B cell response to surface IgM cross-linking identifies different prognostic groups of B-chronic lymphocytic leukemia patients

On the basis of responses to surface IgM (sIgM) cross-linking, B cells from 41 patients with B-chronic lymphocytic leukemia were categorized as 15 nonresponders (group I) and 26 responders (group II). The latter cases were subclassified as those seven where proliferation was induced (subgroup IIa) and the remaining 19 in whom apoptosis occurred (subgroup IIa). Signal disruption in group I was confirmed by the absence of Ca2+ mobilization. Activation of PI3K was constitutive in subgroup IIa, but not in subgroup IIb, and that of Akt induced by anti-mu in subgroup IIa, but not in subgroup IIb. Among the MAPK, ERK was more highly activated relative to p38 in subgroup IIa, whereas activation of p38 predominated over that of ERK in subgroup IIb. For subgroup IIb cells, based on tyrosine phosphorylation and translocation into lipid rafts, sIgM signaling was shown to be enhanced by Zap70. The different consequences of signaling through sIgM were associated with biological prognosis indicators. These included high levels of CD38, lack of mutations in the IgVH chain genes, preferential usage of full-length CD79b, and severe clinical stage. Thus, modification of sIgM-induced signaling could be a therapeutic approach.     

Intraclonal cell expansion and selection driven by B cell receptor in chronic lymphocytic leukemia

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These "stereotyped" BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone.     

Spontaneous apoptosis in chronic lymphocytic leukemia and its relationship to clinical and cell kinetic parameters.

Chronic lymphocytic leukemia (CLL) presents considerable variability in clinical presentation as well as in its evolution. In contrast to the inhibition of apoptosis in vivo, spontaneous apoptosis after short-term culture occurs. We studied the degree of this apoptosis in vitro, and its interactions with several clinical and laboratory parameters. Apoptosis was measured by the annexin V technique. Proliferation rate was evaluated by the AgNOR (nucleolar organizer regions) technique. There were inverse correlations between the percentage of annexin V-positive cells and peripheral lymphocyte count (r = - 0.49), Rai stage (r = - 0.40), Binet stage (r = - 0.50), TTM (total tumor mass score; r = - 0.51), and percentage of cells with one AgNOR cluster (r = - 0.45). Direct correlations were found with hemoglobin values ( r = 0.34) and platelet counts (r = 0.52). The number of CD8-positive cells showed a correlation with peripheral lymphocyte count (r = 0.49). When this variable was held constant, a correlation was detected between CD8-positive cells and staging (r = -0.47), TTM (r = - 0.42), and platelet count (r = 0.67). CD4-positive lymphocytes presented a correlation only with CD8-positive lymphocytes. In a cluster analysis, it was possible to create three groups of patients with different apoptosis rates using the TTM and AgNOR values. We conclude that, with the progression of the disease, together with the increase of tumor mass and proliferation rate, there is a decrease in the susceptibility to apoptosis.     

Detection and characterization of monoclonal antibodies to platelet membrane proteins

We have devised a solid-phase radioimmunoassay for the detection and characterization of monoclonal antibodies directed against platelet surface antigens. Platelet membrane proteins, solubilized with 0.1% Triton X-100, were covalently coupled to cyanogen bromide (CNBr)-activated filter paper disks that were than used as the support in antibody binding assays. SDS PAGE of solubilized membrane proteins taken immediately before and after incubation with activated disks indicated that representative amounts of each membrane protein were bound to the disks. Either monoclonal or heterologous anti-platelet antibody could be detected on disks that had been prepared using as little as 50 micrograms of membrane protein per 100 disks. For the detection of antibody, disks were incubated with test sera for 2 h, washed, and incubated with 125I-labeled anti-immunoglobulin G, and the amount of bound radioactivity was determined. The sensitivity of the disk assay in detecting monoclonal antibodies was far greater than that of a corresponding radioimmunoassay that used whole platelets as the solid phase. By linking other proteins such as fibrinogen or anti-mouse subclass specific antisera to CNBr-activated disks, the method was adapted for antibody characterization. The sensitivity and ease with which the assay can be performed make this technique most suitable for screening and characterizing monoclonal antibodies.     

MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA) expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA) identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+) and IgV(H) unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.     

T cells in B cell chronic lymphocytic leukemia. II. Lack of antigen-specific T suppressor cells and their progenitors

Nylon wool-purified T cells (Tn) of two patients with chronic lymphocytic leukemia of the B cell type were phenotyped and tested in various assays for antigen-specific T helper (Th), T suppressor effector (Tse), T suppressor precursor (Tsp), and T suppressor inducer (Tsi) function. Antigen-specific Th as well as Tsi activity could be effectively generated. Although phenotypically CD8+ T cells, carrying the receptor for the Fc part of IgG, were present in mononuclear blood cells and Tn fractions, no antigen-specific Tse cell activity could be induced. In addition, Tsp cells were found to be functionally absent. These findings are discussed in relation to a tumor-induced limited heterogeneity within the T suppressor (Ts) cell compartment.     

Circulating proangiogenic cytokines and angiogenesis inhibitor endostatin in untreated patients with chronic lymphocytic leukemia

The serum concentration of two pro-angiogenic cytokines: basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1), and anti-angiogenic factor endostatin in the serum of 80 never treated B-cell chronic lymphocytic leukemia (CLL) patients and 27 healthy volunteers was measured using an enzyme linked immunosorbent assay. The serum levels of both bFGF and TGF-beta1 were found to be significantly higher in the CLL group (median 40.5 pg/ml and 38.6 ng/ml respectively) when compared to the control group (median 9.4 pg/ml and 18.9 ng/ml, respectively) (p<0.001). The levels of endostatin were not significantly different in CLL and control groups (median 12.3 ng/ml and 8.4 ng/ml, respectively) (p=0.09). In the group of CLL patients the level of bFGF was significantly higher in patients with progressive disease as compared with patients with stable disease (median 90.5 pg/ml and 40.5 pg/ml respectively) (p<0.001). Patients in Rai stage III and IV also had significantly higher levels of bFGF than patients in Rai stage 0-II (median 100.1 pg/ml and 29.3 pg/ml respectively) (p<0.001). The levels of both TGF-beta1 and endostatin were lower in patients in Rai stage III and IV (median 28.9 ng/ml and 9.1 ng/ml respectively) than in patients in Rai stage 0-II (42.8 ng/ml and 13.1 ng/ml respectively) (p<0.001 and p=0.002 respectively). The level of endostatin was also lower in the group of CLL patients with progressive disease (median 10.0 ng/ml) as compared to patients with stable disease (median 20.5 ng/ml) (p=0.008). In conclusion, the disturbance in the balance between pro- and anti-angiogenic factors may have an important influence on the course of CLL.     

Modulation of NF-kappa B 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-kappaB 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-kappaB activity by electrophoretic mobility shift assay. We consistently observed high levels of nuclear NF-kappaB-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-kappaB activity and prolonged CLL cell survival in vitro. The principle NF-kappaB 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-kappaB 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-kappaB 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.     

Establishment and characterization of a malignant lymphoid cell line from a chronic lymphocytic leukemia patient

A long-term culture Epstein-Barr virus (EBV)-negative malignant lymphoid cell line (NAK) was established from a lymph node biopsy of a chronic lymphocytic leukemia patient. This cell line is of particular interest because it grows as an adherent cell line and depends on the presence of autologous conditioned medium for growth. After 6 months of growth in vitro, doubling time and cell cycle parameters were derived. Doubling time was 48 hours with over 45% cycling cells. Cell viability was over 90%. Expression of B-cell markers (CD19 and CD20) and surface immunoglobulin of the original tumor cell biopsy were roughly the same as in passage 14 (3 months in culture), including the expression of the original patient idiotype and IgM-lambda. Furthermore, binding of antiidiotypic antibodies was only slightly decreased at passage 14. Cytogenetic studies of chromosomal abnormalities in the primary tumor tissue and in later passages indicated similar abnormalities, with no translocations t(8;14), t(14;22), or t(2;8). However, frequent trisomies, deletions, and t(1;4) translocations were observed. Negative results for EBV nuclear antigen indicate that this cell line is an EBV-negative cell line.     

