The Human CD77− Population Represents a Heterogeneous Subset of Cells Comprising Centroblasts, , and Plasmablasts, Prompting Phenotypical This information is current as Revision of September 28, 2021. Carl-Magnus Högerkorp and Carl A. K. Borrebaeck J Immunol 2006; 177:4341-4349; ; doi: 10.4049/jimmunol.177.7.4341 http://www.jimmunol.org/content/177/7/4341 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Human CD77؊ B Cell Population Represents a Heterogeneous Subset of Cells Comprising Centroblasts, Centrocytes, and Plasmablasts, Prompting Phenotypical Revision1

Carl-Magnus Ho¨gerkorp and Carl A. K. Borrebaeck2

The process of becoming an Ig-producing takes the mature B cell through the , where Ig genes are diversified through and class switch recombination. To more clearly define functional characteristics of the germinal center dark zone centroblasts and the light zone centrocytes, we have performed expression analysis of the CD77؉ and CD77؊ populations, because CD77 has been accepted as a discriminator of centroblasts and centrocytes. Our results demonstrated Downloaded from .that the CD77؉ and the CD77؊ populations lack functional associated expression programs discriminating the two populations Both populations are shown to be actively cycling and to share common features associated with cell cycle regulation and DNA maintenance. They are also shown to have an equally active DNA repair program, as well as components involved in somatic hypermutation and class switch recombination. Moreover, the data also demonstrated that the CD77؊ population comprises cells with an already initiated plasma cell differentiation program. Together this demonstrates that CD77 does not discriminate cen- ,troblasts and centrocytes and that the CD77؊ population represents a heterogeneous subset of cells, comprising centroblasts http://www.jimmunol.org/ centrocytes, and plasmablast. The Journal of Immunology, 2006, 177: 4341–4349.

he germinal center (GC)3 constitutes an imperative func- thus suggested that centroblasts were CD77ϩ, whereas the centro- tional niche in the adaptive immune system, where B cell cytes had lost CD77. The IgDϪ/CD38Ϫ subset was defined as Ig genes are diversified through somatic hypermutation memory cells, whereas plasma cells were defined as IgDϪ/ T ϩϩ (SHM) and class switch recombination (CSR) processes. These CD38 . events lead to the formation of high-affinity Abs that will facilitate In previous studies (4, 5), we and others have observed that the eradication of invading pathogens. The current classification of CD77ϩ and the CD77Ϫ populations display only minor differences different stages in human mature B cell differentiation starts with in their expression programs. We have now further analyzed the by guest on September 28, 2021 naive B cells that turn into centroblasts and centrocytes, within the transcriptional profile of the different purified B cell subsets, de- GC. These GC B cells subsequently differentiate into memory cells fined by Liu et al. (1–3), and to functionally discriminate the two or plasma cells, the latter actively secreting Ag-specific Igs. The GC B cell subsets we have now focused on the regulation of spe- most comprehensive classification of the mature B cell subsets was cific programs associated with proliferation, SHM, and CSR. In made already in 1994 (1–3), based on the expression of phenotypic contrast to previous studies (4, 5), we have based our conclusion markers, such as CD38, IgD, IgM, CD23, CD77, and Ki67, as well on transcriptional analysis, using a gene chip covering 22,283 as on the presence of mutations in the variable Ig genes and the probes, including both genes and expressed sequence tags. expression of sterile I␥,I␣, and I␧ transcripts. Thus, naive B cells were defined as IgDϩ/CD38Ϫ B cells, having recombined Ig The global expression profile of all these probes demonstrated that several thousand genes differed between GC and the non-GC genes. A ligand-selected subpopulation of the naive B cells was ϩ Ϫ further defined, on the basis of the low-affinity IgE receptor B cell subsets, whereas the GC CD77 and CD77 subsets again (CD23) expression (1). The GC B cell subsets were the IgDϪ/ only showed minor transcriptional differences. The transcriptional ϩ CD38ϩ/CD77ϩ cells, engaged in the SHM process (2), and the profile of the GC B cells demonstrated that not only the CD77 Ϫ IgDϪ/CD38ϩ/CD77Ϫ cells involved in the CSR process (3). It was cells, but also the CD77 cells are proliferating, which was also validated by cell cycle analysis. The transcriptional analysis fur- ther shows that both GC subsets have an equally active program Department of Immunotechnology, Lund University, Lund, Sweden for SHM and CSR, as exemplified by the expression of activation- Received for publication March 23, 2006. Accepted for publication July 12, 2006. induced cytidine deaminase (AID), uracil-DNA glycosylase 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 (UNG), and several other genes implicated in these processes. with 18 U.S.C. Section 1734 solely to indicate this fact. Analysis of the synthesis pathway of CD77 suggests that the only ϩ Ϫ 1 This study was supported by the Faculty of Technology, Lund University, and in true difference between CD77 and CD77 GC B cell seems to part supported by a Translational Research Grant from The Leukemia & reside in the expression of the B3GALT3 enzyme, which is the Society (Contract 6085-06). enzyme converting CD77 to the P Ag. Moreover, the results also 2 Address correspondence and reprint requests to Dr. Carl A. K. Borrebaeck, Depart- ment of Immunotechnology, Lund University, BMC D13, 221 84 Lund, Sweden. demonstrated that the plasma cell differentiation program is initi- E-mail address: [email protected] ated already in cells included in the CD77Ϫ population. All in all, 3 Abbreviations used in this paper: GC, germinal center; SHM, somatic hypermuta- these findings demonstrate that the current classification of the GC tion; CSR, class switch recombination; AID, activation-induced cytidine deaminase; UNG, uracil-DNA glycosylase; BCL, B cell line; BER, base excision repair; MMR, B cells clearly calls for a reclassification, because CD77 is not a mismatch repair; HR, homologous recombination. discriminator of centroblasts vs centrocytes.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 4342 CD77Ϫ B CELLS ARE A HETEROGENEOUS SUBSET OF CELLS

