Elaine J. Schattner

Modulation of NF-κB Activity and Apoptosis in Chronic Lymphocytic Leukemia B Cells

Chronic lymphocytic leukemia (CLL) is an indolent malignancy of CD5+ B lymphocytes. CLL cells express CD40, a key regulator of B cell proliferation, differentiation, and survival. In nonmalignant B cells, CD40 ligation results in nuclear translocation and activation of NF-κB proteins. Based on observations that in some CLL cases, the tumor cells express both CD40 and its ligand, CD154 (CD40 ligand), we proposed a model for CLL pathogenesis due to CD40 ligation within the tumor. To evaluate this issue, we used freshly isolated CLL B cells to examine constitutive and inducible NF-κB activity by electrophoretic mobility shift assay. We consistently observed high levels of nuclear NF-κB-binding activity in unstimulated CLL B cells relative to that detected in nonmalignant human B cells. In each case examined, CD40 ligation further augmented NF-κB activity and prolonged CLL cell survival in vitro. The principle NF-κB proteins in stimulated CLL cells appear to be quite similar to those in nonmalignant human B cells and include p50, p65, and c-Rel. In a CD154-positive case, blocking CD154 engagement by mAb to CD154 resulted in inhibition of NF-κB activity in the CLL cells. The addition of anti-CD154 mAb resulted in accelerated CLL cell death to a similar degree as was observed in cells exposed to dexamethasone. These data indicate that CD40 engagement has a profound influence on NF-κB activity and survival in CLL B cells, and are consistent with a role for CD154-expressing T and B cells in CLL pathogenesis. The data support the development of novel therapies based on blocking the CD154-CD40 interaction in CLL.

Lymphoma B Cells Evade Apoptosis through the TNF Family Members BAFF/BLyS and APRIL

The mechanisms underlying the autonomous accumulation of malignant B cells remain elusive. We show in this study that non-Hodgkin’s lymphoma (NHL) B cells express B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL), two powerful B cell-activating molecules usually expressed by myeloid cells. In addition, NHL B cells express BAFF receptor, which binds BAFF, as well as transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation Ag (BCMA), which bind both BAFF and APRIL. Neutralization of endogenous BAFF and APRIL by soluble TACI and BCMA decoy receptors attenuates the survival of NHL B cells, decreases activation of the prosurvival transcription factor NF-κB, down-regulates the antiapoptotic proteins Bcl-2 and Bcl-xL, and up-regulates the proapoptotic protein Bax. Conversely, exposure of NHL B cells to recombinant or myeloid cell-derived BAFF and APRIL attenuates apoptosis, increases NF-κB activation, up-regulates Bcl-2 and Bcl-xL, and down-regulates Bax. In some NHLs, exogenous BAFF and APRIL up-regulate c-Myc, an inducer of cell proliferation; down-regulate p53, an inhibitor of cell proliferation; and increase Bcl-6, an inhibitor of B cell differentiation. By showing that nonmalignant B cells up-regulate BAFF and APRIL upon stimulation by T cell CD40 ligand, our findings indicate that NHL B cells deregulate an otherwise physiological autocrine survival pathway to evade apoptosis. Thus, neutralization of BAFF and APRIL by soluble TACI and BCMA decoy receptors could be useful to dampen the accumulation of malignant B cells in NHL patients.

Clonal evolution of B cells in transformation from low‐ to high‐grade lymphoma

An outcome of low‐grade B cell non‐Hodgkinss lymphomas is the transformation to high‐grade diffuse large B cell lymphomas (DLBL). To investigate the mechanisms of clonal evolution in the transformation to DLBL, we performed longitudinal molecular analyses of immunoglobulin (Ig), VHDJH gene sequences expressed in cases of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), and follicular lymphoma (FL) that transformed to DLBL. Among the neoplastic CLL and SLL cells and their respective high‐grade transformants, there was no evidence for a clonotypic shift or acquired mutations in the expressed Ig VHDJH gene segments, as further confirmed by a specific and sensitive PCR‐single strand polymorphism analysis. In contrast, among the FL cells there was a high degree of intraclonal diversification with highly divergent VHDJH gene sequences. Despite this intraclonal heterogeneity, the related DLBL expressed a collinear but unique VHDJH gene sequence. The intraclonal genealogical tree for the FL case demonstrated that the DLBL emerged in association with unique VHDJH gene mutational events. Among the intraclonal FL and related DLBL transformants, the nature and distribution of the Ig VHDJH gene mutations were consistent with antigenic selection. Thus, clonal evolution in the transformation from low‐ to high‐grade B cell lymphoma may involve distinct pathways which vary according to the cellular origin and the type of the progenitor B cell tumor.

