Leukemia (2009) 23, 2075–2080 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE
Defining origins of malignant B cells: a new circulating normal human IgM þ D þ B-cell subset lacking CD27 expression and displaying somatically mutated IGHV genes as a relevant memory population
N Weston-Bell1, M Townsend1, G Di Genova1, F Forconi2 and SS Sahota1
1Cancer Sciences Division, School of Medicine, University of Southampton, Southampton, UK and 2Sezione Ematologia e Trapianti, Universita` di Siena, Siena, Italy
In probing the cell of origin in malignant B cells, an imprint of In normal B cells, a memory of antigen is imprinted somatic hypermutation (SHM) in immunoglobulin (Ig) variable (V) irreversibly by SHM in Ig V genes during T-cell-dependent region genes delineates antigen encounter, and identifying the encounter through germinal centers (GCs) in secondary lym- precise pathway generating SHM in the normal B-cell counterpart 1 becomes relevant. SHM remains the definitive memory imprint in phoid organs. Isotype class switch recombination can also normal human B cells, but CD27 expression also delineates occur in the GCs by deleting specified constant region coding memory. Recently, dye extrusion adenosine triphosphate-bind- sequences.1 Although both SHM and class switch recombina- ing transporter assays identified circulating isotype-switched tion have generally been regarded as markers of memory B cells, memory B cells that lacked CD27, yet exhibited low levels of SHM. a seminal finding was the identification of CD27 as an To extend findings, we report a pre-switched CD27 ve circulating apparently robust phenotypic marker able to discriminate memory B-cell population in normal blood using comparable 2 assays, and isolated CD19 þ IgM þ D þ CD27 ve cells (499% between human naive and memory B cells. The circulating þ purity) for the analysis of IGHV5/IGHV3-IGHM transcripts. Of ‘classical’ CD27 memory pool in that study was shown to these (n ¼ 334), B78% were germ line and naive B cell derived. comprise mutated IgM þ D þ , IgM þ D ve and IgG þ or IgA þ Strikingly, 21.9% of the transcripts were mutated. They B cells.2 However, SHM can also occur through T-independent showed 3–5 mutations (13.5% of sequences) and 45 mutations mechanisms ectopically at non-GC sites,3 and this may impact (8.4% of sequences) per transcript. Accrual of mutations in a subset of CD19 þ IgM þ D þ CD27 ve cells define a new circulating crucially on deciphering a role for instance, for infection-related pre-switched memory B-cell pool, present in substantial antigens in clonal tumor origins. numbers in the population harboring naive B cells. These In malignant B cells, there are instances when the presence of CD19 þ IgM þ D þ CD27 ve memory B cells may have a distinct SHM is confounding, as it does not correlate with the expected lineage and function, and seem relevant to understanding origins classical memory phenotype. This is illustrated by hairy cell of malignant B cells, in particular those of hairy cell leukemia leukemia (HCL) cells that exhibit mutated IGHV genes but are cells, which display mutated V genes yet lack CD27 expression. CD27 ve.4,5 In addition, in IgM-expressing Waldenstrom’s Leukemia (2009) 23, 2075–2080; doi:10.1038/leu.2009.178; ve published online 24 September 2009 macroglobulinemia, mutated tumor cells are seen in CD27 6,7 Keywords: CD27; normal B-cell memory; somatic hypermutation; fractions, and it has been proposed that origins of this disease hairy cell leukemia may be from ‘unusual’ memory B cells.6 Interestingly, more recent observations indicate that the normal human B-cell memory pool may as yet not be mapped fully. These studies have examined the association of CD27 expression as an absolute requirement in normal human Introduction memory B cells. Pivotal to the studies is an assay based on dye extrusion by the adenosine triphosphate-binding cassette Defining the cell of origin that causes B-cell tumors is a central subfamily B1 (ABCB1) transporter pump, which is functionally question in understanding the pathogenesis of malignant B cells. 