Multiple Myeloma-Derived Extracellular Vesicles Modulate the Bone Marrow Immune Microenvironment.

Multiple myeloma (MM), the third most frequent hematological cancer worldwide, is characterized by the proliferation of neoplastic plasma cells in the bone marrow (BM). One of the hallmarks of MM is a permissive BM microenvironment. Increasing evidence suggests that cell-to-cell communication between myeloma and immune cells via tumor cell-derived extracellular vesicles (EV) plays a key role in the pathogenesis of MM. Hence, we aimed to explore BM immune alterations induced by MM-derived EV. For this, we inoculated immunocompetent BALB/cByJ mice with a myeloma cell line, MOPC315.BM, inducing a MM phenotype. Upon tumor establishment, characterization of the BM microenvironment revealed the expression of both activation and suppressive markers by lymphocytes, such as granzyme B and PD-1, respectively. In addition, conditioning of the animals with MOPC315.BM-derived EV, before transplantation of the MOPC315.BM tumor cells, did not anticipate the disease phenotype. However, it induced features of suppression in the BM milieu, such as an increase in PD-1 expression by CD4+ T cells. Overall, our findings reveal the involvement of MOPC315.BM-derived EV protein content as promoters of immune niche remodeling, strengthening the importance of assessing the mechanisms by which MM may impact the immune microenvironment.

Bringing Macrophages to the Frontline against Cancer: Current Immunotherapies Targeting Macrophages.

Macrophages are found in all tissues and display outstanding functional diversity. From embryo to birth and throughout adult life, they play critical roles in development, homeostasis, tissue repair, immunity, and, importantly, in the control of cancer growth. In this review, we will briefly detail the multi-functional, protumoral, and antitumoral roles of macrophages in the tumor microenvironment. Our objective is to focus on the ever-growing therapeutic opportunities, with promising preclinical and clinical results developed in recent years, to modulate the contribution of macrophages in oncologic diseases. While the majority of cancer immunotherapies target T cells, we believe that macrophages have a promising therapeutic potential as tumoricidal effectors and in mobilizing their surroundings towards antitumor immunity to efficiently limit cancer progression.

Chitosan/γ-PGA nanoparticles-based immunotherapy as adjuvant to radiotherapy in breast cancer.

Radiotherapy (RT) is an essential treatment modality for several types of cancer. Despite its therapeutic potential, RT is frequently insufficient to overcome the immunosuppressive nature of the tumor microenvironment, failing to control tumor metastases. Innovative immunomodulatory strategies, like immunostimulatory biomaterials could be used to boost the immunogenic effects of RT. Herein, we addressed the synergistic potential of immunostimulatory chitosan/poly(γ-glutamic acid) nanoparticles (Ch/γ-PGA NPs) combined with RT to induce antitumor immunity in the 4T1 orthotopic breast tumor mouse model. Non-treated animals had progressive primary tumor growth and developed splenomegaly and lung metastases. While RT decreased primary tumor burden, Ch/γ-PGA NPs-treatment decreased systemic immunosuppression and lung metastases. The combination therapy (RT + Ch/γ-PGA NPs) synergistically impaired 4T1 tumor progression, which was associated with a significant primary tumor growth and splenomegaly reduction, a decrease in the percentage of splenic immunosuppressive myeloid cells and an increase in antitumoral CD4+IFN-γ+ population. Notably, animals from the combination therapy presented less and smaller lung metastatic foci and lower levels of the systemic pro-tumor cytokines IL-3, IL-4, IL-10, and of the CCL4 chemokine, in comparison to non-treated animals. Overall, these results evidenced that Ch/γ-PGA NPs potentiate and synergize with RT, headlining their promising role as adjuvant anticancer strategies.

Interactions among myeloid regulatory cells in cancer.

Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells.

Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion.

Interferon-gamma (IFN-γ) is a pleiotropic molecule with associated antiproliferative, pro-apoptotic and antitumor mechanisms. This effector cytokine, often considered as a major effector of immunity, has been used in the treatment of several diseases, despite its adverse effects. Although broad evidence implicating IFN-γ in tumor immune surveillance, IFN-γ-based therapies undergoing clinical trials have been of limited success. In fact, recent reports suggested that it may also play a protumorigenic role, namely, through IFN-γ signaling insensitivity, downregulation of major histocompatibility complexes, and upregulation of indoleamine 2,3-dioxygenase and of checkpoint inhibitors, as programmed cell-death ligand 1. However, the IFN-γ-mediated responses are still positively associated with patient's survival in several cancers. Consequently, major research efforts are required to understand the immune contexture in which IFN-γ induces its intricate and highly regulated effects in the tumor microenvironment. This review discusses the current knowledge on the pro- and antitumorigenic effects of IFN-γ as part of the complex immune response to cancer, highlighting the relevance to identify IFN-γ responsive patients for the improvement of therapies that exploit associated signaling pathways.

