Tumoricidal Function Research Articles

IMMU-21. OVEREXPRESSING CAR T-CELL GLUCOSE TRANSPORTER TO ENHANCE ANTI-TUMOR ACTIVITY IN GLIOBLASTOMA

Abstract INTRODUCTION Despite aggressive interventions, Glioblastoma (GBM) remains as a challenge for neuro-oncologists. Standard treatments are often followed by disease recurrence. Adoptive cell therapies have emerged as a viable therapeutic for brain malignancies. CAR-T cell therapy has generated substantial interest because of its efficacy and specificity in treating various cancers. Despite these efficacies, studies reveal limitations of CAR-T therapy due to the altered metabolic environment in GBM. GBM tumors shift towards aerobic glycolysis, regardless of oxygen availability leading to massive glucose consumption. This results in impeding T-cell glycolysis and activation. OBJECTIVE The GBM microenvironment provides local restraints via metabolic competition suppressing antitumor immunity, specifically inhibiting infiltration and tumoricidal functions of host and adoptively transferred tumor-reactive T-cells. In our study, we manipulated the metabolic fitness of the therapeutic T-cells to enhance their tumoricidal activity, by overexpressing glucose transporter GLUT1 and GLUT3. We show that this manipulation can enhance their anti-tumor activity. METHODS The T-cell glucose metabolic pathway was modulated via glucose transporter overexpression. The functionality of metabolically modified T-cells was investigated in murine and human models. RESULTS We demonstrated that tumor cells impose glucose restriction when competing for glucose with T-cells leading to T-cell hypo responsiveness. Overexpression of glucose transporters such as Glut1 and Glut3 increased T-cell glucose utilization and provide survival advantage leading to enhanced T-cell activation in glucose-restricted conditions. The metabolic modifications regulate T-cell efficacy by enhancing intra-tumoral trafficking. We tested this approach in the context of CD70CAR T cell therapy. It was established that glucose transporter overexpression in CD70 CAR T-cells not only improves intracranial tumor invasion but also elicit functional anti-tumor cell response thus improving survival. CONCLUSIONS This study integrates fundamental concepts of tumor and immune metabolism in the design of immunotherapy and confirms that immunometabolism represents a viable target for new cancer therapy to treat brain tumors.

Consequences of the perivascular niche remodeling for tumoricidal T-cell trafficking into metastasis of ovarian cancer.

The treatment-induced activation level within the perivascular tumor microenvironment (TME) that supports T-cell trafficking and optimal T-cell differentiation is unknown. We investigated the mechanisms by which inflammatory responses generated by tumor-specific T cells delivered to ovarian tumor-bearing mice alone or after oncolytic vaccinia virus-driven immunogenic cancer cell death affect antitumor efficacy. Analyses of the perivascular TME by spatially resolved omics technologies revealed reduced immunosuppression and increased tumoricidal T-cell trafficking and function after moderate inflammatory responses driven by a CXCR4 antagonist-armed oncolytic virus. Neither weak nor high inflammation created a permissive TME for T-cell trafficking. Notably, treatment-mediated differences in T-cell effector programs acquired within the perivascular TME contrasted with comparable antigenic priming in the tumor-draining lymph nodes regardless of the activation mode of antigen-presenting cells. These findings provide new insights into combinatorial treatment strategies that enable tumor-specific T cells to overcome multiple barriers for enhanced trafficking and control of tumor growth. .

Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

Proximity-induction of cell-cell interactions via small molecules represents an emerging field in basic and translational sciences. Covalent anchoring of these small molecules represents a useful chemical strategy to enforce proximity; however, it remains largely unexplored for driving cell-cell interactions. In immunotherapeutic applications, bifunctional small molecules are attractive tools for inducing proximity between immune effector cells like Tcells and tumor cells to induce tumoricidal function. We describe a two-component system composed of electrophilic bifunctional small molecules and paired synthetic antigen receptors (SARs) that elicit Tcell activation. The molecules, termed covalent immune recruiters (CIRs), were designed to affinity label and covalently engage SARs. We evaluated the utility of CIRs to direct anti-tumor function of human Tcells engineered with three biologically distinct classes of SAR. Irrespective of the electrophilic chemistry, tumor-targeting moiety, or SAR design, CIRs outperformed equivalent non-covalent bifunctional adapters, establishing a key role for covalency in maximizing functionality. We determined that covalent linkage enforced early Tcell activation events in a manner that was dependent upon each SARs biology and signaling threshold. These results provide a platform to optimize universal SAR-T cell functionality and more broadly reveal new insights into how covalent adapters modulate cell-cell proximity-induction.