Serum alters the uptake and biologic activity of CpG oligodeoxynucleotides in B cell chronic lymphocytic leukemia

Immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) have a number of effects on B cells, including upregulation of immunogenic molecules, and, therefore, appear attractive as potential components of immunotherapy for B cell chronic lymphocytic leukemia (B-CLL). Previous in vitro studies investigating the effect of CpG ODN on B-CLL cells used serum-low conditions and did not account for the longer-half life of CpG ODN in vitro. The present study was designed to explore how the presence of serum and exposure time affect CpG ODN-mediated changes on B-CLL cells. The optimal concentration for CpG ODN-mediated effects in the presence of 100% serum or plasma was higher (10-20 microg/ml) than for serum-low conditions. Maximal CpG ODN-mediated effects required the presence of ODN for no longer than 3 hours. The inhibition of CpG ODN-mediated effects by serum correlated with lower uptake of ODN into B-CLL cells in the presence of serum. A threshold effect on biologic response was observed, with a given amount of ODN internalized, resulting in phenotypic changes. In conclusion, systemic short-term application of CpG ODN appears to be sufficient to induce phenotypic changes, but higher doses of CpG ODN than previously thought may be necessary because of inhibition of their uptake by serum.     

Chimeric antigen receptor-modified T cell therapy in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL), a common type of B cell chronic lymphoproliferative disorder in adults, has witnessed enormous development in its treatment in recent years. New drugs such as ibrutinib, idelalisib, and venetoclax have achieved great success in treating relapsed and refractory (R/R) CLL. In addition, with the development of immunotherapy, chimeric antigen receptor-engineered T cells (CAR-T) therapy, a novel adoptive immune treatment, has also become more and more important in treating R/R CLL. It combines the advantages of T cells and B cells via ex vivo gene transfer technology and is able to bind targets recognized by specific antibodies without antigen presentation, thus breaking the restriction of major histocompatibility complex. So far, there have been lots of studies exploring the application of CAR-T therapy in CLL. In this review, we describe the structure of chimeric antigen receptor, the preclinical, and clinical results of CAR-T therapy against CLL, along with its adverse events and advances in efficacy.     

TNFR-associated factor 2 deficiency in B lymphocytes predisposes to chronic lymphocytic leukemia/small lymphocytic lymphoma in mice

We have previously shown that transgenic (tg) mice expressing in B lymphocytes both BCL-2 and a TNFR-associated factor 2 (TRAF2) mutant lacking the really interesting new gene and zinc finger domains (TRAF2DN) develop small lymphocytic lymphoma and chronic lymphocytic leukemia with high incidence (Zapata et al. 2004. Proc. Nat. Acad. Sci. USA 101: 16600-16605). Further analysis of the expression of TRAF2 and TRAF2DN in purified B cells demonstrated that expression of both endogenous TRAF2 and tg TRAF2DN was negligible in Traf2DN-tg B cells compared with wild-type mice. This was the result of proteasome-dependent degradation, and rendered TRAF2DN B cells as bona fide TRAF2-deficient B cells. Similar to B cells with targeted Traf2 deletion, Traf2DN-tg mice show expanded marginal zone B cell population and have constitutive p100 NF-κB2 processing. Also, TRAF3, X-linked inhibitor of apoptosis, and Bcl-X(L) expression levels were increased, whereas cellular inhibitors of apoptosis 1 and 2 levels were drastically reduced compared with those found in wild-type B cells. Moreover, consistent with previous results, we also show that TRAF2 was required for efficient JNK and ERK activation in response to CD40 engagement. However, TRAF2 was deleterious for BCR-mediated activation of these kinases. In contrast, TRAF2 deficiency had no effect on CD40-mediated p38 MAPK activation but significantly reduced BCR-mediated p38 activation. Finally, we further confirm that TRAF2 was required for CD40-mediated proliferation, but its absence relieved B cells of the need for B cell activating factor for survival. Altogether, our results suggest that TRAF2 deficiency cooperates with BCL-2 in promoting chronic lymphocytic leukemia/small lymphocytic lymphoma in mice, possibly by specifically enforcing marginal zone B cell accumulation, increasing X-linked inhibitor of apoptosis expression, and rendering B cells independent of B cell activating factor for survival.     

