CD40 Stimulation of Human Peripheral B Lymphocytes: Distinct Response from Naive and Memory Cells

Isolation of human peripheral B lymphocytes

Blood samples were collected from healthy individuals after informed consent in heparinized tubes (Vacutainer; BD Labware, Franklin Lakes, NJ), pooled, and diluted in 1 vol of PBS (10 mM potassium/sodium phosphate buffer with 136 mM NaCl (pH 7.4), Dulbecco’s PBS; Life Technologies, Grand Island, NY). PBMC were prepared by density centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech, Baie D’Urfé, Canada). RBC were removed by lysis with 0.83% (w/v) NH₄Cl and platelets by a second centrifugation over Ficoll-Paque diluted 1/2 with PBS. B lymphocytes were purified by negative selection using the StemSep CD19 mixture according to the manufacturer’s instructions (StemCell Technologies, Vancouver, Canada). Purified human B lymphocytes were >90% CD19⁺ as determined by flow cytometry analysis. CD27⁺ and CD27⁻ B cells were separated by cell sorting, using an Epics Coulter (Beckman Coulter, Burlington, Canada), under sterile conditions. Both purified populations were >90% pure, as determined by flow cytometry.

Culture of human B lymphocytes

Purified B lymphocytes were seeded at 3.75 × 10⁵ cells/ml in Primaria plates (BD Labware) in the presence of 0.5 × 10⁵ cells/cm² gamma-irradiated with 75 Gy (7500 rad) L4.5 expressing CD154 (28). The specificity of the response induced by CD154 expressed on L929 (L4.5 cell line) has been previously confirmed using mock-transfected L929 cells and cytokines as described elsewhere (28). Human B lymphocytes were cultured in IMDM supplemented with 10% heat-inactivated ultra-low IgG FBS (Life Technologies), 5 μg/ml bovine insulin, 5 μg/ml bovine transferrin, antibiotics, and 100 U/ml IL-4 (R&D Systems, Minneapolis, MN) or 25 U/ml IL-10 or 50 U/ml IL-2 (PeproTech, Rocky Hill, NJ) as indicated. Preincubation with 0.01% Staphylococcus aureus (SAC³ Cowan strain I, protein A insoluble; Sigma-Aldrich, Oakville, Ontario, Canada) was used as indicated. Cultures were fed by replacing half of the culture medium every 2–3 days, while irradiated L4.5 cells were renewed every 4–5 days. Cell counts and viability were evaluated in triplicates by trypan blue exclusion using a hemacytometer. B lymphocytes were cultured for short-term (< 10 days) or long-term periods (> 3 wk) as indicated. During these periods, cells were always >96% CD19⁺.

Quantification of IgG and IgM secretion

IgG and IgM concentrations were determined in a standard ELISA using 96-well plates and plastic-adsorbed goat affinity-purified Abs specific to human γ- and μ-chains. The bound Abs were revealed with peroxidase-conjugated goat polyvalent anti-human Ig Abs. All Abs were obtained from The Jackson Laboratory (Mississauga, Ontario, Canada). Tetramethylbenzidine (ScyTek Laboratories, Logan, UT) was used as substrate and OD were measured at 450/630 nm.

Determination of the proportion of Ig-secreting cells

The proportion of cells secreting IgA, IgG, or IgM was determined by a standard ELISPOT assay (29) using the same Ab sandwich described for the ELISA. Cells were washed, seeded in 96-well flat-bottom plates at 100–20,000 cells/ml, and incubated in culture medium for 6 h at 37°C in a 10% CO₂ incubator. Spots were revealed by addition of polyvalent alkaline phosphatase-conjugated goat anti-human specific for κ-, λ-, α-, γ-, and μ-chains. A solution of 1.5 M 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt prepared in 2-amino-2-methyl-1-propanol was used as substrate (Sigma-Aldrich). The spots were counted under the microscope at ×100 magnification.

