We have quantified the level of beta4-galactosyltransferase protein in human B lymphocytes using an ELISA-based assay. Between 1-10ng of beta4-galactosyltransferase was detected per mg total cellular ...protein, indicating that this enzyme constitutes <0.001% of B lymphocyte cellular protein. Akin to previous studies, individuals with rheumatoid arthritis exhibited reduced lymphocytic galactosyltransferase enzyme activity compared with normal controls when using ovalbumin as the acceptor substrate. The levels of enzyme protein present in B lymphocytes from patients with rheumatoid arthritis was, however, not reduced suggesting that the B lymphocyte galactosyltransferase catalytic activity may be regulated post-translationally.
Over the past year, progress has been made in understanding of the physiology and disease associations of CD5+ (B1) B cells, although their exact role in pathogenesis remains unclear. Earlier studies ...on the negative function of CD5 within the B-cell receptor complex have been substantiated, and it seems likely that soon the signaling pathways used by this coreceptor will be elucidated. Progress in diagnosis, physiology, and etiopathogenesis of CD5+ malignancies has been made, particularly in B-cell chronic lymphocytic leukemia. The low-level expression of surface immunoglobulin has been explained by the mutations that occur in the associated CD79b. Two new potential tumor-suppressor genes have been identified in the hot spot of chromosome 13q, which provides an exciting step forward in understanding of the etiopathogenesis of some B-cell chronic lymphocytic leukemia. Activated signal transducers for activation of transcription factors molecules have been shown to be phosphorylated on different amino acids in B1 and chronic lymphocytic leukemia tumors, although the significance of this is, as yet, unclear. Finally, aberrant expression of CD40L by chronic lymphocytic leukemia T cells may contribute to the immunodeficiency that develops in these patients.
The CD5 molecule is expressed by a B cell subset. We have demonstrated that resting B cells do not proliferate in response to CD5 ligation, whereas cells preactivated with anti‐IgM and IL‐2 do so. ...Here, we specifically studied the effects of anti‐CD5 and anti‐IgM on apoptosis of CD5+ B cells. Both ligation of CD5 or of surface IgM (sIgM) resulted in apoptosis. This started earlier following ligation of CD5 than with sIgM, and both responses were time dependent. CD5‐induced apoptosis was independent of the epitope recognized or the way the antibody was presented to the B cells. CD5+ B cells were more sensitive to IgM‐induced apoptosis than CD5− B cells. Engagement of CD5 or CD3 expressed by T cells failed to induce apoptosis. Our data indicate differences in the function of CD5 molecules on tonsillar B cells, compared with blood T cells and suggest that cross‐linking CD5 on B cell activates specific pathways responsible for apoptosis.
Introduction: B-CLL is characterised by the accumulation of long-lived CD5+ B cells. Mature B cells can be activated via TLRs to produce immunoglobulin and proliferate. We have previously reported ...differential surface expression of CD180, TLR-4 associated receptor, by B-CLL cells in relation to IgVh gene status (Porakishvili N et al., 2004; Blood (ASH annual meeting abstracts) 104: 2807). To further characterise subsets of leukemic cells, we assessed B-CLL cells for TLR mRNA expression.
Method: B cells from untreated (n=24) and treated (CHOP n=3, Thalidomide n=1 and chlorambucil and prednisolone n=2) CLL patients, and healthy aged matched controls, (n=14) were isolated by CD19+ selection using iMACs columns (Miltenyi Biotec), total RNA extracted (Qiagen) and primers for TLR2, TLR4, TLR9, CD180 and the adaptor protein MYD88 were used for TaqMan PCR analysis (ABI). GAPDH was used for normalization. Data were expressed as mean delta cycle number (ct) ± standard deviation, giving a value inversely related to the level of expression of the specific gene. Patients were separated into groups according to IgVh gene mutation status or level of Zap 70 expression. Statistical analysis was performed using by Mann-Whitney U test and Spearman rank correlation or Pearson's correlation coefficient.
Results: Overall, there was a heterogeneous pattern of TLR expression in patients and controls with no significant differences between them. Treated patients were characterised by higher mRNA levels for the all TLRs measured (TLR9 p=0.018) and MYD88 (MYD88 p=0.026) compared with untreated patients, despite no significant difference of their GAPDH levels (p>0.05). Significant correlations were seen between mRNA levels for the TLRs within the age matched control group (all p<0.05). Groups of patients were further analysed according to mutated (mutated >2%) or unmutated IgVh genes (unmutated <2%). No significant correlation in the TLR expression levels was found within the mutated patient group (n=8) or Zap 70 low expression group (<0.7 level) (n=11). Preliminary data from patients with an unmutated IgVh gene status (n=4) or according to levels of ZAP 70high expression (n=5) showed correlation of mRNA levels for TLRs and MYD88 expression (all p<0.05).
