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Long-Lived B Cells Are Distinguished by Elevated Expression of A1¹

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

	Abstract

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Only 5% of the 15 million B cells formed daily reach the long-lived peripheral B cell pool, presumably reflecting both negative and positive selection. These selective events occur primarily during late stages of differentiation in the marrow and periphery, when newly formed B cells bear surface IgM (sIgM), but differ from mature B cells in their expression of heat-stable Ag (CD24), B220 (CD45), and sIgD. Because genes of the Bcl-2 family influence longevity, we compared the expression of Bcl-2, Bax, and A1 among immature vs mature peripheral B cells using semiquantitative reverse-transcriptase PCR. While the levels of both Bcl-2 and Bax mRNA remain constant in these two populations, A1 expression is strikingly up-regulated among mature B cells. In addition, A1 expression is low among pro- and pre-B cells, as well as in immature (sIgM⁺) marrow B cells. Together, these data indicate that A1 mRNA expression is low at all stages of B cell development before final maturation in the periphery and, unlike other Bcl-2 family members whose expression changes little after marrow egress, A1 is up-regulated 10-fold as cells are recruited into the long-lived peripheral B cell pool.

	Introduction

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More than 95% of newly formed B cells die in the short interval spanning sIgM³ acquisition in the bone marrow and entry into the long-lived peripheral pool, suggesting that selective events dictating B cell longevity occur at this stage (1, 2, 3, 4, 5). Indeed, numerous studies have shown that ligand-induced deletion occurs at this time, and that as cells transit this window, their sensitivity to deletion decreases (6, 7, 8, 9, 10, 11, 12). Furthermore, the acquisition of longevity can be disrupted by natural mutations (13), and the development of mature B cells interrupted by gene knockouts (14, 15, 16, 17, 18, 19), suggesting recruitment into the long-lived pool involves discrete events, rather than simply a lack of negative selection. Although their nature remains obscure, these events most likely include the differential expression of genes whose products regulate longevity.

The Bcl-2 gene family influences B cell longevity, as evidenced by studies of B cell life span in transgenic and knockout mice, Bcl-2 overexpression in B cell lymphomas, and transfection assays (20, 21, 22, 23, 24, 25, 26, 27, 28, 29). While some Bcl-2 family members promote cell survival, others encourage cell death, and many appear to modulate each other via protein-protein interactions (30, 31, 32, 33). Thus, Bcl-2 family expression in cells undergoing concerted selection has attracted considerable attention. For example, Bcl-2 is expressed at low levels in B cell populations with high death rates, such as pre-B cells, immature marrow B cells, and most germinal center B cells. Conversely, it is up-regulated in pro-B cells and mature B cells, as well as in germinal center B cells that have received death-rescuing signals (34, 35, 36).

We recently showed that immature B cells exit the bone marrow and spend several days in the periphery before final maturation and entrance to the long-lived mature B cell pool (5, 37). These newly emerging B cells remain functionally and phenotypically immature, and can be distinguished from mature peripheral B cells based on HSA (CD24), B220 (CD45), and sIgD expression. Most studies of genes influencing maturation and life span have compared marrow vs peripheral populations, so their expression kinetics have not been assessed at these late differentiative stages in the periphery. We have therefore compared the expression of several Bcl-2 family members among immature vs mature peripheral B cells using HSA, B220, and sIgD expression as phenotypic criteria. The results indicate that: 1) neither Bcl-2 nor Bax mRNA levels change appreciably during this period, suggesting their regulation is not directly responsible for recruitment to the long-lived peripheral pool; and 2) the A1 gene is expressed at low levels among all marrow and peripheral immature B cells, but increases 10-fold upon their recruitment to the long-lived pool. Together, these findings show that the A1 member of the Bcl-2 gene family is a molecular marker for B cells that have entered the long-lived recirculating pool, and suggest that its regulation may be important in determining the fate of immature peripheral B cells.

	Materials and Methods

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Mice

Adult BALB/cJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

Cell preparations

Lymphocytes were flushed from spleens and femurs using ice-cold DMEM, pelleted, and resuspended in 37°C ammonium-Tris-chloride for 5 min to lyse RBCs. Cells were pelleted and resuspended in cold DMEM. Cells from at least three mice were pooled for each experimental preparation.

Antibodies

FITC anti-HSA (M1/69) (38); phycoerythrin, APC, and biotin anti-B220 (RA3-6B2) (39); and phycoerythrin anti-CD43 (S7) were purchased from PharMingen (San Diego, CA). Biotin anti-mouse IgD was purchased from Southern Biotechnology (Birmingham, AL), and FITC anti-mouse IgM and whole mouse and rat Ig were purchased from Jackson Immuno Research Laboratories (Westgrove, PA). RED670-streptavidin was from Life Technologies (Bethesda, MD).

