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.

Stress proteins are intracellular chaperones that also function as immune adjuvants, linking innate and adaptive immune responses. Some stress proteins, such as heat shock protein 70, deliver their chaperoned peptides to professional antigen presenting cells and enhance ensuing immune responses up to four orders of magnitude more than peptide alone. The net result is T cell-mediated protection in animal models of viral and tumor immunity. To gain insights into how stress proteins elicit immune responses in vivo, we have tracked mouse dendritic cells activated by a fusion protein containing ovalbumin linked to heat shock protein 70 derived from Mycobacterium tuberculosis (OVA.TBhsp70). We find that lymph node resident and migrant CD11b+ dendritic cells preferentially internalize OVA.TBhsp70, leading to their activation and enhanced ability to cross-present antigen to antigen-specific CD8+ T cells. We will present data regarding the nature of the dichotomy between the responses of CD11b+ and CD8alpha+ dendritic cells to OVA.TBhsp70. (Supported by NIH grants T32CA009138 (F.O.G.) and R01CA113576 (C.A.P.).

Beta-glucan is a pathogen-associated molecular pattern recognized by a variety of innate immune cells. Beta-1,3/1,6 glucans possess numerous immune potentiating activities. In particular, antitumor activity has been demonstrated with the soluble beta glucan Imprime PGG used in combination with complement-activating antitumor monoclonal antibodies (MAb) in several tumor models. Structurally, beta-1,3/1,6 glucans can vary on the basis of chain length, types of linkages, branching, and tertiary structure. Previously, the antitumor activity of soluble beta-1,3/1,6 glucans in combination with antitumor MAb has only been reported using beta glucans derived from yeast. The primary objective of this study was to compare the antitumor activity of three structurally distinct soluble beta-1,3/1,6 glucans in vivo in a mouse syngeneic lymphoma model. Tumor-bearing mice were administered antitumor MAb plus beta glucan 2x/week for 4 weeks. Antitumor activity was based on inhibition of tumor growth at the tumor implantation site and enhanced long-term survival. In addition, critical innate immune cells responsible for antitumor activity were identified focusing on the role of neutrophils. Also, characterization of relevant human immune cells and their beta glucan receptors will be shown focusing on complement receptor 3 (CR3) and dectin-1. Beta glucan binding to immune cells and identification of relevant receptors were determined by flow cytometry using beta glucan-specific antibody and fluorescently-labeled beta glucans. Key findings are that soluble beta glucans, which differ structurally, do not induce similar antitumor activity, and antitumor activity is abrogated in granulocyte-deficient mice. Finally, soluble glucans bind to human neutrophils in a CR3-dependent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5627.

Beta-1,3/1,6 glucans (BGs) are a diverse class of PAMPs. Structurally, BGs can vary on the basis of chain length, types of linkages, branching, and tertiary structure. BGs have been reported to activate various types of innate immune cells; most studies demonstrate the use of particulate BGs activating murine immune cells. Unlike particulate BGs, most soluble BGs demonstrated minimal activities typically associated with innate immune cell activation, such as induction of cytokines. Hence, little is known regarding the types of human immune cells or receptors that engage soluble BGs, and if structurally distinct BGs differ in their cell selectivity or receptor requirements. The objective of this study was to determine the types of human immune cells that soluble BGs bind to, and to determine the receptors required for binding. Since BGs have been implicated in interacting with Dectin-1 and CR3 expressing cells, we hypothesize that human innate immune cells that express these receptors will bind soluble BGs. Specific binding of BGs was evaluated by flow cytometry on primary human immune cells and cell lines using BG specific antibody and DTAF-labeled BGs. In addition, these studies were conducted using three structurally distinct, purified, and chemically characterized BGs. Key findings demonstrate that on cells that express both CR3 and Dectin-1, CR3 is a critical receptor for binding soluble BGs and not all BGs bind equivalently to human innate immune cells.