Department of Virology and Ludwig Institute for Cancer Research, WrightFleming Institute, Faculty of Medicine, Imperial College London, Norfolk Place, London W2 1PG, UK
Correspondence
Martin J. Allday
m.allday{at}imperial.ac.uk
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ABSTRACT |
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INTRODUCTION |
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In vivo, CD4+ T cells, activated by specific antigen, express CD40 ligand (CD40L/CD154) (Armitage et al., 1992; Casamayor-Palleja et al., 1995
; Lane et al., 1992
; Noelle et al., 1992
), which binds to CD40 on B cells. This interaction, in concert with B cell receptor (BCR) cross-linking and the action of T cell-derived cytokines interleukin (IL)-2, IL-5 and particularly IL-4, stimulates B-cell activation and proliferation (Croft & Swain, 1991
; Gordon & Pound, 2000
; Lohoff et al., 1992
). CD40L is also a critical mediator of B-cell differentiation (Gordon & Pound, 2000
; Schonbeck & Libby, 2001
; van Kooten & Banchereau, 2000
), which either involves formation of a germinal centre, producing either plasma cells (PCs) or memory cells, or direct differentiation of naïve B cells into PCs (Staudt & Brown, 2000
). EBV is thought to mimic T cell-derived signals to stimulate B-cell proliferation and utilize B-cell differentiation to gain access to its in-vivo site of persistence, the memory B cell (Thorley-Lawson, 2001
).
J. Banchereau and colleagues established a system of sustainable B-cell proliferation in vitro by mimicking T cell-dependent stimulation. They demonstrated that CD40 stimulation with monoclonal antibodies could induce primary B cells to proliferate in culture and these blast-like, EBV-free B-cell lines could be maintained for 10 weeks (Banchereau et al., 1991; Banchereau & Rousset, 1991
; Rousset et al., 1995
). Subsequent reports indicated IL-10, but not IL-4, could induce differentiation in CD40-activated B cells (Rousset et al., 1995
) but that B cells stimulated with CD40L in conjunction with IL-4 do not maintain indefinite growth (Jung et al., 2001
; Shvarts et al., 2002
; Urashima et al., 1996
). Current literature describing CD40 stimulation of B cells contains a number of contradictions, with studies reporting that CD40 ligation mediates differentiation into both memory and PCs (Arpin et al., 1995
; Garrone et al., 1995
; Jego et al., 2001
; McCloskey et al., 1999
; Urashima et al., 1996
; Worm et al., 1998
) or blocks differentiation in vitro (Callard et al., 1995
; Randall et al., 1998
). Therefore, mitogenic stimulation of peripheral primary B cells in vitro required further characterization if they were to be compared with EBV-infected isogenic cells. Here, an analysis of cell surface phenotype, cellular morphology and Ig production, revealed that these mitogen-stimulated primary B cells are blast-like 714 days after initial stimulation. However, CD40L/IL-4-stimulated cells ceased to proliferate after a period of 34 weeks and showed features consistent with plasmacytoid differentiation, whereas EBV-infected cells proliferated well beyond this.
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METHODS |
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For CD40 stimulation, 107 primary B cells were cultured with a layer of 1·5x106 gamma-irradiated (50 Gy) CD40L+-L cells (Garrone et al., 1995) and 10 U ml-1 human recombinant IL-4 (R&D). L929 and LTK cells (lacking CD40L) were used as controls. Anti-IgM mAb (BU.1 ascites) (Greipp et al., 1985
) was used at 1 : 2000 dilution. EBV purified by ultrafiltration from B95·8 cells was used to infect primary B cells as previously described (Spender et al., 1999
; Wade & Allday, 2000
).
Cell proliferation and viability.
B cells (2x105) were pulse-labelled for 2 h with 1 µCi [3H]thymidine, collected onto a glass-fibre filter using a semiautomatic cell harvester (Skatron) and scintillations counted using a Beckman LS3801 scintillation counter. Cell viability was monitored by trypan blue staining.
