CD markers variations in chronic lymphocytic leukemia: New insights into prognosis

Chronic lymphocytic leukemia (CLL) is one of the most commonly occurring adult leukemias that is associated with clonal accumulation of mature apoptosis-resistant B-cells in bone marrow, peripheral blood, and specific tissues. Different pathogenesis factors can contribute to the aggression of the clinical course in this disease. Cytogenetic abnormalities and surface biomarkers of neoplastic CLL cells can be effective in the outcome of CLL, and the examination of changing CD markers expressions in the progression of CLL can be related to the prognosis of this disease. Changing expression levels of CD markers on lymphocytes and other cells in CLL patients can play a role in the aggressive clinical outcomes such as organomegaly, immunodeficiency, and advanced disease stages through their interaction with CLL microenvironment. Given the involvement of CD markers in the pathogenesis of CLL, it can be stated that recognizing the expression changes of CD markers in the cells involved in CLL can be a proper approach to evaluate prognosis among these patients.     

Increased autocrine interleukin-6 production is significantly associated with worse clinical outcome in patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) remains incurable with current standard therapy. We have previously reported that an increased expression of interleukin-6 (IL-6) receptor CD126 leads to resistance of CLL cells to chemotherapy and worse prognosis for patients with CLL. In this study, we determine whether autocrine IL-6 production by CLL B cells is associated with poor clinical outcome and explore IL-6-mediated survival mechanism in primary CLL cells. Our results demonstrate that higher levels of autocrine IL-6 are significantly associated with shorter absolute lymphocyte doubling time, patients received treatment, without complete remission, advanced Binet stages, 17p/11q deletion, and shorter time to first time treatment and progression-free survival. IL-6 activated both STAT3 and nuclear factor kappa B (NF-κB) in primary CLL cells. Blocking IL-6 receptor and JAK2 inhibited IL-6-mediated activation of STAT3 and NF-κB. Our study demonstrates that an increased autocrine IL-6 production by CLL B-cells are associated with worse clinical outcome for patients with CLL. IL-6 promotes CLL cell survival by activating both STAT3 and NF-κB through diverse signaling cascades. Neutralizing IL-6 or blocking IL-6 receptor might contribute overcoming the resistance of CLL cells to chemotherapy. We propose that the measurement of autocrine IL-6 could be a useful approach to predict clinical outcome.     

T-cell therapies for T-cell lymphoma

T-cell lymphomas represent a subpopulation of non-Hodgkin lymphomas with poor outcomes when treated with conventional chemotherapy. A variety of novel agents have been introduced as new treatment strategies either as first-line treatment or in conjunction with chemotherapy. Immunotherapy has been demonstrated to be a promising area for new therapeutics, including monoclonal antibodies and adoptive cellular therapeutics. T-cell therapeutics have been shown to have significant success in the treatment of B-cell malignancies and are rapidly expanding as potential treatment options for other cancers including T-cell lymphomas. Although treating T-cell lymphomas with T-cell therapeutics has unique challenges, multiple targets are currently being studied both preclinically and in clinical trials.     

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.     

Loss of CD20 and bound CD20 antibody from opsonized B cells occurs more rapidly because of trogocytosis mediated by Fc receptor-expressing effector cells than direct internalization by the B cells

We previously reported that 1 h after infusion of CD20 mAb rituximab in patients with chronic lymphocytic leukemia (CLL), >80% of CD20 was removed from circulating B cells, and we replicated this finding, based on in vitro models. This reaction occurs via an endocytic process called shaving/trogocytosis, mediated by FcγR on acceptor cells including monocytes/macrophages, which remove and internalize rituximab-CD20 immune complexes from B cells. Beers et al. reported that CD20 mAb-induced antigenic modulation occurs as a result of internalization of B cell-bound mAb-CD20 complexes by the B cells themselves, with internalization of ～40% observed after 2 h at 37°C. These findings raise fundamental questions regarding the relative importance of shaving versus internalization in promoting CD20 loss and have substantial implications for the design of mAb-based cancer therapies. Therefore, we performed direct comparisons, based on flow cytometry, to determine the relative rates and extent of shaving versus internalization. B cells, from cell lines, from patients with CLL, and from normal donors, were opsonized with CD20 mAbs rituximab or ofatumumab and incubated for varying times and then reacted with acceptor THP-1 monocytes to promote shaving. We find that shaving induces considerably greater loss of CD20 and bound mAb from opsonized B cells in much shorter time periods (75-90% in <45 min) than is observed for internalization. Both shaving/trogocytosis and internalization could contribute to CD20 loss when CLL patients receive rituximab therapy, but shaving should occur more rapidly and is most likely to be the key mechanism of CD20 loss.     

