Anti-tumor Effector Functions Research Articles

Potent anti-tumor effector functions in tumor-infiltrating MAIT and γδ T cells isolated from colon cancer patients

Abstract In colon cancer, tumor progression and patient outcome is affected by the cytokine balance in the tumors. IFNγ, TNFα and Granzyme B expression are associated with favorable patient outcome, while high IL-17 expression is associated with accelerated tumor progression. However, knowledge of the regulation and activation of unconventional T cell subsets in colon tumors is limited. The aim of this study was to characterize unconventional T cells in colon tumors and unaffected tissue, determine their capacity to produce cytokines affecting tumor progression as well as the In vitro cytotoxic capabilities of MAIT and γδ T cells. Using flow cytometry, we show that MAIT cells accumulate in colon tumors and that the frequencies of γδ T cells are reduced in the tumor epithelium. Using polyclonal stimulation, we show that IFNγ production by tumour infiltrating MAIT cells is impaired whilst tumour infiltrating γδ T have an increased expression of IFNγ, TNFα and Granzyme B. IL-17 expression was also elevated in tumour infiltrating γδ T cells, but at lower levels than the TH1 - associated cytokines. Tumor infiltrating MAIT cells had an exhausted (PD-1highTim-3+) phenotype compared to MAIT cells from unaffected tissue. Analyzing cytokine expression, we show that while no single molecule is lost the polyfunctional capacity of tumour infiltrating MAIT cells is decreased compared to MAIT cells from unaffected tissue. Altogether, this study shows that γδ T cells and MAIT cells contribute to the cytokine balance in colon tumors with a TH1 – dominated profile and that they have potent cytotoxic capacity, which may reduce tumor progression and improve patient outcome.

Innate Lymphoid Cells: Expression of PD-1 and Other Checkpoints in Normal and Pathological Conditions.

Innate lymphoid cells (ILCs) belong to a family of immune cells. Recently, ILCs have been classified into five different groups that mirror the function of adaptive T cell subsets counterparts. In particular, NK cells mirror CD8+ cytotoxic T cells while ILC1, ILC2, ILC3, and Lymphoid tissue inducer (LTi)-like cells reflect the function of CD4+T helper (Th) cells (Th1, Th2, and Th17 respectively). ILCs are involved in innate host defenses against pathogens and tumors, in lymphoid organogenesis, and in tissue remodeling/repair. In recent years, important molecular inducible checkpoints (PD-1, TIM3, and TIGIT) were shown to control/inactivate different immune cell types. The expression of many of these receptors has been detected on NK cells and subsets of tissue-resident ILCs in both physiological and pathological conditions, including cancer. In particular, it has been demonstrated that the interaction between PD-1+ immune cells and PD-L1/PD-L2+ tumor cells may compromise the anti-tumor effector function leading to tumor immune escape. However, while the effector function of NK cells in tumor is well-established, limited information exists on the other ILC subsets. We will summarize what is known to date on the expression and function of these checkpoint receptors on NK cells and ILCs, with a particular focus on the recent data that reveal an essential contribution of the blockade of PD-1 and TIGIT on NK cells to the immunotherapy of cancer. A better information regarding the presence and the function of different ILCs and of the inhibitory checkpoints in pathological conditions may offer important clues for the development of new immune therapeutic strategies.

Macrophage Origin, Metabolic Reprogramming and IL-1 Signaling: Promises and Pitfalls in Lung Cancer.

Macrophages are tissue-resident cells that act as immune sentinels to maintain tissue integrity, preserve self-tolerance and protect against invading pathogens. Lung macrophages within the distal airways face around 8000–9000 L of air every day and for that reason are continuously exposed to a variety of inhaled particles, allergens or airborne microbes. Chronic exposure to irritant particles can prime macrophages to mediate a smoldering inflammatory response creating a mutagenic environment and favoring cancer initiation. Tumor-associated macrophages (TAMs) represent the majority of the tumor stroma and maintain intricate interactions with malignant cells within the tumor microenvironment (TME) largely influencing the outcome of cancer growth and metastasis. A number of macrophage-centered approaches have been investigated as potential cancer therapy and include strategies to limit their infiltration or exploit their antitumor effector functions. Recently, strategies aimed at targeting IL-1β signaling pathway using a blocking antibody have unexpectedly shown great promise on incident lung cancer. Here, we review the current understanding of the bridge between TAM metabolism, IL-1β signaling, and effector functions in lung adenocarcinoma and address the challenges to successfully incorporating these pathways into current anticancer regimens.

