Combined PD-L1/TGFß blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors.

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Peroxynitrite promotes immune evasion by reducing tumor antigenicity.

A study by Tcyganov et al.1 demonstrates that peroxynitrite, an oxidant abundant in the tumor microenvironment, changes the repertoire of MHC class I peptides presented by tumors and limits immune recognition. Peroxynitrite inhibition in combination with immune checkpoint blockade enhances efficacy preclinically.

Sources of Cancer Neoantigens beyond Single-Nucleotide Variants.

The success of checkpoint blockade therapy against cancer has unequivocally shown that cancer cells can be effectively recognized by the immune system and eliminated. However, the identity of the cancer antigens that elicit protective immunity remains to be fully explored. Over the last decade, most of the focus has been on somatic mutations derived from non-synonymous single-nucleotide variants (SNVs) and small insertion/deletion mutations (indels) that accumulate during cancer progression. Mutated peptides can be presented on MHC molecules and give rise to novel antigens or neoantigens, which have been shown to induce potent anti-tumor immune responses. A limitation with SNV-neoantigens is that they are patient-specific and their accurate prediction is critical for the development of effective immunotherapies. In addition, cancer types with low mutation burden may not display sufficient high-quality [SNV/small indels] neoantigens to alone stimulate effective T cell responses. Accumulating evidence suggests the existence of alternative sources of cancer neoantigens, such as gene fusions, alternative splicing variants, post-translational modifications, and transposable elements, which may be attractive novel targets for immunotherapy. In this review, we describe the recent technological advances in the identification of these novel sources of neoantigens, the experimental evidence for their presentation on MHC molecules and their immunogenicity, as well as the current clinical development stage of immunotherapy targeting these neoantigens.

Effective Treatment of Established Bone Metastases Can Be Achieved by Combinatorial Osteoclast Blockade and Depletion of Granulocytic Subsets.

Osteoclast (OC) blockade has been successful in reducing tumor growth in bone in preclinical settings, but antiresorptive drugs, such as zoledronic acid (ZA), fail to improve the overall survival rate of patients with bone metastasis despite ameliorating skeletal complications. To address this unmet clinical need, we interrogated what other cells modulated tumor growth in bone in addition to OCs. Because myeloid-derived suppressor cells (MDSC)-heterogeneous populations expressing CD11b, Ly6C, and Ly6G markers-originate in the bone marrow and promote tumor progression, we hypothesized that their accumulation hinders ZA antitumor effects. By using a murine model of bone metastasis insensitive to OC blockade, we assessed the antitumor effect of MDSC depletion using anti-Gr1 in mice bearing skeletal lung [Lewis lung carcinoma (LLC)], melanoma (B16-F10), and mammary (4T1) tumors. Differently from soft tissue tumors, anti-Gr1 did not reduce bone metastases and led to the paradoxical accumulation of bone marrow-resident CD11b+Ly6CintLy6Gint cells that differentiated into OCs when cultured in vitro Anti-Gr1-mediated depletion of Ly6G+ granulocytic MDSCs combined with ZA-induced OC blockade reduced growth of established skeletal metastases compared with each agent alone. CD15+ granulocytic populations were increased in patients with breast cancer with progressive bone disease after antiresorptive treatment compared with those with stable bone disease. We provide evidence that antiresorptive therapies fail to reduce bone metastases in the presence of elevated granulocytic populations and that effective treatment of established skeletal metastases requires combinatorial depletion of granulocytes and OC blockade.

Immunization associated with primary tumor growth leads to rejection of commonly used syngeneic tumors upon tumor rechallenge.

The recent success of multiple immunomodulating drugs in oncology highlights the potential of relieving immunosuppression by directly engaging the immune system in the tumor bed to target cancer cells. Durable responses to immune checkpoint inhibitors experienced by some patients may be indicative of the formation of a T cell memory response. This has prompted the search for preclinical evidence of therapy-induced long-term immunity as part of the evaluation of novel therapeutics. A common preclinical method used to document long-term immunity is the use of tumor rechallenge experiments in which tumor growth is assessed in mice that have previously rejected tumors in response to therapy. Failure of rechallenge engraftment, typically alongside successful engraftment of the same tumor in naive animals as a control, is often presented as evidence of therapy-induced tumor immunity. Here, we present evidence that formation of tumor immunity often develops independent of therapy. We observed elevated rates of rechallenge rejection following surgical resection of primary tumors for four of five commonly used models and that such postexcision immunity could be adoptively transferred to treatment-naïve mice. We also show that tumor-specific cytolytic T cells are induced on primary tumor challenge independent of therapeutic intervention. Taken together these data call into question the utility of tumor rechallenge studies and the use of naïve animals as controls to demonstrate therapy-induced formation of long-term tumor immunity.

