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NEOPLASIA
From the Leukemia and Lymphoma Marker Laboratory,
Department of Hematology, and the Finsen Laboratory, Rigshospitalet,
Copenhagen, Denmark.
Interaction between CD40 and the CD40 ligand (CD40L) is critical
for the survival and proliferation of B cells during immunopoiesis. However, the role of CD40L in the pathogenesis of malignant lymphomas is ambiguous. Primary mantle cell lymphoma (MCL) cells were cultured in
the presence of recombinant human CD40L trimer (huCD40LT), and a
significant time- and dose-dependent induction of DNA synthesis was
observed in thymidine incorporation assays (n = 7,
P < .04). The maximal rate of DNA synthesis was reached
at huCD40LT doses of 100 ng/mL and above after 4 days of culture, but a
significant increase of DNA synthesis was detected already at doses of
1 ng/mL (P = .03). HuCD40LT never inhibited the basal
level of DNA synthesis. These findings established 400 ng/mL of
huCD40LT for 4 days as standard conditions in the system. Under
these conditions, huCD40LT significantly increased the proportion of
cells in the S/G2/M phases of the cell cycle in 4 of 7 studied cases, while the fraction of apoptotic cells remained unchanged
(n = 7). HuCD40LT also induced expression of CD80/B7-1, CD86/B7-2,
and CD95/Fas and up-regulated the expression of HLA-DR (n = 6). With
the use of bromodeoxyuridine incorporation in triple-color flow
cytometric analysis, it was found that huCD40LT induced cell-cycle
progression in light chain-restricted cells only, of which a median of
14% (range, 0.5% to 29%; n = 4) returned to G0/1 phase
DNA content after bromodeoxyuridine incorporation, demonstrating
completion of at least one cell cycle in the presence of huCD40LT.
Thus, primary clonal MCL cells are activated and can proliferate in the
presence of huCD40LT as a single agent.
(Blood. 2000;96:2219-2225) Mantle cell lymphoma (MCL) is a distinct
CD5+ B-cell non-Hodgkin lymphoma (NHL) with low
chemosensitivity and a very poor prognosis, even when treated with
high-dose therapy and autologous bone marrow transplantation.1-4 MCL is characterized by the
translocation t(11;14)(q13;q32), leading to overexpression of cyclin D1
and subsequent deregulation of the critical cyclin D1/Retinoblastoma protein pathway of the cell cycle.5-7 Cyclin D1
overexpression, however, is neither tumorigenic per se in mouse models
nor related to the proliferative activity in MCL, suggesting that the
malignant behavior of the disease depends on further growth
deregulation of MCL cells.8,9 CD40/CD40 ligand (CD40L)
interaction is critical for the survival and proliferation of B cells
engaged in secondary immune responses.10-13 In lymphoma
cell lines, activation of CD40 can induce phosphorylation of
Retinoblastoma protein, suggesting a role for CD40L in G1/S
phase transition and in lymphomagenesis.14 The role of
CD40 activation, however, and in particular the data on the effects on
growth and apoptosis in normal and malignant B cells are
conflicting.15 For example, CD40 ligation results in
cell-cycle progression in resting B cells and rescue from apoptosis in
normal germinal center B cells.16,17 By contrast, a direct growth-inhibitory effect was observed in diffuse large cell NHL, Burkitt lymphoma (BL), and Epstein-Barr virus-transformed cell lines
after stimulation using the soluble CD40L.18-21 However, soluble CD40L induced DNA synthesis in primary follicular lymphoma cells.21 At least 2 factors are important for the response
to CD40 ligation. First, different CD40 ligands, agonistic antibodies, or recombinant proteins, as well as different CD40 systems, using transfected and irradiated cell lines for presenting the CD40 ligand,
have been shown to influence the effect of CD40 stimulation differently.22-24 The transfected cell lines influence the
effect of CD40 ligation by expression or secretion of other B-cell
stimulatory molecules. Second, the functional consequences of CD40
signaling appear to be highly dependent on the B-cell type being
triggered.15,25 In 2 previous studies of CD40 ligation in
MCL, agonistic anti-CD40 antibody and CD40L-presenting cell lines both
failed to induce proliferation, as estimated by thymidine incorporation
and relative cell numbers, respectively.26,27 Besides
growth regulation, CD40L plays a critical role in the activation of
antigen-presenting cells, including B cells.28 On normal
and malignant B cells, CD40 ligation induces expression of
co-stimulatory molecules, such as CD80/B7-1 and CD86/B7-2, the
expression of which is essential for appropriate and efficient antigen
presentation during cognate B-cell/T-cell interactions, and the
generation of secondary immune responses.29-31 Therefore,
the possible benefits of ex vivo and in vivo CD40 activation and the
subsequent induction of an antigen-presenting phenotype in malignant B
cells are under investigation in phase 1 trials in low-grade NHL and in
B-cell chronic lymphocytic leukemia (CLL).32-34 Here, we
have used a soluble recombinant human CD40L trimer (huCD40LT) to study
the functional consequences of CD40 ligation in terms of activation,
proliferation, and apoptosis in primary MCL cells in vitro. We
demonstrate that huCD40LT as a single agent induces significant
proliferation of lymphoma cells in primary MCL cell cultures.
