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Serine/threonine‐protein kinase B‐raf (BRAF) mutations are found in 8–15% of colorectal cancer patients and identify a subset of tumors with poor outcome in the metastatic setting. We have previously reported that BRAF‐mutant human cells display a high rate of protein production, causing proteotoxic stress, and are selectively sensitive to the proteasome inhibitors bortezomib and carfilzomib. In this work, we tested whether carfilzomib could restrain the growth of BRAF‐mutant colorectal tumors not only by targeting cancer cells directly, but also by promoting an immune‐mediated antitumor response. In human and mouse colorectal cancer cells, carfilzomib triggered robust endoplasmic reticulum stress and autophagy, followed by the emission of immunogenic‐damage‐associated molecules. Intravenous administration of carfilzomib delayed the growth of BRAF‐mutant murine tumors and mobilized the danger‐signal proteins calreticulin and high mobility group box 1 (HMGB1). Analyses of drug‐treated samples revealed increased intratumor recruitment of activated cytotoxic T cells and natural killers, concomitant with the downregulation of forkhead box protein P3 (Foxp3)+ T‐cell surface glycoprotein CD4 (CD4)+ T cells, indicating that carfilzomib promotes reshaping of the immune microenvironment of BRAF‐mutant murine colorectal tumors. These results will inform the design of clinical trials in BRAF‐mutant colorectal cancer patients. [ABSTRACT FROM AUTHOR]

Summary: Multiple functions of CD38 need exploring to expand clinical application of anti‐CD38 antibodies in multiple myeloma (MM). We investigated membrane dynamics of MM cells and subsequent events when CD38 is targeted by therapeutic antibodies. Human MM cells (BF01) were co‐cultured in vitro with therapeutic antibody (or control immunoglobulin G) and analysed using gene expression profiling. Microvesicles from antibody‐exposed cells were analysed for differential gene and microRNA (miRNA) expression, and for phenotypic characterisation. Exposure of BF01 cells to anti‐CD38 antibody resulted in CD38 membrane redistribution, upregulation of metabolism‐related genes and downregulation of genes involved in cell cycle processes. Microvesicles derived from antibody‐exposed cells showed increased CD73 and CD39 expression, presence of programmed death‐ligand 1 and significant up‐/down‐modulation of miRNAs. Microvesicles accumulated around immunoglobulin Fc receptor‐positive (FcR+) cells. Upon internalisation, natural killer cells displayed significantly increased expression of genes related to activation and immune response, and downregulation of genes involved in the cell cycle. Cells may use microvesicles to transmit signals distally as part of a survival strategy. Microvesicles are equipped on their surface with enzymatic machinery leading to production of tolerogenic adenosine. Further, they are internalised in FcR+ cells with significant functional modifications. These observations have relevance for improving anti‐CD38 therapeutic antibodies through targeting this mechanism and its sequelae. [ABSTRACT FROM AUTHOR]

The advent of immune checkpoint (ICP) blockade has introduced an unprecedented paradigm shift in the treatment of cancer. Though very promising, there is still a substantial proportion of patients who do not respond or develop resistance to ICP blockade. In vitro and in vivo models are eagerly needed to identify mechanisms to maximize the immune potency of ICP blockade and overcome primary and acquired resistance to ICP blockade. Vγ9Vδ2 T cells isolated from the bone marrow (BM) from multiple myeloma (MM) are excellent tools to investigate the mechanisms of resistance to PD-1 blockade and to decipher the network of mutual interactions between PD-1 and the immune suppressive tumor microenvironment (TME). Vγ9Vδ2 T cells can easily be interrogated to dissect the progressive immune competence impairment generated in the TME by the long-lasting exposure to myeloma cellss. BM MM Vγ9Vδ2 T cells are PD-1+ and anergic to phosphoantigen (pAg) stimulation; notably, single agent PD-1 blockade is insufficient to fully recover their anti-tumor activity in vitro indicating that additional players are involved in the anergy of Vγ9Vδ2 T cells. In this mini-review we will discuss the value of Vγ9Vδ2 T cells as investigational tools to improve the potency of ICP blockade and immune interventions in MM. [ABSTRACT FROM AUTHOR]

