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. IntroductionChronic lymphocytic leukemia (CLL) is a low-grade lymphoproliferative disorder with a highly variable course whose clinical features arise through the accumulation of CD5 ϩ CD23 ϩ B cells in the bone marrow, blood, and lymphoid tissue. Although the prognosis of CLL can be predicted by simple clinical parameters that reflect disease burden, such as the extent of lymphadenopathy and presence of bone marrow failure, 1,2 these do not predict outcome for those with early stage disease.A number of biologic markers have recently been described that address this problem and allow the identification of poor risk patients with low bulk disease who might benefit from early more aggressive therapy. One of the most powerful of these biomarkers is the mutational status of the variable portion of the leukemic immunoglobulin heavy chain (IgV H ) genes. 3,4 Within the germinal center, normal B cells undergo somatic hypermutation of their IgV H genes after exposure to T-dependent antigens. Approximately 50% of CLL patients show evidence of somatic hypermutation, arbitrarily defined as a deviation of more than 2% from the germ line IgV H sequence. For reasons that are unclear, these patients have a far better prognosis than those with unmutated IgV H genes.In one of the initial studies of IgV H mutational status in CLL, a high level of membrane CD38 expression by peripheral blood leukemic cells was shown to correlate with unmutated IgV H genes and poor prognosis, and CD38 was thus proposed as a surrogate marker for mutational status. 3 Subsequent studies confirmed that CD38 is an important prognostic factor in CLL; however, expression levels were shown to be largely independent of IgV H gene mutational status. [5][6][7][8][9] During the course of the disease, the level of expression of CD38 may increase, [6][7][8]10 although patients with completely absent CD38 expression at the outset generally never express CD38. 5 The CD38 gene encodes a type II single chain transmembrane protein that is expressed by most cells of the hematopoietic lineage at some time during their differentiation. 11 It has a widespread distribution in other body tissues 12 and may function as both a receptor and an ectoenzyme. 13 In normal B lymphocytes, CD38 acts as an adhesion molecule and a receptor, signaling...

Tumors produce a variety of immunosuppressive factors which can prevent the proliferation and maturation of a number of normal hemopoietic cell types. We have investigated whether primary acute myeloid leukemia (AML) cells have an effect on normal T cell function and signaling. Tumor cell supernatant (TSN) from AML cells inhibited T cell activation and Th1 cytokine production and also prevented activated T cells from entering the cell cycle. These effects occurred in the absence of AML cell-T cell contact. We have demonstrated that AML TSN contained none of the immunosuppressors described to date, namely gangliosides, nitric oxide, TGF-β, IL-10, vascular endothelial growth factor, or PGs. Furthermore, IL-2 did not overcome the block, despite normal IL-2R expression. However, the effect was overcome by preincubation with inhibitors of protein secretion and abolished by trypsinization, indicating that the active substance includes one or more proteins. To determine the mechanism of inhibition, we have studied many of the major pathways involved in T cell activation and proliferation. We show that nuclear translocation of NFATc and NF-κB are markedly reduced in T cells activated in the presence of primary AML cells. In contrast, calcium mobilization and activation of other signal transduction pathways, namely extracellular signal-regulated kinase1/2, p38, and STAT5 were unaffected, but activation of c-Jun N-terminal kinase 1/2 was delayed. Phosphorylation of pRb by cyclin-dependent kinase 6/4-cyclin D and of p130 did not occur and c-Myc, cyclin D3, and p107 were not induced, consistent with cell cycle inhibition early during the transition from G0 to G1. Our data indicate that TSN generated by AML cells induces T cell immunosuppression and provides a mechanism by which the leukemic clone could evade T cell-mediated killing.

Chronic lymphocytic leukemia (CLL) cells rapidly undergo apoptosis in vitro, suggesting that the in vivo microenvironment provides crucial antiapoptotic signals. Overexpression of the antiapoptotic proteins Bcl-2 and Mcl-1 is a hallmark of CLL, and their expression is further enhanced in the lymphoid tissues. However, the high levels of Mcl-1 found in peripheral blood samples, coupled with its short half-life, led us to hypothesize that it must be actively maintained in the peripheral circulation. Coculture of CLL cells with human vascular endothelial cells significantly enhanced tumor cell survival, an effect that was not observed with normal B cells. This was associated with elevated levels of the antiapoptotic proteins Bcl-2, Mcl-1, and Bcl-X L and marked increased expression of CD38 and CD49d, both of which are associated with clinically aggressive disease. Because CD38, CD49d, and some Bcl-2 family genes are transcriptional targets for NF-κB, we assessed NF-κB activation following coculture with endothelial cells. DNA binding of the NF-κB subunit Rel A was significantly increased and strongly correlated with changes in transcription of CD38, CD49d, BCL2, MCL1, and BCLXL, effects that were reversed by a peptide inhibitor of Rel A. These effects were not observed following coculture with nonendothelial cell lines. Therefore, CLL cells receive specific survival signals following interaction with endothelial cells mediated through the activation of NF-κB and the induction of downstream target genes. This type of interaction in the peripheral vasculature may explain the constitutive NF-κB activation and the overexpression of Bcl-2 family proteins commonly seen in this disease.