Novel Mechanism Of Immune Escape Research Articles

AMD3100-Mediated CXCR4 Inhibition Impairs Development of Primary Lymphoma of the Central Nervous System

A hallmark of primary lymphoma of the central nervous system (CNS; PCNSL) is the strong CXCR4 expression of the tumor cells, the function of which is still unknown. Invitro treatment of BAL17CNS lymphoma cells by AMD3100, which inhibits CXCR4-CXCL12 interactions, resulted in the significantly differential expression of 273 genes encoding proteins involved in cell motility, cell-cell signaling and interaction, hematological system development and function, and immunologic disease. Among the genes down-regulated was the one encoding CD200, a regulator of CNS immunologic activity. These data directly translated into the invivo situation; BAL17CNS CD200 expression was down-regulated by 89% (3% versus 28% CD200+ lymphoma cells) in AMD3100-treated versus untreated mice with BAL17CNS-induced PCNSL. Reduced lymphoma cell CD200 expression may contribute to the markedly increased microglial activation in AMD3100-treated mice. AMD3100 also maintained the structural integrity of blood-brain barrier tight junctions and the outer basal lamina of cerebral blood vessels. Subsequently, lymphoma cell invasion of the brain parenchyma was impaired, and maximal parenchymal tumor size was significantly reduced by 82% in the induction phase. Thus, AMD3100 qualified as a potentially attractive candidate to be included into the therapeutic concept of PCNSL. Beyond therapy, CXCR4-induced suppression of microglial activity is of general neuroimmunologic interest and identifies CD200 expressed by the lymphoma cells as a novel mechanism of immune escape in PCNSL.

FGFR1/MAPK-directed brachyury activation drives PD-L1-mediated immune evasion to promote lung cancer progression

Immune checkpoint inhibitors provide promising benefits for patients with cancer. However, efficacy has been encumbered by high resistance rates. It is critical to understand the basic mechanisms of tumor-mediated resistance to this treatment modality. Previous studies have found that the transcription factor brachyury is highly expressed in lung cancer. Here, we show that brachyury activation induces the upregulation of PD-L1 leading to inactivation of T cell proliferation in vitro and inhibited infiltration of CD8+ and CD3+ T cells into tumor in an immunocompetent mouse model. We further demonstrate that FGFR1/MAPK activation regulates brachyury and PD-L1 expressions and promotes immunosuppression. Blocking FGFR1/MAPK suppresses brachyury and PD-L1 expressions, revives immune activity, and reverses the resistance to anti-PD-1 treatment to produce a durable therapeutic response. We also find that lung cancer patients with high activation of the FGFR1-MAPK-brachyury-PD-L1 signature and low expression of CD8A, CD3D, or PDCD1 have worse survival outcomes. These findings elucidate a novel mechanism of immune escape from immune checkpoint therapy and provide an opportunity to enhance its therapeutic efficacy in the treatment of a subset of FGFR1/MAPK/brachyury/PD-L1-driven lung cancer.

Human metapneumovirus M2-2 protein inhibits RIG-I signaling by preventing TRIM25-mediated RIG-I ubiquitination.

Retinoic acid-inducible gene I (RIG-I) is a receptor that senses viral RNA and interacts with mitochondrial antiviral signaling (MAVS) protein, leading to the production of type I interferons and inflammatory cytokines to establish an antiviral state. This signaling axis is initiated by the K63-linked RIG-I ubiquitination, mediated by E3 ubiquitin ligases such as TRIM25. However, many viruses, including several members of the family Paramyxoviridae and human respiratory syncytial virus (HRSV), a member of the family Pneumoviridae, escape the immune system by targeting RIG-I/TRIM25 signaling. In this study, we screened human metapneumovirus (HMPV) open reading frames (ORFs) for their ability to block RIG-I signaling reconstituted in HEK293T cells by transfection with TRIM25 and RIG-I CARD (an N-terminal CARD domain that is constitutively active in RIG-I signaling). HMPV M2-2 was the most potent inhibitor of RIG-I/TRIM25-mediated interferon (IFN)-β activation. M2-2 silencing induced the activation of transcription factors (IRF and NF-kB) downstream of RIG-I signaling in A549 cells. Moreover, M2-2 inhibited RIG-I ubiquitination and CARD-dependent interactions with MAVS. Immunoprecipitation revealed that M2-2 forms a stable complex with RIG-I CARD/TRIM25 via direct interaction with the SPRY domain of TRIM25. Similarly, HRSV NS1 also formed a stable complex with RIG-I CARD/TRIM25 and inhibited RIG-I ubiquitination. Notably, the inhibitory actions of HMPV M2-2 and HRSV NS1 are similar to those of V proteins of several members of the Paramyxoviridae family. In this study, we have identified a novel mechanism of immune escape by HMPV, similar to that of Pneumoviridae and Paramyxoviridae family members.

