A Bacterial and Ganglioside-based Nanoparticle Initiates Reprogramming of Macrophages and Promotes Antitumor Phenotypes.

Macrophages represent the most abundant immune component of the tumor microenvironment and often exhibit protumorigenic (M2-like) phenotypes that contribute to disease progression. Despite their generally accepted protumorigenic role, macrophages can also display tumoricidal (or M1-like) behavior, revealing that macrophages can be functionally reprogrammed, depending on the cues received within the tumor microenvironment. Moreover, such plasticity may be achieved by pharmacologic or biologic interventions. To that end, we previously demonstrated that a novel immunomodulator termed the "very small size particle" (VSSP) facilitates maturation of dendritic cells and differentiation of myeloid-derived suppressor cells to APCs with reduced suppressive activity in cancer models. VSSP was further shown to act in the bone marrow to drive the differentiation of progenitors toward monocytes, macrophages, and dendritic cells during emergency myelopoiesis. However, the underlying mechanisms for VSSP-driven alterations in myeloid differentiation and function remained unclear. In this study, in mouse models, we focused on macrophages and tested the hypothesis that VSSP drives macrophages toward M1-like functional states via IRF8- and PU.1-dependent mechanisms. We further hypothesized that such VSSP-mediated actions would be accompanied by enhanced antitumor responses. Overall, we showed that (1) VSSP drives naive or M2-derived macrophages to M1-like states, (2) the M1-like state induced by VSSP occurs via IRF8- and PU.1-dependent mechanisms, and (3) single-agent VSSP induces an antitumor response that is accompanied by alterations in the intratumoral myeloid compartment. These results provide a deeper mechanistic underpinning of VSSP and strengthen its use to drive M1-like responses in host defense, including cancer.

VSSP-activated macrophages mediate senescence and tumor inhibition in a preclinical model of advanced prostate cancer.

Androgen deprivation therapy (ADT) is a standard therapy for prostate cancer (PCa). Though disseminated disease is initially sensitive to ADT, an important fraction of the patients progresses to castration-resistant prostate cancer (CRPC). For this reason, the identification of novel effective therapies for treating CRPC is needed. Immunotherapeutic strategies focused on macrophages as antitumor effectors, directly enhancing their tumoricidal potential at the tumor microenvironment or their adoptive transfer after ex vivo activation, have arisen as promising therapies in several cancer types. Despite several approaches centered on the activation of tumor-associated macrophages (TAMs) in PCa are under investigation, to date there is no evidence of clinical benefit in patients. In addition, the evidence of the effectiveness of macrophage adoptive transfer on PCa is poor. Here we find that VSSP, an immunomodulator of the myeloid system, decreases TAMs and inhibits prostatic tumor growth when administered to castrated Pten-deficient prostate tumor-bearing mice. In mice bearing castration-resistant Ptenpc-/-; Trp53pc-/- tumors, VSSP administration showed no effect. Nevertheless, adoptive transfer of macrophages activated ex vivo with VSSP inhibited Ptenpc-/-; Trp53pc-/- tumor growth through reduction of angiogenesis and tumor cell proliferation and induction of senescence. Taken together, our results highlight the rationale of exploiting macrophage functional programming as a promising strategy for CRPC therapy, with particular emphasis on ex vivo-activated proinflammatory macrophage adoptive transfer. Video abstract.

Mitigating the prevalence and function of myeloid-derived suppressor cells by redirecting myeloid differentiation using a novel immune modulator.