Intraclonal diversity in the VH genes expressed by CD5- chronic lymphocytic leukemia-producing pathologic IgM rheumatoid factor.

The leukemic cells from 95% of patients with B cell chronic lymphocytic leukemia (CLL) coexpress B cell differentiation antigens and the pan-T lymphocyte surface antigen CD5 (Leu 1). As such, CLL generally may be considered a malignancy of the CD5 B cell, a minor B cell subpopulation implicated in the production of autoantibodies. Recent data indicate that CD5+ leukemic cells may express autoantibody-associated V region genes with little or no somatic mutation. We examined the Heavy chain V genes expressed by an unusual CLL that secretes rheumatoid factor (RF) autoantibodies but does not express the CD5 surface Ag. Nucleic acid sequence analyses of the rearranged VH genes of three independent rDNA clones demonstrated intraclonal diversity not apparent in previously studied cases of CD5+ CLL. Comparison of the rearranged VH genes reveals that they belong to the VH4 gene subgroup and share highest homology with a rearranged VH gene (Ab44) that encodes a polyreactive autoantibody. That these productively rearranged VH genes encode the RF generated by this unusual CLL population is demonstrated by immunoblotting of the RF paraprotein using primary sequence dependent antipeptide antisera. These results indicate that CD5- CLL, like their CD5+ counterparts, may produce RF. However, unlike CD5+ CLL examined to date, CD5- CLL may have intraclonal diversity in their expressed Ig genes.     

Monoclonal antibody therapy of chronic lymphocytic leukemia

Cure of patients with chronic lymphocytic leukemia (CLL) has been an elusive goal. The recent availability of active monoclonal antibodies has rekindled enthusiasm for new and innovative therapeutic approaches. Alemtuzumab, induces responses in about a third of patients with relapsed or refractory CLL following therapy with fludarabine and an alkylating agent. Whereas, rituximab has limited activity in previously treated patients, response rates of 50-70% have been reported in those without prior therapy. Recent data on combinations with rituximab and chemotherapy have shown promise for improving patient outcome. Newer antibodies in development include the primatized monoclonal antibody lumiliximab (IDEC-152), directed against CD23. Other biological approaches include the use of antisense oligonucleotides, proapoptic small molecules, and vaccines directed against the malignant B cells. The rational development of combinations of these promising approaches may eliminate the need for chemotherapy, leading to safer and more effective approaches for patients with CLL.     

Improvement in soluble expression levels of a diabody by exchanging expression vectors

Accumulating evidence suggests that bispecific antibody fragments (BsAbs) seem set to join monoclonal antibodies as powerful therapeutic and diagnostic agents, particularly for targeting cancer. One type of recombinant BsAbs are diabodies, which are constructed from heterogeneous single-chain antibodies and can increase antigen-specific cytotoxicity in T cells by cross-linking tumor antigens with T cell associated antigens. Some diabodies, however, cannot be solubly expressed in sufficient quantities, which limits their use in clinical therapeutics. Previously we constructed an anti-CD20 x CD3 diabody, which effectively directs the lytic potential of cytolytic T cells toward CD20(+) malignant B cells and shows marked antitumor efficacy in vivo. Here, to increase the amount of soluble product for clinical trials, we used an alternative expression vector under the T7 promoter but retained the stII signal sequences to ensure that the expressed protein is secreted to the periplasm of Escherichia coli. We achieved a periplasmic, soluble product by optimizing the conditions for induction with a yield of 8-9mg/L after affinity chromatography purification. This is nearly a five times greater yield than obtained with the previous vector. The diabodies generated from this modified vector retain dual binding specificity for both CD20-positive and CD3-positive cell lines. Taken together, these results suggest that changing expression vectors may be an alternative strategy to accomplish high-level expression of active BsAb proteins from E. coli.     