Flow cytometry analysis

Allophycocyanin-, PerCP-cyanin 5.5-, or FITC-conjugated anti-CD19, PE-conjugated anti-CD27, FITC-conjugated anti-IgG, and allophycocyanin-, PerCP-cyanin 5.5-, PE-, and FITC-conjugated isotype controls were used in double, triple, or quadruple staining procedures. All Abs were IgG1 mouse mAbs obtained from BD PharMingen (Oakville, Ontario, Canada). All stainings were done with 1 μg of each Ab for 1 × 10⁶ cells at 4°C. Cells were fixed with 2% paraformaldehyde. In all analyses, over 95% of cells was double negative with markers set according to isotype-matched negative control staining. The regions containing dead cells were determined using 7-amino-actinomycin D staining, following the manufacturer’s instructions (BD PharMingen). Analyses were done by gating 5,000–10,000 cells with a FACSCalibur flow cytometer and the CellQuest software (BD Biosciences, Mountain View, CA).

Cell proliferation assays

Highly purified CD19⁺, CD27⁺, and CD27⁻ B lymphocytes were seeded at 7.5 × 10⁴/ml in triplicates in flat-bottom 96-well plates in the presence of 2.5 × 10⁴ irradiated L4.5 cells. B lymphocytes were cultured as previously described. Cellular proliferation was monitored using 5-bromo-2′-deoxyuridine (BrdU) incorporation and detection by colorimetric ELISA according to the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN).

RT-PCR amplification of activation-induced cytidine deaminase (AID) mRNA

Total RNA from L4.5, Ramos cell line (CRL-1596; American Type Culture Collection, Manassas, VA) (30), human B lymphocytes, and CD19⁺ and CD27⁻ B lymphocytes cultured for 28 days with L4.5 and IL-4 was isolated using TRIzol reagent (Invitrogen, Burlington, Ontario, Canada). RT-PCR amplification of AID using primer 5′ (5′-GAG GCA AGA AGA CAC TCT GG-3′) and 3′ (5′-CAA AAG GAT GCG CCG AAG CTG TCT GGA G-3′) generated a unique 569-bp fragment. RT-PCR was performed starting from 150 ng total RNA, using the Titan One Tube RT-PCR System according to the manufacturer’s instructions (Roche Diagnostics Canada, Laval, Quebec, Canada). Following a 30-min reverse transcription at 50°C, a PCR composed of 33 cycles of 45 s at 94°C, 45 s at 57°C, and 45 s at 72°C was applied. Cycling was followed by a final elongation period of 10 min at 72°C. No AID mRNA was detected in L4.5 cells. β-Actin amplification was used as internal control in RT-PCR, starting from 150 ng total RNA. The primers used in 5′ (5′-GTG GGG CGC CCC AGG CAC CA-3′) and 3′ (5′-CTC CTT AAT GTC ACG CAC GAT TTC-3′) generated a single 540-bp fragment. Negative control without RNA confirmed theabsence of cross-contamination.

PCR amplification of human IgG1 V_H regions

Total RNA from human B lymphocytes and CD19⁺ and CD27⁻ B lymphocytes cultured for 28 days with L4.5 and IL-4 was prepared and 750 ng was used for RT-PCR as described above. For amplification, a nondegenerated primer (D075: 5′-TGG TGG TAC CCA CTG AAC ACA GAG GAC TCA-3′) was used to hybridize to the cDNA 5′ untranslated region of all human V_H family members, paired with a primer specific for the C_H2 region of human IgG1 (hCγ₁: 5′-GCA TGT GTG AGA CTA GTC ACA AGA TTT GGG-3′). Following a 30-min reverse transcription at 50°C, a touchdown PCR cycling protocol was done as follow: 35 cycles of 30 s at 94°C, 60 s at 65°C (1°C decrease per cycle for the first 10 cycles, and the last 25 cycles at 55°C), and 60 s at 72°C. Cycling was followed by a final elongation period of 10 min at 72°C. Negative controls without RNA or with RNA isolated from the IgM-producing Ramos cell line confirmed the absence of cross-contamination.

Subcloning and sequencing of human IgG1 V_H PCR products

Human IgG1 V_H amplicons were resolved on 1% agarose gel electrophoresis and the band corresponding to amplicon size ranging between 730 and 830 bp was excised. DNA was purified using the QIAquick Gel Extraction kit (Qiagen, Mississauga, Ontario, Canada) and subcloned into the pCRII-TOPO vector (TOPO TA Cloning kit; Invitrogen) according to the manufacturer’s instructions. Plasmid DNA was isolated and purified by standard procedures (31) and inserts were sequenced by automated fluorescent DNA sequencing (ABI 373; PerkinElmer Applied Biosystems, Foster City, CA). From two donors, a total of 18 clones from cultured CD19⁺ and 16 from sorted CD27⁻ B lymphocytes were characterized. To establish the error rate of Taq DNA polymerase mix (Titan One Tube RT-PCR System), the C_H1 and C_H2 domains of the IgG constant region from 10 different clones were compared with sequences from GenBank. From this analysis, the Taq DNA polymerase error frequency was estimated to be 0.34%, which corresponds to 1 bp over 294.

