SummaryThe lymphocyte differentiation marker CD8 acts as a coreceptor with the T cell receptor (TCR) during recognition of peptide presented by major histocompatibility complex (MHC) class I molecules. The functions of CD8 in the TCR complex are thought to be signaling through the association of CD8 with the protein tyrosine kinase p56kk and adhesion to MHC class I through the a3 domain. While the ability of the CD8 a/cx homodimer to bind to classical MHC class I molecules has been shown, it is unclear whether CD8 can also bind nonclassical molecules. Ofparticular interest is human histocompatibility leukocyte antigen (HLA)-G which is expressed on placental cytotrophoblast cells. These cells do not express HLA-A, -B and -C molecules. In this report, we demonstrate that CD8 can bind to HLA-G. It is possible, therefore, that a cell bearing CD8 may interact with HLA-G-expressing cells. I n humans three nonclassical MHC class I molecules have been described human histocompatibility leukocyte antigen (HLA)-E (1, 2), -F (3), and -G (4) which map to the MHC complex and which encode expressible proteins. While mRNA for all three genes has been detected in a number of cell lines (5), only expression of HLA-G on the cell surface was reported (6, 7). Because there are no specific antibodies against HLAE, -F, and -G, specific expression of HLAG can only be identified by analysis of labeled cell surface proteins on two-dimensional polyacrylamide gels (6) . By this approach, HLA-G was the only HLA molecule expressed on fetal cytotrophoblast cells (6) . Cytotrophoblasts invade the maternal decidualized endometrium and are directly exposed to maternal lymphoid cells yet the placenta is not normally rejected by the mother as an allograft . Thus, the ability of HLA-G to be recognized by immune cells is unclear.Because CD8 can bind to MHC class I molecules including HLAA2, we decided to determine whether CD8 could recognize and bind to HLA-G as well . Salter et al . (8) recently identified a conserved negatively charged loop on the MHC class I a3 domain that is important for binding to CD8. Single amino acid substitutions at each position in this loop from residues 223-229 severely reduced binding to CD8 in a cellcell adhesion assay. At position 228 within this loop HLAG has a valine instead of the conserved threonine found in HLA-A, B, and C. Based on this difference as well as other differences, it would not be predicted from sequence comparisons that CD8 would bind to HLA-G. To determine whether CD8 could bind to HLA-G, we obtained a cell line, LCL 221, that was null for expression of classical HLA class I molecules HLA-A, -B, -C and transferents of this cell line that expressed either HLA G or HLAA2 (6) . Using a cell-cell adhesion assay, we found that CD8 could bind to HLAG and that the binding was similar to the binding to HLA-A2 . Materials and MethodsCell Lines. The lymphoblastoid cell lines: the HLA class I null mutant 221 and HLA-A2 and HLA-G transfectants of 221 were obtained from Dr. Robert DeMars . The HLAG and HLA-A...
SummaryAn IgM-binding protein of -60 kD has been identified on activated B cells, but not on resting and activated T cells, monocytes, or granulocytes. Here, we characterize this IgM-binding protein as a receptor for the Fc portion (CH3 and/or CH4 domains) of IgM molecules (FcAR) . The Fct.R can be expressed as a cell surface activation antigen throughout the pre-B and B cell stages in differentiation . Receptor expression is not directly linked with IgM production, as both u-pre-B cells and isotype-switched B cells may express the FcjiR. The receptor molecules produced by both pre-B and B cells are identical in size and are characterized as an acidic sialoglycoprotein with 0-linked, but no Winked, oligosaccharide. The FcIAR is anchored to the surface of B-lineage cells via a glycosyl phosphatidylinositol linkage. The Fcp,R is thus the third member of a family of Fc receptors expressed on B-lineage cells, and its preferential expression on activated B cells suggests a potential role in the response to antigens .
Human leukocytes fixed in suspension were allowed to settle onto poly-L-lysine-coated glass coverslips and prepared for observation with the scanning electron microscope (SEM). The coverslips were dehydrated in ethanol, critical point dried with CO2, and coated with gold-palladium. With the aid of a locator grid, several fields were photographed with light microscopy after the cells had settled onto the poly-L-lysine-coated coverslips and again after completion of the processing before SEM observation. Quantitative comparison of the number of cells present after settling with the number retained for final viewing with the SEM revealed a cell yield approaching 100%. This simple, reproducible, high-yield technique for processing cells fixed in suspension for SEM prevents changes in surface architecture induced by collecting live cells onto various substrates before fixation and also avoids potentially selective cell losses. Such a technique should allow quantitative correlations between SEM and other morphological and functional parameters.
IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both preimmune “natural” and antigen-induced “immune” IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (FcμR). We have recently identified a bona fide FcμR in both humans and mice. In this article we briefly review what we have learned so far about FcμR.
The monocyte chemotactic protein-1 (MCP-1) receptor (MCP-1R) is expressed on monocytes, a subpopulation of memory T lymphocytes, and basophils. Two alternatively spliced forms of MCP-1R, CCR2A and CCR2B, exist and differ only in their carboxyl-terminal tails. To determine whether CCR2A and CCR2B receptors function similarly, Jurkat T cells were stably transfected with plasmids encoding the human CCR2A or CCR2B gene. Nanomolar concentrations of MCP-1 induced chemotaxis in the CCR2B transfectants that express high, intermediate, and low levels of MCP-1R. Peak chemotactic activity was shifted to the right as receptor number decreased. Five-fold more MCP-1 was required to initiate chemotaxis of the CCR2A low transfectant, but the peak of chemotaxis was similar for the CCR2A and CCR2B transfectants expressing similar numbers of receptors. MCP-1-induced chemotaxis was sensitive to pertussis toxin, implying that both CCR2A and CCR2B are Giα protein coupled. MCP-1 induced a transient Ca2+ flux in the CCR2B transfectant that was partially sensitive to pertussis toxin. In contrast, MCP-1 did not induce Ca2+ flux in the CCR2A transfectant. Since MCP-1 can stimulate chemotaxis of the CCR2A transfectant without inducing Ca2+ mobilization, Ca2+ flux may not be required for MCP-1-induced chemotaxis in the Jurkat transfectants. These results indicate that functional differences exist between the CCR2A and CCR2B transfectants that can be attributed solely to differences in the carboxyl-terminal tail.
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