BACKGROUND: Immune suppression is common in neoplasia and a major driver is tumor-induced myeloid dysfunction. Yet, overcoming such myeloid cell defects remains an untapped strategy to reverse suppression and improve host defense. Exposure of bone marrow progenitors to heightened levels of myeloid growth factors in cancer or following certain systemic treatments promote abnormal myelopoiesis characterized by the production of myeloid-derived suppressor cells (MDSCs) and a deficiency in antigen-presenting cell function. We previously showed that a novel immune modulator, termed 'very small size particle' (VSSP), attenuates MDSC function in tumor-bearing mice, which was accompanied by an increase in dendritic cells (DCs) suggesting that VSSP exhibits myeloid differentiating properties. Therefore, here, we addressed two unresolved aspects of the mechanism of action of this unique immunomodulatory agent: (1) does VSSP alter myelopoiesis in the bone marrow to redirect MDSC differentiation toward a monocyte/macrophage or DC fate? and (2) does VSSP mitigate the frequency and suppressive function of human tumor-induced MDSCs? METHODS: To address the first question, we first used a murine model of granulocyte-colony stimulating factor-driven emergency myelopoiesis following chemotherapy-induced myeloablation, which skews myeloid output toward MDSCs, especially the polymorphonuclear (PMN)-MDSC subset. Following VSSP treatment, progenitors and their myeloid progeny were analyzed by immunophenotyping and MDSC function was evaluated by suppression assays. To strengthen rigor, we validated our findings in tumor-bearing mouse models. To address the second question, we conducted a clinical trial in patients with metastatic renal cell carcinoma, wherein 15 patients were treated with VSSP. Endpoints in this study included safety and impact on PMN-MDSC frequency and function. RESULTS: We demonstrated that VSSP diminished PMN-MDSCs by shunting granulocyte-monocyte progenitor differentiation toward monocytes/macrophages and DCs with heightened expression of the myeloid-dependent transcription factors interferon regulatory factor-8 and PU.1. This skewing was at the expense of expansion of granulocytic progenitors and rendered the remaining MDSCs less suppressive. Importantly, these effects were also demonstrated in a clinical setting wherein VSSP monotherapy significantly reduced circulating PMN-MDSCs, and their suppressive function. CONCLUSIONS: Altogether, these data revealed VSSP as a novel regulator of myeloid biology that mitigates MDSCs in cancer patients and reinstates a more normal myeloid phenotype that potentially favors immune activation over immune suppression.

Augmenting antibody response to EGF-depleting immunotherapy: Findings from a phase I trial of CIMAvax-EGF in combination with nivolumab in advanced stage NSCLC.

Background: CIMAvax-EGF is an epidermal growth factor (EGF)-depleting immunotherapy which has shown survival benefit as a switch maintenance treatment after platinum-based chemotherapy in advanced non-small cell lung cancer (NSCLC). The primary objective of this trial is to establish the safety and recommended phase II dose (RP2D) of CIMAvax-EGF in combination with nivolumab as second-line therapy for NSCLC. Methods: Patients with immune checkpoint inhibitor-naive metastatic NSCLC were enrolled using a "3+3" dose-escalation design. Toxicities were graded according to CTCAE V4.03. Thirteen patients (one unevaluable), the majority with PD-L1 0%, were enrolled into two dose levels of CIMAvax-EGF. Findings: The combination was determined to be safe and tolerable. The recommended phase 2 dose of CIMAvax-EGF was 2.4 mg. Humoral response to CIMAvax-EGF was achieved earlier and in a greater number of patients with the combination compared to historical control. Four out of 12 evaluable patients had an objective response.

Murine Mammary Carcinoma Induces Chronic Systemic Inflammation and Immunosuppression in BALB/c Mice.

PURPOSE: The F3II cell line is a highly invasive variant of mammary carcinoma. Although it is frequently used as a model to evaluate the efficacy of immunotherapy, its impact on the immune system remains poorly understood. The main objectives of this study were to evaluate the effects of F3II tumors on the development of chronic inflammation and to characterize tumor-associated immunosuppression. METHODS: Following the experimental implantation of F3II tumors in BALB/c mice, alterations in the liver and spleen anatomy and the numbers of circulating leukocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells were measured using hematological techniques, histopathological analysis, and flow cytometry. The capacity of the F3II tumor-bearing mice to reject MB16F10 allogeneic tumor transplantation was also evaluated. In addition, the restoration of immune parameters in tumor-bearing mice was evaluated after standard breast cancer chemotherapy and surgical tumor excision. RESULTS: F3II tumor implantation increased the levels of chronic inflammatory markers, such as the neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios, and caused myeloid alterations, including extramedullary granulopoiesis and megakaryopoiesis, along with the recruitment of MDSCs to the spleen. Chemotherapy or surgical F3II tumor removal completely rescued the tumor-associated extramedullary granulopoiesis and megakaryopoiesis. Notably, the presence of F3II tumors reduced the capacity of BALB/c mice to reject MB16F10 allogeneic tumor transplantation. CONCLUSION: These results support the occurrence of F3II tumor-mediated immune cell dysfunction, which mimics the immune alterations characterized by chronic systemic inflammation and immunosuppression observed in breast cancer in clinical settings. Thus, the F3II tumor model is relevant for evaluating novel breast cancer immunotherapies and combinations in preclinical studies. This model could also be useful for identifying appropriate therapeutic targets and developing proof-of-concept experiments in the future.