Ongoing In Vivo Immunoglobulin Class Switch DNA Recombination in Chronic Lymphocytic Leukemia B Cells

Chronic lymphocytic leukemia (CLL) results from the expansion of malignant CD5+ B cells that usually express IgD and IgM. These leukemic cells can give rise in vivo to clonally related IgG+ or IgA+ elements. The requirements and modalities of this process remain elusive. Here we show that leukemic B cells from 14 of 20 CLLs contain the hallmarks of ongoing Ig class switch DNA recombination (CSR), including extrachromosomal switch circular DNAs and circle transcripts generated by direct Sμ→Sγ, Sμ→Sα, and Sμ→Sε as well as sequential Sγ→Sα and Sγ→Sε CSR. Similar CLL B cells express transcripts for activation-induced cytidine deaminase, a critical component of the CSR machinery, and contain germline IH-CH and mature VHDJH-CH transcripts encoded by multiple Cγ, Cα, and Cε genes. Ongoing CSR occurs in only a fraction of the CLL clone, as only small proportions of CD5+CD19+ cells express surface IgG or IgA and lack IgM and IgD. In vivo class-switching CLL B cells down-regulate switch circles and circle transcripts in vitro unless exposed to exogenous CD40 ligand and IL-4. In addition, CLL B cells that do not class switch in vivo activate the CSR machinery and secrete IgG, IgA, or IgE upon in vitro exposure to CD40 ligand and IL-4. These findings indicate that in CLL at least some members of the malignant clone actively differentiate in vivo along a pathway that induces CSR. They also suggest that this process is elicited by external stimuli, including CD40 ligand and IL-4, provided by bystander immune cells.

Germinal Center B Cells Regulate Their Capability to Present Antigen by Modulation of HLA-DO

Peptide acquisition by MHC class II molecules is catalyzed by HLA-DM (DM). In B cells, HLA-DO (DO) inhibits or modifies the peptide exchange activity of DM. We show here that DO protein levels are modulated during B cell differentiation. Remarkably, germinal center (GC) B cells, which have low levels of DO relative to naive and memory B cells, are shown to have enhanced antigen presentation capabilities. DM protein levels also were somewhat reduced in GC B cells; however, the ratio of DM to DO in GC B cells was substantially increased, resulting in more free DM in GC B cells. We conclude that modulation of DM and DO in distinct stages of B cell differentiation represents a mechanism by which B cells regulate their capacity to function as antigen-presenting cells. Efficient antigen presentation in GC B cells would promote GC B cell–T cell interactions that are essential for B cells to survive positive selection in the GC.

CD40 Ligand in CLL Pathogenesis and Therapy

Advances in immunology during the past three decades have facilitated our understanding of the biology of specific lymphoid neoplasms including chronic lymphocytic leukemia (CLL). Investigations in our laboratory have focused on CD40, a critical regulator of B cell survival and differentiation, and its ligand, CD154 (CD40L). We have established that in some cases of CLL the malignant cells express both CD40 and CD154, and on the basis of those observations, proposed a model for CLL tumor growth due to CD40-CD154 interactions within and among the malignant cells, and for the occurence of autoimmune syndromes in some cases of CLL. Here, we include an update on our studies regarding CD154 expression in CLL, a review of the data regarding the consequences of CD40 engagement in CLL B cells, and a discussion of these findings in the context of the complex and potentially opposite outcomes that have been reported for CD40-mediated signals in CLL. The implications for therapy, such as by impedance to CD154-CD40 interaction using antibody to CD154, or by selective inhibitors of NF-kB. are considered.

Dysregulation of CD30+ T cells by leukemia impairs isotype switching in normal B cells.

Chronic lymphocytic leukemia (CLL) is associated with impaired immunoglobulin (Ig) class-switching from IgM to IgG and IgA, a defect that leads to recurrent infections. When activated in the presence of leukemic CLL B cells, T cells rapidly up-regulate CD30 through an OX40 ligand and interleukin 4 (IL-4)–dependent mechanism. These leukemia-induced CD30+ T cells inhibit CD40 ligand (CD40L)-mediated Sμ→Sγ and Sμ→Sα class-switch DNA recombination (CSR) by engaging CD30 ligand (CD30L), a molecule that interferes with the assembly of the CD40–tumor necrosis factor receptor–associated factor (TRAF) complex in nonmalignant IgD+ B cells. In addition, engagement of T cell CD30 by CD30L on neoplastic CLL B cells down-regulates the CD3-induced expression of CD40L. These findings indicate that, in CLL, abnormal CD30-CD30L interaction impairs IgG and IgA production by interfering with the CD40-mediated differentiation of nonmalignant B cells.

