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The Journal of Immunology, 2006, 177: 3728-3736.
Copyright © 2006 by The American Association of Immunologists, Inc.

A New Memory CD27–IgG+ B Cell Population in Peripheral Blood Expressing VH Genes with Low Frequency of Somatic Mutation1

Jessie F. Fecteau, Geneviève Côté and Sonia Néron2

Héma-Québec, Recherche et Développement, Sainte-Foy, Quebec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In humans, up to 40% of peripheral B cells express CD27 and have hypermutated variable regions in their Ig genes. The CD27+ B cells are considered to be derived from germinal center following specific antigenic stimulation. Actually, somatic hypermutation in Ig genes and CD27 expression are hallmarks of memory B cells. However, the blood IgM+IgD+CD27+ B cells were recently associated to splenic marginal zone B cells and proposed to be a subset distinct from germinal center-derived memory B cells showing premutated Igs. The results presented herein further weaken this bona fide association because B cells expressing surface IgG, but not CD27, were found in human blood. Representing 1–4% of all peripheral B cells and ~25% of the IgG+ blood B cells, this population expressed mutated IgG genes showing antigenic selection characteristics but with lower mutation frequencies than that of CD27+IgG+ B cells. However, their morphology and phenotype were similar to that of CD27+IgG+ cells. Interestingly, the proportion of IgG2 over IgG3 transcripts was opposite in CD27–IgG+ and CD27+IgG+ cells, suggesting distinct functions or origins. Overall, these findings extend the memory B cell reservoir beyond the CD27+ compartment and could provide further insights into B cell disorders of unknown etiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Memory B cells can be distinguished from naive B cells by the presence of somatic hypermutation in their Ig variable gene sequences (1). Phenotypic characteristics can also be used to identify this B cell subset. Previously, IgD-expressing cells were classified as naive cells and IgD– cells as memory B cells (2, 3). However, the description of an IgD+ subset expressing somatically mutated Ig genes discontinued its use for the identification of memory B cells (4, 5). Currently, CD27, belonging to the TNFR family (6), is widely used as a marker of human memory B cells because its expression correlates with the presence of somatic mutations in Ig genes (4, 5, 7, 8). Based on CD27, IgD, IgM, and CD5 expression, peripheral B cells can be subdivided into four CD27+ memory subsets (IgM+IgD+, IgM+IgD–, IgG+/IgA+, and IgM–IgD+ cells) and two CD27– naive subsets (IgM+IgD+CD5+ and IgM+IgD+CD5–), representing 40 and 60% of peripheral B cells, respectively (4).

During immune responses to T-dependent Ags, immunological memory is established mainly via the generation of memory B cells (9, 10). Following an Ag encounter, naive B cells can differentiate into low-affinity Ig-secreting cells or mature within germinal centers into high-affinity memory cells expressing Ig of various isotypes (11). Activated T cell help, mainly by the engagement of B cell-expressed CD40 by T cell-expressed CD154 (12), is necessary for memory cell generation, including germinal center formation, somatic hypermutation, and isotype switching (reviewed in Refs. 11 and 13). The importance of CD40 stimulation in these events has been demonstrated in patients suffering from X-linked hyper IgM syndrome, in which CD40-CD154 interaction is abrogated (reviewed in Ref. 14). In these patients, even though germinal center formation and memory B cell responses are severely impaired, a peripheral B cell fraction expressing CD27 persists (15, 16, 17), suggesting that this marker does not exclusively identify classical germinal center-derived memory B cells. Recently, this CD27+ compartment was recognized as the peripheral counterpart of splenic marginal zone B cells (16, 18). Although expressing mutated Ig genes, this subset differs from classical memory cells, since in this population, hypermutation can occur in absence of CD40-CD154 interactions (16, 17, 18). Rather, as observed in other species, a developmental mechanism increasing repertoire diversity early in life has been postulated (16, 18). Also, unlike classical memory cells, this CD27+ subset is dedicated to immune responses to T-independent Ags (16, 19, 20), during which they likely act as innate effectors in the first line of defense but not as memory cells (16, 21, 22, 23). These findings reinforce the notion that CD27 expression does not necessarily correlate with memory B cells.

Alternatively, the requirement for CD27 expression on classical memory cells has never been firmly established in humans, nor associated with a well-defined function (24). Indeed, we previously reported that prolonged CD40 stimulation triggered naive B cells to switch to IgG and to express CD27 even in absence of somatic hypermutation, suggesting that these latter events could be independent (25). In mice, rather than a memory marker, CD27 appears to be a surrogate for recent B cell activation and is not absolutely necessary for secondary responses (26).

Overall, questions are still raised concerning the exact identification of human memory B cells based on CD27 expression, and the present study further questions this association. Indeed, a new B cell population expressing hypermutated IgG in the absence of CD27 expression was detected in human blood. Representing 1–4% of all peripheral B cells, these cells correspond to ~25% of IgG+ blood B cells and are morphologically and phenotypically similar to CD27+IgG+ B cells. The identification of this subset extends the human peripheral memory B cell compartment and could suggest novel mechanisms underlying B cell disorders of unknown origin such as hairy cell leukemia (27, 28).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Antibodies

The following Abs conjugated to allophycocyanin, PE, FITC, PE-cyanin 5.1 (PC5), PerCP-Cy5.5, and Alexa Fluor 488 (Alexa-488) were used for flow cytometry analysis or cell sorting: PerCP-Cy5.5- or allophycocyanin-anti-CD19, FITC-, PE-, or PC5-anti-CD27; FITC- or allophycocyanin-anti-IgG, FITC-anti-IgG2, FITC-anti-IgG3, FITC-anti-IgM, allophycocyanin-anti-CD5, allophycocyanin-anti-CD38, PE-anti-IgD, PE-anti-CD126, PE-anti-CD138, Alexa-488-anti-mouse CD45R (B220); and PerCP-Cy5.5-, allophycocyanin-, PE-, PC5-, FITC-, or Alexa-488 isotype controls were used in triple or quadruple staining procedures. Most Abs were mouse monoclonal IgG1 from BD Pharmingen, except PC5 Abs from Beckman Coulter and FITC-anti-IgG2 and anti-IgG3 from Sigma-Aldrich. Alexa-488 Abs were rat monoclonal IgG2a (Caltag Laboratories), and FITC-anti-IgM were polyvalent goat IgG (The Jackson Laboratory).

Peripheral B cell isolation, cell sorting, and flow cytometry analysis

This study has been reviewed and approved by the Héma-Québec Ethical Committee. Blood samples were collected from healthy individuals after informed consent, and PBMC were isolated by density centrifugation as previously described (25) and stored frozen. B cells were purified by negative selection with CD19 mixture according to the manufacturer’s instructions (Stem Sep; Stem Cell Technologies). Purified B cells were >95% CD19+ cells as determined by flow cytometry analysis. CD19+CD27–IgG–, CD19+CD27–IgG+, and CD19+CD27+IgG+ B cells were separated, using PE-anti-CD27 and FITC-anti-IgG, by cell sorting using a BD FACSCalibur low-speed cell sorter (BD Biosciences) or an Epics Elite ESP (Beckman Coulter). Sorted CD27+IgG+ cells were >95%, CD27+ and CD27–IgG+ cells were >95% negative for CD27. IgG2 and IgG3 expression was evaluated in costaining with allophycocyanin-anti-IgG, which recognized all human IgG subclasses. No steric hindrance was observed in these costaining procedures since IgG2- and IgG3-positive cells were both stained by the allophycocyanin-anti-IgG. ATP-binding cassette transporter activity (ABC B1)3 was evaluated as described elsewhere (29), using MitoTracker Green (MTG) (Invitrogen Life Technologies). Briefly, PBMC were incubated at 37°C in IMDM containing 10% FBS and 100 nM MTG for 30 min and in absence of MTG for 40 min and then analyzed for CD19, CD27, and IgG expression. Flow cytometry analysis and staining were done as previously described (25), using FACSCalibur flow cytometer with CellQuest Pro software (BD Biosciences). All analyses with PBMC were done on ≥150,000 events. Data were subsequently analyzed with a computer program (FCS express II; De Novo Software).