Flow cytometry.
Antibodies used for surface antigen analysis were; FITC-conjugated mAbs to CD19 (HD37), CD20, CD23 (MHM6), CD38 (AT 13/5) (Dako), mAbs to CD27 (LT-27) and CD138 (B-B4) (Cymbus Biotechnology Ltd) and mAb to CD40 (B-B20) (Serotec). B cells (5x105) were incubated with primary antibody in 1 % BSA/0·1 % sodium azide/PBS on ice for 1 h. For unconjugated Abs, cells were subsequently incubated with FITC-conjugated goat anti-mouse Ab (Dako) for 1 h.
To analyse cell cycle distribution, 2x106 B cells were fixed in 80 % ethanol, incubated in PI solution [PBS containing 18 µg propidium iodide (PI) ml-1 and 8 µg RNase A ml-1 (Sigma Aldrich)] at 4 °C for 1 h before flow cytometric analysis as previously described (Parker et al., 1996; Wade & Allday, 2000
). To quantify cells in S phase, cells were incubated with 10 µM 5-bromo-2'-deoxyuridine (BrdU) (Sigma Aldrich) for 1 h at 37 °C, fixed in 80 % ethanol and co-stained with FITC-conjugated anti-BrdU mAb and PI (Becton Dickinson) (Wade & Allday, 2000
).
Immunofluorescence and analysis of cellular morphology.
Cytospins of 8x104 B cells were fixed in methanol/acetone (1 : 1) and rehydrated in PBS. For immunofluorescent detection of Ig or EBV nuclear antigen (EBNA) leader protein (EBNA-LP) (EBNA-LP), cytospins were blocked with 1 % BSA/PBS and incubated with FITC-conjugated anti-human IgG or IgG/A/M Ab (Dako) for 1 h or anti-EBNA-LP mAb (JF186) (Finke et al., 1987) followed by FITC-conjugated goat anti-mouse Ab. Stained slides were washed in PBS, mounted in Citifluor and analysed by fluorescence microscopy. To examine cellular morphology, cytospins were stained with Giemsa stain (Accustain; Sigma Diagnostics).
SDS-PAGE and Western blot analysis.
Protein extraction using radioimmuniprecipitation (RIPA) lysis buffer, protein quantification and Western blotting were performed as described previously (Wade & Allday, 2000). Membranes were immunoprobed with the following; mAbs against p21WAF1/CIP1 (SX118) and p53 (DO-1) were kind gifts from Xin Lu; mAbs against Bcl-2 (Dako), cdk6 (C-21) (Santa Cruz), cyclin D2 (G132-4) (BD PharMingen), p18INK4C (Ab-3) (Neomarkers) and polyclonal antibodies (pAbs) to p14ARF (Ab-1) (Oncogene), p21WAF1/CIP1 (N-20) (Santa Cruz) and poly(ADP-ribose)polymerase (PARP) (Roche). Each Ab was used in accordance with the instructions of the supplier. Secondary Abs were rabbit anti-mouse Ig (Dako) followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig (Dako). Bound proteins were visualized by enhanced chemiluminescence (Amersham Biosciences).
Immunoglobulin and cytokine ELISAs.
Secreted human IgG, IgM, IgA and IgE were measured by sandwich ELISA. Briefly, Maxisorp Nunc-Immuno ELISA plates were coated with the appropriate capture pAbs (Dako) diluted in 0·16 % (w/v) Na2CO3, 0·30 % (w/v) NaHCO3, pH 9·6 and incubated with serially diluted B-cell tissue culture supernatants for 2 h at 37 °C in a damp chamber. Plates were washed in PBS/0·05 % Tween 20 and incubated with HRP-conjugated anti-IgE or IgG/A/M (Dako) antibody diluted in 10 % FCS/PBS for 1 h. ELISAs were developed using O-phenylenediamine (OPD)/H2O2 substrate solution, terminated with 2 M H2SO4 and the absorbance read at 492 nm (Anthos Labtec Instruments). Serially diluted, purified human Igs [IgG, IgM, IgA (all from Sigma) and IgE (Serotec)] were used to generate standard curves. To assay secreted human IL-6 and IL-10 in supernatants, human IL-6 or IL-10 OPTEIA ELISA kit II (BD PharMingen) was used, following manufacturer's instructions.