Apoptosis in Lymphocytic Leukemias and Lymphomas

Most current classifications of lymphoid neoplasms define the tumors based on the cell of origin, phenotype, genetic abnormalities, and clinical features. Here it is proposed that human lymphocytic tumors can be categorized based on the propensity and capacity of the tumor cells to undergo apoptosis. The first category is defined by malignant cells that are resistant to apoptosis due to expression of anti-apoptotic factors such as bcl-2 and cellular inhibitors of apoptosis (IAPs). These tumors would include CLL and follicular lymphomas, as well as some malignancies in which the tumor cells are infected by viruses that co-opt cell survival pathways, such as human T-cell leukemia/lymphoma virus (HTLV)-1. The second category, in which the malignant cells are apoptosis-prone, would include tumors arising in the context of impaired cytotoxic T-cell function. These neoplasms would include some human immunodeficiency virus (HIV)-related lymphomas such as Burkitts lymphoma, and post-transplantation lymphomas. The third category would include neoplasms of intermediate sensitivity to apoptosis, some of which are associated with infection such as mucosa-associated lymphoid tissue (MALT) lymphomas of the stomach. Although this classification is tentative, it should evolve in parallel with our understanding of pathogenic mechanisms in lymphoid neoplasia, and provides a novel framework with which to consider the appropriateness of specific therapeutic strategies. Distinctions among lymphocytic tumors in terms of the likelihood of response to therapies such as antisense to bcl-2 related proteins, inhibitors of NF-κB activity, and new approaches aimed at bolstering the hosts immune response, would cross standard classifications based on the T or B-cell origin of the tumor cells.

Functional properties of lymphocytes in idiopathic thrombocytopenic purpura.

Idiopathic or immune thrombocytopenic purpura (ITP) is characterized by antibody-mediated destruction of platelets. The etiology is unknown. We postulated that increased autoantibody production in ITP might be attributable to either increased or prolonged expression of CD40 ligand (CD40L, CD154) in T or B lymphocytes, as has been previously observed in systemic lupus erythematosus (SLE). In addition, we hypothesized that ITP is characterized by increased levels of interleukin 4 (IL-4), a prototypic Th2 cytokine which, along with CD40 ligation, is required for B cell differentiation and production of several IgG subclasses. Cell surface CD154 expression was measured in freshly-isolated and in vitro-activated peripheral blood lymphocytes of sixteen ITP patients and eight healthy volunteers. Plasma levels of IL-4 and the prototypic Th1 cytokine interferon-gamma (IFNgamma) were determined. We observed that CD154 expression in unstimulated and in vitro-activated lymphocytes did not differ between ITP patients and healthy controls. Plasma levels of the Th2 cytokine IL-4 were significantly higher in the ITP patients. These studies indicate that overexpression of CD154 in lymphocytes is unlikely to be a primary pathophysiological defect in most patients with ITP. The data support that in addition to cell membrane antigens such as CD154, soluble cytokines such as IL-4 should be considered as potential targets for therapy in this disease.

Postpartum thrombotic thrombocytopenic purpura (TTP) complicating pregnancy-associated immune thrombocytopenic purpura (ITP)