Patients
Sample preparation
Polymerase chain reaction (PCR) to detect t(11;14) and cyclin D1 RNA overexpression Semi-nested PCR analysis of MCL patients carrying the t(11;14) with breakpoints in the major translocation cluster was performed as described previously.3 Cyclin D1 RNA overexpression was detected using a semiquantitative reverse transcription (RT)-PCR, which competitively amplified cyclin D1, D2, and D3, as described.35 Non-MCL cases of NHL, CLL, and normal donor samples were used as negative controls. Six of 8 patients had the t(11;14) translocation or cyclin D1 RNA overexpression by PCR analysis (Table 1). In the remaining 2 patients (nos. 1 and 2), the diagnosis of MCL was based on pathology and the CD5+/CD19+, CD23 , surface immunoglobulin bright immunophenotype,
which was detected in all 8 patients (Table 1).
Determination of expression of surface membrane molecules Phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against cell surface markers (kappa and lambda light chains, CD19, CD5, CD23, CD40, CD80, CD86, CD95/Fas, CD3, and CD154/CD40L) (Becton Dickinson, Mountain View, CA; Dako, Glostrup, Denmark) and the relevant isotype-matched controls were used in standard flow cytometry analysis, with inclusion of all events larger than 52 on forward scatter and no additional gating (FACSort; Becton Dickinson). A total of 5000 cells were acquired and analyzed using CellQuest software (Becton Dickinson).Cell culture assays Isolated MCL cells were plated at a concentration of 2 × 106 cells per milliliter medium in the presence or absence of various concentrations of purified huCD40LT (Immunex Corporation, Seattle, WA) and anti-CD40L antibodies (Becton Dickinson, Belgium) and incubated at 37°C in a humidified atmosphere containing 5% CO2. For thymidine incorporation, stimulated and unstimulated control cultures were plated in triplicate in 96-well, 200-µL U-bottom plates and pulsed with 1 µCi [methyl-3H] thymidine (3HTdR) (TRK 637; Amersham, Cardiff, UK) 16 hours before harvest, and thymidine incorporation was measured on a direct beta-counter (Matrix 9600; Packard, Meriden, CT). For quantification of cells undergoing programmed cell death (apoptosis) and cells in the S/G2/M phase of the cell cycle, isolated nuclei from cells from single cultures were stained with propidium iodide (PI) and analyzed by flow cytometry, as described.36Viable cell recovery PI exclusion assays of whole-cell preparations from single MCL cell cultures were used to define which cells in the scatter profiles were viable with complete exclusion of PI. Two distinct populations were defined in the scatter profiles based on cells with and without PI exclusion. Viable cells were significantly larger and excluded PI completely. In our recovery assay, isolated cells were plated in triplicate in 50-mL culture flasks at a concentration of 2 × 106 cells/mL with and without huCD40LT in a final volume of 5 mL for up to 2 weeks. Cells sized between 8 and 20 µmol/L were counted 3 times using a Coulter Z1 particle counter (Coulter Electronics Limited, Luton, UK). The averages of these 9 counts were used to calculate the viable cell recovery in the cultures at different time points.Flow cytometric analysis of monoclonality, DNA synthesis, and cell-cycle progression Multiparameter flow cytometry analysis with combined measurement of clonality, DNA synthesis, and DNA content was carried out essentially as described.37,38 In brief, primary MCL cells were cultured for 4 days in single cultures in the presence or absence of 400 ng/mL of huCD40LT and supplemented with 50 µmol/L bromodeoxyuridine (BrdUrd) (Sigma, St Louis, MO) for an additional 24 hours of culture. After harvest, the cells were first stained with PE-conjugated anti-kappa or anti-lambda antibodies. The cells were then permeabilized and fixed in a mixture of 1% paraformaldehyde and 0.05% Nonidet P40. Incorporation of BrdUrd, as evidence of DNA synthesis, was then detected using anti-BrdUrd FITC-labeled antibodies (Becton Dickinson) after 1 mg/mL DNase I (DN-25; Sigma) digestion for 30 minutes. DNA content was measured using the DNA-specific dye 7-amino-actinomycin-D (7-AAD) (Sigma). For each sample, 20 000 events were acquired and analyzed (FACS Vantage; Becton Dickinson). To exclude nuclei without proliferative capacity, we defined an acquisition gate such that only events from the G1 peak and brighter were acquired. This gate contained approximately 70% of the total events. After gating on lymphoma cells expressing the clonally restricted light chain of the particular MCL case, a combined analysis of DNA synthesis and DNA content was performed.Statistical analysis The nonparametric Wilcoxon signed rank sum test was used to compare assay results from samples cultured under different conditions.