Flow-cytometric analysis

Cells were incubated at 2 x 10⁶/ml in staining buffer (PBS with 0.5% BSA) and preincubated on ice with 5 mg/ml each of rat and mouse IgG. Cells were then stained with fluorochrome- or biotin-conjugated Abs, washed in staining buffer and, when appropriate, incubated with RED670-streptavidin, and rewashed, then filtered twice through nitex monofilament cloth. Cells were sorted on a Becton Dickinson (San Jose, CA) FACScan, and the data were acquired and analyzed with LYSIS II and CellQuest software.

Isolating B cell populations

Immature peripheral B cells (HSA^highB220^lowsIgM⁺sIgD^+/-) were obtained from the spleens of sublethally irradiated (500 rad) autoreconstituting animals at day 13.5 postirradiation. Irradiation destroys all peripheral B cells, allowing a synchronous wave of B cell development. The day 13.5 spleen is enriched for immature peripheral B cells that are functionally identical to those in unmanipulated mice (5, 37, 40). Mature B cells (HSA^lowB220^highsIgM⁺sIgD⁺) were obtained from the spleens of unmanipulated adult mice. To isolate only those B cells that had acquired the mature phenotype within a 3-day window, HSA^lowB220^highsIgM⁺sIgD⁺ B cells were sorted from sublethally irradiated autoreconstituting spleens 18 days postirradiation.

Bone marrow populations were FACS purified according to the parameters defined by Hardy et al. (41). Pro (CD43⁺B220⁺sIgM^-)- and pre (CD43^-B220⁺sIgM^-)-B cells were obtained from femurs of unmanipulated mice, while immature bone marrow (CD43^-B220⁺sIgM⁺sIgD^-) cells were obtained from sublethally irradiated autoreconstituting mice on day 11 postirradiation.

Preparation of cDNA

Poly(A) RNA was purified with an oligo(dT) column (Invitrogen FastTrack, San Diego, CA), and first-strand cDNAs were then synthesized by Moloney murine leukemia virus reverse transcriptase (SuperScript; Life Technologies) with random hexamers.

PCR primers

The primers had the following sequences: Bcl-2 sense 5'-tcgctaccgtcgtgacttc-3', antisense 5'-aaacagaggtcgcatgctg-3' (36); Bax sense 5'-atgcgtccaccaagaagctgag-3', antisense 5'-ccccagttgaagttgccatcag-3'; A1 sense 5'-caaatctggctggctgacttttc-3', antisense 5'-caagtgctgataaccattctcgtc-3'; and ß-actin sense 5'-gcattgctgacaggatgcag-3', antisense 5'-cctgcttgctgatccacatc-3'. Bcl-2, Bax, A1, and ß-actin primers yielded 315-, 166-, 123-, and 156-bp products, respectively.

To ensure that amplified sequences corresponded to message expression rather than contaminating genomic DNA, PCR primers for Bcl-2, Bax, and ß-actin were designed to span introns. The genomic structure of A1 is unknown, although amplification using genomic DNA yielded the same size product as cDNA. Although these primers do not span introns, the A1 product detected is unlikely due to the contamination of genomic DNA because amplification of the same cDNA preparations with intron-spanning ß-actin primers did not yield a genomic fragment. Furthermore, amplification of mRNA that had not been reverse transcribed did not yield significant ß-actin or A1 products.

Semiquantitative PCR

PCR was performed with specific primers to Bcl-2, Bax, and A1 using random primed cDNA. PCR products were separated on a denaturing gel (6% acrylamide, 6 M urea, 1x Tris-borate/EDTA), and the presence and quantity of specific amplified sequences were detected by incorporation of [-³⁵S]dCTP. To ensure that all comparisons were made during the exponential phase of amplification, aliquots were removed at progressive cycles, and the amount of product (determined by densitometry with a phosphor imager) was plotted against cycle number. To control for variations in the rate of amplification, each reaction was undertaken in replicate. Relative expression was determined by comparing the amount of product generated in each population at the same cycle. To ensure that the amount of initial cDNA was equal for each population, a semiquantitative PCR using primers specific to ß-actin was performed. Expression levels for the gene of interest were then normalized to the relative ß-actin levels. ß-actin amplification for a typical experiment (A1 amplification in Fig. 3) is shown in Figure 1. When experiments were separated by time, internal standards were used to enable comparisons.

