Immunosuppressive leukocytes are emerging as a critical factor in facilitating tumor progression. These leukocytes are converted by the tumor microenvironment to become tolerogenic, facilitate metastasis, and to aid in neovascularization. The predominant variety of suppressive leukocytes found in human and murine ovarian cancer are called vascular leukocytes (VLC), due to sharing functions and cell surface markers of both dendritic cells and endothelial cells. Using the ID8 murine model of ovarian cancer, the aim of this study was to test the efficacy of VLC elimination as an ovarian tumor therapy. We show that carrageenan-mediated depletion of peritoneal tumor-associated leukocytes inhibits ovarian tumor progression. We then identified scavenger receptor-A

Class-A scavenger receptors (SR-A) are cellular pattern recognition receptors that bind and traffic a variety of endogenous and microbial ligands. However, despite an emerging role for SR-A as a contributor to the innate immune system, little is known of the regulation or function of SR-A on dendritic cells (DCs). Here we show that SR-A expression is upregulated during murine DC differentiation and that SR-A expression levels correlate with the expression of the murine DC marker CD11c. Using bone marrow-derived DCs (BMDCs) from SR-A knockout (SR-A(-/-)) mice, we investigated the contribution of SR-A to BMDC particulate phagocytosis. Functional analyses demonstrated that SR-A is a critical phagocytic receptor for BMDC internalization of the gram-negative bacteria E. coli. SR-A(-/-) BMDCs were impaired in their ability to phagocytose bacteria, and this deficit varied with the bacteria:BMDC cell ratio. Microscopic and biochemical analyses revealed that SR-A is broadly distributed on the surface of BMDCs and is not physically associated with lipid rafts. However, cholesterol depletion demonstrated dependence of SR-A-mediated phagocytosis upon lipid rafts. These data demonstrate a functional contribution for SR-A in the BMDC phagocytic pathway.

Calreticulin (CRT) interaction with cell-surface receptors is integral to its function in escorting associated peptides into the antigen-presenting cell (APC) antigen presentation pathway. Additionally, extracellular CRT is proposed to be required for lung APC interaction with collectins. In both cases, CD91 has been proposed to act as the APC cell-surface receptor requisite for mediating these processes. However, the evidence for a CRT interaction with CD91 is indirect, predicated on partial competition of cellular binding by gp96, of which CD91 has been proposed as the unique endocytic receptor, and by the CD91 ligand a 2 -macroglobulin. Here, we directly investigate the function of CD91 in binding and trafficking CRT. We find that the ability of CRT to interact with APC does not correlate with cellular CD91 expression or function. Additionally, in the first genetic test of CD91 function regarding CRT, CD91 expression neither conferred CRT association nor did CD91-deficient (CD91 -/-) and CD91-expressing cells differ in their ability to traffic CRT. Finally, cellular CRT trafficking did not parallel that of Pseudomonas exotoxin-A, an obligate CD91 ligand, by the criteria of CD91 dependence, cell-type specificity and endocytic itinerary. These data identify that CRT trafficking is not, as previously hypothesized, CD91 dependent and indicate usage of alternative cellular trafficking pathways.

Summary CD8+ T cells (TCD8+) differentiate into effector cells following recognition of specific peptide–major histocompatibility complex (MHC) class I complexes (pMHC‐I) on the surface of professional APCs (pAPCs), such as dendritic cells. Antigenic pMHC‐I can be generated from two spatially distinct sources. The direct presentation pathway involves generation of peptide from protein substrate synthesized within the cell that is presenting the pMHC‐I. Alternatively, the cross presentation pathway involves presentation of antigen that is not synthesized within the presenting cell, but is derived from exogenous proteins synthesized within other donor cells. The mechanisms by which cross presentation of exogenous antigens occur in vivo remain controversial. The C‐type lectin scavenger receptor A (SR‐A) has been implicated in a number of potential cross presentation pathways, including the presentation of peptide bound to heat shock proteins, such as glycoprotein 96 (gp96), and the transfer of pMHC‐I from a donor cell to the pAPC. We demonstrate here that initiation of TCD8+ responses is normal in mice lacking SR‐A, and that the redundancy of ligand binding exhibited by the SR family is likely to be an important mechanism that ensures cross presentation in vivo. These observations emphasize the requirement to target multiple receptors and antigen‐processing pathways during the rational design of vaccines aimed at eliciting protective TCD8+.

Molecular chaperones stimulate the immune system to induce both protective immune responses and therapeutic tumor rejection. However, the underlying basis for this immunogenic activity is not well understood. A variety of chaperones, including calreticulin, hsp70 and grp94, function as vehicles to efficiently traffic associated peptides into professional antigen presenting cells. Importantly, these chaperones have also been proposed to function as adjuvants by stimulating the dendritic cell activation and co-stimulatory responses required to elicit peptide-specific CD8 + T cell cytolytic activity. The efficacy of chaperone-mediated tumor rejection has been attributed to the ability of chaperones to function in both of these capacities. However, purified calreticulin has not previously been assessed for its ability to elicit DC maturation and, moreover, recent data indicates that it is not efficient at inducing Nf-κB activity which often accompanies or stimulates DC maturation. Here we use two complementary methods to produce endotoxin-free calreticulin and demonstrate that it does not measurably mature or activate dendritic cells both in vitro and in vivo. Additionally, a calreticulin/ peptide complex required the addition of an exogenous adjuvant to elicit in vivo cytotoxic CD8 + T cell responses. These data are discussed with respect to current models for chaperone-derived immune responses and in regard to rational vaccine design.