Jiusheng Deng

Toll-like receptor 2–dependent induction of vitamin A–metabolizing enzymes in dendritic cells promotes T regulatory responses and inhibits autoimmunity

Immune sensing of a microbe occurs via multiple receptors. How signals from different receptors are coordinated to yield a specific immune response is poorly understood. We show that two pathogen recognition receptors, Toll-like receptor 2 (TLR2) and dectin-1, recognizing the same microbial stimulus, stimulate distinct innate and adaptive responses. TLR2 signaling induced splenic dendritic cells (DCs) to express the retinoic acid metabolizing enzyme retinaldehyde dehydrogenase type 2 and interleukin-10 (IL-10) and to metabolize vitamin A and stimulate Foxp3+ T regulatory cells (Treg cells). Retinoic acid acted on DCs to induce suppressor of cytokine signaling-3 expression, which suppressed activation of p38 mitogen-activated protein kinase and proinflammatory cytokines. Consistent with this finding, TLR2 signaling induced Treg cells and suppressed IL-23 and T helper type 17 (TH17) and TH1-mediated autoimmune responses in vivo. In contrast, dectin-1 signaling mostly induced IL-23 and proinflammatory cytokines and augmented TH17 and TH1-mediated autoimmune responses in vivo. These data define a new mechanism for the systemic induction of retinoic acid and immune suppression against autoimmunity.

A MCP1 fusokine with CCR2-specific tumoricidal activity.

BackgroundThe CCL2 chemokine is involved in promoting cancer angiogenesis, proliferation and metastasis by malignancies that express CCR2 receptor. Thus the CCL2/CCR2 axis is an attractive molecular target for anticancer drug development.MethodsWe have generated a novel fusion protein using GMCSF and an N-terminal truncated version of MCP1/CCL2 (6-76) [hereafter GMME1] and investigated its utility as a CCR2-specific tumoricidal agent.ResultsWe found that distinct to full length CCL2 or its N-truncated derivative (CCL2 5-76), GMME1 bound to CCR2 on mouse lymphoma EG7, human multiple myeloma cell line U266, or murine and human medulloblastoma cell lines, and led to their death by apoptosis. We demonstrated that GMME1 specifically blocked CCR2-associated STAT3 phosphorylation and up-regulated pro-apoptotic BAX. Furthermore, GMME1 significantly inhibited EG7 tumor growth in C57BL/6 mice, and induced apoptosis of primary myeloma cells from patients.ConclusionOur data demonstrate that GMME1 is a fusokine with a potent, CCR2 receptor-mediated pro-apoptotic effect on tumor cells and could be exploited as a novel biological therapy for CCR2+ malignancies including lymphoid and central nervous system malignancies.

Incorporation of a GPI-anchored engineered cytokine as a molecular adjuvant enhances the immunogenicity of HIV VLPs

HIV vaccines should elicit immune responses at both the mucosal portals of entry to block transmission and systemic compartments to clear disseminated viruses. Co-delivery of mucosal adjuvants has been shown to be essential to induce effective mucosal immunity by non-replicating vaccines. A novel cytokine, GIFT4, engineered by fusing GM-CSF and interleukin-4, was previously found to simulate B cell proliferation and effector function. Herein a membrane-anchored form of GIFT4 was constructed by fusing a glycolipid (GPI)-anchoring sequence and incorporated into Env-enriched HIV virus-like particles (VLPs) as a molecular adjuvant. Guinea pigs were immunized with the resulting HIV VLPs through an intramuscular priming-intranasal boosting immunization route. The GIFT4-containing VLPs induced higher levels of systemic antibody responses with significantly increased binding avidity and improved neutralizing breadth and potency to a panel of selected strains, as well as higher levels of IgG and IgA at several mucosal sites. Thus, the novel GPI-GIFT4-containging VLPs have the potential to be developed into a prophylactic HIV vaccine. Incorporation of GPI-anchored GIFT4 into VLPs as a molecular adjuvant represents a novel approach to increase their immunogenicity.

