SummaryTo determine whether T cells, like B cells, can become clonally expanded in normal individuals as a function of age, we compared the T cell VB repertoire of cord blood to that of peripheral blood from normal donors over 65 yr of age. T cells from elderly subjects contained expanded subsets (greater than the mean + three standard deviations) ofT cell receptor (TCR) VB populations. These expanded subsets were observed primarily among CDS, but not CD4 cells, represented up to 37.5% of all CD8 cells, and were present in most elderly subjects. An expanded V~5.2/3 CD8 subset and a VB6.7a CD8 subset from separate donors were analyzed by reverse transcriptase-polymerase chain reaction, cloning and sequencing of the TCR ~ chain VDJ junction. In both cases the expanded subsets were mono-or oligoclonal while control CD4 populations were polyclonal. Using two-color flow cytometry it was possible to identify the expanded Vf36.7a subset as CD8 + CD28-CDllb + cells. In three of five random old subjects similar expansions of V~ subsets were found specifically in the CD8 + CD28-subpopulation, an interesting subset of cytotoxic T lymphocytes, known to lack proliferative responses to TCR stimuli. It is common practice to use the demonstration of donality as a diagnostic indicator for T cell lymphoma/leukemia. In view of the high frequency of expanded T clones of T cells in normal elderly subjects the diagnostic usefulness of this test should be reexamined.

Following B-cell antigen receptor (BCR) ligation, the cytoplasmic domains of immunoglobulin ␣ (Ig␣) and Ig␤ recruit Syk to initiate signaling cascades. The coupling of Syk to several distal substrates requires linker protein BLNK. However, the mechanism by which BLNK is recruited to the BCR is unknown. Using chimeric receptors with wild-type and mutant Ig␣ cytoplasmic tails we show that the non-immunoreceptor tyrosinebased activation motif (ITAM) tyrosines, Y176 and Y204, are required to activate BLNK-dependent pathways. Subsequent analysis demonstrated that BLNK bound directly to phospho-Y204 and that fusing BLNK to mutated Ig␣ reconstituted downstream signaling events. Moreover, ligation of the endogenous BCR induced Y204 phosphorylation and BLNK recruitment. These data demonstrate that the non-ITAM tyrosines of Ig␣ couple Syk activation to BLNK-dependent pathways.Specific immune responses to foreign antigens require one or more of a family of multimeric receptors including the B-cell antigen receptor (BCR), the T-cell antigen receptor, and the Fc receptors (2, 9). Each consists of an antigen-specific subunit noncovalently associated with signal-transducing subunits (35). Within the BCR complex, membrane-bound immunoglobulin (Ig) recognizes antigen and the Ig␣/Ig␤ heterodimer activates key signaling molecules including phospholipase C-␥ (PLC-␥), Ras, Rac, extracellular signalregulated kinase (ERK), and c-Jun NH 2 -terminal kinase (JNK) (7,11,13,27,32). Coordinated activation of these effectors controls cell differentiation, proliferation, and development.BCR aggregation induces phosphorylation of conserved tyrosines in the immunoreceptor tyrosine-based activation motifs (ITAMs) (8, 48) present in the cytoplasmic tails of Ig␣ and Ig␤ (20,25). ITAM phosphorylation is mediated by Src family tyrosine kinases assembled with the resting BCR (6, 16). Although Ig␣ and Ig␤ both contain ITAMs, Ig␤ may serve to regulate Ig␣ phosphorylation rather than initiate primary signaling (15,36,42,51). Once phosphorylated, the Ig␣ ITAM recruits and activates the tyrosine kinase Syk (50), which is both necessary (38, 49) and sufficient (37) to initiate many BCR-mediated signaling pathways. While the processes regulating Syk activation are well defined, the mechanisms linking Syk to downstream effectors are unclear.The linker protein BLNK (21), also known as SLP-65 (63) and BASH (22), is preferentially expressed in B cells. BLNK phosphorylation is dependent on Syk, and cotransfection studies indicate that BLNK is a direct substrate (21,22,28,63). (33,34,47,64) and has been implicated in human immunodeficiency (43). Deletion of BLNK in DT40 cells blocks BCR-induced JNK and PLC-␥2 activation (30). Expression of BLNK is required for normal B-cell development in miceBLNK contains a carboxy-terminal Src homology 2 (SH2) domain, a proline-rich region, and 13 potential tyrosine phosphorylation sites (21). Six of these tyrosines are part of YXXP motifs, predicted to bind the SH2 domains of PLC-␥, Vav, and Nck (55,56). In addition, the SH2...

PU.1 and Spi-B have previously been implicated in the regulation of genes encoding B cell receptor (BCR) signaling components. Spi-B-/- B lymphocytes respond poorly to BCR stimulation; PU.1-/- mice, however, lack B cells, precluding an analysis of BCR responses. We now show that PU.1+/- Spi-B-/- B cells exhibit more extensive defects than Spi-B-/- B cells, indicating that both PU.1 and Spi-B are required for normal BCR signaling. Strikingly, BCR cross-linking results in substantially reduced protein tyrosine phosphorylation in mutant B cells. Further analysis shows that Igalpha is phosphorylated and syk is recruited and becomes phosphorylated but that BLNK and PLCgamma phosphorylation are defective in mutant cells. Our data support the existence of a novel component coupling syk to downstream targets.

Notch signaling is critical for skeletal muscle development and regeneration, permitting the expansion of progenitor cells by preventing premature differentiation. We have interrogated the pathways through which ligand-mediated signaling inhibits myogenesis by identifying Notch target genes and assessing their impact on differentiation in vitro. Notch activation led to the robust induction of the transcriptional repressors Hey1 and HeyL in myoblasts, but only constitutive expression of Hey1 blocked myogenesis. siRNA-mediated knockdown of Hey1 had no effect on Notch’s ability to inhibit differentiation, suggesting the existence of additional, possibly redundant pathways. We identified 82 genes whose expression was activated when C2C12 myoblasts were cultured in the presence of the Notch ligand Dll4. One of these, MyoR, is a novel Notch-responsive gene, whose protein product is known to repress myogenesis in vitro. siRNA-mediated knockdown of MyoR alone, or in combination with Hey1, was also ineffective at rescuing differentiation in the presence of Dll4. Our data support a model in which Notch signaling inhibits myogenesis through multiple pathways, two of which are defined by the Notch target genes Hey1 and MyoR.

Members of the Hey family of transcriptional repressors are basic helix-loop-helix proteins that are thought to act downstream of Notch in diverse tissues. Although forced expression of Hey1, a target of Notch in myoblasts, is sufficient to recapitulate inhibitory effects of the pathway on differentiation, how Hey1 interferes with myogenic transcription has not been fully elucidated. We provide multiple lines of evidence that Hey1 does not target the intrinsic transcriptional activity of the skeletal muscle master regulator MyoD. Our results indicate instead that Hey1 is recruited to the promoter regions of myogenin and Mef2C, two genes whose induction is critical for myogenesis. Expression of Hey1 in C2C12 myoblasts correlates with reduced recruitment of MyoD to these promoters, arguing that Hey1 inhibits myogenesis by associating with and repressing expression of key myogenic targets.