Vγ9Vδ2 T cells are non-conventional T cells with a natural inclination to recognize and kill cancer cells. Malignant B cells, including myeloma cells, are privileged targets of Vγ9Vδ2 T cells in vitro. However, this inclination is often lost in vivo due to multiple mechanisms mediated by tumor cells and local microenvironment. Multiple myeloma (MM) is a paradigm disease in which antitumor immunity is selectively impaired at the tumor site. By interrogating the immune reactivity of bone marrow (BM) Vγ9Vδ2 T cells to phosphoantigens, we have revealed a very early and long-lasting impairment of Vγ9Vδ2 T-cell immune functions which is already detectable in monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even in clinical remission after autologous stem cell transplantation. Multiple cell subsets [MM cells, myeloid-derived suppressor cells, regulatory T cells, and BM-derived stromal cells (BMSC)] are involved in Vγ9Vδ2 T-cell inhibition via several immune suppressive mechanisms including the redundant expression of multiple immune checkpoints (ICPs). This review will address some aspects related to the dynamics of ICP expression in the BM of MM patients in relationship to the disease status (MGUS, diagnosis, remission, and relapse) and how this multifaceted ICP expression impairs Vγ9Vδ2 T-cell function. We will also provide some suggestions how to rescue Vγ9Vδ2 T cells from the immune suppression operated by ICP and to recover their antimyeloma immune effector functions at the tumor site. [ABSTRACT FROM AUTHOR]

Human Vγ9Vd2 T cells have the capacity to detect supra-physiological concentrations of phosphoantigens (pAgs) generated by the mevalonate (Mev) pathway of mammalian cells under specific circumstances. Isopentenyl pyrophosphate (IPP) is the prototypic pAg recognized by Vγ9Vd2 T cells. B-cell derived tumor cells (i.e., lymp homa and myeloma cells) and dendritic cells (DCs) are privileged targets of Vγ9Vd2 T cells because they generate significant amounts of IPP which can be boosted with zoledronic acid (ZA). ZA is the most potent aminobisphosphonate (NBP) clinically available to inhibit osteoclast activation and a very potent inhibitor of farnesyl pyrophosphate synthase in the Mev pathway. ZA-treated DCs generate and release in the supernatants picomolar IPP concentrations which are sufficient to induce the activation of Vγ9Vd2 T cells. We have recently shown that the ATP-binding cassette transporter A1 (ABCA1) plays a major role in the extracellular release of IPP from ZA-treated DCs. This novel ABCA1 function is fine-tuned by physical interactions with IPP, apolipoprotein A-I (apoA-I), and butyrophilin-3A1 (BTN3A1). The mechanisms by which soluble IPP induces Vγ9Vd2 T-cell activation remain to be elucidated. It is possible that soluble IPP binds to BTN3A1, apoA-I, or other unknown molecules on the cell surface of bystander cells like monocytes, NK cells, Vγ9Vd2 T cells, or any other cell locally present. Investigating this scenario may represent a unique opportunity to further characterize the role of BTN3A1 and other molecules in the recognition of soluble IPP by Vγ9Vd2 T cells. [ABSTRACT FROM AUTHOR]

Vγ9Vδ2 T cells have a natural inclination to recognize malignant B cells in vitro via receptors for stress-induced selfligands and TCR-dependent recognition of phosphoantigens (pAgs) generated in the mevalonate (Mev) pathway. This inclination is continuously challenged in vivo by the immune suppression operated by tumor cells. Multiple myeloma (MM) is a prototypic B-cell malignancy in which myeloma cells subvert the local microenvironment to reshape antitumor immune responses. In this study, we have investigated the immune competence of bone marrow (BM) Vγ9Vδ2 T cells in a large series of MM patients. We have found that the BM microenvironment significantly hampers the pAg-reactivity of BM Vγ9Vδ2 T cells, which become largely PD-1+ and are surrounded by PD-L1+ myeloma cells and increased numbers of PD-L1+ myeloid-derived suppressor cells (MDSC). Vγ9Vδ2 T-cell dysfunction is an early event that can be already detected in individuals with monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even when MM patients achieve clinical remission. Anti-PD-1 treatment increases the cytotoxic potential of Vγ9Vδ2 T cells by almost 5-fold after pAg stimulation, and appears to be a promising strategy for effective immune interventions in MM. [ABSTRACT FROM AUTHOR]

