Anissa Sh Chan

Thrombopoietin cooperates with FLT3-ligand in the generation of plasmacytoid dendritic cell precursors from human hematopoietic progenitors

Type 1 interferon-producing cells (IPCs), also known as plasmacytoid dendritic cell (DC) precursors, represent the key effectors in antiviral innate immunity and triggers for adaptive immune responses. IPCs play important roles in the pathogenesis of systemic lupus erythematosus (SLE) and in modulating immune responses after hematopoietic stem cell transplantation. Understanding IPC development from hematopoietic progenitor cells (HPCs) may provide critical information in controlling viral infection, autoimmune SLE, and graft-versus-host disease. FLT3-ligand (FLT3-L) represents a key IPC differentiation factor from HPCs. Although hematopoietic cytokines such as interleukin-3 (IL-3), IL-7, stem cell factor (SCF), macrophage-colony-stimulating factor (M-CSF), and granulocyte M-CSF (GM-CSF) promote the expansion of CD34+ HPCs in FLT3-L culture, they strongly inhibit HPC differentiation into IPCs. Here we show that thrombopoietin (TPO) cooperates with FLT3-L, inducing CD34+ HPCs to undergo a 400-fold expansion in cell numbers and to generate more than 6 x 10(6) IPCs per 10(6) CD34+ HPCs within 30 days in culture. IPCs derived from HPCs in FLT3-L/TPO cultures display blood IPC phenotype and have the capacity to produce large amounts of interferon-alpha (IFN-alpha) and to differentiate into mature DCs. This culture system, combined with the use of adult peripheral blood CD34+ HPCs purified from G-CSF-mobilized donors, permits the generation of more than 10(9) IPCs from a single blood donor.

Cutting Edge: T Cell Migration Regulated by CXCR4 Chemokine Receptor Signaling to ZAP-70 Tyrosine Kinase

Chemokines regulate the homeostatic trafficking of lymphocytes and lymphocyte influx into sites of injury and inflammation. The signaling pathways by which chemokine receptors regulate lymphocyte migration remain incompletely characterized. We demonstrate that Jurkat T cells lacking the ZAP-70 tyrosine kinase exhibit reduced migration in response to the CXCR4 ligand CXCL12 when compared with wild-type Jurkat T cells. Expression of wild-type, but not kinase-inactive, ZAP-70 resulted in enhanced migration of ZAP-70-deficient Jurkat T cells. The tyrosine residue at position 292 in the interdomain B region of ZAP-70 exerts a negative regulatory effect on ZAP-70-dependent migration. Stimulation of Jurkat T cells with CXCL12 also resulted in ZAP-70-dependent tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) adapter protein. Although CXCL12-dependent migration of SLP-76-deficient Jurkat T cells was impaired, re-expression of SLP-76 did not enhance migration. These results suggest a novel function for ZAP-70, but not SLP-76, in CXCR4 chemokine receptor signaling in human T cells.

Regulation of β1-integrin-mediated cell adhesion by the CbI adaptor protein

BACKGROUND Leukocyte activation results in a rapid increase in adhesion to the extracellular matrix due to the activation of beta 1 integrin receptors. A role for phosphatidylinositol (PI) 3-kinase in integrin activation has been proposed, as activation of integrins by many receptors can be blocked by PI 3-kinase inhibitors. One receptor that regulates integrins is the CD28 surface antigen; here, we investigated the mechanisms responsible for CD28-mediated integrin activation. RESULTS CD28-mediated integrin activation was blocked by mutation of the binding site for the p85 catalytic subunit of PI 3-kinase in the CD28 cytoplasmic domain, and by expression of a dominant-negative form of the p85 subunit. Substitution of the Src homology 2 (SH2)-binding motif in the CD28 cytoplasmic domain for the corresponding motif in the CD28-related CTLA-4 surface antigen also blocked integrin activation but did not affect the recruitment and activation of PI 3-kinase. Mutations of the CD28 cytoplasmic domain that blocked integrin activation also impaired the tyrosine phosphorylation of the Cbl adaptor protein and the activation of the PI 3-kinase that was associated with Cbl. This Cbl-associated PI 3-kinase was distinct from the PI 3-kinase that coprecipitated with the CD28 cytoplasmic domain. CD28-mediated activation of beta 1 integrins was inhibited by expression of a mutant Cbl protein that shows reduced association with PI 3-kinase. CONCLUSIONS Cbl is required for PI-3-kinase-dependent regulation of integrin receptors by CD28. Furthermore, CD28 is coupled to two distinct pools of PI 3-kinase, one directly associated with the CD28 cytoplasmic tail and the other associated with Cbl.