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RESULTS |
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Stimulation of primary B cells with CD40L/IL-4 does not result in cell death but ultimately in cell cycle exit
To determine whether the decrease in [3H]thymidine incorporation consistently observed in CD40L/IL-4-stimulated B cells was due to cell death, the growth characteristics of these cells were investigated over 19 days (Fig. 2A). As before, B-cell proliferation reached a peak and plateaued before declining towards baseline levels. The total number of cells increased following CD40L/IL-4 stimulation and there was no significant decrease in cell viability. Western blotting for PARP cleavage, which is a marker of apoptosis, also showed no correlation between cell death and proliferative decline (Fig. 2B
).
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The effect of prolonged CD40L/IL-4 stimulation on B-cell surface antigen expression
In vitro, continued signalling through CD40 is required to maintain proliferation (Banchereau et al., 1991; data not shown). Therefore, we investigated whether loss of CD40 expression could account for the decrease in proliferation observed. Expression of CD40 on B cells stimulated with CD40L/IL-4 for 26 days was analysed by flow cytometry at intervals corresponding to the different phases of exponential proliferation, plateau and decline (Fig. 3
A). CD40 expression appears to be down-regulated, as the mean fluorescence decreased from 463 (10 days) to 210 (15 days) before falling to 98 (26 days). EBV-infected B cells were also monitored and CD40 expression was consistently higher at each time-point than in CD40L/IL-4-stimulated cells.
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CD40L/IL-4 stimulation induces plasmacytoid-like morphology and immunoglobulin (Ig) production in primary B cells
Plasma cells are morphologically distinct, CD20-, CD38+, immunoglobulin-producing B cells (Harada et al., 1993; Jego et al., 2001
). The effects of EBV infection and CD40L/IL-4 stimulation on the morphology and cytoplasmic Ig production of primary B cells was investigated (Fig. 4
). At 7 days, the morphology of CD40L/IL-4-stimulated B cells is similar to the EBV-infected cells, with both displaying a blast-like appearance of high nucleus/cytoplasm ratio, covered in microvilli-like projections and existing in tight aggregations (Fig. 4A
; upper and lower panels) (Janeway et al., 2001
; Nilsson, 1979
; Nilsson & Ponten, 1975
). In contrast, at 24 days, CD40L/IL-4-stimulated B cells were larger, more ovoid, with a lower nucleus/cytoplasm ratio, fewer cell projections (Fig. 4A
; middle panels) and forming looser clumps in culture. These features are characteristic of plasmacytes, which contain an extended Golgi network in order to produce antibodies.
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The level of Ig secreted by CD40L/IL-4- and EBV-driven B cells was assayed over a period of 28 days (Fig. 4C). All four Ig isotypes (M, G, A and E) were detected in supernatant from both CD40L/IL-4-stimulated and EBV-infected cultures from 712 days post stimulation, with IgM predominant. In mitogen-driven cultures, secreted Ig accumulated to high levels by 23 days and continued to accumulate up to 28 days post-stimulation; however, Ig concentration peaked at 23 days and began to decline in the supernatant of virally driven B cells, consistent with previously reported patterns of Ig secretion following EBV infection of primary B cells (Kataoka et al., 1997
; Nilsson et al., 1971
). It can be assumed that Ig secretion in EBV-infected B cells continues to decline after 28 days, as culture supernatants of two newly established LCLs contained much lower Ig levels (0·0040·010 µg IgM/105 cells and 1·0381·150 µg IgG/105 cells). Therefore, CD40L/IL-4-stimulated cells continue to secrete all isotypes beyond 23 days in culture, whereas Ig secretion peaks in EBV-infected cells at around 20 days.