Wright-GIEMSA staining

Sorted CD19+CD27–IgG+ B cells, CD19+CD27+IgG+ cells, and CD19+CD27–IgG– cells were spun onto glass slides, stained with Wright-Giemsa stain (Sigma-Aldrich), and examined by light microscopy using a Nikon Eclipse TE 2000-S microscope (Nikon) at x400 magnification. Digital pictures were acquired using a Retiga 1300 camera (QImaging) and the SimplePCI 5 Software (Compix).

Cloning and sequencing of IgG VH regions

Total RNA was extracted from 6 to 8 x 104 sorted CD19+CD27+IgG+ and CD19+CD27–IgG+ B cells using TRIzol reagent according to the manufacturer’s instructions (Invitrogen Life Technologies). The total amount of RNA obtained for each cell sample was used to synthesize cDNAs using oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). Reverse transcriptase reaction done with 500 ng of total RNA from CD40-activated B cells (30) was used as positive control. VH and {gamma} 1, 2, 3, and 4 regions of IgG and beta-actin were amplified by PCR. Amplification of VH regions was done using 35 cycles of 94°C for 30s, 45°C for 40 s, and 72°C for 60s. Amplification of {gamma} regions was done as above, except that step 2 was 60 s at 65°C for {gamma} 1, 62.5°C for {gamma} 2, 3, and 4, and 55°C for beta-actin. One to 2 µl of reverse transcriptase mixture was used with Platinum TaqDNA polymerase (Invitrogen Life Technologies). The primers used for VH were 5' IgG-specific C{gamma} (5'-AAGTAGTCCTTGACC) and 3' VH1-, VH2-, VH3-, and VH4-specific primers described elsewhere (31), and for IgG1, IgG2, and IgG3 amplification, the same 5' primer was used (5'-TCCACCAAGGGCCCATCG-3') in combination with the following 3' primers: CG1z (5'-GCATGTACTAGTTTTGTCACAAGATTTGGG-3') for IgG1, CG2a (5'-CTCGACACTAGTTTTGCGCTCAACTGTCTT-3') for IgG2, and CG3a (5'-TGTGTGACTAGTGTCACCAAGTGGGGTTTT-3') for IgG3 (32, 33). IgG4 amplification was done using 5' primer (5'-GCTTCCACCAAGGGCCCATC-3') and in 3' primer CG4a (5'-GCATGAACTAGTTGGGGGACCATATTTGGA-3') (32). beta-actin primers were described elsewhere (25). Amplicon specificity was confirmed on 1% agarose gel electrophoresis using ethidium bromide staining. Densitometry analysis was done using the Alpha EaseFC software from Alpha Innotech, and relative intensity of gene expression was based on the ratio of {gamma} 1, 2, 3, or 4 to beta-actin gene expression. VH amplicons (500–650 nt) were purified from 1% agarose gel using the QIAquick Gel extraction kit (Qiagen) and cloned into pDRIVE vector from the Qiagen PCR cloning kit (Qiagen). Plasmid DNA was purified using conventional techniques (34), and the cloned IgG VH regions were sequenced in both directions using M13 primers by automated fluorescent DNA sequencing (ABI373; Applied Biosystems). Taq polymerase error was estimated at 0.34%, representing one mutation every 294 nt (25).

Mutational analysis of IgG VH genes

Mutational analysis of VH IgG transcripts was done using the ImMunoGeneTics (IMGT) database (available at <http://imgt.cines.fr>) (35). The number of mutated nucleotides was determined for each transcript after their alignment with the germline gene showing the highest homology from framework region (FR) 1 to FR3 for a total of 312 nt. The distribution of replacement (R) and silent (S) mutations and the R:S ratios were evaluated at the amino acid level for each gene region and were used as an index of antigenic selection (36, 37, 38, 39). The distribution of R and S mutations within FR1, FR2, and FR3 and CDR1 and CDR2 was evaluated in each region, comprising 26 aa for FR1, 17 aa for FR2, 39 aa for FR3, 12 aa for CDR1, and 10 aa for CDR2. The classification of each transcript to IgG1, IgG2, IgG3, or IgG4 was done using the applet "Blast 2 sequences" from the National Center for Biotechnology Information web site (<www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi>). The 100-nt constant region amplified from the CH1 region of each transcript was sequentially aligned against sequences representing IgG constant region of each subclass (GenBank accession no. J00228 for IgG1, J00230 for IgG2, D78345, and AF237585 for IgG3, and K01316 for IgG4). The single alignment giving the highest homology (99–100%) was used for identification of subclasses.

Statistical analysis

Statistical significance was evaluated using two-tailed unpaired Student’s t tests or two-tailed Fisher exact tests, using a value of p < 0.05. All results are presented as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD19+CD27–IgG+ cells are present in human blood

Currently, CD27 expression is associated with memory B cells since all CD27+ B cells express hypermutated Ig genes (4, 5, 7, 8) and peripheral B cells expressing isotype switched Ig are usually considered to express CD27 (4, 5). We have detected, in human blood, a distinct B cell population, which did not correspond to these criteria. Indeed, CD19+CD27–IgG+ B cells were detected in PBMC prepared from 15 independent samples (Table I and Fig. 1A). As expected (5), an average of 9.7 ± 4.3% IgG-expressing cells was observed among these blood samples, but IgG+ cells were further separated into 7.3 ± 3.3% CD27+IgG+ cells and 2.4 ± 1.2% CD27–IgG+ cells. The CD19+CD27–IgG+ population represented ~25% of IgG-expressing cells in blood (25.4 ± 5.2%). These results were corroborated using purified CD19+ B cells (Fig. 1B), whereas CD19+IgG+ cells were gated and analyzed for CD27 expression. Three independent samples were showing an average of 28 ± 7% of CD27– cells among IgG-expressing cells (data not shown). This new population within CD19+IgG+ cells resulted in ~75% of the cells coexpressing CD27 instead of the expected 100%. Furthermore, no intracellular CD27 expression wasdetected in these CD27–IgG+ cells (data not shown). When IgG expression was evaluated within CD19+CD27+ and CD19+CD27– populations, 24.3 ± 8% of CD27+ cells expressed IgG whereas 3.4 ± 2% of CD27– cells were IgG+ (Fig. 1C and data not shown). Finally, the mean fluorescence intensity (MFI) of IgG expression was similar in CD27+IgG+ and CD27–IgG+ cells, being of 186 ± 4 and 191 ± 17, respectively (data not shown).


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  		Table I. CD19+CD27–IgG+ B cells are present in peripheral blood

 

Figure 1
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  		FIGURE 1. Surface IgG expression is not restricted to CD27+ B cells in human blood. CD19, CD27, and IgG expression was evaluated by flow cytometry in PBMC and purified blood CD19+ B cells using triple staining. A, IgG and CD27 expression was evaluated in gated CD19+ cells corresponding to 15% of total PBMC. R1 and R2 regions were used to delineate CD27–IgG+ and CD27+IgG+ cells, respectively. These results are representative of 15 independent samples (Table I). B, CD27 expression was evaluated in gated IgG+ cells (~100,000 events, R1). C, IgG expression was evaluated in CD19+CD27+ (~50,000 events, R4) and CD19+CD27– (~50,000 events, R5) cells using purified CD19+ B cells (≥98%). Results presented in B and C are representative of three independent samples.