Tumor-associated neutrophils suppress pro-tumoral IL-17+ γδ T cells through induction of oxidative stress.

Interleukin 17 (IL-17)-producing γδ T cells (γδ17 T cells) have been recently found to promote tumor growth and metastasis formation. How such γδ17 T-cell responses may be regulated in the tumor microenvironment remains, however, largely unknown. Here, we report that tumor-associated neutrophils can display an overt antitumor role by strongly suppressing γδ17 T cells. Tumor-associated neutrophils inhibited the proliferation of murine CD27- Vγ6+ γδ17 T cells via induction of oxidative stress, thereby preventing them from constituting the major source of pro-tumoral IL-17 in the tumor microenvironment. Mechanistically, we found that low expression of the antioxidant glutathione in CD27- γδ17 T cells renders them particularly susceptible to neutrophil-derived reactive oxygen species (ROS). Consistently, superoxide deficiency, or the administration of a glutathione precursor, rescued CD27- Vγ6+ γδ17 T-cell proliferation in vivo. Moreover, human Vδ1+ γδ T cells, which contain most γδ17 T cells found in cancer patients, also displayed low glutathione levels and were potently inhibited by ROS. This work thus identifies an unanticipated, immunosuppressive yet antitumoral, neutrophil/ROS/γδ17 T-cell axis in the tumor microenvironment.

Primary Tumors Limit Metastasis Formation through Induction of IL15-Mediated Cross-Talk between Patrolling Monocytes and NK Cells.

Metastases are responsible for the vast majority of cancer-related deaths. Although tumor cells can become invasive early during cancer progression, metastases formation typically occurs as a late event. How the immune response to primary tumors may dictate this outcome remains poorly understood, which hampers our capacity to manipulate it therapeutically. Here, we used a two-step experimental model, based on the highly aggressive B16F10 melanoma, that temporally segregates the establishment of primary tumors (subcutaneously) and the formation of lung metastases (from intravenous injection). This allowed us to identify a protective innate immune response induced by primary tumors that inhibits experimental metastasis. We found that in the presence of primary tumors, increased numbers of natural killer (NK) cells with enhanced IFNγ, granzyme B, and perforin production were recruited to the lung upon metastasis induction. These changes were mirrored by a local accumulation of patrolling monocytes and macrophages with high expression of MHC class II and NOS2. Critically, the protective effect on metastasis was lost upon patrolling monocyte or NK cell depletion, IL15 neutralization, or IFNγ ablation. The combined analysis of these approaches allowed us to establish a hierarchy in which patrolling monocytes, making IL15 in response to primary tumors, activate NK cells and IFNγ production that then inhibit lung metastasis formation. This work identifies an innate cell network and the molecular determinants responsible for "metastasis immunosurveillance," providing support for using the key molecular mediator, IL15, to improve immunotherapeutic outcomes. Cancer Immunol Res; 5(9); 812-20. ©2017 AACR.

Low-dose ionizing radiation induces therapeutic neovascularization in a pre-clinical model of hindlimb ischemia.

AIMS: We have previously shown that low-dose ionizing radiation (LDIR) induces angiogenesis but there is no evidence that it induces neovascularization in the setting of peripheral arterial disease. Here, we investigated the use of LDIR as an innovative and non-invasive strategy to stimulate therapeutic neovascularization using a model of experimentally induced hindlimb ischemia (HLI). METHODS AND RESULTS: After surgical induction of unilateral HLI, both hindlimbs of female C57BL/6 mice were sham-irradiated or irradiated with four daily fractions of 0.3 Gy, in consecutive days and allowed to recover. We demonstrate that LDIR, significantly improved blood perfusion in the murine ischemic limb by stimulating neovascularization, as assessed by laser Doppler flow, capillary density, and collateral vessel formation. LDIR significantly increased the circulating levels of VEGF, PlGF, and G-CSF, as well as the number of circulating endothelial progenitor cells (EPCs) mediating their incorporation to ischemic muscles. These effects were dependent upon LDIR exposition on the ischemic niche (thigh and shank regions). In irradiated ischemic muscles, these effects were independent of the recruitment of monocytes and macrophages. Importantly, LDIR induced a durable and simultaneous up-regulation of a repertoire of pro-angiogenic factors and their receptors in endothelial cells (ECs), as evident in ECs isolated from the irradiated gastrocnemius muscles by laser capture microdissection. This specific mechanism was mediated via vascular endothelial growth factor (VEGF) receptor signaling, since VEGF receptor inhibition abrogated the LDIR-mediated gene up-regulation and impeded the increase in capillary density. Finally, the vasculature in an irradiated non-ischemic bed was not affected and after 52 week of LDIR exposure no differences in the incidence of morbidity and mortality were seen. CONCLUSIONS: These findings disclose an innovative, non-invasive strategy to induce therapeutic neovascularization in a mouse model of HLI, emerging as a novel approach in the treatment of critical limb ischemia patients.