Materials and Methods Cell cycle analysis T cell-depleted mononuclear tonsil cells were stained with anti-IgD FITC, Anti-IgD FITC, anti-IgD PE, anti-CD3 PE, and anti-CD3 PECy5 were anti-CD38 PECy5, and anti-CD77 (38.13), followed by goat anti-rat IgM PE Abs, or anti-CD77 (5B5), or anti-CD77 (6A2), followed by rat anti- obtained from DakoCytomation. Purified anti-CD38, anti-IgD, anti-CD3 ϩ Ϫ FITC, anti-CD3 PacificBlue, anti-CD14 PacificBlue, anti-CD23 PE, anti- mouse IgM PE Abs. CD77 and CD77 cells were sorted, as previously defined; memory cells were used as reference. Two million cells of each CD38 PECy5, anti-CD38 PECy7, rabbit anti-mouse IgM PE, and rat IgM population were collected. Each population was fixed in cold 70% ethanol Abs were purchased from BD Biosciences. Anti-CD77 (38.13) and goat for 30 min at 4°C, washed twice in cold PBS, and treated with RNase A anti-rat IgM PE Abs were purchased from Beckman Coulter and Serotec, (Sigma-Aldrich) at 100 ␮g/ml, followed by propidium iodide (Sigma- respectively. Abs to P Ag (AME-2) were a gift from I. de Jong (Sanquin, ␮ Amsterdam, The Netherlands). The anti-CD77 (5B5) Ab was provided by Aldrich) at 50 g/ml just before analysis. Sorting and analysis were per- M. Nahm (University of Alabama, Birmingham, AL), and the anti-CD77 formed on a FACSAria (BD Biosciences). (6A2) Ab was derived from hybridoma 424/6A2, which was a gift from P. Histology Bjo¨rk (Active Biotech Research, Lund, Sweden). Cryostat sections (8 ␮m) of tonsil were stained with anti-CD77 (38.13) Cells (Beckman Coulter) or a rat IgM isotype-matched control (BD Biosciences) in PBS at 4°C overnight. After extensive washing in PBS, the tissue sec- Human tonsils were obtained from pediatric patients undergoing routine tions were incubated with a secondary goat anti-rat IgM PE Ab (Serotec) tonsillectomy at the Lund University Hospital or Malmo¨Academic Hos- for 60 min at 4°C. A ScanArray Express HT confocal laser scanner pital. Briefly, tonsils were minced and T cells were removed by rosetting (PerkinElmer) equipped with a 488-nm laser was used for imaging. Image with neuraminidase-treated SRBC. Mononuclear, T cell-depleted cells analysis was performed with the Quantity One software (Bio-Rad). were isolated by density centrifugation, using Ficoll-Isopaque (Amersham Air-dried cytospin preparations of sorted fractions of naive, CD77ϩ, and Ϫ Biosciences). The interphase fraction, containing predominantly B cells, CD77 tonsillar B cells were stained in May-Grunwald eosin (Histolab Downloaded from was washed in PBS containing FBS (10%). To enrich for naive, GC, or Products) for 5 min, washed in distilled water, and stained in Giemsa stain memory B cells, the interphase fraction was subjected to negative selection (Histolab Products) for 20 min. Dried preparations were analyzed in light by incubating with precoated anti-CD38, anti-IgD Dynabeads (sheep anti- microscopy. mouse IgG or pan mouse IgG; Dynal Biotech) for 45 min on ice. Cells depleted for CD38 were stained with anti-CD38 PECy5, anti-CD3 PECy5, Results anti-IgD FITC, and anti-CD77 Abs, followed by goat anti-rat PE Abs. Cells The distribution of CD77 within the GC has been shown to be depleted for IgD were stained with anti-CD38 PECy5, anti-IgD FITC, anti- CD3 FITC, and anti-CD23 PE Abs. Cells depleted for both CD38 and IgD highly dependent on the Ab clone of choice, and the conclusions http://www.jimmunol.org/ were stained with anti-CD38 PECy5, anti-IgD FITC, and anti-CD3 PE. All as to whether CD77 is a dark zone/centroblast marker or not are depleted cell fractions were then positively selected by flow cytometric cell diverging (8–10). To reassess the distribution of CD77 within the ϩ sorting, using a FACSVantage SE (BD Biosciences), as follows: 1) IgD / GC, we stained tissue sections from tonsils for CD77 with the CD23Ϫ/CD38Ϫ/CD3Ϫ;2)IgDϩ/CD23ϩ/CD38Ϫ/CD3Ϫ;3)IgDϪ/CD38ϩ/ ϩ Ϫ Ϫ ϩ Ϫ Ϫ Ϫ Ϫ CD77 clone 38.13, used in previous tissue stainings (9). The anal- CD77 /CD3 ;4)IgDCD38 /CD77 /CD3 ; and 5) IgD /CD38 / CD3Ϫ. These subsets were Ͼ98% pure, as defined by their phenotypic ysis demonstrated that CD77 is evenly distributed in the smaller descriptions. Isotype-matched control rat IgM Ab was used to evaluate the GCs, whereas the larger GCs display a gradual decrease of CD77 unspecific contribution, showing Ͻ0.2% of the cells falling within the going from the dark zone to the light zone (Fig. 1). Thus, in line CD77 sorting gate. with previous studies (9), CD77ϩ cells are enriched in the dark by guest on September 28, 2021 Sample preparation zone of the GC, indicating an association to the centroblasts. How- ever, whether all centroblasts are CD77ϩ is not disclosed by this Freshly isolated cells were lysed in TRIzol (Invitrogen Life Technologies). analysis, implying that the centroblast population may comprise The RNA was extracted from the cell lysate by adding 0.2 vol of chloro- cells also lacking CD77. To further characterize the nature of form. The aqueous phase containing the RNA was separated and subse- ϩ Ϫ quently precipitated with isopropanol and washed in 75% ethanol. The CD77 and CD77 GC B cells, we analyzed the transcriptional