Blood B Cell and Regulatory Subset Content in Multiple Sclerosis Patients

Objective B cell targeted therapies have been effective in slowing multiple sclerosis (MS) disease progression suggesting a direct causal link for this lymphoid subset. A small subset of B cells with regulative properties (Bregs) exists in peripheral blood, and induction of Bregs ameliorates experimental autoimmune encephalomyelitis (EAE), the murine model for MS. Therefore the frequency of B cell subsets and regulatory B cells in particular in peripheral blood of MS patients is of interest. Methods The phenotype and frequency of B cell subsets in peripheral blood from 32 MS patients and 34 healthy controls (HC) were examined using flow cytometry. Results We found that there is an increase in CD19+ cell number in MS 1347 ± 159 cells/μL, (average ± SEM) compared to HC, 935 ± 129 cells/μL and no apparent deficiency in B-cells with a regulatory phenotype. In addition, we observed a loss of correlation between CD19+ B cells and total lymphocyte count in MS. Conclusion These findings suggest altered blood B-cell homeostasis in MS patients.

Engineered Fusokine GIFT4 Licenses the Ability of B Cells to Trigger a Tumoricidal T-cell Response

Engineered chimeric cytokines can generate gain-of-function activity in immune cells. Here, we report potent antitumor activity for a novel fusion cytokine generated by N-terminal coupling of GM-CSF to IL4, generating a fusokine termed GIFT4. B cells treated with GIFT4 clustered GM-CSF and IL4 receptors on the cell surface and displayed a pan-STAT hyperphosphorylation associated with acquisition of a distinct phenotype and function described to date. In C57BL/6J mice, administration of GIFT4 expanded endogenous B cells and suppressed the growth of B16F0 melanoma cells. Furthermore, B16F0 melanoma cells engineered to secrete GIFT4 were rejected immunologically in a B-cell-dependent manner. This effect was abolished when GIFT4-expressing B16F0 cells were implanted in B-cell-deficient mice, confirming a B-cell-dependent antitumor effect. Human GIFT4-licensed B cells primed cytotoxic T cells and specifically killed melanoma cells in vitro and in vivo. Taken together, our results demonstrated that GIFT4 could mediate expansion of B cells with potent antigen-specific effector function. GIFT4 may offer a novel immunotherapeutic tool and define a previously unrecognized potential for B cells in melanoma immunotherapy.

Inhibition of B-Raf/MEK/ERK signaling suppresses DR5 expression and impairs response of cancer cells to DR5-mediated apoptosis and T cell-induced killing

Inhibition of B-Raf/MEK/ERK signaling is an effective therapeutic strategy against certain types of cancers such as melanoma and thyroid cancer. While demonstrated to be effective anticancer agents, B-Raf or MEK inhibitors have also been associated with early tumor progression and development of secondary neoplasms. The ligation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with its receptor, death receptor 5 (DR5), leading to induction of apoptosis, offers a promising anticancer strategy. Importantly, this is also a natural immunosurveillance mechanism against cancer development. We previously demonstrated that activated B-Raf/MEK/ERK signaling positively regulates DR5 expression. Hence, our current work sought to address whether B-Raf/MEK/ERK inhibition and the consequent suppression of DR5 expression impede cancer cell response to DR5 activation-induced apoptosis and activated immune cell-induced killing. We found that both B-Raf (for example, PLX4032) and MEK inhibitors (for example, AZD6244 and PD0325901) effectively inhibited ERK1/2 phosphorylation and reduced DR5 levels in both human thyroid cancer and melanoma cells. Similar to the observed effect of genetic knockdown of the B-Raf gene, pre-treatment of cancer cell lines with either B-Raf or MEK inhibitors attenuated or abolished cellular apoptotic response induced by TRAIL or the DR5 agonistic antibody AMG655 or cell killing by activated T cells. Our findings clearly show that inhibition of B-Raf/MEK/ERK signaling suppresses DR5 expression and impairs DR5 activation-induced apoptosis and T cell-mediated killing of cancer cells. These findings suggest a potential negative impact of B-Raf or MEK inhibition on TRAIL- or DR5-mediated anticancer therapy and on TRAIL/DR5-mediated immune-clearance of cancer cells.