Cancer neoantigens and immunogenicity: mutation position matters.

Cancer mutations can elicit protective immunity. Computational methods are critical for selecting these neoantigens for immunotherapy. While significant progress has been made in the field in predicting peptide presentation, our understanding of which mutated peptide is recognized as foreign by T cells remains limited. We used mouse vaccination studies to examine the features of immunogenic neoantigens and demonstrated that the mutation position is an important criterion for predicting neoantigens.

MAP4K4 negatively regulates CD8 T cell-mediated antitumor and antiviral immunity.

During cytotoxic T cell activation, lymphocyte function-associated antigen-1 (LFA-1) engages its ligands on antigen-presenting cells (APCs) or target cells to enhance T cell priming or lytic activity. Inhibiting LFA-1 dampens T cell-dependent symptoms in inflammation, autoimmune diseases, and graft-versus-host disease. However, the therapeutic potential of augmenting LFA-1 function is less explored. Here, we show that genetic deletion or inhibition of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) enhances LFA-1 activation on CD8 T cells and improves their adherence to APCs or LFA-1 ligand. In addition, loss of Map4k4 increases CD8 T cell priming, which culminates in enhanced antigen-dependent activation, proliferation, cytokine production, and cytotoxic activity, resulting in impaired tumor growth and improved response to viral infection. LFA-1 inhibition reverses these phenotypes. The ERM (ezrin, radixin, and moesin) proteins reportedly regulate T cell-APC conjugation, but the molecular regulator and effector of ERM proteins in T cells have not been defined. In this study, we demonstrate that the ERM proteins serve as mediators between MAP4K4 and LFA-1. Last, systematic analyses of many organs revealed that inducible whole-body deletion of Map4k4 in adult animals is tolerated under homeostatic conditions. Our results uncover MAP4K4 as a potential target to augment antitumor and antiviral immunity.

Mutation position is an important determinant for predicting cancer neoantigens.

Tumor-specific mutations can generate neoantigens that drive CD8 T cell responses against cancer. Next-generation sequencing and computational methods have been successfully applied to identify mutations and predict neoantigens. However, only a small fraction of predicted neoantigens are immunogenic. Currently, predicted peptide binding affinity for MHC-I is often the major criterion for prioritizing neoantigens, although little progress has been made toward understanding the precise functional relationship between affinity and immunogenicity. We therefore systematically assessed the immunogenicity of peptides containing single amino acid mutations in mouse tumor models and divided them into two classes of immunogenic mutations. The first comprises mutations at a nonanchor residue, for which we find that the predicted absolute binding affinity is predictive of immunogenicity. The second involves mutations at an anchor residue; here, predicted relative affinity (compared with the WT counterpart) is a better predictor. Incorporating these features into an immunogenicity model significantly improves neoantigen ranking. Importantly, these properties of neoantigens are also predictive in human datasets, suggesting that they can be used to prioritize neoantigens for individualized neoantigen-specific immunotherapies.

Coexpression of Inhibitory Receptors Enriches for Activated and Functional CD8+ T Cells in Murine Syngeneic Tumor Models.

Exhausted T cells have been described in cancer patients and murine tumor models largely based on their expression of various inhibitory receptors. Understanding of the functional attributes of these cells is limited. Here, we report that among CD8+ T cells in commonly used syngeneic tumor models, the coexpression of inhibitory receptors PD-1, LAG3, and TIM3 defined a group of highly activated and functional effector cells. Coexpression of these receptors further enriched for antigen-specific cells with increased T-cell receptor clonality. Anti-PD-L1 treatment increased the number and activation of these triple-positive CD8+ T cells without affecting the density of PD-1- cells. The intratumoral density of CD8+ T cells coexpressing inhibitory receptors negatively correlated with tumor burden. The density ratio and pretreatment phenotype of CD8+ T cells coexpressing inhibitory receptors was positively correlated with response across a variety of tumor models. Our results demonstrate that coexpression of inhibitory receptors is not a signifier of exhausted T cells, but rather can define a group of activated and functional effector cells in syngeneic tumor models. In the cancer setting, these cells could represent a heterogeneous population of not only exhausted but also highly activated cells responsive to treatment.