Mutation analysis of IgG1 V_H genes

Mutation analysis of the V_H clones was done using the ImMunoGeneTics database available on the internet (the international ImMunoGeneTics database http://imgt.cines.fr:8104 (32, 33, 34)). The number of mutations was determined after the alignment of each sequenced amplicon with the germline gene presenting the highest homology from framework 1 to framework 3 regions, for a total of 312 bp. Mutation proportions were established from the ratio mutated bp:total number of bp.

High level of IgM-secreting cells are generated following CD40 stimulation

In vitro proliferation and differentiation of human B lymphocytes can be achieved by stimulation with CD154 and IL-4 (25). As reported previously, in this system the cell growth is characterized by three phases (35): the initiation period (< 7 days) followed by an exponential growth during 1 or 2 wk and a plateau phase where proliferation is stopped. We have evaluated the viability and cellular expansion of human peripheral blood B lymphocytes when stimulated through CD40 in the presence of IL-4 for 37 days (Fig. 1⇓a). We observed that viability was stable (91 ± 5%) as long as the cellular expansion was high, but decreased rapidly from days 28 to 37 (81–28%) after expansion has reached its maximum (1000-fold) on day 23.

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FIGURE 1.

Human B lymphocytes proliferate and secrete mainly IgM following CD40 stimulation. Purified human B lymphocytes were cultured with irradiated L4.5 cells in complete medium containing 100 U/ml IL-4 for 37 days. Cell numbers and viability were evaluated in triplicates by trypan blue exclusion. Expansion factors and viability (a) were evaluated using viable and dead cell numbers at indicated times. Error bars can be smaller than symbols. In parallel, the proportion of cells secreting IgA, IgG, and IgM (b) was determined using ELISPOT assays. These results are representative of experiments done with cells from three donors.

In parallel, the proportion of IgA-, IgG-, and IgM-secreting cells was evaluated on days 1, 6, 14, 22, 28, and 36 using ELISPOT assays (Fig. 1⇑b). No Ig-secreting cells were detected on days 1 and 6. After 14 days, IgG- and IgM-secreting cells represented 2 and 8% of the total cells, respectively. The proportion of IgA-, IgG-, and IgM-secreting cells increased more rapidly after day 22, whereas expansion was maximum, reaching 7, 16, and 28% of the total cell number, respectively. Until day 28, IgM-secreting cells represented >70% of total Ig-secreting cells. IgA- and IgG-secreting cells increased more rapidly after 28 days, representing ∼50% of total secreting cells. These results could be interpreted in three ways: long-term CD40 stimulation allows the expansion and differentiation of both memory and naive cells with or without isotype switching or no expansion of memory cells but expansion, differentiation, and isotype switching by naive cells.

CD27⁺ B lymphocyte proportion decreases rapidly following CD40 stimulation

In peripheral blood, IgA- and IgG-expressing cells are CD27⁺ memory B lymphocytes, whereas IgM-expressing cells are mainly (75%) CD27⁻ naive B lymphocytes (4, 36). The previous observations showing that the balance between IgM- and isotype-switched Ig-secreting cells changed over time (Fig. 1⇑) led us to analyze the progression of CD27⁺ memory and CD27⁻ naive B lymphocyte populations in the presence of CD154 and IL-4. The expression of CD27 on CD19⁺ B lymphocytes was then evaluated by flow cytometry during a long-term stimulation (28 days; Fig. 2⇓). We observed that the proportion of CD27⁺ memory cells rapidly decreased from 23% on day 0 to 5% on days 6 and 9. Unexpectedly, a CD27⁺ population (25%) reappeared on day 14. This population kept increasing, reaching 46% on day 23 and remained around 40% until day 28. However, the expression level of CD27 on this emerging CD19⁺CD27⁺ population was lower (mean fluorescence intensity (MFI) = 26) than the expression level on peripheral B lymphocytes on day 0 (MFI = 80). These results indicate that memory cells could be able to modulate CD27 expression and to expand or that only CD27⁻ naive B lymphocytes could be able to expand and acquire CD27 expression following long-term stimulation with CD154 and IL-4.