CXCL12 is a costimulator for CD4+ T cell activation and proliferation in chronic lymphocytic leukemia patients

Activated T cells from patients with chronic lymphocytic leukemia (CLL) provide survival and proliferative signals to the leukemic clone within lymphoid tissues. Recruitment of both, CLL cells and T lymphocytes, to this supportive microenvironment greatly depends on CXCL12 production by stromal and myeloid cells. CXCL12 also supplies survival stimuli to leukemic B cells, but whether it exerts stimulatory effects on T lymphocytes from CLL patients is unknown. In order to evaluate the capacity of CXCL12 to increase CD4⁺ T cell activation and proliferation in CLL patients, peripheral blood mononuclear cells were cultured with or without recombinant human CXCL12 or autologous nurse-like cells, and then T cell activation was induced by anti-CD3 mAb. CXCL12 increases the proliferation and the expression of CD25, CD69, CD154, and IFNγ on CD3-stimulated CD4⁺ T cells from CLL patients, similarly in T cells from ZAP-70⁺ to ZAP-70^? patients. Autologous nurse-like cells establish a close contact with CD4⁺ T cells and increase their activation and proliferation partially through a CXCR4-dependent mechanism. In addition, we found that activated T cells in the presence of CXCL12 enhance the activation and proliferation of the leukemic clone. In conclusion, CXCL12 production by lymphoid tissue microenvironment in CLL patients might play a key dual role on T cell physiology, functioning not only as a chemoattractant but also as a costimulatory factor for activated T cells.     

Synergistic inhibition of human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion and infection by an antibody to CD4 domain 2 in combination with anti-gp120 antibodies.

Antibodies to several epitopes of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41) can synergize in inhibiting HIV-1 infection. In the present study we tested the ability of a monoclonal antibody (MAb), 5A8, which interacts with CD4 domain 2, and other CD4-specific MAbs to synergize with antibodies against gp120. We have previously found that 5A8 inhibits HIV-1 entry without interfering with gp120 binding to CD4, presumably by affecting a postbinding membrane fusion event. Because antibodies to the gp120 V3 loop also affect post-CD4-gp120-binding events, 5A8 was first tested in combination with anti-V3 loop antibodies for possible synergy. The anti-V3 loop antibodies 0.5 beta, NEA-9205, and 110.5 acted synergistically with 5A8 in inhibiting syncytium formation between gp120-gp41- and CD4-expressing cells. A human MAb to an epitope of gp120 involved in CD4 binding, IAM 120-1B1, and another anti-CD4 binding site antibody, PC39.13, also exerted synergistic effects in combination with 5A8. Similarly, an antibody against the gp120 binding site on CD4, 6H10, acted synergistically with an anti-V3 loop antibody, NEA-9205. However, a control anti-CD4 antibody, OKT4, which does not significantly inhibit syncytium formation alone, produced only an additive effect when combined with NEA-9205. Serum from HIV-1-infected individuals, which presumably contains antibodies to the V3 loop and the CD4 binding site, exhibited a strong synergistic effect with 5A8 in inhibiting infection by a patient HIV-1 isolate (0104B) and in blocking syncytium formation. These results indicate that therapeutics based on antibodies affecting both non-gp120 binding and gp120 binding epitopes of the target receptor molecule, CD4, could be efficient in patients who already contain anti-gp120 antibodies and could also be used to enhance passive immunization against HIV-1 in combination with anti-gp120 antibodies.     

Differential effects of IL-2 and IL-21 on expansion of the CD4+CD25+Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia

CD4⁺ CD25+ regulatory T cells are expanded in solid and hematological malignancies including Chronic Lymphocytic Leukemia (CLL). Several cytokines and co-stimulatory molecules are required for generation, survival and maintenance of their suppressive effect. We and others have shown direct cytotoxic effect of the novel common gamma chain cytokine interleukin- 21 (IL-21) on primary B cells from CLL patients. Since members of this family of cytokines are known to exhibit their effects on diverse immune cells, we have examined the effects of IL-21 on CLL patient derived regulatory T cell induction, expansion and the inhibitory effect on natural killer cells in vitro. We demonstrate here the expression of IL-21 receptor in CD4⁺CD25^High regulatory cells from CLL patients. In contrast to IL-2, the IL-21 cytokine failed to mediate expansion of regulatory T cells or induced expression of Foxp3 in CD4⁺CD25Intermediate or CD4+CD25^Dim/- T cells in whole blood derived from CLL patients. Interestingly, in contrast to their differential effects on expansion of the CD4⁺CD25⁺Foxp3⁺T cells, IL-2 and IL-21 exhibited a redundant role in T-reg mediated suppression of NK cell mediated antibody dependent cytotoxicity function. Given the infusion related toxicities and pro-survival effect of IL-2 in CLL, these studies provide a rationale to explore IL-21 as an alternate gamma chain cytokine in CLL therapy.     