Modulation of chronic lymphocytic leukemia cells by phorbol ester: increase in Ia expression, IgM secretion and MLR stimulatory capacity

When cultured with 12-O-tetradecanoylphorbol 13-acetate (TPA) at a concentration of 1.6 X 10(-7) M, chronic lymphocytic leukemia (CLL) cells differentiated into mature cells of B lineage and increased their expression of surface Ia antigens when compared with cells cultured in the absence of TPA. Concurrently, TPA enhanced the ability of CLL cells to stimulate in a mixed lymphocyte reaction (MLR). The events induced in vitro by TPA that are characteristic of B cell maturation included morphologic changes, reduction in surface immunoglobulin (Ig), appearance of cytoplasmic Ig, and secretion of IgM. The increase in Ia expression and the enhanced capacity to stimulate in an MLR after incubation with TPA might also be associated with maturation of the CLL cells. The changes induced in vitro by TPA in neoplastic B cells provide new information concerning the terminal events in normal B cell differentiation.     

The use of CD38 expression by monoclonal B lymphocytes as a prognostic factor in B-cell chronic lymphocytic leukemia

In B-cell chronic lymphocytic leukemia (B-CLL) the Rai and Binet staging criteria are not always able to accurately predict the prognosis of each patient. Rapidly evolving, violent disease is often seen in the so-called "good-prognosis" group, which highlights the need of additional and more refined prognostic markers. Several of these markers are described in the literature, with varying abilities to predict patient survival. Among the promising prognostic markers is flowcytometric analysis of CD38 on the monoclonal B cells in CLL. Several studies have shown that expression of CD38 is associated with a decreased overall-, or progression free survival. CD38 expression may be analyzed as percentage positive cells or as antibodies bound per cell. Addition of CD38 to the flow cytometry antibody panel for B-CLL analysis is a relatively easy way to obtain important prognostic information.     

IgM anti-hepatitis C virus in patients with chronic non-A, non-B hepatitis and their relationship to viral replication

Patients with hepatitis C virus (HCV) infection may have different patterns of antibody response to various structural and non-structural viral antigens. We have correlated the serological patterns to the clinical features of chronic infection and to viral replication in 68 HCV-Ab-positive patients with chronic liver disease at different stages (19 with cirrhosis-hepatocellular carcinoma, 38 with chronic active hepatitis and 11 with chronic persistent hepatitis). Serum samples from each patient were assayed for HCV-IgM by enzyme immunoassay and for HCV-RNA by the polymerase chain reaction using primer sets derived from the 5'-non-coding region. The prevalence of HCV-IgM was high (54 patients (79.4%)) and the study showed a good correlation between high values of anti-HCV-IgM and the presence of HCV-RNA in serum, since HCV-RNA was detected in 35 of the 54 IgM-positive patients (64.8%) and notably in 19 of the 20 subjects with high levels of specific IgM. Conversely, all the 35 sera containing HCV-RNA were also reactive for HCV-IgM, while none of the HCV-IgM-negative sera was HCV-RNA reactive. Positivity rates for both HCV-RNA and IgM anti-HCV were higher in the more advanced stages of disease; thus, the clinical pattern of HCV chronic hepatitis seems to be strictly related to the serological pattern and the presence of HCV-RNA.     