Abstract A095: Discovering the immune profiles of a novel antifolate receptor alpha IgE antibody associated with monocyte-mediated antitumor functions

Abstract Monoclonal antibodies are an established modality for the clinical management of many cancers. To date, all antibodies approved for cancer treatment are of the IgG class, and many are targeted towards non-solid tumors. We hypothesized that antibodies of the IgE class may be able to recruit and activate immune cells against tissue resident tumors based on very high affinity of IgE for cognate Fcε Receptors on potent effector cells such as masT-cells, monocytes and macrophages. A novel IgE-based antibody against folate receptor alpha (FRα) for ovarian carcinoma was developed and demonstrated to engender superior anti-tumor effector functions compared with those triggered by its IgG counterpart in different in vivo models of cancer. We have now translated this concept to a first-in-man clinical trial. We have previously reported that monocytes and macrophages are important IgE effector cells against tumors. However, the underlying mechanisms of how IgE exerts antitumor immunity by engaging these effector cells are insufficiently characterized. Here, we sought to investigate the immune profile signatures triggered by the engagement of tumor antigen-specific IgE with Fc-receptors on the surface of monocytes, a key immune effector cell recruited by IgE antibodies. In order to investigate the above aim, the following methods were utilized: Antibody dependenT-cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP) were measured by flow cytometry. Cell stimulation studies with cytokines or by cross-linking of IgE monoclonal antibodies on the surface of monocytes were conducted. Expression and secretion of immune mediators were assessed using quantitative-PCR and ELISA. Toxicity of cytokines towards immune and tumor cells were investigated by cell viability (MTS) assays. As a result, tumor antigen-specific IgE-mediated tumor cell cytotoxicity mediated by human monocytes correlated with increased levels of TNFα, MCP-1 and IL-10. Cross-linking of IgE, but not IgG, on the monocyte surface, triggered up-regulation of the proinflammatory mediator TNFα. TNFα stimulation of monocytes and of tumor cells triggered stimulation of MCP-1 by both cells. Neither cross-linking of IgE or IgG antibodies on tumor cells or monocytes, nor stimulation of these cells with TNFα or MCP-1, was sufficient to upregulate IL-10. However, IL-10 expression was induced by monocytic cells when stimulated by TNFα and MCP-1 in combination, and by stimulation with IL-10 in an autocrine manner. None of these stimulation conditions was sufficient to upregulate IL-10 in cancer cell lines of different origins. None of the three cytokines had direct cytotoxic effects towards immune or tumor cells. However, blockade of TNFα receptor on monocytes reduced ADCC and increased levels of the chemoattractant MCP-1 recruited monocytes in chemotaxis assays in vitro and macrophages in tumor lesions in a rat model of cancer treated with anti-tumor IgE. These findings suggest that IgE-dependent monocyte-mediated tumor cell cytotoxicity triggers TNFα, MCP-1 and IL-10, an axis normally associated with IgE-mediated anti-parasite immune functions. This cascade appears to be responsible for a prominent recruitment of monocytes and macrophages towards tumor cells but may not be solely responsible for IgE-mediated antitumor cytotoxic functions. Identifying the immune profiles involved in IgE-mediated monocyte antitumor mechanisms may help identify markers of effector cell activation and support the development of IgE class as a novel immuno-oncology strategy. Citation Format: Mano Nakamura, Heather J. Bax, James F. Spicer, Katie E. Lacy, Sophia Tsoka, Debra H. Josephs, Sophia Karagiannis. Discovering the immune profiles of a novel antifolate receptor alpha IgE antibody associated with monocyte-mediated antitumor functions [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A095.

Adoptive immunotherapy with CAR modified T cells in cancer: current landscape and future perspectives.