 
CD19+CD27–IgG+ peripheral B cells express hypermutated IgG with an imprint of Ag selection

To further investigate whether these CD19+CD27–IgG+ B cells corresponded to postgerminal center cells or not, the frequency of somatic mutations in their IgG transcripts was evaluated using sorted populations of CD19+CD27–IgG+ and CD19+CD27+IgG+ B cells. A total of 51 and 26 IgG transcripts was analyzed for CD19+CD27–IgG+ and CD19+CD27+IgG+ B cells, respectively (Table II). Two independent samples were used for each targeted population, CD27– and CD27+IgG+ cells, and showed no difference in their level of mutations (p < 0.01; Student’s t test). CD27+IgG+ and CD27–IgG+ cells presented mutation levels significantly different: 22 ± 8 and 11 ± 9 mutated nucleotides per gene, respectively (p < 0,001; Student’s t test, data not shown). All transcripts from CD27+IgG+ B cells showed somatic mutations, with an average frequency of 6.9 ± 2.5%, which is in accordance with our previous observations (25) and the reported 4–12% mutation frequency in peripheral memory B cells (40, 41). Even though CD27–IgG+ subset also showed up to 10.6% mutation frequency, a fraction of these transcripts, 13.7% (7 over 51), showed none or only 1 mutated nucleotide per gene. Excluding these seven unmutated transcripts, the average mutation frequency, 4.1 ± 2.6% instead of 3.6 ± 2.8% for all transcripts, was lower than that of CD27+IgG+ cells. The distribution of mutation frequency in these two subsets further highlighted this difference (Fig. 2A). The proportion of transcripts showing 0–10 mutations was significantly higher in CD27–IgG+ cells (57%) than in CD27+IgG+ cells (7%). Conversely, the proportion of transcripts showing 21–30 mutations was significantly lower in CD27–IgG+ cells (8%) than in CD27+IgG+ cells (42%).


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  		Table II. CD19+CD27–IgG+ blood B cells express hypermutated IgG

 

Figure 2
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  		FIGURE 2. CD27–IgG+ B cells present a lower mutation load than CD27+IgG+ memory cells with an imprint of Ag selection. A, All IgG transcripts (Table II) were sequenced and classified according to the number of mutated nucleotides per gene for each population. * and **, Significant difference between the two populations, with p = 0.00005 and p = 0.0006, respectively, using two-tailed Fisher exact tests. B, The mean frequency of S and R mutations, identified in amino acid sequences of FR1, FR2, and FR3, and CDR1 and CDR2, is presented. Each region was delimited according to IMGT standard. Two-tailed unpaired Student’s t tests were used to compare mutation frequency from CDR and FR regions. *, **, and ***, Significant difference between CDR1 and FR1 (p < 0.01), CDR2 and FR1, FR2, or FR3 (p < 0.001), and CDR1 and FR1 or FR2 (p < 0.001), respectively.

 
Because Ag-selected Ig sequences tend to have a higher frequency of mutations leading to the replacement of amino acids in the CDR, the distribution of replacement vs silent mutations among the FR1, FR2, and FR3 and CDR1 and CDR2 regions of IgG variable regions was compared in both subsets (Fig. 2B). In both populations, the mean frequency of S and R mutations found in FR1, FR2, and FR3 and in CDR1 and CDR2 was similar (Fig. 2B). As expected, the frequency of R mutations was higher in CDR than FR. The frequency of R mutations in CDR2 was significantly higher than that of FR1, FR2, and FR3 in both subsets (p < 0.001; Student’s t test). The R:S ratios for CD27+IgG+ and CD27–IgG+ cells were respectively, 0.8 and 1.8 in FR1, 2.6 and 4.5 in CDR1, 1.9 for both in FR2, 3.9 and 4.5 in CDR2, and 2.0 and 1.8 in FR3. Higher R:S ratios in CDR compared with FR were considered as an indicator of Ag selection, as expected for postgerminal center cells (36, 37, 38, 39). Overall, CD27–IgG+ cells conducted lower mutation frequency in their transcripts but showed antigenic selection characteristics similar to that of CD27+IgG+ cells.

CD19+CD27–IgG+ B cells and CD19+CD27+IgG+ memory B cells show comparable phenotype and morphology

Flow cytometry analysis was used to further characterize the blood CD19+CD27–IgG+ cells in comparison to CD19+CD27+IgG+ cells. Expression of CD5, CD38, CD40, CD70, CD126, CD138, and B220 was monitored among the B cell populations delineated with CD19, CD27, and IgG using quadruple staining. Analyses were done using PBMC prepared from samples obtained from healthy individuals (Table III). The proportion of CD19+ B cells expressing CD5, CD38, CD40, CD70, CD126, CD138, and B220 corroborated previous reports (42, 43, 44, 45, 46). Both CD19+CD27–IgG+ and CD19+CD27+IgG+ cells expressed high levels of CD40 while ~25% of each subset expressed similar level of CD38. The frequency of CD38- and CD40-positive cells was corroborated using purified CD19+ B cells (data not shown). In addition, the marginal zone B cell marker CD1c (23) was not detected in those two populations (data not shown). Although we cannot exclude the possibility of a rapid CD27 modulation or transient CD27 expression (47), CD27 protein was not detected in CD27–IgG+ cells using intracellular staining for flow cytometry analysis (data not shown). As for the CD27+ counterpart, negligible expression of CD5 indicates that the CD19+CD27–IgG+ cells do not belong to B1a cell subset (48).


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  		Table III. CD19+CD27–IgG+ and CD19+CD27+IgG+ blood B cells show comparable phenotype

 
To confirm that the CD27–IgG+ B cells were not CD27–IgD+IgM+ naive B cells that have bound IgG via Fc{gamma}R (49, 50, 51), IgD and IgM expression was verified on purified B cells using quadruple staining (Fig. 3A). Both CD19+CD27–IgG+ and CD19+CD27+IgG+ cells were negative at ≥95% for IgD and IgM expression.


Figure 3
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  		FIGURE 3. Blood CD27–IgG+ and CD27+IgG+ B cells are similarly negative for IgD, IgM, and CD138 expression while differently expressing B220. In each assay, a total of 150,000–200,000 events was analyzed by flow cytometry. A, Purified blood CD19+ B cells were used to delineate CD19+CD27–IgG+ and CD19+CD27+IgG+ cells, as done in Fig. 1C, and IgD and IgM expression was evaluated using quadruple staining. These results are representative of three independent samples. B, PBMC were used to delineate CD19+CD27–IgG+ and CD19+CD27+IgG+ cells, as done in Fig. 1A, and CD138 and B220 expression was evaluated using quadruple staining. These profiles are representative of nine and five independent samples for CD138 and B220 analysis, respectively (Table III).

 
A fraction of peripheral CD19+CD27+IgG+ and CD19+CD27–IgG+ cells, 8.6 ± 5.0 and 9.8 ± 6.8%, respectively, expressed CD138 (Table III). Taken together, these cells represented <1% of the blood CD19+ cells and expressed a low level of CD138 (MFI < 40; Fig. 3B), in comparison to that expected for terminally differentiated blood plasma cells (MFI > 300) (52). These CD138low cells may thus represent IgG+ cells that are in transition toward plasma cell differentiation.

CD27– naive B cells are considered to express B220 (>95%), whereas ~40 ± 10% of isotype-switched CD27+ cells can be B220+ (42, 53). We observed that 24 ± 14% of CD19+CD27+IgG+ and 50 ± 23% of CD19+CD27–IgG+ cells expressed B220 (Table III and Fig. 3B). However, this 2-fold difference in B220 expression was not statistically significant (p < 0.05, Student’s t test), thus reinforcing the similarity between IgG+CD27– and bona fide IgG+CD27+ memory cells.

Significant differences (p < 0.05, Student’s t test) were observed only for the CD126 expression, the {alpha}-subunit of IL-6R (54). Two-fold less CD19+CD27–IgG+ cells were expressing CD126 compared with CD19+CD27+IgG+ cells. These results were confirmed using purified B cells prepared from five independent samples (Fig. 4A). This lower CD126 expression suggests that IL-6 stimulation could be distinct in these populations (55).


Figure 4
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  		FIGURE 4. Expression of CD126 is higher in CD27+IgG+ compared with CD27–IgG+ B cells but both populations are negative for ABC B1 transporter. A, CD126 expression was evaluated using quadruple staining in CD27+, CD27–, CD27–IgG+, and CD27+IgG+ populations using purified B cells (>98% CD19+). B, After MTG incubation, PBMC were stained for CD19, CD27, MTG, and IgG expression, and CD19+IgG+ cells were gated (R3) and analyzed for CD27 expression and MTG content. Gated events (200,000) were analyzed in A and B. Representative profiles of four and three independent samples are shown for A and B, respectively.

