Summary
Progressive chronic lymphocytic leukaemia is characterized by the accumulation of neoplastic B‐cells in the tissues and correlates with the expression of prognostic biomarkers, such as CD38, ...CD49d and matrix metalloproteinase‐9 (MMP9), which are involved in migration and tissue invasion. In this study we investigated the physical relationship between these molecules and demonstrated that CD38, CD49d, MMP9 and CD44 were physically associated in a supramolecular cell surface complex. Our findings provide a molecular basis for the correlation between expression of these proteins and prognosis and, as the complex is not present in normal B‐cells, suggest a novel leukaemia‐specific therapeutic target.
Abstract Bcl-2 family proteins have long been implicated in the pathology of chronic lymphocytic leukaemia (CLL). Indeed, a number of these proteins have been shown to have prognostic importance in ...this disease. The precise ways in which these proteins impact upon CLL and the ways in which they are regulated remain incompletely resolved. However, significant advances have been recently made in our understanding of how these proteins are controlled by genetic, epigenetic and microenvironmental cues. Furthermore, major progress has been made in trying to target these proteins therapeutically. Here we review the current knowledge about this family of apoptosis-regulating proteins and how they impact upon drug resistance and disease progression. We also summarise evolution in the development of Bcl-2 family inhibitors for the treatment of CLL and other cancers.
Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with a highly variable outcome. The prognosis of patients with CLL may be predicted using a number of biomarkers, including the ...level of CD38 expression at the leukemic cell surface. This study investigates the hypothesis that CD38 expression by CLL cells reflects interactions with nonmalignant cells within pseudofollicles in secondary lymphoid tissue where tumor cell proliferation is thought to occur. CD38 expression is higher in tissues that contain pseudofollicles compared with those that do not. In addition, we show that CD38 expression in CLL is dynamic, changes in response to contact with activated CD4+ T cells, and identifies cells that are primed to proliferate. Finally, we demonstrate close contact between activated CD4+ T cells and proliferating tumor in primary patient tissue. Proliferating tumor cells in lymph nodes express CD38, which is in turn associated with an increased number of CD31+ vascular endothelial cells. Although the factors resulting in colocalization of tumor, T cells, and endothelium remain unclear, the existence of these cellular clusters may provide an explanation for the association between CD38 expression and adverse outcome in CLL and suggests novel therapeutic targets.
Summary
Interactions in the tumour microenvironment can promote chronic lymphocytic leukaemia (CLL) cell survival, proliferation and drug resistance. A detailed comparison of three co‐culture systems ...designed to mimic the CLL lymph node and vascular microenvironments were performed; two were mouse fibroblast cell lines transfected with human CD40LG or CD31 and the third was a human microvascular endothelial cell line, HMEC‐1. All three co‐culture systems markedly enhanced CLL cell survival and induced a consistent change in CLL cell phenotype, characterized by increased expression of CD38, CD69, CD44 and ITGA4 (CD49d); this phenotype was absent following co‐culture on untransfected mouse fibroblasts. In contrast to HMEC‐1 cells, the CD40LG and CD31‐expressing fibroblasts also induced ZAP70 expression and marked CLL cell proliferation as evidenced by carboxyfluorescein succinimidyl ester labelling and increased Ki‐67 expression. Taken together, our data show that co‐culture on different stroma induced a remarkably similar activation phenotype in CLL cells but only the CD40LG and CD31‐expressing fibroblasts increased ZAP70 expression and CLL cell proliferation, indicating that ZAP70 may play a critical role in this process. This comparative study reveals a number of striking similarities between the co‐culture systems tested but also highlights important differences that should be considered when selecting which system to use for in‐vitro investigations.
Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node ...(LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4dimCD5bright phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D–related (DR). In addition, LN-CLL cells have higher expression of α4β1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4dimCD5bright with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49dhi CLL cells showed an enhanced capacity to activate T cells compared with CD49dlo subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4+ T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells.
•LN-derived CLL cells have increased capacity for T-cell activation and superior immune synapse formation compared with those from PB.•Enhanced CLL cell immunologic function is also linked to PB circulating cells with the propensity to migrate.