Developmental and Functional Assays to Study Murine and Human γδ T Cells.

The key roles played by gamma-delta (γδ) T cells in immunity to infection and tumors critically depend on their differentiation into effectors capable of secreting cytokines (such as interferon-γ or interleukin-17), and killing infected or transformed cells. Here we detail the main methods used to investigate the differentiation of γδ T cells from murine or human origin. We describe developmental assays, such as thymic organ cultures (TOCs) and coculture of progenitors cells with OP9-DL1 stomal cells, as well as functional assays typically employed to evaluate γδ T cell cytotoxicity and cytokine production.

Effector γδ T Cell Differentiation Relies on Master but Not Auxiliary Th Cell Transcription Factors.

γδ T lymphocytes are programmed into distinct IFN-γ-producing CD27(+) (γδ27(+)) and IL-17-producing CD27(-) (γδ27(-)) subsets that play key roles in protective or pathogenic immune responses. Although the signature cytokines are shared with their αß Th1 (for γδ27(+)) and Th17 (for γδ27(-)) cell counterparts, we dissect in this study similarities and differences in the transcriptional requirements of murine effector γδ27(+), γδ27(-)CCR6(-), and γδ27(-)CCR6(+) γδ T cell subsets and αß T cells. We found they share dependence on the master transcription factors T-bet and RORγt for IFN-γ and IL-17 production, respectively. However, Eomes is fully dispensable for IFN-γ production by γδ T cells. Furthermore, the Th17 cell auxiliary transcription factors RORα and BATF are not required for IL-17 production by γδ27(-) cell subsets. We also show that γδ27(-) (but not γδ27(+)) cells become polyfunctional upon IL-1ß plus IL-23 stimulation, cosecreting IL-17A, IL-17F, IL-22, GM-CSF, and IFN-γ. Collectively, our in vitro and in vivo data firmly establish the molecular segregation between γδ27(+) and γδ27(-) T cell subsets and provide novel insight on the nonoverlapping transcriptional networks that control the differentiation of effector γδ versus αß T cell subsets.

γδ T cells in cancer.

With the promise of T cell-based therapy for cancer finally becoming reality, this Review focuses on the less-studied γδ T cell lineage and its diverse responses to tumours. γδ T cells have well-established protective roles in cancer, largely on the basis of their potent cytotoxicity and interferon-γ production. Besides this, recent studies have revealed a series of tumour-promoting functions that are linked to interleukin-17-producing γδ T cells. Here, we integrate the current knowledge from both human and mouse studies to highlight the potential of γδ T cell modulation to improve cancer immunotherapy.

Molecular Mechanisms of Differentiation of Murine Pro-Inflammatory γδ T Cell Subsets.

γδ T cells are unconventional innate-like lymphocytes that actively participate in protective immunity against tumors and infectious organisms including bacteria, viruses, and parasites. However, γδ T cells are also involved in the development of inflammatory and autoimmune diseases. γδ T cells are functionally characterized by very rapid production of pro-inflammatory cytokines, while also impacting on (slower but long-lasting) adaptive immune responses. This makes it crucial to understand the molecular mechanisms that regulate γδ T cell effector functions. Although they share many similarities with αß T cells, our knowledge of the molecular pathways that control effector functions in γδ T cells still lags significantly behind. In this review, we focus on the segregation of interferon-γ versus interleukin-17 production in murine thymic-derived γδ T cell subsets defined by CD27 and CCR6 expression levels. We summarize the most recent studies that disclose the specific epigenetic and transcriptional mechanisms that govern the stability or plasticity of discrete pro-inflammatory γδ T cell subsets, whose manipulation may be valuable for regulating (auto)immune responses.

Epigenetic and transcriptional signatures of stable versus plastic differentiation of proinflammatory γδ T cell subsets.