Offsetting Low-Affinity Carbohydrate Binding with Covalency to Engage Sugar-Specific Proteins for Tumor-Immune Proximity Induction.

Carbohydrate-binding receptors are often used by the innate immune system to potentiate inflammation, target endocytosis/destruction, and adaptive immunity (e.g., CD206, DC-SIGN, MBL, and anticarbohydrate antibodies). To access this class of receptors for cancer immunotherapy, a growing repertoire of bifunctional proximity-inducing therapeutics use high-avidity multivalent carbohydrate binding domains to offset the intrinsically low affinity associated with monomeric carbohydrate-protein binding interactions (Kd ≈ 10-3-10-6 M). For applications aimed at recruiting anticarbohydrate antibodies to tumor cells, large synthetic scaffolds are used that contain both a tumor-binding domain (TBD) and a multivalent antibody-binding domain (ABD) comprising multiple l-rhamnose monosaccharides. This allows for stable bridging between tumor cells and antibodies, which activates tumoricidal immune function. Problematically, such multivalent macromolecules can face limitations including synthetic and/or structural complexity and the potential for off-target immune engagement. We envisioned that small bifunctional "proximity-inducing" molecules containing a low-affinity monovalent ABD could efficiently engage carbohydrate-binding receptors for tumor-immune proximity by coupling weak binding with covalent engagement. Typical covalent drugs and electrophilic chimeras use high-affinity ligands to promote the fast covalent engagement of target proteins (i.e., large kinact/KI), driven by a favorably small KI for binding. We hypothesized the much less favorable KI associated with carbohydrate-protein binding interactions can be offset by a favorably large kinact for the covalent labeling step. In the current study, we test this hypothesis in the context of a model system that uses rhamnose-specific antibodies to induce tumor-immune proximity and tumoricidal function. We discovered that synthetic chimeric molecules capable of preorganizing an optimal electrophile (i.e., SuFEx vs activated ester) for protein engagement can rapidly covalently engage natural sources of antirhamnose antibody using only a single low-affinity rhamnose monosaccharide ABD. Strikingly, we observe chimeric molecules lacking an electrophile, which can only noncovalently bind the antibody, completely lack tumoricidal function. This is in stark contrast to previous work targeting small molecule hapten and peptide-specific antibodies. Our findings underscore the utility of covalency as a strategy to engage low-affinity carbohydrate-specific proteins for tumor-immune proximity induction.