 
Additionally, based on a recent report (29), we also observed that both CD27+IgG+ and CD27–IgG+ cells were negative for ABC transporter activity (Fig. 4B). As reported, ~75% of CD27– cells were able to expel MTG (MFI 156) while >90% of CD27+ cells were not (MFI 1230). This new physiological marker is considered to delineate more accurately naive vs memory B cells (29) and therefore indicates that CD27–IgG+ cells are memory B cells.

Finally, we investigated the morphology of sorted CD19+CD27–IgG+, CD19+CD27+IgG+, and CD19+CD27–IgG– B cells using Wright-Giemsa staining (Fig. 5). Memory B cells are expected to be larger than CD27– naive cells, with more abundant cytoplasm (7, 56, 57). Indeed, the CD19+CD27–IgG+ cells showed morphology similar to that of CD27+ memory B cells, according to cellular size and cytoplasm abundance. Overall, phenotype and morphology of CD19+CD27–IgG+ B cells indicate that these cells belong to the memory compartment.


Figure 5
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  		FIGURE 5. CD27–IgG+ and CD27+IgG+ B cells are morphologically similar. Blood B cells were sorted according to CD27 and IgG expression and stained using Wright-Giemsa stain. A, CD19+CD27–IgG– naive cells, CD19+CD27+IgG+ memory cells (B), and CD19+CD27–IgG+ cells (C) are presented, next to a 25-µm bar reference (magnification, x400). These results are representative of two independent samples. Cell purity was >95% in all samples.

 
IgG2 and IgG3 are differentially represented in CD19+CD27–IgG+ and CD19+CD27+IgG+ B cells

To further compare CD19+CD27–IgG+ and CD19+CD27+IgG+ B cell populations, we have analyzed the distribution of IgG1, IgG2, IgG3, and IgG4 mRNA in two independent samples (Fig. 6). RT-PCR amplification was done on sorted cells from two samples using beta-actin as control (Fig. 6A). Both CD19+CD27–IgG+ and CD19+CD27+IgG+ B cells expressed all IgG subclasses (Fig. 6B). Densitometry was used to estimate the relative expression level of each IgG subclass. The relative expression levels of IgG1 and IgG4 were similar in both populations, whereas IgG2 gene expression was ~4-fold higher in CD27+IgG+ B cells, and IgG3 gene expression was ~10-fold higher in CD27–IgG+ B cells. The relative expressions of IgG1, IgG2, and IgG3 in these two populations were corroborated by the analysis of the cloned transcripts presented in Table II and in Fig. 2 (Fig. 6C). No IgG4 transcripts have been cloned, probably due to their low expression level (58), but a comparable fraction of IgG1 transcripts was observed in both populations. A significant predominance of IgG2 transcripts was observed in CD27+IgG+ over CD27–IgG+ B cells, representing 30.8 vs 3.9%, respectively (p = 0.002, Fisher exact test). IgG3 transcripts were predominant in CD27–IgG+ over CD27+IgG+ B cells, representing, respectively, 31.4 vs 7.7% (p = 0.02, Fisher exact test). Finally, the distribution of cells positive for surface expression of IgG2 and IgG3 was reverse in CD27–IgG+ vs CD27+IgG+ when analyzing purified B cells by flow cytometry. Indeed, a proportion of 28.4 ± 10.7% of the CD27–IgG+ cells expressed IgG3 compared with 16.4 ± 5.8% within the CD27+IgG+ cells (n = 6; p < 0.05, Student’s t test) (data not shown). Conversely, 12.3 ± 1.2% of CD27+IgG+ cells expressed IgG2 compared with 2.8 ± 1.0% within the CD27–IgG+ cells (n = 3; p < 0.05, Student’s t test) (data not shown). In both subsets, the proportion of IgG3+ cells was higher than IgG2+ cells. Overall, the significant difference in IgG subclass distribution, in accordance to a recent report (29), suggests that CD27+IgG+ and CD27–IgG+ B cells could play distinct roles during secondary immune responses.


Figure 6
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  		FIGURE 6. IgG2 and IgG3 are differentially expressed in CD27–IgG+ and CD27+IgG+ cells. A, IgG1, IgG2, IgG3, IgG4, and beta-actin mRNA were amplified by RT-PCR using total RNA from sorted CD27+IgG+ and CD27–IgG+ B cells, prepared from two independent samples (1 and 2), and from CD40-activated B cells as control. Amplicons of 327, 312, 327, 312, and 540 bp were obtained for IgG1, IgG2, IgG3, IgG4, and beta-actin, respectively. One kilobase Plus DNA ladder m.w. markers were used in lanes 1 and 7. B, Relative intensity of gene expression was determined by densitometry for {gamma} 1, 2, 3, and 4 using beta-actin as a reference, as described in Materials and Methods. C, Subclasses of each IgG transcripts from CD27–IgG+ and CD27+IgG+ cells, respectively, 51 and 26 (Table II), were determined as described in Materials and Methods. * and **, Significant difference between paired observations, with p = 0.002 and p = 0.02, respectively, using two-tailed Fisher exact tests.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Until recently, the presence of CD27–IgG+ B cells showing mutated Ig genes was supported only by observations done within spleen (59, 60) and tonsils (7, 50, 61). For instance in humans, CD27–IgG+ B cells are found in the spleen (59, 60), interfollicular CD27– B cells with somatically mutated Ig genes are detected in lymph nodes (61), and CD27–IgG+ B cells expressing mutated genes and the FcR homolog 4 (FcRH4) are reported in tonsils (50). More recently, a minor population of IgG+ B cells, representing ~4% of CD27– B cells, was observed in human blood (29, 62). We observed a similar frequency of CD27–IgG+ cells within the peripheral B cells (1–4%) and the CD19+CD27– cells (~5%) (29, 62). The fact that these CD27–IgG+ cells were not reported before by many exhaustive studies on human B cell repertoire could be related to their low frequency in PBMC or to the use of IgD to delineate B cell populations (4, 15, 56). Besides, even though the data are not shown in the two recent studies (29, 62), the frequency of CD27–IgG+ cells over IgG+ blood cells can be estimated to a range of 10–16%. We observed that 16–33% of IgG+ cells were CD27– in 15 samples of PBMC (Table I) and also in 6 purified B cell samples (Figs. 1 and 4) following the analysis of ≥150,000 events in both cases. This variation between their observations and ours could result from the parameters used to analyze the IgG+ cells or from the methods used to purify B cells, which was negative selection in our case and positive selection of CD19+ cells in the two others. Despite that, we have all consistently observed a significant population of CD27–IgG+ B cells in human blood. Moreover, we corroborated that the CD27–IgG+ B cells were negative for ABC transporter activity and showed a bias for IgG3 expression at both mRNA and protein levels, as reported by Wirths and Lanzavecchia (29).

The FcRH4+CD27– B cells represent a distinctive tissue-based memory population that is rarely observed in blood (50). These cells are mainly IgG+ and show a mutation frequency of 4.5 ± 3.3% in their VH3 Ig gene regions. Similarities between these FcRH4+CD27– cells and the CD27–IgG+ cells described herein suggest a possible relation between these two and thus justify further investigations.

Furthermore, in reference to the aforementioned studies (29, 50, 62), we have also investigated in more detail the phenotype, morphology and VH repertoire of this blood CD27–IgG+ human B cell population. Overall, the CD27–IgG+ cells, expressing mutated Ig and showing imprints of antigenic selection, were morphologically and phenotypically similar to the blood CD27+IgG+ memory cells, which originated from germinal center (11, 63). However, differences were observed in mutation frequency, IgG subclasses distribution, proportion of CD126+ cells and, obviously, CD27 expression, suggesting a distinct origin or role for these two populations during immune responses.

