Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system ...that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity.
Triggering receptor expressed on myeloid cells (TREM)-1 is an orphan receptor implicated in innate immune activation. Inhibition of TREM-1 reduces sepsis in mouse models, suggesting a role for it in ...immune responses triggered by bacteria. However, the absence of an identified ligand has hampered a full understanding of TREM-1 function. We identified complexes between peptidoglycan recognition protein 1 (PGLYRP1) and bacterially derived peptidoglycan that constitute a potent ligand capable of binding TREM-1 and inducing known TREM-1 functions. Interestingly, multimerization of PGLYRP1 bypassed the need for peptidoglycan in TREM-1 activation, demonstrating that the PGLYRP1/TREM-1 axis can be activated in the absence of bacterial products. The role for PGLYRP1 as a TREM-1 activator provides a new mechanism by which bacteria can trigger myeloid cells, linking two known, but previously unrelated, pathways in innate immunity.
The proinflammatory cytokines IL-17A and IL-17F have a high degree of sequence similarity and share many biological properties. Both have been implicated as factors contributing to the progression of ...inflammatory and autoimmune diseases. Moreover, reagents that neutralize IL-17A significantly ameliorate disease severity in several mouse models of human disease. IL-17A mediates its effects through interaction with its cognate receptor, the IL-17 receptor (IL-17RA). We report here that the IL-17RA-related molecule, IL-17RC is the receptor for IL-17F. Notably, both IL-17A and IL-17F bind to IL-17RC with high affinity, leading us to suggest that a soluble form of this molecule may serve as an effective therapeutic antagonist of IL-17A and IL-17F. We generated a soluble form of IL-17RC and demonstrate that it effectively blocks binding of both IL-17A and IL-17F, and that it inhibits signaling in response to these cytokines. Collectively, our work indicates that IL-17RC functions as a receptor for both IL-17A and IL-17F and that a soluble version of this protein should be an effective antagonist of IL-17A and IL-17F mediated inflammatory diseases.
Members of the CD28 family play important roles in regulating T‐cell functions and share a common gene structure profile. We have identified VSTM3 as a protein whose gene structure matches that of ...the other CD28 family members. This protein (also known as TIGIT and WUCAM) has been previously shown to affect immune responses and is expressed on NK cells, activated and memory T cells, and Tregs. The nectin‐family proteins CD155 and CD112 serve as counter‐structures for VSTM3, and CD155 and CD112 also bind to the activating receptor CD226 on T cells and NK cells. Hence, this group of interacting proteins forms a network of molecules similar to the well‐characterized CD28–CTLA‐4–CD80–CD86 network. In the same way that soluble CTLA‐4 can be used to block T‐cell responses, we show that soluble Vstm3 attenuates T‐cell responses in vitro and in vivo. Moreover, animals deficient in Vstm3 are more sensitive to autoimmune challenges indicating that this new member of the CD28 family is an important regulator of T‐cell responses.
Abstract
We have identified a common gene structure found in all known members of the CD28 family and have used this conserved structure to include the Natural Cytotoxicity Receptor NKp30 as a CD28 ...family member. Soluble forms of NKp30 specifically bound to the chronic myelogenous leukemia cell-line K562 and inhibited cytolysis by the human NK line NK-92. This suggested the presence of a functional counter-structure for NKp30 on the surface of K562 and we employed a proteomic approach to identify this molecule. Our analysis identified an unannontated gene specifically from K562 lysates, which when transfected into P815 cells conveyed NKp30-specific binding activity and sensitivity to NKp30-mediated cytolysis by NK cells. The predicted protein and gene structure of this molecule suggested it was a member of the B7 family and we therefore designated it B7h6. Expression analysis suggested limited expression on normal tissues but expression was apparent on a subset of tumor lines and expression correlated well with sensitivity to NKp30-mediated cytolysis further supporting the notion that this molecule serves as a functional counter-structure for NKp30.