HuCD40LT specifically induces DNA synthesis in primary MCL cell cultures We first applied a thymidine incorporation assay to assess whether huCD40LT could induce DNA synthesis in primary MCL cultures. The quality of the response was homogeneous, and huCD40LT induced dose-dependent thymidine incorporation in all of the 7 MCL samples studied (Figure 1A). The quantity of the response, however, was heterogeneous among the individual cases. The maximal rate of DNA synthesis (mean, 8162 cpm; range, 61-48 000 cpm) was reached at huCD40LT doses between 100 ng/mL and 1000 ng/mL and was significantly higher than the DNA synthesis in the presence of medium alone (mean, 1813 cpm; range, 4-12 500 cpm; P = .03, Wilcoxon) (Figure 1A). We never observed any direct inhibitory effects of huCD40LT on the basal DNA synthesis in primary MCL cell cultures, not even at doses of huCD40LT up to 10 000 ng/mL (Figure 1A). The ability of huCD40LT to induce DNA synthesis in purified leukemic cells was significantly higher in MCL cultures than in cultures from patients with CLL (data not shown). Kinetic studies, using a fixed dose of 400 ng/mL of huCD40LT, showed that the induction of DNA synthesis was also dependent on time and reached a maximal response at day 4 (P < .04, Wilcoxon) (Figure 1B). Significant spontaneous synthesis of DNA could not be demonstrated within the first week of culture (Figure 1C). The specificity of the response was demonstrated in co-cultures, in which the combined presence of 100 µg/mL of anti-CD40L antibody and 400 ng/mL of huCD40LT reduced the huCD40LT-induced DNA synthesis to the level of the unstimulated control (data not shown). On the basis of these results, we chose 400 ng/mL of huCD40LT for 4 days as the standard conditions for further experimentation.
HuCD40LT increases the number of viable and cycling cells, but does not rescue cells from spontaneous apoptosis, in primary MCL cell cultures The ability of huCD40LT to induce DNA synthesis in the system is not necessarily equivalent to proliferation of a majority of the cultured cells. In fact, as little as 0.1% residual cells have been noted to contribute significantly to thymidine incorporation in mixed culture systems like the one applied here.39 PI staining and flow cytometric analysis of DNA content were therefore undertaken to assess the effects of huCD40LT on spontaneous apoptosis, cell-cycle progression, and viable cell recovery. No significant changes in the percentage of cells within the sub-G1 peak of the PI histogram, representing cells undergoing apoptosis, were detected in cells cultured under standard conditions and compared with medium alone (Table 2). The percentage of cells in the S/G2/M region of the PI histogram, representing cells in the S, G2, or M phases of the cell cycle, was increased from a median of 6.4% (range, 1.9% to 11.9%) in medium alone to a median of 10.8% (range, 2.1% to 22.5%) in the presence of huCD40LT (Table 2). Although this difference did not reach statistical significance, patients 2, 3, 4, and 7 showed clear increases in the percentages of cells in S/G2/M phases, suggesting that at least some of the proliferative response occurs within the clonal population of MCL cells. The viability of cells increased significantly from a median of 12.2% (range, 3.8% to 39.9%) in the presence of medium alone to a median of 26.5% (range, 8% to 53%) in the presence of huCD40LT, as assessed by flow cytometry-based PI exclusion (P = .03, n = 6, Wilcoxon) (Table 2). Thus, although the majority of cells were not viable at day 4, huCD40LT induced thymidine incorporation and increased the fraction of cycling and viable cells.