First, the mutation frequency in IgG VH genes expressed by the peripheral CD27–IgG+ cells when only mutated transcripts are analyzed (4.1 ± 2.6%) appears closer to that of CD27+IgD+IgM+ memory B cells from periphery (2–5%) (1, 16, 18, 41, 57) or splenic marginal zone (2–4%) (16, 64, 65) than to germinal center IgG+ cells (4–12.6%) (40, 41, 61). However, marginal zone B cells express CD27 and CD1c (23), which are not expressed by the CD27–IgG+ cells. On the other hand, many studies have investigated the gene expression profiles of germinal center B cell subsets (66, 67, 68, 69). In mice, CD27 is not considered as a memory B cell marker (11) being expressed for short period of time on a limited proportion of cells only (69). Rather, CD27 appears following recent B cell activation and is not absolutely necessary for secondary responses (26). In humans, the requirement for CD27 expression on germinal center memory cells is not yet well characterized, mainly because the genes regulated by its stimulation remain to be defined and because no human disease related to CD27 dysfunction has been reported (24, 69). However, in vitro studies suggested that hypermutation, isotype switching, and CD27 expression can occur independently in germinal center (25, 70). Therefore, blood CD27–IgG+ B cells could reasonably be related to germinal center because of their switched Ig showing mutated VH genes with antigenic selection characteristics.

Approximately 14% of the IgG+CD27– B cells showed unmutated IgG VH gene (7 over 51 transcripts) with a distribution of IgG1 (3 of 7), IgG2 (1 of 7), and IgG3 (3 of 7) similar to that observed for the mutated IgG+CD27– B cells (data not shown and Fig. 6). Interestingly, peripheral IgD+IgM+CD27–, as well as IgD+IgM+CD27+ cells driven to switch in vitro, showed a similar pattern of IgG subclass expression, with higher proportion of IgG3 over IgG2 (71). Such switched human B cells carrying unmutated VH genes could originate from a rapid T cell-independent response as reported elsewhere (72, 73, 74). Additionally, the low frequency of somatic mutation in the remaining CD27–IgG+ cells suggests that this population could emerge independently from T cell help or from CD40-CD154 interaction in humans (17). In fact, the recent finding that IgG+ memory B cells can be generated following T cell independent responses in mice (75) suggests the existence of such cells in humans.

In addition, regardless of CD27 expression, the low frequency of somatic mutation in CD27–IgG+ cells suggests that these cells could represent a first wave of memory B cells as recently proposed (76, 77). Based on mouse models, these studies report the rapid emergence of isotype-switched B cells early after primary immunization. Interestingly, the population of blood (4-hydroxy-3-nitrophenyl)acetyl specific B cells expressing VH genes with low mutation frequency seems tightly regulated (76), suggesting that the CD27–IgG+ human blood B cells could represent, as proposed elsewhere (78), a pool of short-lived memory B cells, while the CD27+IgG+ B cells could be more related to long-lived memory B cells.

In humans, IgG2 are mainly reactive to polysaccharides while IgG3 react with proteins and are more potent than IgG2 in classical complement activation and Ab-dependent cell-mediated cytotoxicity (79). Therefore, the differences between IgG2 and IgG3 expression within CD27+IgG+ and CD27–IgG+ cells further suggest that these cells could be distinct in relation to the nature of the Ags and the outcome of the immunological response, as already proposed for the rotavirus-specific B cell response (29, 80).

CD27 can be triggered by its ligand, CD70, which is expressed on activated B and T cells (69, 81, 82). In human B cells, CD27 triggering promotes mainly differentiation into plasma cells (69). However, interaction between CD27 and CD70 generates negative feedback signal to leukocytes during immune activation (83) and is also involved in T cell homeostasis (84, 85). Disrupted interaction through CD27 and CD70 also correlates with the progression of multiple myeloma and the loss of CD27 expression (86). Therefore, CD27–IgG+ cells, in opposition to CD27+IgG+ cells, are essentially disconnected from any further regulation through CD70+ T or B cells (69, 81, 82), supporting once more their distinct roles in immune response.

We have noted that CD27–IgG+ blood cell characteristics were close to that of hairy cell leukemia cells. These cells are indeed characterized by the lack of CD27 expression (27, 28, 87), the presence of B220 (53), and expression of switched Ig with a dominance in the IgG3 subclass and somatically mutated Ig genes (27, 28, 88, 89, 90, 91), with frequency of mutations ranging from 0 to 5% (28, 88, 89, 90). Interestingly, these cells show heterogeneous mutation frequency, with 6 cases on 32 without mutation (90), which also characterizes CD27–IgG+ cells. Approximately 40% of hairy cell leukemia cases coexpress multiple, clonally related Ig isotypes (reviewed in Ref. 92), which was not observed in the CD27–IgG+ population. Nevertheless, as suggested by Forconi’s work (27, 28), the malignant change could occur during the generation of CD27–IgG+ cells before the deletional recombination events characterizing normal isotype switching, which would allow the expression of several Ig isotypes by a single cell.

Although the exact origin of hairy cell leukemia cells, whether derived from marginal zone, germinal center (28, 89, 90, 92, 93, 94), or T cell-independent reaction (95), is still unclear, this report of a new CD27–IgG+ blood B cell subset showing similar characteristics could help in future investigations. Furthermore, the possible link with a first wave of early memory B cells could also be of interest in a better understanding of hairy cell leukemia

Overall, the characterization of a CD19+CD27–IgG+ population brings a new look in the organization and homeostasis of peripheral B cells in humans, not only by extending the memory B cell reservoir beyond the CD27+ compartment, but also by questioning the role of the lack of interaction with CD70 in B cell homeostasis. We are currently investigating whether this minor CD27–IgG+ B cell population can be stimulated in vitro to express CD27 and to respond to variable level of CD40 stimulation.


   Acknowledgments
 
We thank Drs. André Darveau, Daniel Jung, and Marguerite Massinga-Loembé for helpful discussions and suggestions and Gerry Boucher for his help in sequence analysis. We are thankful to Annie Roy and Claudia Racine for skillful assistance in cell culture. We are also grateful to Jean-François Leblanc for editing the manuscript. Finally, we are thankful to all participants to this study and to Claudine Côté for the coordination of blood sample collection.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 J.F.F. was supported by a Ph.D. fellowship from le Fond Québécois de la Recherche sur la Nature et les Technologies and from Héma-Québec. Back

2 Address correspondence and reprint requests to Dr. Sonia Néron, Héma-Québec, Recherche et Développement, 1009 route du Vallon, Sainte-Foy, Quebec G1V 5C3, Canada. E-mail address: sonia.neron{at}hema-quebec.qc.ca Back

3 Abbreviations used in this paper: ABC, ATP-binding cassette; MTG, MitoTracker Green; IMGT, ImMunoGeneTics; FR, framework region; R, replacement; S, silent; MFI, mean fluorescence intensity; FcRH4, FcR homolog 4. Back