Cellular therapies are a rapidly evolving approach to treat cancer in the light of their unique mechanism of action that potentially overcomes drug resistance and induces durable remissions. Modalities of adoptive cell therapy include gene-modified T cells expressing novel T cell receptors or chimeric antigen receptors (CAR) that modify the immune system to recognize tumor cells and carry out potent anti-tumor effector functions. CAR T cells have shown very promising clinical results and several trials are being conducted worldwide to establish their role in cancer treatment. Most successful results have been observed in lymphoproliferative disorders with the use of CD19-directed CAR T cells, which led to their commercial approval by FDA. In this review, we provide a comprehensive overview of the current role of CAR T cell therapies in hematological malignancies and solid tumors, their associated toxicities and potential future developments in the armamentarium for cancer treatment.

Ex vivo conditioning with IL-12 protects tumor-infiltrating CD8+ T cells from negative regulation by local IFN-γ.

Optimal ex vivo expansion protocols for adoptive cell therapy (ACT) must yield T cells able to effectively home to tumors and survive the inhospitable conditions of the tumor microenvironment (TME), while simultaneously exerting persistent anti-tumor effector functions. Our previous work has shown that ex vivo activation in the presence of IL-12 can induce optimal expansion of murine CD8+ T cells, thus resulting in significant tumor regression after ACT mostly via sustained secretion of IFN-γ. In this report, we further elucidate the mechanism of this potency, showing that IL-12 additionally counteracts the negative regulatory effects of autocrine IFN-γ. IL-12 not only downregulates PD-1 expression by T cells, thus minimizing the effects of IFN-γ-induced PD-L1 upregulation by tumor stromal cells, but also inhibits IFNγR2 expression, thereby protecting T cells from IFN-γ-induced cell death. Thus, the enhanced anti-tumor activity of CD8+ T cells expanded ex vivo in the presence of IL-12 is due not only to the ability of IL-12-stimulated cells to secrete sustained levels of IFN-γ, but also to the additional capacity of IL-12 to counter the negative regulatory effects of autocrine IFN-γ.

Tuning Natural Killer Cell Anti-multiple Myeloma Reactivity by Targeting Inhibitory Signaling via KIR and NKG2A.

Natural killer (NK) cells are attractive candidates for allogeneic cell-based immunotherapy due to their potent antitumor effector function and good safety profile. NK cells express killer immunoglobulin-like receptors (KIRs) and the NKG2A receptor important for NK cells education as well as providing inhibitory signals upon encountering HLA-expressing target cells. Multiple myeloma (MM) is an example of a tumor expressing relatively high levels of HLA molecules. In this review, we discuss the functional relevance of inhibitory KIRs and NKG2A for NK cells anti-MM response and strategies to lower these inhibitory signaling to enhance clinical efficacy of allogeneic NK cells in MM.

Preliminary Clinical Results of a Phase 1 Study Evaluating the Safety and Anti-Tumor Activity of ACTR707 in Combination with Rituximab in Subjects with Relapsed or Refractory CD20+ B-Cell Lymphoma

Preliminary Clinical Results of a Phase 1 Study Evaluating the Safety and Anti-Tumor Activity of ACTR707 in Combination with Rituximab in Subjects with Relapsed or Refractory CD20+ B-Cell Lymphoma

Dendritic Cells Are Critical for the Activation and Expansion of Vδ2+ T Cells After Allogeneic Hematopoietic Transplantation.

γδ T cells perform antitumor and antiviral effector functions and are involved in both innate and adaptive immunity. Vδ2+ T cells represent the predominant γδ T subset in the peripheral blood of healthy subjects. Vδ2+ T cells can be selectively activated and expanded by phosphoantigens (pAgs). Dendritic cells (DCs), as potent antigen-presenting cells, are capable of mediating pAgs–triggered Vδ2+ T cells expansion. However, the association between DCs and Vδ2+ T cell recovery in the context of hematopoietic stem cell transplantation (HSCT) remains unclear. We previously demonstrated that the recovery of Vδ2+ T cells was hampered and inversely correlated with Epstein-Barr virus (EBV) reactivation in patients undergoing haploidentical HSCT (haploHSCT). Whether Vδ2+ T cells from haploHSCT recipients can be expanded by stimulation with aminobisphosphonates or pAg–presenting DCs is of particular interest. Herein, we showed that Vδ2+ T cells recovered after haploHSCT failed to expand after ex-vivo stimulation with pamidronate. In addition, we found that the recovery of DC subsets was significantly decreased, and the concentration of myeloid DCs (mDCs) correlated significantly with Vδ2+ T cell recovery in the setting of allogeneic HSCT. Furthermore, coculture of peripheral lymphocytes from recipients with monocyte-derived and pamidronate-pretreated autologous or allogeneic DCs induced the successful expansion of Vδ2+ T cells. Of note, allogeneic DCs from third-party donors stimulated a significantly higher efficiency of Vδ2+ T cell expansion than autologous DCs. More importantly, the memory features were well-retained and the cytotoxic cytokines-production capacity was significantly enhanced in the expanded Vδ2+ T cells. Taken together, these results suggest that the frequency and function of DCs are critical for the recovery of Vδ2+ T cells after allogeneic HSCT. The fact that vigorous expansions of Vδ2+ T cells were induced by phosphoantigen-pretreated DCs, especially by allogeneic third-party DCs, provides additional options for the development of individualized immunotherapy strategies that utilize the anti-viral and anti-leukemic effects of γδ T cells in the context of hematopoietic transplantation.

