Identification and Characterization of a Human CD5 + Pre-Naive Population Jisoo Lee, Stefan Kuchen, Randy Fischer, Sooghee Chang and Peter E. Lipsky This information is current as of September 23, 2021. J Immunol 2009; 182:4116-4126; ; doi: 10.4049/jimmunol.0803391 http://www.jimmunol.org/content/182/7/4116 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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identification and Characterization of a Human CD5؉ Pre- Population1

Jisoo Lee,*† Stefan Kuchen,† Randy Fischer,† Sooghee Chang,‡ and Peter E. Lipsky2†

We have identified a distinct pre-naive B cell population circulating in human peripheral that exhibits an intermediate phenotype between transitional and naive B cells. Like human transitional B cells, these cells express CD5 but have intermediate densities of CD38, CD10, CD9, and the ABCB1 transporter compared with transitional and naive B cells. These pre-naive B cells account for a majority of circulating human CD5؉ B cells. Importantly, CD5؉ pre-naive B cells could be induced to differentiate into cells with a naive phenotype in vitro. CD5؉ pre-naive B cells show only partial responses to BCR stimulation and CD40 ligation and undergo more spontaneous apoptosis and cell death than do naive B cells, whereas BAFF/BLyS (B cell-activating factor belonging to the TNF family) did not enhance their survival compared with naive B cells. In contrast, CD5؉ pre-naive B cells carry out certain functions comparable to naive B cells, including the capacity to differentiate into plasma cells and the ability Downloaded from to function as APCs. Notably, an increased proportion of CD5؉ pre-naive B cells were found in peripheral blood of patients with systemic lupus erythematosus. These results have identified a unique intermediate in human naive B cell development within the peripheral blood and derangements of its homeostasis in patients with systemic lupus erythematosus. The Journal of Immunol- ogy, 2009, 182: 4116–4126.

