Abstract
Resting mature human B cells undergo a dynamic process of clonal expansion, followed by clonal contraction, during an in vitro response to surrogate C3d-coated Ag and innate immune system cytokines, IL-4 and BAFF. In this study, we explore the mechanism for clonal contraction through following the time- and division-influenced expression of several pro- and anti-apoptotic proteins within CFSE-labeled cultures. Several findings, involving both human and mouse B cells, show that a mitochondria-dependent apoptotic pathway involving p53 contributes to the high activation-induced cell death (AICD) susceptibility of replicating blasts. Activated B cell clones exhibit elevated p53 protein and elevated mRNA/protein of proapoptotic molecules known to be under direct p53 transcriptional control, Bax, Bad, Puma, Bid, and procaspase 6, accompanied by reduced anti-apoptotic Bcl-2. Under these conditions, Bim levels were not increased. The finding that full-length Bid protein significantly declines in AICD-susceptible replicating blasts, whereas Bid mRNA does not, suggests that Bid is actively cleaved to short-lived, proapoptotic truncated Bid. AICD was diminished, albeit not eliminated, by p53 small interfering RNA transfection, genetic deletion of p53, or Bcl-2 overexpression. DNA damage is a likely trigger for p53-dependent AICD because susceptible lymphoblasts expressed significantly elevated levels of both phosphorylated ataxia telangiectasia mutated-Ser1980 and phospho-H2AX-Ser139. Deficiency in activation-induced cytosine deaminase diminishes but does not ablate murine B cell AICD, indicating that activation-induced cytosine deaminase-induced DNA damage is only in part responsible. Evidence for p53-influenced AICD during this route of T cell-independent clonal expansion raises the possibility that progeny bearing p53 mutations might undergo positive selection in peripherally inflamed tissues with elevated levels of IL-4 and BAFF.
The mechanisms regulating the growth of Ag-stimulated B cell clones are complex and involve stimuli from surrounding cells and stroma as well as intracellular pathways for controlling cell cycle and survival. T cells are clearly important for B cell clonal expansion and memory cell development, and only limited B cell memory evolves in their absence (1–4). To better understand how B cell clonal growth is regulated during T cell-independent (TI) responses, this laboratory has probed the dynamic process of clonal expansion and ensuing clonal contraction evidenced during the in vitro response of quiescent human B cells to a set of synergistic stimuli: C3d-coated Ag (as a limiting dose of anti-IgM/anti-CD21/dextran) and the cytokines IL-4/IL-13 and BAFF (5, 6). This in vitro model may mimic the response of naive mature human B cells as they enter inflamed tissues with C3d-coated microbes or self-Ags, for example, IgG complexes or apoptotic cells, and IL-4 and BAFF-producing cells of the innate immune system: mast cells/basophils/eosinophils and dendritic cells/macrophages, respectively. We have reported that progeny from this response are characterized by elevated levels of CD23, CD86, CD38, and CD27 and sustained expression of CD20 (6). Interestingly, they show minimal evidence of plasmablast differentiation (6, 7) and bear some resemblance to the “marginal zone-like” cells observed within salivary glands of BAFF-overexpressing mice (8) and humans with Sjögren’s syndrome (9). Importantly, during this TI response, dividing progeny contemporaneously upregulate activation-induced cytosine deaminase (AID) and several proteins of the cyclooxygenase (COX)-2 pathway (7). The latter, that is, COX-2, downstream PGE2 synthase, mPGES-1, and the PGE2 receptor, EP2, contribute to the progressive rise in AID with each division (7). By day 5 of the response, this TI clonal expansion begins to slow and many of the progeny undergo activation-induced cell death (AICD) (5, 6).
In this study we have examined the mechanisms contributing to clonal contraction of these BCR-triggered, innate immune system-dependent clones. A motivating factor was the potential for valuable insights. First, the study could help illuminate why memory cell formation to TI Ags is impaired, even when prosurvival stimuli from the innate immune system are present. Second, the study should reveal whether AID-induced DNA damage can contribute to the clonal contraction of TI B cell clones, in a manner similar to that recently reported in responses to T cell-dependent (TD) stimuli (10). Third, proapoptotic molecules promoting clonal contraction might be targets for mutation by AID or reactive oxygen species (ROS) generated during clonal expansion. Thus, through understanding the mechanism for clonal contraction, we may be in a better position to understand the etiology of certain B cell disorders characterized by abnormal clonal growth.
Past studies from this laboratory have provided glimpses into possible mechanisms for the demise of human B cell clones during BCR-triggered, innate immune system-driven responses (5, 6). Two findings suggest that mitochondria-dependent intrinsic apoptosis is involved. First, Bcl-2 levels within replicating blasts decline progressively with each division (6), in a manner reminiscent of the low levels of Bcl-2 seen in germinal centers (11, 12). The level of Bcl-2 expressed is inversely related to AICD vulnerability (6). Secondly, when BAFF-, APRIL-, or exogenous PGE2-induced signals are available, dividing cells upregulate Mcl-1, a short-lived Bcl-2 family member, resulting in diminished AICD within replicating blasts (5, 6). Importantly, anti-apoptotic Mcl-1 binds with high affinity to several mitochondrial membrane-disrupting proapoptotic molecules, Bim, Puma, and truncated Bid (tBid) (13–16), suggesting that it is an important controller of mitochondria-dependent cell death.
The identities of the proapoptotic mediators present in the above BCR-triggered, innate immune system-driven clones remain undefined. Although there has been precedent for function of a FOXO3a-driven Bim pathway in promoting B cell AICD (17–20), there are reasons to suspect that a pathway involving DNA damage-promoted p53 contributes to AICD in certain settings. It may be important that replicating blasts in these BCR-triggered, innate immune system-driven clones display high levels of both AID and a marker of DNA damage: serine 1981-phosphorylated ataxia telangiectasia mutated (ATM) protein (7). ATM autophosphorylation at serine 1981 occurs rapidly at sites of DNA double-strand breaks (21) and stabilizes p53, a protein considered the master guardian of the genome (22). p53 can affect survival through nuclear transactivation of genes encoding death receptors, for example, Fas and DR5 (23, 24), and genes encoding molecules that induce mitrochondria-dependent apoptosis, Bax, Puma, Bid, and Bad (13, 25–27), as well as through more direct proapoptotic functions in the cytoplasm (28). To clarify whether AICD in the above clones is mediated by a FOXO-driven Bim pathway involving cytokine growth factor withdrawal (29) and/or a p53 pathway, the present study began by monitoring Bim and known p53-driven proapoptotic molecules within primary lymphoblasts at various stages of a dynamic clonal expansion process. Prompted by our early findings, we have tested the hypothesis that a p53-driven apoptotic pathway contributes to the clonal contraction of BCR-triggered, innate immune system-driven B cell clones.
Materials and Methods
mAb/dextran conjugates
Human B cells were activated with a previously described surrogate for C3dg-coated moderately multivalent Ag: a soluble, high-molecular mass mAb/dextran conjugate, that is, anti-human IgM/anti-human CD21/dextran (BCR:CD21-L), generated with either high-affinity HB57 or intermediate-affinity Mu53 anti-human IgM mAb (30). Cultures were routinely stimulated with mAb/dextran at limiting concentration of 0.01 μg mAb/ml (30, 31). Mouse B cells were activated by a distinct anti-IgM/dextran conjugate (BCR-L) constructed on a high-molecular mass (2 × 106 Da) scaffold of aminoethylcarbamylmethyl (AECM)-dextran with 293 amino groups. For preparation of the latter, AECM-dextran, prepared as previously described (32) (and also available through Fina BioSolutions, Rockville, MD), was covalently linked to streptavidin via its amino groups using the Lightning-Link streptavidin conjugation kit (Novus Biologicals, Littleton, CO). Following conjugation, culture grade BSA (0.2%) was added and the mixture was dialyzed in PBS, sterile filtered, and stored until use. At least 30 min prior to use in culture, streptavidin-AECM-dextran was premixed with biotinylated rat anti-mouse IgM mAb II/41 (eBioscience; catalog no. 13-5790-85) to yield BCR-L (each was present in culture at a final concentration of 2 and 0.5–1 μg/ml, respectively).
Cytokines and culture reagents
2 (Cayman Chemical) was stored as a 2 mM stock in ethyl alcohol at −70°C and diluted in culture medium just before use. Z-VAD-FMK (Sigma-Aldrich) stock (20 mM) was prepared in DMSO and diluted to a final concentration of 40 μM.
B cell sources, purification, and culture
Human follicular cells.