RNA pellet was dissolved in diethyl pyrocarbonate-H2O and further puri- regulation of these two populations in greater detail by gene ex- fied with the RNeasy Mini Kit (Qiagen). The total RNA content was as- pression profiling. sessed by spectroscopy at 260/280 nm (GeneQuant II; Pharmacia Biotech). After a second precipitation step in 2.5 vol of ethanol and subsequent wash, Phenotypic B cell markers correspond to mRNA expression the RNA was resuspended in diethyl pyrocarbonate-H2O. Five micrograms of total RNA was used for the cDNA and cRNA synthesis, as previously Five different subpopulations of tonsillar B cells were isolated by described (4). fluorescence-activated cell sorting and categorized as follows: 1) IgDϩ/CD23Ϫ/CD38Ϫ/CD3Ϫ;2)IgDϩ/CD23ϩ/CD38Ϫ/CD3Ϫ;3) Hybrization and scanning of the DNA chips IgDϪ/CD38ϩ/CD77ϩ/CD3Ϫ;4)IgDϪ/CD38ϩ/CD77Ϫ/CD3Ϫ; and Ϫ Ϫ Ϫ A hybridization mixture was prepared with the biotinylated and fragmented 5) IgD /CD38 /CD3 (Fig. 2A), previously defined as naive, ligand- cRNA at 50 ␮g/ml, as described previously (4), and hybridized onto a selected, centroblast, , and memory cell populations, re- U133A microarray (Affymetrix). The probe array was then stained with a spectively (1–3). Labeled cRNA from all subsets was hybridized to solution of 2 mg/ml acetylated BSA and 10 ␮g/ml streptavidin R-PE (Mo- lecular Probes). A secondary amplification stain was performed with acety- Affymetrix U133A arrays and scanned on a confocal laser scanner. lated BSA, normal goat IgG (Sigma-Aldrich), and biotinylated goat anti- The general behavior of the data was evaluated by correlation streptavidin Ab (Vector Laboratories). A final staining step with analysis, and the data set displayed very high r values of 0.99, streptavidin R-PE was performed before the probe arrays were scanned in 0.98, 0.98, 0.99, and 0.95 between replicates for the five popula- the Gene Array Scanner and checked using the Micro Array Suite 5.0 tions, respectively (Fig. 3A); Fig. 3B also displays the overall gene (Affymetrix), as described previously (4). expression profile of the five populations. To validate the Gene- Data analysis Chip analysis, we analyzed the regulation of known GC and B cell markers (Fig. 2B). Thus, CD23 (FCER2), CD31 (PECAM), CD44, The dataset was analyzed in Micro Array Suite 5.0, Bioconductor (͗www. bioconductor.org͘) and Gene Spring (Agilent Technologies). The signal and IgM (IGHM) were typically absent in the GC B cell subsets, intensity values were analyzed as nonscaled, background corrected, quan- whereas CD9, CD10 (MME), CD27 (TNFRSF7), and CD95 tile normalized (6), and robust multiarray averaged measures, as defined by (TNFRSF6) were present (Figs. 2B and 4A). As expected, B cell Irizarry et al. (7). They were further linearized and correlated to the gene line (BCL)6 was found to be typically linked to GC B cell subsets distribution median to achieve better intergene comparability. Filtering and expression pattern analysis were performed in Gene Spring. The dataset accompanied by a strong down-regulation of BCL2 (Fig. 4A). was annotated based on HGNC (͗www.gene.ucl.ac.uk/nomenclature͘) and Also, AID (AICDA), central in both SHM and CSR, displayed a Unigene (͗www.ncbi.nlm.nih.gov/UniGene/Hs.Home.html͘). strong association to both GC B cell subsets. The Journal of Immunology 4343

FIGURE 1. A, Cryostat section of tonsil stained with anti-CD77 (38.13) demonstrating the distribution of Downloaded from CD77 within the GC. Numbers (1–18) indicate sample points for image analysis in B. B, Quantification of the CD77 distribution within GC structures in tonsil show- ing the average intensity (y-axis) for each sample point selected in A. This analysis demonstrated that CD77 is equally distributed in smaller GC structures, whereas http://www.jimmunol.org/ larger structures show a gradual decrease in CD77 across the GC. by guest on September 28, 2021

CD77Ϫ cells share the CD77ϩ cell proliferation program and EED, involved in proliferation (12) were also highly up-reg- The transcriptional regulation of cell cycle regulators outlines the ulated in the GC B subsets, whereas BMI1 was equally signifi- proliferative status of the different B cell populations. In the cantly down-regulated (Fig. 4C) in both subsets (13). This transcriptional profile of the cell cycle regulators was non-GC subsets, the G1 regulators cyclin D1 (CCND1) and D2 (CCND2) were predominant (Fig. 4C). Unexpectedly, both the shown to correspond to the actual cell cycle status of both the GC ϩ B cell subsets. Cell cycle analysis by flow cytometry of the sorted proliferating CD77 as well as the alleged nonproliferating ϩ Ϫ populations demonstrated that both the CD77 cells as well as the CD77 expressed genes involved in an active cell division pro- Ϫ Ϫ gram (Fig. 4C). Genes, including cyclin D3 (CCND3), E1 CD77 cells are actively cycling, whereas CD38 naive and (CCNE1), E2 (CCNE2), A2 (CCNA2), B1 (CCNB1), and B2 memory cells are residing in G0/G1 (Fig. 5A and data not shown). Collectively, these data do not support the traditional conception (CCNB2), all regulators of the G1-S, S, and G2-M phase transi- ϩ Ϫ tions, were expressed in both of these subsets. This was also true that centroblasts are CD77 and centrocytes are CD77 . for other components involved in cell cycle progression, such as The GC genomic integrity and DNA maintenance programs are CDK4, CDK2, CDC2, and the E2F family of transcription factors ϩ Ϫ (E2F1–3) (11). Furthermore, the inhibitors of CDK2, p21Cip active in both the CD77 and CD77 population (CDKN1A) and p27Kip (CDKN1B), were effectively down-regu- The high proliferation rate within the GC jeopardizes the genome lated, and among the inhibitors of CDK4 class of proteins (INK4) integrity of the proliferating centroblast and will, thus, require only p18 (CDKN2C) displayed an increased expression in the GC active DNA-damage and replication checkpoint systems (14). We B cell subsets. The members of the polycomb group of genes, ENX therefore looked into whether the regulation of components in 4344 CD77Ϫ B CELLS ARE A HETEROGENEOUS SUBSET OF CELLS