8 restricted to naive B cells. When loaded with a suitable dye, Allied to this are questions of a role for antigen in selecting such ABCB1-positive naive B cells extrude the dye and retain a low a cell, what the nature of antigen might be and whether it is þ level of dye fluorescence, whereas CD27 memory B cells are presented in the context of T-cell help or through T-independent 8 pump negative and retain much higher levels of dye. Using this pathways. We have been addressing these questions by focusing þ ve assay, a population of IgG but CD27 B cells could be on the analysis of immunoglobulin (Ig) variable (V) region genes identified as ABCB1-negative in normal human blood lympho- in malignant B cells, as these genes encode the antigen-binding ve þ ve cytes. A subset of this ABCB1 IgG CD27 population was domains of the surface Ig molecule and can reveal important þ þ indeed shown to be functionally comparable with IgG CD27 aspects of clonal history of the cell of origin. The most striking 8 þ ve memory B cells. It is noteworthy that these IgG CD27 cells aspect is whether such a cell has undergone somatic hyper- were not initially examined for V gene mutations. However, two mutation (SHM) in response to antigen, then generating þ ve parallel studies established that the new memory IgG CD27 questions of where SHM may have occurred and in response population indeed displayed SHM, but at a lower level than did to which stimuli. 9,10 classical switched memory B cells. A relevance for these IgG þ CD27 ve cells also emerged in B-cell disease, specifically Correspondence: Dr SS Sahota, Cancer Sciences Division, School of for systemic lupus erythematosus in which they represent a Medicine, Southampton University Hospitals, Tremona Road, South- major circulating compartment.10 ampton SO16 6YD, UK. E-mail: [email protected] As yet, however, it is not known whether a pre-switched ve Received 23 December 2008; revised 3 July 2009; accepted 13 July CD27 component also exists in the normal human circula- 2009; published online 24 September 2009 tion, with a parallel mutational imprint. This is of relevance Human IgM þ D þ memory B cells lacking CD27 N Weston-Bell et al 2076 for understanding the CD27 ve memory B-cell pool more 5 mg/ml (Dako) and streptavidin-perCP-cy5.5 at 2 mg/ml (BD) at fully as well as for defining origins of B cells in malignancy. 4 1C for 30 min. Isotype antibody control labeling was In this study, we report the IGHV mutational status performed as described above. Separation of both ABCB1- of CD19 þ IgM þ D þ CD27 ve cells in normal circulating positive and ABCB1-negative cells was established using the B lymphocytes. ABCB1 transporter inhibitor. As MTG was never entirely extruded from cells, MTG-stained cells were spiked with unstained cells to confirm the correct acquisition voltage. Materials and methods CD19 þ IgM þ D þ cells were then analyzed by MTG retention and CD27 expression as reported.8 Analysis and isolation of circulating B lymphocytes by flow cytometry procedures Peripheral blood mononuclear cells were isolated from healthy Isolation of RNA and DNA for IGHV analysis donors after obtaining informed consent using Lymphoprep The highly purified CD19 þ IgD þ CD27 ve,CD19þ IgD þ CD27 þ (Axis Shield, Bicton Industrial Park, UK), and were analyzed and and CD19 þ IgD veCD27 þ populations were used for RNA cell sorted by flow cytometry using a FACSAria (BD Biosciences, extraction using the RNeasy Mini Kit (Qiagen, Crawley, UK), Oxford, UK). The two donors for cell sorted fractions for and cDNA was synthesized using SuperScript II Reverse molecular analysis were aged 23 and 36 years, respectively. Transcriptase (Invitrogen), both in accordance with the For flow cytometry, 2–4 107 peripheral blood mononuclear manufacturers’ instructions. Using cDNA, IGHM transcripts cells were incubated with a-CD19-perCP-cy5.5 at 1:10 (BD were specifically amplified with downstream Cm primers. Pharmingen, Oxford, UK), a-IgM-phycoerythrin (PE) at 10 mg/ml IGHV5-IGHM gene transcripts were amplified from (Dako, Ely, UK), a-IgD-FITC (a-IgD-fluorescein isothiocyanate) sorted CD19 þ IgD þ CD27 ve, CD19 þ IgD þ CD27 þ and at 16 mg/ml (Dako) and a-CD27-allophycocyanin (APC) at 1:10 CD19 þ IgD veCD27 þ fractions by semi-nested reverse tran- (Biolegend, San Diego, CA, USA), or with relevant isotype scriptase-PCR, using in the first round a combination of IGHV5 antibody controls