VSSP abrogates murine ovarian tumor-associated myeloid cell-driven immune suppression and induces M1 polarization in tumor-associated macrophages from ovarian cancer patients.

The ovarian tumor microenvironment (TME) is characterized by the accumulation of immunosuppressive tumor-associated macrophages (TAMs) and granulocytic cells. Very small size particles (VSSP), comprised of the ganglioside NAcGM3 and Neisseria meningitidis derived outer membrane vesicles, is being developed as a nanoparticulated modulator of innate immunity. Prior studies have shown that VSSP enhanced antigen-specific cytotoxic T cell responses and reduced the suppressive phenotype of splenic granulocytic cells in tumor-bearing mice. Here, we hypothesized that intraperitoneal VSSP would modify myeloid cell accumulation and phenotypes in the ovarian TME and abrogate suppressor function of TAMs and tumor-associated granulocytic cells. In the ID8 syngeneic model of epithelial ovarian cancer, VSSP reduced peritoneal TAMs and induced M1-like polarization in TAMs. In addition, VSSP stimulated peritoneal inflammation characterized by increased granulocytes and monocytes, including inflammatory monocytic cells. VSSP treatment resulted in peritoneal TAMs and granulocytic cells being less suppressive of ex vivo stimulated CD8+ T cell responses. VSSP alone and combined with anti-PD-1 modestly but significantly prolonged survival in tumor-bearing mice. In addition, ex vivo treatment with VSSP induced M1-like polarization in TAMs from patients with metastatic ovarian cancer and variably abrogated their suppressor phenotype. VSSP treatment also partially abrogated the induction of suppressor function in healthy donor neutrophils exposed to ascites supernatants from patients with ovarian cancer. Together, these results point to VSSP reprogramming myeloid responses resulting in abrogation of suppressive pathways and raise the potential for administration of VSSP into the TME to enhance anti-tumor immunity.

Sticholysins, pore-forming proteins from a marine anemone can induce maturation of dendritic cells through a TLR4 dependent-pathway.

Sticholysins (Sts) I and II (StI and StII) are pore-forming proteins (PFPs), purified from the Caribbean Sea anemone Stichodactyla helianthus. StII encapsulated into liposomes induces a robust antigen-specific cytotoxic CD8+ T lymphocytes (CTL) response and in its free form the maturation of bone marrow-derived dendritic cells (BM-DCs). It is probable that the latter is partially supporting in part the immunomodulatory effect on the CTL response induced by StII-containing liposomes. In the present work, we demonstrate that the StII's ability of inducing maturation of BM-DCs is also shared by StI, an isoform of StII. Using heat-denatured Sts we observed a significant reduction in the up-regulation of maturation markers indicating that both PFP's ability to promote maturation of BM-DCs is dependent on their conformational characteristics. StII-mediated DC maturation was abrogated in BM-DCs from toll-like receptor (TLR) 4 and myeloid differentiation primary response gene 88 (MyD88)-knockout mice but not in cells from TLR2-knockout mice. Furthermore, the antigen-specific CTL response induced by StII-containing liposomes was reduced in TLR4-knockout mice. These results indicate that StII, and probably by extension StI, has the ability to induce maturation of DCs through a TLR4/MyD88-dependent pathway, and that this activation contributes to the CTL response generated by StII-containing liposomes.

A rationally-engineered IL-2 improves the antitumor effect of anti-CD20 therapy.

Anti-CD20 treatment represents a therapeutic benefit for patients with B-cell lymphomas, although more efficient therapies are needed for refractory or relapsing patients. Among them, the combination of anti-CD20 and IL-2 that induces T cell response has been hampered by the expansion of FoxP3+ Tregs that strongly express the high affinity IL-2 receptor (IL-2R αßγ). We explore here the anti-tumor effect of an anti-CD20 antibody combined with a mutated IL-2 (no-alpha mutein) which has a disrupted affinity for the IL-2R αßγ. We demonstrate that anti-CD20/no-alpha mutein combination significantly augments the survival rate of mice challenged with huCD20+ cells as compared to animals treated with anti-CD20 ± IL-2. Moreover, the combination with no-alpha mutein but not IL-2 provokes an increase of granzyme B and perforin in splenic NK and CD8+ T cells, a reduction of Tregs and an increase in activated macrophages. The former combination also induces a T helper profile different from that obtained with IL-2, with an earlier polarization to Th1 and no increase in Th17. The therapeutic effect of anti-CD20/no-alpha mutein was accompanied by an expansion of peripheral central (TCM) and effector (TEM) memory CD8+ T cell compartments. Last, as opposed to IL-2, no-alpha mutein administered at the beginning of anti-CD20 treatment did not dampen the long-term protection of surviving mice after tumor rechallenge. Thus, this study shows that the combination of anti-tumor antibodies and no-alpha mutein is a promising approach to improve the therapeutic effect of these antibodies by potentiating NK/macrophage-mediated innate immunity and the adaptive T-cell response.