Characterizing neoantigens for personalized cancer immunotherapy.

Somatic mutations can generate neoantigens that are presented on MHC molecules and drive effective T cells responses against cancer. Mutation load in cancer patients predicts response to immune checkpoint blockade therapy. Additionally, vaccination targeting neoantigens controls established tumor growth in preclinical models. These recent findings led to a renewed interest in the field of cancer vaccines and the development of antigen-targeted cancer immunotherapies. However, targeting neoantigens is challenging, as most mutations are unique to each cancer patient. In addition, only a small fraction of the mutations are immunogenic and therefore their accurate prediction is critical. In this review, we discuss the properties of neoantigens that influence their immunogenicity, along with questions that remain to be addressed in order to improve prediction algorithms.

Novel ERα positive breast cancer model with estrogen independent growth in the bone microenvironment.

Despite successful therapeutic options for estrogen receptor-α (ERα)+ breast cancer, resistance to endocrine therapy frequently occurs leading to tumor recurrence. In addition to intrinsic changes in the cancer cells, herein we demonstrate that tumor cell-microenvironment interactions can drive recurrence at specific sites. By using two ERα+ cell lines derived from spontaneous mammary carcinomas in STAT1-/- mice (SSM2, SSM3), we establish that the bone microenvironment offers growth advantage over primary site or lung in the absence of ovarian hormones. While SSM3 did not engraft at primary and skeletal locations in the absence of estrogen, SSM2 selectively grew in bone of ovariectomized mice and following administration of aromatase inhibitors. However, SSM2 growth remained hormone-dependent at extraskeletal sites. Unexpectedly, bone-residing SSM2 cells retained ERα expression and JAK2/STAT3 activation regardless of the hormonal status. These data position the bone microenvironment as a unique site for acquisition of tumor/estrogen independency and identify the first ERα+ hormone-independent tumor model in immunocompetent mice.

Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis.

Age is a significant risk factor for the development of cancer. However, the mechanisms that drive age-related increases in cancer remain poorly understood. To determine if senescent stromal cells influence tumorigenesis, we develop a mouse model that mimics the aged skin microenvironment. Using this model, here we find that senescent stromal cells are sufficient to drive localized increases in suppressive myeloid cells that contributed to tumour promotion. Further, we find that the stromal-derived senescence-associated secretory phenotype factor interleukin-6 orchestrates both increases in suppressive myeloid cells and their ability to inhibit anti-tumour T-cell responses. Significantly, in aged, cancer-free individuals, we find similar increases in immune cells that also localize near senescent stromal cells. This work provides evidence that the accumulation of senescent stromal cells is sufficient to establish a tumour-permissive, chronic inflammatory microenvironment that can shelter incipient tumour cells, thus allowing them to proliferate and progress unabated by the immune system.

Dickkopf-related protein 1 (Dkk1) regulates the accumulation and function of myeloid derived suppressor cells in cancer.

Tumor-stroma interactions contribute to tumorigenesis. Tumor cells can educate the stroma at primary and distant sites to facilitate the recruitment of heterogeneous populations of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs). MDSCs suppress T cell responses and promote tumor proliferation. One outstanding question is how the local and distant stroma modulate MDSCs during tumor progression. Down-regulation of ß-catenin is critical for MDSC accumulation and immune suppressive functions in mice and humans. Here, we demonstrate that stroma-derived Dickkopf-1 (Dkk1) targets ß-catenin in MDSCs, thus exerting immune suppressive effects during tumor progression. Mice bearing extraskeletal tumors show significantly elevated levels of Dkk1 in bone microenvironment relative to tumor site. Strikingly, Dkk1 neutralization decreases tumor growth and MDSC numbers by rescuing ß-catenin in these cells and restores T cell recruitment at the tumor site. Recombinant Dkk1 suppresses ß-catenin target genes in MDSCs from mice and humans and anti-Dkk1 loses its antitumor effects in mice lacking ß-catenin in myeloid cells or after depletion of MDSCs, demonstrating that Dkk1 directly targets MDSCs. Furthermore, we find a correlation between CD15(+) myeloid cells and Dkk1 in pancreatic cancer patients. We establish a novel immunomodulatory role for Dkk1 in regulating tumor-induced immune suppression via targeting ß-catenin in MDSCs.

