Inhibition of human breast carcinoma growth by a soluble recombinant human CD40 ligand

CD40 is present on B cells, monocytes, dendritic cells, and endothelial cells, as well as a variety of neoplastic cell types, including carcinomas. CD40 stimulation by an antibody has previously been demonstrated to induce activation-induced cell death in aggressive histology human B-cell lymphoma cell lines. Therefore, we wanted to assess the effects of a recombinant soluble human CD40 ligand (srhCD40L) on human breast carcinoma cell lines. Human breast carcinoma cell lines were examined for CD40 expression by flow cytometry. CD40 expression could be detected on several human breast cancer cell lines and this could be augmented with interferon-gamma. The cell lines were then incubated with a srhCD40L to assess effects on in vitro growth. srhCD40L significantly inhibited the proliferation of the CD40(+) human breast cancer cell lines. This inhibition could also be augmented with interferon-gamma. Viability was also affected and this was shown to be due to increased apoptosis of the cell lines in response to the ligand. Treatment of tumor-bearing mice was then performed to assess the in vivo efficacy of the ligand. Treatment of tumor-bearing SCID mice with the ligand resulted in significant increases in survival. Thus, CD40 stimulation by its ligand directly inhibits human breast carcinoma cells in vitro and in vivo. These results suggest that srhCD40L may be of clinical use to inhibit human breast carcinoma growth.     

Antibodies to CD40 prevent Epstein-Barr virus-mediated human B-cell lymphomagenesis in severe combined immune deficient mice given human peripheral blood lymphocytes

CD40 is expressed on both normal and neoplastic B lymphocytes. Signal transduction through CD40 in vitro has been shown to exert stimulatory effects on normal B cells and inhibitory effects on Epstein-Barr virus (EBV)-induced B-cell lymphoma lines and some other cell lines derived from patients with aggressive histology lymphoma. The transfer of normal human peripheral blood lymphocytes (huPBL) from EBV-seropositive donors into severe combined immune deficient (SCID) mice has been previously shown to result in the generation of human B-cell lymphomas. These tumors are similar to the highly aggressive EBV-induced lymphomas that can arise clinically after transplantation or in the setting of immunodeficiency. Treatment of huPBL-SCID chimeric mice with anti-CD40 or anti-CD20 monoclonal antibodies (MoAb) significantly delayed the development of EBV-induced B-cell lymphoma. However, the effects of the two MoAb were mechanistically distinct. Anti-CD40 treatment prevented lymphoma generation, while still allowing for functional human B-cell engraftment in the huPBL-SCID mice compared with mice receiving no treatment, all of which succumbed to lymphoma. By contrast, treatment with anti-CD20 significantly inhibited total human B-cell engraftment in the SCID recipients, which accounted for the absence of lymphomas. In vitro assays examining the transformation of human B cells by EBV also indicated that anti-CD40 could directly inhibit EBV- transformation, whereas anti-CD20 antibodies had no effect. Thus, anti- CD40 exerts selective effects to allow for the engraftment of normal human B cells and prevent the emergence of EBV lymphomas. Stimulation of CD40 by antibodies or its physiologic ligand may, therefore, be of significant clinical use in the prevention of EBV-induced B lymphomas that may arise when EBV-seropositive individuals receive immunosuppressive regimens after transplantation or in immune deficiency states, such as acquired immune deficiency syndrome.     

Inhibition of human B-cell lymphoma growth by CD40 stimulation

CD40 is a molecule present on B lymphocyte lineage cells that is important in B-cell differentiation and activation. Signaling through CD40 has been shown to exert costimulatory signals on normal B cells resulting in proliferative and differentiation responses. Examination of several B-cell lymphomas showed cell-surface expression of the CD40 molecule. Incubation of these lymphomas with anti-CD40 antibodies resulted in significant growth inhibition in vitro. Cross-linking of the CD40 antibodies resulted in even greater inhibition of proliferation. A recombinant soluble human CD40 ligand was also shown to inhibit lymphoma proliferation. When various human B-cell lymphomas were transferred into mice with severe combined immune deficiency, the treatment of the mice with anti-CD40 antibodies resulted in significant increases in survival showing that anti-CD40 is efficacious after in vivo administration. Thus, CD40 stimulation by either the antibody or soluble ligand directly inhibits human B-cell lymphoma growth and therefore, may be of significant clinical use in their treatment.     