Developing “Off-the-Shelf” CLL1 CAR-DNT Therapeutics for the R/R Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is one of the most common and highly heterogeneous hematological malignancy. The prognosis is poor especially for the elderly patients. For Relapsed/Refractory(R/R) AML patients, the one-year overall survival rate is merely 20%. CAR-T cells have shown tremendous therapeutic benefits for ALL, DLBCL and MM. One of the limitations of current CAR-T therapies is the use of autologous T cells. This can be limiting due to the cost, complicated manufacturing process, rapid disease progression, and manufacturing failures etc. Therefore, there is a need to develop the ready-to-use allogeneic cell product. Double negative T (DNT) cells are the ideal candidate for AML cell therapy as they naturally kill AML cells, and have been successfully administered to AML patients with therapeutic benefit without causing GvHD. Relying on their own natural immune activated receptors, DNT cells could lyse a wide range of tumors independent of TAA expression, which have more advantages compared to the conventional CAR-T cells. In our study, we developed the allogeneic product of CAR-DNT cells targeting C-type lectin-like molecule-1 (CLL1), which is a promising target due to its over-expression in primary AML cells and leukemia stem cells, while absence on hematopoietic stem/progenitor cells. Methods: We developed a humanized single chain variable fragment (scFv) with specific recognition of human CLL1 antigen by screening from 62 murine-derived CLL1 antibodies. Then, we designed a second-generation CLL1 CAR structure containing 4-1BB-CD3ζ and transduced it into the DNT cells by lentivirus. To clarify the GvH and HvG reactions of DNT cells, we performed the mixed lymphocyte reaction experiments in vitro and compared DNT cells with TCR and B2M double knockout T(DKO) cells and TCR knockout T(TKO) cells. We evaluated the anti-tumor function of CLL1 CAR-DNT cells through killing experiments by different effector-to-target ratios and multiple stimulations with AML cell lines (THP1 and HL60). Furthermore, we speculated that the CAR structure plays a critical role in both tumoricidal function and persistence of CAR-DNT cells. We designed nearly 20 enhanced CAR structures based on the signaling pathway of DNT cells. These newly designed structures have been tested in vitro and will be confirmed in vivo. Results: The humanized anti-CLL1 scFv candidate H1-3Q2 demonstrated high specificity toward human CLL1 antigen at the Membrane Protein Array. H1-3Q2 based CAR-T cells showed good tumor suppression in vivo. However, due to the heterogenous expression of CLL1 in AML, CLL1 CAR-T cells may not be able to eliminate AML cancer cells completely. While DNT cells could naturally lyse tumor cells through the receptors like NKG2D and DNAM-1, providing them with lots of advantages. Moreover, DNT cells have the potential to be used as allogenic cell therapy product. Co-culturing allogenic PBMCs or umbilical cord blood with DNT cells for up to 72 hours resulted in little or no killing reaction, indicated a low risk of GvHD. Conversely, the PBMCs from three different donors were used to attack allogenic DNT cells or DKO/TKO T cells in vitro. The killing effect on DKO/TKO T cells gradually increased over time, while no effect on DNT cells was observed even after 192 hours. This suggested that DNT cells are not easily cleared by host immune cells. DNT cells could efficiently lyse various hematological and solid tumor cell lines, particularly showing high sensitivity towards AML cells. The introduction of the CAR moiety further enhanced the specificity of DNT cells. The in vitro killing results demonstrated that CLL1 CAR-DNT cells exhibited stronger and faster AML resistance compared with CAR-T cells, and even eliminated tumor cells that have lost TAA. However, CLL1 CAR-DNT cells showed limited proliferation in the multiple-stimulation assays. To address this, we designed enhanced CAR structures. Screening by multiple-challenge assays, the fifth-generation CAR exhibited outstanding proliferation and superior tumor lysis ability in vitro. Conclusions: Our findings demonstrate that CAR-DNT product is an ideal candidate for allogeneic adoptive immunotherapy and exhibits superior tumoricidal function. This study underscores the potential of the “off-the-shelf” CLL1 CAR-DNT cell product for treating R/R AML.

Macrophage Membrane-Coated Nano-Gemcitabine Promotes Lymphocyte Infiltration and Synergizes AntiPD-L1 to Restore the Tumoricidal Function.

The limited lymphocyte infiltration and exhaustion of tumoricidal functions in solid tumors remain a formidable obstacle to cancer immunotherapy. Herein, we designed a macrophage membrane-coated nano-gemcitabine system (MNGs) to promote lymphocyte infiltration and then synergized anti-programmed death ligand 1 (antiPD-L1) to reinvigorate the exhausted lymphocytes. MNGs exhibited effective intratumor-permeating and responsive drug-releasing capacity, produced notable elimination of versatile immunosuppressive cells, and promoted lymphocyte infiltration into cancer cell regions in tumors, but over 50% of these infiltrated lymphocytes were in the exhausted state. Compared with MNG monotherapy, the MNGs+antiPD-L1 combination produced 31.77% and 30.63% reduction of exhausted CD3+CD8+ T cells and natural killer (NK) cells and 2.83- and 3.17-fold increases of interferon-γ (IFN-γ)-positive subtypes, respectively, thereby resulting in considerable therapeutic benefits in several tumor models. Thus, MNGs provide an encouraging strategy to promote lymphocyte infiltration and synergize antiPD-L1 to restore their tumoricidal function for cancer immunotherapy.