B cells induced to differentiate with CD40L/IL-4 can be infected but not immortalized with EBV
To determine whether CD40L/IL-4-mediated proliferative decline could be rescued by EBV, B cells stimulated with CD40L/IL-4 for 19 days were infected with EBV and returned to culture with CD40L/IL-4. Cytospins prepared after 48 h were stained with an anti-EBNA-LP mAb (Fig. 5A). The presence of positive staining for EBNA-LP (
25 %) demonstrated that CD40L/IL-4-stimulated B cells could be successfully infected after 19 days. This was confirmed by Western blot analysis, showing that EBNA-LP is only expressed in infected cultures (Fig. 5B
). As expected, proliferation continued at a low level in the uninfected CD40L/IL-4-stimulated cells but, surprisingly, in the infected population, proliferation decreased dramatically 2 days post-infection and completely ceased by 7 days (Fig. 5C
). Therefore, EBV is unable to induce proliferation in these cells and override the differentiation-associated halt in proliferation in primary B cells stimulated with CD40L/IL-4.
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The effect of chronic CD40L/IL-4 stimulation on the expression of key cell cycle regulatory proteins
The above data show that prolonged CD40L/IL-4 stimulation induces phenotypic changes in primary B cells consistent with differentiation, accompanied by cell cycle arrest in G1/G0. Protein extracts were prepared from CD40L/IL-4-stimulated B cells at intervals between 10 and 22 days and G1 regulatory proteins were analysed by Western blot (Fig. 7A). Extracts from isogenic, EBV-infected cells at 15 or 22 days were included for comparison as non-differentiating, EBV-positive B-blasts. The proliferation of these two populations is shown in Fig. 7(B)
. Normal B cells possess functional p53, which accumulates in response to stress (Allday et al., 1995
; O'Nions & Allday, 2003
; Vousden & Lu, 2002
). Once the cells were activated, no change in the level of expression of p53 was detected in either EBV-infected or CD40L/IL-4-stimulated cells, consistent with differentiation, rather than stress, triggering the arrest observed after chronic CD40L/IL-4 stimulation. The expression of cyclin-dependent kinase inhibitors (CKIs), which regulate cell cycle progression and mediate G1 arrest, was also investigated. No variation in the level of p27KIP1 was detected in either population; however, p21WAF1/CIP1 accumulated in CD40L/IL-4-stimulated B cells after prolonged culture. A slower migrating band (indicated by * in Fig. 7A
) was detected after 15 days using a pAb raised against the N-terminus of p21WAF1/CIP1, which could represent a modification to p21WAF1/CIP1.
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DISCUSSION |
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Here we show that prolonged stimulation of primary B cells with CD40L/IL-4 can be characterized by three individual phases; a period of exponential proliferation, a plateau phase and a final decline. No correlation was found between this decline and cell death; instead, flow cytometric analysis indicated that the decrease in proliferation corresponded to an arrest in G0/G1. Furthermore, during the decline phase, CD40L/IL-4-stimulated B cells showed increased synthesis of Ig and changes in morphology and cell surface phenotype consistent with differentiation from blastoid towards plasmacytoid, antibody-secreting cells. This is consistent with studies showing B-cell differentiation coupled to cell cycle arrest, including a recent study reporting that B cells chronically stimulated with CD40L/IL-4 show progressive decrease in proliferation concurrent with Ig accumulation (Altmeyer et al., 1997; Morse et al., 1997
; Jung et al., 2001
). Importantly, these changes were largely absent from the EBV-driven population, which maintained a blast-like phenotype and sustained proliferation.
