Fc Receptor Gamma Chain Research Articles

Tumor immunity and autoimmunity induced by immunization with homologous DNA.

The immune system can recognize self antigens expressed by cancer cells. Differentiation antigens are prototypes of these self antigens, being expressed by cancer cells and their normal cell counterparts. The tyrosinase family proteins are well characterized differentiation antigens recognized by antibodies and T cells of patients with melanoma. However, immune tolerance may prevent immunity directed against these antigens. Immunity to the brown locus protein, gp75/ tyrosinase-related protein-1, was investigated in a syngeneic mouse model. C57BL/6 mice, which are tolerant to gp75, generated autoantibodies against gp75 after immunization with DNA encoding human gp75 but not syngeneic mouse gp75. Priming with human gp75 DNA broke tolerance to mouse gp75. Immunity against mouse gp75 provided significant tumor protection. Manifestations of autoimmunity were observed, characterized by coat depigmentation. Rejection of tumor challenge required CD4(+) and NK1.1(+) cells and Fc receptor gamma-chain, but depigmentation did not require these components. Thus, immunization with homologous DNA broke tolerance against mouse gp75, possibly by providing help from CD4(+) T cells. Mechanisms required for tumor protection were not necessary for autoimmunity, demonstrating that tumor immunity can be uncoupled from autoimmune manifestations.

The collagen-platelet interaction.

Collagen-platelet interaction, occurring in hemostasis but also a cause of thrombosis, is a two-step process of adhesion and activation involving the sequential recognition of distinct receptors. Adhesion involves first the reversible recognition of collagen-bound von Willebrand factor by the platelet receptor complex Gp Ib/IX/V, followed by direct interaction between collagen and the platelet integrin receptor alpha2beta1, which binds to specific sequences in collagen in which the GER motif appears important. Platelet activation then follows from the recognition by the receptor Gp VI of GPP* sequences in collagen, culminating in signalling events unique to collagen as a platelet agonist: Gp VI leads via the novel platelet Fc receptor gamma-chain to p72syk and phospholipase Cgamma2.

Intestinal intraepithelial lymphocytes include precursors committed to the T cell receptor alpha beta lineage.

The majority of T cells develop in the thymus and exhibit well characterized phenotypic changes associated with their maturation. Previous analysis of intestinal intraepithelial lymphocytes (IEL) from nude mice and a variety of experimentally manipulated models led to the view that at least a portion of these cells represent a distinct T cell population that matures extrathymically. The IEL that are postulated to mature within the intestine include both T cell receptor (TCR) alpha beta- and gamma delta-bearing subpopulations. They can be distinguished from conventional thymically derived T cells in that they express an unusual coreceptor, a CD8alpha homodimer. In addition, they can utilize the Fc receptor gamma-chain in place of the CD3-associated zeta-chain for TCR signaling and their maturation depends on the interleukin 2 receptor beta-chain. Moreover, TCRalpha beta+CD8alpha alpha+ IEL are not subject to conventional thymic selection processes. To determine whether CD3(-)CD8alpha alpha+ IEL represent precursors of T cells developing extrathymically, we examined IEL from knockout mice lacking the recombination activating gene-1 (rag-1), CD3epsilon, or both Lck and Fyn, in which thymic T cell development is arrested. CD3(-)CD8alpha alpha+CD16(+) IEL from all three mutant strains, as well as from nude mice, included cells that express pre-TCRalpha transcripts, a marker of T cell commitment. These IEL from lck-/-fyn-/- animals exhibited TCR beta-gene rearrangement. However, CD3(-)CD8alpha alpha+CD16(+) IEL from epsilon-deficient mice had not undergone Dbeta-Jbeta joining, despite normal rearrangement at the TCRbeta locus in thymocytes from these animals. These results revealed another distinction between thymocytes and IEL, and suggested an unexpectedly early role for CD3epsilon in IEL maturation.

Syk and Fyn Are Required by Mouse Megakaryocytes for the Rise in Intracellular Calcium Induced by a Collagen-related Peptide

Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recently shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor gamma chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream of tyrosine phosphorylation of phospholipase Cgamma2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the G-protein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.