in FACS buffer (1% bovine serum albumin, leader (50-atggggtcaaccgccatcctcg) or IGHV3 leader (50-ccatg 4mM EDTA (ethylenediaminetetraacetic acid) and 0.15 mM gagtttgggctgagctgg) primers, respectively, with downstream sodium azide in phosphate-buffered saline) for 30 min at 4 1C. IGHM100 30 (50-ggagaaagtgatggagtcgg), followed in the second 0 Isotype antibody control labeling was performed with mIgG1,k- round of amplification by IGHV5 framework-1 (5 -gaggtg 0 perCP-cy5.5 (BD, Pharmingen), mIgG1,k-APC (BD, Pharmin- cagctgngtcagtctg) or IGHV3 framework-1 (5 -gaggtgcagctggtg 4,7,11 gen), rF(ab)2-FITC (Dako, Pharmingen) and rF(ab)2-PE (Dako) at sagtcyg) with IGHM100 as previously described. PCR equivalent concentrations to relevant staining antibody. Quad- products were cloned and individual plasmids with IGHV- rants defining positive and negative staining were positioned IGHM inserts were sequenced as described.4,7,11 Taq error was according to isotype control staining. calculated in a 350–400 bp stretch of the IGHM constant region Cells were gated first as lymphocytes by forward and side (excluding primer sequences) using semi-nested IGHV5 or scatter, and then CD19 þ IgM þ / ve B cells were analyzed for IGHV3 upstream primers, as described above, with IGHM370 CD27 and IgD expression. Stringent sorting gates were (50-tggacttgctgcgggggttg) or IGHM430 (50-ggacacctgaatctgccg). positioned within CD27/IgD quadrants, and CD19 þ IgM þ / ve Data analysis, using Immunogenetics (IMGT) databases,12 is cells were sorted into four populations according to CD27 and based on pooled clones obtained from replicate PCRs. Data IgD expression. Cell-sorted populations were immediately from the two CD19 þ CD27 þ fractions (IgD þ and IgD ve) were returned for analysis under the same gating strategy to pooled to give the full spectrum of conventional IgM þ CD27 þ establish purity. Sorted CD19 þ IgD þ CD27 ve (comprising memory B cells. To identify the germline donor IGHV5 gene, IgM þ D þ , as discussed further under the ‘Results’ section), genomic DNA was extracted from sorted CD19 veCD3 þ T cells CD19 þ IgD þ CD27 þ (comprising IgM þ D þ ) and CD19 þ IgD ve using the QIAamp DNA Blood Mini Kit (Qiagen), according to CD27 þ (comprising IgM þ D ve) populations were harvested for the manufacturer’s instructions. The germline IGHV5 gene RNA extraction. Amplification of IGHV-IGHM gene transcripts, segment was amplified using IGHV5-leader and 30 recombina- detailed below, then confined our analysis to CD19 þ IgM þ cells tion site-specific primers (50-tcggggctggtttctctcactgtg, 50-ggctcg within the CD27/IgD-sorted populations. gggctggtttctctcact) before cloning and analysis. Donor T cells (2 106) were isolated for genomic DNA analysis using a-CD19-perCP-cy5.5 (BD) and a-CD3-PE (BD) at 1:10 and cell sorted as a CD19 veCD3 þ fraction, with isotype Results controls mIgG1, k-perCP-cy5.5 and mIgG1, k-PE (BD) used at appropriate concentrations. To examine the nature of IgM þ D þ CD27 ve cells, two separate investigations were undertaken. First, we used a MTG dye extrusion assay to seek evidence for ABCB1 pump-negative ABCB1 transporter assay putative CD27 ve memory B cells. Second, we isolated The ABCB1 transporter assay was based on the use of the CD19 þ IgM þ D þ CD27 ve cells to virtual purity to allow a Mitotracker Green-FM dye (MTG; Invitrogen, Paisley, UK). molecular analysis of IGHV genes, in the absence of any dye use Approximately 1 107 peripheral blood mononuclear cells during isolation. were incubated in medium with 100 nM MTG at 37 1C for To facilitate the use of the MTG dye, the extrusion assay was 30 min in the presence or absence of 10 nM ABCB1 transporter optimized as reported to distinguish between naive and memory inhibitor, PGP-4008 (Alexis, Lausen, Switzerland), followed by a B cells in normal lymphocytes.8 CD19 þ -gated B cells incubated 2-h chase in medium alone. Cells were then washed in the FACS with MTG comprised a mixture of cells able to extrude and buffer before staining with a-IgM-biotin at 50 mg/ml (Southern retain dye (Figure 1a; top panel). These two populations were Biotech, Birmingham, AL, USA) at 4 1C for 30 min. A further separable using the ABC-transporter inhibitor PGP-4008 wash in the FACS buffer preceded staining with a-CD19-Pacific (Figure 1a; middle panel), in which high fluorescence indicates Blue at 1:50 (BD), a-CD27-APC at 1:10 (Biolegend), a-IgD-PE at retention of dye as cells are ABCB1 ve. These ABCB1 ve cells,