Antitumor effects of the GM3(Neu5Gc) ganglioside-specific humanized antibody 14F7hT against Cmah-transfected cancer cells.

The GM3(Neu5Gc) ganglioside represents a tumor-specific antigen that is considered a promising target for cancer immunotherapy. We previously demonstrated that the humanized antibody 14F7hT, specific for this ganglioside, exhibited significant antitumor effects in preclinical hematological tumor models. As this antibody recognizes human tumor tissues from several origins, we addressed its potential effect on different tumor types. The use of cell lines for testing GM3(Neu5Gc)-targeting strategies, in particular for human malignancies, is complicated by the absence in humans of functional cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMAH), the enzyme required for Neu5Gc sialic acid biosynthesis. Quantitative flow cytometry revealed the absence of surface GM3(Neu5Gc) in several human but also mouse cell lines, in the last case due to low expression of the enzyme. Hypoxia-induced expression of this ganglioside on human SKOV3 cells was observed upon culture in Neu5Gc-containing medium without evidence for CMAH-independent biosynthesis. However, only transfection of the mouse Cmah gene into human SKOV3 and mouse 3LL cells induced a stable expression of GM3(Neu5Gc) on the cancer cell surface, resulting in effective models to evaluate the antitumor responses by 14F7hT in vitro and in vivo. This antibody exerted antibody-dependent cell-mediated cytotoxicity (ADCC) and in vivo antitumor effects on these Cmah-transfected non-hematological tumors from both mouse and human origin. These results contribute to validate GM3(Neu5Gc) as a relevant target for cancer immunotherapy and reinforces the value of 14F7hT as a novel anti-cancer drug.

Cancer-induced systemic myeloid dysfunction: Implications for treatment and a novel nanoparticle approach for its correction.

Unlike other regulatory circuits, cancer-induced myeloid dysfunction involves more than an accumulation of impaired dendritic cells, protumoral macrophages, and myeloid derived suppressor cells in the tumor microenvironment. It is also characterized by "aberrant" myelopoiesis that results in the accumulation and expansion of immature myeloid precursors with a suppressive phenotype in the systemic circulation. The first part of this review briefly describes the evidence for and consequences of this systemic dysfunctional myelopoiesis and the possible reinforcement of this phenomenon by conventional treatments used in patients with cancer, in particular chemotherapy and granulocyte-colony stimulating factor. The second half of this review describes very small size particles, a novel immune-modulatory nanoparticle, and the evidence indicating a possible role of this agent in correcting or re-programming the dysfunctional myelopoiesis in different scenarios.

Novel Adjuvant Based on the Pore-Forming Protein Sticholysin II Encapsulated into Liposomes Effectively Enhances the Antigen-Specific CTL-Mediated Immune Response.

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.

Antitumor and cytotoxic properties of a humanized antibody specific for the GM3(Neu5Gc) ganglioside.

Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors.

Cancer intelligence acquired (CIA): tumor glycosylation and sialylation codes dismantling antitumor defense.

Aberrant glycosylation is a key feature of malignant transformation and reflects epigenetic and genetic anomalies among the multitude of molecules involved in glycan biosynthesis. Although glycan biosynthesis is not template bound, altered tumor glycosylation is not random, but associated with common glycosylation patterns. Evidence suggests that acquisition of distinct glycosylation patterns evolves from a 'microevolutionary' process conferring advantages in terms of tumor growth, tumor dissemination, and immune escape. Such glycosylation modifications also involve xeno- and hypersialylation. Xeno-autoantigens such as Neu5Gc-gangliosides provide potential targets for immunotherapy. Hypersialylation may display 'enhanced self' to escape immunosurveillance and involves several not mutually exclusive inhibitory pathways that all rely on protein-glycan interactions. A better understanding of tumor 'glycan codes' as deciphered by lectins, such as siglecs, selectins, C-type lectins and galectins, may lead to novel treatment strategies, not only in cancer, but also in autoimmune disease or transplantation.