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FIGURE 2.

The proportion of CD27⁺ memory B lymphocytes decreases rapidly following CD40 stimulation. Purified human B lymphocytes were cultured, as described in Fig. 1⇑, for 28 days (d). The expression of CD19 and CD27 was evaluated by flow cytometry using direct double staining with PE-conjugated anti-CD27 and allophycocyanin-conjugated anti-CD19 Abs. These results are representative of experiments done with cells from three donors.

Proliferation of CD27⁺ memory B lymphocytes is engaged following CD40 stimulation, but does not lead to expansion

The decrease of CD27⁺ memory B lymphocytes (Fig. 2⇑) could be caused by a modulation of CD27 expression or a loss of this population. To discriminate between these possibilities, the expansion of sorted CD27⁺ (92%) and CD27⁻ (97%) B lymphocytes was evaluated in parallel with total CD19⁺ cells (93%) as control by viable cell counts (Fig. 3⇓, a and b). The total cell number of all populations was stable during the first 5 days, with a viability of 92 ± 1%. On day 9, total CD19⁺ and CD27⁻ naive cells started to expand at a similar rate with a high viability level (94 ± 2%). In contrast, the number of CD27⁺ cells decreased by almost 10-fold after 9 days and mortality was increased to 52%. These results suggest that CD27⁺ memory B lymphocytes are lost following CD40 stimulation as a result of cell death.

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FIGURE 3.

CD27⁺ memory B lymphocytes do not expand following CD40 stimulation. Total CD19⁺ and sorted CD27⁺ memory and CD27⁻ naive B lymphocytes were cultured, as described in Fig. 1⇑, for 22 (b) or 7 (c) days. The expression of CD19 and CD27 (a) was evaluated on total CD19⁺ and on sorted CD27⁺ and CD27⁻ B lymphocytes by flow cytometry using direct double staining with PE-conjugated anti-CD27 and FITC-conjugated anti-CD19 Abs. These profiles are representative of the two samples used in b and c. Expansion factors (b) were determined as described in Fig. 1⇑ for total CD19⁺, CD27⁺, and CD27⁻ B lymphocytes. Proliferation (c) was measured by BrdU incorporation, as described in Materials and Methods, on days 1, 4, and 7 for all subsets. Error bars can be smaller than symbols. These results are representative of experiments done with cells from three donors.

To further characterize the outcome of these B lymphocyte populations during the initiation phase, their proliferation was evaluated using BrdU incorporation after 1, 4, and 7 days of CD40 stimulation (Fig. 3⇑c). As observed above (Fig. 3⇑b), total CD19⁺, CD27⁻ naïve, and CD27⁺ memory B lymphocytes showed similar proliferation levels during the first 4 days. After 7 days, the proliferation rate was increased in total and naive cells, while CD27⁺ cell proliferation remained as low as that of day 4. Then, the dramatic decrease observed in the CD27⁺ population can be attributed to a low proliferation combined with an increased cell death and not to the modulation of CD27 expression. These results suggest that CD27⁻ naive B lymphocytes were advantaged over CD27⁺ memory B lymphocytes and represented the only subset able to expand in these culture conditions.

Memory B lymphocytes differentiate into Ig-secreting cells earlier than naive cells after CD40 stimulation

To evaluate the capacity of total CD19⁺, CD27⁻, and CD27⁺ B lymphocytes to differentiate into Ig-secreting cells following CD40 stimulation, we used standard ELISA. IgG and IgM secretion was monitored after short (5 days)- and long-term (14 and 22 days) stimulation of the sorted populations (Table I⇓). After 5 days, only CD27⁺ memory B lymphocytes differentiated into IgG- and IgM-secreting cells. After 14 days, while sorted CD27⁺ cells were all dead, CD27⁻ cells started to differentiate into IgM- and IgG-secreting cells. On day 22, as indicated by IgG and IgM secretion, the differentiation level was indistinguishable in sorted CD27⁻ and total CD19⁺ B lymphocytes. Consequently, CD27⁻ cells were the only subset able to respond to the long-term CD40 stimulation by expansion and differentiation. In addition, these results show that memory B lymphocytes differentiate more rapidly than naive cells following CD40 stimulation. Also, it suggests the capacity of naive cells to perform isotype switching.

