Sungyoul Hong

Cross talk between the bone and immune systems: osteoclasts function as antigen-presenting cells and activate CD4+ and CD8+ T cells

The bone and immune systems are closely related through cellular and molecular interactions. Because bone-resorbing osteoclasts (OCs) are derived from the monocyte/macrophage lineage, similar to dendritic cells (DCs), we hypothesized that OCs could serve as antigen-presenting cells (APCs) to activate T cells. In this study, OCs were generated from human monocytes with stimulation by receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Results showed that, similar to DCs, OCs express major histocompatibility complex (MHC) classes I and II, and CD80, CD86, and CD40; and uptake soluble antigens. OCs secrete interleukin-10 (IL-10), transforming growth factor-beta (TGF-beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha), but not IL-12p70. OCs present allogeneic antigens and activate both CD4+ and CD8+ alloreactive T cells in an MHC-restricted fashion. OCs also present soluble protein tetanus toxoid to activate autologous CD4+ T cells. These findings indicate that OCs can function as APCs and activate both CD4+ and CD8+ T cells. Thus, our study provides new insight into the effect of OCs on the immune system and may help develop novel strategies for treating diseases such as rheumatoid arthritis and multiple myeloma, which affect both the bone and immune systems.

Th9 cells promote antitumor immune responses in vivo

Th9 cells are a subset of CD4+ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8+ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8+ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell-mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8+ CTL-mediated antitumor immunity and indicate that Th9 cell-based cancer immunotherapy may be a promising therapeutic approach.

Macrophages are an abundant component of myeloma microenvironment and protect myeloma cells from chemotherapy drug-induced apoptosis

Multiple myeloma remains an incurable disease. One of the major problems is that myeloma cells develop drug resistance on interaction with bone marrow stromal cells. In this study, we examined the effects of macrophages (Mvarphis), a type of stromal cells, on myeloma cell survival and response to chemotherapy. We showed that Mvarphi, in particular tumor-associated Mvarphi, is a protector of myeloma cells. The protective effect was dependent on direct contact between Mvarphis and myeloma cells. Mvarphis protected both myeloma cell lines and primary myeloma cells from spontaneous and chemotherapy drug-induced apoptosis by attenuating the activation and cleavage of caspase-dependent apoptotic signaling. These findings are clinically relevant because we found that CD68+ Mvarphis heavily infiltrate the bone marrow of patients with myeloma but not the bone marrow of control patients. Thus, our results indicate that Mvarphis may contribute to myeloma cell survival and resistance to chemotherapeutic treatment in vivo.

Active vaccination with Dickkopf-1 induces protective and therapeutic antitumor immunity in murine multiple myeloma

Dickkopf-1 (DKK1), broadly expressed in myeloma cells but highly restricted in normal tissues, together with its functional roles as an osteoblast formation inhibitor, may be an ideal target for immunotherapy in myeloma. Our previous studies have shown that DKK1 (peptide)-specific CTLs can effectively lyse primary myeloma cells in vitro. The goal of this study was to examine whether DKK1 can be used as a tumor vaccine to elicit DKK1-specific immunity that can control myeloma growth or even eradicate established myeloma in vivo. We used DKK1-DNA vaccine in the murine MOPC-21 myeloma model, and the results clearly showed that active vaccination using the DKK1 vaccine not only was able to protect mice from developing myeloma, but it was also therapeutic against established myeloma. Furthermore, the addition of CpG as an adjuvant, or injection of B7H1-blocking or OX40-agonist Abs, further enhanced the therapeutic effects of the vaccine. Mechanistic studies revealed that DKK1 vaccine elicited a strong DKK1- and tumor-specific CD4+ and CD8+ immune responses, and treatment with B7H1 or OX40 Abs significantly reduced the numbers of IL-10-expressing and Foxp3+ regulatory T cells in vaccinated mice. Thus, our studies provide strong rationale for targeting DKK1 for immunotherapy of myeloma patients.

Novel phosphatidylinositol 3-kinase inhibitor NVP-BKM120 induces apoptosis in myeloma cells and shows synergistic anti-myeloma activity with dexamethasone.