Role of the CD40 and CD95 (APO-1/Fas) antigens in the apoptosis of human B-cell malignancies

Ligation of CD40 inhibits apoptosis and stimulates proliferation of normal B cells, whereas ligation of CD95 (APO-1/Fas) induces apoptosis of activated lymphocytes. Aberrant signalling through the CD40 and CD95 antigens could thus participate in the pathogenesis of lymphoid malignancies. The expression and function of CD40 and CD95 on neoplastic B cells from patients with acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL) and non-Hodgkin’s lymphoma (NHL) were examined. CD40 was expressed by all 30 B-cell tumours, whereas CD95 was detected on neoplastic B cells in only one of 10 cases of ALL, two of 10 cases of CLL, and three of 10 cases of NHL. Incubation with an agonistic CD95 monoclonal antibody (MoAb) did not augment apoptosis in any of the unstimulated B-cell neoplasms. CD40 triggering did not consistently inhibit spontaneous apoptosis, but ultimately stimulated the growth of neoplastic B cells in most cases. Furthermore, CD40 activation led to up-regulation of the CD95 antigen in all 30 B-cell neoplasms. Ligation of CD95 on CD40-activated tumour cells augmented apoptosis in five of 10 ALL, three of 10 CLL, and nine of 10 NHL cases. The degree of apoptosis induced by CD95 triggering was greater for NHL cells than for ALL cells or CLL cells. Bcl-2 expression by ALL and NHL cells was substantially decreased after in vitro culture, whereas Bcl-2 expression by CLL cells was not significantly changed. However, there was no correlation between the level of Bcl-2 expression and sensitivity to CD95-mediated apoptosis. Thus, factors other than levels of CD95 and Bcl-2 determine susceptibility of malignant B cells to apoptosis after CD95 triggering. CD40-activated lymphoma cells appear to be very sensitive to CD95-mediated apoptosis, suggesting potential strategies for treatment of NHL. Elucidation of the mechanisms underlying resistance of ALL and CLL cells to CD95 triggering may facilitate the development of novel therapeutic approaches to these diseases as well.     

Differentiating germinal center‐derived lymphomas through their cellular microenvironment

Recent studies on normal and malignant B‐cells have provided evidence that the germinal center (GC) of lymphoid follicles exerts a role in B‐cell physiology and malignancy. GC‐derived lymphomas include both B‐cell and T‐cell lymphomas. Remarkably, tumor cells of GC‐derived lymphomas proliferate in close association with cellular environment that retains key features of normal GC cellular microenvironment. Neoplastic follicles in follicular lymphoma contain, in addition to follicular dendritic cells (FDC) other non‐neoplastic cells including macrophages and GC T‐cells. In addition to aggregates of FDCs, the background infiltrate of nodular lymphocyte predominant Hodgkin lymphoma includes small B‐cells, T‐cells, and histiocytes. Typically, most of the lymphocyte predominant (LP) cells are ringed by CD3+/CD4+ T‐cells expressing CD57, PD1, BCL6, and MUM1/IRF4. By contrast, Reed–Sternberg cells of classic Hodgkin lymphoma (cHL) are surrounded by CD3+/CD4+ T‐cells expressing CD40L. Unlike cHL and other peripheral T‐cell lymphomas, the AITL microenvironment characteristically contain a prominent proliferation of high endothelial venules and FDC. Thus, these findings shed new light on the characterization of GC‐derived lymphomas and may help in the differential diagnosis and acknowledge several novel pathogenetic mechanisms on these lymphomas. Am. J. Hematol., 2009. © 2009 Wiley‐Liss, Inc.     

Rescue of "crippled" germinal center B cells from apoptosis by Epstein-Barr virus

Epstein-Barr virus (EBV) is associated with B-cell lymphomas such as Hodgkin lymphoma, Burkitt lymphoma, and post-transplantation lymphoma, which originate from clonal germinal center (GC) B cells. During the process of somatic hypermutation, GC B cells can acquire deleterious or nonsense mutations in the heavy and light immunoglobulin genes. Such mutations abrogate the cell surface expression of the B-cell receptor (BCR), which results in the elimination of these nonfunctional B cells by immediate apoptosis. EBV encodes several latent genes, among them latent membrane protein 1 (LMP1) and LMP2A, which are regularly expressed in EBV-positive Hodgkin lymphoma and posttransplantation lymphomas. Since LMP1 and LMP2A mimic the function of 2 key receptors on B cells, CD40 and BCR, respectively, we wanted to learn whether EBV infection can rescue proapoptotic GC B cells with crippling mutations in the heavy chain immunoglobulin locus from apoptosis. We show here that BCR-negative GC B cells readily enter the cell cycle upon infection with EBV in vitro and yield clonal lymphoblastoid cell lines that are incapable of expressing a functional BCR because the rearranged and formerly functional heavy chain immunoglobulin alleles carry deleterious mutations. Our findings imply an important role for EBV in the process of lymphomagenesis in certain cases of Hodgkin lymphoma and posttransplantation lymphomas.     

Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation

We identified the human germinal center-associated lymphoma (HGAL) in gene-expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL correlated with survival in patients with DLBCL. The HGAL gene is the human homolog of M17, a mouse gene expressed specifically in normal germinal center (GC) B cells. We generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC lymphocytes and in lymphomas of GC derivation. Among 727 lymphomas tested by immunohistochemistry on tissue microarrays, HGAL staining was found in follicular lymphomas (103 of 107), Burkitt lymphomas (40 of 40), mediastinal large B lymphomas (7 of 8), and in DLBCLs (103 of 151). Most marginal zone lymphomas lacked HGAL staining. Lymphocyte-predominant Hodgkin lymphomas (12 of 17) and, surprisingly, classical Hodgkin lymphomas (78 of 107) were found to be positive. Hierarchical clustering of comparative immunohistologic results in DLBCLs demonstrates that the expression of HGAL is similar to 2 other GC-associated proteins, BCL6 and CD10, but different from 2 markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.     

Lymphoma development in mice and humans: diversity of initiation is followed by convergent cytogenetic evolution.

Human B cell lymphoma and murine T cell leukemia can be initiated by several agents. The present paper formulates some thoughts on the role of cytogenetic changes in the subsequent neoplastic process. Initiation creates long-lived preneoplastic cells. In some respects, they are comparable to in vitro-transformed ("immortalized") cell lines that maintain a diploid karyotype and are not tumorigenic in vivo. The development of a tumorigenic ("autonomous") clone is dependent on additional changes at the genetic level. In human B and murine T cell lymphoma, there are characteristic nonrandom chromosomal changes. The 14q+ marker appears to play a key role in human B cell lymphomas. The reciprocal 8;14 translocation in Burkitt lymphoma is a specialized subclass within this category. In murine T cell leukemia, trisomy 15 is the predominant change. The clustering of these nonrandom changes to tumors derived from a certain cell type rather than to tumors induced by a given etiological agent has important implications for the understanding of the genetic control of cellular responsiveness to growth-regulating forces in vivo.     

Mediastinal lymphoma of clear cell type is a tumor corresponding to terminal steps of B cell differentiation

This article reports eight primary mediastinal tumors occurring in young adults (19 to 43 years, mean 29.4 years), predominantly female (six of eight) adults. Most patients responded badly to aggressive therapy. Progression is presently noted in one patient; five patients died 10, 11, 13, 18, and 22 months after diagnosis. No patient developed leukemia. The tumors were highly proliferative, had a diffuse growth pattern, and comprised clear cells of variable size. They could not be classified histologically, but could, however, be immunohistologically characterized as B cell lymphomas. In all cases, the immunophenotype was LC+, cALLa-, CD19+, CD20+, CD21-, Ig (surface/cytoplasm)-, and PC-1+. In addition, the neoplastic cells exhibited variable defects in the expression of HLA-A,B,C and HLA-DR and inconstant expression of other B cell-restricted/associated antigens. This combination of immunophenotypical and clinical features suggests that the mediastinal clear cell lymphoma (MCCL) is a previously undescribed type of B cell lymphoma corresponding to the terminal steps of B cell differentiation.     

Definition and characterization of the systemic T-cell dysregulation in untreated indolent B-cell lymphoma and very early CLL

