Biomedical subjects

A Aruffo

Publications and source records attributed to A Aruffo.

At least 91 records · Page 5Linked to original sources

PubMed1995

The human gp39 promoter. Two distinct nuclear factors of activated T cell protein-binding elements contribute independently to transcriptional activation.

gp39, a cytokine expressed on the surface of activated T cells, is essential for T cell-dependent antibody responses in vivo. We cloned and sequenced 1.2 kilobases of the 5' flank region of the human gp39 gene promoter and determined its transcription start site. When used in reporter gene assays, this DNA segment conferred promoter activity in response to T cell activation. gp39 promoter function in transfectants was inhibited by cyclosporin A, as is expression of the endogenous gp39 gene in T-lineage cells. At least 0.5 kilobase of the 5' flank region was required for promoter activity. Two putative binding sites for the NF-AT family of transcriptional activator proteins were identified at -259 to -265 and -62 to -69 with respect to the transcription start site. Both sites contributed significantly and independently to promoter activity in response to T cell activation. Additionally, when incubated in vitro with nuclear protein purified from activated human CD4 T cells, both of these sites preferentially bound the NF-AT family member, NF-ATp. These results suggest that NF-ATp, via binding to at least two cis-elements, is essential for the induction of gp39 gene expression in response to T cell activation.

Base Sequence↗

PubMed1995

A template for generation and comparison of three-dimensional selectin models.

We have complemented multiple sequence alignments of the lectin domains of the selectins with an analysis of structurally invariant regions in X-ray structures of the mannose-binding protein (MBP) and E-selectin. The analysis shows that regions of structural conservation between MBP and E-selectin extend beyond regions of rigorous sequence conservation within the selectin family and suggests that reliable three-dimensional models of selectins from different species can be generated by modification of only a few backbone segments in E-selectin. A model of the L-selectin lectin domain is built and discussed with regard to observed differences in selectin specificity.

Amino Acid Sequence↗

PubMed1995

Stimulation of CD40 with purified soluble gp39 induces proinflammatory responses in human monocytes.

CD40 is a glycoprotein of about 50 kDa that plays a crucial role in B cell growth and differentiation. It is found on the surface of B cells, follicular dendritic cells, monocytes, and some endothelial, epithelial, and carcinoma cells. Engagement of CD40 with anti-CD40 mAbs, gp39 expressed on the cell surface or soluble forms of gp39, primes B cells to efficiently respond to subsequent stimulatory signals leading to B cell proliferation, differentiation, and isotype switching. Peripheral monocytes also express CD40 on the cell surface and expression in increased following treatment with IFN-gamma. Using a soluble murine CD8/human gp39 fusion protein (sgp39) we have found that CD40 plays a crucial role in the regulation of monocyte function. Stimulation of human peripheral monocytes with sgp39 induced homotypic aggregation and significantly increased the expression of several cell-surface proteins including CD54, MHC class II, CD86, and CD40. Soluble gp39 also dramatically enhanced monocyte survival, preventing the onset of apoptosis that normally occurs upon withdrawal of serum. Finally, in the absence of any costimulatory molecules, sgp39 stimulated monocytes to produce TNF-alpha, IL-1 beta, IL-6, and IL-8. These results suggest that ligation of CD40 on human monocytes induces phenotypic changes that would be expected to influence T cell activation by the monocyte and also to enhance or prolong inflammatory responses.

Animals↗

PubMed1995

Trypanosoma cruzi amastigote adhesion to macrophages is facilitated by the mannose receptor.

Trypanosoma cruzi is an obligate intracellular protozoan parasite. The mammalian stage of the parasite life cycle describes amastigotes as an intracellular form that replicates, and trypomastigotes as an extracellular form that disseminates and invades cells. Recent studies, however, have demonstrated that amastigotes circulate in the blood of infected mammals and can invade mammalian cells. In this report, a T. cruzi surface glycoprotein gene, SA85-1.1, was expressed as an immunoglobulin chimera, and this recombinant globulin was used to screen normal mouse tissues for adhesive interactions. This approach identified a subset of macrophages in the skin and peripheral lymph node that bind the T. cruzi surface glycoproteins through the mannose receptor. To further examine the T. cruzi mannose receptor carbohydrate ligands, the interaction between T. cruzi and the mannose-binding protein, a mammalian lectin with similar carbohydrate binding specificities as the mannose receptor, was examined. These studies demonstrated that the mannose-binding protein recognized amastigotes, but not trypomastigotes or epimastigotes, and suggested that amastigotes would also be recognized by the mannose receptor. Therefore, amastigote adhesion to macrophages was investigated, and these experiments demonstrated that the mannose receptor contributes to amastigote adhesion. The data identify the first mammalian lectins that bind to T. cruzi, and are involved in T. cruzi invasion of mammalian cells. The data suggest that amastigotes and trypomastigotes may have developed different mechanisms to adhere to and invade host cells. In addition, it has been established that IFN-gamma-activated macrophages express low levels of the mannose receptor and are trypanocidal; this suggests that the interaction between amastigotes and the mannose receptor enables amastigotes to increase their adherence with a population of macrophages that are nontrypanocidal and permissive for their intracellular replication.