The proteasome deubiquitinase inhibitor b-AP15 enhances DR5 activation-induced apoptosis through stabilizing DR5

Abstractb-AP15 and its derivatives block proteasome deubiquitinase (DUB) activity and have been developed and tested in the clinic as potential cancer therapeutic agents. b-AP15 induces apoptosis in cancer cells, but the underlying mechanisms are largely undefined. The current study focuses on studying the modulatory effects of b-AP15 on death receptor 5 (DR5) levels and DR5 activation-induced apoptosis as well as on understanding the underlying mechanisms. Treatment with b-AP15 potently increased DR5 levels including cell surface DR5 in different cancer cell lines with limited or no effects on the levels of other related proteins including DR4, c-FLIP, FADD, and caspase-8. b-AP15 substantially slowed the degradation of DR5, suggesting that it stabilizes DR5. Moreover, b-AP15 effectively augmented apoptosis when combined with TRAIL or the DR5 agonistic antibody AMG655; these effects are DR5-dependent because DR5 deficiency abolished the ability of b-AP15 to enhance TRAIL- or AMG655-induced apoptosis. Therefore, it is clear that b-AP15, and possibly its derivatives, can stabilize DR5 and increase functional cell surface DR5 levels, resulting in enhancement of DR5 activation-induced apoptosis. Our findings suggest that b-AP15 and its derivatives may have potential in sensitizing cancer cells to DR5 activation-based cancer therapy.

Regulatory B cells induce formation of IL-10 expressing T cells in mice with autoimmune neuroinflammation

Although B cells are traditionally known for their role in propagating proinflammatory immune responses, their immunosuppressive effects have only recently begun to be appreciated. How these regulatory B cells (Bregs) suppress the immune response remains to be worked out in detail. In this article, we show that Bregs can induce the formation of conventional FoxP3+ regulatory T cells (Tregs), as well as a more recently described CD49b+CD223+ regulatory T-cell subset, known as type 1 regulatory T cells (Tr1s). When Bregs are transferred into mice with experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, they home to the spleen and mesenteric lymph nodes, leading to an expansion of Tregs and Tr1 in vivo. Tregs and Tr1s are also found in greater proportions in the CNS of mice with EAE treated with Bregs and are correlated with the remission of symptoms. The discovery that Bregs induce the formation of regulatory T-cell subsets in vivo may herald their use as immunosuppressive agents in adoptive cellular therapies for autoimmune pathologies. SIGNIFICANCE STATEMENT Although B cells are traditionally known for their role in propagating proinflammatory immune responses, their immunosuppressive effects have only recently begun to be appreciated. How regulatory B cells (Bregs) suppress the immune response remains to be fully understood. In this article, we show that Bregs can induce the formation of conventional regulatory T cells (Tregs) as well as type 1 regulatory T cells (Tr1s). When Bregs are transferred into mice with experimental autoimmune encephalomyelitis (EAE), they home to secondary lymphoid organs, leading to an expansion of Tregs and Tr1s in vivo. Tregs and Tr1s are also found in greater proportions in the CNS of mice with EAE treated with Bregs and are correlated with the remission of symptoms.

BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth

Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy.

Maltose-binding protein fusion allows for high level bacterial expression and purification of bioactive mammalian cytokine derivatives.

Fusokines are chimeric proteins generated by the physical coupling of cytokines in a single polypeptide, resulting in proteins with highly pleiotropic activity and the potential to treat cancer and autoimmune ailments. For instance, the fusokine GIFT15 (GM-CSF and Interleukin 15 Fusion Transgene) has been shown to be a powerful immunosuppressive protein able to convert naïve B cells into IL-10-producing B cells. To date, the mammalian cell systems used for the expression of GIFT15 allow for secretion of the protein in the culturing media, an inefficient system for producing GMP-compliant fusokines. In this study we report the bacterial expression of bioactive recombinant GIFT15 (rGIFT15). Indeed, there is a constant demand to improve the expression systems for therapeutic proteins. Expression of a maltose-binding protein (MBP) fusion protein efficiently allowed the accumulation of soluble protein in the intracellular milieu. Optimizing the bacterial culture significantly increased the yield of recombinant protein. The biological activity of rGIFT15 was comparable to that of fusokine derived from a mammalian source. This approach led to the production of soluble, endotoxin-free functional protein, averaging 5 mg of rGIFT15 per liter of culture. This process is amenable to scale up for the development of Food and Drug Administration (FDA)-compliant immune-modulatory rGIFT15.