presence of tumor CD70 or CD80 expression, and further defined the optimal efficacy requirements of cytokines that directly activate NK cells.

Functional dissection of the granzyme family: cell death and inflammation

Summary:u2002 Cytotoxic lymphocytes rapidly respond and destroy both malignant cells and cells infected with intracellular pathogens. One mechanism, known as granule exocytosis, employs the secretory granules of these lymphocytes. These include the pore‐forming protein perforin (pfp) and a family of serine proteases known as granzymes that cleave and activate effector molecules within the target cell. Over the past two decades, the study of granzymes has largely focused on the ability of these serine proteases to induce cell death. More recently, sophisticated mouse models of disease coupled with gene‐targeted mice have allowed investigators to ask why granzyme subfamilies are encoded on different chromosomal loci and what broader role these enzymes might play in inflammation and immune response. Here, we provide a brief overview of the granzyme superfamily, their relationship to pfp, and their reported functions in apoptosis. This overview is followed by a comprehensive analysis of the less characterized and developing field regarding the non‐apoptotic functions of granzymes.

Application of CD27 as a marker for distinguishing human NK cell subsets.

It has long been recognized that human NK cells can be divided into two phenotypically and functionally distinct subsets, based on their levels of expression of CD56. We recently found that CD27 distinguishes subsets of mature mouse NK cells. Here we report that CD27 can be used as a marker to discriminate human NK cell subsets. The majority of peripheral blood human NK cells were CD27(lo)/CD56(dim) NK cells, whereas the minor CD27(hi) NK cell population correspondingly displayed a CD56(bright) phenotype. Distinctions between CD27(lo) and CD27(hi) NK cells in their receptor expression and typical NK cell functions such as cytotoxicity and cytokine production can be easily delineated. Therefore, we propose the dual use of CD27 and CD56 as maturation/subset markers for human NK cells. The identification of CD27 subsets in both mice and humans will allow more accurate projections of the role of NK cell subsets in murine models of human pathologies where NK cells are involved.

IFN-γ-Dependent Recruitment of Mature CD27high NK Cells to Lymph Nodes Primed by Dendritic Cells

NK cells have been proposed to be an initial source of IFN-γ that supports either Th1 or CTL priming. Although NK cells reside in naive lymph nodes (LN) at a very low frequency, they can be recruited into LN draining sites of infection, inflammation, or immunization where they potentially influence adaptive immunity. In this study, we report that mature CD27high NK cells are predominantly recruited into the draining LN following dendritic cell (DC) challenge. Importantly, the recruitment of the CD27high NK cell subset in the draining LN was dependent on host IFN-γ and the activation status of NK cells. Endogenous epidermal DC migration induced by hapten challenge also triggers NK cell recruitment to the draining LN in an IFN-γ-dependent mechanism. Thus, our results identify that CD27high NK cells are the dominant population recruited to the draining LN and NK cell recruitment requires endogenous IFN-γ in coordinating with DC migration.

Failed CTL/NK cell killing and cytokine hypersecretion are directly linked through prolonged synapse time

Jenkins et al. discover that failure of perforin and granzyme cytotoxicity by human and mouse CTLs/NK cells prolongs the immunological synapse, leading to repetitive calcium signaling and hypersecretion of inflammatory mediators that subsequently activate macrophages. Disengagement from target cells is dependent on apoptotic caspase signaling. The findings may provide mechanistic understanding for immunopathology in familial hemophagocytic lymphohistiocytosis.

Cancer vaccines for established cancer: how to make them better?