Vaccination with dendritic cells loaded with tumor apoptotic bodies (Apo-DC) in patients with chronic lymphocytic leukemia: effects of various adjuvants and definition of immune response criteria

We previously demonstrated that autologous dendritic cells that have endocytosed apoptotic bodies of chronic lymphocytic leukemia (CLL) cells (Apo-DC) can stimulate antileukemic T cell responses in vitro. In this phase I study, we vaccinated 15 asymptomatic CLL patients at five time points with Apo-DC administered intradermally either alone (cohort I), or in combination with subcutaneous granulocyte-macrophage-colony-stimulating-factor (GM-CSF) (cohort II) or with GM-CSF and intravenous low-dose cyclophosphamide (cohort III). Aim of the study was to evaluate the safety and immunogenicity of Apo-DC alone or in combination with GM-CSF and low-dose cyclophosphamide in CLL patients. All patients completed the vaccination schedule without dose-limiting toxicity. No objective clinical responses were seen. Vaccine-induced leukemia-specific immune responses were evaluated by IFN-γ ELISpot and proliferation assays over a 52 weeks observation period and immune response criteria were defined. According to these criteria, 10/15 patients were defined as immune responders. The frequency of immune-responding patients was higher in cohorts II (3/5) and III (5/5) than in cohort I (2/5). In order to further characterize the induced immune response, estimation of secreted cytokines and CD107-degranulation assay were performed. Clustering of T and CLL cells was observed in CD107-degranulation assay and visualized by confocal microscopy. Additionally, assessment of regulatory T cells (T(regs)) revealed their significantly lower frequencies in immune responders versus non-responders (P?相似文献   

Chronic lymphocytic leukemia cells bind and present the erythrocyte protein band 3: possible role as initiators of autoimmune hemolytic anemia

The mechanisms underlying the frequent association between chronic lymphocytic leukemia (CLL) and autoimmune hemolytic anemia are currently unclear. The erythrocyte protein band 3 (B3) is one of the most frequently targeted Ags in autoimmune hemolytic anemia. In this study, we show that CLL cells specifically recognize B3 through a still unidentified receptor. B3 interaction with CLL cells involves the recognition of its N-terminal domain and leads to its internalization. Interestingly, when binding of erythrocyte-derived vesicles as found physiologically in blood was assessed, we observed that CLL cells could only interact with inside-out vesicles, being this interaction strongly dependent on the recognition of the N-terminal portion of B3. We then examined T cell responses to B3 using circulating CLL cells as APCs. Resting B3-pulsed CLL cells were unable to induce T cell proliferation. However, when deficient costimulation was overcome by CD40 engagement, B3-pulsed CLL cells were capable of activating CD4(+) T cells in a HLA-DR-dependent fashion. Therefore, our work shows that CLL cells can specifically bind, capture, and present B3 to T cells when in an activated state, an ability that could allow the neoplastic clone to trigger the autoaggressive process against erythrocytes.     

Evolution of anti-CD20 monoclonal antibody therapeutics in oncology

Approval of an anti-CD20 chimeric monoclonal antibody, rituximab, has revolutionized cancer treatment and also validated CD20 targeting for providing benefit and improvement of overall response rate in B cell malignancies. Although many patients have benefited from the treatment of rituximab, there are still significant numbers of patients who are refractory or develop resistance to the treatment. Here we discuss pre-clinically well-defined potential mechanisms of action for rituximab and review the ways next generation anti-CD20 monoclonal antibodies can potentially exploit them to further enhance the treatment of B cell malignancies. Although the relative importance of each of these mechanism remains to be established in the clinic, well-designed clinical trials will help to define the efficacy and understanding of which effector activity of modified next generation anti-CD20 mAb will be important in the treatment of B-cell malignancies.Key words: CD20, NHL, CLL, monoclonal antibody, next generation anti-CD20 antibodies, ADCC, CDC, ADCP, PCD, rituximab     