Immune regulation of bone metastasis.

Metastases to bone occur in about 70% of patients with metastatic prostate and breast cancers. Unfortunately, bone metastases result in significant morbidity and mortality and treatment options are limited. Thus, significant effort has focused on understanding the mechanisms that drive tumor dissemination to bone. Bone metastases are typically characterized by a self-perpetuating 'vicious' cycle wherein tumor cells and bone-resorbing cells (osteoclasts) are locked in a cycle that leads to osteoclast-driven bone destruction and the release of bone-stored factors that in turn stimulate tumor cell proliferation and survival. To break this 'vicious' cycle, potent antiresorptive agents such as zoledronic acid (ZOL) have been used. However, in the clinical setting, ZOL failed to improve the overall survival of cancer patients even though it inhibited osteoclast resorptive activity. Thus, other cells in addition to osteoclasts are likely involved in modulating tumor growth in the bone. The immune system has the ability to eliminate tumor cells. Nevertheless, tumor cells can acquire the ability to escape immune control. Our recent observations indicated that a decline in the ability of the immune cells to recognize and kill the tumor drives tumor dissemination to bone even when osteoclasts are inhibited by potent antiresorptive agents. This review focuses on the antitumor and protumor effects of various immune cell populations involved in the bone metastatic process. We also discuss strategies to enhance antitumor immune responses and bypass cancer immune resistance.

Down-regulation of PLCγ2-ß-catenin pathway promotes activation and expansion of myeloid-derived suppressor cells in cancer.

Myeloid-derived suppressor cells (MDSCs) favor tumor promotion, mainly by suppressing antitumor T cell responses in many cancers. Although the mechanism of T cell inhibition is established, the pathways leading to MDSC accumulation in bone marrow and secondary lymphoid organs of tumor-bearing hosts remain unclear. We demonstrate that down-regulation of PLCγ2 signaling in MDSCs is responsible for their aberrant expansion during tumor progression. PLCγ2(-/-) MDSCs show stronger immune-suppressive activity against CD8(+) T cells than WT MDSCs and potently promote tumor growth when adoptively transferred into WT mice. Mechanistically, PLCγ2(-/-) MDSCs display reduced ß-catenin levels, and restoration of ß-catenin expression decreases their expansion and tumor growth. Consistent with a negative role for ß-catenin in MDSCs, its deletion in the myeloid population leads to MDSC accumulation and supports tumor progression, whereas expression of ß-catenin constitutively active reduces MDSC numbers and protects from tumor growth. Further emphasizing the clinical relevance of these findings, MDSCs isolated from pancreatic cancer patients show reduced p-PLCγ2 and ß-catenin levels compared with healthy controls, similar to tumor-bearing mice. Thus, for the first time, we demonstrate that down-regulation of PLCγ2-ß-catenin pathway occurs in mice and humans and leads to MDSC-mediated tumor expansion, raising concerns about the efficacy of systemic ß-catenin blockade as anti-cancer therapy.

Stimulated γδ T cells increase the in vivo efficacy of trastuzumab in HER-2+ breast cancer.

One fourth of women with HER-2(+) metastatic breast carcinoma are treated with a combination regimen with trastuzumab, but the frequent resistance to this Ab requires definition of new means to improve its bioactivity. The mechanisms of action of trastuzumab involve several pathways including Ab-dependent cellular cytotoxicity. Because human γδ T lymphocytes mediate Ab-dependent cellular cytotoxicity and can be activated further by phosphoantigens, these cells are prone to improve the efficacy of Abs, as recently demonstrated for CD20(+) B cell lymphomas. Whether this concept applies as well with carcinomas remained to be demonstrated in vivo, however. In this study, we asked whether a combination of trastuzumab and phosphoantigen-stimulated γδ lymphocytes increases the efficacy of trastuzumab against HER-2(+) breast carcinoma cell lines in vivo. We report that repeated infusions of this combination had a better efficacy than that of trastuzumab alone against HER-2(+) mammary carcinoma xenografts in mice. In these models, reduction of tumor growth was observed together with trastuzumab opsonization of HER-2(+) cells and tumor infiltration by γδ lymphocytes. In addition in humans, the mammary carcinomas of 27 of 30 patients showed significant γδ T cell infiltrates. Altogether, these findings indicate that combination of trastuzumab and stimulated γδ cells represents a new strategy to improve the efficacy of Herceptin (trastuzumab) in HER-2(+) breast cancer.