Background: Multidrug resistant cancer cells are hard to eradicate for the inefficacy of conventional anticancer drugs. Besides escaping the cytotoxic effects of chemotherapy, they also bypass the pro-immunogenic effects induced by anticancer drugs: indeed they are not well recognized by host dendritic cells and do not elicit a durable anti-tumor immunity. It has not yet been investigated whether multidrug resistant cells have a different ability to induce immunosuppression than chemosensitive ones. We addressed this issue in human and murine chemosensitive and multidrug resistant cancer cells. Results: We found that the activity and expression of indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the conversion of tryptophan into the immunosuppressive metabolite kynurenine, was higher in all the multidrug resistant cells analyzed and that IDO1 inhibition reduced the growth of drug-resistant tumors in immunocompetent animals. In chemoresistant cells the basal activity of JAK1/STAT1 and JAK1/STAT3 signaling was higher, the STAT3 inhibitor PIAS3 was down-regulated, and the autocrine production of STAT3-target and IDO1-inducers cytokines IL-6, IL-4, IL-1β, IL-13, TNF-α and CD40L, was increased. The disruption of the JAK/STAT signaling lowered the IDO1 activity and reversed the kynurenine-induced pro-immunosuppressive effects, as revealed by the restored proliferation of T-lymphocytes in STAT-silenced chemoresistant cells. Conclusions: Our work shows that multidrug resistant cells have a stronger immunosuppressive attitude than chemosensitive cells, due to the constitutive activation of the JAK/STAT/IDO1 axis, thus resulting chemo- and immune-evasive. Disrupting this axis may significantly improve the efficacy of chemo-immunotherapy protocols against resistant tumors. [ABSTRACT FROM AUTHOR]

Background The activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors. Methods We studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells. Results MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA reactivated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumorimmune system recognition in response to chemotherapy in MDR cells. Conclusions Our work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells. [ABSTRACT FROM AUTHOR]

Durable tumor cell eradication by chemotherapy is challenged by the development of multidrug-resistance (MDR) and the failure to induce immunogenic cell death. The aim of this work was to investigate whether MDR and immunogenic cell death share a common biochemical pathway eventually amenable to therapeutic intervention. We found that mevalonate pathway activity, Ras and RhoA protein isoprenylation, Ras- and RhoA-downstream signalling pathway activities, Hypoxia Inducible Factor-1alpha activation were significantly higher in MDR+ compared with MDR− human cancer cells, leading to increased P-glycoprotein expression, and protection from doxorubicin-induced cytotoxicity and immunogenic cell death. Zoledronic acid, a potent aminobisphosphonate targeting the mevalonate pathway, interrupted Ras- and RhoA-dependent downstream signalling pathways, abrogated the Hypoxia Inducible Factor-1alpha-driven P-glycoprotein expression, and restored doxorubicin-induced cytotoxicity and immunogenic cell death in MDR+ cells. Immunogenic cell death recovery was documented by the ability of dendritic cells to phagocytise MDR+ cells treated with zoledronic acid plus doxorubicin, and to recruit anti-tumor cytotoxic CD8+ T lymphocytes. These data indicate that MDR+ cells have an hyper-active mevalonate pathway which is targetable with zoledronic acid to antagonize their ability to withstand chemotherapy-induced cytotoxicity and escape immunogenic cell death. [ABSTRACT FROM AUTHOR]

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much legs is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer. We have addressed this issue in multiple myeloma (MM), a prototypic disease with several immune dysfunctions that also affect γδ T cells and DC. ZA-treated MM DC were highly effective in activating autologous γδ T cells, even in patients refractory to stimulation with ZA-treated monocytes. ZA inhibited the mevalonate pathway of MM DC and induced the intracellular accumulation and release into the supernatant of isopentenyl pyrophosphate, a selective γδ T cell activator, in sufficient amounts to induce the proliferation of γδ T cells. Immune responses against the tumor-associated Ag survivin (SRV) by MHC-restricted, SRV-specific CD8+ αβ T cells were amplified by the concurrent activation of αβ T cells driven by autologous DC copulsed with ZA and SRV-derived peptides. Ancillary to the isopentenyl pyrophosphate-induced γδ T cell proliferation was the mevalonate-independent ZA ability to directly antagonize regulatory T cells and downregulate PD-L2 expression on the DC cell surface. In conclusion, ZA has multiple immune modulatory activities that allow MM DC to effectively handle the concurrent activation of γδ T cells and MHC-restricted CD8+ αβ antitumor effector T cells. [ABSTRACT FROM AUTHOR]