Animals↗

PubMed1995

An essential role for gp39, the ligand for CD40, in thymic selection.

The interactions between CD40 on B cells and its ligand gp39 on activated T helper cells are known to be essential for the development of thymus-dependent humoral immunity. However, CD40 is also functionally expressed on thymic epithelial cells and dendritic cells, suggesting that gp39-CD40 interactions may also play a role in thymic education, the process by which self-reactive cells are deleted from the T cell repertoire. Six systems of negative selection were studied for their reliance on gp39-CD40 interactions to mediate negative selection. In all cases, when the antigen/superantigen was endogenously expressed (in contrast to exogenously administered), negative selection was blocked by loss of gp39 function. Specifically, blockade of gp39-CD40 interactions prevented the deletion of thymocytes expressing V beta 3, V beta 11, and V beta 12, specificities normally deleted in BALB/c mice because of the endogenous expression of minor lymphocyte-stimulating determinants. Independent verification of a role of gp39 in negative selection was provided by studies in gp39-deficient mice where alterations in T cell receptor (TCR) V beta expression were also observed. Studies were also performed in the AND TCR transgenic (Tg) mice, which bear the V alpha 11, V beta 3 TCR and recognize both pigeon cytochrome c (PCC)/IEk and H-2As. Neonatal administration of anti-gp39 to AND TCR Tg mice that endogenously express H-2As or endogenously produce PCC prevented the deletion of TCR Tg T cells. In contrast, deletion mediated by high-dose PCC peptide antigen (administered exogenously) in AND TCR mice was unaltered by administration of anti-gp39. In addition, deletion by Staphylococcus enterotoxin B in conventional mice was also unaffected by anti-gp39 administration. gp39 expression was induced on thymocytes by mitogens or by antigen on TCR Tg thymocytes. Immunohistochemical analysis of B7-2 expression in the thymus indicated that, in the absence of gp39, B7-2 expression was substantially reduced. Taken together, these data suggest that gp39 may influence negative selection through the regulation of costimulatory molecule expression. Moreover, the data support the hypothesis that, for negative selection to some endogenously produced antigens, negative selection may be dependent on TCR engagement and costimulation.

Amino Acid Sequence↗

PubMed1995

Cloning of the murine counterpart of the tumor-associated antigen H-L6: epitope mapping of the human and murine L6 antigens.

The murine monoclonal antibody (mAb) L6 was raised against human lung carcinoma cells and found to recognize an antigen which is highly expressed on lung, breast, colon, and ovarian carcinomas. Promising results in phase 1 clinical studies with this antibody or its chimerized counterpart suggest the antigen recognized by mAb L6 (H-L6) is an attractive target for monoclonal antibody-based cancer therapy. Further development of L6 as an anti-tumor-targeting agent would benefit from the development of a murine model. However, initial attempts to develop such a model were hampered by our inability to generate antibodies against the murine homologue of the L6 antigen, M-L6. Here we describe the preparation of the mAb 12A8, which was raised against murine thymic epithelial cells, the tissue distribution of the murine antigen recognized by 12A8, the cloning of a cDNA encoding the 12A8 target antigen, and the demonstration that this antigen is M-L6. Using H-L6/M-L6 chimeric proteins, we show that the region of the M-L6 protein recognized by mAb 12A8 corresponds to the region of H-L6 recognized by mAb L6. There are five amino acid differences in the regions of the H-L6 and M-L6 proteins recognized by L6 and 12A8, respectively. We further mapped the protein epitope recognized by L6 by individually exchanging each of these residues in H-L6 with the corresponding residue found in M-L6. Substitution of the single H-L6 residue Leu122 with Ser resulted in the H-L6 mutant HL6-L122S which failed to bind L6. The HL6-L122S mutant also failed to bind 12A8.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Differential colon cancer cell adhesion to E-, P-, and L-selectin: role of mucin-type glycoproteins.