How tumors might withstand γδ T-cell attack.

Several clinical trials are currently assessing the therapeutic activity of human TCRVγ9Vδ2(+) lymphocytes in cancer. Growing tumors usually follow a triphasic "Elimination, Equilibrium, Escape" evolution in patients. Thus, at diagnostic, most tumors have already developed some means to escape to immune protection. We review here the conventional immunoescape mechanisms which might also protect against cytolytic TCRVγ9Vδ2(+) lymphocytes activated by phosphoantigens. Neutralization of these deleterious processes might prove highly valuable to improve the efficacy of ongoing γδ cell-based cancer immunotherapies.

Phosphoantigens overcome human TCRVgamma9+ gammadelta Cell immunosuppression by TGF-beta: relevance for cancer immunotherapy.

Human gammadelta cells expressing TCRVgamma9 are HLA-unrestricted CTLs with high relevance for cancer immunotherapy. Many tumor cell types produce TGF-beta, however, a cytokine strongly immunosuppressive for conventional T CD4, CD8, and NK cells. Whether TGF-beta also inhibits TCRVgamma9+ lymphocytes was unknown. Because phosphoantigens (PAgs), such as bromohydrin pyrophosphate, selectively activate the antitumor functions of TCRVgamma9+ T cells, in this study, we investigated whether TGF-beta modulates these functions. We report that TGF-beta does not block activation of TCRVgamma9+ T cells but inhibits their PAg/IL-2-induced proliferation and maturation into effector cells and finally reduces the cytotoxic activity of these gammadelta T cells when exposed to lymphoma target cells. TGF-beta did not bias their differentiation pattern toward gammadelta Th17 or gammadelta regulatory T cells. Nevertheless, increasing doses of PAg stimulus countered TGF-beta inhibition. So, although TGF-beta impairs TCRVgamma9+ gammadelta cells like other cytolytic lymphocytes, PAg alone or combined to therapeutic mAb has the ability to bypass its immunosuppressive activity.

Emerging concepts for the treatment of hematological malignancies with therapeutic monoclonal antibodies.

The development of therapeutic monoclonal antibodies (mAbs) has revolutionized the treatment of cancer along the last ten years. The best examples of their therapeutic efficacies have been obtained with rituximab for the treatment of CD20+ B-cell Non-Hodgkin Lymphoma (B-NHL), and several others antibodies with optimized bioactivities are now being developed for the treatment of various malignant hemopathies. We review here the main drugs developed in this field, and present some emerging concepts able to improve the bioactivities of the next generation of therapeutic mAbs.

Bromohydrin pyrophosphate enhances antibody-dependent cell-mediated cytotoxicity induced by therapeutic antibodies.

In human blood, 1% to 5% of lymphocytes are gammadelta T cells; they mostly express the gammadelta T-cell receptor (TCR)Vgamma9, recognize nonpeptide phosphoantigens (PAgs) produced by microbes and tumor cells, and mediate different modes of lytic activities directed against tumor target cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by cytolytic lymphoid cells is essential for the clinical activity of anticancer monoclonal antibodies (mAbs), but whether PAgs affect ADCC by gammadelta T cells is unknown. Here we report that, in association with the CD20(+)-specific mAb rituximab (RTX), the synthetic PAg bromohydrin pyrophosphate (BrHPP) increased TCRVgamma9(+) cell binding to CD20(+) lymphoma cells in vitro. This combination activated phospho-ZAP70 and phospho-ERK1/2 signaling in TCRVgamma9(+) cells and strongly enhanced their ADCC activity. We obtained similar results with BrHPP in the context of the mAbs alemtuzumab and trastuzumab. Furthermore, BrHPP enhanced RTX-mediated depletion of CD20(+) cells in vitro from peripheral blood mononuclear cells of healthy subjects and enhanced ADCC by gammadelta T cells from patients with chronic lymphocytic leukemia. In cynomolgus macaques, a regimen combining RTX, BrHPP, and IL2 activated TCRVgamma9(+) lymphocytes and enhanced B-cell depletion from blood and lymph nodes. Thus, the combination with BrHPP PAg is able to improve the efficacy of cancer immunotherapy by therapeutic mAbs.