Chuanlin Ding

Yeast β-Glucan Amplifies Phagocyte Killing of iC3b-Opsonized Tumor Cells via Complement Receptor 3-Syk-Phosphatidylinositol 3-Kinase Pathway

Anti-tumor mAbs hold promise for cancer therapy, but are relatively inefficient. Therefore, there is a need for agents that might amplify the effectiveness of these mAbs. One such agent is β-glucan, a polysaccharide produced by fungi, yeast, and grains, but not mammalian cells. β-Glucans are bound by C receptor 3 (CR3) and, in concert with target-associated complement fragment iC3b, elicit phagocytosis and killing of yeast. β-Glucans may also promote killing of iC3b-opsonized tumor cells engendered by administration of anti-tumor mAbs. In this study, we report that tumor-bearing mice treated with a combination of β-glucan and an anti-tumor mAb show almost complete cessation of tumor growth. This activity evidently derives from a 25-kDa fragment of β-glucan released by macrophage processing of the parent polysaccharide. This fragment, but not parent β-glucan, binds to neutrophil CR3, induces CBRM 1/5 neoepitope expression, and elicits CR3-dependent cytotoxicity. These events require phosphorylation of the tyrosine kinase, Syk, and consequent PI3K activation because β-glucan-mediated CR3-dependent cytotoxicity is greatly decreased by inhibition of these signaling molecules. Thus, β-glucan enhances tumor killing through a cascade of events, including in vivo macrophage cleavage of the polysaccharide, dual CR3 ligation, and CR3-Syk-PI3K signaling. These results are important inasmuch as β-glucan, an agent without evident toxicity, may be used to amplify tumor cell killing and may open new opportunities in the immunotherapy of cancer.

Differential pathways regulating innate and adaptive antitumor immune responses by particulate and soluble yeast-derived β-glucans

β-glucans have been reported to function as a potent adjuvant to stimulate innate and adaptive immune responses. However, β-glucans from different sources are differential in their structure, conformation, and thus biologic activity. Different preparations of β-glucans, soluble versus particulate, further complicate their mechanism of action. Here we show that yeast-derived particulate β-glucan activated dendritic cells (DCs) and macrophages via a C-type lectin receptor dectin-1 pathway. Activated DCs by particulate β-glucan promoted Th1 and cytotoxic T-lymphocyte priming and differentiation in vitro. Treatment of orally administered yeast-derived particulate β-glucan elicited potent antitumor immune responses and drastically down-regulated immunosuppressive cells, leading to the delayed tumor progression. Deficiency of the dectin-1 receptor completely abrogated particulate β-glucan-mediated antitumor effects. In contrast, yeast-derived soluble β-glucan bound to DCs and macrophages independent of the dectin-1 receptor and did not activate DCs. Soluble β-glucan alone had no therapeutic effect but significantly augmented antitumor monoclonal antibody-mediated therapeutic efficacy via a complement activation pathway but independent of dectin-1 receptor. These findings reveal the importance of different preparations of β-glucans in the adjuvant therapy and allow for the rational design of immunotherapeutic protocols usable in clinical trials.

Orally Administered Particulate β-Glucan Modulates Tumor-Capturing Dendritic Cells and Improves Antitumor T-Cell Responses in Cancer

Purpose: The beneficial properties of β-glucans have been recognized for centuries. Their proposed mechanisms of action in cancer therapy occur via stimulation of macrophages and priming of innate neutrophil complement receptor 3 for eliciting complement receptor 3–dependent cellular cytotoxicity of iC3b-opsonized tumor cells. The current study is to investigate whether β-glucan therapy has any effect on antitumor adaptive T-cell responses. Experimental Design: We first examined the trafficking of orally administered particulate yeast-derived β-glucan and its interaction with dendritic cells (DC) that captured tumor materials. Antigen-specific T cells were adoptively transferred into recipient mice to determine whether oral β-glucan therapy induces augmented T-cell responses. Lewis lung carcinoma and RAM-S lymphoma models were used to test oral β-glucan therapeutic effect. Further mechanistic studies including tumor-infiltrating T cells and cytokine profiles within the tumor milieu were determined. Results: Orally administered particulate β-glucan trafficked into spleen and lymph nodes and activated DCs that captured dying tumor cells in vivo, leading to the expansion and activation of antigen-specific CD4 and CD8 T cells. In addition, IFN-γ production of tumor-infiltrating T cells and CTL responses were significantly enhanced on β-glucan treatment, which ultimately resulted in significantly reduced tumor burden. Moreover, β-glucan–treated tumors had significantly more DC infiltration with the activated phenotype and significant levels of Th1-biased cytokines within the tumor microenvironment. Conclusions: These data highlight the ability of yeast-derived β-glucan to bridge innate and adaptive antitumor immunity and suggest that it can be used as an adjuvant for tumor immunotherapy. Clin Cancer Res; 16(21); 5153–64. ©2010 AACR.