• Metabolites released from mitochondria help cancer cells to evade immune system. • Effector and immune-suppressive cells have a different mitochondria metabolism. • Targeting mitochondria metabolism in cancer cells can reverse immune-resistance. • Modulating mitochondria metabolism activate or suppress immune cells activity. • A precision medicine targeting mitochondria of cancer or immune subsets is needed. Mitochondria have been considered for a long time only as the principal source of building blocks and energy upon aerobic conditions. Recently they emerged as key players in cell proliferation, invasion and resistance to therapy. The most aggressive tumors are able to evade the immune-surveillance. Alterations in the mitochondria metabolism either in cancer cells or in host immune system cells are involved in such tumor-induced immune-suppression. This review will focus on the main mitochondrial dysfunctions in tumor and immune cell populations determining immune-resistance, and on the therapies that may target mitochondrial metabolism and restore a powerful anti-tumor immune-activity. [ABSTRACT FROM AUTHOR]

Vγ9Vδ2 T cells are activated by phosphoantigens, such as isopentenyl pyrophosphate (IPP), which is generated in the mevalonate pathway of antigen-presenting cells. IPP is released in the extracellular microenvironment via unknown mechanisms. Here we show that the ATP-binding cassette transporter A1 (ABCA1) mediates extracellular IPP release from dendritic cells (DC) in cooperation with apolipoprotein A-I (apoA-I) and butyrophilin-3A1. IPP concentrations in the supernatants are sufficient to induce Vγ9Vδ2 T cell proliferation after DC mevalonate pathway inhibition with zoledronic acid (ZA). ZA treatment increases ABCA1 and apoA-I expression via IPP-dependent LXRα nuclear translocation and PI3K/Akt/mTOR pathway inhibition. These results close the mechanistic gap in our understanding of extracellular IPP release from DC and provide a framework to fine-tune Vγ9Vδ2 T cell activation via mevalonate and PI3K/Akt/mTOR pathway modulation. [ABSTRACT FROM AUTHOR]

This study investigates themechanism(s) underlying the immunoregulatory activities of placenta-derived human amnion epithelial cells (hAEC). The working hypothesis is that NAD+ and ATP, along with ectoenzymes involved in their metabolism, play a significant role in hAEC-mediated immune regulation. Proof of principle of the hypothesis was obtained by analyzing the interactions between hAEC and the main human leukocyte populations. The results obtained indicate that hAEC constitutively express a unique combination of functional ectoenzymes, driving the production of adenosine (ADO) via canonical (CD39, CD73) and alternative (CD38, CD203a/PC-1, CD73) pathways. Further, the picture is completed by the observation that hAEC express A1, A2a, and A2b ADO receptors as well as ADO deaminase, the enzyme involved in ADO catabolism. The contribution of the purinergic mediator to immunomodulation was confirmed by exposing in vitro different immune effector cells to the action of primary hAECs. B cells showed an enhanced proliferation and diminished spontaneous apoptosis when in contact with hAEC. T cell proliferation was partially inhibited by hAEC through ADO production, as confirmed by using specific ectoenzyme inhibitors. Further, hAEC induced an expansion of both Tand B regulatory cells. Last, hAEC inhibited NK cell proliferation. However, the involvement of ADO-producing ectoenzymes is less apparent in this context. In conclusion, hAEC exert different in vitro immunoregulatory effects, per se, as a result of interactions with different populations of immune effector cells. These results support the view that hAEC are instrumental for regenerative medicine as well as in therapeutic applications for immune-related diseases. [ABSTRACT FROM AUTHOR]