De-identified tonsils from elective tonsillectomy were used according to Institutional Review Board guidelines (with the cooperation of the Department of Pathology, New York Eye and Ear Infirmary, New York, NY and the Department of Pathology, North Shore University Hospital, Manhasset, NY). De-identified spleens were obtained from the National Disease Research Interchange and Cooperative Human Tissue Network, processed, and stored at −150°C (6). The follicular (FO) subset designation is used in this study to represent cells of the conventional mature B cell subset, as distinguished from B1 cells and MZ cells. As we described earlier (5, 6), human FO (B2) cells were selected from human tonsil, or occasionally from normal human spleen, on the basis of their high density, and hence relatively resting state, upon Percoll density gradient centrifugation, as well as their CD27 and CD43 negativity upon depletion of CD27- and CD43-positive cells by magnetic bead separation (Miltenyi Biotec). To monitor replication, FO B cells were labeled with 1 μM CFSE and cultured in 96-well plates at a concentration of 0.5 × 105 to 105 cells per 200 μl, in an enriched medium (5). For lysate preparation, cells were cultured in 24-well plates at 1 to 3 × 106 cells/2 ml. In some experiments, purified resting B cells from the peripheral blood of one of the authors was obtained by negative depletion using a magnetic bead purification kit (Miltenyi Biotec).
Mouse B cells.
Studies with mice were performed following review and in concordance with Institutional Animal Use and Care Committee guidelines. Age-matched male wild-type C57BL/6nTac mice and congenic B6.129-Trp53tmlBrd N12 mice with a homozygous p53 mutation (33) were obtained from Taconic Farms. Mice of the congenic C57BL/6 mouse strain with deletion of gene for AID (AICDA) generated in the laboratory of Dr. Tasuko Honjo (34) (also on the C57BL/6 background) were the gift of Dr. Matthew Scharff. BALB/c × EμBcl-2.22 mice (35) with an inserted Bcl-2 transgene (TG) were provided by Dr. Betty Diamond. All mouse spleen cell suspensions were subjected to density gradient centrifugation as above to obtain high-density lymphocytes. B cells were subsequently purified by negative selection (Miltenyi Biotec B cell isolation kit; catalog no. 130-090-862), labeled with CFSE, and cultured as above.
Assessment of culture viability and replication
Methods described previously were employed (5, 6). In brief, CFSE-labeled cells were harvested into cold PBS, fixed in 1–2% formaldehyde; and analyzed by flow cytometry (FACScan or FACSCalibur with CellQuest data analysis software or FlowJo 7.6.1 software) following exclusion of very low forward light scatter (FSC) events (debris), with an exception shown in Fig. 1A. The relative yield and relative viability of cells within each division subset were calculated by gating total cells (viable and apoptotic) into various division subsets and subsequently determining percentage viability within each subset on the basis of FSC/SSC (3).
Intracellular staining of cultured CFSE-labeled B cells
Cells were fixed in EM grade formaldehyde (Polysciences) (2% in PBS, pH 7.2), washed, and preincubated in PBS-HEPES plus 30% heat-inactivated human AB serum plus 0.1% saponin, as previously described (6), or exposed to Fix/Perm and Perm/Wash solutions (BD Biosciences). For detection of p53, p-ATM, and p-H2AX cells were exposed to 90% methanol (−20°C for 2 h to overnight) prior to washing and emersion in permeabilizing buffer (with phosphatase inhibitor in the case of p-ATM and p-H2AX staining). Cells were stained intracellularly by exposure to specific Ab (or with control IgG) for 20–40 min, washed, and, if needed, exposed to secondary labeled anti-IgG for 20–30 min. Washed cells were refixed in 2% formaldehyde prior to flow cytometry. Abs used for the above assays included: anti-Bid (mouse mAb 7A3; Cell Signaling Technology), anti–p-H2AX-Ser139/140 mouse mAb 3F2 (Thermo Scientific), followed by PE-goat F(ab′)2 anti-mouse IgG preabsorbed against human IgG; anti-p53 (PE-anti-p53 [clone DO-7] and PE-IgG2b mAb control [clone 27-35]; BD Pharmingen staining kit); PE-conjugated anti–p-ATM-Ser1981 (mouse mAb 10H11-E12; Millipore) and anti–Mcl-1 (mouse mAb clone 22; BD Pharmingen) or MOPC-21 mouse IgG1 mAb control (directly labeled with Zenon Alexa Fluor 647-R-PE mouse IgG1 labeling reagent).
Immunoblotting studies
Lysates were prepared in M-PER lysis solution (Pierce) or in RIPA lysis buffer plus 1 mM EDTA plus protease/phosphatase inhibitor mix (Roche). Lysates were quantified for protein, electrophoresed, and blotted onto nitrocellulose or polyvinylidene difluoride membranes as described (6). Western blotting was performed with the following: rabbit anti-Bax and rabbit anti-Bim polyclonal Abs (pAbs; BD Pharmingen); rabbit anti-Bad pAb and mouse anti-Bid (mAb 7A3) (Cell Signaling Technology); anti-Puma (rabbit mAb EP512Y; Epitomics); anti-p53 (mouse mAb DO-1) (a gift of Dr. Roger Grand, University of Birmingham, Birmingham, U.K.); anti-total H2AX (rabbit mAb D17A3; Cell Signaling Technology); anti–phospho-H2AXS139 (mouse mAb 3F2; Thermo Scientific) and loading control anti-actin mAb (Novus Biologicals) or anti-catalase pAb (6). In each experiment, blots were sequentially stripped and reanalyzed for additional proapoptotic molecules and loading control protein. The latter was detected with standard ECL detection reagent; other blots used SuperSignal Pico or Femto high-sensitivity substrate (Pierce). In analysis of Bax, Bad, Bid, Bim, and Puma, standardized values of a given protein were obtained as described (36) and then expressed as a percentage of the maximum level attained within that experiment.
Quantitative RT-PCR of Bim, Bid, Bax, and p53 mRNA levels
For quantitative analysis of mRNA levels within B cells, mRNA was isolated from harvested cells (≥106) using a Mini-Prep Qiagen kit. From each mRNA, cDNA synthesis was performed using oligo(dT) primers from a cDNA kit (Invitrogen). Primers and probes were designed from universal probe library (Roche). Bim, probe no. 63 and primer sequence (forward, 5′-GCTGTGGAGGCTGAATCC-3′ and reverse, 5′-TCGGCTGCTTGGTAATTATTC-3′); Bid, probe no. 44 and primer sequence (forward, 5′-TGTGAACCAGGAGTGAGTCG-3′ and reverse, 5′-GGCTGGAACCGTTGTTGA-3′); Bax, probe no. 55 and primer sequence (forward, 5′-CAAGACCAGGGTGGTTGG-3′ and reverse, 5′-CACTCCCGCCACAAAGAT-3′); p53, probe no. 12 and primer sequence (forward, 5′-AGGCCTTGGAACTCAAGGAT-3′ and reverse, 5′-CCCTTTTTGGACTTCAGGTG-3′). Quantitative RT-PCR was executed at least in triplicates using cDNA as a template with Eurogentec MasterMix. The conditions for quantitative PCR were as follows: initial uracil-N-glycosylase activation at 50°C for 2 min; HotGoldStar activation (and uracil-N-glycosylase inactivation) at 95°C for 10 min; followed by 45 cycles, each at 95°C for 15 s (denaturation) and 60°C for 1 min (annealing and extension). Fold change was calculated by comparing treated versus untreated groups. Endogenous control for assay was β-actin, used as described previously (10). Data were analyzed using RQ Manager version 1.2. Fold change was calculated by comparing treated versus untreated groups.
p53 small interfering RNA treatment
Prior to small interfering RNA (siRNA) transfection, B cells were preactivated with low-dose BCR:CD21-L plus IL-4 (with or without BAFF) for 2–3 d. Recovered cells (in some cases enriched for viable cells by Ficoll-Hypaque centrifugation) were washed, and 1–5 × 106 cells were subjected to nucleofection with 2 μg p53 siRNA (Qiagen-validated siRNA [Hs_TP53_9]) or 2 μg AllStars control siRNA (Qiagen) (or, alternatively, 1.5 μg siRNA representing p53 Stealth RNA interference [TP53HSS110905-7] and GC-rich Stealth control RNA interference [Invitrogen]) using program U-15 of an Amaxa nucleofection device. Following re-equilibration (6–12 h in RPMI 1640 plus 10% FCS plus GlutaMAX plus PenStrep), nucleofected cells were restimulated with BCR:CD21-L plus IL-4 plus BAFF for an additional 3 d in medium containing Primocin (Amaxa). At that time, cell cultures were assessed by flow cytometry for viability and cell size (light scatter). Cell recovery was determined in most experiments by introducing a known number of standardizing Calibrite beads into each assay tube prior to analysis. In early optimization experiments, nucleofection of Amaxa pmaxGFP plasmid under the above conditions resulted in 40–70% GFP+ cells within the gated viable subset at 24 h following nucleofection.