A Novel Association of Fc Receptor γ-Chain with Glycoprotein VI and Their Co-expression as a Collagen Receptor in Human Platelets

The mechanism by which occupancy of collagen receptors is coupled to platelet activation has been uncertain. Our group previously demonstrated that glycoprotein (GP) VI, an uncharacterized platelet membrane protein, is specifically required for collagen-platelet interaction leading to activation of protein-tyrosine kinase Syk. Since collagen stimulation of platelets has recently been found to induce tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, a signal-generating subunit of FcR, we further investigated the relationships between FcR gamma-chain and GPVI in human platelets. Our present study revealed the following. FcR gamma-chain was physically and stably associated with GPVI in human platelets; both FcR gamma-chain and GPVI were proportionally absent in GPVI-deficient platelets; GPVI cross-linking or collagen stimulation of platelets resulted in tyrosine phosphorylation of GPVI-associated FcR gamma-chain accompanied by Syk association and activation. These findings strongly suggest that the associated complex of GPVI and FcR gamma-chain is a collagen receptor featuring the signaling through immune receptors.

Amplified follicular immune complex deposition in mice lacking the Fc receptor gamma-chain does not alter maturation of the B cell response.

The role of Ag-Ab complexes (or immune complexes; ICs) in the regulation of the maturation of the B cell immune response was investigated in mice perturbed in the deposition and retention of such complexes. Loss of surface expression of Fc gammaRI and Fc gammaRIII due to targeted disruption of the common FcR gamma-chain gene results in dramatically increased deposition of ICs on follicular dendritic cells (FDCs) in germinal centers (GCs), attributed to altered clearance of circulating ICs. Despite these changes in the trapping of ICs by FDCs, serum Ab production, V gene hypermutation, isotype class switching and Ab affinity maturation are overtly unaltered. Thus, substantially augmenting B cell cognate Ag density on FDCs does not alter the outcome of the maturation of the B cell response. The significance of this finding in terms of the currently accepted model for the generation of B cell memory is discussed.

The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.

Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor gamma-chain, the tyrosine kinase Syk and phospholipase Cgamma2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor gamma-chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cgamma2 by collagen stimulation is absent in mice lacking the Fc receptor gamma-chain. Tyrosine phosphorylation of phospholipase Cgamma2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor gamma-chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor gamma-chain or Syk. The absence of Fc receptor gamma-chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus.

Platelet Activation and Signal Transduction by Convulxin, a C-type Lectin from Crotalus durissus terrificus (Tropical Rattlesnake) Venom via the p62/GPVI Collagen Receptor

Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent tyrosine phosphorylation of platelet proteins, and binding studies with 125I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor gamma chain, phospholipase Cgamma2, p72(SYK), c-Cbl, and p36-38. However, unlike collagen, pp125(FAK) and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72(SYK), showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the alpha2beta1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor gamma chain and, by a mechanism previously described in T- and B-cells, activate p72(SYK) that is critical for downstream activation of platelets.

Xenograft rejection of porcine islet-like cell clusters in immunoglobulin- or Fc-receptor gamma-deficient mice.

The aim of the present study was to evaluate the role of xenoreactive antibodies in islet-like cell cluster (ICC) xenograft rejection. For this purpose, normal mice, mice with a targeted disruption of the Fc-receptor (FcR) gamma-chain, or the membrane exon of the immunoglobulin mu-chain gene, were transplanted with fetal porcine ICC under the kidney capsule. Mice lacking the FcR gamma have no functional FcR for IgG or IgE. Mice with disruption of the immunoglobulin mu-chain cannot produce antibodies, because B cell development is arrested at the stage of preB cells. All animals, irrespective of recipient group, readily rejected the ICC xenograft. Analyses of the pattern of cellular infiltration revealed only minor dissimilarities between the different experimental groups. Xenograft destruction was evident on day 6 after transplantation, and a large number of mononuclear cells were found to be evenly distributed throughout the ICC graft. The majority of the infiltrating cells were large, macrophage-like cells expressing the macrophage-specific phenotype marker F4/80. CD3-positive T lymphocytes were found to be mainly accumulated in the peripheral parts of the ICC xenograft. This study has demonstrated that xenoreactive antibodies are not crucial to ICC xenograft rejection in the pig-to-mouse model.

Tyrosine Phosphorylation of the Fc Receptor γ-Chain in Collagen-stimulated Platelets

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor gamma-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor gamma-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor gamma-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin alpha2beta1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcgammaRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor gamma-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin alpha2beta1 and involves phosphorylation of the Fc receptor gamma-chain, its association with Syk and subsequent phosphorylation of phospholipase Cgamma2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.