TOSO, the Fcmicro receptor, is highly expressed on chronic lymphocytic leukemia B cells, internalizes upon IgM binding, shuttles to the lysosome, and is downregulated in response to TLR activation

TOSO/FAIM3 recently has been identified as the long-sought-after FcR for IgM (FcμR). FcμR is expressed on human CD19(+) B cells, CD4(+)/CD8(+) T cells, and CD56(+)/CD3(-) NK cells and has been shown to be overexpressed in chronic lymphocytic leukemia (CLL) cells. CLL is a malignancy of mature IgM(+) B lymphocytes that display features of polyreactive, partially anergized B cells related to memory B cells. In this article, we report that FcμR is O-glycosylated in its extracellular domain and identify the major sites of O-glycosylation. By using immunofluorescence confocal microscopy, we found that FcμR localized to the cell membrane but also found that large pools of FcμR accumulate in the trans-Golgi network. Aggregation of FcμR on CLL cells by IgM prompted rapid internalization of both IgM and FcμR, reaching half-maximal internalization of cell-bound IgM within 1 min. Upon internalization, FcμR transported IgM through the endocytic pathway to the lysosome, where it was degraded. Using a series of FcμR deletion mutants, we identified a proline-rich domain essential for cell surface expression of FcμR and a second domain, containing a YXXΦ motif, that controls internalization. Although it has been reported that BCR activation increases FcμR expression, we found that activation of TLRs strongly downregulated FcμR at both the mRNA and protein levels. Through internalization of IgM bound immune complexes, FcμR may play a role in immune surveillance and contribute to B cell activation. In addition, FcμR deserves study as a potential pathway for the delivery of therapeutic Ab-drug conjugates into CLL cells.     

Phenotypic modulation of chronic lymphocytic leukemia cells by phorbol ester: induction of IgM secretion and changes in the expression of B cell-associated surface antigens

Freshly explanted neoplastic populations from 22 cases of phenotypically well-characterized chronic type B lymphocytic leukemia were studied for their capacity to respond to the phorbol ester TPA in vitro. In all but four cases the secretion of IgM was either induced or increased, often to a high level. In contrast, the export of free immunoglobulin (Ig) light chains, an almost consistent feature of the B lymphocytic leukemias, remained relatively constant after TPA treatment. Parallel changes in leukemic cell surface phenotype were probed with both "conventional" and monoclonal antibodies, revealing some modulation of markers in every case investigated. A diminution in the level of surface Ig (preferentially IgD) and the accumulation of cytoplasmic Ig observed after phorbol ester treatment were accompanied by a corresponding reduction or loss of the B1 antigen and usually of B2 when present. The most consistent change induced by TPA was the appearance of BB-1, a marker of activated B lymphocytes, which was rarely expressed on fresh leukemic cells. Another marker of activated lymphocytes, LB-1, was also often induced or increased in its expression after exposure of the cells to TPA. The magnitude of the TPA response appeared to relate to the stage of maturation arrest of the individual leukemic clones rather than to any clinical parameter explored. The significance of the findings to normal B cell differentiation and their potential clinical utility are discussed.     

Flagellar membrane and paraxial rod proteins of Leishmania: characterization employing monoclonal antibodies

Flagella-specific proteins of Leishmania have been identified employing the monoclonal antibody technique. Six monoclonal antibodies recognized 3 different proteins. A doublet of protein of Mr 69,000 and 74,000 Da identified by monoclonal antibodies F-3, F-4 and F-6 is continuously distributed along the flagellum by immunofluorescence. Immunocytochemical electron microscopic studies localize these molecules to the paraxial rod of the flagellum. A single protein of Mr 13,200 Da is recognized by monoclonal antibodies F-1, F-2 and F-5. The distribution of the Mr 13,200 protein appears irregular, occurring in localized patches along the length of the flagellum, especially at the flagellar tip. Immunocytochemical electron microscopic experiments show that the Mr 13,200 molecule is associated with the membrane of the flagellum. Indirect immunofluorescence experiments demonstrated these monoclonal antibodies cross-reacted with members of the Kinetoplastida family (Endotrypanum, trypanosoma, Leishmania) suggesting that these molecules may be evolutionarily conserved.