Results
Kinetics of AICD in human FO lymphocytes stimulated by surrogate C3d-coated Ag and innate immune system cytokines
The representative experiment in Fig. 1A reveals the time course for the dynamic clonal expansion/contraction response of CFSE-labeled quiescent human tonsil FO (B2) cells upon culture with a limiting dose of surrogate C3d-Ag (BCR:CD21-L) and IL-4 plus BAFF. The histograms confirm our prior findings that the greatest proportional yield of viable progeny in this TI response is seen between days 4 and 5 of the response (6). At day 5, apoptotic progeny (shrunken cells with low CFSE) are clearly evident. Following day 5 of activation, the proportion of gated intact apoptotic cells increases with time, as does the detection of fragmented cells of very low FSC (debris, or D) (Fig. 1A). The rise in debris indicates that percentage viability values, calculated on the basis of gated intact viable and apoptotic cells (Fig. 1B), are undoubtedly an overestimate at late culture intervals. Nevertheless, as we have described earlier (5, 6), through evaluating percentage viability as a function of division it is readily apparent that AICD affects primarily cells with two or more divisions (Fig. 1B). The AICD of progeny is not unique to tonsil FO cells following in vitro activation, but it is also evidenced during BCR:CD21-L plus IL-4 plus BAFF-induced clonal expansion of purified peripheral blood B cells and isolated splenic FO B cells (Fig. 1C) (6). In an earlier study we demonstrated that progeny gated as apoptotic, on the basis of FSC/SSC, express high levels of active caspase 3 (23). The importance of intracellular caspases in mediating the above AICD is presently shown by the significantly greater recovery of viable progeny in cultures pulsed on day 4 with Z-VAD, a pan-caspase inhibitor (Fig. 1D).
Kinetics of caspase-dependent AICD following human B cell clonal expansion in response to surrogate C3d-coated Ag (BCR:CD21-L), IL-4, and BAFF. (A) CFSE-labeled, resting B cells were cultured with a limiting dose of BCR:CD21-L (0.01 μg/ml) plus IL-4 and BAFF. Following varying culture intervals, cultures were pulsed with a known quantity of AutoCOMP beads, harvested, fixed with 2% formaldehyde, and analyzed by flow cytometry. Twenty thousand total events were acquired at each time point, and FSC and SSC were evaluated on a log scale (A, left column). This enabled detection of AutoCOMP beads (which have a lower FCS than do the standardization beads used for assessing cell recovery in all other experiments [CountBright absolute counting beads; Invitrogen]). It also revealed that subcellular particles (D, debris) rise substantially after day 5 (6). For analysis of the divisions represented within intact viable and apoptotic cells (right column), events were gated on the basis of light scatter (as seen in left column) and analyzed for level of CFSE fluorescence: dark lines, viable; tinted histogram, apoptotic. As described earlier (6), a fraction of undivided cells succumbs to death during the first 2 d culture (far right apoptotic peak in CFSE histograms). These retain the high levels of CFSE present at the time of their demise and are unlikely related to activation, since they are seen at greater levels within nonstimulated cultures containing medium alone (6). (B) Calculations for percentage viability within each division subset of total intact cells (viable plus apoptotic) were made from the cultures in (A), as described in our earlier studies (5, 6) and in the present Materials and Methods. Note that these percentage viability determinations are not fully accurate because they do not account for the significant number of apoptotic cells that fragment into debris (D) and are excluded from consideration. Nevertheless, the percentage viability calculations are valuable because they demonstrate that survival of highly divided progeny diminishes with time. Note that the percentage viability as determined by FSC/SSC-based gating of these cultured cells is comparable to percentage viability determinations on the basis of FITC-annexin V/PI staining (37) or PE-annexin/7-aminoactinomycin D staining (Supplemental Fig. 1). (C) CFSE-labeled peripheral blood B cells and splenic FO B cells were cultured with BCR:CD21-L plus IL-4 plus BAFF for 5 d, gated into apoptotic and viable subsets as shown, and evaluated for CFSE fluorescence. (In these and all subsequent experiments, FSC and SSC are plotted on linear scales, with the FSC threshold setting excluding most debris.) Similarly to activated tonsil FO (B2) cells, both of these latter populations show evidence of significant AICD in replicating blasts, (D) Cultures of activated B cells received a day 4 pulse of pan-caspase inhibitor, Z-VAD-FMK (40 μM), or DMSO vehicle control. Cultures were harvested on day 6, fixed, and analyzed by flow cytometry, Scatter plots to the left show the gating of viable and apoptotic cells and the percentage viability values of replicate cultures within the experiment (mean ± SEM). Histograms to right represent day 6 CFSE fluorescence profiles for viable (solid) and apoptotic (dashed) cells. Results are representative of more than three experiments.
Importantly, several possibilities for this in vitro AICD have been excluded. The AICD is not due to limiting nutrients following the burst of proliferation: cultures pulsed with CD40-L or with TLR9-engaging CpG (oligodeoxynucleotide 2006) manifest considerably greater cycling and substantially lesser AICD through day 7 (data not shown). Furthermore, supplementing cultures with fresh medium or reducing the cell density does not affect AICD (data not shown). Additionally, membrane death receptors, Fas, TNFR, and DR, do not appear relevant: in several experiments, Fas was undetectable by immunostaining with Ab that detected Fas on CD40-L–stimulated cells (36), and AICD was unaffected by neutralizing anti–TNF-α mAb or TRAIL-Fc (data not shown). In the following experiments, we explore other explanations for the declining viability of replicating blasts in these TI clones.
Expression of several proapoptotic Bcl-2 family members rises during BCR-triggered, innate immune system-driven clonal expansion
To obtain greater insights into the mechanism for the above AICD, we monitored cultures prior to (days 3–4) and after (days 5–6) AICD was evident for expression of several molecules implicated in intrinsic, mitochondria-dependent apoptosis. This involved immunoblotting and/or immunostaining for FOXO-regulated Bim (38, 39) and p53-regulated Bax, Bid, Bad, Puma, and caspase 6 (13, 25–27). Expression of proapoptotic molecules within lysates of cells exposed to low-dose BCR:CD21-L plus growth-promoting IL-4 with or without BAFF was compared with that within cultures exposed to BCR:CD21-L alone (Fig. 2A–D). B cells exposed solely to this low dose of BCR:CD21-L typically manifest sustained viability through day 6, but minimal growth (6, 31). We additionally examined proapoptotic molecules, alongside anti-apoptotic Bcl-2 and Mcl-2, by two-color intracellular immunofluorescent staining of CFSE-labeled cultures (Fig. 2E–G). In this case, we compared relative expression of apoptosis-regulating proteins within dividing, apoptosis-vulnerable blasts and nondividing, apoptosis-resistant blasts (6).
Profile of pro- and anti-apoptotic molecules displayed in BCR:CD21-L–triggered B cells receiving costimuli from IL-4 with or without BAFF. (A–D) Immunoblotting analyses of SDS-PAGE separated proteins in lysates of human FO cells cultured for 3–4 or 5–6 d with BCR:CD21-L and medium, IL-4, or IL-4 plus BAFF. (A) Lysates from a representative single experiment were analyzed for Bax, Bid, Bim, and Bad by sequential stripping and reblotting. In this experiment, day 3 and day 5 lysates were contemporaneously transferred to different blots, with subsequent simultaneous analysis. (B) Immunoblots from two separate experiments evaluating Puma within day 4 lysates. (C) Pooled analysis of the relative expression of Bax, Bid, Bad, Puma, and BimEL and BimL isoforms, within multiple experimental lysates collected at days 3–4 (prior to AICD) and at days 5–6 (AICD evident). Within each experiment, densitometric data for expression of the test molecule and loading control protein were obtained; values for the given test molecule were standardized on the basis of loading control in each experiment and calculated as percentage of maximum observed; and finally, all the latter values were from multiple experiments and are expressed as means ± SEM. The p values for significance from the Student t test are shown. The shaded bar below each set of pooled data indicates the relative propensity of cultures to display AICD, as shown in Fig. 1 and elsewhere (6). (D) Bcl-2/Bax expression ratios were obtained for the differing culture conditions by dividing the mean values for Bcl-2 expression (as percentage of maximum from a past analysis (6) of the same lysates here analyzed for Bax) by the mean values (percentage of max) for Bax expression. (E and F) Immunocytofluorimetric analysis of selected proapoptotic and anti-apoptotic molecules within CFSE-labeled blasts in 5 d cultures activated by BCR:CD21-L plus IL-4 plus BAFF. (E) Following intracellular staining with mAb to intact Bid, caspase 6, Puma (red), or Bcl-2 or Mcl-1 (green), or, alternatively, IgG control (black), viable-gated cells were analyzed for expression of each protein within cells of differing division status by two-color flow cytometry. (F) Shown are the calculated values for PE fluorescence intensity above IgG control background, for each CFSE-determined division. Results are representative of a minimum of three experiments. (G) Means ± SEM of percentage of maximum values for Bid expression in diverse division subsets (n = 6 experiments).
Profile prior to AICD.