Epidemiologic data show that the immune system may control or promote the emergence and growth of neoplastic lymphomatous clones. Conversely, systemic lymphomas, especially myeloma and chronic lymphocytic leukemia (CLL), are associated with clinical immunodeficiency. This prospective controlled study demonstrates substantially reduced circulating T helper cells, predominantly naive CD4(+) cells, in patients with nonleukemic follicular lymphoma and extranodal marginal zone lymphoma, but not in monoclonal gammopathy and early CLL. These changes were correlated with a preactivated phenotype, hyperreactivity in vitro, pre-senescence, and a T helper 2 shift of peripheral T helper cells. No prominent alterations existed in the regulatory T-cell compartment. Gene expression profiling of in vitro-stimulated CD4(+) cells revealed an independent second alteration of T helper cell physiology, which was most pronounced in early CLL but also detectable in follicular lymphoma/extranodal marginal zone lymphoma. This pattern consisted of down-regulation of T-cell receptor signaling cascades and globally reduced cytokine secretion. Both types of T-cell dysfunction may contribute to significant immunodeficiency in nonleukemic indolent B-cell lymphomas as demonstrated by unresponsiveness to hepatitis B vaccination. The precise definition of systemic T-cell dysfunction serves as the basis to study its prognostic impact, its relationship to the established influence of the lymphoma microenvironment, and its therapeutic manipulation.     

Flow cytometry using the monoclonal antibody CD10-Pe/Cy5 is a useful tool to identify follicular lymphoma cells

Follicular lymphoma (FL) is a specific entity defined by characteristic histology, phenotype and molecular rearrangements. Classically, reactivity for CD19, CD10, and strong positivity for the surface light chain immunoglobulin (SIg) are considered to be phenotypic signs typically expressed in FL. In practice, this pattern is difficult to identify since most neoplastic cells analysed by flow cytometry (FC) show weak intensity for CD19-Pe/Cy5 and for SIg and negativity for CD10-FITC. We used triple antigen combinations including two monoclonal antibodies (MoAbs) against CD10 (CD10-FITC and CD10-Pe/Cy5) and a long-distance polymerase chain reaction (PCR) approach to establish the phenotypic pattern of neoplastic cells carrying t(14;18)(q32;q21). Neoplastic cells showed the following immunophenotype: stronger reactivity against CD20 than against CD19, positivity for CD22 and SIg and negativity for CD5, CD11c and CD10-FITC. Characteristically, CD10-Pe/Cy5 was expressed in all the samples with positive bcl-2/JH rearrangements. In FL, there was a high correlation between histologic diagnosis and reactivity against CD10-Pe/Cy5 (96% cases). In diffuse large cell lymphomas (DLCL), CD10-Pe/Cy5 identified positive cases with t(14;18)(q32;q21) chromosomal translocation, whereas Burkitt lymphomas showed all cases reactivity against CD10-Pe/Cy5. In conclusion, CD10-Pe/Cy5 is a useful antibody for identifying neoplastic cells carrying t(14;18)(q32;q21) in FL and DLCL. In combination with other MoAbs, anti-CD10 (HI10a, Cy-Chrome) can be used to identify a characteristic phenotypic profile of FL against other lymphoproliferative disorders.     

Telomerase activation in normal B lymphocytes and non-Hodgkin's lymphomas

Activation of telomerase seems to be a prerequisite for immortalization and is found in permanent cell lines and most malignant tumors. Normal somatic cells are generally telomerase negative, except for bone marrow stem cells. Weak activity is also present in peripheral blood cells. In the present study strong telomerase activity was demonstrated in vivo in normal mature cells of the immune system, as well as in malignant lymphomas. Benign lymph nodes had lower telomerase activity than benign tonsils, which exhibited intermediate to high activity comparable with findings in malignant lymphomas. In benign tonsils the activity seemed to be restricted to germinal center B cells. In benign lymphoid tissues telomerase activity correlated with B-cell numbers and cell proliferation, but this was not observed in the lymphoma group. High- grade lymphomas exhibited higher levels of telomerase compared with low- grade cases. The data showed that in vivo activation of telomerase is a characteristic feature of germinal center B cells. Different signals for activation of telomerase are likely to exist, one of them being immune stimulation. The data suggest that telomerase activity in malignant lymphomas can be explained by an "induction and retention" model, ie, transformation occurs in a normal, mature B cell with reactivated telomerase, which is retained in the neoplastic clone.     