Naive B lymphocytes can acquire CD27 expression following CD40 stimulation

Based on the results presented in Fig. 3⇑ and Table I⇑, we concluded that CD27⁻ naive B lymphocytes were the only cell subset responding to prolonged CD40 stimulation. Since CD27⁺ cells emerged in high proportion (>40%) following a sustained stimulation (>23 days; Fig. 2⇑), we assessed whether CD27 expression could be induced on naive B lymphocytes. Total CD19⁺ and sorted CD27⁻ B lymphocytes were stimulated with CD154 and IL-4, and the expression of CD27 was evaluated by flow cytometry (Fig. 4⇓). Total CD19⁺ and CD27⁻ B lymphocytes showed a similar progression of CD27 expression on days 9, 19, and 28 (Fig. 4⇓). On day 28, populations issued from total CD19⁺ and naive CD27⁻ B lymphocytes showed 31 and 37% CD19⁺CD27⁺ cells, respectively. The MFI was similar in both populations, but lower in emerging CD27⁺ B lymphocytes (MFI = 29) than in the initial peripheral populations (MFI = 61). In human tonsils, low expression of CD27 has been observed on a B cell subset (CD20⁺CD38^low/−) in germinal center (37). We have also observed that these newly formed CD27⁺ B lymphocytes expressed a normal level of CD19 and a low level of CD38 (data not shown). In addition, most (>98%) of the cells were CD138⁻ (data not shown). These results indicate that CD27 expression can be induced on naive B lymphocytes, confirming that the newly formed CD27⁺ population can emerge from CD27⁻cells following long-term CD40 stimulation.

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FIGURE 4.

CD27⁺ B lymphocytes are generated from naive CD27⁻ B lymphocytes following long-term CD40 stimulation. Total CD19⁺ B lymphocytes and sorted CD27⁻ naive B lymphocytes were cultured, as described in Fig. 1⇑, for 28 days. The expression of CD19 and CD27 was evaluated on days 0, 9, 19, and 28 by flow cytometry, as described in Figs. 2⇑ and 3⇑. These results are representative of experiments done with cells from three donors.

Long-term CD40 stimulation of naive B lymphocytes can induce isotype switching

Following long-term stimulation, we observed a high level of IgG secretion from remaining naive B lymphocytes (Table I⇑). To verify the occurrence of isotype switching, the proportion of IgG⁺ cells was monitored in total CD19⁺ and sorted CD27⁻ populations following CD40 stimulation (Fig. 5⇓). As observed for CD27⁺ cells (Fig. 4⇑), the IgG⁺ population decreased rapidly within the total CD19⁺ population and reappeared gradually during the culture. Total CD19⁺ and naive CD27⁻ B lymphocytes showed a similar progression of IgG⁺ cells, reaching 7 and 9% on day 28, respectively, indicating that naive B lymphocytes have been able to switch.

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FIGURE 5.

IgG⁺ B lymphocytes are generated from naive CD27⁻ lymphocytes following long-term CD40 stimulation. Total CD19⁺ B lymphocytes and sorted CD27⁻ naive B lymphocytes were cultured for 28 days as described in Fig. 1⇑. The expression of CD19 and IgG was evaluated on days 0, 9, 19, and 28 by flow cytometry using direct double staining with FITC-conjugated anti-IgG and allophycocyanin-conjugated anti-CD19 Abs. These results are representative of experiments done with cells from three donors.

To further investigate, the expression of AID mRNA, essential for isotype switching and somatic hypermutation (38), was monitored in comparison to β-actin mRNA in resting and CD40-activated B lymphocytes (Fig. 6⇓). Amplification conditions allowed the detection of AID mRNA in the Ramos cell line, which expresses low levels of AID (30). In comparison to the low level observed in Ramos, the expression of AID was high and similarly induced in both sorted naive and total CD19⁺ cells. This further supports that naive B lymphocytes can carry out isotype switching.

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FIGURE 6.

Expression of AID is induced in total CD19⁺ cells and sorted CD27⁻ cells after long-term CD40 stimulation. Total CD19⁺ and sorted CD27⁻ B lymphocytes were cultured for 28 days as described in Fig. 1⇑. In parallel with β-actin mRNA, AID mRNA was amplified by RT-PCR and analyzed on a 1% agarose gel electrophoresis for both populations on day 28 as described in Materials and Methods (lanes 4 and 5). A 100-bp DNA ladder was used as marker (lane 1). Ramos cell line mRNA (lane 2) and human resting B lymphocytes mRNA (lane 3) were used as control. These results are representative of experiments done with cells from two donors.