Prolonged co-culture with CD40L/IL-4 resulted in down-regulation of all surface markers analysed, except CD38. During PC generation, B cells pass through pre-plasmablast (CD20±, CD38±, CD138-), plasmablast (CD20-, CD38++, CD138-) and early PCs (CD20-, CD38+++, CD138+++) (Jego et al., 2001). However, following prolonged culture with CD40L/IL-4, the expression of CD38 or CD138 was not increased, indicating that CD40L/IL-4 stimulation induces activation and differentiation of primary B cells to the plasmablast stage but that additional signals are probably required to produce CD138-expressing PCs. This is consistent with the findings of Jung and colleagues, who reported that CD40L/IL-4-stimulated B cells do not acquire a full PC-surface phenotype (Jung et al., 2001
). Differentiation of B-blasts to memory B cells also involves cell cycle exit, and CD40L induces differentiation of tonsillar germinal centre (GC) B cells to memory cells in vitro (Arpin et al., 1995
). Memory B cells are CD27+, CD20+ (Agematsu, 2000
); however, CD20 expression decreased and CD27 was not expressed at any stage following CD40L/IL-4 stimulation, indicating memory cells were not produced. Memory-cell production is thought to require GC formation, whereas PC production can occur independently of this. No expression of the GC cell marker, CD10, was detected on CD40L/IL-4-stimulated cells (not shown). Therefore, differentiation to antibody-producing PCs could represent a default pathway for peripheral primary B cells following prolonged stimulation and differentiation to memory cells may require additional signals.
Another marker of plasmacytoid differentiation is increased Ig synthesis. Cytoplasmic Ig increased in CD40L/IL-4-stimulated B cells. Neither high levels of cytoplasmic Ig nor morphological changes indicative of antibody-producing PCs were visible in comparable EBV-infected cells. However, the Ig level in culture supernatant did increase, consistent with previous studies (Crawford, 1986; Kataoka et al., 1997
; Nilsson et al., 1971
). This suggests that more cells are producing a lower amount of Ig in the EBV-infected cultures as B-blasts also secrete Ig, albeit at levels below that of PCs.
All Ig isotypes were present in both CD40L/IL-4- and EBV-driven cultures, consistent with previous reports (Kataoka et al., 1997; Urashima et al., 1996
) and can be explained by isotype switching occurring in culture (perhaps due to cytokine action) or stimulation of memory cells, which have already undergone isotype switching. Both naïve and memory cells were present in our quiescent B-cell population, demonstrated by the presence of CD27+ cells (Fig. 3B
, day 0). The latter hypothesis is supported by recent work indicating that EBV can infect both memory and naïve B cells in vitro with similar efficiency (Ehlin-Henriksson et al., 2003
).
CD40L/IL-4-stimulated B cells secreted high levels of IL-6, correlating with proliferative decline, development of plasmacytoid morphology and increases in cytoplasmic and secreted Ig. It has been reported that CD40L/IL-4 induces Ig production, in part due to the autocrine action of IL-6 and induces differentiation of B-blasts (Chen-Kiang, 1995; Jung et al., 2001
; Smeland et al., 1989
; Urashima et al., 1996
), consistent with our data. The EBV-infected cells, which did not differentiate, secreted threefold less IL-6 than CD40L/IL-4-stimulated B cells; however, the addition of IL-6 to certain LCLs induces differentiation (Altmeyer et al., 1997
; Morse et al., 1997
), therefore, either a threshold level of IL-6 is required or only some LCLs are responsive. Conversely, EBV-driven cells secreted high levels of cellular IL-10, not seen in mitogenically stimulated cells, which correlated with Ig secretion. This is consistent with previous studies showing IL-10 induces Ig secretion (Rousset et al., 1995
; Urashima et al., 1996
). The use of B cells from mixed donor buffy coats removes the possibility that these effects are influenced by cytokine production variability between individuals. The differing production of IL-6 and IL-10 in EBV- and mitogen-stimulated B cells suggests they may have roles in the different behaviour of the two populations; however, culture with LCL-conditioned supernatant did not extend proliferation of CD40L/IL-4-stimulated B cells.