Two distinct subsets of γδ T cells that produce interleukin 17 (IL-17) (CD27(-) γδ T cells) or interferon-γ (IFN-γ) (CD27(+) γδ T cells) develop in the mouse thymus, but the molecular determinants of their functional potential in the periphery remain unknown. Here we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory networks of peripheral IFN-γ-producing or IL-17-producing γδ T cell subsets in vivo. We found that CD27(+) γδ T cells were committed to the expression of Ifng but not Il17, whereas CD27(-) γδ T cells displayed permissive chromatin configurations at loci encoding both cytokines and their regulatory transcription factors and differentiated into cells that produced both IL-17 and IFN-γ in a tumor microenvironment.

Selective effects of NF-κB1 deficiency in CD4⁺ T cells on Th2 and TFh induction by alum-precipitated protein vaccines.

NF-κB1-dependent signaling directs the development of CD4(+) Th2 cells during allergic airway inflammation and protective responses to helminth infection. Here, we show that IL-4 and IL-13 production is NF-κB1-dependent in mouse OVA-specific CD4(+) (OTII) T cells responding to alum-precipitated OVA (alumOVA) immunization. More surprisingly, we found that NF-κB1 deficiency in OTII cells also selectively impairs their CXCR5 induction by alumOVA without affecting upregulation of BCL6, IL-21, OX40 and CXCR4 mRNA and PD-1 protein. This results in functional impairment of follicular helper T cells. Thus, fewer germinal center B cells develop in LN responses to alumOVA in T-cell-deficient mice reconstituted with NF-κB1(-/-) OTII cells as opposed to NF-κB1(+/+) OTII cells, while plasma cell numbers are comparable. Unlike CXCR5 induction in CD4(+) T cells, NF-κB1-deficient recirculating follicular B cells are shown to express normal levels of CXCR5. The selective effects of NF-κB1-deficiency on Th2 and follicular helper T cell induction do not appear to be due to altered expression of the Th2-associated transcription factors - GATA-3, c-Maf and Ikaros. Altogether, these results suggest that NF-κB1 regulates the expression of CXCR5 on CD4(+) T cells primed in vivo, and thus selectively controls the T-cell-dependent germinal center component of B-cell response to alumOVA.

Ontogeny of stromal organizer cells during lymph node development.

The development of secondary lymphoid organs, such as lymph nodes (LNs), in the embryo results from the reciprocal action between lymphoid tissue inducer (LTi) cells and stromal cells. However, the initial events inducing LN anlagen formation before the LTi stromal cells cross-talk interactions take place are not fully elucidated. In this study, we show that the inguinal LN anlagen in mouse embryos developed from mesenchymal cells surrounding the lymph sacs, spherical structures of endothelial cells that bud from veins. Using inguinal and mesenteric LNs (mLNs), we provide evidence supporting a two-step maturation model for stromal cells: first, ICAM-1(-)VCAM-1(-) mesenchymal precursor cells become ICAM-1(int)VCAM-1(int) cells, in a process independent of LTi cells and lymphotoxin beta receptor (LTbetaR) signaling. The second step involves the maturation of ICAM-1(int)VCAM-1(int) cells to ICAM-1(high)VCAM-1(high) mucosal addressin cell adhesion molecule-1(+) organizer cells and depends on both LTi cells and LTbetaR. Addition of alphaLTbetaR agonist to LN organ cultures was sufficient to induce ICAM-1(int)VCAM-1(int) cells to mature. In LtbetaR(-/-) embryos, both inguinal and mLN stromal cells showed a block at the ICAM-1(int)VCAM-1(int) stage, and, contrary to inguinal LNs, mLNs persist longer and contained LTi cells, which correlated with the sustained gene expression of Il-7, Cxcl13, and, to a lesser degree, Ccl21. Taken together, these results highlight the importance of the signals and cellular interactions that induce the maturation of stromal cells and ultimately lead to the formation of lymphoid tissues.

Early simultaneous production of intranodal CD4 Th2 effectors and recirculating rapidly responding central-memory-like CD4 T cells.

This study characterizes the diversity of CD4 Th cells produced during a Th2 response in vivo. Kinetics of effector and memory cell differentiation by mouse OVA-specific CD4 T cells was followed during primary responses to alum-precipitated OVA. The complexity of the CD4 T response was assessed in nodes draining and distant from the site of immunization for phenotype, location and function. By 3 days IL-4-producing effector CD4 T cells developed in the draining node and a proportion of the responding cells had migrated to B-cell follicles, while yet others had left the draining node. Some of these early migrant cells were recirculating cells with a central memory phenotype. These had divided four or more times in the draining node before migrating to distant nodes not exposed to antigen. We questioned the responsiveness of these early central-memory-like cells on secondary antigen challenge at sites distant to the primary immunization. They re-entered cell cycle and migrated to B-cell follicles, much more rapidly than naive CD4 T cells and could still be induced to produce IL-4. Their production and survival were independent of the starting frequency of antigen-specific CD4 T cells. Thus intranodal effector cells and recirculating, rapidly responding central-memory-like cells emerged simultaneously from the third day of a primary Th2 response.