P-085: Resistance to immunogenic cell death as a novel mechanism of immune escape in high-risk multiple myeloma

P-085: Resistance to immunogenic cell death as a novel mechanism of immune escape in high-risk multiple myeloma

Epstein-Barr Virus-Encoded Circular RNA CircBART2.2 Promotes Immune Escape of Nasopharyngeal Carcinoma by Regulating PD-L1.

Epstein-Barr virus (EBV) infection is an established cause of nasopharyngeal carcinoma (NPC) and is involved in a variety of malignant phenotypes, including tumor immune escape. EBV can encode a variety of circular RNAs (circRNA), however, little is known regarding the biological functions of these circRNAs in NPC. In this study, EBV-encoded circBART2.2 was found to be highly expressed in NPC where it upregulated PD-L1 expression and inhibited T-cell function in vitro and in vivo. circBART2.2 promoted transcription of PD-L1 by binding the helicase domain of RIG-I and activating transcription factors IRF3 and NF-κB, resulting in tumor immune escape. These results elucidate the biological function of circBART2.2, explain a novel mechanism of immune escape caused by EBV infection, and provide a new immunotherapy target for treating NPC. SIGNIFICANCE: This work demonstrates that circBART2.2 binding to RIG-I is essential for the regulation of PD-L1 and subsequent immune escape in nasopharyngeal carcinoma.

Quantitative and Functional Alterations of 6-Sulfo Lacnac Dendritic Cells in Multiple Myeloma