Despite the relatively high cell viability at days 3–4 in cultures supplemented with growth-promoting cytokines (Fig. 1) (6), these cells showed a pronounced upregulation of several proapoptotic molecules reported to be downstream of p53, that is, Bax, Bid, Bad, and Puma (Fig. 2A–C). As discussed in an earlier report (6), it is highly likely that apoptosis is thwarted in these day 3 lymphoblasts by the concomitant high expression of several anti-apoptotic members of the Bcl-2 family: Bcl-2, Bcl-xL, and Mcl-1. The levels of FOXO-regulated Bim were not significantly increased. Indeed, the most proapoptotic isoforms, BimL and BimS, were less evident in stimulated cultures containing growth-promoting cytokines than those without (Fig. 2A, 2C, bottom row).
Profile when AICD is manifest (days 5–6).
Further insights into the mechanism for AICD came from analyzing the repertoire of expressed proapoptotic proteins, in the period after the initial burst of replication. First, Bim levels generally declined in late stage cytokine-supplemented cultures, as compared with cultures with BCR:CD21-L alone (Fig. 2A, 2C, bottom row), further suggesting that Bim is not a primary inducer of AICD late in these cultures. This was preceded by significantly diminished Bim mRNA on day 4 (Fig. 3). Second, both Bax and Bad appear elevated compared with levels detected in cells responding to BCR:CD21-L alone (Fig. 2A). Although these differences did not reach statistical significance in the pooled experiments (Fig. 2C), we attribute this to 1) the notably greater cell death and ensuing protein degradation within the cytokine-supplemented cultures at this late interval (cells with most elevated levels have succumbed to apoptosis), and 2) the fewer experimental lysates analyzed at this late interval. In support of this conclusion, Bax mRNA was significantly elevated within day 4 cultures (Fig. 3), just prior to obvious AICD (Fig. 1). Third, proapoptotic Puma was not only upregulated at day 4, as assessed by immunoblotting (Fig. 2B, 2C, top row), but also strongly expressed within viable day 5 replicating lymphoblasts, as discerned by intracellular staining (Fig. 2E, 2F). Fourth, caspase 6, a protease reported to lower the threshold for apoptosis, cleave nuclear lamin, and activate caspase 8 (27, 40) was found highly expressed in day 5 replicating blasts (Fig. 2E, 2F). Finally, tBid, a truncated highly proapoptotic form of Bid (13), appeared to be actively formed within the replicating clones, as detailed below.
Replicating B cells stimulated with BCR:CD21-L plus IL-4 plus BAFF exhibit elevated mRNA levels of Bid and Bax, but repressed Bim mRNA. (A) Cultures stimulated with BCR:CD21-L alone or with growth-promoting IL-4 and BAFF were harvested on day 4, mRNA isolated, and cDNA was prepared with oligo(dT) primer. Quantitative PCR (q-PCR) was performed with primers specific for Bim, Bid, Bax, and β-actin. ΔCt values for each of the above were obtained through comparison with β-actin. Values for fold difference (Δ) were obtained by comparing ΔCt values for each proapoptotic mRNA in cytokine-supplemented cultures with the respective ΔCt values in control cultures with BCR:CD21-L alone. The p values represent the significance of comparisons between Δ values in cytokine-supplemented cultures versus those in control cultures in a total of four replicate experiments, using a two-tailed Student t test. (B) Bid mRNA levels in divided blasts are comparable to Bid mRNA in undivided blasts. For semiquantitative RT-PCR of Bid, cells were stimulated for 5 d with BCR:CD21-L (0.01 μg/ml) plus IL-4 plus BAFF and sorted on the basis of CFSE fluorescence into two populations: undivided blasts and divided blasts (two to five divisions). mRNA was isolated and cDNA prepared as above. PCR amplification was performed with Bid or β-actin–specific primers. Varying microliter amounts of the amplicons were loaded onto 1.5% agarose gels and electrophoresed. NC, negative control for PCR; PC, positive control for PCR. Similar results obtained from a second experiment evaluating levels of Bid mRNA in undivided versus divided blasts.
Evidence for tBid generation in replicating blasts.
Although indirect, there is substantial evidence suggesting that Bid is fragmented to tBid during this TI B cell clonal expansion. First, prior to the proliferative burst, the nonapoptotic Bid proform (21 kDa) was highly expressed in cytokine-supplemented cultures (particularly those with both IL-4 and BAFF). This major 21-kDa band vanished 2 d later when AICD was prominent; instead, weak bands approximating tBid (14–17 kDa) were occasionally evident (Fig. 2A, 2C). Difficulty in discerning tBid is expected due to 1) asynchrony of AICD in these cultures, 2) the short intracellular half-life of tBid (<30 min) (41), 3) size heterogeneity in tBid fragments, due to variable protease cleavage sites (42), and 4) preferential specificity of the immunoblotting mAb for Bid over tBid. A second line of evidence supporting Bid fragmentation to tBid derives from findings that Bid mRNA levels remained high within IL-4 plus BAFF-supplemented cultures on day 4, just prior to prominent AICD (Fig. 3A). Furthermore, semiquantitative RT-PCR of RNA from sorted nondivided and divided blasts on day 5 of activation showed Bid mRNA levels to be sustained during division (Fig. 3B). A third line of evidence supporting the formation of tBid is that levels of intact Bid significantly decline in viable divided blasts, as compared with the undivided blasts within the same cultures (Fig. 2E–G). Finally, there clearly exists an inverse relationship between level of intact Bid and lymphoblast susceptibility to AICD: cells with the most extensive division manifest the lowest levels of intact Bid (Fig. 2G) and the greatest susceptibility to AICD (5, 6). Taken together, the above findings strongly suggest that cleavage of intact Bid into labile, but highly proapoptotic, tBid contributes to the high apoptosis susceptibility of progeny within these TI human B cell clones.
Significance of diminished Bcl-2 expression for AICD
In the context of a notable upregulation of several proapoptotic molecules within replicating blasts, it is warranted to reconsider expression of the opposing anti-apoptotic molecules, namely Bcl-2 and Mcl-1. We have previously shown that Bcl-2 levels precipitously decline with each successive division, whereas Mcl-1 levels rise, in a BAFF-dependent manner (6). The opposing trends are also evident within the new experiment in Fig. 2E and 2F. Given previous evidence that the Bcl-2/Bax ratio is strongly linked to B cell survival (43), we compared levels of Bcl-2 and Bax in cultures before and after AICD had commenced (day 3 versus day 5). Fig. 2D shows that whereas the Bcl-2/Bax ratio in cells exposed only to BCR:CD21-L remains high throughout 5–6 d culture, this ratio notably drops in cytokine-supplemented cultures beginning to manifest AICD. Thus, despite the BAFF-driven elevation in Mcl-1, the decline in Bcl-2 appears to jeopardize the survival of replicating blasts under these stimulation conditions.
To further test this contention, we established similar culture conditions for stimulating a TI response from B cells from wild-type (WT) and Bcl-2 transgenic mice (35). In these latter experiments, cultures were stimulated with a limiting dose of BCR-L (anti-mouse IgM/dextran at 0.5–1 μg/ml) plus IL-4 and BAFF. The data in Fig. 4 clearly demonstrate that presence of the Bcl-2 TG augments the yield of viable day 5 progeny. The bonus effect of Bcl-2 overexpression in these mouse B cell cultures was most prominent in responses to BCR-L plus IL-4 (Fig. 4, left panels), but nevertheless was also evident in responses to BCR-L plus IL-4 plus BAFF (Fig. 4, right panels). Taken together, we conclude that a rise in proapoptotic molecules, combined with declining Bcl-2 expression, makes these TI clones highly susceptible to AICD.
Overexpression of Bcl-2 augments the recovery of viable lymphoblasts in mouse B cell TI clones. CFSE-labeled splenic B cells from BALB/c WT mice, and BALB/c mice overexpressing Bcl-2, owing to insertion of a Bcl-2 TG, were stimulated for 5 d with BCR-L plus IL-4 with or without BAFF. Cultures were harvested following the addition of standardization beads to each well, fixed, and analyzed by flow cytometry. (A) Shown are overlays of CFSE fluorescence within the viable-gated cells recovered from each set of congenic cultures, stimulated under the indicated conditions. Results are representative of two identical experiments. (B) Bcl-2 TG/WT ratio for total viable cell recovery within each division subset. Total viable cell recovery was computed, with the use of CountBright standardization beads, from each of six replicates. A ratio was obtained by dividing mean value for Bcl-2 TG cultures by mean value for WT cultures in a representative experiment.
p53 is upregulated during TI stimulation of human B cells
Although levels of p53 protein in normal cells are typically quite low due to ubiquitination of p53 by Mdm2 (44, 45), the evidence for heightened expression of molecules known to be direct transcriptional targets of p53 led us to monitor p53 protein expression. Elevated p53 expression was confirmed though two separate approaches. First, p53 immunoblotting of day 3 and day 5 lysates showed that this transcription factor was significantly upregulated in cytokine-supplemented cultures, as compared with those exposed to BCR:CD21-L or medium alone (Fig. 5A). Interestingly, when freshly isolated blots were probed with p53 and subjected to longer periods of exposure, several bands other than the p53 kDa band were evident (as seen for T662). The stack of weak bands above p53 may represent ubiquitinated forms of p53, as a pattern similar to the one we observed was attributed to multimonoubiquitinated forms of p53 in a recent study (46). The band below the major p53 band may be 46- to 48-kDa protein isoforms of p53 known to represent multiple splice variants, as mAb D01 used for immunoblotting can recognize some of the latter (47). These bands were less evident when the blots were reprobed with less film exposure (as seen with T663). Note that the level of expressed p53 protein in activated normal human B cells was always substantially less than expressed within the Ramos human B cell line (Fig. 5B), which bears a mutated, less functional p53 (48).
