The complexity of immune evasion mechanisms throughout the metastatic cascade.

Metastasis, the spread of cancer from a primary site to distant organs, is an important challenge in oncology. This Review explores the complexities of immune escape mechanisms used throughout the metastatic cascade to promote tumor cell dissemination and affect organotropism. Specifically, we focus on adaptive plasticity of disseminated epithelial tumor cells to understand how they undergo phenotypic transitions to survive microenvironmental conditions encountered during metastasis. The interaction of tumor cells and their microenvironment is analyzed, highlighting the local and systemic effects that innate and adaptive immune systems have in shaping an immunosuppressive milieu to foster aggressive metastatic tumors. Effectively managing metastatic disease demands a multipronged approach to target the parallel and sequential mechanisms that suppress anti-tumor immunity. This management necessitates a deep understanding of the complex interplay between tumor cells, their microenvironment and immune responses that we provide with this Review.

Lethal giant larvae-1 deficiency enhances the CD8(+) effector T-cell response to antigen challenge in vivo.

Lethal giant larvae-1 (Lgl-1) is an evolutionary conserved protein that regulates cell polarity in diverse lineages; however, the role of Lgl-1 in the polarity and function of immune cells remains to be elucidated. To assess the role of Lgl-1 in T cells, we generated chimeric mice with a hematopoietic system deficient for Lgl-1. Lgl-1 deficiency did not impair the activation or function of peripheral CD8(+) T cells in response to antigen presentation in vitro, but did skew effector and memory T-cell differentiation. When challenged with antigen-expressing virus or tumor, Lgl-1-deficient mice displayed altered T-cell responses. This manifested in a stronger antiviral and antitumor effector CD8(+) T-cell response, the latter resulting in enhanced control of MC38-OVA tumors. These results reveal a novel role for Lgl-1 in the regulation of virus-specific T-cell responses and antitumor immunity.

Membrane-bound Fas ligand only is essential for Fas-induced apoptosis.

Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasL(Deltas/Deltas)) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasL(Deltam/Deltam)) could not kill cells through Fas activation. FasL(Deltam/Deltam) mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL(gld/gld) mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL(Deltam/Deltam) mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL(gld/gld) mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

Chemotherapy and radiotherapy: cryptic anticancer vaccines.

An attractive, yet hitherto unproven concept predicts that the promotion of tumor regression should elicit the host's immune response against residual tumor cells to achieve an optimal therapeutic effect. In a way, chemo- or radiotherapy must trigger "danger signals" emitted from immunogenic cell death and hence elicit "danger associated molecular patterns" to stimulate powerful anticancer immune responses. Here, based on the recent experimental and clinical evidence, we will discuss the molecular identity of the multiple checkpoints that dictate the success of "immunogenic chemotherapy" at the levels of the drug, of the tumor cell and of the host immune system.

Eradication of solid tumors using histone deacetylase inhibitors combined with immune-stimulating antibodies.

Histone deacetylase inhibitors (HDACi) have been successfully used as monotherapies for the treatment of hematological malignancies; however, the single agent effects of HDACi against solid tumors are less robust. Using preclinical models of lymphoma, we have recently demonstrated that HDACi induce tumor cell-specific apoptosis and that this is essential for the therapeutic effects of these agents. Herein, we demonstrate that HDACi can be combined with immune-activating antibodies designed to promote the function of antigen-presenting cells (APCs) and enhance proliferation and survival of cytotoxic T cells (CTL) to stimulate a host antitumor immune response resulting in eradication of established solid tumors. This unique combination therapy was dependent on tumor cell apoptosis mediated by HDACi that stimulated the uptake of dead tumor cells by APCs. Tumor eradication was mediated by CD8(+) CTL that used perforin as the key immune effector molecule. This combination therapy was well tolerated and induced long-term immunological antitumor memory capable of mediating spontaneous tumor eradication upon rechallenge. These studies indicate that the ability of HDACi to mediate subtherapeutic levels of tumor cell apoptosis can be exploited by combining with antibodies that augment host antitumor immune responses to mediate robust and prolonged eradication of solid tumors.

Context-restricted PD-(L)1 checkpoint agonism by CTLA4-Ig therapies inhibits T cell activity.

