Nolz, Jeffrey C.

Abstract The protein tyrosine kinase ZAP-70, known to be critical for T cell development and T cell receptor signaling, has become a potential prognostic tool for B cell chronic lymphocytic leukemia (B-CLL). Recent studies have suggested that those patients with ZAP-70 positive leukemic cells have a poorer disease prognosis than do patients whose leukemic cells lack ZAP-70 expression. In addition, ZAP-70 expression occurs more frequently in CLL B cells that express unmutated immunoglobulin (Ig) VH region genes. However, several questions remain regarding ZAP-70 expression in B-CLL. First, is ZAP-70 expression in some patients with B-CLL the consequence of malignant transformation or do normal human B cells express ZAP-70 under certain conditions of stimulation? Secondly, does the expression of ZAP-70 reflect a more activated phenotype, thus resulting in the more aggressive phenotype of ZAP-70+ B-CLL? To begin to address these questions, we focused our studies on ZAP-70 expression in normal, human B cells. CD19+ B cells were isolated by magnetic bead separation from normal human peripheral blood (PB) or tonsil and spleen surgical waste tissue. We first used three-color flow cytometry to evaluate ZAP-70 expression in CD19+ versus CD3+ lymphocytes. While PB B cells were negative for ZAP-70 expression, we were able to detect a subset of CD19+/ZAP-70+ cells in both spleen and tonsil and this subset expressed ZAP-70 at levels comparable to those of CD3+ T cells. To verify the flow cytometry results, we evaluated total ZAP-70 expression by immunoblotting and observed readily detectable ZAP-70 in both splenic and tonsillar CD19+ cells. Based on the observation that ZAP-70 was found in B cells of tonsil and spleen, but not PB, we hypothesized that ZAP-70 may be associated with activation status since tonsillar and splenic B cells will include a population of activated B cells. To examine this issue, we sorted CD19+/CD38+ from CD19+/CD38− tonsillar cells and again evaluated ZAP-70 expression. Whereas both populations expressed ZAP-70, CD38+ B cells expressed ZAP-70 at a higher level suggesting that ZAP-70 may be associated with a more activated phenotype. To address the issue of activation, we activated CD19+ PB B cells with a cocktail of CD40L, IL-10, IL-4 and IL-6 for 3 days and analyzed ZAP-70 expression. Indeed, we were able to induce ZAP-70 expression in a subpopulation of blood B cells. We also assessed ZAP-70 phosphorylation following tonsillar B cell receptor (BCR) ligation and we observed that phosphorylation of ZAP-70 (tyrosine 493) occurred within 10 min. of BCR stimulation. Finally, we assessed whether ZAP-70 expression in B-CLL could be associated with an activated cell phenotype. By stimulating Ig VH non-mutated and mutated B-CLL patient samples with the CD40L cocktail, we observed an increase in ZAP-70 expression in non-mutated CLL by immunoblotting. These results demonstrate expression of ZAP-70 in a subset of normal B cells that express an activated phenotype. Moreover, our results also suggest that ZAP-70 levels in CLL B cells may also be increased following cellular activation. Given that non-mutated type B-CLL expresses ZAP-70 more frequently than mutated type B-CLL, ZAP-70 expression may simply reflect a more activated population of leukemic cells that may correlate with progressive disease. Continuing investigation into the role of ZAP-70 in normal B cell development and B-CLL is clearly warranted.

Abstract TCR/CD28 engagement triggers the initiation of a variety of signal transduction pathways that lead to changes in gene transcription. Although reorganization of the actin cytoskeleton is required for T cell activation, the molecular pathways controlled by the actin cytoskeleton are ill defined. To this end, we analyzed TCR/CD28-stimulated signaling pathways in cytochalasin D-treated T cells to determine the cytoskeletal requirements for T cell activation. Cytochalasin D treatment impaired T cell activation by causing a reduction in TCR/CD28-mediated calcium flux, and blocked activation of two regulatory elements within the IL-2 promoter, NFAT/AP-1 and CD28RE/AP. Treatment had no effect on signaling leading to the activation of either AP-1 or NF-κB. Significantly, we found that NFAT1 is required for optimal c-rel up-regulation in response to TCR/CD28 stimulation. In fact, NFAT1 could be detected bound at the c-rel promoter in response to TCR/CD28 stimulation, and targeting of NFAT1 using RNA interference in human CD4+ T cells abrogated c-rel transcription. Overall, these findings establish that disrupting actin cytoskeletal dynamics impairs TCR/CD28-mediated calcium flux required for NFAT1-mediated c-rel transcription and, thus, activation of the CD28RE/AP.

WAVE2 regulates T cell receptor (TCR)–stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL–C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL–C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.