Multiple myeloma (MM) is characterized by expansion of abnormal plasma cells in the bone marrow (BM). Monocytes and macrophages have been shown to accumulate in the microenvironment / BM of MM patients while promoting MM cells survival and proliferation. Recently, another population of myeloid cells expressing the M-DC8 antigen containing a 6-sulfo LacNAc structure, called 6-sulfo LacNac dendritic cells (Slan-DCs) has been described. Functionally, Slan-DCs have potent pro-inflammatory properties based on their capacity to produce large amounts of Tumor Necrosis Factor-α and Interleukin (IL) 12p70 upon stimulation with Toll-like receptor ligands such as R848. They also express C-X-C chemokine receptor type 4 which is important in homing to the BM. Nevertheless, little is known about microenvironmental interactions between MM cells and Slan-DCs.To gain insights on the Slan-DCs' potential role in MM pathophysiology, we evaluated their phenotype and functions in the BM and blood of patients with newly diagnosed symptomatic MM. Using multicolor flow cytometry, we performed quantitative and functional analyses. Compared to healthy donors (n=11), the percentages and absolute numbers of circulating HLA-DR+ M-DC8+ Slan-DCs were significantly decreased in the blood of MM patients (n=23) (for absolute number: 20.64 vs 7.61 Slan-DCs/μL, p=0.0015 and for percentage: 1.06 vs 0.49%, p=0.029). In contrast, there were no significant difference in the frequencies of Slan-DCs in the patients BM.We next addressed their functional properties, in particular cytokine production. We observed a significant decrease in IL-12-producing Slan-DCs from MM patients as compared to healthy donors both in blood (25.88% versus 56.86% respectively, p<0.0001) and BM (25.11% versus 48.83% respectively, p=0.016). In order to investigate whether the decrease in IL-12 secretion was due to the MM cells, circulating Slan-DCs from healthy controls were stimulated with R848 in the presence of 4 different MM cell lines. The capacity of Slan-DCs from healthy controls to secrete IL-12 under this stimulation was measured by ELISA. We observed that the RPMI-8226, KMS-12-PE and LP-1 MM cell lines inhibited R848-induced IL-12 secretion by Slan-DCs. Furthermore, we could show that primary MM cells from patients at diagnosis (magnetically sorted CD138+ cells from patients) were able to inhibit IL-12 secretion by Slan-DCs from healthy donors (n=5).Since MM cells inhibited Slan-DCs secretion of IL-12 and given the pivotal role of IL-12 in Th1 polarization, we investigated whether MM cells could modulate the capacity of Slan-DCs to induce T cell proliferation and Th1 differentiation. To this aim, we co-cultured T cells and Slan-DCs (n=5). Here, we found that normal Slan-DCs can efficiently induce the proliferation of T cells. In addition, their ability to promote T cell proliferation was more pronounced after R848 priming. However, after co-culture with RPMI-8226 MM cell line, the fraction of proliferating T cells at day 7 was significantly decreased (58.14% versus 28.37% ; p=0.03). Moreover, RPMI-8226 MM cell line markedly impaired the capacity of Slan-DCs to induce the differentiation of naïve CD4+ T cells into IFNγ-producing Th1 cells (34.59% versus 7.89% ; p=0.04).Based on the above results, we hypothesized that Slan-DCs' pro-inflammatory phenotype could also be impacted by MM cells. Thus, freshly-sorted Slan-DCs were cultured with or without MM cell lines and their phenotype was analyzed by flow cytometry (n=9). When Slan-DCs were cultured in the presence of MM cells, expression of CD14 and CD16, but not CD11b, was induced, in line with a phenotype of an “intermediate” monocytes population. Moreover, in the presence of MM cells, the expression of the costimulatory molecule CD86 was weakly induced on Slan-DCs (Mean of Fluorescence Intensity = 102049 versus 199470, p=0.004).In conclusion, these results provide evidence that MM cells are able to significantly impair the capacity of Slan-DCs to secrete IL-12 and stimulate CD3+ T cell proliferation and to polarize naïve CD4+ T cell into Th1 cells. The latter suggests that MM cells are able to strongly suppress the pro-inflammatory function of Slan-DCs, directing them towards a more tolerogenic profile. These findings reveal a novel mechanism of immune escape in MM, and may pave the way for newer immune-intervention strategies in combination with other currently available therapies. DisclosuresGarderet:Amgen: Honoraria; Takeda: Honoraria. Mohty:Sanofi: Honoraria, Speakers Bureau.

Extramedullary leukemia relapse after allogeneic stem cell transplantation: a novel mechanism of immune escape?

Background: Relapse is a significant cause of treatment failure after allogeneic stem cell transplantation. In many cases relapse occurs when leukemic cells escape from immune surveillance. Methods &results: In the setting of haploidentical transplantation, immune escape is usually the result of the loss of the mismatched haplotype from leukemic cells, while downregulation of HLA-expression has been postulated as a significant cause of immune escape after transplantation with the use of HLA-matched donors. We observed that patients with acute leukemia who relapse at the time of active graft-versus-host-disease, usually develop extramedullary leukemia while they remain free of leukemia in peripheral blood and bone marrow. Conclusion: Our observation points toward a novel mechanism of immune escape which is microenvironment-specific.

Loss of the Immune Checkpoint CD85j/LILRB1 on Malignant Plasma Cells Contributes to Immune Escape in Multiple Myeloma.

Mechanisms of immune regulation may control proliferation of aberrant plasma cells (PCs) in patients with monoclonal gammopathy of undetermined significance (MGUS) preventing progression to active multiple myeloma (MM). We hypothesized that CD85j (LILRB1), an inhibitory immune checkpoint for B cell function, may play a role in MM pathogenesis. In this study, we report that patients with active MM had significantly lower levels of CD85j and its ligand S100A9. Decreased CD85j expression could also be detected in the premalignant condition MGUS, suggesting that loss of CD85j may be an early event promoting tumor immune escape. To gain insight into the molecular mechanisms underlying CD85j functions, we next enforced expression of CD85j in human myeloma cell lines by lentiviral transduction. Interestingly, gene expression profiling of CD85j-overexpressing cells revealed a set of downregulated genes with crucial functions in MM pathogenesis. Furthermore, in vitro functional assays demonstrated that CD85j overexpression increased susceptibility to T cell- and NK-mediated killing. Consistently, ligation of CD85j decreased the number of PCs from individuals with MGUS but not from patients with MM. In conclusion, downregulation of inhibitory immune checkpoints on malignant PCs may provide a novel mechanism of immune escape associated with myeloma pathogenesis.