E-, P-, and L-selectin support the adhesion of leukocytes to the vessel wall through the recognition of specific carbohydrate ligands, which often contain sialylated, fucosylated lactosamines such as sialyl Lewis x [sLex; Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-]. E-selectin expressed by activated endothelium has been shown to support the adhesion of sLex-bearing colon cancer cells. In the present study, we examine the interactions of multiple colon cancer cell lines with all three selectins. Three colon cancer cell lines (LS 180, T84, and COLO 205) bound to recombinant purified E-, P-, and L-selectin. The colon cancer line COLO 320 bound to P- and L-selectin but not E-selectin; conversely, HT-29 cells bound E-selectin but not P- and L-selectin. Caco-2 showed little or no interaction with any of the three selectins. Treatment of the cells with O-sialoglycoprotease from Pasteurella haemolytica, an enzyme that selectively cleaves mucin-type O-linked glycoproteins, reduced binding to purified P- and L-selectin in all cases. In addition, recombinant soluble P- and L-selectin bound to affinity-purified mucins from all adherent tumor cell lines. Of the four tumor cell lines that interacted with E-selectin, O-glycoprotease treatment substantially diminished adhesion of LS 180 and T84, had little effect on COLO 205, and failed to inhibit the binding of HT-29. As predicted by these data, E-selectin showed substantial binding only to mucins purified from LS 180 and T84. These findings suggest that L- and P-selectin interact primarily with mucin-type ligands on colon cancers, whereas E-selectin can recognize both mucin and nonmucin ligands. Binding of the colon cancer lines to purified selectins correlates with their adhesion to activated endothelial cells (E-selectin-dependent), platelets (P-selectin-dependent), and neutrophils (L-selectin-dependent). These differential tumor cell-selectin interactions may influence metastatic spread and may also contribute to the observed variability in host response to tumor progression.

Animals↗

PubMed1995

Ig domains 1 and 2 of murine CD22 constitute the ligand-binding domain and bind multiple sialylated ligands expressed on B and T cells.

Baby hamster kidney cells transfected with murine CD22 (mCD22) mediate adhesion to B- and T-lineage cells. To further characterize mCD22-mediated cell adhesion, we generated a panel of recombinant globulins (Rg) consisting of different extracellular Ig-like (Ig) domains of mCD22. FACS analysis using these mCD22.Rgs revealed that ligands for mCD22 are expressed on both B and T cell lines and also normal B and T cells. In B-lineage cells, the expression of mCD22 ligands began on sIgM- pre-B cells in bone marrow. The ligand-binding site of mCD22 for ligands was mapped to Ig domains 1 and 2: mCD22.Rgs containing Ig domains 1 and 2 bound target cells and immunoprecipitated sets of glycoproteins similar to Rgs containing Ig domains 1 to 3 or all 7 CD22 Ig domains, whereas Rgs containing Ig domains 2 to 3 or 3 to 7 did not bind either B or T cells. Furthermore, B cells apparently expressed higher levels of mCD22 ligands than that of T cells, suggesting a potential competition for CD22 binding between ligands expressed on the same B cell and those expressed on another B cell or T cells. Immunoprecipitation experiments using the mCD22.Rgs identified mCD22 itself and the B cell-specific isoform of mCD45RA (B220) as two of the mCD22 ligands expressed on B cells. Thus, mCD22 may potentially regulate B cell activation through interactions with itself or mCD45RA/B220.

Amino Acid Sequence↗

PubMed1995

Both extracellular immunoglobin-like domains of CD80 contain residues critical for binding T cell surface receptors CTLA-4 and CD28.