Leukemia Human IgM þ D þ memory B cells lacking CD27 N Weston-Bell et al 2077 as defined by Lanzavecchia’s laboratory,8 consist of transitional dye despite efflux (Figure 1a; lower panel), and this would be a and memory B cells. Inclusion of unstained cells with MTG- limiting factor in obtaining highly pure populations of cells if a labeled cells showed that ABCB1 þ cells do retain a low level of panel of markers is used that include dye. Focusing on CD19 þ expressing IgM þ D þ cells (Figure 1b), we evaluated MTG retention in both CD27 ve and CD27 þ populations (Figure 1c). Notably, we identified a subset of IgM þ D þ CD27 ve cells that was ABCB1 ve in 4/4 healthy individuals, suggestive of a new potential memory population (Figure 1c). This subset comprised 18.9 (±2.6 s.d.)% of the CD19 þ IgM þ D þ pool (Figure 1c). Naive IgM þ D þ CD27 ve B cells, in contrast, were ABCB1 þ as reported (Figure 1c).8 To examine whether IGHV gene mutation analysis could substantiate the putative memory status of a subset of CD19 þ expressing IgM þ D þ CD27 ve B cells suggested by the ABCB1 transporter assay, we next isolated IgM þ D þ CD27 ve cells to virtual purity (n ¼ 2 donors) using flow cytometry-based cell sorting, and avoiding MTG dye use. CD19 þ IgM þ / ve B cells were readily separated by IgD/CD27 expression to identify IgM þ D þ CD27 ve, IgM þ D þ CD27 þ and IgM þ D veCD27 þ populations (Figure 2a–c). IgD expression is largely coupled as dual IgM þ D þ expression in mature circulating B cells. In Figure 2c, the IgM þ D þ CD27 þ cells shown in the top right-hand quadrant correspond to classical pre-switched memory. Any IgD þ CD27 þ memory IgD B cells that lack IgM in this quadrant will be a very minor component, and remain controversial as a circulating population (see the review by Marti et al.13). The IgD veCD27 þ population is composed of conventional IgM þ D ve and isotype-switched memory B cells (Figure 2c). The IgM þ D þ CD27 ve fractions were harvested by high- resolution cell sorting (Figure 2d). Potential contamination by CD19 þ CD27 þ B cells was o0.4% when CD27 ve cells were re-sorted to check purity (Figure 2d; bottom right-hand quad- rant). Populations of CD19 þ IgM þ / veCD27 þ cells comprising conventional memory cells were also purified for IGHV gene comparisons (Figure 2c). The molecular analysis of IGHV genes in sorted fractions focused entirely on IGHM transcripts. In the CD19 þ IgM þ D þ CD27 ve fraction, these transcripts are derived from IgM þ D þ B cells, whereas in the combined CD27 þ comparator fractions, Cm transcripts are derived either from IgM þ D ve or IgM þ D þ B cells. We examined IGHV5 and IGHV3 genes to probe the smallest (HV5) and largest (HV3) families to obtain a representative view of repertoire use. As these fractions contain mature B cells, mRNA levels encoding
Figure 1 CD19 þ IgM þ D þ CD27 ve B cells include ABCB1 transpor- ter-negative cells that retain high levels of Mitotracker green-FM (MTG) dye, revealing a potential memory B-cell pool. MTG dye readily permeates B cells and is only extruded by naive B cells which express the ABCB1 transporter, whereas memory B cells lacking ABCB1 transporter activity retain dye and exhibit high fluorescence.7 MTG permeates all cells and is then specifically extruded to give a broad spectrum of fluorescence (a, top-most profile), and when coupled with the use of the ABCB1 transporter inhibitor PGP-4008, allowed gating to separate ABCB1 þ B cells which expel dye and ABCB1 ve B cells which retain dye (panel a, middle profile), and spiking with unstained cells confirmed a dye-related gating (panel a, bottom profile). CD19 þ B cells gated on IgM þ D þ expression (b) contain ABCB1 veIgM þ Dþ CD27 ve and ABCB1 þ IgM þ D þ CD27 ve B cells (c, bottom right and left quadrants). The potential memory B-cell subset delineated by ABCB1 veIgM þ D þ CD27 ve expression is clearly visible (panel c, bottom right quadrant): percentages are from a representative individual normal donor, and overall (n ¼ 4) this subset was 18.9 (±2.6)%.
