The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. Here we showed that TIM-4 is expressed on human and mouse macrophages and dendritic cells, and both TIM-4 and TIM-1 specifically bound phosphatidylserine (PS) on the surface of apoptotic cells but not any other phospholipid tested. TIM-4(+) peritoneal macrophages, TIM-1(+) kidney cells, and TIM-4- or TIM-1-transfected cells efficiently phagocytosed apoptotic cells, and phagocytosis could be blocked by TIM-4 or TIM-1 monoclonal antibodies. Mutations in the unique cavity of TIM-4 eliminated PS binding and phagocytosis. TIM-4 mAbs that blocked PS binding and phagocytosis mapped to epitopes in this binding cavity. These results show that TIM-4 and TIM-1 are immunologically restricted members of the group of receptors whose recognition of PS is critical for the efficient clearance of apoptotic cells and prevention of autoimmunity.

CD1 activates T cells, but the functions and size of possible human T cell repertoires recognizing each of the CD1 antigen presenting molecules remain unknown. Using an experimental system that bypasses major histocompatibility complex (MHC) restriction and the requirement for defined antigens, we found that polyclonal T cells responded at higher rates to cells expressing CD1a compared to CD1b, CD1c or CD1d. Unlike invariant NKT cells, the CD1a-autoreactive repertoire contains diverse T cell receptors. Functionally, many CD1a-autoreactive T cells home to skin, where they produce interleukin 22 (IL-22) in response to CD1a on Langerhans cells. The strong and frequent responses among genetically diverse donors define CD1a-autoreactive cells as a normal part of the human T cell repertoire and CD1a as a target of TH22 cells.

CD1a autoreactive T cells are common in human blood and skin, but the search for natural autoantigens has been confounded by background T cell responses to CD1 proteins and self lipids. After capturing CD1a-lipid complexes, we gently eluted ligands, while preserving unliganded CD1a for testing lipids from tissues. CD1a released hundreds of ligands of two types. Inhibitory ligands were ubiquitous membrane lipids with polar headgroups, whereas stimulatory compounds were apolar oils. CD1a autoantigens naturally accumulate in epidermis and sebum, where they were identified as squalene and skin waxes. T cell activation by skin oils suggests that headless mini-antigens nest within CD1a and displace non-antigenic resident lipids with large head groups. Oily autoantigens naturally coat the skin's surface, pointing to a new mechanism of barrier immunity.

Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as in vitro and in vivo functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances β-catenin–mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling.

Numerous studies have pointed to the role of programmed death-1 ligand 1 (PD-L1) in regulating tolerance, chronic infection, and tumor immunity. Recently, we have identified murine B7-1 as a new binding partner for murine PD-L1. Human and mouse B7-1 share only 46% identity, leading us to question whether human B7-1 and PD-L1 can participate in a similar interaction. Here we show that human B7-1 can interact with human PD-L1 with affinity greater than that of B7-1 with CD28, but less than that of B7-1 with CTLA-4 or of PD-L1 with PD-1. We characterize a series of anti-human PD-L1 monoclonal antibodies and identify antibodies that can block interactions of PD-L1 with B7-1, PD-1, or both. Since PD-L1 and CD28 on T cells may compete for B7-1 as a binding partner and CD8 T cells may express high or low levels of CD28, we examined when PD-L1 and CD28 are co-expressed on CD8 T cells. We compared the time-course and extent of PD-L1 induction on CD8 CD28high versus CD28low T cells following stimulation with anti-CD3. We show that PD-L1 is induced to a higher level on CD28high T cells than on CD28low T cells upon activation. These results suggest that PD-L1 may play an important and undervalued role on human T cells.

The appearance of newly translated group 1 CD1 proteins (CD1a, CD1b, CD1c) on maturing myeloid DC to effective lipid antigen presenting cells. Here we show that Borrelia burgdorferi, the causative agent of Lyme disease, triggers appearance of group 1 CD1 proteins at high density on the surface of human myeloid DC during infection. Within human skin, CD1b and CD1c expression was low or absent prior to infection, but increased significantly after experimental infections and in erythema migrans lesions from Lyme Disease patients. The induction of CD1 was initiated by borrelial lipids acting through TLR-2 within minutes, but required 3 days for maximum effect. The delay in CD1 protein appearance involved a multi-step process whereby TLR-2 stimulated cells release soluble factors, which are sufficient to transfer the CD1-inducing effect in trans to other cells. Analysis of these soluble factors identified IL-1β as a previously unknown pathway leading to group 1 CD1 protein function. These studies establish that upregulation of group 1 CD1 proteins is an early event in B. burgdorferi infection and suggest a stepwise mechanism whereby bacterial cell walls, TLR activation and cytokine release cause DC precursors to express group 1 CD1 proteins.

Iatrogenic cutaneous infection with vaccinia virus (VV) and naturally occurring systemic infection with variola virus both lead to the characteristic skin "pox" lesions. Despite significant medical experience with both viruses, surprisingly little is understood about the interactions between these poxviruses and healthy resident skin cells. In recent years, it has become clear that skin plays an essential role in modulating both innate and adaptive immune responses, in part by producing and responding to a variety of cytokines and chemokines upon stimulation. Antagonists of many of these compounds are encoded in poxvirus genomes. Infection of skin cells with poxvirus may lead to a unique pattern of cytokine and chemokine production that might alter the cutaneous immune surveillance function. In this study, we infected primary cultures of human skin cells with VV and monitored antigen expression, virus replication, and cytokine production from the infected cells. While T cells, Langerhans cells, and dermal dendritic cells were infected abortively, keratinocytes, dermal fibroblasts, and dermal microvascular endothelial cells (HMVEC-d) all supported the complete virus life cycle. In contrast to the robust viral replication in fibroblasts and HMVEC-d, only limited viral replication was observed in keratinocytes. Importantly, VV infection of keratinocytes led to up-regulation of immunoregulatory and Th2 cytokines, including transforming growth factor ␤, interleukin-10 (IL-10), and IL-13. We propose that the rapid induction of keratinocyte Th2 and immunoregulatory cytokines represents a poxvirus strategy to evade immune surveillance, and the limited viral multiplication in keratinocytes may be a protective mechanism to help the immune system "win the race

To explore the feasibility of preparing a human immune globulin specific for respiratory syncytial virus (RSV) by screening plasma donors, the ability of seven RSV antibody assays to identify plasma-yielding IgG with high virus-neutralizing and animal-protective activities was compared. IgG prepared from plasma units selected by microneutralization assay had significantly higher activity in protecting mice from respiratory RSV challenge than did IgGs prepared from plasmas selected by three direct ELISAs using purified F protein, G protein, or RSV-infected cell lysate, by two competitive ELISAs with RSV neutralizing monoclonal antibodies directed to the F2 or F3 epitopes of the F protein, or by plaque reduction neutralization. Relative to IgG made from unselected plasma, microneutralization-screened IgG was enriched fivefold by plaque-reduction neutralization assays done with or without complement. The microneutralization assay identified RSV antibodies with highest animal protective activity. This assay will be useful for identifying plasma donors for the preparation of a human immune globulin with high protective activity against RSV and deserves further evaluation for prediction of protective antibody concentrations in children.