FIGURE 2. A, The five mature B cell populations were sorted according to the model by Pascual et al. (2) at a purity typically of 90% of the original sortgate. Lower right panel, Total CD23 distribution of the CD38Ϫ/IgDϩ population (black line), sorted CD38Ϫ/ IgDϩ/CD23Ϫ subset, and CD38Ϫ/IgDϩ/CD23ϩ subset (shaded areas); filled bar, indicates original sort gate and beneath the sort purity in percentages. Upper right panel, Total CD77 distribution of the CD38ϩ/IgDϪ pop- ulation (black line), sorted CD38ϩ/IgDϪ/CD77ϩ subset, and sorted CD38ϩ/IgDϪ/CD77Ϫ subset (shaded areas); filled bar, indicates original sort gate and beneath the sort purity in percentages. Lower left panel, Sorted Downloaded from CD38Ϫ/IgDϪ subset; square box, indicates original sort gate and above the sort purity in percentages. B, The transcriptional expression pattern corresponds well with protein expression, as validated by flow cytometry for several surface-expressed proteins represented by CD9, CD10, CD23, CD27, CD31, CD44, CD95, IgM (x-axis), http://www.jimmunol.org/ and CD38 (y-axis). The onset of CD38 presents a dis- tinct change in the transcriptional program, which is ev- ident, both at the transcript and protein level. by guest on September 28, 2021

these systems gives any clue to the nature of the two GC B cell Transcriptional regulation of SHM and CSR does not separate populations. Although the sensor kinases ATM and ATR showed no CD77ϩ and CD77Ϫ indication of being induced in either of the GC B cell subsets, Centroblasts and centrocytes have earlier been suggested to be func- several members of the initial damage recognition system, such as tionally separated based on their ability to carry out SHM and CSR, RAD17, RFC2-5, and RAD1, and most of the downstream effectors respectively (2, 3). To characterize the two GC B cell subsets from a of ATM and ATR, such as Chk1 (CHEK1), Chk2 (CHEK2), and ϩ Ϫ functional perspective, we then looked more carefully into the tran- BRCA1, were up-regulated in both the CD77 and CD77 popu- scriptional regulation of components implicated in these processes. lations (Fig. 4D). This was also true for the respective Chk1 and This included members of base excision repair (BER), mismatch re- Chk2 targets CDC25C and CDC25A. Notable was that p53 pair (MMR), homologous recombination (HR), and error-prone trans- (TP53), another target of ATM, displayed a baseline expression pattern across all B cell subsets. However, it was apparent that the lesion DNA polymerases (19, 20) or nonhomologous end joining and p53 activity was functionally restricted in the two GC B cell sub- nucleotide excision repair implied in CSR (21, 22). Thus, as can be expected for replicating cells, many HR components were induced in sets. This was evident as suggested by the absence of typical p53 ϩ Ϫ targets, such as BID, NOXA (PMAIP1), PUMA (BBC3), APAF1, the CD77 population, but to a similar degree also in the CD77 and p21Cip (CDKN1A), as well as the lack of transcriptional re- population (Fig. 6A). This transcriptional regulation was seen also pression from p53 of the antiapoptotic and prosurvival proteins among components participating in MMR, such as the MutS homo- survivin (BIRC5) and strathmin (STMN1). Interestingly, a recent logue 2 (MSH2), MutS homologue 6 (MSH6), and EXO1 (Fig. 6C), study demonstrated that this restriction may be mediated by a di- as well as for the BER enzyme UNG (Fig. 6D), which is noteworthy rect repression of the p53 gene transcription by BCL6 (15). This, considering the specific implication of these particular MMR and however, is not supported by our data, as suggested by the detec- BER members in SHM (19). Interestingly, another group of BER tion calls in the Affymetrix MAS 5.0, which still indicate a pres- enzymes displaying an induced expression in both GC B cell subsets ence of p53 transcript in all B cell subsets, also reported by others included OGG1, HFPG1 (NEIL1), and HFPG2 (NEIL3). These en- (16). The p53 restriction within the GC may, however, be ex- zymes are involved in the excision of mutagenic 7,8-dihydro-8-ox- plained by the presence of selective inhibitors of p53, such as oguanine formed or incorporated in the DNA during replication. The MDM2 and GTSE1, which are up-regulated in the two GC B cell presence of these enzymes was substantiated also by the induced subsets (Fig. 4D). These inhibitors affect both the trans-activating 8-oxo-dGTPase MTH1 (NUDT1) (23), which clears 7,8-dihydro-8- functions as well as the protein level of p53 (17, 18). oxoguanine from the nucleotide pool (Fig. 6D). The Journal of Immunology 4345