The B7-related molecules CD80 and CD86 are expressed on antigen-presenting cells, bind the homologous T cell receptors CD28 and CTLA-4, and trigger costimulatory signals important for optimal T cell activation. All four molecules are immunoglobulin superfamily members, each comprising an extracellular Ig variable-like (IgV) domain, with CD80 and CD86 containing an additional Ig constant-like (IgC) domain. Despite limited sequence identity, CD80 and CD86 share similar overall receptor binding properties and effector functions. We have identified, by site-directed mutagenesis of soluble forms of CD80 and CD86, residues in both the IgV and IgC domains that are important for CTLA4Ig and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11 amino acids that support receptor binding. Many of these residues are conserved in the B7 family, are hydrophobic, and approximately map to the GFCC'C" beta-sheet face of an IgV fold. Mutagenesis of corresponding residues in CD86 established that some, but not all, of these residues also played a role in CD86 receptor binding. In general, mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby indicating that both receptors bind to overlapping sites on CD80 and CD86. Further, mutagenesis of several conserved residues in the ABED beta-sheet face of the IgC domain of CD80 completely ablated receptor binding. Point mutagenesis had a more pronounced effect than complete truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to B7 molecules is dependent upon residues in the GFC'C" face of the IgV domain and the ABED face of the IgC domain.

Abatacept↗

PubMed1995

Analysis of gp39/CD40 interactions using molecular models and site-directed mutagenesis.

The interaction between gp39 (CD40L, TRAP, T-BAM) on activated T cells and mast cells and CD40 on antigen-presenting cells modulates immune responses. Gp39 and CD40 are homologous to tumor necrosis factor (TNF) and its receptor (TNFR), respectively. The TNF-beta/TNFR interaction has been analyzed on the basis of mutagenesis experiments and crystal structures. Using the interaction of TNF-beta/TNFR as a guide, we previously reported a site-directed mutagenesis study in which we identified residues in gp39 (K143, Y145) and CD40 (Y82, D84, N86) involved in gp39/CD40 interactions. Here we describe the use of the TNF-beta/TNFR complex crystal structure as a template to prepare molecular models of gp39, CD40, and their approximate interaction. The application of these models has allowed us to extend our mutagenesis analysis of gp39/CD40 interactions. These experiments have led to the identification of additional gp39 (Y146, R203, Q220) and CD40 (E74, E117) residues that contribute to the gp39/CD40 interaction. We also further explored the importance of gp39 residue Y145 and CD40 residue Y82 for the gp39/CD40 interaction by conservatively replacing these residues with Phe. The results of these studies have enabled us to approximately outline the binding sites in gp39 and CD40. It appears that the gp39/CD40 interaction is centered on at least two clusters of residues and involves residues of two adjacent gp39 monomers. The molecular regions involved in the gp39/CD40 interaction essentially correspond to those in the homologous TNF-beta/TNFR system.

Amino Acid Sequence↗

PubMed1995

The membrane-proximal scavenger receptor cysteine-rich domain of CD6 contains the activated leukocyte cell adhesion molecule binding site.

Binding studies with a CD6 immunoglobulin fusion protein (CD6 Rg) resulted in the identification and cloning of a CD6 ligand. This ligand was found to be a member of the immunoglobulin supergene family and was named ALCAM (activated leukocyte cell adhesion molecule). Cell adhesion assays showed that CD6-ALCAM interactions mediate thymocyte-thymic epithelium cell binding. ALCAM is also expressed by activated leukocytes and neurons and may be involved in interactions between T cells and activated leukocytes and between cells of the immune and nervous systems, respectively. Herein we describe the preparation of domain-specific murine CD6 Rg fusion proteins and show that the membrane-proximal SRCR (scavenger receptor cysteine-rich) domain of CD6 contains the ALCAM binding site. We also show that mAbs which bind to this domain preferentially block CD6-ALCAM binding. These results demonstrate that the membrane-proximal SRCR domain of CD6 is necessary for CD6 binding to ALCAM and provide the first direct evidence for the interaction of an SRCR domain with a ligand.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed1995

Expression of functional CD40 by vascular endothelial cells.

The interaction between activated vascular endothelium and T cells has been shown to play an important role in the recruitment and activation of T cells at sites of inflammation. Here we report the expression of CD40 by vascular endothelial cells and its regulation by inflammatory agents. Using the soluble recombinant CD40 ligand, sgp39, we show that the interaction of CD40 with its ligand can lead to endothelial cell activation, which in turn leads to leukocyte adhesion. This adhesion is partly mediated by the expression of E-selectin. In addition to E-selectin expression, sgp39 induces the expression of intercellular adhesion molecule 1 and augments the tumor necrosis factor alpha-induced expression of vascular cell adhesion molecule 1. The effects of sgp39 on endothelial cells can be blocked with anti-gp39 monoclonal antibody (mAb), anti-CD40 mAb, or soluble CD40. Staining of tissues from healthy human skin using anti-CD40 mAb showed very weak expression of CD40 by the endothelium, while skin involved in inflammatory disease showed marked upregulation of CD40 expression. These studies suggest that interactions between cell surface proteins expressed by activated T cells with their receptors on vascular endothelium can stimulate the vasculature at sites of inflammation and may be involved in normal inflammatory responses and in inflammatory disease.