Abstract The world of chronic lymphocytic leukemia (CLL) research is awash with prognostic markers. However, very few of the current group play a clearly defined role in the pathology of this disease ...and even fewer represent a tractable therapeutic target. One such marker that fulfils both of these criteria is the integrin CD49d. This molecule been implicated in the capacity of CLL cells to migrate into lymphoid tissues and there is a CD49d blocking antibody, Natalizumab, currently in clinical trials. Here we carried out the largest multi-centre evaluation of CD49d as a prognostic marker in 652 primary CLL samples. We confirm that CD49d is predictive for time to first treatment ( P < 0.0001) and overall survival ( P < 0.0001) and increases the prognostic power of CD38, ZAP-70 and IGHV gene mutation status in concordant cases. Furthermore, CD49d retained independent prognostic significance in multivariate analysis. In contrast to previous studies, we showed no correlation between CD49d expression and in vitro resistance to fludarabine in liquid cultures ( P = 0.28) but CD49dhi cells were significantly more resistant than CD49dlo cells when assays were carried out on fibronectin-coated plates ( P = 0.03). Furthermore, we showed for the first time that the expression of CD49d is strongly associated with expression of the chemokine receptor CXCR4 suggesting a co-ordinated role for these molecules in the trafficking of CLL cells to the lymphoid tissues. Taken together, our data support the introduction of CD49d into routine immunophenotyping panels for CLL and indicate that the therapeutic targeting of this molecule may prove useful in this disease.
CAMPATH antibodies recognize CD52, a phosphatidylinositol-linked membrane protein expressed by mature lymphocytes and monocytes. Since some antigen-presenting dendritic cells (DCs) differentiate from ...a monocytic progenitor, we investigated the expression of CD52 on dendritic cell subsets. Four-color staining for lineage markers (CD3, 14, 16, 19, 20, 34, and 56), HLA-DR, CD52, and CD123 or CD11c demonstrated that myeloid peripheral blood (PB) DCs, defined as lineage−HLA-DR+CD11c+, express CD52, while expression by CD123+ lymphoid DCs was variable. Depletion of CD52+ cells from normal PB strongly inhibited their stimulatory activity in an allogeneic mixed lymphocyte reaction and also reduced the primary autologous response to the potent neoantigen keyhole limpet hemocyanin. CD52 is thus expressed by a myeloid subset of PBDCs that is strongly allostimulatory and capable of initiating a primary immune response to soluble antigen. Administration of alemtuzumab, a humanized monoclonal antibody against CD52, to patients with lymphoproliferative disorders or as conditioning for hematopoietic stem cell transplantation resulted in a marked reduction in circulating lineage−HLA-DR+ DCs (mean 31-fold reduction,P = .043). Analysis of monocyte-derived DCs in vitro revealed a reduction in CD52 expression during culture in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4, with complete loss following activation-induced maturation with lipopolysaccharide. In contrast to the findings in PB, epidermal and small-intestine DCs did not express CD52, suggesting either that transit from blood to epidermis and gut is associated with loss of CD52 or that DCs in these tissues originate from another population of cells.
We showed previously that tumor-derived supernatant (TSN) from acute myeloid leukemia (AML) myeloblasts inhibits peripheral blood T cell activation and proliferation, rendering the T cells ...functionally incompetent. We show here that the AML TSN also significantly delays apoptosis of both resting and stimulated T cells, as judged by reduction in annexin V/propidium iodide staining. In addition, we show that this is not unique to T cells and that AML TSN inhibits apoptosis of peripheral B cells, neutrophils, and monocytes. Furthermore, it also enhances the survival of other AML myeloblasts with lower viability. Investigations into the mechanism demonstrate a reduction in the cleavage of procaspase-3, -8, and -9 and the caspase substrate, poly(ADP-ribose)polymerase (PARP). This may be due to Bcl-2, which is normally down-regulated in CD3/CD28-stimulated T cells, but is maintained in the presence of AML TSN. We conclude that AML cells generate an antiapoptotic microenvironment that favors the survival of malignant cells, but also inhibits apoptosis of other normal hemopoietic cells. Reversal of these immunosuppressive effects and restoration of normal immune responses in patients with AML would improve the success of immunotherapy protocols.