FIGURE 3. A, The behavior of the data set was eval- uated by Pearson correlation of replicate 1 (x-axis) vs replicate 2 (y-axis) for the following: 1, IgDϩ/CD23Ϫ/ CD38Ϫ/CD3Ϫ; 2, IgDϩ/CD23ϩ/CD38Ϫ/CD3Ϫ; 3, IgDϪ/CD38ϩ/CD77ϩ/CD3Ϫ; 4, IgDϪ/CD38ϩ/CD77Ϫ/ CD3Ϫ; and 5, IgDϪ/CD38Ϫ/CD3Ϫ sorted populations. The plots display normalized and background corrected robust multiarray averaged measures. The r values within each respective scatter plot show that the corre- lation coefficient was typically very high between rep- licates. B, Line graph representing the mean relative ex- pressional change (y-axis) of all 22,283 genes and expressed sequence tags across all populations, 1–5 (x- axis). Genes up-regulated in the GC are colored red; genes down-regulated in the GC are colored green. The general transcription pattern demonstrates a profound change in the transcriptional program for the GC sub- sets. This transcription program is prevailing, displaying Downloaded from no distinctive difference between the two GC B cell subsets. http://www.jimmunol.org/

The successive stage of BER involves either a short or a long polymerases (19). In our analysis, the long patch repair pathway, patch repair of the single strand break generated by the base ex- represented by DNA polymerase ␦ (POLD1 and POLD3), DNA cision, a step that also may include error-prone translesion DNA polymerase ␧ (POLE and POLE2), proliferating cell nuclear Ag by guest on September 28, 2021

FIGURE 4. Heat map representation of expression measures for B cell control genes (A), plasma cell signature (B), cell cycle (C), and DNA maintenance and checkpoint control (D) for the following: 1, IgDϩ/CD23Ϫ/CD38Ϫ/CD3Ϫ; 2, IgDϩ/CD23ϩ/CD38Ϫ/CD3Ϫ; 3, IgDϪ/CD38ϩ/CD77ϩ/CD3Ϫ; 4, IgDϪ/ CD38ϩ/CD77Ϫ/CD3Ϫ; and 5, IgDϪ/CD38Ϫ/CD3Ϫ sorted populations. Color range from Ͼ2-fold up-regulated (red) to Ͼ2-fold down-regulated (green) expression relative to the mean relative intensity for each gene. Genes are ordered based on a functional relationship within each group. 4346 CD77Ϫ B CELLS ARE A HETEROGENEOUS SUBSET OF CELLS

FIGURE 5. A, Cell cycle analysis of sorted CD38ϩ/IgDϪ/CD77ϩ, CD38ϩ/IgDϪ/CD77Ϫ, and CD38Ϫ/IgDϪ (memory) populations. The dia- grams represent 50,000 analyzed cells, assessed using three different anti-CD77 Ab clones: 38.13, 5B5, 6A2. The cell cycle analysis substantiates the expression data, demonstrating that both CD38ϩ/IgDϪ/CD77ϩ and CD38ϩ/IgDϪ/CD77Ϫ are actively cycling and proliferating. B, Tran- script levels of A4GALT (textured) and B3GALT3 (striped) across the following: 1, IgDϩ/CD23Ϫ/CD38Ϫ/CD3Ϫ; 2, IgDϩ/CD23ϩ/ Ϫ Ϫ Ϫ ϩ ϩ Ϫ

CD38 /CD3 ; 3, IgD /CD38 /CD77 /CD3 ; Downloaded from 4, IgDϪ/CD38ϩ/CD77Ϫ/CD3Ϫ; and 5, IgDϪ/ CD38Ϫ/CD3Ϫ sorted populations. The transcrip- tional analysis demonstrated that in contrast to A4GALT, involved in the conversion of lactacyl- cermide to CD77, the B3GALT3, implicated in the successive conversion of CD77 to P Ag, dis- played a several fold differential expression be- http://www.jimmunol.org/ tween the CD77ϩ and the CD77Ϫ population. by guest on September 28, 2021

(PCNA), flap endonuclease 1 (FEN1), and DNA ligase I (LIG1), Differential gene expression reveals a GC-specific marker showed a considerable induction in both the GC B cell subsets Although the majority of the functionally associated transcripts do (Fig. 6E). The short patch repair pathway, in contrast, was neither ϩ Ϫ not differ between CD77 and CD77 populations, several dif- induced nor repressed (Fig. 6E) and, thus, not indicating any spe- ferences did become apparent. cific linkage to SHM. Interestingly, this was also the case for other At first, the expressional regulation of the CD77 synthase factors that have been implicated in SHM, namely the error-prone (A14GALT) between the two GC B cell subsets did not offer any Y family of DNA polymerases, including Pol ␨ (REV3L) (24), Pol explanation to the differential display of the glycosphingolipid ␩ (POLH) (25), and Pol ␫ (POLI) (26), which all displayed no CD77 (Fig. 5B), as it is equally expressed in both CD77ϩ and association to the GC B cell subsets (Fig. 6B). In fact, apart from CD77Ϫ cells. However, looking into the regulation of other mem- the major replicative B family polymerases, the only polymerase bers in the globoside metabolic pathway (͗www.genome.jp/kegg/ that showed any induced expression in the GC B cell subsets was ␪ pathway/map/map00603.html͘) revealed that the differential dis- the A family member, polymerase (POLQ) (Fig. 6B), which is k interesting with reference to the recent association of polymerase play of CD77 (i.e., globotriaocylceramide: Gb3/P bloodgroup Ag) ␪ to SHM (27, 28). is likely to be mediated by a differential regulation of the enzyme As for the regulation of the repair pathways implicated in CSR, downstream of the CD77 synthase. The transcript of the down- stream enzyme, P synthase (B3GALT3), was in fact Ͼ2-fold the nonhomologous end joining members H2AX (H2AFX) and Ϫ DNA-PKcs PRKDC up-regulated in the CD77 population as compared with the ( ) (14) together with XRCC4 demonstrated an ϩ activation-induced expression seen in both GC B cell subsets (Fig. CD77 population (Fig. 4A), which is indicating that the dis- 6G), and the only members of the nucleotide excision repair path- play of CD77 is reflecting a transition stage, to become P Ag way that changed were the DDB2, which increased, and the XPG positive. Consequently, we also analyzed the presence of the P (ERCC5), which surprisingly decreased in the GC subsets (Fig. Ag (i.e., globoside: Gb4) by flow cytometry, and it was clear ϩ 6E). Noteworthy was that the H2AX (H2AFX), a component vital that P Ag is displayed on a majority of the CD38 B cells (Fig. in suppressing translocations and tumor formation (29, 30), was up 7A). In fact, the P Ag is displayed on a greater number of the ϩ to 5-fold up-regulated (Fig. 6G). CD38 cells than CD77 is, and it further appears that CD77 and Together, this demonstrates the need for active HR, MMR, and the P Ag can be displayed on the same cell (data not shown), BER programs not only in the CD77ϩ, but also in the CD77Ϫ indicating that the 2-fold expression of the P synthase in the population, further validating that these events are present in both CD77Ϫ cells reflects a fully developed ability to convert CD77 GC subpopulations. to P Ag. Although the P Ag is present on a majority of the The Journal of Immunology 4347 Downloaded from FIGURE 6. Heat map representation of expression measures for functional groups associated with DNA repair across the fol- lowing: 1, IgDϩ/CD23Ϫ/CD38Ϫ/CD3Ϫ; 2, IgDϩ/CD23ϩ/CD38Ϫ/CD3Ϫ; 3, IgDϪ/ CD38ϩ/CD77ϩ/CD3Ϫ; 4, IgDϪ/CD38ϩ/ Ϫ Ϫ Ϫ Ϫ Ϫ