Binding of Soluble Yeast β-Glucan to Human Neutrophils and Monocytes is Complement-Dependent.

The immunomodulatory properties of yeast β-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate β-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble β-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast β-glucan, this study evaluated and characterized the binding of soluble β-glucan to human neutrophils and monocytes. The results demonstrated that soluble β-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble β-glucan in these cells. Binding of soluble β-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble β-glucan was demonstrated by detection of iC3b, the complement opsonin on β-glucan-bound cells, as well as by the direct binding of iC3b to β-glucan in the absence of cells. Binding of β-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding.

Differential regulation of oxidative burst by distinct β-glucan-binding receptors and signaling pathways in human peripheral blood mononuclear cells.

β-Glucans possess broad immunomodulatory properties, including activation of innate immune functions such as oxidative burst activity. The differential roles of complement receptor type 3 (CR3) and Dectin-1, the known β-glucan receptors, and their associated signaling pathways in the generation of oxidative burst induced by different physical forms of Saccharomyces cerevisiae-derived β-glucan were examined in human peripheral blood mononuclear cells (PBMC). In this study whole glucan particle (WGP) or immobilized soluble β-glucan (ISG) was used to represent the phagocytizable or the nonphagocytizable form of a fungus, respectively. Oxidative burst as measured by the formation of superoxide (SO) was detected in PBMC in response to WGP and ISG. SO induction with WGP was concluded to be Dectin-1-mediated and required Src family kinases, phosphatidylinositol-3 kinase and protein kinase B/Akt. In contrast, the SO induction generated by ISG was CR3-mediated and required focal adhesion kinase, spleen tyrosine kinase, phosphatidylinositol-3 kinase, Akt, p38 mitogen activated protein kinase, phospholipase C and protein kinase C. The study results support the hypothesis that human PBMC, specifically monocytes, utilize distinct receptors and overlapping, but distinct, signaling pathways for the oxidative burst in response to challenge by different physical forms of β-glucan.

Imprime PGG-Mediated Anti-Cancer Immune Activation Requires Immune Complex Formation

Imprime PGG (Imprime), an intravenously-administered, soluble β-glucan, has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically, Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells, triggering a coordinated anti-cancer immune response. Herein, using whole blood from healthy human subjects, we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring, anti-β glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement, primarily via the classical complement pathway, and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa, eliciting phenotypic activation of, and enhanced chemokine production by, neutrophils and monocytes, enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly, these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together, these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy.

Imprime PGG, a yeast β-glucan immunomodulator, has the potential to repolarize human monocyte-derived M2 macrophages to M1 phenotype

Meeting abstracts Imprime PGG (IPGG) has shown promising clinical efficacy in combination with monoclonal antibody treatment of cancer [[1][1]]. In mice, complement receptor 3 (CR3) on innate immune cells (neutrophils and macrophages) is required for IPGGs anti-tumor activity [[2][2], [3][3]]. In

Abstract LB-225: Imprime PGG modulates the function of monocyte-derived M2 macrophages and dendritic cells to drive T-cell expansion