Abstract A73: Optogenetic regulation of T cell metabolism in the tumor microenvironment

Abstract The tumor microenvironment presents significant metabolic challenges to T cells by depleting oxygen and glucose, as well as limiting the uptake of key nutrients. Therefore, T cells and tumor cells engage in fierce metabolic competition, as the demand for both oxygen and glucose in the niche is extremely high. The transition from a resting naïve T cell into an activated and highly proliferative effector T cell requires substantial metabolic reprogramming from relying primarily on oxidative phosphorylation (OxPhos) to the rapid induction of aerobic glycolysis. However, evidence suggests that tumor infiltrating CD8+ T cells show defects in glycolytic functions. In addition, our data indicates that actively migrating effector CD8+ T cells have greater levels of OxPhos activity than stationary cells, imposing an increasing demand for oxygen during T cell migration to the tumor site. To overcome the glycolytic deficiency of the tumor-targeting T cells and boost anti-tumor effector functions at the tumor microenvironment, we developed a genetically encoded light-activated proton pump (fungal proton pump, “Mac”), namely photoactivatable OxPhos (PA-OxPhos) that is expressed in the inner mitochondrial membrane. During OxPhos, electrons enter the electron transport chain (ETC), causing protons to be pumped across the inner mitochondrial membrane to establish a proton gradient. The gradient is then used to generate ATP through complex V (CxV). Therefore, the outward proton pumping through the inner mitochondrial membrane by light stimulation of PA-OxPhos mimics the ETC function and boosts ATP generation in T cells, even in the presence of low levels of oxygen and substrates, giving T cells a metabolic competitive advantage in the tumor microenvironment. PA-OxPhos is tagged with GFP and is expressed in the mitochondria of transfected 293T cells and in activated mouse CD8+ T cells. When cells were treated with Rotenone (an inhibitor of complex I of the ETC), ATP production was decreased after 90 minutes. Importantly, light stimulation of 293T cells expressing PA-OxPhos successfully increased ATP production even in the presence of Rotenone. Our data suggests that PA-OxPhos can remotely provide a competitive metabolic advantage and boost T cell functions in the tumor microenvironment. The utilization of an alternative mechanism for ATP production in T cells could potentially dissipate the failures of T-cell-based cancer immunotherapies in destroying malignant cells of solid tumors. Citation Format: Andrea Amitrano, Brandon Walling, Kyun Do Kim, Brandon Berry, Adam Trewin, Andrew Wojtovich, Minsoo Kim. Optogenetic regulation of T cell metabolism in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A73.

Molecular Recalibration of PD-1+ Antigen-Specific T Cells from Blood and Liver.