Adjuvants and myeloid-derived suppressor cells: enemies or allies in therapeutic cancer vaccination.

Adjuvants are a critical but largely overlooked and poorly understood component included in vaccine formulations to stimulate and modulate the desired immune responses to an antigen. However, unlike in the protective infectious disease vaccines, adjuvants for cancer vaccines also need to overcome the effect of tumor-induced suppressive immune populations circulating in tumor-bearing individuals. Myeloid-derived suppressor cells (MDSC) are considered to be one of the key immunosuppressive populations that inhibit tumor-specific T cell responses in cancer patients. This review focuses on the different signals for the activation of the immune system induced by adjuvants, and the close relationship to the mechanisms of recruitment and activation of MDSC. This work explores the possibility that a cancer vaccine adjuvant may either strengthen or weaken the effect of tumor-induced MDSC, and the crucial need to address this in present and future cancer vaccines.

Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are among the major obstacles that adjuvants for cancer vaccines have to overcome. These cells cross-present tumor-associated antigens (TAA) to naive T lymphocytes with a tolerogenic outcome. Very Small Size Proteoliposomes (VSSP) is used as adjuvant by four therapeutic cancer vaccines currently in Phase I and II clinical trials. We previously found that VSSP reduces the suppressive function of MDSCs, then activating antigen-specific CTL responses in tumor-bearing (TB) mice, with the consequent reduction of tumor growth. However the mechanistic explanation for the immunomodulatory effect of this adjuvant in TB hosts has not been addressed before. METHODS: TB mice were inoculated with VSSP and MDSCs isolated and characterized by their expression of Arg1 and Nos2 genes by RT-PCR. The effect of VSSP on antigen cross-presentation by MDSCs, regulatory T cells (Tregs) expansion and MDSCs differentiation towards dendritic cells (DCs) was analyzed by FACS. Student's t test or ANOVA and Tukey's tests were used for statistical analyses. RESULTS: After inoculating VSSP into TB mice, a significant reduction of Arg1 and Nos2 gene expression was observed in recovered MDSCs. Concurrently the ability of these cells to induce down-regulation of CD3ζ chain on T cells was lost. Likewise in mice inoculated with the adjuvant lower percentages of Tregs were detected. In vitro, VSSP treatment was enough to differentiate MDSCs into phenotypically mature DCs, eliminating the former suppressive effect. Noteworthy, in vivo administration of VSSP to OVA-expressing (EG.7) TB mice abrogated this model antigen cross-presentation by splenic MDSCs. Similar results were obtained even when OVA antigen was administered into these TB mice formulated in VSSP. On the contrary, immunization with the same protein in polyI:C did not change the percentage of MDSCs expressing SIINFEKL/H-2K(b) complexes, whereas a concomitant injection of VSSP aborted the limitations of polyI:C in this setting. CONCLUSIONS: Altogether, these results indicate that VSSP has the peculiar capacity of inhibiting TAA cross-presentation and certain suppressive mechanisms on MDSCs which in turn, combined with the ability to induce differentiation of these cells into antigen-presenting cells (APCs), sustains this adjuvant as an ideal immunomodulator for cancer immunotherapy.

Very small size proteoliposomes derived from Neisseria meningitidis: an effective adjuvant for antigen-specific cytotoxic T lymphocyte response stimulation under leukopenic conditions.

Leukopenia is a severe condition resulting from both pathological processes and some treatments, like chemotherapy in cancer patients. However, the activation of the patient immune system is required for the success of immunotherapeutic strategies, as cancer vaccines. In this regard, leukopenia constitutes a major hurdle to overcome, mainly due to the impairment of cytotoxic T lymphocyte (CTL) responses. Adjuvants are basic components of vaccine formulations, which might be useful to stimulate immunity under this immunosuppressed condition. To this aim, we tested the capacity of a novel nanoparticulated complex, very small size proteoliposomes (VSSP), to promote CTL even in a leukopenic scenario. Noteworthy, we observed that a VSSP-based OVA vaccine induced a normal antigen-specific CTL response in mice rendered leukopenia by the administration of high doses of the chemotherapeutic agent cyclophosphamide (CY), while under the same conditions the OVA antigen formulated in the TLR-3 agonist polyinosinic-polycytidylic acid (P(I:C)) was ineffective. Moreover, an appropriate combination of VSSP with the P(I:C) vaccine was able to restore the CD8(+) T cell effector function in leukopenic mice. VSSP induced not only a faster repopulation of immune cells in CY-receiving animals, but also enhanced the recovery of memory T lymphocytes and myeloid dendritic cells (DCs) while simultaneously abrogated the immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs). Our results suggest that VSSP could be a particularly suitable immunomodulator to be used in CTL-promoting active immunotherapy strategies operating in severe immune compromised scenarios.