Therefore, we next assessed whether huCD40LT could also increase the absolute number of cells in primary MCL cultures. Compared with day 0, the absolute number of viably recovered cells from patients 3 and 4 doubled within 1 week of culture in the presence of huCD40LT (data not shown). We were unable to extend the cell growth in these cultures beyond day 8, and washing, replating, and restimulation with huCD40LT did not induce any further increase in viable cell recovery. This failure to induce responses to huCD40LT after 1 week of culture may be a result of MCL lymphoma cell differentiation and CD40 rewiring.13 HuCD40LT induces expression of CD80/B7-1, CD86/B7-2, and CD95/Fas in primary MCL cell cultures To explore whether the failure to induce extended growth could be caused by loss of CD40 expression, we analyzed the expression of a number of surface membrane markers (see Materials and methods) in primary MCL cell cultures. The vast majority of CD19+ cells expressed readily detectable CD40 throughout 4 days of culture in the presence of huCD40LT in all of the 6 MCL cases studied (data not shown). We never observed coexpression of CD154/CD40L on CD19+ cells regardless of the culture conditions. Consistent with previous reports,29,30 huCD40LT induced significant expression of the costimulatory molecule CD80/B7-1 on CD19+ cells regardless of the proliferative response of the particular MCL case (Figure 2). CD86/B7-2 was similarly induced on CD19+ cells. Along with the up-regulation of the activation markers, we also noted an induced expression of the death receptor CD95/Fas and increased expression of the major histocompatibility complex molecule HLA-DR. All samples remained CD23 regardless of the culture conditions (data
not shown).
HuCD40LT induces selective cell-cycle progression in clonal MCL cells in primary MCL cell cultures To verify that huCD40LT-induced cell-cycle progression took place in the clonal cells of the primary MCL cultures and not in any residual nonleukemic cells, in particular residual T cells receiving costimulatory signals from MCL cells activated by huCD40LT, we applied a multiparameter flow cytometric assay. In this assay, simultaneous measurement of clonality (light-chain restriction, FL2), DNA synthesis (BrdUrd incorporation, FL1), and DNA content (7-AAD, FL3) was carried out. The kappa and lambda light-chain markers were first used to identify cells in which BrdUrd incorporation occurred in the presence of huCD40LT. As shown in Figure 3A, the majority of cells incorporating BrdUrd after 4 days of culture stained positive for a restricted light chain. These results indicate that the cells belonging to the MCL population, and not residual nonclonal cells in the culture system, are the major contributors to the DNA synthesis observed in the other proliferation assays. Simultaneous analysis of BrdUrd incorporation and DNA content (7-AAD) in light chain-restricted cells from the primary MCL cell cultures yielded 2 populations of BrdUrd-positive cells, based on 7-AAD fluorescence intensity: one population of cells in S/G2/M phase and another population in post-mitotic G0/1 phase. As seen in Figure 3B, huCD40LT induced at least a 3-fold increase of clonal BrdUrd-positive G1 cells, which have progressed through and completed the cell cycle within the last 24 hours of the BrdUrd incubation period of the 5-day culture. HuCD40LT induced a median of 14% (range, 0.5% to 29%; n = 4) of G1/BrdUrd-positive cells as compared with the unstimulated control cultures. Because the cells in this analysis were gated for light-chain restriction, this observation can be explained only by huCD40LT-induced proliferation of primary MCL cells.