Checkpoint inhibitors and adoptive cell therapy provide promising options for treating solid cancers such as HBV-related HCC, but they have limitations. We tested the potential to combine advantages of each approach, genetically reprogramming T cells specific for viral tumor antigens to overcome exhaustion by down-modulating the co-inhibitory receptor PD-1. We developed a novel lentiviral transduction protocol to achieve preferential targeting of endogenous or TCR-redirected, antigen-specific CD8 T cells for shRNA knockdown of PD-1 and tested functional consequences for antitumor immunity. Antigen-specific and intrahepatic CD8 T cells transduced with lentiviral (LV)-shPD-1 consistently had a marked reduction in PD-1 compared to those transduced with a control lentiviral vector. PD-1 knockdown of human T cells rescued antitumor effector function and promoted killing of hepatoma cells in a 3D microdevice recapitulating the pro-inflammatory PD-L1hi liver microenvironment. However, upon repetitive stimulation, PD-1 knockdown drove T cell senescence and induction of other co-inhibitory pathways. We provide the proof of principle that T cells with endogenous or genetically engineered specificity for HBV-associated HCC viral antigens can be targeted for functional genetic editing. We show that PD-1 knockdown enhances immediate tumor killing but is limited by compensatory engagement of alternative co-inhibitory and senescence program upon repetitive stimulation.

Checkpoint Blockade Rescues the Repressive Effect of Histone Deacetylases Inhibitors on γδ T Cell Function

Histone deacetylases (HDAC) are one of the key epigenetic modifiers that control chromatin accessibility and gene expression. Their role in tumorigenesis is well established and HDAC inhibitors have emerged as an effective treatment modality. HDAC inhibitors have been investigated for their specific antitumor activities and also clinically evaluated in treatment of various malignancies. In the present study, we have investigated the effect of HDAC inhibitors on the effector functions of human γδ T cells. HDAC inhibitors inhibit the antigen-specific proliferative response of γδ T cells and cell cycle progression. In antigen-activated γδ T cells, the expression of transcription factors (Eomes and Tbet) and effector molecules (perforin and granzyme B) were decreased upon treatment with HDAC inhibitors. Treatment with HDAC inhibitors attenuated the antitumor cytotoxic potential of γδ T cells, which correlated with the enhanced expression of immune checkpoints programmed death-1 (PD-1) and programmed death ligand-1 in γδ T cells. Interestingly, PD-1 blockade improves the antitumor effector functions of HDAC inhibitor-treated γδ T cells, which is reflected in the increased expression of Granzyme B and Lamp-1. This study provides a rationale for designing HDAC inhibitor and immune check point blockade as a combinatorial treatment modality for cancer.

Abstract 122: Hypoxia-enhanced proliferation and effector function of NK cells

Abstract Hypoxia promotes tumor cell survival while providing immune cell shaping and resistance. Although NK cells function within hypoxic lymphatics and inflamed tissues, they were shown to mount impaired anti-tumor effector functions in ex-vivo hypoxic environment. This raises a question that additional cues exist in vivo to allow full NK cell activation. By varying the percentage and time of pO2 exposure ex-vivo, we were able to fine-tune a condition that reverses hypoxia-induced NK cell suppression to become highly proliferative and enhanced cytolytic phenotypes. We found that exposing NK cells to moderate hypoxia in the presence of IL-2 and feeder cells allowed HIF-1α stabilization and its target gene expression, metabolic changes toward glycolysis, specific upregulation of NKp44 receptor, and increased STAT3 phosphorylation with concomitant reduction of apoptosis and p16 senescence pathways. Therefore, the hypoxic exposure allows HIF-1α-mediated adaptation of mature NK cells toward highly proliferative and cytolytic NK phenotypes, which may occur in vivo at the time of tumor growth and inflammation. [This work was supported by the National Research Foundation of Korea (NRF), grants NRF-2013M3A9D3046248] Citation Format: Yunwon Moon, Seon Ah Lim, Kyung-Mi Lee, Hyunsung Park. Hypoxia-enhanced proliferation and effector function of NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 122.

Abstract CT132: Safety and dose selection for LYC-55716, a first-in-class RORγ agonist to treat solid tumors: Phase I results from an open-label, multicenter Phase I/IIa trial