Antibodies, Monoclonal↗

PubMed1995

HER4 receptor activation and phosphorylation of Shc proteins by recombinant heregulin-Fc fusion proteins.

Heregulins (HRGs) are mosaic glycoproteins that bind to and induce the tyrosine phosphorylation of the HER4/p180erbB4 receptor. This work was aimed at studying the biological effects induced by recombinant epidermal growth factor (EGF)-like domains of HRGs as well as identifying intracellular molecules involved in HER4 signaling. To this end, we cloned the EGF-like domains of HRG-alpha, -beta 2, and -beta 3 into a eukaryotic expression vector in frame with sequences encoding a thrombin cleavage site followed by the Fc portion of a human IgG1. These chimeric genes directed the expression of recombinant fusion proteins, rHRGs-T-Fc, which specifically stimulated the phosphorylation of HER4/p180erbB4. We also show that rHRG-alpha-T-Fc bound to human breast cancer cells that express HER4 receptors and induced the expression of intercellular adhesion molecule-1. After thrombin protease cleavage of rHRGs-T-Fc, their EGF-like domains were purified and shown to stimulate protein phosphorylation in HER4-expressing cells. Moreover, the rHRG-beta 2 EGF-like domain markedly induced the phosphorylation of Shc proteins on tyrosine, suggesting a role for these adaptor molecules in HRG-mediated signaling.

Amino Acid Sequence↗

PubMed1995

Effects of chemical modification on the binding activities of P-selectin mutants.

P-Selectin (GMP140, CD62P, PADGEM), a 140-kDa glycoprotein found on activated platelets and endothelial cells, is involved in one of the early events in the inflammatory response due to its role in initiating the recruitment of circulating leukocytes. From a three-dimensional model of P-selectin and site-specific mutagenesis studies, a number of residues were previously identified as critical for the binding of P-selectin to HL-60 cells, a human myeloid cell line. Included among them were lysines 111 and 113 (K111 and K113). In this study, the roles of K111 and K113 were further characterized by the generation and specific chemical modification of two cysteine mutants, K111C and K113C, of a P-selectin-immunoglobulin fusion protein (P-selectin-Rg). Both K111C and K113C displayed significantly reduced binding activity compared to the wild-type P-selectin from which they were derived, further illustrating the importance of these particular lysines for ligand binding. Reaction of K111C with aziridine or nipsylcysteamine resulted in the formation of K111C-AZ and K111C-CY, both of which displayed significant increases in HL-60 binding activity. No such increase took place upon reaction of K111C with N-ethylmaleimide, indicating that a free amine at position 111 is important for binding. Residue length at position 111 is not critical, since the synthetic side chains are 0.5-2.0 A longer than lysine yet still impart binding activity. Similar modification studies of K111A and K113C did not lead to any detectable increase in binding of these proteins to HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Identification and characterization of a 100-kD ligand for CD6 on human thymic epithelial cells.

CD6 is a 130-kD glycoprotein expressed on the surface of thymocytes and peripheral blood T cells that is involved in TCR-mediated T cell activation. In thymus, CD6 mediates interactions between thymocytes and thymic epithelial (TE) cells. In indirect immunofluorescence assays, a recombinant CD6-immunoglobulin fusion protein (CD6-Rg) bound to cultured human TE cells and to thymic fibroblasts. CD6-Rg binding to TF and TE cells was trypsin sensitive, and 54 +/- 4% of binding was divalent cation dependent. By screening the blind panel of 479 monoclonal antibodies (mAbs) from the 5th International Workshop on Human Leukocyte Differentiation Antigens for expression on human TE cells and for the ability to block CD6-Rg binding to TE cells, we found one mAb (J4-81) that significantly inhibited the binding of CD6-Rg to TE cells (60 +/- 7% inhibition). A second mAb to the surface antigen identified by mAb J4-81, J3-119, enhanced the binding of CD6-Rg to TE cells by 48 +/- 5%. Using covalent cross-linking and trypsin digestion, we found that mAb J4-81 and CD6-Rg both bound to the same 100-kD glycoprotein (CD6L-100) on the surface of TE cells. These data demonstrate that a 100-kD glycoprotein on TE cells detected by mAb J4-81 is a ligand for CD6.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed1995

Identification of residues on CD40 and its ligand which are critical for the receptor-ligand interaction.