TMET-21. ENHANCING THE METABOLIC FITNESS OF T CELLS TO TREAT BRAIN TUMORS

Abstract INTRODUCTION Glioblastoma is a challenge for neuro-oncologists and current therapies are minimally effective. Standard-of- care treatment is almost inevitably followed by disease recurrence. Adoptive T cell transfer has emerged as a viable therapeutic for brain malignancies. While promising, the efficacy of this approach is often limited by a complex immunosuppressive tumor microenvironment. These complexities mean that more sophisticated T cell products are required. Objectives: The brain tumor microenvironment provides local restraints via metabolic competition suppressing antitumor immunity, specifically inhibiting infiltration and tumoricidal functions of host and adoptively transferred tumor-reactive T cells. The overall goal of this project is to test new treatments to reverse immune dysfunction in cancer through the regulation of T cell metabolic signaling. We propose that modulating the glucose pathway in T cells can potentiate their anti-tumor activity once adoptively transferred. METHODS The glucose metabolic pathway of T cells was modulated via overexpression of glucose transporters. The functionality of metabolically modified T cells was investigated in murine and human models. RESULTS We demonstrated the existence of a competition for glucose between T cells and tumor cells, with tumor cells imposing glucose restriction mediating T cell hyporesponsiveness. Overexpression of glucose transporters such as Glut1 and Glut3 increased T cell glucose utilization and provided a survival/growth advantage and enhanced T cell activation in glucose-restricted conditions. We also established that glucose transporter overexpression improves intratumoral infiltration and expansion of adoptively transferred T cells, resulting in improved survival. CONCLUSION This project integrates fundamental concepts of tumor and immune metabolism in the design of immunotherapy and confirms that immunometabolism represents a viable target for new cancer therapy to treat brain tumors.

Tumoricidal effects of unprimed and curcumin-primed adipose-derived stem cells on human hepatoma HepG2 cells under oxidative conditions

IntroductionAdipose-derived stem cells (ASCs) have been proven to have tumoricidal effects against hepatic cancer cell lines. However, it appears that exposure to oxidative microenvironment compromises the potential outcome of ASCs in real hepatoma. Herein, we aimed to examine the tumoricidal effects of ASCs under oxidative conditions and to investigate the impact of curcumin priming on ASCs’ therapeutic potential. MethodsWe used human hepatoma (HepG2) cells in a coculture system with unprimed or curcumin-primed ASCs (Cur-ASCs) under H2O2-induced oxidative conditions. To investigate HepG2 proliferation and death, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) and annexin V staining assays were performed. To determine the HepG2 migration and invasion potential, the scratch healing and the transwell invasion assays were performed. To evaluate the expression of apoptosis-protein markers, Western blotting was performed. ResultsCur-ASCs suppressed HepG2 proliferation, migration, and invasion as well as prompted apoptosis more significantly compared to unprimed ASCs under oxidative conditions. Expressional studies also revealed an obvious decline in the BCL-2/BAX ratio in HepG2 cocultured with Cur-ASCs. In addition, we noticed a marked elevation of apoptosis and senescence in unprimed ASCs compared to Cur-ASCs after coculture experiments, which demonstrated that curcumin priming preserved the survival and growth potential of ASCs; hence, Cur-ASCs performed better tumoricidal functions under oxidative conditions. ConclusionOur findings suggest that ASCs have the intrinsic ability to induce cell death in HepG2 cells; however, their functions can be compromised under oxidative conditions. We believe that curcumin priming is an effective approach for improving the therapeutic effectiveness of ASCs in the cancerous microenvironment.

Down-regulation of MLLT1 super elongation complex subunit impairs the anti-tumor activity of natural killer cells in esophageal cancer

Natural killer (NK) cells actively participate in anti-tumor immunity and are thus regarded as a promising tool in immunotherapy against esophageal cancer (EC). However, the mechanisms regulating NK cell activation and exhaustion have not been completely elucidated. In this study, we characterized the expression and function of MLLT1 super elongation complex subunit (MLLT1) in esophageal NK cells in a mouse EC model. MLLT1 was down-regulated in esophageal NK cells, especially NK cells expressing both T cell immunoglobulin and mucin-domain containing-3 (TIM-3) and lymphocyte activation gene3(LAG-3). In vitro knockdown of MLLT1 in NK cells resulted in significant decreases in the expression of IFN-γ and perforin, as well as impaired NK cell cytotoxicity on tumor cells. Adoptive transfer of MLLT-deficient NK cells into EC-bearing mice showed consistent impairment of NK cell anti-tumor activity, as evidenced by decreases in IFN-γ and perforin but not granzyme B. Furthermore, EC tissue cells, which were enriched from the esophagus of EC-bearing mice, induced down-regulation of MLLT1 in splenic NK cells. This down-regulation was partially restored by a TIM-3 blocking antibody. Therefore, this study indicated that TIM-3 signaling down-regulated MLLT1 in esophageal NK cells, and MLLT1 down-regulation undermined the tumoricidal function of NK cells in EC. Our study unveils a novel mechanism underlying NK cell exhaustion/dysfunction in the EC microenvironment. MLLT1 could be a potential target in future NK cell-mediated immunotherapy against EC.

