Concurrent blockade of IL-13 and IL-17A may improve control of asthma.

The activation of protein tyrosine kinases is a critical event in T cell antigen receptor (TCR)-mediated signaling. One substrate of the TCR-activated protein tyrosine kinase pathway is a 76-kDa protein (pp76) that associates with the adaptor protein Grb2. In this report we describe the purification of pp76 and the molecular cloning of its cDNA, which encodes a novel 533-amino acid protein with a single carboxyl-terminal Src homology 2 (SH2) domain. Although no recognizable motifs related to tyrosine, serine/threonine, or lipid kinase domains are present in the predicted amino acid sequence, it contains several potential motifs recognized by SH2 and SH3 domains. A cDNA encoding the murine homologue of pp76 was also isolated and predicts a protein with 84% amino acid identity to human pp76. Northern analysis demonstrates that pp76 mRNA is expressed solely in peripheral blood leukocytes, thymus, and spleen; and in human T cell, B cell and monocytic cell lines. In vitro translation of pp76 cDNA gives rise to a single product of 76 kDa that associates with a GST/Grb2 fusion protein, demonstrating a direct association between these two molecules. Additionally, a GST fusion protein consisting of the predicted SH2 domain of pp76 precipitates two tyrosine phosphoproteins from Jurkat cell lysates, and antiserum directed against phospholipase C-gamma 1 coprecipitates a tyrosine phosphoprotein with an electrophoretic mobility identical to that of pp76. These results demonstrate that this novel protein, which we term SLP-76 (SH2 domain-containing Leukocyte Protein of 76 kDa), is likely to play an important role in TCR-mediated intracellular signal transduction.

Engagement of the T cell antigen receptor (TCR)1 results in the sequential activation of the Src (p56 lck /p59 fyn ) and Syk (Syk/ZAP-70) families of protein-tyrosine kinases (PTKs) (1-3). Both families of PTKs are required for normal T cell development and function (4 -9). In resting T cells, the TCR chain is constitutively phosphorylated and associated with 11). Tyrosine phosphorylation of the receptor-associated ZAP-70 by p56 lck is a requisite modification resulting in the up-regulation of ZAP-70 catalytic activity (12, 13). Therefore, the recruitment of the CD4 and CD8 co-receptors into the TCR complex positions p56 lck to trans-phosphorylate ZAP-70. Phosphorylation and activation of ZAP-70, in turn, is required for both an increase in phosphoinositide metabolism and activation of the ras pathway.2 The integration of these downstream signals gives rise to transcriptional activation of cytokine genes and a resultant elevation in cytokine synthesis and secretion (15). While the up-regulation of ZAP-70 catalytic activity is required for TCR function, little is known about the cellular proteins which serve as substrates for this PTK.SLP-76 is a recently identified molecule which undergoes tyrosine phosphorylation upon TCR cross-linking (16 -19). This protein associates with both the SH3 domain of Grb2 and an SH2 domain of PLC␥. SLP-76 is structurally characterized by a C-terminal SH2 domain, a region enriched in proline residues which probably serves as the site for Grb2 binding, and an N-terminal motif which contains three tandemly repeated DYE(S/P)P sequences. We demonstrate here that SLP-76 is phosphorylated by ZAP-70 and that phosphorylation of these repeated tyrosine motifs is required for optimal generation of IL-2 in response to TCR ligation. In addition, overexpression of SLP-76 augments TCR-mediated transcriptional activation of the IL-2 gene, while mutation of the SH2 domain attenuates this response. Together, these studies identify SLP-76 as a physiologic substrate for ZAP-70 and suggest a mechanism by which TCR-induced activation of ZAP-70 regulates both the calcium and ras pathways. EXPERIMENTAL PROCEDURESCells and Antibodies-Jurkat and Sf9 cells (Pharmingen) were maintained as described previously (12). The mouse monoclonal antibody (mAb) H3 was generated against the SLP-76 SH2 domain. SLP-76 polyclonal antisera (22652) was generated against a peptide spanning amino acids 301-318 of human SLP-76. C305 is an anti-Jurkat Ti␣/␤-mAb (20); 4G10 (UBI) and PY20 (Santa Cruz) are anti-phosphotyrosine mAbs; 2F3.2 is an anti-ZAP-70 mAb (UBI), and 9E10 is an anti-myc mAb. Anti-GST mAb was purchased from Santa Cruz.Construction of Plasmids-A full-length SLP-76 cDNA was generated by reverse transcription PCR from murine T-cell mRNA. This cDNA was appended with a myc-epitope at the 3Ј end of the coding cDNA. Mutations were generated by PCR-directed mutagenesis and confirmed using standard dideoxy sequencing methods. These constructs were then subcloned into the pApuro vector (21) and the baculoviral vector pVL13...

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IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1′ domain, and using genetically modified mice that contain the human M1′ domain inserted into the mouse IgE locus, we demonstrated that M1′-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1′-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1′-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1′-specific antibodies may be a novel treatment for asthma and allergy.