ABSTRACT Class switching consists in the substitution of the heavy-chain constant region of immunoglobulin M (IgM) with that of IgG, IgA, or IgE. This enables antibodies to acquire new effector functions that are crucial to combat invading pathogens. Class switching usually requires engagement of CD40 on B cells by CD40 ligand (CD40L) on antigen-activated CD4 + T cells and the production of cytokines. The process must be regulated tightly because abnormal IgG and IgA production favors the onset of autoimmunity, whereas increased switching to IgE leads to atopy. These inflammatory disorders can be triggered or exacerbated by costimulatory signals. Although thoroughly investigated on T cells, the roles of the inhibitory receptors CD85j, LAIR-1, and CD152 on B-cell functions have not been fully elucidated. In this study we show that cross-linking of the B-cell inhibitory receptors by specific monoclonal antibodies inhibits IgG and IgE production, reduces the percentage of IgG- and IgE-expressing B cells, and down-regulates interleukin 8 (IL-8), IL-10, and tumor necrosis factor alpha production. These effects were demonstrated using different B-cell stimulatory pathways (recall antigens, CD40L-transfected cells plus IL-4, and lipopolysaccharide plus IL-4). It thus appears that CD85j, LAIR-1, and CD152 play a central role for the control of IL-4-driven isotype switching.
A polyclonal CD3+4-8-WT31- cell line (termed SFG) was utilized for mice immunization in order to produce monoclonal antibodies (mAb) specific for the T cell receptor (TcR) gamma/delta. Hybrid supernatants were screened for their ability to induce SFG cells (but not conventional TcR alpha/beta + CTL lines) to kill the murine Fc receptor-positive P815 target cell line. Three hybrids, termed G1, A13 and F11, were isolated according to this screening. By indirect immunofluorescence G1 mAb reacted with 65% of SFG cells, while A13 stained 26% and F11 75% of cells. Double-fluorescence analysis revealed that G1 and A13 mAb identify two distinct, non-overlapping subsets of cells present in the SFG cell line. The reactivity of the mAb was also analyzed on a panel of representative TcR gamma/delta clones. G1 mAb reacted with 5 clones, that were also stained by the previously described BB3 mAb (recognizing the disulfide-linked form of TcR gamma/delta). These clones failed to react with A13 and delta-TCS-1 mAb (the latter of which is known to react with a non-disulfide-linked form of TcR gamma/delta). Out of six clones that reacted with A13 mAb, four were also delta-TCS-1+, whereas two were delta-TCS-1- and none of them reacted with G1, (or BB3) mAb. In contrast to the mAb above, F11 brightly stained the G1+A13- clones and more weakly the G1-A13+ clones. Moreover, F11 efficiently triggered both types of clones to kill the P815 target cells while G1 and A13 were able to trigger only G1+ or A13+ clones, respectively. None of the mAb above reacted with a large number of CD3+WT31+ clones. Antibody-induced surface antigen modulation experiments indicated that molecules recognized by G1, A13 and F11 were physically associated on cell surface with CD3 determinants. In addition, immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (performed on 125I-surface-labeled TcR gamma/delta+ clones) revealed that molecules recognized by G1, A13 and F11 displayed an apparent mol. wt. corresponding to that of CD3-associated TcR molecules, immunoprecipitated by anti-CD3 mAb from the same clones.
Abstract Immature dendritic cells (DCs) derived from freshly isolated human monocytes were used to evaluate the effect of the inhibiting receptor CD85j (leukocyte Ig-like receptor-1/ILT2) on activation induced by cross-linking of the human osteoclast-associated receptor (hOSCAR). CD85j and hOSCAR were expressed consistently at the same density on monocytes and on monocyte-derived DCs (both immature and mature). Cross-linking of hOSCAR, which activates via the FcR-associated γ-chain, induced Ca2+ flux in DCs. Concomitant cross-linking of anti-CD85j mAb abolished this early activation event. Likewise, CD85j stimulation strongly reduced IL-8 and IL-12 production by hOSCAR-activated DCs. Inhibition of DCs via CD85j also impaired their ability to enhance Ag-specific T cell proliferation induced by hOSCAR. Finally, because hOSCAR prevents apoptosis of DCs in the absence of growth/survival factors, CD85j cross-linking was able to counteract completely this antiapoptotic effect and to reduce Bcl-2 expression enhanced by hOSCAR stimulation. Thus, CD85j is an inhibiting receptor that is functional in human DCs.