NVP-BKM120 is a novel phosphatidylinositol 3-kinase (PI3K) inhibitor and is currently being investigated in phase I clinical trials in solid tumors. This study aimed to evaluate the therapeutic efficacy of BKM120 in multiple myeloma (MM). BKM120 induces cell growth inhibition and apoptosis in both MM cell lines and freshly isolated primary MM cells. However, BKM120 only shows limited cytotoxicity toward normal lymphocytes. The presence of MM bone marrow stromal cells, insulin-like growth factor, or interleukin-6 does not affect BKM120-induced tumor cell apoptosis. More importantly, BKM120 treatment significantly inhibits tumor growth in vivo and prolongs the survival of myeloma-bearing mice. In addition, BKM120 shows synergistic cytotoxicity with dexamethasone in dexamethasone-sensitive MM cells. Low doses of BKM120 and dexamethasone, each of which alone has limited cytotoxicity, induce significant cell apoptosis in MM.1S and ARP-1. Mechanistic study shows that BKM120 exposure causes cell cycle arrest by upregulating p27 (Kip1) and downregulating cyclin D1 and induces caspase-dependent apoptosis by downregulating antiapoptotic XIAP and upregulating expression of cytotoxic small isoform of Bim, BimS. In summary, our findings demonstrate the in vitro and in vivo anti-MM activity of BKM120 and suggest that BKM120 alone or together with other MM chemotherapeutics, particularly dexamethasone, may be a promising treatment for MM.

Roles of Idiotype-Specific T Cells in Myeloma Cell Growth and Survival: Th1 and CTL Cells Are Tumoricidal while Th2 Cells Promote Tumor Growth

Idiotype (Id) protein, secreted by myeloma cells, is a tumor-specific antigen. Id-based immunotherapy has been explored in patients with myeloma, and results were disappointing. Although previous studies have shown that Id-specific CTLs are able to lyse myeloma cells, it is unclear whether other types of Id-specific T cells, such as type-1 T-helper (Th1) and type-2 T-helper (Th2) cells, are also able to suppress or kill myeloma cells. Using a 5T murine myeloma model, we generated T-cell clones of different subsets and examined their function in the context of myeloma cells. Id-specific CTLs specifically lysed myeloma cells via MHC class I, perforin, and Fas ligand (FasL), and Th1, but not Th2, cells lysed the myeloma cells by FasL-Fas interaction. CTL and Th1 cells also suppressed the growth and function of myeloma cells, whereas Th2 cells promoted the proliferation and enhanced the secretion of Id protein and cytokines by myeloma cells. CTL and Th1, but not Th2, cells were able to eradicate established myeloma in vivo after adoptive transfer. These results show that Id-specific CTL and Th1 are promising effector cells, whereas Th2 provide no protection and may even promote tumor progression in vivo.

Myeloma cell line-derived, pooled heat shock proteins as a universal vaccine for immunotherapy of multiple myeloma

Tumor cell-derived heat shock proteins are used as vaccines for immunotherapy of cancer patients. However, current approaches require the generation of custom-made products and are clinically ineffective. To improve the applicability of heat shock protein-based immunotherapy in cancers and to enhance clinical efficacy, we explored combinational treatments in a myeloma setting using pooled heterogeneous or allogeneic myeloma cell line-derived glycoprotein 96 (gp96) as universal vaccines, and clearly demonstrated that pooled but not single gp96 from heterogeneous or allogeneic myeloma cell lines was as effective as autologous gp96 in protecting mice from tumor challenge and rechallenge and in treating established myeloma. We showed that interferon gamma and CD4+ and CD8+ T cells were required for gp96-induced antimyeloma responses and that pooled gp96 induced broader immune responses that protected mice from developing different myeloma. Furthermore, pooled gp96 plus CpG in combination with anti-B7H1 or anti-interleukin-10 monoclonal antibodies were effective in treating mice with large tumor burdens. Thus, this study strongly suggests that pooled gp96 vaccines from myeloma cell lines can replace gp96 vaccines from autologous tumors for immunotherapy and induce immune responses against broader tumor antigens that may protect against tumor recurrence and development of unrelated tumors in vaccinated myeloma patients.

Optimizing dendritic cell vaccine for immunotherapy in multiple myeloma: tumour lysates are more potent tumour antigens than idiotype protein to promote anti-tumour immunity.