Chronic lymphocytic leukemia (CLL) is a highly variable disorder whose outcome can be predicted by a number of clinical and biological markers. The level of expression of CD38 by peripheral blood ...leukemic cells is one such prognostic biomarker however despite widespread use in clinical practice, its role in the pathogenesis of CLL remains unclear and an area of active research. A relationship between CD38 expression and proliferation of CLL cells both in the blood and tissues has been demonstrated by several groups and we recently reported that expression is higher in tissues that contain proliferation centers such as splenic white pulp and bone marrow compared to red pulp and peripheral blood which do not. Using an in-vitro model designed to mimic some of the cellular interactions that take place in proliferation centers, we showed that contact with activated autologous CD4+ T cells causes upregulation of CD38 and proliferation of the tumor. In addition, staining of CLL lymph node sections demonstrated that the highest CD38 levels are found in perivascular areas and that overall there are more microvessels in lymph nodes from CD38 positive patients. CD38 expression in CLL is thus linked to tumor proliferation and regulated by interactions in the microenvironment that involve activated T cells and the microvasculature.
In order to investigate these interactions, we further developed the in-vitro proliferation center model by incorporating the human microvascular endothelial cell line HMEC-1. CLL cells were incubated for 24–48 hours in the absence or presence of HMEC-1 and/or activated autologous T cells prior to staining for CD19 and CD38 and analysis by flow cytometry. In the presence of HMEC-1, activated T cells or both, CD38 expression by CLL cells increased from an unstimulated mean fluorescence intensity of 3693 to 4747 (n=10, p = 0.001), 18,691 (n= 10, p = 0.03) and 20,729 (n=10, p = 0.01) respectively confirming our previous results and demonstrating an additional effect of endothelial cells. During the course of these experiments it was also noted that HMEC-1 cells markedly improved the viability of the tumor cells. After 7 days, CLL cells purified by negative selection and co-cultured with HMEC-1 had a viability of 86.5%, assessed by flow cytometric measurement of CD19, annexin-V and 7AAD staining, compared to 25.5% in control medium (n=15, p<0.0001). Identical experiments using selected B cells from two normal donors resulted in no viable cells after 7 days in culture regardless of the presence endothelial cells.
It is well recognized that anti-apoptotic proteins such as Bcl-2 are up-regulated in CLL however the mechanism is unclear. Analysis of CLL cells purified by negative selection following co-culture with HMEC-1 revealed a marked increase in the expression of the anti-apoptotic proteins Bcl-2 (in 6/6 patients) and Bcl-XL (5/6) as well as inhibition of PARP cleavage (6/6) compared to CLL cells cultured in control medium. These results demonstrate that factors derived from the microvasculature enhance the viability of CLL cells by up-regulating Bcl-2 family members. Interfering with these interactions, for example using drugs that target angiogenesis, might thus be a useful adjunct to the therapy of CLL.
There is growing evidence that lymphocyte trafficking contributes to the clinical course of chronic lymphocytic leukemia (CLL), but to date, only static in vitro cultures have been used to study ...these phenomena. To address this lack of data, we have developed a dynamic in vitro model in which CLL cells experience shear forces equivalent to those in capillary beds and are made to flow through capillary-like hollow fibers lined with endothelial cells. CLL cells treated in this way increased their expression of CD62L and CXCR4 (both P < .0001) and of CD49d and CD5 (both P = .003) directly as a result of the shear force. Furthermore, CLL cells migrated through the endothelium into the “extravascular” space (mean migration, 1.37% ± 2.14%; n = 21). Migrated CLL cells had significantly higher expression of CD49d (P = .02), matrix metallopeptidase-9 (P = .004), CD38 (P = .009), CD80 (P = .04), and CD69 (P = .04) compared with CLL cells that remained in the circulation. The degree of migration observed strongly correlated with CD49d expression (r2, 0.47; P = .01), and treatment with the CD49d-blocking antibody natalizumab resulted in significantly decreased migration (P = .01). Taken together, our data provide evidence for a novel, dynamic, and tractable in vitro model of lymphocyte migration and confirm that CD49d is a critical regulator of this process in CLL.
•We have developed a novel in vitro system to model how shear force and transient interaction with endothelial cells alter chronic lymphocytic leukemia cell phenotype and behavior.•We have used our model to investigate chronic lymphocytic leukemia cell migration and have determined the critical role for integrin α4β1 in this process.
PDF