CD77 /CD3 ; and 5, IgD /CD38 /CD3 http://www.jimmunol.org/ sorted populations. The functional groups present: homologous recombination repair (A), DNA polymerase (B), mismatch repair (C), base excision repair (D), short and long patch repair (E), nucleotide excision repair (F), and non-homologous end joining (G). Color range from Ͼ2-fold up-regulated (red) to Ͼ2-fold down-regulated (green) expres- sion relative to the mean relative intensity for each gene. Genes are ordered based on a by guest on September 28, 2021 functional relationship within each group.

CD38ϩ cells, a fraction is still negative for the P Ag (depicted expression (Fig. 4B). Interestingly, concomitant with the Ig gene as yellow in Fig. 7A). The implication of this is that the P Ag expression, the plasma cell-specific XBP1 (31, 32) was shown to may constitute a specific GC B cell marker, distinguishing be up-regulated in the CD77Ϫ subset (Fig. 4B). This was also true CD38ϩ GC B cells from CD38ϩ non-GC B cells, i.e., the P for the plasma cell-specific Blimp1 (PRDM1), as well as several Ag-negative cells. members of the unfolded protein response, such as GRP78 The expressional analysis of Ig genes revealed a clear-cut reg- (HSPA5), GRP94 (TRA1) (33), ARMET, HSP70 (HSPA4), p58 ulatory diversion between the CD77ϩ and the CD77Ϫ populations. (DNAJC3), ERdj4 (DNAJB9), RAMP4 (SERP1), P5 (34), and two Within the CD77ϩ cells, the Ig gene expression was typically low, other transcripts previously reported in tonsillar plasma cells, whereas the CD77Ϫ cells had a significantly up-regulated Ig gene STHC and FRZB (35) (Fig. 4B). 4348 CD77Ϫ B CELLS ARE A HETEROGENEOUS SUBSET OF CELLS

7B). Thus, this population contains cells actively involved in re- combination and transcription, in which the difference in size re- flects the DNA content. The CD77Ϫ population had the same cel- lular composition of both small and large cells having decondensed chromatin. However, in addition, this population also contained cells with a plasmacytoid appearance (Fig. 7B). Taken together, this morphological analysis is supporting the findings, from both the transcriptional analysis as well as the cell cycle analysis, in that both the CD77ϩ as well as the CD77Ϫ populations contain actively cycling cells. Furthermore, the CD77Ϫ population contains cells with an already initiated plasma cell differentiation program.

Discussion A prevailing concept has for many years been that the human GC can be subdivided based on the presence of the glycosphingolipid CD77. The generally accepted view of the GC and its functional

contributors has lately been challenged (36), and it has been ques- Downloaded from tioned whether the unique GC functionalities can be spatially sep- arated in a dark and light zone, as suggested. In this study, we have looked into the transcriptional regulation of the five mature human B cell subsets, defined by Liu et al. (1–3), with a special focus on the GC B cell subsets.