HLA dependent immune escape mechanisms in B-cell lymphomas: Implications for immune checkpoint inhibitor therapy?

ABSTRACTAntigen presentation by tumor cells in the context of Human Leukocyte Antigen (HLA) is generally considered to be a prerequisite for effective immune checkpoint inhibitor therapy. We evaluated cell surface HLA class I, HLA class II and cytoplasmic HLA-DM staining by immunohistochemistry (IHC) in 389 classical Hodgkin lymphomas (cHL), 22 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL), 137 diffuse large B-cell lymphomas (DLBCL), 39 primary central nervous system lymphomas (PCNSL) and 19 testicular lymphomas. We describe a novel mechanism of immune escape in which loss of HLA-DM expression results in aberrant membranous invariant chain peptide (CLIP) expression in HLA class II cell surface positive lymphoma cells, preventing presentation of antigenic peptides. In HLA class II positive cases, HLA-DM expression was lost in 49% of cHL, 0% of NLPHL, 14% of DLBCL, 3% of PCNSL and 0% of testicular lymphomas. Considering HLA class I, HLA class II and HLA-DM together, 88% of cHL, 10% of NLPHL, 62% of DLBCL, 77% of PCNSL and 87% of testicular lymphoma cases had abnormal HLA expression patterns. In conclusion, an HLA expression pattern incompatible with normal antigen presentation is common in cHL, DLBCL, PCNSL and testicular lymphoma. Retention of CLIP in HLA class II caused by loss of HLA-DM is a novel immune escape mechanism, especially prevalent in cHL. Aberrant HLA expression should be taken into account when evaluating efficacy of checkpoint inhibitors in B-cell lymphomas.

Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape.

H9N2 avian influenza virus is a major cause of poultry production loss across Asia leading to the wide use of vaccines. Efficacy of vaccines is often compromised due to the rapid emergence of antigenic variants. To improve the effectiveness of vaccines in the field, a better understanding of the antigenic epitopes of the major antigen, hemagglutinin, is required. To address this, a panel of nine monoclonal antibodies were generated against a contemporary Pakistani H9N2 isolate, which represents a major Asian H9N2 viral lineage. Antibodies were characterized in detail and used to select a total of 26 unique ‘escape’ mutants with substitutions across nine different amino acid residues in hemagglutinin including seven that have not been described as antigenic determinants for H9N2 viruses before. Competition assays and structural mapping revealed two novel, discrete antigenic sites “H9-A” and “H9-B”. Additionally, a second subset of escape mutants contained amino acid deletions within the hemagglutinin receptor binding site. This constitutes a novel method of escape for group 1 hemagglutinins and could represent an alternative means for H9N2 viruses to overcome vaccine induced immunity. These results will guide surveillance efforts for arising antigenic variants as well as evidence based vaccine seed selection and vaccine design.

Abstract 4062: Role of CD137 signaling in immune evasion of Hodgkin and Reed-Sternberg cells