Leukemia Human IgM þ D þ memory B cells lacking CD27 N Weston-Bell et al 2078
Figure 2 (a) lymphocyte gate using forward (FSC) and side scatter (SSC). Analysis of CD27 þ / ve B cells by flow cytometry and isolation of highly purified CD19 þ IgM þ D þ CD27 ve B cells for IGVH gene analysis. Peripheral blood mononuclear cells (PBMCs) were gated by CD19 and IgM expression (b), selecting CD19 þ B cells, which were IgM þ / ve. These were then analyzed for IgD and CD27 expression (c). The quadrants in panel c are: bottom right IgD þ CD27 ve which will include IgM þ cells; top right IgD þ CD27 þ cells which are essentially IgM þ D þ CD27 þ cells as IgM veD þ CD27 þ cells are rare (reviewed in Marti et al.13); top left IgD veCD27 þ which comprise IgM þ only and isotype switched CD27 þ B cells. High-resolution procedures allowed purification of the IgD þ CD27 ve fraction (comprising IgM þ D þ CD27 ve), and back-sorting this purified fraction (d) confirmed virtual purity (contamination by CD27 þ cells o0.4%).
IGHV rearrangements are comparable between cells, and do not 45 mutations (Figure 3b). Taken together, of IGHV5-IGHM and bias cloning analysis. Only individual potentially functionally IGHV3-IGHM transcripts in CD19 þ IgM þ D þ CD27 ve B cells, rearrangements with distinct CDR3 domains were included in 13.5% exhibited 3–5 mutations and 8.4% 45 mutations, to total the analysis, and rare repeat sequences were excluded. 21.9% of all IgM þ D þ CD27 ve transcripts (Figure 3c). These Importantly, to assess Taq error rate accurately, we simulta- data reveal a CD19 þ IgM þ D þ CD27 ve subset with a mutated neously examined a 350–400 bp region of the Cm constant memory imprint. region by nested reverse transcriptase-PCR followed by cloning The level of mutation in Cm transcripts was compared in the and sequence analysis using the same cDNA as used for IGHV CD27 ve versus CD27 þ cells obtained from the same donor analysis from each fraction. Data are based on pooled population (Figure 4). The frequency of CD27 ve transcripts sequences from replicate nested PCRs. exhibiting between 16 and 20 mutations decreased markedly, From two donors, we analyzed IGHV5-IGHM (n ¼ 191) and and there were no CD27 ve transcripts that displayed 420 IGHV3-IGHM (n ¼ 143) transcripts from CD19 þ IgM þ mutations, a level of mutation restricted solely to CD27 þ cells D þ CD27 ve fractions, and compared their mutational imprints (Figure 4a). Similarly, the number of transcripts displaying with IGHV5-IGHM (n ¼ 113) and IGHV3-IGHM (n ¼ 74) 16–20 and 420 mutations diminished substantially in the study transcripts from conventional CD19 þ IgM þ D þ / veCD27 þ of IgG þ CD27 ve memory B cells, whereas the 420 level of memory B cells. Analysis of individual donors yielded very mutation was highly prevalent in classical switched CD27 þ similar data, and pooled data sets are shown (Figures 3 and 4). B cells.9,10 Consequently, a restricted level of IGHV mutation In the CD19 þ IgM þ D þ CD27 ve populations, 12% of appears to be a parallel feature in both pre-switched and IGHV5-IGHM transcripts exhibited 3–5 mutations in potentially switched subsets of CD27 ve memory B cells. functional V(D)J sequences and 7.9% displayed 45 mutations Patterns of IGHV3 family gene segment use were similar in (Figure 3a). The frequency of 3–5 mutations was sixfold higher IgM-expressing cells derived from the CD19 þ IgM þ than observed in the ‘control’ Cm region transcripts (2%; D þ CD27 ve and CD19 þ IgM þ D þ / veCD27 þ memory pools n ¼ 101), analyzed in parallel in this population to assess Taq (Figure 4b), with 17 (CD27 ve memory) and 18 (CD27 þ error. Only a maximum of three mutations per Cm region were memory) of 22 known IGHV3 gene segments used, indicating observed, and more than five mutations were never seen in no apparent restrictive bias in recruitment to this mutational these ‘control’ Cm sequences (Figures 3a and b). The remainder pathway. of CD19 þ IgM þ D þ CD27 ve transcripts (B78%) revealed unmutated 100% germline IGHV genes (data not shown), reflecting naive B-cell derivation, and confirming a very low Discussion Taq error rate. In donor 1, we also tracked the germline IGHV5 gene using genomic DNA from T cells and specific primers Our observations show a new circulating subset of flanking the gene segment, and identified the same allele as IgM þ D þ CD27 ve cells which are memory B cells by IGHV determined by IMGT database alignment (data not shown). This gene mutational status, the ‘gold standard,’ existing at a verified that the low level of mutation seen in IGHV5 was not frequency of B20% in a population previously regarded as caused by polymorphic variation. In comparison, of the IGHV5- entirely composed of naive B cells. The identification of IGHM transcripts from conventional IgM þ D þ / veCD27 þ circulating mutated IgM þ D þ CD27 ve B cells extends the memory B cells, 20.4% displayed 3–5 mutations and 54.9% repertoire of unusual memory B cells which include isotype- with 45 mutations (Figure 3a) (remaining 24.7% transcripts switched mutated CD27 ve B cells in the normal periphery,9,10 displaying o3 mutations; data not shown). and taken together, suggests distinctive origins and functions. IGHV3 family gene usage is most frequent in the normal As yet, the source of these CD27 ve memory B cells is B-cell repertoire, and was also assessed for mutations and gene unknown. Interestingly, there are CD27 ve-mutated B-cell segment use in the CD19 þ IgM þ D þ CD27 ve pool. Of the subsets that are tissue localized, but their relationship IGHV3-IGHM transcripts described in this study, 15.4% to the circulating memory CD27 ve B lymphocytes is displayed 3–5 mutations, and 9.1% 45 mutations (Figure 3b). currently obscure. The tissue-localized subsets may differ. In CD27 þ B cells, 9.5% displayed 3–5 mutations and 79.7% IgD þ CD27 veCD38 ve ‘pro/pre-GC’ B cells exist as a tonsillar
Leukemia Human IgM þ D þ memory B cells lacking CD27 N Weston-Bell et al 2079
Figure 4 Comparison of levels of somatic hypermutation and IGHV3 family gene segment usage in mutated CD19 þ IgM þ D þ CD27 ve versus CD19 þ IgM þ D þ / veCD27 þ memory B cells. The number of mutations per transcript were compared in the two subsets, and are shown in the ranges 6–10, 11–15, 16–20 and 420 mutations per transcript (a). Differences in the frequency of transcripts displaying 16–20 mutations are apparent, with much fewer CD27 ve transcripts revealing this level of mutation than CD27 þ transcripts. This is more marked when evaluating 420 mutations, a level of mutation not observed in any CD27 ve transcript but displayed by 5.8% of CD27 þ sequences. To examine the repertoire of B cells recruited into the CD27 ve/ þ memory pools, IGHV3 family gene segments were evaluated (b), revealing a comparable repertoire of gene segment usage in both subsets.