Although immune thrombocytopenic purpura in pregnancy is common, thrombotic thrombocytopenic purpura is rare and requires aggressive therapy. We report the case of a young pregnant woman with refractory immune thrombocytopenic purpura who developed postpartum thrombotic thrombocytopenic purpura and was successfully treated with plasmapheresis. The case emphasizes the importance of reviewing peripheral blood smears for evaluation of thrombocytopenia even when the diagnosis is felt to be clear. The coexistence of these two disorders has not been previously reported in the English-language literature. Case Report A 15-year-old girl, gravida 1, para 0, presented at 11 weeks of pregnancy with hyperemesis gravidarum. A manual platelet count was 56 109/L. The patient had had no previous blood counts and no history of bleeding or bruising. She was normotensive and had no petechiae, bruises, or organomegaly. The hemoglobin was 107 g/L; hematocrit, 0.33; mean corpuscular volume, 83 fL; and leukocyte count, 11.5 109/L. The peripheral blood smear showed normal red cell structure, with some platelet clumping and an overall decreased platelet count. The prothrombin time was 12.1 s (normal range, 9 to 13 s), and the activated partial thromboplastin time was 31.8 s (normal range, 23 to 38 s). Circulating antiplatelet antibody assays were negative, and renal and liver functions were normal. Idiopathic thrombocytopenic purpura was diagnosed, based on persistent thrombocytopenia during an otherwise normal pregnancy. The patient was followed until week 36 without pharmacologic intervention. Her platelet count ranged from 40 109/L to 68 109/L, and she had no clinical evidence of bleeding. At 36 weeks the patient was hospitalized with early labor, cervical dilatation, and a platelet count of 20 109/L. Peripheral blood smears still showed normal red cell structure and thrombocytopenia (Figure 1, top). The patient was treated with intravenous methylprednisolone, 20 mg every 8 hours, and gammaglobulin, 0.5 g/kg body weight daily for 4 days. Because she did not respond to therapy, bone marrow aspiration and biopsy were done to confirm the diagnosis of immune thrombocytopenic purpura. The aspirate showed fully maturing granulocytic and erythroid elements, with abundant megakaryocytes and decreased iron stores. She refused administration of vincristine, and a second course of methylprednisolone, 1 g/d for 2 days, and gammaglobulin, 2 g/kg for 3 days was given. Figure 1. Peripheral blood smear showing conversion to microangiopathic hemolytic anemia. Top. Bottom. On day 13, the fetus developed late cardiac decelerations. The patient went into active labor, and an uncomplicated cesarean section was done. She received 12 units of platelets intraoperatively. The patient delivered a normal infant boy whose initial platelet count was 100 109/L. After the cesarean section the patient appeared well despite a decrease of 2 g/L in hemoglobin that was probably caused by intraoperative blood loss. The platelet count increased to 41 109/L. Pneumovax 0.23 [Merck and Company; West Point, Pennsylvania] was given in preparation for a possible splenectomy. On postoperative day 3 she was afebrile and asymptomatic except for some ecchymoses and petechiae, but her hemoglobin decreased to 56 g/L and her platelet count, to 7 109/L. Review of the peripheral blood smear showed a new, marked schistocytosis with nucleated red blood cells and polychromasia (Figure 1, bottom). The lactate dehydrogenase level was 19.32 kat/L (Table 1). Table 1. Laboratory Data for a Patient with Idiopathic Thrombocytopenic Purpura Complicated by Thrombotic Thrombocytopenic Purpura On the basis of the postpartum development of microangiopathic hemolysis and profound thrombocytopenia, combined with the absence of hypertension, uremia, or disseminated intravascular coagulation, we diagnosed thrombotic thrombocytopenic purpura. Although overt neurologic symptoms were absent, trace proteinuria (0.3 g/L) and microscopic hematuria were present. Fever may have been masked by the high dose of steroids she received. We treated her with plasmapheresis, antibiotics, and continued steroids. The hemolysis cleared, and her platelet count increased steadily. After 4 days the platelet count was more than 100 109/L, and the steroids were tapered. During the next month, while not receiving steroids, her platelet count decreased to 64 109/L, but hemoglobin and lactate dehydrogenase levels remained normal. The peripheral blood smear showed no schistocytes. Subsequent outpatient evaluation has included a negative antinuclear antibody test, normal C3 and C4 levels, and a negative test for human immunodeficiency virus (HIV-1). The patient has chronic immune thrombocytopenic purpura, is asymptomatic, and currently requires no therapy. Discussion This case shows the importance of reviewing peripheral blood smears for pregnant patients with thrombocytopenia, even when the diagnosis seems to be clear. Burrows and Kelton [1] have shown that immune thrombocytopenic purpura is relatively common in pregnancy, occurring in 11 of 6715 consecutive deliveries at McMaster University, or approximately 1 in 500 deliveries. The condition is usually detected incidentally and usually does not require medical intervention. In contrast, thrombotic thrombocytopenic purpura is a rare complication of pregnancy. The McMaster series had no cases of thrombotic thrombocytopenic purpura. Thrombotic thrombocytopenic purpura must be treated aggressively, as illustrated by Weiners review of 45 cases of pregnancy-associated thrombotic thrombocytopenic purpura reported in the literature. He found a mortality rate of 44% for the mothers and of 80% for the infants [2]. Most cases of pregnancy-associated thrombotic thrombocytopenic purpura occur antepartum; in Weiners series, only 5 (11%) presented after delivery. We speculate that the occurrence of these two disorders in one person was not accidental. The fact that both immune and thrombotic thrombocytopenic purpura occur with increased frequency among persons with systemic lupus erythematosus, HIV disease, or pregnancy supports the hypothesis that some pathophysiologic factors are shared. Circulating antibodies or antigen-antibody complexes, caused by a primary autoimmune aberration, might induce endothelial cell damage and, consequently, thrombotic thrombocytopenic purpura. Alternatively, both immune and thrombotic purpura may represent separate hematologic manifestations of one underlying process. The distinctions among various microangiopathic hemolytic anemias associated with pregnancy are subtle [3, 4]. The diagnosis of thrombotic thrombocytopenic purpura can be obscured by signs of eclampsia or the hemolytic-uremic syndrome. In one case that may have been similar to ours, a woman had severe preeclampsia with persistent postpartum hemolysis and thrombocytopenia, which responded to plasmapheresis [5]. Similarly, our patient developed thrombotic thrombocytopenic purpura superimposed on a relatively common disorder, idiopathic thrombocytopenic purpura. The prognosis and management of these conditions differ completely; accurate diagnosis with review of the peripheral blood smear is crucial.