In this study, we demonstrate that activation of CD40 by a purified recombinant human CD40 ligand trimer as a single agent induces proliferation of lymphoma cells in primary MCL cell cultures. This conclusion is based on several lines of evidence from 4 different proliferation assays: (1) huCD40LT induced dose- and time-dependent thymidine incorporation; (2) huCD40LT increased the percentage of cells in S, G2, or M phases of the cell cycle; (3) huCD40LT increased the viable cell recovery; (4) essentially all huCD40LT-induced BrdUrd incorporation occurred in cells expressing a restricted light chain; (5) a significant proportion of these BrdUrd-positive, light chain-restricted clonal MCL cells completed the cell cycle within 24 hours, between day 4 and day 5 of the culture period; and (6) we never observed any growth inhibition, not even at very high doses of huCD40LT. The huCD40LT-induced proliferation occurred without any concomitant effects on the degree of spontaneous apoptosis. The implications of these results are important for 3 aspects of MCL biology: the interpretation of data from different CD40 systems, the response elicited by CD40 in heterogeneous primary cultures, and the possible role of CD40 in the pathobiology and treatment of MCL. Our results are contradictory to previous studies of the role of CD40 activation in MCL and other NHL lymphomas.18-21,26,27 In NHL cell lines, CD40 ligation induced a direct growth inhibition,18-21 and in 2 MCL studies, the proliferative response of CD40 activation was very weak or absent.26,27 These contradictions stress that the interpretation of studies of CD40 function must reflect the applied CD40 activation system as well as the developmental stage of the triggered B cell.15 For example, the agonistic antibody used in one of the MCL studies may significantly underestimate the full effect of CD40 ligation when compared with the huCD40LT.22 The use of irradiated stimulatory cell lines presenting either CD40 ligand or agonistic CD40 antibodies introduces the risk that other presently identified or unidentified biologic substances may influence the observed responses.23,24 Our results clearly demonstrate that a purified recombinant CD40L trimer induces proliferation in primary MCL clonal cells. Although the MCL cells did respond to additional activation of the interleukin-4 receptor (data not shown), activation through CD40 alone was sufficient to trigger the proliferative response without any requirement for supplementary stimulation through cytokine receptors. This is important because proliferation assays may measure the results of activation of residual T cells in the mixed system.39 In particular, the present study confirms that huCD40LT induced phenotypic changes of MCL cells associated with acquisition of an effective antigen-presenting phenotype. Thus, DNA synthesis in mixed primary cell cultures like the one used in this study could be the result of direct huCD40LT-induced growth of MCL cells or, alternatively, could be the result of CD40-induced phenotypic changes and subsequent activation and proliferation of residual T cells, responding to the antigen presented by CD40-activated MCL cells with coexpression of CD80/B7-1 and CD86/B7-2. The simultaneous analysis of phenotype, DNA synthesis, and DNA content did not support the latter possibility. Essentially all BrdUrd incorporation occurred in cells with restricted light-chain expression, which strongly suggests that these cells belong to the malignant clone. Thus, the results of this study are in keeping with a recent study in which primary follicular lymphoma cells were found to undergo proliferation in the presence of huCD40LT.21 This conclusion raises concern for the application of CD40L therapy in vivo in MCL. On the other hand, ex vivo expansion of primary MCL cells with an effective antigen-presenting phenotype may be a useful tool for the application of adaptive immune therapy in MCL. One unexplained finding in this study was our failure to extend absolute expansion of primary MCL cells beyond 1 week of culture. Despite restimulation, washing, dilution, and replating, the cells from the 2 patients studied with the recovery assay (patients 3 and 4) ceased proliferation within 1 week of culture. This suggests that stimulation through CD40 somehow alters subsequent restimulation through CD40. Whether this phenomenon is caused by MCL lymphoma cell differentiation after huCD40LT stimulation and altered signaling by the receptor or altered gene-expression profiles following the primary CD40-derived signal is not known. Although the quality of the response was comparable among the 8 patients studied, the magnitude of the response was highly heterogeneous (Figures 1 and 3, Table 2). This difference could also be the result of either differential signal transduction through CD40 or differential gene-expression profiles between low- and high-responder cases. To address these issues, we are currently investigating the kinetics and array of gene expression, as well as CD40-mediated signal transduction after huCD40LT stimulation, in low- and high-responder cases of MCL. Finally, our results demonstrate that activation through CD40 is sufficient to commit a fraction of primary MCL lymphoma cells to cell-cycle progression in vitro, although the size of this fraction is highly variable. In vivo CD40L is induced on activated T cells at different stages of immunopoiesis in secondary lymphoid tissues. The abundance of follicular dendritic cells in MCL-infiltrated lymph nodes may lead to higher numbers of activated T cells and increased CD40L expression, as have been observed in BL.40,41 In line with this, we have previously observed somatic mutations in the heavy chain of the immunoglobulin gene in clonal MCL cells, indicating that antigen selection can take place in at least some cases of MCL.3 To do so, the B cell, in which the final transformation took place, must have had the capacity to interact productively with a T cell. Together with the results of the present study, these data suggest that cooperation between inherent cell-cycle deregulatory genetic lesions in MCL and a CD40L-rich microenvironment may be sufficient to induce cell-cycle progression of importance in the pathogenesis of MCL.