Targeting MDK Abrogates IFN-γ-Elicited Metastasis inCancers of Various Origins.

IFN-γ is a pleiotropic cytokine with immunomodulatory and tumoricidal functions. It has been used as an anti-tumor agent in adjuvant therapies for various cancers. Paradoxically, recent advances have also demonstrated pro-tumorigenic effects of IFN-γ, especially in promoting cancer metastasis, with the mechanism remains unclear. This will undoubtedly hinder the application of IFN-γ in cancer treatment. Here, we verified that IFN-γ treatment led to activation of the epithelial-to-mesenchymal transition (EMT) programme and metastasis in cell lines of various cancers, including the kidney cancer cell line Caki-1, the lung cancer cell line A549, the cervical carcinoma cell line CaSki, the breast cancer cell line BT549 and the colon cancer cell line HCT116. We further disclosed that midkine (MDK), an emerging oncoprotein and EMT inducer, is a common responsive target of IFN-γ in these cell lines. Mechanistically, IFN-γ upregulated MDK via STAT1, a principle downstream effector in the IFN-γ signalling. MDK is elevated in the majority of cancer types in the TCGA database, and its overexpression drove EMT activation and cancer metastasis in all examined cell lines. Targeting MDK using a specific MDK inhibitor (iMDK) broadly reversed IFN-γ-activated EMT, and subsequently abrogated IFN-γ-triggered metastasis. Collectively, our data uncover a MDK-dependent EMT inducing mechanism underlying IFN-γ-driven metastasis across cancers which could be attenuated by pharmacological inhibition of MDK. Based on these findings, we propose that MDK may be used as a potential therapeutic target to eliminate IFN-γ-elicited pro-metastatic adverse effect, and that combined MDK utilization may expand the application of IFN-γ in cancer and improve the clinical benefits from IFN-γ-based therapies.

Activatable Cancer Sono-Immunotherapy using Semiconducting Polymer Nanobodies.

Despite the great promises of sonodynamic therapy (SDT) in combination cancer therapy, its clinical applications are hindered by the "always-on" pharmacological activities of therapeutic agents and the lack of efficient sonosensitizers. Herein, the development of semiconducting polymers as efficient sonosensitizers and further development of sono-immunotherapeutic nanobodies (SPNAb ) for activatable cancer sono-immunotherapy are reported. Conjugation of anti-CTLA-4 antibodies onto the polymer nanoparticles through a 1 O2 -cleavable linker affords SPNAb with relatively low CTLA-4 binding affinity. Upon sono-irradiation, SPNAb generates 1 O2 not only to elicit a sonodynamic effect to induce immunogenic cell death, but also to release anti-CTLA-4 antibodies and trigger in situ checkpoint blockade. Such a synergistic therapeutic action mediated by SPNAb modulates the tumoricidal function of T-cell immunity by promoting the proliferation of cytotoxic T lymphocytes and depleting immunosuppressive regulatory T cells, resulting in effective tumor regression, metastasis inhibition, durable immunological memory, and prevention of relapse. Therefore, this study represents a proof-of-concept sonodynamic strategy using semiconducting polymers for precise spatiotemporal control over immunotherapy.

Immunomodulatory Properties of Immune Checkpoint Inhibitors-More than Boosting T-Cell Responses?