Imprime PGG is a soluble, yeast β-1,3/1,6 glucan currently in phase 3 clinical trial for the treatment of cancer in conjunction with complement-activating, therapeutic monoclonal antibodies (e.g. cetuximab). Imprime PGG is a pathogen- associated molecular pattern (PAMP) that complexes with endogenous anti-β-glucan antibodies, then binds and primes innate immune cells (including neutrophils and monocytes) to kill antibody-targeted cancer cells via a complement receptor 3-dependent mechanism. The early response to a PAMP requires innate immune effector cells (neutrophils, monocytes, macrophages, dendritic cells) and is a prerequisite for subsequent activation of adaptive immune effector cells. Given that macrophages and dendritic cells are the two key antigen presenting cell types that bridge innate and adaptive immunity, the objective of this study was to evaluate the phenotypic and functional effect of Imprime PGG on human monocyte-derived macrophages and dendritic cells (MoDC). Monocytes enriched from Imprime PGG- or vehicle-treated whole blood were cultured in media containing the appropriate cytokines for differentiation of the different cell types: GM-CSF for M1 macrohages; M-CSF for M2 macrophages; M-CSF plus IL-4 for M2a macrophages; and GM-CSF plus IL-4 for dendritic cells. Although Imprime PGG treatment did not affect the expression of CD206, CD209, CD163, HLA-DR, CD80, and CD86 on M1 macrophages, Imprime PGG treatment of whole blood did elicit a substantial reduction in surface expression of the scavenger receptor CD163 on M2 and M2a macrophages. Further, both M2 and M2a macrophages derived from Imprime PGG-treated whole blood substantially enhanced CD3/CD28-stimulated CD4 T cell proliferation and IFNγ production, whereas those from vehicle-treated whole blood did not. MoDC from Imprime PGG-treated whole blood showed increased surface expression of the maturation and co-stimulatory markers CD80, CD83, CD86 as well as HLA-DR. Furthermore, these MoDC also showed enhanced function in an allogeneic mixed lymphocyte reaction, triggering increased CD4 and CD8 T cell expansion and increased IFNγ production versus MoDC from vehicle treated whole blood. Imprime PGG9s ability to enhance M2 mediated T cell proliferation and MoDC maturation was maintained even in the presence of tumor conditioned media. These results demonstrate that Imprime PGG treatment drives a coordinated immune response, modulating the function of M2 macrophages and MoDC, enabling the expansion of CD4 and CD8 T cell effector cells, and driving Th1 polarization. These data thereby suggest the potential of combining Imprime PGG treatment with the modalities that relieve tumor-mediated T cell immunosuppression. Citation Format: Anissa SH Chan, Xiaohong Qiu, Adria Jonas, Takashi Kangas, Nadine R. Ottoson, Nandita Bose. Imprime PGG modulates the function of monocyte-derived M2 macrophages and dendritic cells to drive T-cell expansion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-225. doi:10.1158/1538-7445.AM2015-LB-225

Abstract LB-228: Imprime PGG treatment elicits a coordinated antitumor immune response that triggers enhanced expression of PD-L1 on tumor cells as well as monocyte-derived macrophages and dendritic cells

Immune checkpoint inhibitors, including anti-PD-1 and anti-PD-L1 antibodies are emerging as an important therapeutic modality in NSCLC as well as other cancers, as these therapies block the tumor-induced T cell suppression. Translational studies from clinical trials with PD-1 and PD-L1 targeted therapies have demonstrated that patients whose cancers show PD-L1 on the surface of tumor cells and infiltrating immune cells, or PD-1 on T cells (i.e. “adaptive immune resistance”) derived greatest benefit from these therapies. It has therefore been suggested that the efficacy of these anti-PD-1/PD-L1 immunotherapies could be enhanced by combinations with agents that can adaptively induce PD-L1 expression as a consequence of de novo immune responses within the tumor microenvironment. Here we show that Imprime PGG, a yeast β-1,3/1,6 glucan currently in phase 3 development in combination with complement-activating monoclonal antibodies (e.g. cetuximab), can elicit a coordinated, anti-cancer immune response that prompts a tumor response akin to adaptive immune resistance. Monocytes derived from human whole blood treated with Imprime PGG or vehicle were cultured in media with the appropriate cytokines to foster differentiation of M2 macrophages (M-CSF), M2a macrophages (M-CSF plus IL-4) or dendritic cells (GM-CSF plus IL-4). At the end of differentiation, both M2 and M2a macrophages showed lower surface expression of the scavenger receptor CD163 and elevated levels of surface PD-L1. Imprime PGG treatment also enhanced the ability of both M2 and M2a macrophages to augment CD3/ CD28-stimulated expansion of effector T cells and increased production of IFNγ. Imprime PGG similarly affected monocyte-derived dendritic cells (MoDC), eliciting increased surface expression of the maturation and antigen presentation markers CD80, CD83, CD86, HLA-DR and PD-L1. Furthermore, the MoDC increased CD4 and CD8 T cell proliferation and IFNγ production in an allogeneic mixed lymphocyte reaction. After confirming the ability of Imprime PGG to drive Th1 polarizing immunity, the supernatants from the M2/Mo-DC and T cell co-cultures were incubated with cell lines from numerous cancer types, including NSCLC, breast, pancreatic, colon, and B cell lymphoma. PD-L1 expression was substantially upregulated on A549 (NSCLC), MiaPaCa2 (pancreatic), and SKBR3 (breast) cancer cell lines but not on the colon cancer cell line (HT-29). These results demonstrate that Imprime PGG has the potential to drive PD-L1-upregulating adaptive immune responses by modulating the function of M2 macrophages and MoDC and suggest that further studies to evaluate potential combinatorial approaches of Imprime PGG with immune checkpoint inhibitors are warranted. Citation Format: Nandita Bose, Anissa SH Chan, Adria Jonas, Xiaohong Qiu, Nadine R. Ottoson, Takashi Kangas, Jeremy R. Graff. Imprime PGG treatment elicits a coordinated antitumor immune response that triggers enhanced expression of PD-L1 on tumor cells as well as monocyte-derived macrophages and dendritic cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-228. doi:10.1158/1538-7445.AM2015-LB-228