Levels of p53 are upregulated in human B cells triggered by BCR:CD21-L plus IL-4 plus BAFF. (A) Immunoblotting evidence for p53. Top, Day 3 and 5 lysates from tonsil FO cells stimulated with BCR:CD21-L with or without IL-4 with or without BAFF were analyzed for p53 and for β-actin loading control (following stripping and reblotting). A fresh blot and longer film exposure was used for p53 analysis of T662 lysates, whereas a restripped blot with shorter film exposure was used for p53 analysis of T663 lysates. p53 upregulation is representative of six experiments. (B) Comparative p53 levels within lysates of the human Ramos B cell line (expressing mutated p53) or day 3 normal human B cell cultures stimulated with BCR:CD21-L plus IL-4 plus BAFF. Note that the Ramos lysate was loaded at one-twentieth of the total protein as the day 3-stimulated normal B cell lysate. (C) Flow cytometric evidence for p53. Top row, Histograms representing CFSE fluorescence in cells stimulated for 4 d with BCR:CD21-L with or without IL-4 with or withut BAFF, prior to intracellular staining. Subsequent rows, Stimulated cells were gated on the basis of CFSE fluorescence (division status) and assessed for PE fluorescence indicative of p53 staining (line) or IgG2b control staining (tinted histogram). Shown is the ratio of MFI values (RMFI) obtained when anti-p53 MFI is divided by control MFI. The proportion of viable cells following into the 0, 1, 2, and 3 division subgroups in this experiment were as follows: BCR:CD21-L only, 92, 7, 1, and 0%; BCR:CD21-L plus IL-4, 72, 18, 8, and 1%; BCR:CD21-L plus IL-4 plus BAFF, 49, 27, 19, and 5%, respectively. (D) Pooled data from 15 experiments evaluating the relative expression of p53 within the varying division subsets. The left plot shows data expressed as RMFI; the right plot shows data expressed as percentage of the maximal MFI above background (ΔMFI) within each experiment. The values above the bars show the p values for level of significance between p53 expression in undivided cells and cells with the indicated division.
Heightened p53 protein in these TI cultures was further evidenced by staining CFSE-labeled cells for intracellular p53 (Fig. 5C, 5D). Both the representative experiment shown in Fig. 5C and the results from pooled experiments in Fig. 5D indicate that p53 was maximally expressed in divided lymphoblasts. Cytokines clearly augmented p53 expression, but do not appear to be obligatory given that the very infrequent progeny within cultures exposed to BCR:CD21-L alone also showed elevated p53 (Fig. 5C). Cells exposed solely to IL-4 did not manifest elevated p53 (data not shown). Taken together, these findings indicate that p53 protein is upregulated within low-dose BCR:CD21-L–triggered B cells, particularly those blasts undergoing IL-4 with or without BAFF-driven growth.
p53 loss impairs AICD
Two approaches were taken to evaluate whether p53 expression contributes to AICD observed under these TI activation conditions. In one approach, we treated activated human B cells with p53 siRNA and control siRNA. Nucleofection of day 2 preactivated B cells with specific siRNA only partially inhibited p53 expression in restimulated B cells, as indicated both by diminished, but not ablated, p53 mRNA expression, as assessed by quantitative PCR of cDNA (Fig. 6A) and by a 40% decline in p53 protein (as reflected by mean fluorescence intensity [MFI] of PE-anti-p53 intracellular staining) (Fig. 6B). Nevertheless, this partial decline in p53 expression was associated with a significant increase in culture viability, cell size, and cell yield (Fig. 6B). Treatment with specific vimentin siRNA failed to affect p53 levels, while effectively downregulating vimentin (Fig. 6A), and it had no effects on cell viability or size, as compared with parallel cultures treated with control siRNA (data not shown). These findings are consistent with a p53 effect on B cell viability and/or division. We were unable to reliably monitor the effects of p53 siRNA transfection on CFSE-labeled cultures given that even control nucleofected cells failed to regain the strong proliferative burst seen in unmanipulated cultures.
p53 siRNA treatment of preactivated human FO cells diminishes p53 protein expression but augments B cell viability, cell size, and total cell yield. Purified FO cells from tonsils or spleens were preactivated for 2 d (with either BCR:CD21-L, BCR:CD21-L plus IL-4, or BCR:CD21-L plus IL-4 plus BAFF) prior to nucleofection with p53 siRNA or control siRNA (or vimentin siRNA) as described in Materials and Methods. (A) mRNA was isolated from cells at 24–32 h after nucleofection. cDNA was prepared and analyzed for p53 and actin levels by quantitative PCR with specific primers. Values for ΔCt values were obtained as described for Fig. 3A; fold change was calculated by comparing ΔCt values within p53 siRNA-treated cultures with respective ΔCt values in cultures treated with control siRNA. Shown are fold change values from transfections performed with two distinct activated B cell populations and two different sources of p53 siRNA (mean ± SEM values from replicate quantitative PCR assays are shown). (B) Cells were harvested at 3 d following nucleofection with the indicated siRNA and fixed, permeabilized, and stained intracellularly with PE-anti-p53 Ab, PE-anti-vimentin, or PE-IgG control. Shown is PE fluorescence histograms on a 4-log scale. (C) At 3 to 4 d after nucleofection, fixed cells were analyzed for cell viability (by light scatter), cell size (by FSC of viable subset), and total cell yield (as determined with standardization beads, or in the first experiment, by comparison of “events per minute” during acquisition of cells from control and p53 siRNA-treated cultures). To standardize data from multiple experiments, values for each parameter within an experiment were expressed as a percentage of the value obtained with control siRNA-treated cells. Shown are the mean ± SEM values of such values. A two-tailed, non-paired Student t test was used to compare the responses in cultures treated with p53 siRNA versus control siRNA (p values of significance are shown).
As an alternative test for assessing the functional relevance of p53 in mediating AICD, we compared viability and viable cell yield within cultures of CFSE-labeled B cells from p53-deficient (p53 knockout [KO]) or WT mice. Purified high-density splenic B cells were stimulated either with mouse-specific BCR-L and IL-4 (Fig. 7A, 7C, 7D, 7G) or with BCR-L plus IL-4 and BAFF (Fig. 7B, 7E, 7F, 7H). (In Supplemental Fig. 2, an experiment testing various BCL-L doses is shown; a dose of 0.5 or 1 μg/ml was used for all subsequent experiments.) These studies with p53 KO B cells consistently showed (five of five experiments) that overall culture viability was increased when p53 was absent (Fig. 7A, 7B). When CFSE-labeled division subsets were analyzed, heightened viability was typically seen at all divisions, but this was statistically significant only in those cells that had divided minimally (Fig. 7C, 7E). Furthermore, the yield of viable lymphoblasts was greater within all division subsets of p53-deficient B cell cultures, as compared with WT B cell cultures, at levels that were either statistically significant or of borderline significance (Fig. 7D, 7F). Thus, overall survival is heightened when B lymphoblasts lack the p53 gene.
Stimulated cultures of p53-deficient mouse B cells exhibit greater viability and greater total lymphoblast yield than do similarly activated cultures of WT mouse B cells. CFSE-labeled high-density splenic B cells from C57BL/6 WT and p53 KO congenic mice were stimulated for 5 d with BCR-L plus IL-4 with or without BAFF. Cultures were harvested with standardization beads, fixed, and analyzed by flow cytometry for percentage viability within each division subset, as well as total viable cell recovery, within each gated division subset (6). (A and B) Top dot plots, Comparisons of total culture viability in each of the treated cell groups, on the basis of FSC/SSC gating (mean ± SEM for five replicate cultures in an experiment is shown). Bottom histograms, Overlays of CFSE fluorescence within gated viable (dark line) and apoptotic (gray shadow) cells from cultures of WT or p53 KO B cells in a representative experiment (of five). Cultures were seeded with equivalent numbers of cells and each plot represents a total of 40,000 events collected, using an identical scale for cell number. (C and E) Percentage viability values for each division subset in p53 KO and WT B cell cultures were calculated for each of five experiments and expressed as means ± SEM. The p values were calculated with the Student t test. Darkened p values show the division subsets where statistical significance was reached. Lighter p values show those of borderline statistical significance. (D and F) p53 deficiency provides a bonus for viable cell yield. Total number of viable cells recovered within each division subset was determined, with the use of standardization beads. A ratio of the total cells recovered in p53 KO cultures versus the recovery in WT cultures was calculated for each experiment. Shown are the means ± SEM of these values from five experiments. The p values are shown for the division subsets in which the p53 KO/WT ratio was significantly (dark) or nearly significantly (light) different from the control value of 1. (G and H) Kinetic analysis of total viable cell yield within p3 KO and WT cultures stimulated with either BCR-L plus IL-4 (G) or BCR-L plus IL4 and BAFF (H). Note that the greater yield of viable blasts in p53 KO cultures is most significant from days 4 to 5; by day 6, the number of viable highly divided blasts in the p53 KO cultures drops, possibly reflecting induction of “mitotic catastrophe” as discussed in the text.