Dendritic cells and monocyte/macrophages that create the IL-6/APRIL-rich lymph node microenvironments where plasmablasts mature.

IL-6 and APRIL influence the growth, differentiation, and survival of normal and neoplastic Ab-forming cells (AFC). In this study, we identify two subsets of myeloid cells that associate with the AFC and are the main producers of these factors during a T-dependent Ab response to alum-precipitated protein in mouse lymph nodes. First CD11c(+)CD8alpha(-) dendritic cells located in the perivascular area of the T zone provide about half of the IL-6 mRNA produced in the node together with significant amounts of APRIL mRNA. The number of these cells increases during the response, at least in part due to local proliferation. The second subset comprises Gr1(+)CD11b(+)F4/80(+) monocyte/macrophages. These colonize the medullary cords during the response and are the other main IL-6 mRNA producers and the greatest source of APRIL mRNA. This medullary cord monocyte/macrophage subset results in local increase of APRIL mRNA that mirrors the polarity of CXCL12 expression in the node. The distribution of these myeloid cell subsets correlates with a gradient of AFC maturation assessed by progressive loss of Ki67 as AFC pass from the B cell follicle along the perivascular areas to the medullary cords.

Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells.

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.

Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection.

T-dependent Ab responses are characterized by parallel extrafollicular plasmablast growth and germinal center (GC) formation. This study identifies that, in mice, the Ab response against Salmonella is novel in its kinetics and its regulation. It demonstrates that viable, attenuated Salmonella induce a massive early T-dependent extrafollicular response, whereas GC formation is delayed until 1 mo after infection. The extrafollicular Ab response with switching to IgG2c, the IgG2a equivalent in C57BL/6 mice, is well established by day 3 and persists through 5 wk. Switching is strongly T dependent, and the outer membrane proteins are shown to be major targets of the early switched IgG2c response, whereas flagellin and LPS are not. GC responses are associated with affinity maturation of IgG2c, and their induction is associated with bacterial burden because GC could be induced earlier by treating with antibiotics. Clearance of these bacteria is not a consequence of high-affinity Ab production, for clearance occurs equally in CD154-deficient mice, which do not develop GC, and wild-type mice. Nevertheless, transferred low- and high-affinity IgG2c and less efficiently IgM were shown to impede Salmonella colonization of splenic macrophages. Furthermore, Ab induced during the infection markedly reduces bacteremia. Thus, although Ab does not prevent the progress of established splenic infection, it can prevent primary infection and impedes secondary hemogenous spread of the disease. These results may explain why attenuated Salmonella-induced B cell responses are protective in secondary, but not primary infections.

Loss of CD154 impairs the Th2 extrafollicular plasma cell response but not early T cell proliferation and interleukin-4 induction.

Ligation of CD40 by CD4 T cells through CD154 is key both to germinal centre induction and follicular T-dependent Ig class switching, but its requirement for aspects of T cell priming and extrafollicular antibody responses is less clear. Here comparison of the T helper (Th) type 2 response in lymph nodes from wild-type mice and CD154-deficient mice after immunization with alum-precipitated antigen reveals selective effects of this immunodeficiency. The timing and magnitude of the early interleukin (IL)-4 induction and proliferation in T cells of the T zone were unaltered by CD154 deficiency. As expected, germinal centres were not induced. Additionally the T-dependent extrafollicular antibody response, which initially requires T cell help but expands without further T cell involvement, was severely curtailed. The median number of extrafollicular antigen-specific plasma cells was 370-fold lower in CD154-deficient mice. Of these plasma cells the median proportion that had switched to IgG1 was <5%, while in wild-type mice the proportion was 89%. Surprisingly, some CD154-deficient lymph nodes showed substantial switching to IgG1. Commensurately, increases in gamma1 germline transcripts and Blimp-1 mRNA were observed, albeit significantly lower than in controls, but activation-induced cytidine deaminase mRNA was undetectable in CD154-deficient mice. These experiments demonstrate that the acquisition of some T cell priming characteristics can be CD154-independent; in contrast, T-dependent extrafollicular responses require CD154. Thus functional CD154 ligation during the first encounter of T cells and B cells in the T zone is critical for follicular and extrafollicular antibody responses.