We thank the Nordic Lymphoma Group and Dr Erik Segel for supplying patient samples. Human recombinant CD40 ligand trimer was kindly provided by Immunex Corporation, Seattle, WA.
Submitted March 10, 2000; accepted May 9, 2000.
Supported by grants from the Danish Medical Research Council (96-018-28), the Danish Cancer Society (94-100-62 and 96-100-09), the John and Birthe Meyer Foundation, Katrine and Vigo Skovgaards Foundation, and A. L. Vengs Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Niels S. Andersen, Department of Hematology L-4041, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark; e-mail: nanders{at}rh.dk.
1.
Harris NL, Jaffe ES, Stein H, et al.
A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.
Blood.
1994;84:1361-1392
2.
Hiddemann W, Longo DL, Coiffier B, et al.
Lymphoma classification
3.
Andersen NS, Donovan JW, Borus JS, et al.
Failure of immunologic purging in mantle cell lymphoma assessed by polymerase chain reaction detection of minimal residual disease.
Blood.
1997;90:4212-4221
4.
Freedman AS, Neuberg D, Gribben JG, et al.
High-dose chemoradiotherapy and anti-B-cell monoclonal antibody-purged autologous bone marrow transplantation in mantle-cell lymphoma: no evidence for long-term remission.
J Clin Oncol.
1998;16:13-18
5.
Rosenberg CL, Wong E, Petty EM, et al.
PRAD1, a candidate BCL1 oncogene: mapping and expression in centrocytic lymphoma.
Proc Natl Acad Sci U S A.
1991;88:9638-9642 6. Oka K, Ohno T, Kita K, et al. PRAD1 gene over-expression in mantle-cell lymphoma but not in other low-grade B-cell lymphomas, including extranodal lymphoma. Br J Haematol. 1994;86:786-791[Medline] [Order article via Infotrieve]. 7. Bartek J, Bartkova J, Lukas J. The retinoblastoma protein pathway in cell cycle control and cancer. Exp Cell Res. 1997;237:1-6[Medline] [Order article via Infotrieve]. 8. Bodrug SE, Warner BJ, Bath ML, Lindeman GJ, Harris AW, Adams JM. Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene. EMBO J. 1994;13:2124-2130[Medline] [Order article via Infotrieve]. 9. Ott G, Ott MM, Kalla J, et al. Genetic lesions in mantle cell lymphoma. Recent Results Cancer Res. 1997;143:307-319[Medline] [Order article via Infotrieve]. 10. Allen RC, Armitage RJ, Conley ME, et al. CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science. 1993;259:990-993[Abstract]. 11. Korthauer U, Graf D, Mages HW, et al. Defective expression of T cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM. Nature. 1993;361:539-541[Medline] [Order article via Infotrieve]. 12. Banchereau J, Bazan F, Blanchard D, et al. The CD40 antigen and its ligand. Annu Rev Immunol. 1994;12:881-922[Medline] [Order article via Infotrieve]. 13. Gordon J. CD40 and its ligand: central players in B lymphocyte survival, growth, and differentiation. Blood Rev. 1995;9:53-56[Medline] [Order article via Infotrieve]. 14. Wang H, Grand RJ, Milner AE, Armitage RJ, Gordon J, Gregory CD. Repression of apoptosis in human B-lymphoma cells by CD40-ligand and Bcl-2: relationship to the cell-cycle and role of the retinoblastoma protein. Oncogene. 1996;13:373-379[Medline] [Order article via Infotrieve]. 15. Durie FH, Foy TM, Masters SR, Laman JD, Noelle RJ. The role of CD40 in the regulation of humoral and cell-mediated immunity. Immunol Today. 1994;15:406-411[Medline] [Order article via Infotrieve].
16.
Spriggs MK, Armitage RJ, Strockbine L, et al.
Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion.
J Exp Med.
1992;176:1543-1550 17. Lui YJ, Joshua DE, Williams GT, Smith CA, Gordon J, MacLennan IC. Mechanism of antigen-driven selection in germinal centres. Nature. 1989;342:929-931[Medline] [Order article via Infotrieve].
18.
Henriquez NV, Floettmann E, Salmon M, Rowe M, Rickinson AB.
Differential responses to CD40 ligation among Burkitt lymphoma lines that are uniformly responsive to Epstein-Barr virus latent membrane protein 1.
J Immunol.