Conclusion: Our data indicates a direct relationship between MYD88 and TLRs transcription levels in healthy control individuals. Lack of significant correlation amongst TLR mRNA levels in CLL patients with mutated IgVh genes and Zap70low expression levels suggests aberrant regulation of TLRs in this group.
Introduction: An expansion of CD4+ T cells expressing perforin (PF) has recently been described in B-CLL and we have previously demonstrated anti-cytomegalovirus (CMV) reactivity within this ...population (Walton et al; 2004; Blood ASH annual meeting abstracts 104:4787). Here we further characterise the anti-CMV response of CD4+PF+ T cells in B-CLL and investigate the role of CMV in CD4+PF+ T cell expansion.
Methods: Peripheral blood mononuclear cells (PBMC's) from 24 untreated B-CLL patients (17 CMV seropositive SP, 7 CMV seronegative SN), 2 SP treated (Campath) patients and 12 healthy age-matched control individuals (8 SP, 4 SN) were fixed, permeabilised and stained with anti-CD4PerCP, anti-IFN-γ-APC and anti-PF-FITC monoclonal antibodies (mABs) (BD). PBMC were cultured for 18 hrs with DOWNE cell lysate (Dade Behring) containing CMV-antigen or lysate alone and with anti-CD28 and anti-CD49d mAbs (BD), in the presence of Brefeldin A (eBiosciences). In blocking experiments PBMC were pre-incubated with anti-HLA DR,DP,DQ mAb (BD) for 1 hour. The CMV specific response was assessed by flow cytometry (Dako Cyan, Summit software) as the percentage of IFN-γ+ cells in PF+ and PF− CD4+ T cell populations. Statistical analysis was performed using the Mann-Whitney U test and Spearman rank correlation.
Results: The proportion of CD4+ T cells expressing PF directly ex vivo was significantly higher in SP B-CLL patients (17.5±18.6%) compared to SN patients (2.0±2.3%, p=0.019). In seropositive aged matched controls the percentage of CD4+ cells expressing perforin was positively correlated with the percentage of CMV-reactive CD4+ cells (r=0.976, p<0.01). In contrast, there was no significant correlation in the patient group. However, two patients with relatively large expansions of CD4+PF+ cells (37.7±3.39%) post-Campath treatment had high percentages of CMV-reactive CD4+ cells (10.93±0.62%) compared to SP B-CLL patients (1.34±1.19%) and SP controls (1.31±1.14%), implying Campath related CMV reactivation. The addition of anti-HLA-DR,DP,DQ mAb to patients' PBMCs, prior to CMV stimulation, led to an 80% (from 3.26% to 0.79%) and 90% (from 3.9% to 0.45%) reduction in the proportion of antigen reactive CD4+ and CD4+PF+ cells respectively.
Conclusions: A population of major histocompatibility complex (MHC) class II restricted, CMV reactive, CD4+PF+ T cells exists peripherally, in a large group of CMV SP B-CLL patients. Furthermore, CMV is associated with CD4+PF+ T cell expansion in patients and controls. Our data implies that high numbers of B-CLL cells inhibit anti-viral effector function, leading to increased viral activity and chronic antigenic exposure, potentially driving CD4+PF+ T cell expansion.
A subset of B cells expressing the CD5 marker, a 67 KD molecule, has been implicated in the pathogenesis of autoimmune disease. To study the immunoglobulin repertoire of CD5+ B cells we investigated ...chronic lymphocytic leukemic (CLL) cells, since the majority of the malignant clones express CD5. CLL were induced to secrete their IgM in vitro by phorbol 12-myristate 13-acetate (PMA) and the supernatants screened for binding to a panel of autoantigens. Twelve out of 14 CLL clones were autoreactive, binding to Fc of IgG, ssDNA, dsDNA, histones, cardiolipin, or cytoskeletal components. Many also bound to more than one antigen tested for, showing multispecificity. Our data suggest that a high proportion of CD5+ B cells are programmed to secrete multispecific autoantibodies.
Introduction B cell Chronic lymphocytic leukemia (BCLL) is characterized by the accumulation of monoclonal malignant CD5+ B cells that show resistance to apoptosis in vivo, but are susceptible to ...apoptosis when incubated in vitro. A number of cytokines have been reported to protect against apoptosis in vitro, but it is not clear whether the effects of these cytokines are mediated in similar ways. We have therefore studied the regulation of apoptosis in B-CLL cells in vitro, in response to various cytokines.