The GL183 mAb was obtained by immunizing BALB/c mice with the E57 clone (CD7+CD2+CD3-CD16+CD56+) derived from human peripheral blood NK cells. In human peripheral blood, GL183-reactive cells ranged between 2 and 12% (mean 6.5%) in 10 different donors. Double fluorescence and FACS analysis showed that GL183+ cells were consistently included in the CD56+ or CD16+ cell populations. Moreover, since only a fraction of CD56+ or CD16+ cells (approximately 40%) coexpressed GL183 surface antigen, reactivity with GL183 mAb appears to define two subsets within the CD3- lymphocyte population expressing NK cell markers. Although, the majority of GL183+ cells were CD3-, approximately 1% expressed CD3 surface antigens. As shown by clonal analysis, these infrequent CD3+GL183+ cells coexpressed CD56 and CD16 antigens. Cloning of CD3-GL183+ or CD3-GL183- cell populations under limiting dilution conditions yielded clonal progenies that maintained their original surface phenotype. Therefore, expression or lack of expression of GL183 surface antigens represents a stable phenotypic property of a subset of human CD3- NK cells. Immunoprecipitation experiments and two-dimensional PAGE analysis indicated that GL183-reactive molecules were represented in different clones either by a single 58-kD chain or, more frequently, by two chains of approximately 55 and approximately 58 kD, respectively. Analysis of GL183+ or GL183- NK clones for their ability to lyse human (IGROV I) or murine (P815) tumor target cells indicated that GL183- clones were, on average, fivefold more efficient in inducing target cell lysis. GL183+ and GL183- clones produced comparable levels of TNF-alpha in response to PHA plus PMA or anti-CD16 mAb plus PMA. Importantly, production of TNF-alpha was also induced by stimulation of GL183+ clones with GL183 mAb plus PMA. These data indicated that GL183 antigen could mediate cell triggering. This concept was confirmed by the analysis of Ca2+ mobilization, as GL183 mAb induced (in GL183+ clones) increments of [Ca2+]i comparable with those induced by PHA. Moreover, GL183 mAb, or its F(ab')2 fragments, strongly enhanced the cytolytic activity of GL183+ clones against a panel of human tumor target cells, including U937, Raji, IGROV I, M14, and A549. In contrast, GL183 mAb, but not the F(ab')2 fragments, sharply inhibited the cytolytic activity of the same clones against P815, M12, and P3U1 murine target cells. In this case, the effect of GL183 mAb (inhibition) was opposite that of PHA or of stimulatory anti-CD2 or anti-CD16 mAbs, which consistently enhanced the target cell lysis.(ABSTRACT TRUNCATED AT 400 WORDS)
Increased proportions of the small lymphoid subset expressing T cell receptor (TCR) gamma delta occur in different infectious diseases, particularly in mycobacterial infections. In this study the two main subsets of TCR gamma delta+ cells in peripheral blood mononuclear cells (PBMC) of 54 patients with human immunodeficiency virus type 1 infection were analyzed. These subsets were defined by indirect immunofluorescence techniques and FACS analysis using BB3 and A13 monoclonal antibodies, which specifically react with V delta 2- and V delta 1-encoded forms of TCR gamma delta. The proportion of BB3+(V delta 2+) and A13+(V delta 1+) cells was analyzed in purified PBMC populations. Patients were stratified according to Walter Reed (WR) clinical stage. A sharp increase in percentage of A13+(V delta 1+) cells was observed in all stages of the disease. In addition, a strict correlation was found with stage of the disease and percentage of CD8+ PBMC. An inverse correlation was found with the proportion of CD4+ PBMC. An early (WR2) inversion of the V delta 2-to-V delta 1 ratio was consistently detected even before the inversion of the CD4-to-CD8 ratio.
A minor subset of T lymphocytes express a TCR composed of gamma and delta chains. This subset differs from conventional T cells for a number of phenotypic and functional characteristics. TCR gamma/delta + cells simultaneously lack both CD4 and CD8 antigens. Cloning of CD4-8-peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which efficiently lyse tumor target cells. Formal proofs have been provided that TCR gamma/delta + cells are able to recognize antigens. For example, they proliferated in response to allogeneic mixed lymphocyte culture (MLC), in addition MLC-derived TCR gamma/delta + cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The selection of monoclonal antibodies (MoAbs) specific for TCR gamma/delta molecules allowed to identify two distinct subsets of TCR gamma/delta + cells. These MoAbs, termed BB3 and delta-TCS-1 (or the equivalent A13) respectively, induced specific activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca2+ and/or lymphokine production of cytolytic activity). Analysis of the distribution of subsets expressing different forms of TCR gamma/delta, showed that the BB3-reactive form is prevalent in the peripheral blood. In contrast, delta-TCS-1-reactive cells are relatively infrequent in peripheral blood, but represent the majority of TCR gamma/delta + in the tissues.