Simple SummaryTumor immune evasion is mediated in large part by the inhibition of anti-tumor T cell responses. Both the induction of tumor antigen-specific T cells and the activation state of T effector cells may be attenuated by surface receptors, which upon binding to counter receptors on immunoregulatory cell types and tumor cells, induce inhibitory T cell signaling. Immune checkpoint inhibitors (ICI) are antibodies that block interaction of these receptor pairs and thereby prevent T cell inhibition. Only a small percentage of tumor patients are responsive to treatment with ICI, which on the one hand raises the issue of possible reasons for failure, and on the other hand has spurred the development of additional ICI targeting other T cell inhibitory receptors. Our review aims to summarize knowledge on the functional role of these (inhibitory) receptors by additional types of leukocytes, and consequences of receptor blockade by ICI as a potential cause for unwanted side effects limiting the success of therapy. Deeper knowledge in this regard is a prerequisite for the development of more refined combination therapies.The approval of immune checkpoint inhibitors (ICI) that serve to enhance effector T-cell anti-tumor responses has strongly improved success rates in the treatment of metastatic melanoma and other tumor types. The currently approved ICI constitute monoclonal antibodies blocking cytotoxic T-lymphocyte-associated protein (CTLA)-4 and anti-programmed cell death (PD)-1. By this, the T-cell-inhibitory CTLA-4/CD80/86 and PD-1/PD-1L/2L signaling axes are inhibited. This leads to sustained effector T-cell activity and circumvents the immune evasion of tumor cells, which frequently upregulate PD-L1 expression and modulate immune checkpoint molecule expression on leukocytes. As a result, profound clinical responses are observed in 40–60% of metastatic melanoma patients. Despite the pivotal role of T effector cells for triggering anti-tumor immunity, mounting evidence indicates that ICI efficacy may also be attributable to other cell types than T effector cells. In particular, emerging research has shown that ICI also impacts innate immune cells, such as myeloid cells, natural killer cells and innate lymphoid cells, which may amplify tumoricidal functions beyond triggering T effector cells, and thus improves clinical efficacy. Effects of ICI on non-T cells may additionally explain, in part, the character and extent of adverse effects associated with treatment. Deeper knowledge of these effects is required to further develop ICI treatment in terms of responsiveness of patients to treatment, to overcome resistance to ICI and to alleviate adverse effects. In this review we give an overview into the currently known immunomodulatory effects of ICI treatment in immune cell types other than the T cell compartment.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

TAMI-27. METABOLIC MANIPULATION OF T CELLS TO TREAT BRAIN TUMORS

Abstract INTRODUCTION Glioblastoma are a challenge for neuro-oncologists and current therapies are minimally effective. Standard-of- care treatment is almost inevitably followed by disease recurrence. Adoptive T cell transfer has emerged as a viable therapeutic for brain malignancies. While promising, the efficacy of this approach is often limited by a complex immunosuppressive tumor microenvironment. These complexities mean that more sophisticated T cell products are required. OBJECTIVES The brain tumor microenvironment provides local restraints via metabolic competition suppressing antitumor immunity, specifically inhibiting infiltration and tumoricidal functions of host and adoptively transferred tumor-reactive T cells. The overall goal of this project is to test new treatments to reverse immune dysfunction in cancer through the regulation of T cell metabolic signaling. We propose that modulating glucose pathway in T cells can potentiate their anti-tumor activity once adoptively transferred. METHODS T cells glucose metabolic pathway was modulated via glucose transporters overexpression. The functionality of metabolically modified T cells was investigated in murine and human models. RESULTS We demonstrated the existence of a competition for glucose between T cells and tumor cells, with tumor cells imposing glucose restriction mediating T cell hyporesponsiveness. Overexpression of glucose transporters such as Glut1 and Glut3 increased T cell glucose utilization and provide survival/growth advantage and enhanced T cell activation in glucose-restricted conditions. We also established that glucose transporter overexpression improves intratumoral infiltration of adoptively transferred T cells. CONCLUSION This project integrates fundamental concepts of tumor and immune metabolism in the design of immunotherapy and confirms that immunometabolism represents a viable target for new cancer therapy to treat brain tumors.