View larger version (46K): [in this window] [in a new window]   FIGURE 3. Expression of Bcl-2, Bax, and A1message in immature peripheral and long-lived mature B cells. Bcl-2, Bax, and A1 sequences were amplified in triplicate from HSAhighB220lowIgM+IgD- (open circles) and HSAlowB220highIgM+IgD+ (open squares) cDNA by semiquantitative RT-PCR in the presence of [-35S]dCTP. Aliquots were removed at progressive cycles during exponential amplification and run on a denaturing gel. The amount of product was determined by densitometry and plotted against cycle number. Expression levels were normalized for ß-actin expression, which was also determined by semiquantitative PCR (see Fig. 1).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Comparison of ß-actin expression between different cell preparations. ß-actin sequences were amplified in triplicate from different cDNA preparations by semiquantitative RT-PCR in the presence of [-35S]dCTP (this experiment shows the HSAhighB220lowIgM+IgD- (open circles) and HSAlowB220highIgM+IgD+ (open squares) preparations used in Fig. 3). Aliquots were removed at progressive cycles during exponential amplification and run on a denaturing gel. The amount of product was determined by densitometry and plotted against cycle number. To estimate the relative amounts of cDNA in each sample, differences in log band intensity were compared.

	Results

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Bcl-2 and Bax mRNAs are expressed at similar levels in immature peripheral and mature splenic B cells

In the spleen, mature B cells are distinguished from their precursors by both turnover rate and surface marker expression (5, 37). Immature peripheral B cells either die or mature within 4 days and are HSA^highB220^lowsIgM⁺sIgD^+/-. In contrast, mature B cells are long-lived and are HSA^low B220^high sIgM⁺sIgD⁺. Because members of the Bcl-2 family modulate B lymphocyte survival (20, 22, 23, 24, 25, 43), we compared Bcl-2 family expression within these two splenic B cell populations (Fig. 2).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Isolation of immature peripheral and long-lived mature splenic B cells. A, Immature peripheral HSAhighB220lowIgD-, and B, mature HSAlowB220high (IgD+) splenic B cells were purified from BALB/c spleens by FACS within a live lymphocyte gate. This figure demonstrates the FACS profiles before and after purification.

A1 mRNA is expressed by murine B cells

Another Bcl-2 family member is A1, which was first described as a hemopoietic lineage-specific gene (42). A1 promotes cell survival (42), perhaps by decreasing available Bax (33). A1 shares structural homology with other Bcl-2 family members, including BH1 and 2 domains, but lacks an N-terminal region rich in charged residues that is shared by most other members.

Although previous studies have not detected A1 mRNA in cultured B cell lines, we identified multiple clones expressing A1 while using a degenerate RT-PCR strategy to identify Bcl-2 family genes expressed among normal splenic B cells (data not shown).

A1 mRNA levels increase more than 10-fold upon recruitment to the long-lived mature B cell pool

We compared the expression of A1 mRNA in immature peripheral vs mature splenic B cells using the same RT-PCR approach described above. While detectable in both cell populations, A1 mRNA levels in mature peripheral B cells were consistently >10-fold higher than in immature peripheral B cells (Fig. 3). These results were confirmed by competitive PCR (data not shown). Taken together, these data show that although unexpressed in some cultured B cell lines (42), A1 is indeed expressed by normal B lineage cells.

To more precisely determine the timing of A1 mRNA up-regulation, we examined its expression in several additional populations (Fig. 4). We first assessed A1 mRNA expression in the bone marrow, using the phenotypic criteria of Hardy to delineate early stages of B cell development (41). The results show that A1 mRNA expression is low throughout marrow B cell development, never rising significantly higher than among newly emerging immature peripheral B cells (Fig. 4).

View larger version (14K): [in this window] [in a new window]   FIGURE 4. A1 mRNA expression throughout bone marrow and peripheral B cell differentiation. Several marrow and splenic B cell subpopulations were compared for relative A1 mRNA expression using semiquantitative PCR. Each point represents the relative expression of an independent cell preparation. Average expression for early immature peripheral B cells was arbitrarily set at 1.

Because of their slow turnover rate (t_1/2 > 8 wk), mature (HSA^lowB220^high) splenic B cells from unmanipulated mice contain both newly recruited cells and long-term residents. It was therefore unclear whether A1 up-regulation occurs concomitant with recruitment to the long-lived peripheral pool or later. To isolate B cells recruited to the long-lived pool no more than 3 days previously, we purified HSA^lowB220^high B cells from autoreconstituting mice 18 days postirradiation. Irradiation destroys all peripheral B cells, and no HSA^lowB220^high B cells are evident until 15 days afterward. Semiquantitative PCR analysis demonstrated that these incipient mature cells express at least fivefold more A1 mRNA than their immature progenitors (Fig. 4), indicating that A1 up-regulation is tightly correlated with maturation and the acquisition of longevity.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
These studies have compared the expression of several Bcl-2 family members among immature vs mature peripheral B cells. The results show that the Bcl-2 family member A1 is regulated developmentally in the B cell lineage and, unlike any other Bcl-2 member studied to date, distinguishes long-lived mature B cells from their immature peripheral precursors. Furthermore, they show that A1 mRNA is expressed at low levels among all immature B cell differentiation stages in the marrow and periphery, but is up-regulated 10-fold as cells enter the long-lived pool.