Abstract
Members of the B7-CD28 family play critical roles in the response of T cells. We have identified the transmembrane protein Vstm3 as a member of the CD28 family based on gene structure. It ...has two immunoreceptor tyrosine inhibitory motifs in its cytoplasmic domain and is expressed on NK cells and a subset of T cells. Antibodies against VSTM3 antagonize multiple aspects of T cell activation including proliferation and cytokine production indicating it plays an inhibitory role in T cell activation. We have also identified two counter-structures for this protein, the previously identified proteins CD155 and CD112, which are expressed on activated antigen presenting cells. CD155 and CD112 also bind to the activating receptor CD226 and hence this extended family of interacting proteins forms a network of molecules similar to the well characterized CD28-CTLA4-CD80-CD86 network. A soluble form of Vstm3 binds to CD155 and CD112 and disrupts their ability to trigger an activating response through CD226, and hence antagonizes immune responses in vitro and in vivo. On the other hand, an antibody that specifically blocks Vstm3 interaction with CD155 and CD112 tends to enhance T cell responses in vitro and in vivo. Finally, mice that lack Vstm3 are more sensitive to the development of autoimmune diseases. Our data indicate that this novel member of the CD28 family negatively regulates T cell responses.
The receptor (Fms) for macrophage colony-stimulating factor (M-CSF) is a member of the tyrosine kinase class of growth factor receptors. It maintains survival, stimulates growth, and drives ...differentiation of the macrophage lineage of hematopoietic cells. Fms accumulates on the cell surface and becomes activated for signal transduction after M-CSF binding and is then internalized via endocytosis for eventual degradation in lysosomes. We have investigated the mechanism of endocytosis as part of the overall signaling process of this receptor and have identified an amino acid segment near the cytoplasmic juxtamembrane region surrounding tyrosine 569 that is important for internalization. Mutation of tyrosine 569 to alanine (Y569A) eliminates ligand-induced rapid endocytosis of receptor molecules. The mutant Fms Y569A also lacks tyrosine kinase activity; however, tyrosine kinase activity is not essential for endocytosis because the kinase inactive receptor Fms K614A does undergo ligand-induced endocytosis, albeit at a reduced rate. Mutation of tyrosine 569 to phenylalanine had no effect on the M-CSF-induced endocytosis of Fms, and a four-amino-acid sequence containing Y-569 could support endocytosis when transferred into the cytoplasmic juxtamembrane region of a glycophorin A construct. These results indicate that tyrosine 569 within the juxtamembrane region of Fms is part of a signal recognition sequence for endocytosis that does not require tyrosine phosphorylation at this site and that this domain also influences the kinase activity of the receptor. These results are consistent with a ligand-dependent step in recognition of the potential cryptic internalization signal.
The c-fms gene encodes the receptor for the macrophage colony-stimulating factor (M-CSF), and its extracellular domain consists of five immunoglobulin-like subdomains. To identify which of the five ...immunoglobulin-like regions are involved in ligand binding, we polymerase chain reaction-cloned five segments of the extracellular domain of the murine c-fms gene, each starting with the normal initiation codon and containing successive additions of the immunoglobulin-like subdomains. These protein segments are designated A, B, C, D, and E and contain, from the N-terminal end, either one, two, three, four, or all five immunoglobulin-like subdomains, respectively. Each segment was expressed as a secreted soluble protein from a baculovirus expression vector in Sf9 insect cells. In addition, segments A, B, C, and E were produced as soluble alkaline phosphatase fusion proteins, as was a segment containing only the fourth and fifth immunoglobulin domains. These segments of the Fms extracellular domain were used to assess M-CSF binding by competition radioimmunoassays, plate binding immunoassays, and immunoprecipitation analyses. The results indicated that the first two N-terminal immunoglobulin-like domains did not interact with M-CSF but, in combination with the third immunoglobulin-like domain, provided high-affinity M-CSF binding. The fourth and fifth immunoglobulin-like domains near the cell membrane did not exhibit M-CSF binding and may inhibit interaction of M-CSF with the first three immunoglobulin domains. These results suggest that the three N-terminal immunoglobulin-like domains constitute the high-affinity M-CSF binding region and that the fourth and fifth immunoglobulin-like domains may perform functions other than ligand binding.
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