Despite the above, it was also evident that at least half of the p53-deficient, highly divided lymphoblasts succumbed to death in cultures optimally stimulated with BCR-L plus IL-4 plus BAFF. This finding might suggest that p53 has only a minor proapoptotic function. Nevertheless, an alternative possibility is that the highly divided lymphoblasts within the p53-deficient cultures are dying by “mitotic catastrophe.” The latter is a death that occurs in cells with defective checkpoints and has been observed in replicating p53-deficient cells during mitosis, due to the lack of p53-imposed cell cycle brakes that permit repair of DNA damage (49–51).
Although not definitive, there is some support for mitotic catastrophe within p53-deficient B cell cultures activated with BCR-L plus IL-4 plus BAFF. First, the viability bonus attributed to p53 deficiency was less apparent in day 6 cultures as compared with day 4 and day 5 cultures (Fig. 7G, 7H). Second, greater levels of cyclin B1 were evident in a lysate of activated day 5 p53 KO cultures as compared with WT cultures upon immunoblotting (Supplemental Fig. 3). Cyclin B1 is characteristically elevated in cells undergoing mitotic catastrophe due to loss of a cell cycle brake in G2/M (52). Although further studies are necessary before we can unequivocally conclude that p53-deficient clones die by mitotic catastrophe, this process termed the “Achilles heel” of p53-deficient cells (51) may contribute to the eventual demise of p53-deficient blasts that avoid earlier apoptosis. Taken together, our results indicate that a p53-mediated proapoptotic pathway is at least in part responsible for the reduced viability of lymphoblasts in WT cultures.
Viable replicating B lymphoblasts in TI clones express high levels of phosphorylated ATM and H2AX
One primary route whereby p53 protein levels rise is through p53 phosphorylation (stabilization) by ATM. Once phosphorylated, p53 is resistant to ubiquitination by Mdm2 (53, 54). Thus, recent findings that activated (Ser1981-phosphorylated) ATM is highly upregulated within blasts of these TI cultures (55) is consistent with an active p53 axis. Insomuch as ATM can be activated both through its detection of DNA double strand breaks and through oxidative stress (54), in this study we probed TI blasts for a more definitive indicator of DNA damage: phosphorylation of the histone H2AX (56). Results from two representative experiments in which CFSE-labeled blasts in day 5 cultures were stained for p-ATM-Ser1981 and p-H2AX-Ser139 are shown in Fig. 8A (left panels, human B cells activated with BCR:CD21-L plus IL-4 plus BAFF; right panels, mouse B cells activated with BCR-L plus IL-4 plus BAFF). Although all cells were positive for p-ATM and p-H2AX, dividing lymphoblasts clearly exhibited the highest levels of these phosphoproteins, both in human and mouse cultures.
p-ATM-Ser1981 and p-H2AX-Ser139 expression is elevated in human and mouse B lymphoblasts undergoing TI activation. (A) Cultures of human and mouse CFSE-labeled B cells were stimulated as indicated, harvested on day 5, and stained intracellularly for molecules indicative of DNA double strand breaks (phosphorylated ATM and H2AX) in the presence of phosphatase inhibitors. The human B cell cultures were pulsed with the pan-caspase inhibitor Z-VAD (20 μM) on day 4 to diminish apoptosis of activated B cells with DNA damage. (Z-VAD was absent in the mouse cultures.) Plots of human B cells are representative of four experiments; plots of mouse B cells are representative of two experiments. ATM is known to be autophosphorylated and activated upon binding DNA double stand breaks; activated p-ATM-Ser1981 targets histone H2AX for phosphorylation at Ser139 (56, 57). The dotted horizontal line indicates the PE intensity below which are >95% of the cells stained with IgG control. (B) Comparison of the time course of p53, p-ATM-Ser1981, and p-H2AX-Ser139 expression in CFSE-labeled cells exposed to BCR:CD21-L alone or in combination with IL-4 and BAFF. Purified unlabeled cells prior to culture, or after CFSE-labeling and 2 and 3 d culture with the above stimuli, were frozen in an optimal DMSO/FSC-based cell freezing solution until simultaneous defrosting for analysis of p53, p-ATM, and p-H2AX by flow cytometry. Shown are the overlaid histograms of viable-gated cells stained with the indicated specific mAb or with the respective IgG control. Values shown represent RMFI, as calculated in Fig. 5. (C) Evidence for upregulation of p-H2AX-Ser139 by immunoblotting. Lysates from activated cultures were immunoblotted for p53, as in Fig. 5A. Following stripping, they were reblotted for the Ser139 phosphorylated form of H2AX, total H2AX, and β-actin. Shown are ratiometric values for the relative degree of H2AX phosphorylation (an indicator of DNA damage) within the various cultures. Not considered in this calculation is an undefined band of high molecular mass that was quite prominent in this and a replicate experiment upon immunoblotting with the anti–p-H2AX-Ser139 mAb. As discussed in the text, the latter might represent a complex of p-H2AX with other protein(s) known to cluster in areas of DNA damage. It may be of relevance that both experimental sets of lysates, a low ratio in day 3 cultures supplemented with both IL-4 and BAFF, was accompanied by evidence of a very prominent band between the 52- and 225-kDa markers in the p-H2AX blots (the intensity of this upper band always exceeded that seen under other stimulation conditions, both at day 3 and day 5). (D) Bar graph showing ratios of p-H2AX densitometric intensity to total H2AX intensity (means ± SEM of the 15-kDa band in two replicate experiments).
To better understand the timing of DNA damage within these cultures, B cells were stained for p-ATM-Ser1981 and p-H2AX-Ser139 both prior to culture and at days 2–3 following culture with BCR:CD21-L, with or without supplementary cytokines. The representative experiments in Fig. 8B show that whereas levels of p-H2AX remain low in cultures exposed to BCR:CD21-L alone, expression of this indicator of DNA damage significantly rises in cultures with added IL-4 and BAFF. When a small proportion of blasts has begun to divide (day 3), a subset of undivided cells and all the divided blasts express high levels of p-H2AX. Interestingly, p-ATM was more prominent in freshly isolated cells than was p-H2AX, perhaps reflecting the capacity of oxidants such as H2O2 to activate ATM phosphorylation (54). Levels of p-ATM did not further increase in cells exposed to BCR:CD21-L alone, but rose substantially in cells receiving growth-promoting stimuli from IL-4 and BAFF, particularly in dividing blasts. Taken together, the representative experiment in Fig. 8B shows that B cells exhibit some ATM activation even prior to culture, but DNA damage, as shown by p-H2AX, is minimal until cells begin showing signs of DNA replication/division.
The immunoblotting evidence in Fig. 8C and 8D provides further support for the conclusion that H2AX phosphorylation is most marked in BCR:CD21-L–triggered B cells exposed to growth-promoting cytokines. Although the ratio of p-H2AX to total H2AX was elevated in IL-4–supplemented cultures at day 3, evidence for p-H2AX phosphorylation was substantially more pronounced at day 5 when many cells had divided. Interestingly, blotting with the anti–p-H2AX mAb not only revealed an expected 15-kDa band for H2AX, but also a relatively intense band between the 52- and 225-kDa molecular mass markers. In both experiments, the band was most notable within cultures exposed to IL-4 and BAFF. Although its identity remains unclear, there is a possibility that the high-molecular mass band reflects a complex of p-H2AX and other proteins known to aggregate at sites of DNA damage (58, 59). This possibility is consistent with the decline in 15-kDa total H2AX, as well as 15-kDa pH2AX (as compared with actin loading control) within cultures supplemented with both IL-4 and BAFF (Fig. 8C). Although a similar high-molecular mass band was not noted with the anti-H2AX immunoblotting mAb that recognizes total H2AX (data not shown), it is possible that the epitope engaged by the latter mAb is modified or blocked in the putative complex. Taken together, although the identity of the molecule(s) in the upper molecular mass band is unclear, both the available evidence from intracellular staining and the ratiometric analysis of monomeric (15-kDa) pH2AX/H2AX levels show that B cells within cytokine-supplemented cultures exhibit greater p-H2AX-Ser193 phosphorylation and hence greater DNA damage. Several PI3K-like kinases, ATM, ATR, and DNA-PK, could be involved in initiating the phosphorylation of the H2AX histone at sites of DNA damage. Evidence of the concomitant upregulation of p-ATM-Ser1981 by both immunostaining (Fig. 8A, 8B) (7) and immunoblotting (Supplemental Fig. 4) suggests that activated ATM at a minimum is involved.
