Search

SummaryAs antigenic stimulation of the B cell antigen receptor (BCR) is key to chroniclymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinasesinvolved in BCR signalling is an attractive therapeutic approach. We studiedthe effects of the Src/c-Abl tyrosine kinase inhibitor dasatinib on BCR signaltransduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinibinduced apoptosis by an average reduction in viability of 33AE7% at 48 h, withdasatinib sensitivity correlating with inhibition of Syk Y348 phosphorylation.Dasatinib inhibited calcium flux, phosphatidylinositol-3-kinase and mitogen-activated protein kinase activation following BCR crosslinking, and blockedthe Mcl-1-dependent increase in CLL cell survival on prolonged BCRstimulation. However, the pro-apoptotic effect of dasatinib was abrogated bystromal cell contact alone or in the presence of CD154 and interleukin (IL)-4(CD154L/IL-4 system). Whilst dasatinib retained the ability to sensitize CLLcells in stromal co-culture to both fludarabine and chlorambucil, the additionof CD154 and IL-4 rendered cells resistant to these drug combinations. Wedemonstrate that the HSP90 inhibitor 17-DMAG exhibited synergy withdasatinib in vitro, and moreover, induced apoptosis of CLL cells in theCD154L/IL-4 system. Our data provide evidence that dasatinib would bemost clinically effective in combination with agents able to target antigen-independent microenvironmental signals.Keywords: chronic lymphocytic leukaemia, dasatinib, B cell antigen receptorsignalling, HSP90 inhibition, microenvironment.

Summary T-cell immune dysfunction in patients with malignant tumours has been attributed to the altered expression of components of the T-cell receptor (TCR)/CD3 complex and their associated intracellular protein tyrosine kinases. In this study, four-colour flow cytometry was applied to study the surface bound molecules TCRab, CD28, CD152 and CD154 involved in T-cell signalling and the signal transduction molecules CD3f, p56 lck , p59 fyn , 1 ZAP-70 and phosphatidyl-inositol-3 kinase (PI3-k) as well as the intracellular cytokines interferon-c (IFN-c), interleukin (IL)-4 and IL-2 as a functional read-out of non-stimulated and superantigen (staphylococcus enterotoxin B)-stimulated blood T cells of multiple myeloma (MM) patients at different stages of the disease. Multiple abnormalities were demonstrated in the CD4 and CD8 populations, both under non-stimulated and superantigenstimulated conditions. There was a marked reduction, particular in advanced stage MM, in the proportion of CD4 and CD8 cells expressing CD28, CD152, CD3f, p56 lck , ZAP-70 and PI3-k. The level of intracellular T-cell cytokines (IFN-c, IL-2 and IL-4) was normal or increased in non-stimulated cells but activation-induced cytokine production was impaired. These results illustrated profound and multiple T-cell signalling defects, from the surface and down-stream, consistent with involvement of a master T-cell function, especially in advanced stage MM. These data should be taken into consideration when developing immune-based therapeutic approaches and when applying new emerging technologies that aim to restore T-cell functions.

Neoplastic plasma cells from patients with myeloma fail to stimulate an effective anti-myeloma immune response, which may be in part due to their deficient expression of immune accessory molecules. Attempting to alter this, we infected myeloma cell lines and patient-derived primary myeloma cells with an adenovirus encoding CD154 (Ad-CD154). Myeloma cells were made to express the CD154 transgene at multiplicity of infection (MOI) between 10 and 1000. Furthermore, infection of CD40(positive) myeloma cells with Ad-CD154, but not an adenovirus encoding an irrelevant transgene, beta-galactosidase (Ad-LacZ), induced enhanced expression of immune accessory molecules, such as CD54, HLA-DR and CD70. In addition, Ad-CD154-infected myeloma cells could activate bystander CD40(positive) antigen-presenting cells to express immune accessory molecules. Consequently, Ad-CD154 infected myeloma cells stimulated proliferation in allogeneic mixed lymphocyte reactions (MLR). Finally, co-infection of CD40(negative) myeloma cells with Ad-CD154 and an adenovirus encoding CD40 (Ad-CD40) induced expression of immune accessory molecules and enhanced the MLR stimulatory capacity of transduced myeloma cells. Collectively, these results indicate that infection of myeloma cells with Ad-CD154 or Ad-CD154/Ad-CD40 can induce changes in myeloma cells that enhance their ability to induce cellular immune activation. As such, this approach may have potential application for immune therapy of patients with this disease.

B-cell chronic lymphocytic leukaemia (B-CLL) is characterized by an accumulation of clonal malignant B cells. The intrinsic characteristics that permit this accumulation have been extensively studied and described. However, it is possible that proliferation and survival of this malignant clone is facilitated by a disruption in the interaction between B and T cells that normally regulate the immune system. In this study, using flow cytometry and cell culture techniques, marked abnormalities of the expression of certain key activation and interaction molecules on the peripheral blood T cells of patients with B-CLL were demonstrated. In particular, on comparison with normal controls, there was a marked reduction in the number of circulating T cells expressing CD25 (interleukin 2 receptor) (P = 0·007), CD28 (P = 0·01) and CD152 (CTLA-4) (P = 0·001). There was also a reduction in the number of circulating T cells expressing CD4 (P = 0·03), CD5 (P = 0·05) and CD11a (P = 0·01). There was no difference in the number expressing T-cell receptor αβ (P = 0·1), CD8 (P = 0·4), CD54 (P = 0·4) and CD154 (P = 0·5), and the only marker expressed on a greater number of circulating T cells in B-CLL patients was HLA-DR (P = 0·05). These results suggest that there is a profound T-cell dysregulation that may contribute to the survival of the malignant B cells in patients with B-CLL and to the related autoimmune phenomena of the disease.