1999;162:3298-3307
19.
Baker MP, Eliopoulos AG, Young LS, Armitage RJ, Gregory CD, Gordon J.
Prolonged phenotypic, functional and molecular change in group 1 Burkitt lymphoma cells on short-term exposure to CD40 ligand.
Blood.
1998;92:2830-2843
20.
Funakoshi S, Longo DL, Beckwith M, et al.
Inhibition of human B-cell lymphoma growth by CD40 stimulation.
Blood.
1994;83:2787-2794 21. Johnson P, Gribben JG, Nadler LM, Schultze JL. Soluble CD40 ligand induces proliferation rather than apoptosis in primary human low-grade lymphomas: implications for immunotherapy [abstract]. Blood. 1998;92:484a.
22.
Pound JD, Challa A, Holder MJ, et al.
Minimal cross-linking and epitope requirements for CD40-dependent suppression of apoptosis contrast with those for promotion of the cell cycle and homotypic adhesions in human B cells.
Int Immunol.
1999;11:11-20
23.
DeMilito A.
Nerve growth factor released by CD40 ligand-transfected L cells: implications for functional and phenotypic studies on CD40+ cells.
Blood.
1998;92:4482-4483 24. Buske C, Gogowski G, Schreiber K, Rave FM, Hiddemann W, Wormann B. Stimulation of B-chronic lymphocytic leukemia cells by murine fibroblasts, IL-4, anti-CD40 antibodies, and the soluble CD40 ligand. Exp Hematol. 1997;25:329-337[Medline] [Order article via Infotrieve]. 25. Rathmell JC, Townsend SE, Xu JC, Flavell RA, Goodnow CC. Expansion or elimination of B cells in vivo: dual roles for CD40- and Fas (CD95)-ligands modulated by the B cell antigen receptor. Cell. 1996;87:319-329[Medline] [Order article via Infotrieve]. 26. Planken EV, Dijkstra NH, Willemze R, Kluin NJ. Proliferation of B cell malignancies in all stages of differentiation upon stimulation in the `CD40 system.' Leukemia. 1996;10:488-493[Medline] [Order article via Infotrieve]. 27. Castillo R, Mascarenhas J, Telford W, Chadburn A, Friedman SM, Schattner EJ. Proliferative response of mantle cell lymphoma stimulated by CD40 ligation and IL-4. Leukemia. 2000;14:292-298[Medline] [Order article via Infotrieve]. 28. Hollenbaugh D, Grosmaire LS, Kullas CD, et al. The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EMBO J. 1992;11:4313-4321[Medline] [Order article via Infotrieve].
29.
Ranheim EA, Kipps TJ.
Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal.
J Exp Med.
1993;177:925-935
30.
Schultze JL, Cardoso AA, Freeman GJ, et al.
Follicular lymphomas can be induced to present alloantigen efficiently: a conceptual model to improve their tumor immunogenicity.
Proc Natl Acad Sci U S A.
1995;92:8200-8204 31. Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF, Heath WR. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature. 1998;393:478-480[Medline] [Order article via Infotrieve]. 32. Vonderheide R, Dutcher J, Eckhardt G, et al. Phase I study of rhuCD40 ligand in cancer patients [abstract]. Blood. 1999;94:63a. 33. Wierda W, Cantwell MJ, Rassenti LZ, Avery E, Johnson TA, Kipps TJ. CD154 (CD40-ligand) gene immunization of chronic lymphocytic leukemia: a phase I study [abstract]. Blood. 1999;92:489a. 34. Fisher DC, Abbeele A, Singer S, et al. Phase 1 trial with CD40-activated follicular lymphoma cells: a novel cellular vaccine strategy for B cell malignancies [abstract]. Blood. 1998;92:247a.
35.
Uchimaru K, Taniguchi T, Yoshikawa M, et al.
Detection of cyclin D1 (bcl-1, PRAD1) overexpression by a simple competitive reverse transcription-polymerase chain reaction assay in t(11;14)(q13;q32)-bearing B-cell malignancies and/or mantle cell lymphoma.
Blood.