Abstract Hodgkin lymphoma (HL), a lymphoid malignancy, is a frequently occurring cancer among adolescents and young adults. HL is marked by the presence of malignant Hodgkin and Reed-Sternberg (HRS) cells, which constitute only a small fraction of the tumor mass. HRS cells are present in a complex stroma of inflammatory leukocytes, but are often able to evade the host immune system. These cells express various receptors to recruit leukocytes and hijack them for supporting the tumor growth. Our group has reported the overexpression of CD137, a member of the tumor necrosis factor receptor (TNFR) superfamily, on the HRS cells of HL tumor samples. CD137 is a co-stimulatory molecule expressed by T cells, only upon activation. We speculated that the expression of CD137 on the HRS cells may be associated with its malignancy, as HRS cells are clonally derived from B cells which are not expressing CD137. Our study aims to analyse the functional role of CD137 expression behind the survival, progression, and cytokine release of the malignant HRS cells. We found that the induction of CD137 signaling in HRS cell lines enhanced the secretion of cytokines such as IL-6, TNF-α and IL-13. In order to investigate the effect of these cytokines on the immune microenvironment, we cultured peripheral blood mononuclear cells (PBMCs) in the supernatants derived from CD137-induced HRS cells. PBMCs, when cultured in the above mentioned conditioned supernatants, secreted lesser levels of IFN-γ (a Th1 cytokine essential for T cell and NK cell cytotoxicity), implying a suppressed state of immune cells. Further, neutralisation of IL-13 (a Th2 cytokine) in the conditioned supernatants reversed the reduction of IFN-γ observed in PBMCs. This shows that IL-13 secreted by the HRS cells upon CD137 signaling is responsible for the inhibition of IFN-γ secretion by PBMCs. To summarize, CD137 signaling in HRS cells leads to the secretion of cytokines, which drive the polarization of helper T cells towards Th2 type, thus reducing Th1 immune responses against cancers. Taken together, our findings reveal the role of CD137 signaling as a novel mechanism of immune escape in HRS cells. Hence, targeting CD137 can be a potential therapeutic option for HL patients. Citation Format: Sakthi Rajendran, Herbert Schwarz. Role of CD137 signaling in immune evasion of Hodgkin and Reed-Sternberg cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4062. doi:10.1158/1538-7445.AM2015-4062

ME-19 * ANALYSIS OF TREG RECRUITMENT AND FOXP3 EXPRESSION IN GLIOMAS REGARDING THE INTRAOPERATIVE FLUORESCENCE OF 5-ALA

Not only are regulatory T cells (Tregs) an essential component of the immune system in maintaining immunologic self-tolerance but they are also involved in the suppression of anti-tumor immune responses. The transcription factor forkhead box P3 (FOXP3) is widely used as a marker for the identification of Tregs being essential for the functionality. Oral 5 aminolevulinic acid (5-ALA) is used to assist surgical resection of high-grade gliomas, being known as an indicator of malignancy as it is able to pass the blood brain barrier in high-grade gliomas. Gaining more knowledge regarding the pathogenesis of gliomas, the tumor microenvironment came to the fore. In this study, patterns of Treg recruitment and FOXP3 expression in the microenvironment of gliomas were characterized, regarding the fluorescence of 5-ALA. Glioma tissue positive and negative for intraoperative fluorescence of 5-ALA was analyzed and compared. Tregs present in the tumor microenvironment were detected by double-immunohistochemistry for FOXP3 and the glioma marker, anti- glial fibrillary acidic protein (GFAP). FOXP3 mRNA expression was examined, using quantitative real-time-PCR. Tregs were found in the tumor microenvironment as well as within tumor cell islets, and FOXP3 mRNA expression in the tumors was significantly higher than in normal brain. Intriguingly, single FOXP3+ cells exhibited morphologic characteristics of glioma cells. 5-ALA positive tumor tissue showed a higher accumulation of Tregs than 5-ALA negative tissue. Gliomas recruit Tregs into their microenvironment, presumably in order to suppress immunosurveillance, thus avoiding destruction by the immune system. Endogenous FOXP3 expression in glioma cells might present a novel mechanism of immune escape. Our study showed differences between the amount of Treg and FOPX3 expression in 5-ALA positive and negative glioma tissue indicating that Tregs are more present in high-grade malignant gliomas. These findings suggest FOXP3 and Tregs as a possible therapeutic target in the therapy of gliomas.

Gangliosides drive the tumor infiltration and function of myeloid-derived suppressor cells.