Received for publication February 7, 2006. Accepted for publication June 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Klein, U., T. Goossens, M. Fischer, H. Kanzler, A. Braeuninger, K. Rajewsky, R. Kuppers. 1998. Somatic hypermutation in normal and transformed human B cells. Immunol. Rev. 162: 261-280. [Medline]
  2. Black, S. J., W. van der Loo, M. R. Loken, L. A. Herzenberg. 1978. Expression of IgD by murine lymphocytes: loss of surface IgD indicates maturation of memory B cells. J. Exp. Med. 147: 984-996. [Abstract/Free Full Text]
  3. Hayakawa, K., R. Ishii, K. Yamasaki, T. Kishimoto, R. R. Hardy. 1987. Isolation of high-affinity memory B cells: phycoerythrin as a probe for antigen-binding cells. Proc. Natl. Acad. Sci. USA 84: 1379-1383. [Abstract/Free Full Text]
  4. Klein, U., K. Rajewsky, R. Kuppers. 1998. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J. Exp. Med. 188: 1679-1689. [Abstract/Free Full Text]
  5. Kuppers, R., U. Klein, M. L. Hansmann, K. Rajewsky. 1999. Cellular origin of human B cell lymphomas. N. Engl. J. Med. 341: 1520-1529. [Free Full Text]
  6. Goodwin, R. G., M. R. Alderson, C. A. Smith, R. J. Armitage, T. VandenBos, R. Jerzy, T. W. Tough, M. A. Schoenborn, T. Davis-Smith, K. Hennen. 1993. Molecular and biological characterization of a ligand for CD27 defines a new family of cytokines with homology to tumor necrosis factor. Cell 73: 447-456. [Medline]
  7. Agematsu, K., S. Hokibara, H. Nagumo, A. Komiyama. 2000. CD27: a memory B cell marker. Immunol. Today 21: 204-206. [Medline]
  8. Tangye, S. G., Y. J. Liu, G. Aversa, J. H. Phillips, J. E. de Vries. 1998. Identification of functional human splenic memory B cells by expression of CD148 and CD27. J. Exp. Med. 188: 1691-1703. [Abstract/Free Full Text]
  9. Zandvoort, A., W. Timens. 2002. The dual function of the splenic marginal zone: essential for initiation of anti-TI-2 responses but also vital in the general first-line defense against blood-borne antigens. Clin. Exp. Immunol. 130: 4-11. [Medline]
  10. Gourley, T. S., E. J. Wherry, D. Masopust, R. Ahmed. 2004. Generation and maintenance of immunological memory. Semin. Immunol. 12: 323-333.
  11. Wolniak, K. L., S. M. Shinall, T. J. Waldschmidt. 2004. The germinal center response. Crit. Rev. Immunol. 24: 39-65. [Medline]
  12. van Kooten, C., J. Banchereau. 2000. CD40-CD40 ligand. J. Leukocyte Biol. 67: 2-17. [Abstract]
  13. Grewal, I. S., R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16: 111-135. [Medline]
  14. Lougaris, V., R. Badolato, S. Ferrari, A. Plebani. 2005. Hyper immunoglobulin M syndrome due to CD40 deficiency: clinical, molecular, and immunological features. Immunol. Rev. 203: 48-66. [Medline]
  15. Agematsu, K., H. Nagumo, K. Shinozaki, S. Hokibara, K. Yasui, K. Terada, N. Kawamura, T. Toba, S. Nonoyama, H. D. Ochs, A. Komiyama. 1998. Absence of IgD–CD27+ memory B cell population in X-linked hyper-IgM syndrome. J. Clin. Invest. 102: 853-860. [Medline]
  16. Weller, S., M. C. Braun, B. K. Tan, A. Rosenwald, C. Cordier, M. E. Conley, A. Plebani, D. S. Kumararatne, D. Bonnet, O. Tournilhac, et al 2004. Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood 104: 3647-3654. [Abstract/Free Full Text]
  17. Weller, S., A. Faili, C. Garcia, M. C. Braun, F. Le Deist, G. de Saint Basileqq, O. Hermine, A. Fischer, C. A. Reynaud, J. C. Weill. 2001. CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proc. Natl. Acad. Sci. USA 98: 1166-1170. [Abstract/Free Full Text]
  18. Weller, S., A. Faili, S. Aoufouchi, Q. Gueranger, M. Braun, C. A. Reynaud, J. C. Weill. 2003. Hypermutation in human B cells in vivo and in vitro. Ann. NY Acad. Sci. 987: 158-165. [Medline]
  19. Kruetzmann, S., M. M. Rosado, H. Weber, U. Germing, O. Tournilhac, H. H. Peter, R. Berner, A. Peters, T. Boehm, A. Plebani, et al 2003. Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. J. Exp. Med. 197: 939-945. [Abstract/Free Full Text]
  20. Spencer, J., M. E. Perry, D. K. Dunn-Walters. 1998. Human marginal-zone B cells. Immunol. Today 19: 421-426. [Medline]
  21. Weller, S., C. A. Reynaud, J. C. Weill. 2005. Vaccination against encapsulated bacteria in humans: paradoxes. Trends Immunol. 26: 85-89. [Medline]
  22. Pillai, S., A. Cariappa, S. T. Moran. 2004. Positive selection and lineage commitment during peripheral B lymphocyte development. Immunol. Rev. 197: 206-218. [Medline]
  23. Pillai, S., A. Cariappa, S. T. Moran. 2005. Marginal zone B cells. Annu. Rev. Immunol. 23: 161-196. [Medline]
  24. Tangye, S. G., P. D. Hodgkin. 2004. Divide and conquer: the importance of cell division in regulating B cell responses. Immunol. 112: 509-520. [Medline]
  25. Fecteau, J. F., S. Néron. 2003. CD40 stimulation of human peripheral B lymphocytes: distinct response from naïve and memory cells. J. Immunol. 171: 4621-4629. [Abstract/Free Full Text]
  26. Xiao, Y., J. Hendriks, P. Langerak, H. Jacobs, J. Borst. 2004. CD27 is acquired by primed B cells at the centroblast stage and promotes germinal center formation. J. Immunol. 172: 7432-7441. [Abstract/Free Full Text]
  27. Forconi, F., S. S. Sahota, D. Raspadori, C. I. Mockridge, F. Lauria, F. K. Stevenson. 2001. Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing. Blood 98: 1174-1181. [Abstract/Free Full Text]
  28. Forconi, F., S. S. Sahota, D. Raspadori, M. Ippoliti, G. Babbage, F. Lauria, F. K. Stevenson. 2004. Hairy cell leukemia: at the crossroad of somatic mutation and isotype switch. Blood 104: 3312-3317. [Abstract/Free Full Text]
  29. Wirths, S., A. Lanzavecchia. 2005. ABCB1 transporter discriminates human resting naive B cells from cycling transitional and memory B cells. Eur. J. Immunol. 35: 3433-3441. [Medline]
  30. Néron, S., C. Racine, A. Roy, M. Guerin. 2005. Differential responses of human B lymphocyte subpopulations to graded levels of CD40-CD154 interaction. Immunol. 116: 454-463. [Medline]
  31. Hamblin, T. J., Z. Davis, A. Gardiner, D. G. Oscier, F. K. Stevenson. 1999. Unmutated Ig VH genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 94: 1848-1854. [Abstract/Free Full Text]
  32. Barbas, I. C. F., D. R. Burton. 1994. Human antibodies from combinational libraries. C. S. Harbor, ed. Advances in Immunology 1st ed.191-280. Cold Spring Harbor Laboratory Press, Woodbury, New York.
  33. Barbas, I. C. F., D. R. Burton, J. K. Scott, G.J. Silverman. 2001. Appendix 1: human primers for Falo amplification, original set. C. S. Harbor, ed. Phage Display: A Laboratory Manual 1st ed.A1.6 Cold Spring Harbor Laboratory Press, New York.
  34. Maniatis, T., E. F. Fritsch, J. Sambrook. 1989. Extraction and purification of plasmid DNA. C. Nolan, ed. Molecular Cloning: A Laboratory Manual 2nd ed.1.21-1.32. Cold Spring Harbor Laboratory Press, New York.
  35. Giudicelli, V., D. Chaume, M. P. Lefranc. 2004. IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res. 32: W435-W440. [Abstract/Free Full Text]