Differential developmental requirement and peripheral regulation for dermal Vγ4 and Vγ6T17 cells in health and inflammation.

Dermal IL-17-producing γδT cells play a critical role in skin inflammation. However, their development and peripheral regulation have not been fully elucidated. Here we demonstrate that dermal γδT cells develop from the embryonic thymus and undergo homeostatic proliferation after birth with diversified TCR repertoire. Vγ6T cells are bona fide resident but precursors of dermal Vγ4T cells may require extrathymic environment for imprinting skin homing properties. Thymic Vγ6T cells are more competitive than Vγ4 for dermal γδT cell reconstitution and TCRδ−/− mice reconstituted with Vγ6 develop psoriasis-like inflammation after IMQ-application. Although both IL-23 and IL-1β promote Vγ4 and Vγ6 proliferation, Vγ4 are the main source of IL-17 production, which requires IL-1 signaling. Mice with deficiency of IL-1RI signaling have significantly decreased skin inflammation. These studies reveal a differential developmental requirement and peripheral regulation for dermal Vγ6 and Vγ4 γδT cells, implying a new mechanism that may be involved in skin inflammation.

Plasmacytoid Dendritic Cells Regulate Autoreactive B Cell Activation via Soluble Factors and in a Cell-to-Cell Contact Manner

Plasmacytoid dendritic cells (pDCs) are specialized type I IFN producers, which play an important role in pathogenesis of autoimmune disorders. Dysregulated autoreactive B cell activation is a hallmark in most autoimmune diseases. This study was undertaken to investigate interactions between pDCs and autoreactive B cells. After coculture of autoreactive B cells that recognize self-Ag small nuclear ribonucleoprotein particles with activated pDCs, we found that pDCs significantly enhance autoreactive B cell proliferation, autoantibody production, and survival in response to TLR and BCR stimulation. Neutralization of IFN-α/β and IL-6 abrogated partially pDC-mediated enhancement of autoreactive B cell activation. Transwell studies demonstrated that pDCs could provide activation signals to autoreactive B cells via a cell-to-cell contact manner. The involvement of the ICAM-1-LFA-1 pathway was revealed as contributing to this effect. This in vitro enhancement effect was further demonstrated by an in vivo B cell adoptive transfer experiment, which showed that autoreactive B cell proliferation and activation were significantly decreased in MyD88-deficient mice compared with wild-type mice. These data suggest the dynamic interplay between pDCs and B cells is required for full activation of autoreactive B cells upon TLR or BCR stimulation.

Opposing Roles for Complement Component C5a in Tumor Progression and the Tumor Microenvironment

Promoting complement (C) activation may enhance immunological mechanisms of anti-tumor Abs for tumor destruction. However, C activation components, such as C5a, trigger inflammation, which can promote tumor growth. We addressed the role of C5a on tumor growth by transfecting both human carcinoma and murine lymphoma with mouse C5a. In vitro growth kinetics of C5a, control vector, or parental cells revealed no significant differences. Tumor-bearing mice with C5a-transfected xenografted tumor cells had significantly less tumor burden as compared with control vector tumors. NK cells and macrophages infiltrated C5a-expressing tumors with significantly greater frequency, whereas vascular endothelial growth factor, arginase, and TNF-α production were significantly less. Tumor-bearing mice with high C5a-producing syngeneic lymphoma cells had significantly accelerated tumor progression with more Gr-1+CD11b+ myeloid cells in the spleen and overall decreased CD4+ and CD8+ T cells in the tumor, tumor-draining lymph nodes, and the spleen. In contrast, tumor-bearing mice with low C5a-producing lymphoma cells had a significantly reduced tumor burden with increased IFN-γ–producing CD4+ and CD8+ T cells in the spleen and tumor-draining lymph nodes. These studies suggest concentration of local C5a within the tumor microenvironment is critical in determining its role in tumor progression.

Hampering immune suppressors: therapeutic targeting of myeloid-derived suppressor cells in cancer.