Dendritic cells (DCs) are the most potent antigen‐presenting cells and are the mediators of T cell immunity. Many investigators have explored the potential of using DCs as a vaccine for tumour‐derived antigens in immunotherapy of B cell malignancies, and the results have been disappointing. To search for better tumour antigens to improve the efficacy of DC‐based immunotherapy in myeloma, we evaluated and compared the efficacy of the vaccination of DCs pulsed with idiotype (Id) or tumour lysate in the 5TGM1 myeloma mouse model. Our results showed that Id‐ or tumour lysate‐pulsed DC vaccines protected mice efficiently against developing myeloma, retarded tumour growth, induced tumour regression against established tumour and protected surviving mice from tumour rechallenge. The therapeutic responses were associated with an induction of strong humoral immune responses, including anti‐Id or anti‐lysate antibodies, and cellular immune responses including myeloma‐specific CD8+ cytotoxic T lymphocytes, CD4+ type 1 T helper cells and memory T cells in mice receiving Id‐ or tumour lysate‐pulsed DC vaccines. In addition, our studies showed that tumour lysate‐pulsed DCs were more potent vaccines than the Id‐pulsed DC vaccines to promote anti‐tumour immunity in the model. This information will be important for improving the strategies of DC‐based immunotherapy for patients with myeloma and other B cell tumours.

A severe combined immunodeficient-hu in vivo mouse model of human primary mantle cell lymphoma

Purpose: To establish a severe combined immunodeficient (SCID)-hu in vivo mouse model of human primary mantle cell lymphoma (MCL) for the study of the biology and novel therapy of human MCL. Experimental Design: Primary MCL cells were isolated from spleen, lymph node, bone marrow aspirates, or peripheral blood of six different patients and injected respectively into human bone chips, which had been s.c. implanted in SCID-hu. Circulating human β2-microglobulin in mouse serum was used to monitor the engraftment and growth of patients MCL cells. H&E staining and immunohistochemical staining with anti-human CD20 and cyclin D1 antibodies were used to confirm the tumor growth and migration. Results: Increasing levels of circulating human β2-microglobulin in mouse serum indicated that the patients MCL cells were engrafted successfully into human bone chip of SCID-hu mice. The engraftment and growth of patients MCL cells were dependent on human bone marrow microenvironment. Immunohistochemical staining with anti-human CD20 and cyclin D1 antibodies confirmed that patients MCL cells were able to not only survive and propagate in the bone marrow microenvironment of the human fetal bone chips, but also similar to the human disease, migrate to lymph nodes, spleen, bone marrow, and gastrointestinal tract of host mice. Treatment of MCL-bearing SCID-hu mice with atiprimod, a novel antitumor compound against the protection of bone marrow stromal cells, induced tumor regression. Conclusion: This is the first human primary MCL animal model that should be useful for the biological and therapeutic research on MCL.

CpG or IFN-α are more potent adjuvants than GM-CSF to promote anti-tumor immunity following idiotype vaccine in multiple myeloma

Idiotype (Id) protein in combination with GM-CSF has been used as vaccines for immunotherapy of patients with myeloma and B-cell tumors and the results have been disappointing. To search for better immune adjuvants to improve the efficacy of Id-based immunotherapy in myeloma, we evaluated and compared the efficacy of vaccination of Id protein in combination with CpG or IFN-α, or GM-CSF as a control, in the 5TGM1 myeloma mouse model. Our results showed that Id vaccine combined with CpG or IFN-α, but not GM-CSF, not only efficiently protected mice from developing myeloma but also eradicated established myeloma. The therapeutic responses were associated with an induction of strong humoral immune responses including anti-Id antibodies, and cellular immune responses including Id- and myeloma-specific CD8+ cytotoxic T lymphocytes (CTLs), CD4+ type-1 T-helper (Th1) cells and memory T cells in mice receiving Id vaccine combined with CpG or IFN-α. Furthermore, Id vaccine combined with CpG or IFN-α induced Id- and tumor-specific memory immune responses that protected surviving mice from tumor rechallenge. Thus, our study clearly shows that CpG or IFN-α are better immune adjuvants than GM-CSF. This information will be important for improving the strategies of Id-based immunotherapy for patients with myeloma and other B-cell tumors.