Although it is now widely appreciated that antitumor immunity is critical to impede tumor growth and progression, there remain significant gaps in knowledge about the mechanisms used by tumors to escape immune control. In tumor cells, we hypothesized that one mechanism of immune escape used by tumors involves the synthesis and extracellular shedding of gangliosides, a class of biologically active cell surface glycosphingolipids with known immunosuppressive properties. In this study, we report that tumor cells engineered to be ganglioside deficient exhibit impaired tumorigenicity, supporting a link between ganglioside-dependent immune escape and tumor outgrowth. Notably, we documented a dramatic reduction in the numbers and function of tumor-infiltrating myeloid-derived suppressor cells (MDSC) in ganglioside-deficient tumors, in contrast with the large MDSC infiltrates seen in ganglioside-rich littermate control tumors. Transient ganglioside reconstitution of the tumor cell inoculum was sufficient to increase MDSC infiltration, supporting a direct connection between ganglioside production by tumor cells and the recruitment of immunosuppressive MDSC into the tumor microenvironment. Our results reveal a novel mechanism of immune escape that supports tumor growth, with broad implications given that many human tumors produce and shed high levels of gangliosides.

Cutaneous and oral squamous cell carcinoma–dual immunosuppression via recruitment of FOXP3+ regulatory T cells and endogenous tumour FOXP3 expression?

Regulatory T cells (Tregs) are an essential component of the immune system, but are also involved in the suppression of anti-tumour immune responses. The study examines their immunoregulatory role including their transcription factor, FOXP3, in oral and cutaneous SCC.Tregs were detected by double-immunohistochemistry. FOXP3-mRNA-expression was examined in tumour tissue, as well as in skin-derived primary cells and cell lines of different malignancy.Tregs were found in the tumour microenvironment, and FOXP3-mRNA-expression was significantly higher than in normal skin. Intriguingly, single FOXP3+ cells exhibited morphologic characteristics of SCC cells. Consistent with this, endogenous FOXP3-mRNA-expression was indeed detected in the epidermal cell lineage and dramatically increased with increasing malignancy of the cells.SCCs recruit Tregs into their microenvironment, presumably in order to suppress immunosurveillance, thus avoiding destruction by the immune system. Endogenous FOXP3-expression in malignant epidermoid cells might present a novel mechanism of immune escape.

Metalloprotease-mediated tumor cell shedding of B7-H6, the ligand of the natural killer cell-activating receptor NKp30.

Natural killer (NK) cells are potent immune effector cells capable of mediating antitumor responses. Thus, during immunoediting, tumor cell populations evolve strategies to escape NK-cell-mediated recognition. In this study, we report a novel mechanism of immune escape involving tumor cell shedding of B7-H6, a ligand for the activating receptor NKp30 that mediates NK-cell binding and NK-cell-mediated killing. Tumor cells from different cancer entities released B7-H6 by ectodomain shedding mediated by the cell surface proteases "a disintegrin and metalloproteases" (ADAM)-10 and ADAM-17, as demonstrated through the use of pharmacologic inhibitors or siRNA-mediated gene attenuation. Inhibiting this proteolytic shedding process increased the levels of B7-H6 expressed on the surface of tumor cells, enhancing NKp30-mediated activation of NK cells. Notably, we documented elevated levels of soluble B7-H6 levels in blood sera obtained from a subset of patients with malignant melanoma, compared with healthy control individuals, along with evidence of elevated B7-H6 expression in melanoma specimens in situ. Taken together, our results illustrated a novel mechanism of immune escape in which tumor cells impede NK-mediated recognition by metalloprotease-mediated shedding of B7-H6. One implication of our findings is that therapeutic inhibition of specific metalloproteases may help support NK-cell-based cancer therapy.

API5 confers tumoral immune escape through FGF2-dependent cell survival pathway.

Identifying immune escape mechanisms used by tumors may define strategies to sensitize them to immunotherapies to which they are otherwise resistant. In this study, we show that the antiapoptotic gene API5 acts as an immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specific T cells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immune sensitivity. Conversely, introducing API5 into API5(low) cells conferred immune resistance. Mechanistic investigations revealed that API5 mediated resistance by upregulating FGF2 signaling through a FGFR1/PKCδ/ERK effector pathway that triggered degradation of the proapoptotic molecule BIM. Blockade of FGF2, PKCδ, or ERK phenocopied the effect of API5 silencing in tumor cells expressing high levels of API5 to either murine or human antigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited to potentiate the efficacy of targeted active immunotherapies.