Identification of B-cell growth factors (interleukin-14; high molecular weight-B-cell growth factors) in effusion fluids from patients with aggressive B-cell lymphomas

The molecular basis of neoplastic B-cell growth is complex and poorly understood. Cytokines have been postulated to contribute to neoplastic cell growth, and many in vitro studies have confirmed this prediction, but little is known about the in vivo role of these growth factors. We have examined the production of interleukin-14 (IL-14) (high molecular weight [HMW], B-cell growth factor [BCGF]) by aggressive intermediate (diffuse large cell) lymphomas of the B-cell type non-Hodgkin's lymphoma (NHL-B) in four patients with lymphomatous effusions. In these studies, IL-14 was detected in the effusion fluids by Western blots and IL-14 mRNA was constitutively expressed in the freshly isolated lymphoma cells that also expressed the receptor for IL-14 (IL14R). Lymphoma B cells placed at low serum and cell density proliferated in vitro to either purified IL-14 or IL-14 derived from effusion fluids. Antibodies to IL-14 removed the growth-stimulating cytokine(s) from the effusions. Cell lines developed from these patients produced IL-14 in vitro and antisense oligos to IL-14 blocked their growth in vitro. Thus, autocrine or paracrine production of IL-14 may play a significant role in the rapid proliferation of aggressive NHL-B. Interrupting this pathway could be a useful goal of therapy for patients resistant to conventional chemotherapy.     

Lack of vimentin occurring during the intrafollicular stages of B cell development characterizes follicular center cell lymphomas

Reactive germinal-center B cells completely lack vimentin. This observation made by using monoclonal vimentin antibody V9 and a sensitive immunoperoxidase technique led to the investigation of vimentin expression in an unselected series of 73 immunohistochemically proven B cell lymphomas. While small lymphocytic lymphomas regularly expressed vimentin, the neoplastic population of mixed, small and large, and large cell lymphomas often contained various proportions of vimentin-negative tumor cells or failed to express it at all. Against the background of the Lukes-Collins classification and the Kiel classification, both defining and subdividing follicular center-cell lymphomas, there were statistically highly significant correlations between the absence of vimentin and the histology, suggesting a germinal center origin. Our data suggest that loss of vimentin expression is indicative of the transient, follicular center-cell stage of normal and malignant B lymphocytic transformation. The complete absence of vimentin within the neoplastic population of all Burkitt's lymphoma cases examined may serve as an argument for its follicular center stage of differentiation, as was asserted by Lukes and Collins.     

Monoclonal antibody against a Burkitt lymphoma-associated antigen.

A monoclonal antibody, referred to as 38.13, was obtained by fusing murine myeloma cells with Lewis rat splenocytes sensitized with Daudi cells (human Burkitt lymphoma containing Epstein--Barr virus genome but lacking HLA-A, -B, and -C and beta 2-microglobulin molecules at the cell surface). 38.13 antibody was demonstrated to be a rat IgM. By complement-dependent microcytotoxicity and indirect immunofluorescence assays, 38.13 antibody was shown to react specifically with cells derived from Burkitt tumors, including both Epstein--Barr virus genome-carrying and Epstein--Barr virus-negative Burkitt lymphoma. By contrast, Epstein--Barr virus-containing lymphoblastoid cell lines derived from normal B lymphocytes were not recognized by 38.13 antibody. Fresh malignant cells from patients affected with various lymphoproliferative disorders were negative, except 4/8 having abdominal Burkitt-like lymphomas. Normal lymphocytes from peripheral blood, spleen, lymph node, tonsil, and bone marrow and mitogen (phytohemagglutinin, pokeweed mitogen, and concanavalin A)-activated blasts were also negative. Thus, 38.13 antibody apparently recognized a Burkitt-associated antigen that is not related to Epstein--Barr virus. The pattern of reactivity of 38.13 antibody with various Burkitt lymphoma cells appeared quite heterogenous and some Burkitt cells were consistently negative. 38.13 antibody thus defines a subset of Burkitt lymphomas.     

From the bench to the bedside: ways to improve rituximab efficacy

Rituximab (MabThera, Rituxan) is a chimeric IgG1 monoclonal antibody that specifically targets the CD20 surface antigen expressed on normal and neoplastic B-lymphoid cells. Rituximab is currently used in the treatment of both follicular and aggressive B-cell non-Hodgkin lymphomas. Despite its demonstrated clinical effectiveness, its in vivo mechanisms of action remain unknown and could differ by subtype of lymphoma. Rituximab has been shown to induce apoptosis, complement-mediated lysis, and antibody-dependent cellular cytotoxicity in vitro, and some evidence points toward an involvement of these mechanisms in vivo. Rituximab also has a delayed therapeutic effect as well as a potential "vaccinal" effect. Here, we review the current understanding of the mechanism of action of rituximab and discuss approaches that could increase its clinical activity. A better understanding of how rituximab acts in vivo should make it possible to develop new and more effective therapeutic strategies.     