T cell surface CTLA4 sequesters the costimulatory ligands CD80 and CD86 on antigen-presenting cells (APCs) to prevent autoimmunity. Therapeutic immunosuppression by recombinant CTLA4-immunoglobulin (Ig) fusion proteins, including abatacept, is also attributed to CD80/CD86 blockade. Recent studies show that CTLA4-Ig binding to APC surface cis-CD80:PD-L1 complexes can release the inhibitory ligand PD-L1, but whether this contributes to T cell inhibition remains unclear. Here, we show that PD-L1 liberation by CTLA4-Ig is strictly limited, both in extent and context, relative to PD-L1-competing anti-CD80 antibodies. At APC surface CD80:PD-L1 ratios exceeding 2:1, CTLA4-Ig therapies fail to release PD-L1 regardless of their CD80 affinity. Additionally, introducing flexibility into CTLA4-Ig by modifying its rigid homodimer interface produces biologics that retain bivalent CD80 binding without dissociating cis-bound PD-L1. These findings demonstrate that CTLA4-Ig therapies liberate PD-L1 through a CD80 reorientation mechanism that imposes a strict context dependence to their PD-1 checkpoint agonism and resultant T cell inhibition.

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.

Plasma cell S1P1 expression determines secondary lymphoid organ retention versus bone marrow tropism.

After induction in secondary lymphoid organs, a subset of antibody-secreting cells (ASCs) homes to the bone marrow (BM) and contributes to long-term antibody production. The factors determining secondary lymphoid organ residence versus BM tropism have been unclear. Here we demonstrate that in mice treated with FTY720 or that lack sphingosine-1-phosphate (S1P) receptor-1 (S1P1) in B cells, IgG ASCs are induced and localize normally in secondary lymphoid organs but they are reduced in numbers in blood and BM. Many IgG ASCs home to BM on day 3 of the secondary response and day 3 splenic ASCs exhibit S1P responsiveness, whereas the cells remaining at day 5 are unable to respond. S1P1 mRNA abundance is higher in ASCs isolated from blood compared to spleen, whereas CXCR4 expression is lower. Blood ASCs also express higher amounts of Kruppel-like factor (KLF)2, a regulator of S1P1 gene expression. These findings establish an essential role for S1P1 in IgG plasma cell homing and they suggest that differential regulation of S1P1 expression in differentiating plasma cells may determine whether they remain in secondary lymphoid organs or home to BM.

CD11c+ dendritic cells and B cells contribute to the tumoricidal activity of anti-DR5 antibody therapy in established tumors.

The selective targeting of the tumor-associated death-inducing receptors DR4 and DR5 with agonistic mAbs has demonstrated preclinical and clinical antitumor activity. However, the cellular and molecular mechanisms contributing to this efficacy remain poorly understood. In this study, using the first described C57BL/6 (B6) TRAIL-sensitive experimental tumor models, we have characterized the innate and adaptive immune components involved in the primary rejection phase of an anti-mouse DR5 (mDR5) mAb, MD5-1 in established MC38 colon adenocarcinomas. FcR mediated cross-linking of MD5-1 significantly inhibited the growth of MC38 colon adenocarcinomas through the induction of TRAIL-R-dependent tumor cell apoptosis. The loss of host DR5, TRAIL, perforin, FasL, or TNF did not compromise anti-DR5 therapy in vivo. By contrast, anti-DR5 therapy was completely abrogated in mice deficient of B cells or CD11c(+) dendritic cells (DCs), providing the first direct evidence that these cells play a critical role. Importantly, the requirement for an intact B cell compartment for optimal anti-DR5 antitumor efficacy was also observed in established AT-3 mammary tumors. Interestingly, MD5-1-mediated apoptosis as measured by early TUNEL activity was completely lost in B cell-deficient microMT mice, but intact in mice deficient in CD11c(+) DCs. Overall, these data show that Ab-mediated targeting of DR5 triggers tumor cell apoptosis in established tumors in a B cell-dependent manner and that CD11c(+) DCs make a critical downstream contribution to anti-DR5 antitumor activity.

Basic cancer immunology for radiation oncologists.