Summary: If one envisions dendritic cells (DCs) as natures adjuvant, then it is easy to predict that they would be advantageous for cancer immunotherapy. Advances in culture processes that generate large numbers of purified and functionally mature DCs raised the possibility that DCs might be promising clinical agents to generate effective immune responses against cancer. The use of mature DCs as cellular vaccines was proposed to be superior to conventional strategies aimed at treating cancer, yet a phase III clinical trial in patients with melanoma demonstrated no increased benefit of DCs over standard therapy. Despite this and other apparent failures, we propose that DC‐based therapy should not be discarded but rather reassessed. The heterogeneity of DCs and their interaction with other innate cells and regulatory and effector pathways must be clearly understood before the full therapeutic benefit of DCs are recognized. Several aspects of DC vaccination require optimization including the following: effective delivery of vaccines to DCs in lymphoid tissues; incorporation of components that induce appropriate DC activation; and facilitation of innate and adaptive interactions and reduction of regulatory T‐cell networks or suppressive microenvironments that hinder the function of immune effectors. Application of this knowledge is resulting in encouraging new data in pre‐clinical settings, where multiple arms of the immune system are targeted for cancer therapy.

Can NK cells be a therapeutic target in human diseases

Our current knowledge of NK‐cell recognition and effector function suggests that it will be possible to design various new NK‐cell‐based immunotherapies against human cancer. The application of NK cells is already showing promise using HLA‐mismatched haematopoietic stem cell transplantation for treatment of haematological malignancies. A better understanding of NK‐cell heterogeneity and function will only broaden the applications for human cancer. Here we review the key developments that will propel this field.

A Critical Role for Granzymes in Antigen Cross-Presentation through Regulating Phagocytosis of Killed Tumor Cells

Granzymes A and B (GrAB) are known principally for their role in mediating perforin-dependent death of virus-infected or malignant cells targeted by CTL. In this study, we show that granzymes also play a critical role as inducers of Ag cross-presentation by dendritic cells (DC). This was demonstrated by the markedly reduced priming of naive CD8+ T cells specific for the model Ag OVA both in vitro and in vivo in response to tumor cells killed in the absence of granzymes. Reduced cross-priming was due to impairment of phagocytosis of tumor cell corpses by CD8α+ DC but not CD8α− DC, demonstrating the importance of granzymes in inducing the exposure of prophagocytic “eat-me” signals on the dying target cell. Our data reveal a critical and previously unsuspected role for granzymes A and B in dictating immunogenicity by influencing the mode of tumor cell death and indicate that granzymes contribute to the efficient generation of immune effector pathways in addition to their well-known role in apoptosis induction.

Loss of Host Type-I IFN Signaling Accelerates Metastasis and Impairs NK-cell Antitumor Function in Multiple Models of Breast Cancer

Type-I interferon immune signaling plays a critical role during the antimetastatic immune response. The authors show that loss of this pathway can promote bone metastasis in three different breast cancer models, revealing its importance across different models. Metastatic progression is the major cause of breast cancer–related mortality. By examining multiple syngeneic preclinical breast cancer models in mice lacking a functional type-I interferon receptor (Ifnar1−/− mice), we show that host-derived type-I interferon (IFN) signaling is a critical determinant of metastatic spread that is independent of primary tumor growth. In particular, we show that bone metastasis can be accelerated in Balb/c Ifnar1−/− mice bearing either 4T1 or 66cl4 orthotopic tumors and, for the first time, present data showing the development of bone metastasis in the C57Bl/6 spontaneous MMTV-PyMT–driven model of tumorigenesis. Further exploration of these results revealed that endogenous type-I IFN signaling to the host hematopoietic system is a key determinant of metastasis-free survival and critical to the responsiveness of the circulating natural killer (NK)–cell population. We find that in vivo–stimulated NK cells derived from wild-type, but not Ifnar1−/−, mice can eliminate the 4T1 and 66cl4 breast tumor lines with varying kinetics in vitro. Together, this study indicates that the dysregulated immunity resulting from a loss of host type-I IFN signaling is sufficient to drive metastasis, and provides a rationale for targeting the endogenous type-I IFN pathway as an antimetastatic strategy. Cancer Immunol Res; 3(11); 1207–17. ©2015 AACR.