CD40L/IL-4-induced differentiation was associated with an increase in p18INK4C, p21WAF1/CIP1, cyclin D2 and p14ARF. This is consistent with previous reports that p18INK4C is induced in differentiating B cells (Franklin et al., 1998; Morse et al., 1997
; Schrantz et al., 2000
; Tourigny et al., 2002
). In addition, IL-6-mediated up-regulation of p18INK4C has been proposed to mediate differentiation-associated arrest in the LCL, CESS (Morse et al., 1997
). p21WAF1/CIP1 is up-regulated in differentiated cells of many lineages (Parker et al., 1995
), including B cells (Morse et al., 1997
), consistent with the increased levels of this protein following long-term CD40L/IL-4 stimulation. The concomitant increase in cyclin D2 levels in CD40L/IL-4-stimulated B cells is interesting in the light of a recent study demonstrating that p21WAF1/CIP1 is stabilized by interaction with cyclin D1 (Coleman et al., 2003
; and our unpublished data). Cyclin D2 also binds and hence could stabilize p21WAF1/CIP1. Interestingly, p27KIP1 did not increase, a phenomenon generally associated with cell cycle exit; however, these data are consistent with another study showing no increase in p27KIP1 in differentiating B cells (Schrantz et al., 2000
). p14ARF is not a CKI, but negatively regulates growth through p53 stabilization, leading to growth arrest (Kamijo et al., 1998
; Quelle et al., 1995
). p14ARF accumulation in CD40L/IL-4-treated B cells suggests either it is involved in B-cell differentiation or it accumulates as the cells arrest. In addition, the slower migrating band, detectable with the anti-p21WAF1/CIP1 antibody, may play a role in the differentiation phenotype.
Data presented here indicate that the replicative lifespan of normal B cells stimulated by CD40L/IL-4 in culture is determined by plasmacytoid differentiation. Since isogenic EBV-infected cells do not differentiate, latent EBV may suppress differentiation, at least in vitro. EBNA-2 has been reported to block c-myc-induced B-cell differentiation in vitro (Polack et al., 1996). It is unlikely that EBV abrogates B-cell differentiation per se, since studies on the latent virus life cycle in vivo suggest that EBV-infected cells differentiate to gain access to their site of persistence, the memory B-cell compartment (Thorley-Lawson, 2001
). Furthermore, in-vitro studies have shown exogenous signals, such as IL-6 and CD40L, can induce differentiation of EBV-positive B-blasts (Chen-Kiang, 1995
; Fukuda et al., 2000
; Pokrovskaja et al., 2002
). Infection of CD40L/IL-4-stimulated B cells with EBV could not reverse proliferative decline (Fig. 5
), consistent with early reports that only small, resting (not large, activated) B cells can be immortalized by EBV (Aman et al., 1984
). It should be remembered that B cells receive a plethora of additional signals in the GC milieu and it is possible that EBV cannot override these.
Cells undergoing senescence display a progressive reduction of proliferation and eventually arrest, characterized by an increase in the levels of markers including p53, p21WAF1/CIP1, p14ARFand p16INK4A (Campisi, 2003; Mathon & Lloyd, 2001
; Sherr & DePinho, 2000
). It remains possible that senescent cells could provide the increases seen in p14ARF and p21WAF1/CIP1, thus altering the interpretation of these data; however, induction of p53 or p16INK4A was not seen in B cells chronically stimulated with CD40L/IL-4.
Collectively, the data presented here indicate that differentiation, rather than senescence or cell death, determines the replicative lifespan of CD40L/IL-4-stimulated B cells in culture. These data also suggest that EBV infection either provides additional signals to extend proliferation, which is lacking in CD40L/IL-4 stimulation, or overrides a pre-programmed, differentiation pathway at least in vitro. In addition, this characterization of B-cell proliferation has highlighted CD40L/IL-4 stimulation as a useful system to enable comparisons to be made between isogenic populations of EBV-infected and -uninfected cycling B cells. The window of opportunity in which the two isogenic populations proliferate at a similar rate, and are at comparable stages of differentiation, is 714 days after stimulation.
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ACKNOWLEDGEMENTS |
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Received 8 October 2003;
accepted 7 January 2004.