Interactions between gp39 (CD40L, TRAP, T-BAM) on activated T cells and CD40 on antigen-presenting cells play an important role in regulating antibody production by B cells, cytokine production by monocytes, and other immune responses which require T cell "help". Using structure-based sequence alignments, a molecular model of gp39, site-directed mutagenesis, and receptor-ligand binding assays, we have identified CD40 and gp39 surface residues which are important for receptor-ligand binding. Binding studies with CD40 or gp39 proteins containing single and double amino acid substitutions showed that CD40 residues Y82, D84, and N86 are involved in gp39 binding, while gp39 residues K143 and Y145 are important for CD40 binding. Analysis of the location of amino acid substitutions in the naturally occurring gp39 mutants expressed by the X-linked hyper-IgM (X-HIM) patients studied to date indicated the E129/G substitution found in the S128/R-E129/G double mutant affects a solvent-accessible residue which might participate in CD40/gp39 binding. Binding studies with E129/G and E129/A gp39 point mutants showed that this residue does not contribute directly to CD40/gp39 binding but that its substitution with a glycine disrupts the gp39 structure. Comparison of the gp39 and CD40 residues involved in receptor-ligand contacts with those previously identified as playing an important role in TNF-beta/TNFR binding suggests that some of the identified residues from contacts similar to those found in the TNF-beta/TNFR while others are unique to the CD40-gp39 interaction.

Amino Acid Sequence↗

PubMed1995

Studies on the interdependence of gp39 and B7 expression and function during antigen-specific immune responses.

Interactions between T and B cells are dynamic and regulated by interacting receptor: co-receptors. Interactions between CD40 and its ligand, gp39, and the CD28/CTLA-4 and B7 family members play a decisive role in regulating the progression of cognate interactions. The interdependence of gp39-CD40 and CD28/CTLA-B7 expression and function was studied in vitro during an antigen-induced immune response using T cells from mice expressing a transgenic T cell receptor (TCR). gp39 was induced on pigeon cytochrome c (PCC)-transgenic T cells in the presence of antigen and antigen-presenting cells. The antigen-induced expression of gp39 on transgenic T cells was inhibited by antibodies to class II major histocompatibility complex, CD4 and LFA-1, but not by CTLA-4 Ig, anti-B7-1 or anti-B7-2. These data established that the antigen-induced expression of gp39 was not dependent on co-stimulation via CD28/CTLA-4. The addition of PCC also resulted in the modest expression of B7-1 and a more robust expression of B7-2 on the cognate B cells. The addition of anti-gp39 blocked the up-regulated expression of B7-1 and partially blocked the up-regulated expression of B7-2. The addition of anti-gp39 and anti-interleukin-4 inhibited antigen-induced expression of B7-2 on B cells to near background levels. Studies on the up-regulation of B7-1 and B7-2 on resting B cells showed that soluble gp39 up-regulated B7-1 and B7-2 expression on B cells. In addition, interleukin-4 and interferon-gamma up-regulated B7-2 expression on B cells. Taken together, these data demonstrate that the antigen-induced expression of gp39 is dependent on TCR-derived signals, yet independent of CD28/CTLA-4 co-stimulatory signals. Cognate interactions also resulted in the modest enhancement of B7-1 expression and a more profound expression of B7-2 which were completely or partially dependent on gp39-CD40 interactions.

Abatacept↗

PubMed1995

Specific binding of Fyn and phosphatidylinositol 3-kinase to the B cell surface glycoprotein CD19 through their src homology 2 domains.

CD19 is a B cell surface protein capable of forming non-covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3 kinase (PI-3 kinase). These associations are predicted to occur via the src-homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (YPO4-X-X-M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (YPO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-domain fusion proteins. In activated B cells, both Fyn and PI-3 kinase SH2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-phosphorylated peptides comprising each of the CD19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD19 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y484 and Y515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y405 and Y445) bound preferentially to the Fyn SH2-domain fusion protein via a novel sequence, Y-E-N-D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y484 was able to compete with tyrosine phosphorylated CD19 specifically for binding to the PI-3 kinase SH2 domain fusion proteins, while peptides Y405 and Y445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 signal transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase.

Amino Acid Sequence↗