Modulating signal transduction pathways represents a promising approach for altering the biological behaviour of haemopoietic malignancies. B-cell chronic lymphocytic leukaemia (B-CLL) cells were treated in vitro with CD40-ligand (CD40L) (CD154) or the protein kinase C modulator Bryostatin-1, exploring the effects on: (a) sensitivity to apoptosis induction by chemotherapeutic drugs (fludarabine, dexamethasone) or anti-Fas antibody; (b) expression of apoptosis-regulatory proteins (Bcl-2, Bcl-X, Mcl-1, Bax, Bak, BAG-1, Flip, XIAP); (c) expression of cell surface co-stimulatory antigens (CD80 [B7.1]; CD54 [ICAM-1]; CD70); and (d) expression of immune modulatory receptors (CD27, CD40, CD95 [Fas]). CD40L and Bryostatin decreased both spontaneous and drug-induced apoptosis in most B-CLL specimens tested. Apoptosis resistance was associated with CD40L- and Bryostatin-induced elevations in the anti-apoptotic Bcl-2 family protein Mcl-1. CD40L also induced striking increases in the levels of the anti-apoptotic protein Bcl-XL in B-CLLs. CD40L stimulated increases in the surface expression of CD40, CD54, CD69, CD70, CD80 and CD95, whereas Bryostatin induced expression of CD40, CD54, CD69 and CD95 but not the co-stimulatory molecules CD70 and CD80. Despite elevations in the expression of CD95 (Fas), anti-Fas antibodies failed to induce apoptosis of CD40L- and Bryostatin-treated B-CLL cells. This Fas-resistance was associated with increased expression of the Fas-antagonist Flip in CD40L-treated, and with elevations in the caspase inhibitor XIAP in Bryostatin-treated B-CLLs. The potential anti-apoptotic properties of CD40L and Bryostatin should be taken into consideration when employing these agents in clinical trials involving patients with B-CLL.

Chronic lymphocytic leukaemia (CLL) cells encounter T-cells and proliferate in response to T-cell signals in the lymph node microenvironment. In this report we determined interleukin 21 (IL21) function in CLL and showed that IL21 and interleukin 4 (IL4) act co-operatively to promote leukaemic cell proliferation without apoptosis or differentiation We further show that IL21 increased side population (SP) cells, which are associated with resistance to chemotherapy and increased self-renewal capacity in CLL. IL21 and IL4 are the major cytokines produced by the recently described CD4(+) T follicular helper (Tfh) cell subset. Determination of Tfh cells in peripheral blood showed that patients had significantly increased numbers as compared to normal subjects although no association was found between Tfh numbers and IGHV gene mutational status or clinical stage. Our data suggests that the Tfh cytokines, IL4 and IL21, contribute to driving leukaemic cell proliferation in the lymph node microenvironment, and may contribute to the specific production of cells resistant to conventional chemotherapy. We suggest that increased circulating Tfh cells is a component of T-cell dysregulation in CLL. Our findings have implications for the therapeutic use of IL21.

Cytomegalovirus (CMV) infections post-haematopoietic stem cell transplantation (HSCT) can be effectively controlled through the adoptive transfer of donor-derived CMV-specific T cells (CMV-T). Current strategies involve a second leukapheresis collection from the original donor to manufacture CMV-T, which is often not possible in the unrelated donor setting. To overcome these limitations we have investigated the use of a small aliquot of the original granulocyte-colony stimulating factor (G-CSF) mobilized HSCT graft to manufacture CMV-T. We explored the T cell response to CMVpp65 peptide stimulation in G-CSF mobilized peripheral blood mononuclear cells (PBMC) and subsequently examined isolation of CMV-T based on the activation markers CD154 and CD25. CD25 + enriched CMV-T from G-CSF mobilized PBMC contained a higher proportion of FoxP3 expression than non-mobilized PBMC and showed superior suppression of T cell proliferation. Expanded CMV-T enriched through CD154 were CD4 + and CD8 + , demonstrated a high specificity for CMV, secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. These data provide the first known evidence that CMV-T can be effectively manufactured from G-CSF mobilized PBMC and that they share the same characteristics as CMV-T isolated in an identical manner from conventional non-mobilized PBMC. This provides a novel strategy for adoptive immunotherapy that abrogates the need for successive donation. © 2012 Blackwell Publishing Ltd.

Chronic lymphocytic leukaemia cells survive and proliferate in patients but rapidly die in culture. The microenvironment that sustains leukaemic cells in vivo contains both stromal cell elements and T cells. We defined changes in Bcl-2 family protein expression on culture with CD40 ligand (CD154) expressed on mouse fibroblast L cells, and interleukin-4 (IL-4; CD154/IL-4 system): conditions that support survival and proliferation. Unexpectedly, Bcl-2 protein expression decreased whilst pro-survival Bcl-x(L) (as well as A1 and Mcl-1) increased. However, the CD154-L cell/IL-4 system also increased the pro-apoptotic proteins, Bid and Noxa, suggesting that an increased pool of pro-survival factors and not the effects of a single protein mediate survival. Most pro-apoptotic proteins were not induced in drug or spontaneous apoptosis, but expression of Bcl-x(S), a pro-apoptotic BCL2L1 isoform, was associated with cell death. This was post-transcriptionally controlled, and, therefore, alternative splicing at the Bcl-x locus appears to have a role in the regulation of chronic lymphocytic leukaemia (CLL) cell survival. This study demonstrated a switch in pro-survival proteins associated with the transition from quiescence to CD154-driven proliferation. CLL therapies targeting Bcl-2 may need to be modified to antagonize proliferation centre-specific pro-survival proteins.