Specific Interaction of Topoisomerase IIβ and the CD3ε Chain of the T Cell Receptor Complex

T cell antigen receptor (TCR)-CD3 complex is composed of six different subunits: TCR alpha and TCR beta and CD3 gamma, CD3 delta, CD3 epsilon, and CD3 eta. Antigen recognition signals are transduced from TCR to the cytoplasm through the cytoplasmic domain of the CD3 chains. To understand the downstream signal transduction pathways, we cloned genes encoding proteins capable of binding to CD3 epsilon with a probe of glutathione S-transferase fused to the cytoplasmic region of CD3 epsilon. One of these clones was found to encode topoisomerase II beta (topoII beta). The binding region of CD3 epsilon is located within the N-terminal 12 amino acids containing the motif of a basic amino acid cluster. A similar motif was found in the gamma chain of Fc receptors (FcR gamma) but not in the CD33 eta chain, and indeed, FcR gamma but not CD3 eta bound to topoII beta. The binding region of topoII beta was determined to be the C terminus. Since this region appears to be the regulatory region of the enzymatic activity, the binding of CD3 epsilon might affect the function of topoII beta. Although topoII beta is localized mainly in the nucleus and CD3E is a membrane protein, we demonstrated the presence of CD3 epsilon in the nuclear fraction of thymocytes, which increased upon T cell activation. The specific interaction in cells was evidenced by co-immunoprecipitation of topoII beta and CD3E from the nuclear fraction of T cells. The possible function of this interaction is discussed.

Functional Association between the Human Myeloid Immunoglobulin A Fc Receptor (CD89) and FcR γChain: MOLECULAR BASIS FOR CD89/FcR γCHAIN ASSOCIATION

FcR gamma chain has previously been shown to interact with the TCR-CD3 complex, the IgE Fc receptor I (Fc epsilon RI), and the class I and IIIA IgG receptors (Fc gamma RI and Fc gamma RIIIa). Here, we demonstrate that the Fc receptor gamma chain associates with Fc alpha R in transfected IIA1.6 B lymphocytes. Fc alpha R could be expressed at the surface of IIA1.6 B cells by itself, but was devoid of signaling capacity. Upon co-expression of FcR gamma chain, a physical interaction with Fc alpha R could be demonstrated. This association proved crucial for the triggering of both proximal (intracellular calcium increase and tyrosine phosphorylation), as well as distal (IL-2 release), signal transduction responses. We next tested the hypothesis that a positively charged arginine residue (Arg209) within the transmembrane domain of Fc alpha R promotes association with FcR gamma chain. We therefore constructed Fc alpha R molecules where Arg209 was mutated to either a positively charged histidine, a negatively charged aspartic acid, or an uncharged leucine. A functional association between Fc alpha R and FcR gamma chain was observed only with a positively charged residue (Arg209 or His209) present within the Fc alpha R transmembrane domain. These data show that transmembrane signal transduction by the Fc alpha R is mediated via FcR gamma chain, and that Fc alpha R requires a positively charged residue within the transmembrane domain to promote functional association.

Preferential usage of the Fc receptor gamma chain in the T cell antigen receptor complex by gamma/delta T cells localized in epithelia.

zeta and eta chains of the T cell antigen receptor (TCR) complex and the gamma chain of Fc receptors (FcR gamma) constitute a family of proteins important for the expression of, and signal transduction through, these receptors in hematopoietic cells. In zeta-deficient mice, TCR expression was reduced in most T cells. By contrast, CD8 alpha alpha + TCR-gamma/delta + intestinal intraepithelial lymphocytes in these mice expressed a normal level of TCR. Biochemical analysis of the TCR complex in these cells from zeta-deficient as well as normal mice revealed the predominant usage of FcR gamma. Furthermore, gamma/delta + T cells in epithelia of the skin and female reproductive organs from zeta-deficient mice also showed relatively high TCR expression, indicating the usage of FcR gamma. These observations demonstrate the preferential usage of FcR gamma by gamma/delta + T cells localized in epithelia of normal mice.

Lysis of ovarian cancer cells by human lymphocytes redirected with a chimeric gene composed of an antibody variable region and the Fc receptor gamma chain.

To expand the spectrum of recognition of effector lymphocytes and to redirect them towards predefined targets, we have altered the specificity of human tumor-infiltrating lymphocytes (TIL) through stable modification with chimeric receptor genes consisting of single-chain antibody variable regions linked to the gamma subunit common to the immunoglobulin (Ig)G and IgE Fc receptors. Using either hapten or ovarian carcinoma-specific monoclonal antibodies, we constructed chimeric receptor genes and retrovirally introduced them into CD8+ TIL. Redirected TIL specifically lysed trinitrophenyl-labeled Daudi or a human ovarian carcinoma cell line (IGROV-1), and secreted granulocyte/macrophage colony-stimulating factor upon stimulation with the appropriate antigen. This strategy may allow new approaches towards the adoptive immunotherapy of cancer in humans.