Considering the skewing of Ig gene usage (1, 3, 4) and the fact that a high proportion of CLL B cells display natural autoantibody activity (37), it follows that the most likely candidate is an autoantigen. Taking all of these data into account, it appears that antigen stimulation may have a central role in the natural PF-03084014 history of CLL and especially of U-CLL with a poor prognosis. signal cascade triggered by B cell receptor (BCR) stimulation, we studied its pattern of expression following BCR engagement. Normal mature B cells stimulated by anti-IgM shifted the non- or less-phosphorylated form of HS1 toward the more phosphorylated form. Naive B cells showed both HS1 forms while memory B cells expressed mainly the phosphorylated fraction. These data indicate a central role for antigen stimulation in CLL and suggest a new therapeutic target for patients with aggressive disease. Introduction Chronic lymphocytic leukemia (CLL), the most common B cell malignancy in adults, is characterized by the relentless expansion of monoclonal mature B lymphocytes. The biased expression of certain (somatic mutations together with the typical expression of gene products associated with B cell signaling and activation (9) raise the possibility that an antigenic drive may be instrumental in malignant cell growth. The analysis of somatic mutations, which track the clonal history to an in vivo activation of B cell receptorCmediated (BCR-mediated) activation, differentiates 2 distinct CLL subsets, 1 with somatically mutated and 1 with unmutated genes (1, 4). The 2 2 subsets have a markedly different prognosis, with unmutated CLL (U-CLL) patients presenting a considerably shorter survival period (2, 10). The presence of somatic mutations suggests a role for BCR activation in the natural history of mutated CLL (M-CLL), though these cases are typically unresponsive to BCR stimulation in vitro (11, 12) and thus resemble B cells that have undergone receptor desensitization following chronic stimulation by antigen (13). Conversely, though expressing germline genes, U-CLL strongly responds in vitro to anti-IgM stimulation, as shown by an Mouse monoclonal to CD40 increase in global tyrosine phosphorylation (11, 12), which suggests that U-CLL carry a more competent BCR, able to receive signals for maintenance or proliferation. In addition, CD38, a signaling molecule that may influence the outcome of BCR signaling once a specific surface expression threshold is reached (e.g., in the presence of IL-2) (14), also tends to be better represented in U-CLL (10). CD38 too has a significant prognostic value, which is not related to the presence or absence of somatic mutations (15). Interestingly, a strong correlation between CD38 expression and responsiveness to signaling via surface IgM (16, 17) has been reported and appears to be enhanced by the expression of the signaling molecule chainCassociated protein of 70 kDa (ZAP-70) (11, 18). The expression of ZAP-70 (19), a kinase that shares functions with the spleen tyrosine kinase PF-03084014 Syk, is also associated with unmutated mutational status (unmutated vs. mutated), CD38 expression (positive vs. negative), and clinical behavior (progressive vs. stable disease) PF-03084014 (Table ?(Table1,1, patients 1C14). Seven patients carried unmutated genes, were positive for CD38, and experienced clinical progression requiring treatment during their clinical course (subset with poor prognoses). The other 7 patients had mutated genes, were negative for CD38, and had stable disease throughout a long follow-up period (median time, 102 months) (subset with good prognoses). Table 1 Clinical and biological features of the CLL patients Open in a separate window CD19+CD5+ purified leukemic cells were lysed and proteins resolved on 2-DE and visualized by silver staining. The protein profile analysis on silver-stained gels showed a number of protein spots differentially expressed in the samples obtained from the 2 2 CLL subsets. We focused our attention on 2 close protein spots with the same relative molecular mass (Mr) of 79 kDa and different isoelectric points (pIs; 4.83 and 4.86; Figure ?Figure1A)1A) that were both expressed in the 2-DE gels obtained from most patients with good prognoses (Figure ?(Figure1B1B and Table ?Table1)1) while the more acidic protein prevailed in the preparations from the patients with poor prognoses (Figure ?(Figure1B1B and Table ?Table1).1). Though the number of patients studied was small, 5 of 7 progressive but only 1 1 of 7 stable patients had 1 protein spot. Differences in protein expression pattern were statistically significant (< 0.05 using a 2 test). Open in a separate window Figure 1 2-DE proteomic analysis of purified CLL cells. (A) The square identifies 2 close protein spots with the same Mr of 79 kDa and different pIs, 4.83 and 4.86, which were identified as HS1 proteins by MALDI-TOF MS analysis (see Results). IEF, isoelectric focusing. (B) Both right and left protein spots.