Abstract 4844: A novel mechanism of immune escape in cancer and its regulation by nitric oxide

Abstract One key to malignant progression is the acquired ability of tumor cells to escape immune-mediated lysis. Whereas tumor hypoxia is known to play a causal role in cancer metastasis and resistance to therapy, the link between hypoxia and immune escape in cancer remains poorly understood. Previously, we showed that hypoxia induces resistance to natural killer (NK) cell-mediated lysis in tumour cells through a mechanism that involves the shedding of the NK cell-activating ligand, MHC Class I Chain Related molecule A (MICA), from the cell surface. Additionally, we reported that activation of NO signalling in the tumour cells was able to block this hypoxia-induced shedding of MICA, as well as resistance to NK-mediated lysis. Here, we show that hypoxia-induced tumor resistance to lysis is mediated by a HIF-1-dependent increased expression of the metalloproteinase ADAM10, which is required for the hypoxia-induced shedding of MICA. Our findings demonstrate that HIF-1α knock down attenuated the hypoxia-induced expression of ADAM10 and MICA in tumour cells. Also, siRNA-mediated knockdown of HIF-1α or ADAM10 prevented the hypoxia-induced resistance of DU145 cells to innate immune effector cell-mediated lysis. Treatment with nitric oxide mimetic agents interfered with the hypoxia-induced accumulation of HIF-1α, and with the hypoxia-induced up-regulation of ADAM10 expression required for decreased surface MICA expression and resistance to lysis. To validate the in vitro results, we adopted a model in which human DU145 cells were injected subcutaneously into Swiss nude mice (T and B cell-deficient). Continuous transdermal delivery of nitroglycerine (1.8 µg/h for up to 62 days) significantly attenuated attenuated the tumor growth (P &amp;lt; 0.05). This tumor growth-inhibitory effect of nitroglycerine was absent in mice depleted of innate immune effector cells. Within the tumours, areas of hypoxia co-localized with the metalloproteinase ADAM10 expression. Our results demonstrate a novel mechanism by which hypoxia contributes to immune escape in tumor cells and provide evidence that activation of nitric oxide signalling interferes with this mechanism. The findings described here are important because they indicate that nitric oxide mimetics could potentially be used as immunosensitizers in the treatment and/or prevention of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4844. doi:1538-7445.AM2012-4844

Tumor-Derived Chemokine CCL5 Enhances TGF-β–Mediated Killing of CD8+ T Cells in Colon Cancer by T-Regulatory Cells

Chemokine CCL5/RANTES is highly expressed in cancer where it contributes to inflammation and malignant progression. In this study, we show that CCL5 plays a critical role in immune escape in colorectal cancer. We found that higher levels of CCL5 expression in human and murine colon tumor cells correlated with higher levels of apoptosis of CD8+ T cells and infiltration of T-regulatory cells (T(reg)). In mouse cells, RNA interference (RNAi)-mediated knockdown of CCL5 delayed tumor growth in immunocompetent syngeneic hosts but had no effect on tumor growth in immunodeficient hosts. Reduced tumor growth was correlated with a reduction in T(reg) infiltration and CD8(+) T-cell apoptosis in tumors. Notably, we found that CCL5 enhanced the cytotoxicity of T(reg) against CD8(+) T cells. We also found tumor growth to be diminished in mice lacking CCR5, a CCL5 receptor, where a similar decrease in both T(reg) cell infiltration and CD8(+) T-cell apoptosis was noted. TGF-β signaling blockade diminished apoptosis of CD8(+) T cells, implicating TGF-β as an effector of CCL5 action. In support of this concept, CCL5 failed to enhance the production of TGF-β by CCR5-deficient T(reg) or to enhance their cytotoxic effects against CD8(+) T cells. CCR5 signaling blockade also diminished the in vivo suppressive capacity of T(reg) in inhibiting the antitumor responses of CD8(+) T cells, in the same way as CCL5 signaling blockade. Together, our findings establish that CCL5/CCR5 signaling recruits T(reg) to tumors and enhances their ability to kill antitumor CD8(+) T cells, thereby defining a novel mechanism of immune escape in colorectal cancer.