Abstract Background: LYC-55716, a first-in-class, oral, small-molecule agonist of the retinoic acid receptor-related orphan receptor γ (RORγ), is under development as a novel immuno-oncology agent for solid tumors. Preclinical data show that LYC-55716 modulates gene expression to reprogram immune cells for improved anti-tumor effector function as well as decreased immunosuppressive mechanisms, resulting in slower tumor growth and enhanced survival. An ongoing, open-label, Phase I/IIa trial is evaluating the safety and tolerability of the investigational agent LYC-55716 in patients with advanced solid tumors. Methods: The Phase I portion of the trial (NCT02929862) enrolled adults with locally advanced or metastatic solid tumors (no specific exclusions) who had progressed on standard therapy. Patients received 28-day treatment cycles of LYC-55716 in which dose and dosing regimen were determined according to PK profile and safety. Primary endpoints were safety (monitoring of adverse events [AEs], physical examination, lab results) and incidence of dose-limiting (Grade 3-4) toxicities during the first treatment cycle. Secondary endpoints included pharmacokinetics (PK) and response assessment. Pharmacodynamic markers of RORγ activation were also evaluated. Results: Data to date: Thirty-two patients were enrolled (23-79 y; 63% female); 27 completed ≥1 (28-day) treatment cycle. Treatment-related AEs occurring in ≥3 patients included diarrhea (n=7 [22%]); fatigue (n=5 [16%]); and anemia, poor appetite, and dry mouth (n=3 [9%] each). No dose-limiting toxicities were observed during Phase 1. The majority of treatment-related AEs were Grade 1-2; Grade 3 treatment-related AEs were reported only for anemia, elevated gamma-glutamine transferase, and hypophosphatemia; no Grade 4 treatment-related AEs occurred. None of the 5 deaths or 14 serious AEs (in 11 patients) were considered treatment-related, and no subjects withdrew due to AEs. LYC-55716 demonstrated an estimated elimination half-life of ~12 h. Twice daily dosing resulted in minimum plasma concentrations that were consistently higher than QD dosing and the highest BID dose resulted in a median minimum plasma concentration that exceeded by ~24-fold the 50% effective concentration that preclinical studies had indicated was required for target gene regulation. Pharmacodynamic data indicated target engagement, consistent with PK evidence of exposure levels in the predicted efficacious range. Updated data, including details of Phase II dose selection and response assessment, will be presented. Conclusion: Phase I data support the safety of LYC-55716 and selection of a BID dosing regimen for the ongoing Phase 2a phase of the trial in patients with non-small cell lung, head and neck, gastroesophageal, renal cell, urothelial, and ovarian cancers. Citation Format: Devalingam Mahalingam, Judy S. Wang, Erika P. Hamilton, Garry Weems, Marshall Schreeder, H. Jeffrey Wilkins. Safety and dose selection for LYC-55716, a first-in-class RORγ agonist to treat solid tumors: Phase I results from an open-label, multicenter Phase I/IIa trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT132.

Targeting Macrophages in Cancer: From Bench to Bedside.

Macrophages are a major component of the tumor microenvironment and orchestrate various aspects of immunity. Within tumors, macrophages can reversibly alter their endotype in response to environmental cues, including hypoxia and stimuli derived from other immune cells, as well as the extracellular matrix. Depending on their activation status, macrophages can exert dual influences on tumorigenesis by either antagonizing the cytotoxic activity immune cells or by enhancing antitumor responses. In most solid cancers, increased infiltration with tumor-associated macrophages (TAMs) has long been associated with poor patient prognosis, highlighting their value as potential diagnostic and prognostic biomarkers in cancer. A number of macrophage-centered approaches to anticancer therapy have been investigated, and include strategies to block their tumor-promoting activities or exploit their antitumor effector functions. Integrating therapeutic strategies to target TAMs to complement conventional therapies has yielded promising results in preclinical trials and warrants further investigation to determine its translational benefit in human cancer patients. In this review, we discuss the molecular mechanisms underlying the pro-tumorigenic programming of macrophages and provide a comprehensive update of macrophage-targeted therapies for the treatment of solid cancers.

Ex Vivo-expanded Natural Killer Cells Derived From Long-term Cryopreserved Cord Blood are Cytotoxic Against Primary Breast Cancer Cells.

