The expression of CD38 by B cells chronic lymphocytic leukemia (B-CLL) was studied in 20 untreated patients. The cells expressed abundant CD38 (relative fluorescence intensity range, 6 to 15) in 6 cases (group I patients), whereas CD38 expression was low to absent (relative fluorescence intensity range, 0 to 3) in the remaining cases (group II patients). Exposure of the cells from group I patients to goat antihuman mu chain antibodies (Ga mu-ab) resulted in the elevation of intracellular free Ca2+ concentration([Ca2+]i) followed by apoptosis. In contrast, exposure of group II cells to Ga mu-ab was not followed by increased levels of [Ca2+]i, programmed cell death or cell proliferation. No differences in the expression of surface IgM were noted in the two groups of B-CLL cells. Normal peripheral blood B cells, which expressed low to absent CD38, were capable of mobilizing [Ca2+]i and of proliferating after exposure to Ga mu-ab. The collected data suggest that, although group I B-CLL cells were able to transduce the signals delivered by IgM crosslinking, this pathway was severely impaired in group II B-CLL cells. However, unlike that observed in normal circulating B cells, stimulation of group I cells with Ga mu-ab resulted in apoptosis rather than proliferation. CD38 did not appear to be directly involved in [Ca2+]i mobilization induced by Ga mu-ab in group I B-CLL cells because their exposure to anti-CD38 monoclonal antibodies failed to cause [Ca2+]i mobilization or to block the [Ca2+]i response induced by Ga mu-ab. These data indicate that CD38 expression identified a particular subset of B-CLL cells with defined functional properties, including the propensity to undergo apoptosis.

This study investigates the main functional features of subepithelial (SE) B cells and compares them with those of purified germinal center (GC) and follicular mantle (FM) B cells isolated from the same tonsils. Unlike FM B cells, SE B cells failed to produce polyspecific antibodies in vitro; unlike GC B cells, SE B cells expressed high levels of Bcl-2 and failed to undergo spontaneous apoptosis in vitro. The most striking function of SE B cells was their ability to produce IgM antibodies to T cell-independent type-2 (TI-2) (but not to TI-1) antigens (Ag). These antibodies could not be detected when both FM and GC B cells were stimulated with TI-2 Ag in vitro. Moreover, B cells isolated from peripheral blood were unable to mount a response to TI-2 Ag. The latter finding is consistent with the observation that B cells with the phenotypic features of SE B cells were virtually absent in the peripheral blood and emphasizes the notion that SE B cells belong to a subset of non-recirculating B cells. SE B cells were by far superior to FM B cells in mixed lymphocyte reaction (MLR) stimulation of allogeneic T cells in vitro, although they were not as efficient as dendritic cells (DC). In order to stimulate T cells efficiently, SE B cells had to be exposed to anti-mu antibody, a treatment which induced expression of activation markers such as CD80, CD86, CD69 and CD39, usually absent in resting SE B cells. CD80 and CD86 molecules expressed by SE B cells participated in the chain of events required to promote the proliferation of allogeneic T cells as demonstrated by inhibition tests with the appropriate mAb. The expression of CD80 and CD86 by anti-mu-treated SE B cells was not, however, the sole explanation for their good antigen presenting capacities since the exposure of FM B cells to anti-mu antibody also induced expression of these surface structures. Nevertheless, these cells failed to become good MLR stimulators. Collectively, the above data contribute further to the characterization of a distinct subset of tonsillar B cells which resemble, both phenotypically and functionally, the B cells of the splenic marginal zone.

This study describes the features of the marginal zone (MZ) B cells of human tonsils and spleens and compares them with those of the follicular mantle (FM) B cells from the same tissues. The two B cell subpopulations displayed marked differences in phenotype, in response capacity to T cell-independent antigens and polyclonal B cell activators, and in presentation of antigens to T cells. FM B cells expressed surface CD5, and hence should be considered as B1 cells by current nomenclature. Fractionation of MZ B cells according to the presence or absence of surface IgD revealed the presence of two subsets. These subsets were characterized by different properties, including the presence of Ig V(H) gene mutations and the response capacity to TI-2 antigens, this latter property being associated with IgD-positive cells. Comparison of the data with those reported for mice revealed that human MZ B cells had strong analogies with both the murine MZ and B1 cells. In contrast, human B1 cells (that is, CD5-positive FM cells) were considerably different, an observation that should prompt further studies. Indeed, B cells with characteristics analogous to those of murine B1 cells were detected in small but definite proportions in the peripheral blood and tonsils. If the current distinction into B1 and B2 cells has to be maintained also for humans, it is likely that only these CD5-positive cells rather than the FM B cells should be called B1 cells.

The effects of short-chain fatty acids (SCFA) produced by anaerobic bacteria, namely propionic, butyric and iso-butyric on T-cell proliferation was investigated. A dose-dependent inhibition of both phytohemagglutinin-induced blastogenesis and mixed lymphocyte culture was observed in the millimolar range of SCFA concentrations. The tested SCFA displayed different levels of suppression. The degree of activity was in the following order: butyrate greater than propionate greater than isobutyrate. T-cell inhibition was partially reversed, at least for propionic and isobutyric acids, by increasing the concentration of macrophages in the assay system. Furthermore, butyric acid displayed an interesting biphasic stimulation of monocyte interleukin-1 beta production, a cytokine with a powerful bone-resorbing activity. Since millimolar concentrations of SCFA are present in gingival fluid from periodontal pockets, the observed results support the role of these by-products of anaerobic metabolism in the pathogenesis of periodontal diseases.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.