B cells genetically deficient in AID exhibit lesser, but not ablated, AICD
Although H2AX phosphorylation in part undoubtedly reflects DNA strand breaks known to occur during the process of DNA replication (60), the evidence that AID is highly expressed and active within replicating human blasts stimulated within BCR:CD21-L plus IL-4 with or without BAFF (7) strongly suggests that a component of the evidenced DNA damage reflects AID-induced enzymatic activity. To test the hypothesis that AID function is contributing to AICD within TI clones, we compared the in vitro responses of B cells from WT mice and mice with deleted Aicda (AID-KO) to a similar mode of TI activation. The data in Fig. 9 show that although AICD remains prominent in AID-KO cultures, AID deficiency does have a minor, yet reproducible, impact on the percentage viability of highly divided lymphoblasts and their total yield. This is weakly apparent upon the overlay of histograms from viable and apoptotic CFSE-labeled blasts (Fig. 9A, 9B). It is further evidenced by the greater percentage viability (Fig. 9C, 9E) and greater total viable cell yield (Fig. 9D, 9F) of AID-KO cultures versus WT cultures. Whereas in BCR-L plus IL-4–activated cultures, the prosurvival effects of AID deficiency are consistently noted only in the most highly divided B lymphoblasts (Fig. 9C, 9D), within cultures exposed to both IL-4 and BAFF, the prosurvival effects of AID deficiency appear to extend to both undivided and divided lymphoblasts (Fig. 9E, 9F). This might relate to the fact that BAFF synergizes with IL-4 in promoting AID expression/activity (7, 61, 62). Taken together, these mouse B cell studies indicate that although AID function contributes to AICD within BCR-triggered, IL-4 and BAFF-driven clones, AID function is not obligatory for AICD of the replicating B lymphoblasts.
TI stimulation of AID-deficient mouse B cells yields a slight, but significant, increase in viable lymphoblast recovery as compared with WT mouse B cells. CFSE-labeled high-density splenic B cells from C57BL/6 WT and AID-KO congenic mice were cultured at the same density for 5 d with BCR-L plus IL-4 with or without BAFF. Cultures were processed as in Fig. 7. (A and B) Overlays of the CFSE fluorescence histograms from a representative experiment (of three) with WT and AID-KO B cells, gated as viable (red) or apoptotic (black). (C and E) Percentage viability values for cells in each gated division subset were calculated for the four to six replicates within one experiment. Shown is the intraexperimental mean ± SEM values for percentage viability in the AID-KO and WT B cell cultures. The p values show that percentage viability values from the two sets of cultures are significantly different. (D and F) The total number of recovered viable cells within each division subgroup was determined for each of three experiments and expressed as a ratio of the yield in AID-KO versus WT cultures (as in Fig. 7). Any bonus in viable cell recovery attributed to AID deficiency is indicated by a ratio >1. The p values are shown for divisions in which these interexperimental ratios were statistically significant (p = 0.03) or approached statistical significance (p = 0.06) as compared with WT control values of 1. Note that although the ratios for infrequent viable cells reflecting four divisions were not significantly different in the cultures with BCR-L plus IL-4, this reflected the wide spread in ratios between the three experiments (1.84, 1.28, and 6.53); in all experiments the AID-KO/WT ratio was greater than the WT control value of 1. Mean ± SEM values for the absolute number of viable cells representing four divisions (in five to six replicate cultures per each of three experiments) were 154 ± 69, 2205 ± 90, and 270 ± 37 (WT cultures) as compared with 284 ± 96, 2816 ± 274, and 1764 ± 222 (AID-KO), respectively.
Discussion
The present study provides novel evidence that p53—a transcription factor, coined “guardian of the genome and policeman of the oncogenes” (63, 64)—has a functional role in regulating lymphoblast survival during an in vitro TI immune response of mature B lymphocytes. Importantly, the synergistic stimuli that generate susceptible clones, Ag, C3d, IL-4, and BAFF are abundant in certain in vivo settings where B cell foci are prominent (65–67), suggesting that these findings may have physiologic relevance. A functional role for p53 in promoting AICD was supported by several lines of data: 1) elevated levels of p53 protein, as well as upregulated mRNA and/or protein of several proapoptotic molecules known to be under the positive transcriptional control of p53, that is, Bax, Bad, Puma, Bid, and caspase 6 (25–27, 68); 2) augmented lymphoblast survival upon p53 siRNA treatment (human) or p53 gene deletion (mouse); and 3) correlation of p53 pathway upregulation with heightened levels of DNA damage. p53 can transactivate genes for proapoptotic proteins involved in mitochondria-dependent intrinsic apoptosis as well as genes for membrane death receptors, Fas and DR. Nevertheless, these studies with B lymphoblasts from TI clones showed evidence for the former but not the latter. Such findings are consistent with evidence that p53 transcriptional targeting is highly regulated (69). Importantly, in recent years it has become clear that p53 can also promote mitochondria-dependent apoptosis through its direct functions in the cytoplasm (28, 70, 71). This latter function of p53 may also influence TI clonal contraction. Future studies are needed to clarify its relative importance.
Until now, direct evidence that p53 influences B lymphoblast survival during a normal immune response has been limited. This is perhaps surprising given the established roles of p53 in regulating cell cycle progression and survival following cell stress in many lineages (64). Certainly, p53 function is anticipated within T cell-dependent germinal centers given the high levels of AID-induced DNA damage and oxidative stress within rapidly replicating centroblasts. Quite consistent with this, a study by Martinez-Valdez et al. (12) found that germinal centers express elevated p53 protein as well as proapoptotic proteins such as Bax. Nevertheless, the importance of p53 in such T cell-dependent foci became obscured by other findings suggesting that p53 function within germinal centers was under strong negative control. First, p53 mutations were rare in most B cell malignancies of germinal center origin compared with an average rate of ∼50% in non-B cell malignancies (72). Second, the major germinal center master regulator, Bcl-6, was found to actively repress transcription of p53 mRNA (73), as well as transcription of ATR, a kinase that phosphorylates and stabilizes p53 protein following single-stranded DNA damage (74). Third, there was evidence that FOXO-regulated Bim, but not p53-regulated Bid, was important in germinal center apoptosis (18). Finally, mice with genetic deficiency in p53 readily succumbed to tumors of non-B cell lineages, rather than B cell tumors (75), suggesting that p53 function in B cells is distinctly regulated. A possible reconciling explanation is that transcription from the p53 gene is significantly downregulated within germinal centers (making the p53 gene less vulnerable to mutation), but that low levels of translated p53 protein are significantly stabilized by p53-modifying kinases, ATM and DNA-PKA. The latter are not subject to Bcl-6 repression and are activated by DNA damage/cell stress (21, 76, 77).
Of relevance to TI responses and p53 activity, two earlier studies with p53-deficient murine B cells showed that p53 can slow cell cycle progression in response to LPS and IL-4 (78, 79). Because no difference was observed in the viability of cultured p53-deficient and WT B cells at day 3, the authors concluded that p53 did not influence mature B lymphocyte survival. Consistent with the above, we also observed no difference in viability between WT and p53-deficient B cells at this early culture interval. Nevertheless, significant differences emerged as clonal expansion progressed through day 5 of the response.
Thus, the present report provides the first direct evidence, to our knowledge, that p53 has a functional role in regulating B lymphoblast viability during clonal expansion. Findings reported by Hao et al. (80) as this manuscript was in revision are consistent with our conclusions that p53 plays a role in regulating the survival of B cells during TI B cell responses and, furthermore, suggest that this occurs in vivo. In the latter study, mice with genetic ablation of Mule (a ubiquitin ligase which targets p53, as well as other proteins such as Mcl-1; see Ref. 81) exhibited significantly diminished levels of serum IgM and IgG3 in response to the TI stimulus, TNP-Ficoll, while exhibiting relatively normal IgM and IgG responses to the TD stimulus, NP-CGG. Interestingly, cultures of mule-deficient B cells yielded lesser highly divided lymphoblasts in response to LPS plus IL-4 and showed an abnormal increase in Puma mRNA as compared with WT B cells, which was not apparent in the absence of p53 (80). Taken together, the data from the current study involving both human and mouse B cells and the recent in vivo and in vitro murine studies of Hao et al. (80) strongly argue that p53 has a functional role in regulating B cell survival during a TI clonal expansion.