  36. Chang, B., P. Casali. 1994. The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement. Immunol. Today 15: 367-373. [Medline]
  37. Lossos, I. S., R. Tibshirani, B. Narasimhan, R. Levy. 2000. The inference of antigen selection on Ig genes. J. Immunol. 165: 5122-5126. [Abstract/Free Full Text]
  38. Messmer, B. T., E. Albesiano, D. Messmer, N. Chiorazzi. 2004. The pattern and distribution of immunoglobulin VH gene mutations in chronic lymphocytic leukemia B cells are consistent with the canonical somatic hypermutation process. Blood 103: 3490-3495. [Abstract/Free Full Text]
  39. Bose, B., S. Sinha. 2005. Problems in using statistical analysis of replacement and silent mutations in antibody genes for determining antigen-driven affinity selection. Immunology 116: 172-183. [Medline]
  40. Klein, U., R. Kuppers, K. Rajewsky. 1997. Evidence for a large compartment of IgM-expressing memory B cells in humans. Blood 89: 1288-1298. [Abstract/Free Full Text]
  41. Chong, Y., H. Ikematsu, K. Yamaji, M. Nishimura, S. Kashiwagi, J. Hayashi. 2003. Age-related accumulation of Ig V-H gene somatic mutations in peripheral B cells from aged humans. Clin. Exp. Immunol. 133: 59-66. [Medline]
  42. Bleesing, J. J. H., T. A. Fleisher. 2003. Human B cells express a CD45 isoform that is similar to murine B220 and is downregulated with acquisition of the memory B cell marker CD27. Cytometry 51B: 1-8.
  43. Deneys, V., A. M. Mazzon, J. L. Marques, H. Benoit, M. De Bruyere. 2001. Reference values for peripheral blood B-lymphocyte subpopulations: a basis for multiparametric immunophenotyping of abnormal lymphocytes. J. Immunol. Methods 253: 23-36. [Medline]
  44. Lens, S. M., R. de Jong, B. Hooibrink, G. Koopman, S. T. Pals, M. H. van Oers, R. A. van Lier. 1996. Phenotype and function of human B cells expressing CD70 (CD27 ligand). Eur. J. Immunol. 26: 2964-2971. [Medline]
  45. Jego, G., N. Robillard, D. Puthier, M. Amiot, F. Accard, D. Pineau, J. L. Harousseau, R. Bataille, C. Pellat-Deceunynck. 1999. Reactive plasmacytoses are expansions of plasmablasts retaining the capacity to differentiate into plasma cells. Blood 94: 701-712. [Abstract/Free Full Text]
  46. van der Meijden, M., J. Gage, E. C. Breen, T. Taga, T. Kishimoto, O. Martinez-Maza. 1998. IL-6 receptor (CD126'IL-6R') expression is increased on monocytes and B lymphocytes in HIV infection. Cell. Immunol. 190: 156-166. [Medline]
  47. Loenen, W. A., E. de Vries, L. A. Gravestein, R. Q. Hintzen, R. A. Van Lier, J. Borst. 1992. The CD27 membrane receptor, a lymphocyte-specific member of the nerve growth factor receptor family, gives rise to a soluble form by protein processing that does not involve receptor endocytosis. Eur. J. Immunol. 22: 447-455. [Medline]
  48. Fischer, M., U. Klein, R. Kuppers. 1997. Molecular single-cell analysis reveals that CD5-positive peripheral blood B cells in healthy humans are characterized by rearranged V{kappa} genes lacking somatic mutation. J. Clin. Invest. 100: 1667-1676. [Medline]
  49. Heyman, B.. 2000. Regulation of antibody responses via antibodies, complement, and Fc receptors. Annu. Rev. Immunol. 18: 709-738. [Medline]
  50. Ehrhardt, G. R., J. T. Hsu, L. Gartland, C. M. Leu, S. Zhang, R. S. Davis, M. D. Cooper. 2005. Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells. J. Exp. Med. 202: 783-791. [Abstract/Free Full Text]
  51. Davis, R. S., G. Dennis, Jr, H. Kubagawa, M. D. Cooper. 2002. Fc receptor homologs (FcRH1–5) extend the Fc receptor family. Curr. Top. Microbiol. Immunol. 266: 85-112. [Medline]
  52. Medina, F., C. Segundo, A. Campos-Caro, I. Gonzalez-Garcia, J. A. Brieva. 2002. The heterogeneity shown by human plasma cells from tonsil, blood, and bone marrow reveals graded stages of increasing maturity, but local profiles of adhesion molecule expression. Blood 99: 2154-2161.
  53. Rodig, S. J., M. Shahsafaei, B. Li, D. M. Dorfman. 2005. The CD45 isoform B220 identifies select subsets of human B cells and B cell lymphoproliferative disorders. Hum. Pathol. 36: 51-57. [Medline]
  54. Kishimoto, T., S. Akira, M. Narazaki, T. Taga. 1995. Interleukin-6 family of cytokines and gp130. Blood 86: 1243-1254. [Free Full Text]
  55. Breen, E. C., J. R. Gage, B. C. Guo, L. Magpantay, M. Narazaki, T. Kishimoto, S. Miles, O. Martinez-Maza. 2001. Viral interleukin 6 stimulates human peripheral blood B cells that are unresponsive to human interleukin 6. Cell. Immunol. 212: 118-125. [Medline]
  56. Agematsu, K., H. Nagumo, F. C. Yang, T. Nakazawa, K. Fukushima, S. Ito, K. Sugita, T. Mori, T. Kobata, C. Morimoto, A. Komiyama. 1997. B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production. Eur. J. Immunol. 27: 2073-2079. [Medline]
  57. Shi, Y., K. Agematsu, H. D. Ochs, K. Sugane. 2003. Functional analysis of human memory B cell subpopulations: IgD+CD27+ B cells are crucial in secondary immune response by producing high affinity IgM. Clin. Immunol. 108: 128-137. [Medline]
  58. Kitani, A., W. Strober. 1993. Regulation of C{gamma} subclass germline transcripts in human peripheral blood B cells. J. Immunol. 151: 3478-3488. [Abstract]
  59. Tangye, S. G., B. C. M. van de Weerdt, D. T. Avery, P. D. Hodgkin. 2002. CD84 is up-regulated on a major population of human memory B cells and recruits the SH2 domain containing proteins SAP and EAT-2. Eur. J. Immunol. 32: 1640-1649. [Medline]
  60. Pene, J., J. F. Gauchat, S. Lecart, E. Drouet, P. Guglielmi, V. Boulay, A. Delwail, D. Foster, J. C. Lecron, H. Yssel. 2004. IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells. J. Immunol. 172: 5154-5157. [Abstract/Free Full Text]
  61. Marafioti, T., M. Jones, F. Facchetti, T. C. Diss, M. Q. Du, P. G. Isaacson, M. Pozzobon, S. A. Pileri, A. J. Strickson, S. Y. Tan, et al 2003. Phenotype and genotype of interfollicular large B cells, a subpopulation of lymphocytes often with dendritic morphology. Blood 102: 2868-2876. [Abstract/Free Full Text]
  62. Ma, C. S., S. Pittaluga, D. T. Avery, N. J. Hare, I. Maric, A. D. Klion, K. E. Nichols, S. G. Tangye. 2006. Selective generation of functional somatically mutated IgM+CD27+, but not Ig isotype-switched, memory B cells in X-linked lymphoproliferative disease. J. Clin. Invest. 116: 322-333. [Medline]
  63. Manser, T.. 2004. Textbook germinal centers?. J. Immunol. 172: 3369-3375. [Abstract/Free Full Text]
  64. Dunn-Walters, D. K., P. G. Isaacson, J. Spencer. 1995. Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells. J. Exp. Med. 182: 559-566. [Abstract/Free Full Text]
  65. Tierens, A., J. Delabie, L. Michiels, P. Vandenberghe, C. De Wolf-Peeters. 1999. Marginal-zone B cells in the human lymph node and spleen show somatic hypermutations and display clonal expansion. Blood 93: 226-234. [Abstract/Free Full Text]