In addition, triple staining was used to monitor CD27 and IgG expression within the CD27⁻ population (Fig. 7⇓). The first IgG⁺ cells detected were not expressing CD27. This IgG⁺CD27⁻ population represented 77 and 68% of all IgG⁺ cells on days 9 and 19, respectively. In contrast, on day 28, the majority (69%) of the IgG⁺ population was expressing CD27. Similar results were obtained with total CD19⁺ B lymphocytes following long-term stimulation (data not shown). This last observation indicates that isotype switching and CD27 expression in naive B lymphocytes can occur independently on different populations or that IgG switching occurs before CD27 expression following long-term stimulation.

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FIGURE 7.

IgG⁺ and CD27⁺ B lymphocytes are two distinct populations until 28 days of CD40 stimulation. CD27⁻ naive B lymphocytes were cultured for 28 days as described in Fig. 1⇑. The expression of CD27 and IgG was evaluated on days 0, 9, 19, and 28 by flow cytometry using direct double staining with PE-conjugated anti-CD27 and FITC-conjugated anti-IgG Abs. These results are representative of experiments done with cells from three donors.

Emerging IgG⁺ B lymphocytes are characterized by the absence of somatic mutations in their V_H genes

To rule out the possibility that residual CD27⁺ memory B lymphocytes could be responsible for the emerging CD27⁺ population, the presence of somatic mutations in IgG1 V_H genes was evaluated for total CD19⁺ and sorted CD27⁻ naive B lymphocytes after a 28-day CD40 stimulation (Table II⇓). All sequences amplified were part of the larger V_H family, V_H3. The number of mutations per clone (312 nt) ranged from 0 to 3 (<0.96%) and 0 to 5 (<1.6%) in total CD19⁺ and naive CD27⁻ B lymphocytes, respectively. Considering a Taq DNA polymerase error frequency of 0.34%, the number of unmutated clones was 89 and 69% for total CD19⁺ and CD27⁻ B lymphocytes, respectively. The remaining mutated clones showed 5 or less mutations, which can be expected for this in vitro stimulation (39, 40) but is not significant when compared with 13–34 mutations obtained from peripheral memory B lymphocytes. Despite the fact that AID mRNA was expressed in both populations (Fig. 6⇑), it was not sufficient to achieve somatic hypermutation. Overall, the low frequency of somatic mutations in IgG⁺ cells confirmed the disappearance of memory B lymphocytes and the switching to IgG by naive cells following long-term CD40 stimulation. It also suggests that CD27 expression can be acquired without somatic hypermutation and that the rapid loss of the CD27⁺ population corresponds to the loss of memory B lymphocytes.

Naive B lymphocytes could be advantaged over memory cells by L4.5

In vivo, CD40 stimulation of memory and naive B lymphocytes occurs in a complex environment involving Ag, cytokines, and accessory cells. In vitro, simple signals such as IL-4 and CD154 (20, 24, 25) and more recently IL-10 and CD154 (41) have been reported to induce proliferation in tonsillar and splenic naive and memory B lymphocytes. To verify that CD154 and IL-4 were not limiting for memory B lymphocyte response, we used additional signals, including IL-2, IL-10, and SAC, for 3–5 days. Based on the relationship observed between the rapid disappearance of CD27⁺ cells and the absence of somatically mutated cells (Fig. 4⇑ and Table II⇑), we used as an indicator the expression of CD27 and IgG to follow memory cells (Table III⇓). SAC and IL-2, known to activate memory B lymphocytes (42), allowed their proliferation and maintenance. Similar results were obtained with SAC and IL-4 stimulation, suggesting that IL-4 was not limiting for memory cell response. However, the addition of L4.5 and IL-10 to SAC and IL-2, as well as L4.5 and IL-10 signals did not allow the maintenance of CD27⁺ and IgG⁺ cells. These results suggest that stimulation through the B cell receptor (SAC) without L4.5 activated significantly memory B lymphocytes, while L4.5 addition to these signals seemed to favor naive cell expansion. This advantage of naive cells over memory cells could result from the nature of the CD154 signal provided by the L4.5 cell line.