Our analysis demonstrates that a profound change occurs in the http://www.jimmunol.org/ transcriptional program as the B cell enters the GC (see Fig. 3B), which is the point in which the cells start to rapidly proliferate, expand, and go through a process of affinity maturation. These events are manifested in the transcriptional program of the GC B cells and are unexpectedly shared between both the CD77ϩ and the CD77Ϫ populations, previously suggested to be the centroblasts and centrocytes, respectively. Flow cytometric analysis of DNA content, as well as the transcriptional regulation of cell cycle me- diators, demonstrates that both these populations are highly pro- by guest on September 28, 2021 liferative and that the mitotic program activates several other sys- tems required for a correct passage through the cell cycle. This is evident by the induced expression of factors participating in DNA maintenance checkpoints and DNA repair. Apart from the defined plasma cell program seen in the CD77Ϫ population, very small differences are disclosed in the transcrip- tional regulation between the proposed centroblast population and FIGURE 7. A, Three-dimensional dot plot of P Ag, CD38, and IgD the centrocyte population. Both populations show an equivalent displaying naive cells (IgDϩ/CD38Ϫ/P AgϪ) in red, GC cells (IgDϪ/ ϩ ϩ Ϫ Ϫ Ϫ proliferation status, and components involved in DNA mainte- CD38 /P Ag ) in blue, memory cells (IgD /CD38 /P Ag ) in orange, nance checkpoints and repair are present at similar transcript lev- plasma blasts (IgDϪ/CD38ϩ/P AgϪ) in yellow, and plasma cells (IgDϪ/ ϩ Ϫ els. Furthermore, components coupled to SHM and CSR reveal no CD382 /P Ag ) in purple. Flow cytometric analysis of the presence of functional difference between the two subsets. surface P Ag revealed that this marker discriminates a large and distinctive ϩ ϩ The basis for the definition of the centroblast as CD77 is de- subset of cells from the CD38 population (blue). B, Morphologic analysis ϩ of May-Grunwald/Giemsa-stained cytospin preparations of sorted IgDϩ/ rived from the observation of CD77 cells in the GC dark zone, CD38Ϫ/CD3Ϫ cells (naive, upper right), IgDϪ/CD38ϩ/CD77ϩ/CD3Ϫ cells referring to stained tissue sections (1, 9). If correlating this tissue- (CD77ϩ, lower right), and IgDϪ/CD38ϩ/CD77Ϫ/CD3Ϫ cells (CD77Ϫ, left) staining pattern to the traditional GC definition by Hanna (37), at ϫ100 magnification. All cells are at scale. The morphological analysis who stated that most lymphoid cells in the light zone of the GC are ϩ revealed that the CD77 population consists of large and small cells with slowly proliferating derivatives from the actively cycling precur- Ϫ decondensed chromatin. The CD77 population had a similar content of sors in the dark zone, it could suggest that all GC B cells lacking small and large cells (lower left), but did also contain cells with a plasma- CD77 are nonproliferating light zone centrocytes. On the basis of cytoid appearance (upper left). this categorization, it was suggested that CSR occurs after the on- set of SHM (3), because both CD77ϩ as well as the CD77Ϫ cells

ϩ Ϫ had accumulated a large number of somatic mutations (2). How- Morphological analysis of CD77 and CD77 cells reveals a ever, SHM has never yet been demonstrated to exclusively occur cellular heterogeneity in the CD77ϩ population despite several characterizations of this To further validate our finding, we sorted naive, CD77ϩ, and population (2, 3, 5, 38). CD77Ϫ cells and analyzed the general morphology of these cells in This indicates that a functional dichotomy coupled to the CD77 each population. This analysis revealed that the CD77ϩ population display cannot be made, and that CD77ϩ cells are not, necessarily, contained both large and small lymphocytes, with a typical decon- the only cells to participate in SHM or to make up the GC dark densed chromatin structure, as compared with naive cells (Fig. zone. In fact, both subsets may participate in the SHM and CSR The Journal of Immunology 4349 programs, because they are both expressing AID, UNG, and sev- 11. Trimarchi, J. M., and J. A. Lees. 2002. Sibling rivalry in the E2F family. Nat. eral other transcripts related to these processes. Two other findings Rev. Mol. Cell Biol. 3: 11–20. 12. Jacobs, J. J., and M. van Lohuizen. 2002. Polycomb repression: from cellular in support of our conclusion are as follows: first, the observations memory to cellular proliferation and cancer. Biochim. Biophys. Acta 1602: that both CD77ϩ and CD77Ϫ cells are actively cycling; second, 151–161. 13. Raaphorst, F. M., F. J. van Kemenade, E. Fieret, K. M. Hamer, D. P. Satijn, the observation that both these populations express ENX and EED, A. P. Otte, and C. J. Meijer. 2000. Cutting edge: polycomb gene expression and lack BMI-1. The polycomb genes ENX and EED have, in patterns reflect distinct B cell differentiation stages in human germinal centers. tissue sections, been demonstrated to separate typical dark zone J. Immunol. 164: 1–4. 14. Hoeijmakers, J. H. 2001. Genome maintenance mechanisms for preventing can- centroblasts from the light zone centrocytes, which, in contrast are cer. Nature 411: 366–374. BMI-1 positive (13). This separation between the centroblast, as 15. Phan, R. T., and R. Dalla-Favera. 2004. The BCL6 proto-oncogene suppresses ENX and EED positive, and centrocytes, as BMI-1 positive, is not p53 expression in germinal-centre B cells. Nature 432: 635–639. 16. Martinez-Valdez, H., C. Guret, O. de Bouteiller, I. Fugier, J. Banchereau, and seen in our analysis. Thus, to separate dark zone centroblasts and Y. J. Liu. 1996. Human germinal center B cells express the apoptosis-inducing light zone centrocytes, other markers are needed, and in this re- genes Fas, c-myc, P53, and Bax but not the survival gene bcl-2. J. Exp. Med. 183: spect, the distinctive staining pattern with anti-P Abs needs to be 971–977. 17. Meek, D. W. 2004. The p53 response to DNA damage. DNA Repair 3: further elucidated. 1049–1056. In summary, we have in this study, based on microarray data 18. Monte, M., R. Benetti, G. Buscemi, P. Sandy, G. Del Sal, and C. Schneider. 2003. and flow cytometry, revealed that CD77ϩ and CD77Ϫ cells lack The cell cycle-regulated protein human GTSE-1 controls DNA damage-induced apoptosis by affecting p53 function. J. Biol. Chem. 278: 30356–30364. transcription programs that can be associated with functional char- 19. Petersen-Mahrt, S. K., R. S. Harris, and M. S. Neuberger. 2002. AID mutates E. acteristics of centroblasts and centrocytes. Furthermore, we found coli suggesting a DNA deamination mechanism for diversification. Na- that both populations are actively cycling, which is a description ture 418: 99–103. Downloaded from Ϫ 20. Zan, H., X. Wu, A. Komori, W. K. Holloman, and P. Casali. 2003. AID-depen- not fitting with the accepted definitions of centrocytes. The CD77 dent generation of resected double-strand DNA breaks and recruitment of Rad52/ population also contains cells with an already initiated plasma cell Rad51 in somatic hypermutation. Immunity 18: 727–738. 21. Manis, J. P., D. Dudley, L. Kaylor, and F. W. Alt. 2002. IgH class switch re- differentiation program, and finally, we demonstrated that P Ag combination to IgG1 in DNA-PKcs-deficient B cells. Immunity 16: 607–617. constitutes a GC-specific B cell marker. Based on this, we con- 22. Wu, X., P. Geraldes, J. L. Platt, and M. Cascalho. 2005. The double-edged sword clude that CD77 is not a reliable marker discriminating centro- of activation-induced cytidine deaminase. J. Immunol. 174: 934–941. ϩ Ϫ 23. Meyer, F., E. Fiala, and J. Westendorf. 2000. Induction of 8-oxo-dGTPase ac- blasts and centrocytes, and further that the CD38 CD77 popu- tivity in human lymphoid cells and normal fibroblasts by oxidative stress. Tox- http://www.jimmunol.org/ lation is heterogeneous, comprising several different cells ranging icology 146: 83–92. from proliferating centroblasts to Ig-producing plasma blasts. 24. Zan, H., A. Komori, Z. Li, A. Cerutti, A. Schaffer, M. F. Flajnik, M. Diaz, and P. Casali. 2001. The translesion DNA polymerase ␨ plays a major role in Ig and bcl-6 somatic hypermutation. Immunity 14: 643–653. Acknowledgments 25. Yavuz, S., A. S. Yavuz, K. H. Kraemer, and P. E. Lipsky. 2002. The role of We thank Ann-Charlotte Olsson for expert technical assistance; Dr. Martin polymerase ␩ in somatic hypermutation determined by analysis of mutations in L. Olsson (Division of Hematology and Transfusion Medicine, Lund Uni- a patient with xeroderma pigmentosum variant. J. Immunol. 169: 3825–3830. 26. Faili, A., S. Aoufouchi, E. Flatter, Q. Gueranger, C. A. Reynaud, and J. C. Weill. versity, Lund, Sweden) for helpful discussions on the P Ag; and Ingbritt 2002. Induction of somatic hypermutation in immunoglobulin genes is dependent Åstrand-Grundstro¨m (Hemopoietic Stem Cell Laboratory, Lund Univer- on DNA polymerase ␫. Nature 419: 944–947.