The relationship between CD204 M2-polarized tumour-associated macrophages (TAMs), tumour-infiltrating lymphocytes (TILs), and microglial activation in glioblastoma microenvironment: a novel immune checkpoint receptor target

BackgroundTumour associated macrophages (TAMs) and tumour infiltrating lymphocytes (TILs) are considered dominant cells in glioblastoma microenvironment.AimThe purpose of this study was to assess the expression of CD204+ M2-polarized TAMs in glioblastomas and their relationship with CD4+TILs, Iba+microglia, and IDH1 mutation. We also exploreed the prognostic value of these markers on the recurrence-free interval (RFI).MethodsThe expressions of CD204+TAMs, CD4+TILs, and Iba1+microglia were quantitively assessed in 45 glioblastomas using immunohistochemistry. Kaplan–Meier analysis and Cox hazards were used to examine the relationship between these factors.ResultsCD204+TAMs were highly expressed in 32 tumours (71%) and the remaining 13 tumours (29%) had reduced expression. CD4+TILs were highly expressed in 10 cases (22%) and 35 cases (77.8%) had low expression. There was an inverse correlation between CD204+TAMs and CD4+TILs, in which 85% of tumours had a high expression of CD204+TAMs and a low expression of CD4+TILs. Nevertheless, there was no significant difference in IDH1 mutation status between the two groups (p = 0.779). There was a significant difference in Iba1+microglial activation between IDH1mutant and IDH1wildtype groups (p = 0.031). For cases with a high expression of CD204+TAMs and a low expression of CD4+TILs, there was a significant difference in RFI after treatment with chemoradiotherapy or radiotherapy (p = 0.030).ConclusionGlioblastoma with a dense CD204+TAMs and few CD4+TILs is associated with IDH1wildtype. These findings suggest that TAMs masks tumour cell and suppress T-cell tumoricidal functions via immunomodulatory mechanisms. Blockade of the CD204-TAM receptor may prevent this mechanism and allow the evolution of TILs.

“Cytokine-microfactories” recruit DCs and deliver tumor antigens via gap junctions for immunotherapy

The majority (~80%) of patients with cancer do not derive clinical benefit from current immunotherapy, largely due to attenuation of immune responses imposed by robust immunosuppression at tumor sites. Here, a cell-based tumor antigen delivery strategy was developed to boost tumor-specific immunity. Notably, the platform constructing ferric oxide nanoparticle-trained macrophages loading tumor antigens (MFe-N) acquired an immunostimulatory program and functioned as the tumoritropic “cytokine-microfactories” to sustainably produce high levels of multiple therapeutic cytokines (GM-CSF, TNFα, and MIP-1α), which are important in activation of immune cells with antitumor potential. Indeed, MFe-N markedly enhanced recruitment of the professional antigen-presenting cells, dendritic cells (DCs), to the tumor sites of an established B16F10 mouse melanoma model. Subsequently, MFe-N effectively delivered tumor antigens to DCs by gap junction-mediated cell-to-cell transmission. And this trafficking was critical for DC maturation to augment antitumor T-cell responses. Simultaneously, the “cytokine-microfactories” elicited high production of the tumoricidal effectors, and in turn blunted the pro-angiogenic activity of tumor-associated macrophages, resulting in conversion of the tumor-supporting milieu to a tumoricidal function that favored infiltration of antitumor T-cells. The findings provided a novel “cytokine-microfactories” harnessing effective delivery of tumor antigens and production of therapeutic cytokines to robustly promote antigen presentation and reshape the tumor immune milieu for priming antitumor immunity. This can enhance existing T-cell mediated immunotherapeutic potency and extend the curative potential immunotherapy to a broader range of patients.

FABP4 deactivates NF-κB-IL1α pathway by ubiquitinating ATPB in tumor-associated macrophages and promotes neuroblastoma progression.

Neuroblastoma (NB) is the most common and deadliest pediatric solid tumor. Targeting and reactivating tumor‐associated macrophages (TAMs) is necessary for reversing immune suppressive state and stimulating immune defense to exert tumoricidal function. However, studies on the function and regulation of TAMs in NB progression are still limited. Fatty acid binding protein 4 (FABP4) in TAMs was correlated with advanced clinical stages and unfavorable histology of NB. FABP4‐mediated macrophages increased migration, invasion, and tumor growth of NB cells. Mechanically, FABP4 could directly bind to ATPB to accelerate ATPB ubiquitination in macrophages. The consequently decreased ATP levels could deactivate NF‐κB/RelA‐IL1α pathway, which subsequently results in macrophages reprogrammed to an anti‐inflammatory phenotype. We also demonstrated that FABP4‐enhanced migration and invasion were significantly suppressed by IL1α blocking antibody. Furthermore, circulating FABP4 was also associated with the clinical stages of NB. Our findings suggest that FABP4‐mediated macrophages may promote proliferation and migration phenotypes in NB cells through deactivating NF‐κB‐IL1α pathway by ubiquitinating ATPB. This study reveals the pathologic and biologic role of FABP4‐mediated macrophages in NB development and exhibits a novel application of targeting FABP4 in macrophages for NB treatment.