Together, these data demonstrate critical differences in the expression patterns of A1 vs other Bcl-2 family members during the last stages of B cell maturation in the periphery. Others have shown that Bcl-2 expression is tightly regulated during B cell differentiation in the bone marrow (35, 36) and germinal centers (34), and that Bcl-2 expression is down-regulated in many death-susceptible B cell subpopulations. Our findings extend these observations, and show that while elevated Bcl-2 expression indeed parallels marrow egress, it does not change as B cells enter the long-lived peripheral pool. Similarly, Bax expression is identical among both short-lived immature and long-lived mature peripheral B cells, and Bcl-x_L protein is undetectable in all resting sIgM⁺ cells (21). Thus, since two-thirds of immature peripheral B cells die before entering the mature B cell pool (5, 37), none of these Bcl-2 family members displays expression patterns concordant with the acquisition of longevity. As our experiments have examined expression among populations rather than within individual cells, it remains possible that in the immature peripheral pool, only those B cells destined for longevity have up-regulated Bcl-2. However, since the average Bcl-2 levels in the immature and mature peripheral pools are equivalent, and since more than half of the immature cells are destined for death, Bcl-2 expression would have to be at least twofold higher among long-lived immature vs mature peripheral cells to accommodate this explanation.

Negative selection is thought to occur during late marrow and early peripheral differentation stages (9, 10, 44, 45), but recruitment to the long-lived pool may require discrete events in the periphery (12, 13). In contrast to the behavior of other Bcl-2 family members, A1 expression is low and relatively constant among immature B cells in both the marrow and periphery, but is up-regulated profoundly as cells enter the long-lived pool. This up-regulation occurs within a relatively short time frame, as B cells developing synchronously during autoreconstitution up-regulate A1 mRNA at least fivefold within 3 days of their recruitment to the long-lived pool. Based on this chronology, it is tempting to speculate that Bcl-2 primarily mediates resistance to ligand-induced deletion, whereas increased A1 expression follows the receipt of signals necessary for extended life in the periphery.

Whether A1 directly affords the lengthy t_1/2 characteristic of mature B cells remains unclear. A1 has been characterized as a life span-promoting molecule (46), and like many other Bcl-2 family members, forms heterodimers with Bax (33). While A1 shares much structural homology with other Bcl-2 family members, including the BH1 and BH2 transmembrane domains, there is one notable structural deviation: A1 lacks a stretch of charged amino acids toward the N terminus shared by most other members. This loop is a site of proteolytic cleavage in Bcl-2 (47), suggesting that it may be important in regulating cellular turnover of these proteins. Thus, A1 might provide more potent or protracted life span-promoting activity related to this structural difference. Consistent with this hypothesis is the observation that loop deletion mutants of Bcl-2 and Bcl-x_L have stronger antiapoptotic ability than do wild type (48). It will prove intriguing to assess A1 expression among B cells with different life span properties, such as the B1 subset or various germinal center subpopulations, as well as in mutant, knockout, or transgenic animals with altered B cell life span (13, 14, 15, 24).

Whatever proves to be its role in life span determination, these findings show that A1 is a bona fide molecular marker for transit to the long-lived peripheral pool, and its increased expression indicates a developmental program for longevity has been triggered.

	Acknowledgments

 
The authors thank Drs. D. Gay and D. S. Navaratnam for valuable advice and critical reading of the manuscript, and C. H. Pletcher, The University of Pennsylvania Cancer Center Flow Cytometry and Cell Sorter Shared Resource, and the Lucille P. Markey Trust for providing excellent FACS support.

	Footnotes

 
¹ This work was supported by a grant to M.P.C. from the Arthritis Foundation.

² Address correspondence and reprint requests to Dr. Michael P. Cancro, 233 John Morgan Building, 36th and Hamilton Walk, Philadelphia, PA 19104. E-mail address:

³ Abbreviations used in this paper: sIg, surface immunoglobulin; HSA, heat-stable antigen; RT-PCR, reverse-transcriptase polymerase chain reaction.

Received for publication June 5, 1997. Accepted for publication September 18, 1997.

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