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Ho¨gerkorp, C.-M., and C. A. K. Borrebaeck. 2006. The human CD77Ϫ B cell population represents a heterogeneous subset of cells comprising centroblasts, centrocytes, and plasmablasts, prompting phenotypical revision. J. Immunol. 177: 4341–4349.

In Results, under the heading Phenotypic B cell markers correspond to mRNA expression, in the second paragraph, sentence four, the designation “B cell line (BCL)6” is incorrect. The corrected sentence is shown below. As expected, BCL6 was found to be typically linked to GC B cell subsets accompanied by a strong down- regulation of BCL2 (Fig. 4A).

Under the heading CD77Ϫ cells share the CD77Ϫ cell proliferation program, in sentence four, six, and seven, cyclin D3, E1, E2, A2, B1, B2, p27Kip, p18, and BMI1 should be italicized. The corrected sentences are shown below. Genes, including cyclin D3 (CCND3), E1 (CCNE1), E2 (CCNE2), A2 (CCNA2), B1 (CCNB1), and B2

(CCNB2), all regulators of the G1-S, S, and G2-M phase transitions, were expressed in both of these subsets.... Furthermore, the inhibitors of CDK2, p21Cip (CDKN1A) and p27Kip (CDKN1B), were effectively down-regu- lated, and among the inhibitors of CDK4 class of proteins (INK4) only p18 (CDKN2C) displayed an increased expression in the GC B cell subsets. The members of the polycomb group of genes, ENX and EED, involved in proliferation (12) were also highly up-regulated in the GC B subsets, whereas BMI1 was equally significantly down-regulated (Fig. 4C) in both subsets (13).

Under the heading The GC genomic integrity and DNA maintenance programs are active in both the CD77Ϫ and CD77Ϫ population, in sentence five, p53 (TP53) should be italicized. Notable was that p53 (TP53), another target of ATM, displayed a baseline expression pattern across all B cell subsets.

Under the heading Transcriptional regulation of SHM and CSR does not separate CD77Ϫ and CD77Ϫ, in the first paragraph, sentence five, “MutS homologue 2 (MSH2), MutS homologue 6 (MSH6)” are incorrect; and EXO1 and UNG should be italicized. In the first sentence of the third paragraph, H2AX, XRCC4 DDB2, and XPG should be italicized. The corrected sentences are shown below. This transcriptional regulation was seen also among components participating in MMR, such as the MSH2, MSH6, and EXO1 (Fig. 6C), as well as for the BER enzyme UNG (Fig. 6D), which is noteworthy considering the specific implication of these particular MMR and BER members in SHM (19). As for the regulation of the repair pathways implicated in CSR, the nonhomologous end joining members H2AX (H2AFX) and DNA-PKcs (PRKDC) (14) together with XRCC4 demonstrated an activation-induced expression seen in both GC B cell subsets (Fig. 6G), and the only members of the nucleotide excision repair pathway that changed were the DDB2, which increased, and the XPG (ERCC5), which surprisingly decreased in the GC subsets (Fig. 6E).

Kawasaki, T., W. J. Hubbard, M. A. Choudhry, M. G. Schwacha, K. I. Bland, and I. H. Chaudry. 2006. Trauma- hemorrhage induces depressed splenic dendritic cell functions in mice. J. Immunol. 177: 4514–4520.

Two authors’ names were inadvertently omitted from the article. The corrected author and affiliation lines are shown below.

Takashi Kawasaki,* Satoshi Fujimi,† James A. Lederer,† William J. Hubbard,* Mashkoor A. Choudhry,* Martin G. Schwacha,* Kirby I. Bland,* and Irshad H. Chaudry*

*Center for Surgical Research and Department of Surgery, University of Alabama, Birmingham, AL 35294; and †Department of Surgery (Immunology), Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115