Abstract Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with suppressive properties that preferentially expand in cancer. Myeloid-derived suppressor cells mainly suppress T-cell proliferation and cytotoxicity, inhibit natural killer cell activation, and induce the differentiation and expansion of regulatory T cells. The wide spectrum of MDSC suppressive activity in cancer and its role in tumor progression have rendered these cells a promising target for effective cancer immunotherapy. In this review we briefly discuss the origin of MDSCs and their main mechanisms of suppression and focus more on the approaches developed up to date targeting of MDSCs in tumor-bearing animals and cancer patients.

Therapeutic potential of various β-glucan sources in conjunction with anti-tumor monoclonal antibody in cancer therapy

Combined β-glucan with anti-tumor mAb therapy has demonstrated therapeutic efficacy in murine tumor models. The current study was designed to compare the therapeutic efficacy of various sources of β-glucans. Our studies demonstrated that yeast β-glucan, in combination with anti-tumor mAb, resulted in significantly smaller tumor burdens and achieved enhanced long-term survival compared to mAb alone or β-glucan extracts from mushrooms. Further studies indicated that yeast β-glucan particle was superior to mushroom extracts in inducing cytokine secretion, particularly IL-12 production in dendritic cells (DCs). In addition, results showed that cytokine production was markedly decreased in MyD88-deficient macrophages and DCs but not in complement receptor 3 (CR3)-deficient mice. Our data suggest that yeast β-glucan demonstrates much stronger adjuvant activity compared to mushroom β-glucan extracts in tumor therapy. This effect of yeast β-glucan may be in part ascribed to the cytokine secretion by DCs and macrophages and bioavailability of active β-glucan moiety.

Targeting of antigens to B cells augments antigen-specific T-cell responses and breaks immune tolerance to tumor-associated antigen MUC1

B cells are antibody (Ab)-secreting cells as well as potent antigen (Ag)-presenting cells that prime T-cell activation, which evokes great interest in their use for vaccine development. Here, we targeted ovalbumin (OVA) to B cells via CD19 and found that a single low dose of anti-CD19-OVA conjugates, but not isotype mAb-OVA, stimulated augmented CD4 and CD8 T-cell proliferation and expansion. Administration of TLR9 agonist CpG could significantly enhance long-term T-cell survival. Similar results were obtained when the tumor-associated Ag MUC1 was delivered to B cells. MUC1 transgenic (Tg) mice were previously found to lack effective T-cell help and produce low-titer of anti-MUC1 Abs after vaccination. Targeting MUC1 to B cells elicited high titer of anti-MUC1 Abs with different isotypes, predominantly IgG2a and IgG2b, in MUC1 Tg mice. The isotype switching of anti-MUC1 Ab was CD4 dependent. In addition, IFN-gamma-producing CD8 T cells and in vivo cytolytic activity were significantly increased in these mice. The mice also showed significant resistance to MUC1(+) lymphoma cell challenge both in the prophylactic and therapeutic settings. We conclude that Ags targeting to B cells stimulate CD4 and CD8 T-cell responses as well as Th-dependent humoral immune responses.

Dectin-1 Activation by a Natural Product β-Glucan Converts Immunosuppressive Macrophages into an M1-like Phenotype.

Tumor-associated macrophages (TAM) with an alternatively activated phenotype have been linked to tumor-elicited inflammation, immunosuppression, and resistance to chemotherapies in cancer, thus representing an attractive target for an effective cancer immunotherapy. In this study, we demonstrate that particulate yeast-derived β-glucan, a natural polysaccharide compound, converts polarized alternatively activated macrophages or immunosuppressive TAM into a classically activated phenotype with potent immunostimulating activity. This process is associated with macrophage metabolic reprograming with enhanced glycolysis, Krebs cycle, and glutamine utilization. In addition, particulate β-glucan converts immunosuppressive TAM via the C-type lectin receptor dectin-1–induced spleen tyrosine kinase–Card9–Erk pathway. Further in vivo studies show that oral particulate β-glucan treatment significantly delays tumor growth, which is associated with in vivo TAM phenotype conversion and enhanced effector T cell activation. Mice injected with particulate β-glucan–treated TAM mixed with tumor cells have significantly reduced tumor burden with less blood vascular vessels compared with those with TAM plus tumor cell injection. In addition, macrophage depletion significantly reduced the therapeutic efficacy of particulate β-glucan in tumor-bearing mice. These findings have established a new paradigm for macrophage polarization and immunosuppressive TAM conversion and shed light on the action mode of β-glucan treatment in cancer.