1997;89:965-974 36. Nicoletti I, Migliorati G, Pagliacci M, Grignagi F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 1991;139:271-275[Medline] [Order article via Infotrieve]. 37. Holm M, Thomsen M, Høyer M, Hokland P. Optimization of a flow cytometric method for the simultaneous measurement of cell surface antigen, DNA content, and in-vitro BrdUrd incorporation into normal and malignant hematopoietic cells. Cytometry. 1998;32:28-36[Medline] [Order article via Infotrieve]. 38. Carayon P, Bord A. Combined staining of BrdUrd (DNA synthesis) and cell surface antigen in PFA/detergent-fixed hematological cells. J Immunol Methods. 1992;147:225-230[Medline] [Order article via Infotrieve]. 39. Kluin NJ, Hakwoort HWJ, Falkenburg JH, Herion JC, Willemze R. Pseudo-proliferation of hairy cell leukemia (HCL) in vitro [abstract]. Blood. 1987;70:262a. 40. Jaffe ES, Campo E, Raffeld M. Mantle cell lymphoma: biology, diagnosis and management American Society of Hematology Annual Meeting: Educational Program Book. University of Washington Seattle: WA.; 1999:319-325. 41. Gregory CD, Tursz T, Edwards CF, et al. Identification of a subset of normal B cells with a Burkitt's lymphoma (BL)-like phenotype. J Immunol. 1987;139:313-318[Abstract].
© 2000 by The American Society of Hematology.
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  H. Maby-El Hajjami, P. Ame-Thomas, C. Pangault, O. Tribut, J. DeVos, R. Jean, N. Bescher, C. Monvoisin, J. Dulong, T. Lamy, et al. Functional Alteration of the Lymphoma Stromal Cell Niche by the Cytokine Context: Role of Indoleamine-2,3 Dioxygenase Cancer Res., April 1, 2009; 69(7): 3228 - 3237. [Abstract] [Full Text] [PDF]
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 M. Luqman, S. Klabunde, K. Lin, G. V. Georgakis, A. Cherukuri, J. Holash, C. Goldbeck, X. Xu, E. E. Kadel III, S. H. Lee, et al. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells Blood, August 1, 2008; 112(3): 711 - 720. [Abstract] [Full Text] [PDF]
|
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J. Dal Col, P. Zancai, L. Terrin, M. Guidoboni, M. Ponzoni, A. Pavan, M. Spina, S. Bergamin, S. Rizzo, U. Tirelli, et al. Distinct functional significance of Akt and mTOR constitutive activation in mantle cell lymphoma Blood, May 15, 2008; 111(10): 5142 - 5151. [Abstract] [Full Text] [PDF]
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N. Kawamata, J. Chen, and H. P. Koeffler Suberoylanilide hydroxamic acid (SAHA; vorinostat) suppresses translation of cyclin D1 in mantle cell lymphoma cells Blood, October 1, 2007; 110(7): 2667 - 2673. [Abstract] [Full Text] [PDF]
|
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M. Guidoboni, P. Zancai, R. Cariati, S. Rizzo, J. Dal Col, A. Pavan, A. Gloghini, M. Spina, A. Cuneo, F. Pomponi, et al. Retinoic Acid Inhibits the Proliferative Response Induced by CD40 Activation and Interleukin-4 in Mantle Cell Lymphoma Cancer Res., January 15, 2005; 65(2): 587 - 595. [Abstract] [Full Text] [PDF]
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 A. Younes and M. E. Kadin Emerging Applications of the Tumor Necrosis Factor Family of Ligands and Receptors in Cancer Therapy J. Clin. Oncol., September 15, 2003; 21(18): 3526 - 3534. [Abstract] [Full Text] [PDF]
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 R. Greil, G. Anether, K. Johrer, and I. Tinhofer Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools J. Leukoc. Biol., September 1, 2003; 74(3): 311 - 330. [Abstract] [Full Text] [PDF]
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A. Challa, A. G. Eliopoulos, M. J. Holder, A. S. Burguete, J. D. Pound, A. Chamba, G. Grafton, R. J. Armitage, C. D. Gregory, H. Martinez-Valdez, et al. Population depletion activates autonomous CD154-dependent survival in biopsylike Burkitt lymphoma cells Blood, May 1, 2002; 99(9): 3411 - 3418. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
80°C using an automated cell freezer (FTS-systems,
Stone Ridge, NY) and stored in liquid nitrogen. Before use, the cells
were thawed, washed, and resuspended in RPMI 1640 medium containing
10% fetal calf serum and antibiotics. The percentage of
CD5+/CD19+ MCL cells in the mononuclear
fraction was always higher than 90%, and no additional non-B-cell
depletion was performed.
the gap between biology and clinical management is closing.
Blood.
1996;88:4085-4089