Introduction: Bone marrow (BM) Vγ9Vδ2 T cells are intrinsically predisposed to sense the immune fitness of the tumor microenvironment (TME) in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS)., Methods: In this work, we have used BM Vγ9Vδ2 T cells to interrogate the role of the immune checkpoint/immune checkpoint-ligand (ICP/ICP-L) network in the immune suppressive TME of MM patients., Results: PD-1+ BM MM Vγ9Vδ2 T cells combine phenotypic, functional, and TCR-associated alterations consistent with chronic exhaustion and immune senescence. When challenged by zoledronic acid (ZA) as a surrogate assay to interrogate the reactivity to their natural ligands, BM MM Vγ9Vδ2 T cells further up-regulate PD-1 and TIM-3 and worsen TCR-associated alterations. BM MM Vγ9Vδ2 T cells up-regulate TIM-3 after stimulation with ZA in combination with αPD-1, whereas PD-1 is not up-regulated after ZA stimulation with αTIM-3, indicating a hierarchical regulation of inducible ICP expression. Dual αPD-1/αTIM-3 blockade improves the immune functions of BM Vγ9Vδ2 T cells in MM at diagnosis (MM-dia), whereas single PD-1 blockade is sufficient to rescue BM Vγ9Vδ2 T cells in MM in remission (MM-rem). By contrast, ZA stimulation induces LAG-3 up-regulation in BM Vγ9Vδ2 T cells from MM in relapse (MM-rel) and dual PD-1/LAG-3 blockade is the most effective combination in this setting., Discussion: These data indicate that: 1) inappropriate immune interventions can exacerbate Vγ9Vδ2 T-cell dysfunction 2) ICP blockade should be tailored to the disease status to get the most of its beneficial effect., Competing Interests: MM reports advisory boards for AbbVie, Janssen-Cilag, Sanofi, and research funding from Sanofi; MG reports advisory boards for Amgen, Bristol Myers Squibb, and Janssen-Cilag; MDA reports honoraria for lectures and advisory boards for GlaxoSmithKline, and Sanofi; AL reports honoraria and advisory boards for Janssen-Gilag, Bristol Myers Squibb, Amgen, Takeda, Oncopeptides, GlaxoSmithKline, Sanofi, and Karyopharm. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Giannotta, Castella, Tripoli, Grimaldi, Avonto, D’Agostino, Larocca, Kopecka, Grasso, Riganti and Massaia.)

Vγ9Vδ2 T cells are immune effector cells very well-suited for immunotherapy, but clinical results have been disappointing in multiple myeloma (MM) and other cancers. We have shown that Vg9Vd2 T cells are victimized prematurely by the immune suppressive tumor microenvironment (TME) established by myeloma and neighbouring cells in the bone marrow (BM) of MM patients. One major mechanism is the highly redundant expression of multiple immunecheckpoints/immune checkpoint-ligands (ICP/ICP-L) in the TME impairing antimyeloma Vg9Vd2 T-cell immune responses. Another major immune suppressive mechanism is the metabolic reset driven by myeloma cells in the TME to satisfy their energetic needs to the detriment of effector cells. Recently, it has become clear that the ICP/ICP-L circuitry and metabolic checkpoints (MCP) jointly operate in the TME of cancer patients to promote tumor cell growth and suppress antitumor immune responses. In this review, we discuss the possible interactions between ICP/ICP-L and MCP in the TME of MM patients that may compromise the immune competence of BM Vγ9Vδ2 T cells, envisaging novel combination therapies to improve the outcome of immune-based interventions.

In chronic lymphocytic leukemia (CLL) the occurrence and the impact of antibody responses toward tumor-derived antigens are largely unexplored. Our serological proteomic data show that antibodies toward 47 identified antigens are detectable in 29 out of 35 patients (83%) with untreated CLL. The glycolytic enzyme alpha-enolase (ENO1) is the most frequently recognized antigen (i.e. 54% of CLL sera). We show that ENO1 is upregulated in the proliferating B-cell fraction of CLL lymph nodes. In CLL cells of the peripheral blood, ENO1 is exclusively expressed at the intracellular level, whereas it is exposed on the surface of apoptotic leukemic cells.From the clinical standpoint, patients with progressive CLL show a higher number of antigen recognitions compared to patients with stable disease. Consistently, the anti-ENO1 antibodies are prevalent in sera from patients with progressive disease and their presence is predictive of a shorter time to first treatment. This clinical inefficacy associates with the inability of patients' sera to trigger complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against leukemic cells.Together, these results indicate that antibody responses toward tumor-derived antigens are frequently detectable in sera from patients with CLL, but they are expression of a disrupted immune system and unable to hamper disease progression.