Abstract A015: Imprime PGG treatment enhances antibody-dependent cellular phagocytosis (ADCP) of tumor cells by monocyte-derived macrophages

Imprime PGG (Imprime), a soluble, yeast β-1,3/1,6 glucan is currently in clinical development for the treatment of cancer in conjunction with several therapeutic monoclonal antibodies (MAbs). Some of the MAbs that are being evaluated in combination with Imprime include, rituximab (anti-CD20) in an investigator initiated study for chronic lymphocytic leukemia, bevacizumab in a phase 2 study for non small cell lung cancer, and erbitux in a phase 3 study for metastatic colorectal cancer. Previous studies have demonstrated that Imprime, a pathogen- associated molecular pattern (PAMP) forms an immune complex with endogenous anti-β-glucan antibodies, then binds and primes innate immune cells (including macrophages, monocytes and neutrophils) to kill antibody-targeted cancer cells via a complement-dependent cellular mechanism (CDCC). The objective of this study was to evaluate the effect of Imprime on other innate immune effector mechanisms that are employed by the tumor-targeting MAbs, including antibody dependent cellular phagocytosis (ADCP)- a function largely attributed to macrophages. We therefore sought to investigate whether Imprime PGG treatment might enhance ADCP. Monocytes enriched from Imprime PGG- or vehicle-treated whole blood were cultured in media containing the appropriate cytokines for differentiation of different macrophage subtypes: GM-CSF for M1 macrophages; M-CSF for M2 macrophages; M-CSF plus IL-4 for M2a macrophages; and M-CSF plus IL-10 for M2c macrophages. ADCP was evaluated by using specific macrophage subtypes with various tumor cell lines, including Raji (Burkitt9s lymphoma) and Z138 (Mantle cell lymphoma) in the presence of rituximab, ofatumumab, and obinutuzumab (anti-CD20 MAbs), or SKBR3 (Her2 positive breast cancer) and MDA-MB-231 (Her2 negative breast cancer) in the presence of trastuzumab (anti-Her2). A flow cytometric phagocytosis assay was performed by incubating macrophages and tumor cell lines that are labeled with different flourochromes, and subsequently quantitating the phagocytosed cells that are rendered double positive. The results demonstrated that among all the different sub-types of macrophages, M1 and M2c macrophages have higher phagocytic capacity. In comparison to the vehicle control, Imprime treatment enhanced the ability of M2c macrophages to phagocytose both Raji and Z138 tumor cells in the presence of each of the anti-CD20 antibodies- rituximab, ofatumumab, and obinutuzumab. For the breast cancer cell lines, Imprime treatment enhanced the ability of M1 macrophages to phagocytose trastuzumab- decorated SKBR3 cells. Mechanistic investigation demonstrated that increased surface expression of Fc receptors, especially CD16 and CD32, on Imprime-treated macrophages may drive increased tumor cell phagocytosis. Further, preliminary data have suggested that CD47-SIRPa and FcgRIIb may regulate Imprime-mediated ADCP. Together, these data indicate that Imprime can potentiate the anti-tumor activity of tumor-targeting antibodies by enhancing macrophage-mediated ADCP. Citation Format: Nandita Bose, Adria Jonas, Xiaohong Qiu, Anissa SH Chan, Nadine R. Ottoson, Jeremy R. Graff. Imprime PGG treatment enhances antibody-dependent cellular phagocytosis (ADCP) of tumor cells by monocyte-derived macrophages. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A015.