Although the impressive clinical responses seen with modern cancer immunotherapy are currently limited to a subset of patients, the underlying paradigm shift has resulted in now hardly a segment in oncology that has not been touched by the immuno-oncology revolution. A growing body of data indicates that radiation therapy (RT) can modulate the tumour immune microenvironment and complement cancer immunotherapy via non-overlapping mechanisms to reinvigorate immunity against cancer. Thus, increasingly RT is viewed as a highly unique partner for immunotherapy across the spectrum of cancer settings, as radiobiology and cancer immunology foreseeably become more intertwined. Considering these developments, this review summarises the key concepts and terminology in immunology for the radiation oncologist, with a focus on the cancer setting and with reference to important recent advances. These concepts will provide a starting point for understanding the strategies that underlie current and emerging immunotherapy trials, as well as the indirect effects of RT by which immune responses against cancer are shaped.

Estrogen-induced immune changes within the normal mammary gland.

Breast cancer (BCa) incidence increases following aberrant hormone exposure, which has been linked to direct effects on estrogen receptor (ER)+ mammary epithelium. While estrogen exposure during mammary involution has been shown to drive tumour growth via neutrophils, the potential for the ER + immune microenvironment to mediate part (in addition to mammary epithelial cells) of hormonally controlled BCa risk during normal development has not been assessed. We collected mammary tissue, lymph nodes and blood from tumour naïve mice treated with, oophorectomy, estrogen (17ß estradiol) or Fulvestrant. Flow cytometry was used to examine the impact on the frequency of innate and adaptive immune cells. Oophorectomy and fulvestrant decreased the proportion of macrophages, particularly pro-tumour polarized M2 macrophages and neutrophils. Conversely, dendritic cells were increased by these therapies, as were eosinophils. Estrogen increased the proportion of M2 macrophages and to a lesser extent CD4-CD8- double negative and FoxP3+ regulatory T cells but decreased CD8 + T cells and B cells. Excluding eosinophils, these changes were restricted to the mammary tissue. This suggests that inhibiting estrogen action lowers the immune suppressive myeloid cells, increases in antigen presentation and eosinophil-mediated direct or indirect cytotoxic effects. In contrast, estrogen exposure, which drives BCa risk, increases the suppressive myeloid cells and reduces anti-tumour cytotoxic T cells. The impact of hormonal exposure on BCa risk, may in part be linked to its immune modulatory activity.

Immunogenic anti-cancer chemotherapy as an emerging concept.

Tumors can acquire mutations or hijack regulatory pathways of the host immune system to render them resistant to immune attack. Standard first line therapies such as chemotherapy and radiation were not thought to provoke natural immunity to cancer, but recent findings demonstrating that dying tumor cells present and release key signals to stimulate or evade neighboring leukocytes are challenging that view. Killing tumor cells in a manner that provides danger signals and tumor antigens in the right context promotes the engagement of innate and adaptive immunity; however, this response alone will not be effective against established cancer. Coincidently driving the immune response with specific monoclonal antibodies and other immunomodulators that activate and mature dendritic cells and co-stimulate T cells and other lymphocytes is one approach. Additionally releasing immune checkpoints and inhibiting tumor-derived molecules that prevent effective tumor immunity is another. Combined these approaches have enormous potential to improve the current outcomes from conventional cancer therapy.

Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist.

Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.

Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease.

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

CAR-T Plus Radiotherapy: A Promising Combination for Immunosuppressive Tumors.

Radiotherapy (RT) is the standard-of-care treatment for more than half of cancer patients with localized tumors and is also used as palliative care to facilitate symptom relief in metastatic cancers. In addition, RT can alter the immunosuppressive tumor microenvironment (TME) of solid tumors to augment the anti-tumor immune response of immune checkpoint blockade (ICB). The rationale of this combination therapy can also be extended to other forms of immunotherapy, such as chimeric antigen receptor T cell (CAR-T) therapy. Similar to ICB, the efficacy of CAR-T therapy is also significantly impacted by the immunosuppressive TME, leading to compromised T cell function and/or insufficient T cell infiltration. In this review, we will discuss some of the key barriers to the activity of CAR-T cells in the immunosuppressive TME and focus on how RT can be used to eliminate or bypass these barriers. We will present the challenges to achieving success with this therapeutic partnership. Looking forward, we will also provide strategies currently being investigated to ensure the success of this combination strategy in the clinic.

Regulatory T Cells Shape the Differential Impact of Radiation Dose-Fractionation Schedules on Host Innate and Adaptive Antitumor Immune Defenses.