Morphological and immunophenotypic features of chronic lymphocytic leukemia

In this review, we summarize the morphological features and immunophenotypic profile of chronic lymphocytic leukemia (CLL) cells, discuss the value of these investigations as front line diagnostic tests, and emphasize their correlation with the clinical features, disease progression, molecular genetics and pathogenesis of CLL. In CLL, the morphology of the circulating cells is characteristic and typical in the majority of cases. However, 15% of patients, either at diagnosis or during the course of the disease, show atypical morphology reflected by either (1) an increased (> 10%) number of circulating prolymphocytes, designated CLL/PL, or (2) an increased (> 15%) number of circulating lymphoplasmacytic and cleaved cells, designated 'atypical' CLL. There is strong evidence of a close association between atypical morphology (CLL/PL) and atypical (CLL) and clinical features, e.g. disease progression, advanced stage and survival, molecular genetics, particularly trisomy 12, but also the rare cases with t(11;14) or t(14;19), p53 abnormalities, unmutated immunoglobulin (Ig) VH genes and origin of the cell (naive, pregerminal center cell). CLL cells have a distinct immunological repertoire different from that of other lymphoproliferative disorders. The typical CLL phenotype is CD5+, CD23+, FMC7-, weak expression of surface Ig (sIg) and weak or absent expression of membrane CD22 and CD79b. The latter marker identifies an extracellular epitope of the B-cell receptor (BCR) beta chain and its weak or absent expression in CLL may derive from the expression of a truncated form. This, together with the low expression of CD22, might explain the abnormal signal transduction of CLL cells similar to that of anergic B lymphocytes. Because no single marker is specific for CLL, a composite phenotype considering this set of 5 or 6 markers compounded into a scoring system helps to distinguish CLL from the other B-cell malignancies. Immunophenotypic analysis has also been shown to be useful for minimal residual disease detection and adds valuable prognostic information because the expression of certain markers, such as FMC7 or CD38, seems to be associated with a poor outcome. In addition, CLL cells express a variety of Bcl-2 family proteins with a profile that favors inhibition of apoptosis which, together with the interaction with microenvironmental (e.g. stromal) cells and the release of cytokines, explains the long life span and subsequent accumulation of CLL cells in various organs. Despite controversies relating to the expression of adhesion molecules (selectins and integrins) in CLL cells, it appears that some of these molecules do play a role in the pathogenesis, biology and clinical patterns of the disease. In conclusion, morphology and immunophenotype are the two essential investigations, which must be carried out in all cases of CLL. Both provide relevant information in terms of diagnosis, course of the disease, prognosis and pathogenesis.     

54例慢性淋巴细胞白血病的临床回顾性分析

目的：本研究旨在探讨慢性淋巴细胞白血病（CLL）的实验室检查特点及特征性临床表现。方法：利用血细胞分析仪、流式细胞术、骨髓形态分析及R显带技术等对我院2002年4月．2012年4月收治的54例慢性淋巴细胞白血病患者的相关临床资料如血细胞计数、骨髓形态、染色体及免疫表型等进行检测并对结果进行回顾性分析。结果：CLL多发于老年患者,男性多见,中位年龄65岁（45．82岁）。大部分患者初诊时可出现典型的临床表现,37例（68％）患者初诊时淋巴结大,49例（91％）初诊时白细胞≥10×109、L,淋巴细胞绝对值≥5×109／L。13例（24％）初诊时肝脾或者脾大,17例（31％）初诊时乏力、消瘦。34（63％）例患者可见典型的CLL免疫表型,CD5、CDl9．CD23、CD20的阳性率分别为90％、87％、72％、67％。32例患者染色体检测结果表明：13q-2例,17p．2例,11q-1例,＋12有1例,6q-1例,t（14,16）1例。2例患者发生了自身免疫性溶血性贫血（AIHA）。1例患者发生了Richter转化,肿大淋巴结活检显示部分区域为弥漫性大B细胞淋巴瘤,其高表达CD20、CDl9、CD22。结论：慢性淋巴细胞白血病具有其典型的临床表现、免疫表型及遗传学改变,并且对诊断及治疗有重要意义。     