  66. Shaffer, A. L., A. Rosenwald, E. M. Hurt, J. M. Giltnane, L. T. Lam, O. K. Pickeral, L. M. Staudt. 2001. Signatures of the immune response. Immunity 15: 375-385. [Medline]
  67. Feldhahn, N., I. Schwering, S. Lee, M. Wartenberg, F. Klein, H. Wang, G. L. Zhou, S. M. Wang, J. D. Rowley, J. Hescheler, et al 2002. Silencing of B cell receptor signals in human naive B cells. J. Exp. Med. 196: 1291-1305. [Abstract/Free Full Text]
  68. Klein, U., Y. H. Tu, G. A. Stolovitzky, J. L. Keller, J. Haddad, V. Miljkovic, G. Cattoretti, A. Califano, R. Dalla-Favera. 2003. Transcriptional analysis of the B cell germinal center reaction. Proc. Natl. Acad. Sci. USA 100: 2639-2644. [Abstract/Free Full Text]
  69. Borst, J., J. Hendriks, Y. Xiao. 2005. CD27 and CD70 in T cell and B cell activation. Curr. Opin. Immunol. 17: 275-281. [Medline]
  70. Nagumo, H., K. Agematsu, N. Kobayashi, K. Shinozaki, S. Hokibara, H. Nagase, M. Takamoto, K. Yasui, K. Sugane, A. Komiyama. 2002. The different process of class switching and somatic hypermutation; a novel analysis by (CD27–) naïve B cells. Blood 99: 567-575. [Abstract/Free Full Text]
  71. Werner-Favre, C., F. Bovia, P. Schneider, N. Holler, M. Barnet, V. Kindler, J. Tschopp, R. H. Zubler. 2001. IgG subclass switch capacity is low in switched and in IgM-only, but high in IgD+IgM+, post-germinal center (CD27+) human B cells. Eur. J. Immunol. 31: 243-249. [Medline]
  72. Litinskiy, M. B., B. Nardelli, D. M. Hilbert, B. He, A. Schaffer, P. Casali, A. Cerutti. 2002. DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL. Nat. Immunol. 3: 822-829. [Medline]
  73. He, B., X. Qiao, P. J. Klasse, A. Chiu, A. Chadburn, D. M. Knowles, J. P. Moore, A. Cerutti. 2006. HIV-1 envelope triggers polyclonal Ig class switch recombination through a CD40-independent mechanism involving BAFF and C-type lectin receptors. J. Immunol. 176: 3931-3941. [Abstract/Free Full Text]
  74. He, B., X. Qiao, A. Cerutti. 2004. CpG DNA induces IgG class switch DNA recombination by activating human B cells through an innate pathway that requires TLR9 and cooperates with IL-10. J. Immunol. 173: 4479-4491. [Abstract/Free Full Text]
  75. Obukhanych, T. V., M. C. Nussenzweig. 2006. T-independent type II immune responses generate memory B cells. J. Exp. Med. 203: 305-310. [Abstract/Free Full Text]
  76. Blink, E. J., A. Light, A. Kallies, S. L. Nutt, P. D. Hodgkin, D. M. Tarlinton. 2005. Early appearance of germinal center-derived memory B cells and plasma cells in blood after primary immunization. J. Exp. Med. 201: 545-554. [Abstract/Free Full Text]
  77. Inamine, A., Y. Takahashi, N. Baba, K. Miyake, T. Tokuhisa, T. Takemori, R. Abe. 2005. Two waves of memory B cell generation in the primary immune response. Int. Immunol. 17: 581-589. [Abstract/Free Full Text]
  78. Dorner, T., A. Radbruch. 2005. Selecting B cells and plasma cells to memory. J. Exp. Med. 201: 497-499. [Abstract/Free Full Text]
  79. Bruggemann, M., G. T. Williams, C. I. Bindon, M. R. Clark, M. R. Walker, R. Jefferis, H. Waldmann, M. S. Neuberger. 1987. Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J. Exp. Med. 166: 1351-1361. [Abstract/Free Full Text]
  80. Jaimes, M. C., O. L. Rojas, E. J. Kunkel, N. H. Lazarus, D. Soler, E. C. Butcher, D. Bass, J. Angel, M. A. Franco, H. B. Greenberg. 2004. Maturation and trafficking markers on rotavirus-specific B cells during acute infection and convalescence in children. J. Virol. 78: 10967-10976. [Abstract/Free Full Text]
  81. Agematsu, K., T. Kobata, K. Sugita, T. Hirose, S. F. Schlossman, C. Morimoto. 1995. Direct cellular communications between CD45R0 and CD45RA T cell subsets via CD27/CD70. J. Immunol. 154: 3627-3635. [Abstract]
  82. Lens, S. M., K. Tesselaar, M. H. van Oers, R. A. van Lier. 1998. Control of lymphocyte function through CD27-CD70 interactions. Semin. Immunol. 10: 491-499. [Medline]
  83. Nolte, M. A., R. Arens, R. van Os, M. van Oosterwijk, B. Hooibrink, R. A. van Lier, M. H. van Oers. 2005. Immune activation modulates hematopoiesis through interactions between CD27 and CD70. Nat. Immunol. 6: 412-418. [Medline]
  84. Ochsenbein, A. F., S. R. Riddell, M. Brown, L. Corey, G. M. Baerlocher, P. M. Lansdorp, P. D. Greenberg. 2004. CD27 expression promotes long-term survival of functional effector-memory CD8+ cytotoxic T lymphocytes in HIV-infected patients. J. Exp. Med. 200: 1407-1417. [Abstract/Free Full Text]
  85. Laouar, A., V. Haridas, D. Vargas, Z. N. Xia, D. Chaplin, R. A. W. van Lier, N. Manjunath. 2005. CD70+ antigen-presenting cells control the proliferation and differentiation of T cells in the intestinal mucosa. Nat. Immunol. 6: 698-706. [Medline]
  86. Katayama, Y., A. Sakai, N. Oue, H. Asaoku, T. Otsuki, T. Shiomomura, R. Masuda, N. Hino, Y. Takimoto, F. Imanaka, et al 2003. A possible role for the loss of CD27-CD70 interaction in myelomagenesis. Br. J. Haematol. 120: 223-234. [Medline]
  87. Forconi, F., S. S. Sahota, F. Lauria, F. K. Stevenson. 2004. Revisiting the definition of somatic mutational status in B cell tumors: does 98% homology mean that a V-H-gene is unmutated?. Leukemia 18: 882-883. [Medline]
  88. Maloum, K., C. Magnac, Z. Azgui, C. Cau, F. Charlotte, J. L. Binet, H. Merle-Beral, G. Dighiero. 1998. VH gene expression in hairy cell leukaemia. Br. J. Haematol. 101: 171-178. [Medline]
  89. Vanhentenrijk, V., A. Tierens, I. Wlodarska, G. Verhoef, C. D. Wolf-Peeters. 2004. V-H gene analysis of hairy cell leukemia reveals a homogeneous mutation status and suggests its marginal zone B cell origin. Leukemia 18: 1729-1732. [Medline]
  90. Thorselius, M., S. H. Walsh, U. Thunberg, H. Hagberg, C. Sundstrom, R. Rosenquist. 2005. Heterogeneous somatic hypermutation status confounds the cell of origin in hairy cell leukemia. Leukemia Res. 29: 153-158. [Medline]
  91. Kluin-Nelemans, H. C., M. M. Krouwels, J. H. Jansen, K. Dijkstra, M. J. van Tol, G. J. den Ottolander, E. J. Dreef, P. M. Kluin. 1990. Hairy cell leukemia preferentially expresses the IgG3-subclass. Blood 75: 972-975. [Abstract/Free Full Text]
  92. Tiacci, E., A. Liso, M. Piris, B. Falini. 2006. Evolving concepts in the pathogenesis of hairy-cell leukemia. Nat. Rev. Cancer 6: 437-448. [Medline]
  93. Klein, U., R. Dalla-Favera. 2005. New insights into the phenotype and cell derivation of B cell chronic lymphocytic leukemia. Curr. Top. Microbiol. Immunol. 294: 31-49. [Medline]
  94. Basso, K., A. Liso, E. Tiacci, R. Benedetti, A. Pulsoni, R. Foa, F. Di Raimondo, A. Ambrosetti, A. Califano, U. Klein, et al 2004. Gene expression profiling of hairy cell leukemia reveals a phenotype related to memory B cells with altered expression of chemokine and adhesion receptors. J. Exp. Med. 199: 59-68. [Abstract/Free Full Text]
  95. Burthem, J., M. Zuzel, J. C. Cawley. 1997. What is the nature of the hairy cell and why should we be interested?. Br. J. Haematol. 97: 511-514. [Medline]



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