Figure 3 Analysis of somatic hypermutation (SHM) levels in 14 CD19 þ IgM þ D þ CD27 ve memory B cells versus conventional influenced by any infection at this site. Isotype-switched CD19 þ IgM þ IgD þ / veCD27 þ memory B lymphocytes. IGHV5/ CD27 veCD38 veFcRH4 þ B cells with a comparably low level IGHV3-IGHM sequences were analyzed for somatic mutations in of mutation have also been described in the tonsil, but these the respective populations, and pooled data obtained from two normal remain tissue localized and are not observed in the blood.15 donors is shown as the level of mutation per transcript. This was FcRH4, Fc receptor homolog 4, is a immunoregulatory mole- quantified as 3–5 mutations per transcript and 45 mutations per 15 transcript in the CD19 þ IgM þ D þ CD27 ve and the CD19 þ IgM þ cule, and based on these observations seemed to be a relevant ve D þ / veCD27 þ subsets. The data shows the frequency of transcripts marker for mutated CD27 cells. However, normal circulating (%/all sequences analyzed) revealing stated number of mutations, first IgG þ CD27 ve memory B cells lack FcRH4 expression,10 indicat- in IGHV5-IGHM in respective populations (a), next in IGHV3-IGHM ing divergent origins from the tonsillar B-cell subset. Lymph nodes sequences (b) and then as combined (IGHV5 þ IGHV3)-IGHV also harbor a predominantly isotype-switched CD27 ve pool of sequences (c). PCR error was assessed in the CD19 þ IgM þ þ ve large inter-follicular B cells, but in marked contrast, these nodal B D CD27 B-cell population by amplifying a 350–400 bp constant 16 region of the Cm transcripts, a region known not to be targeted by cells are extensively mutated. It seems then that multiple ve SHM, and herein, a very low number of transcripts displaying Taq pathways generate a repertoire of CD27 memory B cells at error nucleotide changes (3–5 range) were identified (2.0% of 101 different sites, with as yet unknown functions. sequences analyzed) (panels a–c). In these ‘control’ Cm region It is worth noting that even in relation to conventional transcripts, only a maximum of 3 nucleotide errors per transcript mutated IgM þ D þ CD27 þ cells, the precise pathway(s) of their were observed, and 45 changes resulting from Taq mistakes were never observed. origins is by no means fully ascertained. Although early observations suggested that IgM þ D þ CD27 þ are GC-derived B cells, it has also been argued that they are generated in the subset and display a very low level of mutations in IgM pre-immune repertoire in the marginal zone.17 However, more transcripts, but it is unclear whether these cells exit into recent experiments in which human hematopoietic stem cells the circulation, and whether their origins may have been have been grafted in immunodeficient mice reveal an induction
Leukemia Human IgM þ D þ memory B cells lacking CD27 N Weston-Bell et al 2080 of SHM in IgM þ D þ CD27 þ B cells in a T-cell-independent References manner, but appear to exclude marginal zone origins.18 Nevertheless, these observations suggest that separate pathways 1 Honjo T, Kinoshita K, Muramatsu M. Molecular mechanism of could lead to pre-switched CD27 þ memory B cells and isotype- class switch recombination: linkage with somatic hypermutation. switched CD27 þ cells. In speculating, this bifurcation may also Annu Rev Immunol 2002; 20: 165–196. hold for both the pre-switched and the switched CD27 ve 2 Klein U, Rajewsky K, Kuppers R. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell memory pool. surface antigen carry somatically mutated variable region genes: The lower level of mutation seen in both pre- and post- CD27 as a general marker for somatically mutated (memory) switched CD27 ve B cells argues for a common mechanism. It is B cells. J Exp Med 1998; 188: 1679–1689. evident that CD27–CD70 interactions are not required to 3 Toellner KM, Jenkinson WE, Taylor DR, Khan M, Sze DM, Sansom mediate SHM in these CD27 ve cells. This suggests either DM et al. Low-level hypermutation in T cell-independent germinal non-GC sites of mutational activity, such as those that generate a centers compared with high mutation rates associated with T cell- dependent germinal centers. J Exp Med 2002; 195: 383–389. low level of SHM activity in response to T-independent 3 ve 4 Forconi F, Sozzi E, Rossi D, Sahota SS, Amato T, Raspadori D et al. antigens, or that CD27 memory cells may in fact represent Selective influences in the expressed immunoglobulin heavy and early e´migre´s from GC-initiated events, as postulated for light chain gene repertoire in hairy cell leukemia. Haematologica IgG þ CD27 ve memory B cells.9 These possibilities remain 2008; 93: 