Inhibition of tumor-induced myeloid-derived suppressor cell function by a nanoparticulated adjuvant.

The interaction between cancer vaccine adjuvants and myeloid-derived suppressor cells (MDSCs) is currently poorly understood. Very small size proteoliposomes (VSSP) are a nanoparticulated adjuvant under investigation in clinical trials in patients with renal carcinoma, breast cancer, prostate cancer, and cervical intraepithelial neoplasia grade III. We found that VSSP adjuvant induced a significant splenomegaly due to accumulation of CD11b(+)Gr-1(+) cells. However, VSSP-derived MDSCs showed a reduced capacity to suppress both allogeneic and Ag-specific CTL response compared with that of tumor-induced MDSCs. Moreover, splenic MDSCs isolated from tumor-bearing mice treated with VSSP were phenotypically more similar to those isolated from VSSP-treated tumor-free mice and much less suppressive than tumor-induced MDSCs, both in vitro and in vivo. Furthermore, different from dendritic cell vaccination, inoculation of VSSP-based vaccine in EG.7-OVA tumor-bearing mice was sufficient to avoid tumor-induced tolerance and stimulate an immune response against OVA Ag, similar to that observed in tumor-free mice. This effect correlated with an accelerated differentiation of MDSCs into mature APCs that was promoted by VSSP. VSSP used as a cancer vaccine adjuvant might thus improve antitumor efficacy not only by stimulating a potent immune response against tumor Ags but also by reducing tumor-induced immunosuppression.

Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor.

Tumor growth is associated with a profound alteration in myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). We showed that among factors produced by various experimental tumors, the cytokines GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse and human bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by the ability to impair the priming of CD8(+) T cells and allow long term acceptance of pancreatic islet allografts. Cytokines inducing MDSCs acted on a common molecular pathway and the immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on the C/EBPbeta transcription factor. Adoptive transfer of tumor antigen-specific CD8(+) T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBPbeta in the myeloid compartment, suggesting that C/EBPbeta is a critical regulator of the immunosuppressive environment created by growing cancers.

Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF.

CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8+ T-cells. Two major MDSC subsets were recently shown to play an equal role in MDSC-induced immune dysfunctions: monocytic- and granulocytic-like. We isolated three fractions of MDSC, i.e. CD11b+/Gr-1high, CD11b+/Gr-1int, and CD11b+/Gr-1low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b+/Gr-1int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8+ T-cell activation, while CD11b+/Gr-1high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b+/Gr-1int but not CD11b+/Gr-1high cells were also immunosuppressive in vivo following adoptive transfer. CD11b+/Gr-1low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM-CSF was necessary to induce preferential expansion of both CD11b+/Gr-1int and CD11b+/Gr-1low subsets in the spleen of tumor-bearing mice and mediate tumor-induced tolerance whereas G-CSF, which preferentially expanded CD11b+/Gr-1high cells, did not create such immunosuppressive environment. GM-CSF also acted on granulocyte-macrophage progenitors in the bone marrow inducing local expansion of CD11b+/Gr-1low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor-released GM-CSF.

Role of lipopolysaccharide in the induction of type I interferon-dependent cross-priming and IL-10 production in mice by meningococcal outer membrane vesicles.

We investigated the contribution of lipopolysaccharide (LPS) to adjuvant properties of native outer membrane vesicles (NOMV), a vaccine candidate for meningococcal B disease. NOMV induce the maturation of and cytokine production by murine bone marrow-derived dendritic cells through both toll-like receptors (TLR) 2 and 4 which are mostly dependent on the signalling adaptor MyD88. NOMV are also able to induce B cell proliferation in splenocytes from LPS-hyporesponsive mice. However, induction of IL-10 and type I interferon-dependent, antigen-specific and IFN(gamma)-secreting CD8(+) cytotoxic T lymphocyte responses in vivo by NOMV requires LPS. The importance of LPS in the induction of IL-10 and functional cross-priming has implications for NOMV-based vaccine and adjuvant development.