Hyposialated 185 kDa CD45RA+ molecules attain a high concentration in B lymphoma cells and in activated human B cells

Alternate splicing of exons of the CD45 molecule generates multiple isoforms differing in their molecular weights (MWs). In B-lymphocytes the CD45RA isoform was previously shown to be expressed on glycoproteins with MWs of 220 and 205 kDa, while the CD45RO isoform was expressed on glycoproteins with MW of 180 kDa. The present study demonstrated that B cell lymphomas and activated B-cells contain CD45 molecules with a MW of 185 kDa that express the CD45RA and CD45RC specificities but neither the CD45RB nor the CD45RO specificities. 185 kDa CD45RA+ molecules were detected in B cell lymphoma B lines, in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, and in tonsillar B cells, but not in normal, unstimulated peripheral blood B cells. These molecules were not detected in neoplastic and normal T cells. CD45RA+ 185 kDa molecules were present in B cells from three non-Hodgkin's patients in leukemic phase were not detected in B lymphocytes of seven of nine CLL patients tested. Trypsin treatment eliminated only 220 kDa CD45RA+ molecules but not 185 kDa CD45RA+ molecules, indicating that the 185 kDa CD45RA+ molecules are not expressed on the cell surface. Pulse-chase experiments, and studies on the effects of tunicamycin, neuraminidase and O-glycosidase, indicated that the 185 kDa molecules are partially glycosylated CD45RABC molecules that constitute precursors of the 220 kDa molecules. The high concentration of 185 kDa CD45RA+ molecules in B lymphoma cells and in activated B cells seems to reflect a high turnover of CD45RA+ molecules characteristic for these cells.     

Isolated follicular lymphoma cells are resistant to apoptosis and can be grown in vitro in the CD40/stromal cell system

Low-grade follicular non-Hodgkin's lymphomas are characterized by the presence of a t(14;18) chromosomal translocation that results in deregulation of the B-cell lymphoma (Bcl-2) gene. Studies in cell lines and transgenic animal models have suggested that this results in the suppression of apoptotic cell death in germinal centers. B lymphocytes from normal germinal centers and lymph nodes infiltrated by follicular lymphoma were isolated by immunomagnetic depletion of cells bearing CD4, CD8, or slgD for study in vitro. Follicular lymphoma cells expressing Bcl-2 protein were shown to resist apoptosis after isolation, and could be induced to proliferate in a culture system previously described for the growth of normal B lymphocytes. By the use of a mouse fibroblast monolayer transfected with the CDw32 Fc receptor to present CD40 monoclonal antibody in the presence of interleukin-4, prolonged culture was possible. Karyotypic analysis of cultured lymphoma cells showed the t(14;18) translocation, with clonal identity confirmed by polymerase chain reaction amplification of the breakpoints and direct sequence analysis. These findings support the hypothesis that resistance to apoptosis is an influence on the initiation of follicular lymphoma, and provide a novel means of studying in vitro the intercellular reactions that may be important in progression of the disease.     

An essential role for Bruton''s [corrected] tyrosine kinase in the regulation of B-cell apoptosis.

Mutations of the Bruton's tyrosine kinase (btk) gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (Xid) in mice. To establish the BTK role in B-cell activation we examined the responses of wild-type and Xid B cells to stimulation through surface IgM and CD40, the transducers of thymus independent-type 2 and thymus-dependent activation, respectively. Wild-type BTK was necessary for proliferation induced by soluble anti-IgM (a prototype for thymus independent-type 2 antigen), but not for responses to soluble CD40 ligand (CD40L, the B-cell activating ligand expressed on T-helper cells). In the absence of wild-type BTK, B cells underwent apoptotic death after stimulation with anti-IgM. In the presence of wild-type but not mutated BTK, anti-IgM stimulation reduced apoptotic cell death. In contrast, CD40L increased viability of both wild-type and Xid B cells. Importantly, viability after stimulation correlated with the induced expression of bcl-XL. In fresh ex vivo small resting B cells from wild-type mice there was only barely detectable bcl-XL protein, but there was more in the larger, low-density ("activated") splenic B cells and peritoneal B cells. In vitro Bcl-XL induction following ligation of sIgM-required BTK, was cyclosporin A (CsA)-sensitive and dependent on extracellular Ca2+. CD40-mediated induction of bcl-x required neither wild-type BTK nor extracellular Ca2+ and was insensitive to CsA. These results indicate that BTK lies upstream of bcl-XL in the sIgM but not the CD40 activation pathway. bcl-XL is the first induced protein to be placed downstream of BTK.