PURPOSE: We examined how radiation dose per fraction (DPF) and total dose, as represented by biological effective dose (BED), can independently and differentially affect the immunomodulatory capacity of radiation therapy (RT). METHODS AND MATERIALS: AT3-OVA mammary and MC38 colorectal tumors in C57BL/6 mice were irradiated with rationally selected dose-fractionation schedules, alone or with immune-modulating or -depleting agents. Tumor growth was monitored as a readout of therapeutic response. Flow cytometry and RNA sequencing of mouse tumors and analysis of transcriptomic data sets from irradiated human cancers were used to examine the immunomodulatory effects of the different radiation schedules. RESULTS: In AT3-OVA tumors, radiation DPF rather than BED determined the ability of RT to evoke local antitumor CD8+ T cell responses and synergize with anti-PD-1 therapy. Natural killer cell-mediated control of irradiated tumors was more sensitive to radiation BED. Radiation-induced regulatory T cell (Treg) responses, which were detected in both mouse and human tumors, were a major factor underlying the differential activation of adaptive immunity by radiation DPF and the activity of natural killer cells during the early phase of response to RT. Targeted inhibition of Treg responses within irradiated tumors rescued and enhanced local tumor control by RT and permitted the generation of abscopal and immunologic memory responses, irrespective of radiation schedule. MC38 tumors did not support the induction of an amplified Treg response to RT and were highly vulnerable to its immunoadjuvant effects. CONCLUSIONS: Local radiation-induced Treg responses are influenced by radiation schedule and tumor type and are a critical determinant of the immunoadjuvant potential of RT and its ability to synergize with T cell-targeted immunotherapy.

Combined BRAF, MEK, and CDK4/6 Inhibition Depletes Intratumoral Immune-Potentiating Myeloid Populations in Melanoma.

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a-/-Pten-/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

CDK4/6 Inhibition Promotes Antitumor Immunity through the Induction of T-cell Memory.

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor-positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. SIGNIFICANCE: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer.This article is highlighted in the In This Issue feature, p. 2355.

Molecular and genomic characterisation of a panel of human anal cancer cell lines.

Anal cancer is a rare disease that has doubled in incidence over the last four decades. Current treatment and survival of patients with this disease has not changed substantially over this period of time, due, in part, to a paucity of preclinical models to assess new therapeutic options. To address this hiatus, we set-out to establish, validate and characterise a panel of human anal squamous cell carcinoma (ASCC) cell lines by employing an explant technique using fresh human ASCC tumour tissue. The panel of five human ASCC cell lines were validated to confirm their origin, squamous features and tumourigenicity, followed by molecular and genomic (whole-exome sequencing) characterisation. This panel recapitulates the genetic and molecular characteristics previously described in ASCC including phosphoinositide-3-kinase (PI3K) mutations in three of the human papillomavirus (HPV) positive lines and TP53 mutations in the HPV negative line. The cell lines demonstrate the ability to form tumouroids and retain their tumourigenic potential upon xenotransplantation, with varied inducible expression of major histocompatibility complex class I (MHC class I) and Programmed cell death ligand 1 (PD-L1). We observed differential responses to standard chemotherapy, radiotherapy and a PI3K specific molecular targeted agent in vitro, which correlated with the clinical response of the patient tumours from which they were derived. We anticipate this novel panel of human ASCC cell lines will form a valuable resource for future studies into the biology and therapeutics of this rare disease.

Characterizing the anti-tumor function of adoptively transferred NK cells in vivo.

Natural killer (NK) cells represent a promising cell type to utilize for effective adoptive immunotherapy. However, little is known about the important cytolytic molecules and signaling pathways used by NK cells in the adoptive transfer setting. To address this issue, we developed a novel mouse model to investigate the trafficking and mechanism of action of these cells. We demonstrate that methylcholanthrene-induced RKIK sarcoma cells were susceptible to NK cell-mediated lysis in vitro and in vivo following adoptive transfer of NK cells in C57BL/6 RAG-2(-/-)gammac(-/-) mice. Cytotoxic molecules perforin, granzymes B and M as well as the death ligand TRAIL and pro-inflammatory cytokine IFN-gamma were found to be important in the anti-tumor effect mediated by adoptively transferred NK cells. Importantly, we demonstrate that adoptively transferred NK cells could traffic to the tumor site and persisted in vivo which correlated with the anti-tumor effect observed. Overall, the results of this study have important implications for enhancing NK cell-based immunotherapies.