A predilection for TI B cell clones to succumb to p53-mediated apoptosis, a result of sustained DNA damage, sheds some light on several past observations. Mice deficient in p53 exhibit an expanded splenic marginal zone and increased incidence of marginal zone lymphoma (75). p53-deficient lymphoblasts switch more effectively to IgG2a than do WT B cells following in vitro stimulation with LPS plus cytokines or in vivo stimulation of mice with polyoma virus (82). Ig-associated gene translocations occur more frequently in LPS plus IL-4–stimulated B cell cultures from p53-deficient mice than in similarly stimulated normal B cells or B cells from mice genetically deficient in both p53 and AID (83). Finally, in humans, p53 mutations are more common in malignancies that likely originate outside of germinal centers: B-chronic lymphocytic leukemia and related prolymphocytic leukemia (72, 84–86), MALT lymphoma (72, 87, 88), and marginal zone lymphoma (89, 90).
In the present model for TI B cell clonal expansion, it appears likely that Puma and tBid are the important triggers for the oligomerization of apoptosis inducers, Bax or Bak, within the mitochondrial membrane and ensuing AICD (91). Although the evidence for Puma involvement is only indirect, other recent evidence shows that Puma has a critical role in determining the fate of mitogen-stimulated B cells in vitro and the development of B cell memory in vivo (16). Interestingly, although Puma is transcriptionally upregulated by p53 upon DNA damage, it can also be upregulated during cytokine growth withdrawal by FOXO3a (29). The latter pathway may be less relevant in our IL-4– and BAFF-supplemented cultures given that mRNA and protein for another FOXO3a-regulated molecule, the apoptosis-activator Bim (92), is downregulated in these cultures. Diminished Bim mRNA may represent optimal activation of the PI3K/Akt pathway by IL-4 (93) since Akt phosphorylates FOXO3a and promotes its exclusion from the nucleus (94, 95). Consistent with the above interpretation, lysates of cultures stimulated with BCR:CD21-L plus BAFF, in the absence of IL-4, expressed significantly greater levels of the various Bim isoforms (P.K.A. Mongini, unpublished observations).
The evidence for involvement of tBid in promoting AICD is also indirect, yet worth noting. Although tBid levels were difficult to reliably observe, intact Bid protein (but not mRNA) precipitously declined during successive divisions within IL-4– and BAFF-supplemented cultures. Bid cleavage to the highly proapoptotic, labile tBid can be achieved by number of proteases, for example, caspases 8 and 10, calpain, granzyme B, and cathepsins (96). Importantly, all have been reported in activated B cells (97–99). Thus, although in vivo studies with Bid-deficient mice suggest that tBid has minimal to no effect on germinal center formation (18), there is a strong possibility that this molecule is relevant in precipitating the clonal contraction of TI clones.
In the context of Puma and tBid, it is undoubtedly relevant that Mcl-1 can bind both with high affinity and thereby block intrinsic apoptosis (13, 100). The significance of Mcl-1 is made more apparent by the precipitous decline of Bcl-2. Whereas the latter anti-apoptotic protein is highly expressed in resting B cells and nondividing activated blasts, its levels drop with each successive division (present study and Ref. 6). This phenomenon may help explain why an in vivo-expressed Bcl-2 TG helped prevent deletion of autoreactive mouse B cells within peripheral tissues (101, 102) and, furthermore, had a greater effect at augmenting B cell memory to TI Ags, as compared with TD Ags (103). Prior findings from this laboratory suggest that deletion of autoreactive B cells by p53-driven AICD might be averted and TI memory increased in tissues with elevated concentrations of Mcl-1–augmenting BAFF, APRIL and PGE2 (5). Such a scenario might in part explain why salivary gland-targeted Sjögren’s syndrome is highly linked to APRIL titers (104) and why SLE autoantibody titers decline in mice treated with inhibitors of Mcl-1–upregulating COX-2 (105).
Note that p53 has been implicated in the negative regulation of Bcl-2 both through direct repressive effects at the Bcl-2 promoter (106) and through induction of Bcl-2 inhibitory micro-RNA: miR-15, miR-16, and miR-34 (107–109). Nevertheless, because Bcl-2 is subject to other means of regulation (110–112), one cannot conclude that the decline in Bcl-2 reflects an augmented p53 axis. This will require further study.
In this study, we made efforts to replicate the human TI response to low-dose BCR:CD21-L plus IL-4 with or without BAFF with mouse cultures stimulated with higher dose BCR-L plus IL-4 with or without BAFF to more readily study the effects of AID on the process of AICD. The resulting experiments showed that AID function only slightly contributes to the clonal contraction of mouse B cell clones. Interestingly, although reduced AICD in p53-deficient cultures extended to all division subsets, the protective effect of AID deletion was noted predominantly in the most highly divided cells. This is consistent with the strong linkage between AID expression/function and cell division (7, 113).
There is a strong possibility that the present findings with mouse B cells do not adequately mirror the AICD-inducing effects of functional AID within human B cell clones. First, dividing mouse lymphoblasts recovered from BCR-L–, IL-4–, and BAFF-activated cultures manifest little, if any, intracellular staining with an anti-mouse AID mAb (clone ZA001) (H. Lee and P.K.A. Mongini, unpublished results), whereas replicating blasts of human B cell cultures show high AID expression upon staining with the same mAb or an additional human AID-specific rat mAb (7). One possible explanation for these findings is that stronger BCR engagement in the mouse B cell cultures may have dampened AID transcription (114). (Mouse cultures were stimulated with 1 μg/ml anti-IgM/dextran, whereas human B cell cultures were stimulated with 0.01 μg/ml anti-IgM/anti-CD21/dextran.) Second, in addition to diminished AID protein, AID function is less evident in the mouse cultures than in human cultures: substantially fewer IgG+ switched progeny are typically recovered from the above TI-activated mouse B cell cultures (P.K.A. Mongini, unpublished results) than from activated human B cell cultures (7). Thus, taken together, it remains quite possible that AID activity has a significantly greater contribution to p53-mediated AICD within the studied TI human B cell clones than revealed in the present mouse cultures. Importantly, replicating mouse B cells are not resistant to AICD following AID-induced DNA damage. Zaheen et al. (10) recently demonstrated that AID activity significantly constrains clonal size within BCR and CD40-stimulated mouse B cell cultures.
In conclusion, the present evidence that a p53 axis governs the survival of replicating B lymphoblasts during the above TI response may illuminate the stimulatory environment where premalignant or transformed B cells with altered genes of the p53 axis arise. We propose that inflamed peripheral tissues laden with C3dg-coated foreign or self Ag and IL-4(IL-13)– and BAFF-releasing cells of the innate immune system (65, 115–121) may be such a milieu. In these sites, TI B cell clonal expansion would be promoted, but there would be strong positive selection pressure for B lymphocytes to escape p53-mediated AICD. Both the high AID expression/activity elicited by these stimuli (7) as well as the oxidative stress of the proliferative burst should induce mutations. Those within p53 and other genes of the p53 axis will be selected for and may represent one step in a series toward full malignancy, or alternatively the defining event. Such a stimulatory milieu might explain why the p53 gene is deleted in ∼30% of patients with benign CD5− monoclonal B cell lymphocytosis (122). It might also help explain why a high proportion of CD5+ B-chronic lymphocytic leukemia clones display alterations in genes encoding p53 pathway proteins (84, 123, 124) and/or have lost one of several p53-regulated miRNA genes that negatively control Bcl-2 (125–128).
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
This study is dedicated to the memory of the late Dr. Leon T. Rosenberg, who inspired both creative thought and tenacity and remained captivated by life’s complexities to his end. We are grateful to Dr. Betty Diamond for the Bcl-2 mice and Dr. Matthew Scharff for the AID-KO mice used in the study. We additionally acknowledge the assistance of Bill Kennedy in obtaining tonsil specimens. Comments from Dr. Tatjana Stankovic regarding mitotic catastrophe and Drs. Janet Stavnezer and Betty Diamond concerning the manuscript were most useful.
Footnotes
This work was supported in part by National Institutes of Health Grant R01 AI052189, M01 General Clinical Research Center Grant RR018535 from the National Center for Research Resources, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases intramural program, and grants from the Karches Foundation, the Peter Jay Sharp Foundation, and the Marks Family Foundation.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- AECM
- aminoethylcarbamylmethyl
- AICD
- activation-induced cell death
- AID
- activation-induced cytosine deaminase
- ATM
- ataxia telangiectasia mutated
- BCR:CD21-L
- anti-IgM/anti-CD21/dextran conjugate
- BCR-L
- anti-IgM/dextran conjugate
- COX
- cyclooxygenase
- FO
- follicular
- FSC
- forward light scatter
- KO
- knockout
- MFI
- mean fluorescence intensity
- pAb
- polyclonal Ab
- RMFI
- ratio of mean fluorescence intensity value
- ROS
- reactive oxygen species
- siRNA
- small interfering RNA
- SSC
- side light scatter
- tBid
- truncated Bid
- TD
- T cell dependent
- TG
- transgene
- TI
- T cell independent
- WT
- wild-type.
- Received October 21, 2011.
- Accepted April 13, 2012.



