Methods B-CLL cells were purified and incubated with the following agents; Interferon-gamma, IL-4, IL-10, IL-13, TNF-alpha and GM-CSF, each at 3 different concentrations. Apoptosis was measured after 70 hours by Annexin/PI staining and analysed for early apoptosis (Annexin V positive), late apoptotic (Annexin V and PI positive), and necrotic (PI positive).
Results The percentage of live cells in unstimulated B-CLL cells was 42.4±2.3%. Co-incubation of B-CLL cells with IL-4 at 100ng/ml increased the percentage of live cells to 63.2±8.4%, while in the presence of interferon-gamma the percentage of live cells was 48.7±5.9%. In contrast, co-incubation with TNF-alpha reduced the percentage of live cells to 30.8±6.8%. IL-10, IL-13 and GM-CSF had no effect on B-CLL cells survival at any of the concentrations used.. No significant differences in the pattern of early/late apoptosis or necrosis were seen between the different cytokines used.
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Introduction: Perforin (PF) expressing CD4+ T cells are expanded in B-CLL patients 1 and are able to induce PF-mediated apoptosis of autologous leukaemic cells in the presence of bispecific ...anti-CD3/CD19 antibodies 2. However, the role of the expanded PF+CD4+ T population in B-CLL remains unclear. In this study we have examined possible involvement of this cell subset in immune responses to cytomegalovirus (CMV).
Methods: Blood mononuclear cells from 11 CMV seropositive (SP) and 6 seronegative (SN) B-CLL patients were cultured for 4 and 18 hours in presence of Downe cell lysates containing CMV-antigen or cell lysates alone (control) and brefeldin and co-stimulated with anti-CD28 and anti-CD49 monoclonal antibodies. Anti-CMV response was assessed by flow cytometry as percentages of CD69+ and IFNγ+ cells in PF+ and PF- CD4+T cell populations.
Results: CD4+ T cells from 9 of 11 SP patients showed a strong increase in percentages of IFNγ+ cells after 18h, but not 4h, in culture with CMV antigens, compared to the control lysates and SN patients (Table) and healthy age-matched SP subjects (1.78±0.45, p=0.012). In contrast, none of the 6 SN patients as well as 2 out of 11 SP patients responded to CMV by increased IFNγ expression. In responders IFNγ+ cells were proportionally distributed between PF+ and PF- CD4+ T cells. High levels of cell activation measured by CD69 expression, especially in PF+ population, were mainly due to the Downe cell lysate since expression was significantly lower in lysate free cell cultures (8.3±11.5%, p=0.0018 for SP B-CLL patients). Anti-CMV response was accompanied by a decrease in percentages of CD4+PF+ cells suggesting antigen-induced degranulation.
Conclusion: PF+CD4+ T cells respond to CMV antigens suggesting an expansion of mature CMV specific cytotoxic CD4+ T cells in the majority of CMV seropositive B-CLL patients.
IFN-γ and CD69 expression by CD4+PF+ and PF- T cells after 18h exposure to CMV+ and CMV- lysates in CMV seropositive and seronegative B-CLL patients
CD4+PF+ CD4+PF-CD4+SNSPSNSPSNSPIFN- γ, %CMV+lys0.7±0.589.1±7.92.0±3.0 p=0.01314.4±13.60.61±0.56 p=0.0089.0±8.1CMV−lys0.26±0.340.9±1.7 p=0.0071.1±1.73.9±6.9 p=0.0290.24±0.271.46±2.55 p=0.013CD69+cells, %CMV+lys16.3±14.221.4±13.041.9±35.151.2±30.314.9±13.420.4±13.2CMV−lys14.4±12.89.7±7.828.6±34.839.8±33.013.8±12.112.9±15.0
CD5 expression in human B-cell populations Youinou, Pierre; Jamin, Christophe; Lydyard, Peter M
Immunology today (Amsterdam. Regular ed.),
07/1999, Letnik:
20, Številka:
7
Journal Article
The origin of CD5
+ B cells remains controversial. The differential response to ligation of CD5 resulting in apoptosis or proliferation provides insight into its roles in distinct human B cells. ...Here, Pierre Youinou, Christophe Jamin and Peter Lydyard review current knowledge of B-1 and B-2 cells, and propose that CD5 has different functions when expressed by different B-cell subpopulations.
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