Arsenite suppresses IL-2-dependent tumoricidal activities of natural killer cells

Chronic exposure to arsenic causes cancers in various organs including the skin, liver, lung, and bladder in humans, but the mechanisms of the multi-organ carcinogenicity of arsenic remain unknown. Natural killer (NK) cells play important roles in the immune surveillance and elimination of tumor cells. Although accumulating evidence has indicated that arsenic has immunosuppressive properties, little is known about the effects of arsenic on the tumoricidal functions of NK cells. We examined the effects of arsenite on the cytotoxic activities of human and mouse NK cells toward target tumor cells. Exposure of human NK-92 cells and primary mouse NK cells to sublethal doses of arsenite reduced the IL-2-activated cytotoxic activities toward human K562 cells and murine YAC-1 cells, respectively. NK cells recognize target cells via integrated signals from both activating and inhibitory receptors and induce apoptosis of target cells via a granzyme/perforin system. We found that exposure of NK-92 cells to arsenite diminished the IL-2-activated down-regulation of the inhibitory receptors, KIR2DL2 and KIR2DL3, and the up-regulation of granzyme B and lymphotoxin-α. The IL-2-activated increases in secretion of interferon-γ and IL-10 were also slightly reduced by arsenite. Thus, arsenite suppressed the IL-2-activated cytotoxic activity of NK cells by disrupting multiple pathways required for the recognition and killing of target tumor cells. Our findings provide new insights into the roles of NK cell-mediated tumor immunity in cancer development by arsenic.

An IL6-Adenosine Positive Feedback Loop between CD73+ γδTregs and CAFs Promotes Tumor Progression in Human Breast Cancer.

The tumor microenvironment induces immunosuppression via recruiting and expanding suppressive immune cells such as regulatory T cells (Treg) to promote cancer progression. In this study, we documented that tumor-infiltrating CD73+ γδTregs were the predominant Tregs in human breast cancer and exerted more potent immunosuppressive activity than CD4+ or CD8+ Tregs. We further demonstrated that cancer-associated fibroblast (CAF)-derived IL6, rather than TGFβ1, induced CD73+ γδTreg differentiation from paired normal breast tissues via the IL6/STAT3 pathway to produce more adenosine and become potent immunosuppressive T cells. CD73+ γδTregs could in turn promote IL6 secretion by CAFs through adenosine/A2BR/p38MAPK signaling, thereby forming an IL6-adenosine positive feedback loop. CD73+ γδTreg infiltration also impaired the tumoricidal functions of CD8+ T cells and significantly correlated with worse prognosis of patients. The data indicate that the IL6-adenosine loop between CD73+ γδTregs and CAFs is important to promote immunosuppression and tumor progression in human breast cancer, which may be critical for tumor immunotherapy.

Natural killer cell engineering – a new hope for cancer immunotherapy

Natural killer (NK) cells are an important component of the innate immune system, particularly for metastasis immunosurveillance. They can rapidly recognize and kill transformed cells without the requirement of specific neo-antigen recognition. Their effector functions are modulated by a range of stimulatory and inhibitory surface receptors that regulate their cellular activation, differentiation and homeostasis. However, cancer cells can evade NK cell detection by receptor interaction or secretion of soluble immunosuppressant molecules. Therefore, genetic reprogramming of these immune suppressing or activating receptors of NK cells is a promising strategy to augment NK cell tumoricidal functions. In this review, we highlight the current clinical trials of chimeric antigen receptor engineered NK cells with redirected antigen specificity to eliminate hematological cancers and solid tumors. New alternative strategies that are advancing NK cell engineering for cancer treatment are also outlined. Lastly, different NK cell transgenesis approaches are reviewed and compared, and we discuss how these methods can be employed to maximize their anti-tumor effector functions.