With over 600,000 units of umbilical cord blood (CB) stored on a global scale, it is important to elucidate the therapeutic abilities of this cryopreserved reservoir. In the advancing field of natural killer (NK) cell cancer immunotherapy, CB has proven to be a promising and noninvasive source of therapeutic NK cells. Although studies have proven the clinical efficacy of using long-term cryopreserved CB in the context of hematopoietic stem cell transplantations, little is known about its use for the ex vivo expansion of effector immune cells. Therefore, our group sought to derive ex vivo-expanded NK cells from long-term cryopreserved CB, using an artificial antigen presenting cell-mediated expansion technique. We compared the expansion potential and antitumor effector function of CB-derived NK (CB-NK) cells expanded from fresh (n=4), short-term cryopreserved (<1-year old, n=5), and long-term cryopreserved (1-10-year old, n=5) CB. Here, we demonstrated it is possible to obtain an exponential amount of expanded CB-NK cells from long-term cryopreserved CB. Ex vivo-expanded CB-NK cells had an increased surface expression of activating markers and showed potent antitumor function by producing robust levels of proinflammatory cytokines, interferon-γ, and tumor necrosis factor-α. Moreover, expanded CB-NK cells (n=3-5) demonstrated cytotoxicity towards primary breast cancer cells (n=2) derived from a triple-negative breast cancer and an estrogen receptor-positive/progesterone receptor-positive breast cancer patient. Long-term cryopreservation had no effect on the expansion potential or effector function of expanded CB-NK cells. Therefore, we propose that long-term cryopreserved CB remains clinically useful for the ex vivo expansion of therapeutic NK cells.

Modulation of antitumor immunity with histone deacetylase inhibitors.

Histone deacetylase inhibitors possess a broad array of antitumor activities; however, their net impact on the evolving antitumor immune response is highly dependent on the inhibitors used and the histone deacetylases they target. Herein, we sequentially focus on each stage of the antitumor immune response - from dendritic cell activation and migration, antigen uptake and presentation, T-cell activation and differentiation and the enactment of antitumor effector functions within the tumor microenvironment. In particular, we will discuss how various inhibitors have different effects depending on cellular activation, experimental design and specific histone deacetylases being targeted - and how these changes impact the outcome of an antitumor immune response. At last, we consider the impact these inhibitors may have on T-cell exhaustion and implications for combination with other immunomodulating therapies.

Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response

BackgroundIt remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies.MethodsWe employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods.ResultsWe demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors.ConclusionsSingle-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.

Iron Induces Anti-tumor Activity in Tumor-Associated Macrophages.

Tumor-associated macrophages (TAMs) frequently help to sustain tumor growth and mediate immune suppression in the tumor microenvironment (TME). Here, we identified a subset of iron-loaded, pro-inflammatory TAMs localized in hemorrhagic areas of the TME. The occurrence of iron-loaded TAMs (iTAMs) correlated with reduced tumor size in patients with non-small cell lung cancer. Ex vivo experiments established that TAMs exposed to hemolytic red blood cells (RBCs) were converted into pro-inflammatory macrophages capable of directly killing tumor cells. This anti-tumor effect could also be elicited via iron oxide nanoparticles. When tested in vivo, tumors injected with such iron oxide nanoparticles led to significantly smaller tumor sizes compared to controls. These results identify hemolytic RBCs and iron as novel players in the TME that repolarize TAMs to exert direct anti-tumor effector function. Thus, the delivery of iron to TAMs emerges as a simple adjuvant therapeutic strategy to promote anti-cancer immune responses.

Tumor-associated myeloid cells: new understandings on their metabolic regulation and their influence in cancer immunotherapy.

Tumor-associated myeloid cells (TAMCs), mainly represented by tumor-associated macrophages and myeloid-derived suppressor cells, can promote tumor growth directly, by favoring tumor cell proliferation and survival, and indirectly, by creating an immunosuppressive microenvironment. Myeloid cells are characterized by an extreme phenotypical and functional plasticity. Immunometabolism is now emerging as a crucial aspect of TAMCs skewing toward pro-tumoral activities. The metabolic re-education of myeloid cells is a new strategy to boost their antitumor effector functions. Several anticancer therapies targeting TAMCs are already under investigation. Nowadays, the hot topic of cancer immunotherapy is represented by immune checkpoint inhibitors. These drugs unrestrain T-cell-mediated tumor elimination by removing suppressive signals delivered by tumor-associated cells. The efficacy of immune checkpoint blockade can be enhanced using coordinated strategies to counteract the TAMCs-dependent impairment of immune adaptive responses. In the first part of the review, we will describe the association between metabolic reprogramming and TAMCs biological activities. In the second part, we will illustrate the potential of combination therapies associating TAMC-targeting drugs with immune checkpoint inhibitors.