A Combination of Cytokines Rescues Highly Purified Leukemic CLL B-Cells from Spontaneous Apoptosis In Vitro

B-chronic lymphocytic leukemia (B-CLL), the most common human leukemia, is characterized by predominantly non-dividing malignant mature CD5+ B lymphocytes with an apoptosis defect. Various microenvironmental stimuli confer a growth advantage on these leukemic cells and extend their survival in vivo. Nevertheless, when cultured in vitro, CLL B-cells rapidly die from apoptosis. Certain cytokines may extend the survival capacity of CLL B-cells in vitro and individual anti-apoptotic effects of several cytokines have been reported. The potential cumulative effect of such cytokines has not been studied. We therefore investigated the effects on CLL B-cells survival in vitro of humoral factors, polyclonal lymphocyte activators and a combination of cytokines known for their anti-apoptotic effects. Purified CLL B-cells were cultured in the presence or absence of various soluble molecules and the leukemic cell response was assessed in terms of viability. Apoptotic cell death was detected by flow cytometry using annexinV and 7-amino-actinomycin. The survival of CLL B-cells in vitro was highly variable. When tested separately, cytokines (IL-2, -6, -10, -12, -15, -21, BAFF and APRIL) improved CLL B cell survival moderately; in combination, they significantly enhanced survival of these cells, even up to 7 days of culture. We also report that humoral factors from autologous serum are important for survival of these malignant cells. Our findings support the concept that the CLL microenvironment is critical and suggest that soluble factors may contribute directly to the prolonged survival of CLL B-cells. Therefore, the combination of cytokines we describe as providing strong resistance to apoptosis in vitro might be used to improve the treatment of CLL.     

Circulating monoclonal IgM proteins in B cell chronic lymphocytic leukemia: their identification, characterization and relationship to membrane IgM

Accumulated evidence indicates that there is a circulating monoclonal Ig protein related to the leukemic cell-associated Ig in the majority of patients with B cell chronic lymphocytic leukemia (CLL) despite the failure to demonstrate such a protein by conventional serum electrophoresis. Methodology has been developed to reveal these hidden monoclonal bands and to show that they are related to the leukemia-associated membrane Ig (mIg). Of nine CLL cases with stainable mIgM and without discernable plasma Ig bands, marked hypogammaglobulinemia was evident in six. In the other three, a significant amount of protein was present in the gamma region. IgM was isolated from the plasma of these patients by affinity chromatography with Sepharose-4B, conjugated with affinity purified anti-human IgM antibodies. One to 3 mg were isolated from 20 to 40 ml of plasma. Agarose electrophoresis revealed a monoclonal Ig band in the isolated IgM in all cases. Eight of these IgM proteins were analyzed by high-pressure liquid chromatography. Five were found to be pentameric IgM. In the remaining three, various amounts of monomeric IgM were detected. Attempts to make anti-idiotypic antibodies to the isolated proteins have been successful. Thus far, a rabbit anti-idiotypic antiserum was obtained in one case and two mouse monoclonal anti-idiotypic antibodies in two additional cases. Immunofluorescence analysis revealed that plasma IgM and mIgM shared similar idiotypic determinants. One other monoclonal antibody was shown to be specific for a V region marker of a minor Ig population. These findings indicate that B leukemic lymphocytes do secrete a small amount of IgM and lend further support to the thesis that the maturation defect in CLL is incomplete. It is also feasible to isolate the secreted IgM and to produce anti-idiotypic antibodies to them. In view of the potential therapeutic effect of anti-idiotypic antibodies, this may offer an alternative and efficient approach to generate a large panel of anti-idiotypic antibodies for clinical trials. The possibility also exists that this approach is applicable to other B cell proliferative disorders such as the non-Hodgkin B cell lymphomas.     

Exhaustion of cytotoxic effector systems may limit monoclonal antibody-based immunotherapy in cancer patients

The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.     

Natural phosphorylation of CD5 in chronic lymphocytic leukemia B cells and analysis of CD5-regulated genes in a B cell line suggest a role for CD5 in malignant phenotype

Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-kappaB (RELB, BCL3), Wnt, TGFbeta, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.