697–705. unresolved, but the precise tissue site(s) will have relevance for 5 Forconi F, Raspadori D, Lenoci M, Lauria F. Absence of surface dissecting a role in disease. CD27 distinguishes hairy cell leukemia from other leukemic B-cell malignancies. Haematologica 2005; 90: 266–268. This study has implications for understanding origins of 6 Kriangkum J, Taylor BJ, Treon SP, Mant MJ, Belch AR, Pilarski LM. malignant B cells, and in particular, for HCL. We had previously Clonotypic IgM V/D/J sequence analysis in Waldenstrom shown that IgM-expressing HCL displayed mutated IGHV genes macroglobulinemia suggests an unusual B-cell origin and an yet lacked CD27, and frequently co-expressed multiple expansion of polyclonal B cells in peripheral blood. Blood 2004; switched isotypes (mult-HCL).4,5,11 Our large cohort study had 104: 2134–2142. shown that mult-HCLs comprised the major subset of disease, as 7 Babbage G, Townsend M, Zojer N, Mockridge IC, Garand R, 4 Barlogie B et al. IgM-expressing Waldenstrom’s macroglobuline- compared with single isotype-expressing HCL. It is conceivable mia tumor cells reveal a potential for isotype switch events in vivo. that IgM-co-expressing mult-HCL derives from the novel Leukemia 2007; 21: 827–830. mutated IgM þ D þ CD27 ve B cells described in this study. 8 Wirths S, Lanzavecchia A. ABCB1 transporter discriminates human mult-HCL tumor cells also initiate atypical switch events, resting naive B cells from cycling transitional and memory B cells. which seem to exclude deletional class switch recombination Eur J Immunol 2005; 35: 3433–3441. and generate variant isotype transcripts most likely through 9 Fecteau JF, Coˆte´ G, Ne´ron S. A new memory CD27-IgG+ B-cell population in peripheral blood expressing VH genes with low RNA processing.11 In single isotype-expressing HCL þ ve frequency of somatic mutation. J Immunol 2006; 177: 3728–3736. expressing only IgG, origins from IgG CD27 cells are also 10 Wei C, Anolik J, Cappione A, Zheng B, Pugh-Bernard A, Brooks J plausible.9,10 Ongoing mutational activity targeting IGHV in et al. A new population of cells lacking expression of CD27 HCL is also a recurrent feature of leukemic cells, in which AID represents a notable component of the B cell memory compart- transcripts are constitutively expressed,11 and may lead to the ment in systemic lupus erythematosus. J Immunol 2007; 178: accrual of higher levels of mutations observed in this tumor.4,11 6624–6633. 11 Forconi F, Sahota SS, Raspadori D, Ippoliti M, Babbage G, Lauria F IGHV-mutated CD27 ve cells also comprise part of the tumor 6,7 et al. Hairy cell leukemia: at the crossroad of somatic mutation and clone in Waldenstrom’s macroglobulinemia, but here it is isotype switch. Blood 2004; 104: 3312–3317. unknown whether malignant cells derive from unusual 12 Giudicelli V, Chaume D, Lefranc MP. IMGT/V-QUEST, an IgM þ D þ CD27 ve memory B cells that we describe herein or integrated software program for immunoglobulin and T-cell from conventional CD27 þ memory B cells that subsequently receptor V-J and V-D-J rearrangement analysis. Nucleic Acids lose CD27 expression. If the latter, it will then also be relevant to Res 2004; 32: W435–W440. þ þ þ 13 Marti G, Abbasi F, Raveche E, Rawstron AC, Ghia P, Aurran T et al. know whether IgM D CD27 -mutated B cells are GC- or Overview of monoclonal B-cell lymphocytosis. Br J Haematol non-GC-derived in defining origins of Waldenstrom’s macro- 2007; 139 (5): 701–708. globulinemia. 14 Kolar GR, Mehta D, Pelayo R, Capra JD. A novel human B-cell In conclusion, delineating a new IgM þ D þ CD27 ve circula- subpopulation representing the initial germinal center population ting memory B-cell population has relevance to understanding to express AID. Blood 2007; 109: 2545–2552. origins of B cells in health and disease. 15 Ehrhardt GR, Hsu JT, Gartland L, Leu CM, Zhang S, Davis RS et al. Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells. J Exp Med 2005; 202: 783–791. Conflict of interest 16 Marafioti T, Jones M, Facchetti F, Diss TC, Du MQ, Isaacson PG et al. Phenotype and genotype of interfollicular large B cells, a subpopulation of lymphocytes often with dendritic morphology. The authors declare no conflict of interest. Blood 2003; 102: 2868–2876. 17 Tangye SG, Good KL. Human IgM+CD27+ B cells: memory B cells or ‘‘memory’’ B cells? J Immunol 2007; 179: 13–19. 18 Scheeren FA, Nagasawa M, Weijer K, Cupedo T, Kirberg J, Legrand Acknowledgements N et al. T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+ CD27+ B cells. J Exp Med This work was funded by the Leukaemia Research Fund (UK). 2008; 205: 2033–2042.
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