Tumor-associated macrophages suffocate NKT cells: a novel tumor escape mechanism and a target for therapy (48.7)

Abstract Vα24-invariant Natural Killer T cells (NKTs) are important for antitumor immunity and immunotherapy. We have demonstrated that NKTs inhibit tumor growth indirectly via targeting tumor-associated monocytes/macrophages (TAMs) in neuroblastoma (NB), suggesting that tumor progression requires an escape from NKTs via yet unknown mechanism. Here, we demonstrate that NB cells via membrane-bound TNFα induce CCL20 production in monocytes and this effect is amplified in hypoxia. Anti-CCL20 neutralizing mAb strongly inhibited NKT-cell in vitro migration towards tumor-conditioned hypoxic monocytes and in vivo localization in NB/TAM xenografts in NOD/SCID mice. The immunofluorescent staining of primary NB tumors revealed selective CCL20 accumulation in TAMs. We also found that hypoxia impairs NKT-cell function, suggesting that NKT-cell trafficking toward CCL20-producing TAMs may serve as a hypoxic trap for tumor-infiltrating NKTs and other T cells. IL-15 protected NKTs from hypoxia in vitro and IL-15-transduced NKTs more efficiently inhibited tumor growth in metastatic NB model in humanized NOD/SCID/IL2rgamma(null) mice compared with parental NKTs. Thus, CCL20 production in hypoxic TAMs and consequent inhibition of NKTs reveals what we believe to be a novel mechanism of immune escape that can be reversed by adoptive immunotherapy with IL-15-transduced NKTs.

Phenotypic and Functional Features of Vγ9/Vδ2 T Cells in Chronic Lymphocytic Leukemia Are Strongly Correlated with the Mutational Status of the Immunoglobulin Heavy Chain Variable Region (IgVH) and Metabolic Activity of the Mevalonate Pathway in Tumor Cells.

Vγ9/Vδ2 (γδ) T cells have an important effector and regulatory role in innate and adaptive immunity against microbes, stressed cells and tumor cells. They represent less than 5% of peripheral blood lymphocytes, but can be activated and expanded in vitro by aminobisphosphonates (ABP) like zoledronic acid (Zol), as surrogates of their natural ligands. In this study, we have investigated the proliferative response of γδ T cells to Zol in 59 patients with chronic lymphocytic leukemia (CLL) at diagnosis. Proliferation of γδ T cells was observed in 30 patients (51%)(responders, R), whereas 29 patients (49%) were non-responders (NR). γδ T-cell subset distribution [e.g., naive (TN), central memory (TCM), effector memory (TEM), and terminally differentiated effector memory (TEMRA)] was well balanced in R patients, whereas TEM and TEMRA were largely predominant in NR patients. TEMRA of NR patients had a unique profile of NK receptors expression, characterized by the expression of the ILT4 inhibitory receptor, whereas R patients tended to have an higher expression of the costimulatory molecule NKG2D. Notably, HLA-G, a negative prognosticator in CLL and a well known ILT4 ligand, was more expressed in tumor cells of NR patients. This correlation prompted us to determine whether other tumor cell features were different in R and NR patients. Indeed, the latter showed significantly higher tumor cell counts, higher frequency of poor-risk cytogenetic abnormalities, and a significantly shorter median time to first treatment. Lastly, a very significant association was observed with the mutational status of the tumor immunoglobulin heavy chain variable region (IgVH), which was mutated (M) in 96% of R patients, and unmutated (UM) in 74% of NR patients. To gain further insight into this association, we evaluated the activity of the mevalonate pathway in tumor cells of M and UM patients. This pathway, that can be targeted by Zol, generates the phosphoantigens naturally recognized by γδ T cells. Bioinformatic and biochemical approaches showed that this pathway is more active in tumor cells of UM than M patients. Based on these data, we propose that tumor cells of UM CLL patients can more easily engage γδ T cells and, in the long run, exhaust their immune surveillance potential by driving their differentiation into functionally impaired TEMRA. In conclusion, we have identified a novel mechanism of immune escape which can contribute to the disease progression in UM CLL patients.