he BCRs are generated by random recombination of Ig V, B cell tolerance checkpoints remove self-reactive B cells that http://www.jimmunol.org/ D, and J gene segments during B cell development (1). arise during development. However, the presence of certain auto- T The randomness of the process generates BCRs that have reactive B cells in normal individuals is revealed by the identifi- affinity for self Ags. However, most of the autoreactive B cells are cation of serum polyreactive referred to as natural eliminated or silenced at several tolerance checkpoints during B Abs (9, 10). Persistence of autoreactive B cells that secrete natural cell maturation, and only B cells that survive the checkpoints can Abs seems paradoxical to the concept of B cell tolerance, and it continue on in the maturation process. To develop into a naive B raises the possibility that autoantibodies may perform some useful cell, a B cell must survive at least two checkpoints. The first check- functions in immune regulation. point is at the immature B cell stage in the where In mice, natural Ab-secreting B cells belong to a specific B cell by guest on September 23, 2021 most B cells that react with autoantigens present in the bone mar- lineage called B-1 B cells, which reside in the peritoneal cavity (11). row are lost by mechanisms that include deletion, anergy, or re- B-1 B cells were identified initially by their expression of the CD5 ceptor editing (2–5). The second checkpoint is in the periphery surface glycoprotein, but subsequently CD5Ϫ B cells similar to B-1 B before transition of new emigrant immature B cells into the mature cells were found, and, thus, CD5ϩ B-1 cells are referred to as B-1a naive B cell subset. Tolerance mechanisms operating at the sec- cells, and CD5Ϫ B-1 cells as B-1b cells (11, 12). B-1 B cells have ond checkpoint are less clear, although anergy and deletion been shown to participate in thymus-independent type 2 responses by have been proposed as major mechanisms (6–9). Failure to re- rapid production of Ab before induction of adaptive immune re- move autoantibodies at either stage would increase the number sponses (13–15). Like murine B-1a cells, human peripheral blood of circulating self-reactive naive B cells that can participate in CD5ϩ B cells have been reported to produce polyspecific autoreactive immune responses, and can result in increased susceptibility to Ab, such as rheumatoid factor and anti-ssDNA (16, 17). However, . human CD5ϩ B cells have not been clearly characterized, and whether these cells are equivalent to B-1a cells in mice is unclear. In humans, most of the mature B cells in fetal circulation express CD5 *Division of Rheumatology, Department of Internal Medicine, Ewha Womans Uni- (18), but they decrease to 11–49% in the adult circulation (19). Re- versity School of Medicine, Seoul, Korea; †Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, cently, human transitional T1 B cells were shown to express CD5, Bethesda, MD 20892; and ‡Rheumatism Research Center, Catholic University Col- although the murine counterpart does not express this molecule (20). lege of Medicine, Seoul, Korea However, human transitional B cells account for only 1–2% of total Received for publication October 9, 2008. Accepted for publication January 28, ϩ 2009. circulating B cells, suggesting the existence of other CD5 B cell subsets. 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 Since human transitional B cells express the CD5 molecule, it is with 18 U.S.C. Section 1734 solely to indicate this fact. possible that CD5 may serve some functional role during peripheral B 1 This research was supported in part by the National Institute of Arthritis and Mus- cell maturation. During a systemic analysis to address this issue, we culoskeletal and Skin Diseases, National Institutes of Health. J.L. was supported by have identified a distinct pre-naive B cell population in human pe- the Korea Research Foundation Grant KRF-2005-331-E00123. S.K. was supported by the Jean et Linette Warnery Foundation and the Swiss Foundation for Medical-Bio- ripheral blood that exhibits an intermediate phenotype between tran- logical Scholarships. sitional and naive B cells. Functionally, pre-naive B cells exhibited 2 Address correspondence and reprint requests to Dr. Peter E. Lipsky, National In- some B cell functions in common with naive B cells, including the stitute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Building 10, Room 6D47C, Bethesda, MD 20892. E-mail address: capacity to differentiate into plasma cells and Ag presentation, [email protected] whereas they were distinctly more prone to undergo apoptosis and www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803391 The Journal of Immunology 4117 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. Characterization of CD27ϪCD38intIgDϩ B cells. A, Peripheral blood CD19ϩCD27Ϫ B cells were gated into CD38low (naive), CD38int (intermediate), and CD38high (transitional) B cell subsets and the differential expression of CD5 was assessed. Numbers represent mean fluorescence intensity of each population (naive, intermediate, and transitional B cells noted from left to right). Data are representative results from a total of 12 donors. B, Expression of a variety of B cell markers by CD27ϪCD38lowIgDϩ naive, CD27ϪCD38intIgDϩ intermediate, and CD27ϪCD38highIgDϩ populations. Numbers represent mean fluorescence intensity of each population (naive, intermediate, and transitional B cells noted from left to right). Data are representative results from a total of 12 donors. The mean (ϮSE) frequencies of naive B cells, intermediate B cells, and transitional B cells of the total CD19ϩ B cells from the 12 donors were 63.0 Ϯ 2.8% (range, 44–82%), 7.0 Ϯ 0.9% (range, 3–16%), and 2.0 Ϯ 2.9% (range, 1–4%), respectively. C, Expression of the ABCB1 transporter was assessed by R123 dye extrusion. CD27ϪCD38intIgDϩ intermediate B cells express an intermediate degree of ABCB1 transporter activity between CD27ϪCD38lowIgDϩ naive B cells and CD27ϪCD38highIgDϩ transitional B cell populations. Data are represen- tative results from three donors. 4118 HUMAN CD5ϩ PRE-NAIVE B CELL POPULATION IDENTIFIED Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. CD27ϪCD38intIgDϩ intermediate B cells are enriched in cord blood, but not in normal bone marrow. A, Adult blood and cord blood B cells were assessed for the distribution of transitional, intermediate, and naive B cells after staining for expression of CD19, CD27, IgD, and CD38. B, B cells from bone marrow of a normal donor were stained with anti-CD19, anti-CD27, anti-CD38, anti-IgD, anti-CD20, anti-IgM, and anti-CD5 to examine the frequency of naive, intermediate, transitional, immature, and pro- and pre-B cells and the differential expression of CD5 by each population. responded poorly to ligation of either CD40 or surface IgM. Of in- Materials and Methods terest, this pre-naive B cell population accounted for the majority of Cell preparation circulating human CD5ϩ B cells, and an increase in the proportion of Blood samples are obtained from healthy adult donors of various ages, this population was found in peripheral blood of patients with sys- 3 sexes, and races (39 from the National Institutes of Health, 10 from Ewha temic lupus erythematosus (SLE). Our data suggest that increased Womans University) and from 6 patients with active SLE (all from Seoul, production or lack of further maturation of these pre-naive B cells can Korea). The mean (ϮSD) ages of the normal donors were 41 Ϯ 14 (range, occur in autoimmune diseases, and may contribute to the development 20–68) years, and 22 men and 27 women were included. SLE patients Ϯ Ϯ of autoimmunity. were all women, and the mean ( SD) age was 35 10 (range, 26–50) years. At the time of the blood collection, three patients were untreated, and the other three were treated with hydroxychloroquine with or without low- 3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; BAFF/BlyS, dose glucocorticoids. Bone marrow aspirates were obtained from healthy B cell-activating factor belonging to the TNF family. donors. Cord blood was obtained from healthy newborns. These studies The Journal of Immunology 4119 have been approved by the National Institute of Arthritis and Musculo- 37°C. R123 stained cells were chased for 3 h and analyzed for green flu- skeletal and Skin Diseases Institutional Review Board and the Ethics Com- orescence by flow cytometry. mittee of Ewha Womans University School of Medicine, and informed consent was obtained according to the Declaration of Helsinki. B cells and Determination of Ig levels ϩ CD4 T cells were enriched by negative selection from buffy coats or Secreted Ig in the culture supernatant was quantitated by ELISA. Briefly, leukapheresis using RosetteSep or StemSep B cell purification Ab cocktails MaxiSorp 96-well ELISA plates (Nalge Nunc International) were coated following the manufacturer’s instructions (StemCell Technologies). overnight at 4°C with 1 ␮g/ml (50 ng/well) affinity-purified goat anti- Flow cytometry and cell sorting human IgG-Fc, IgM, or IgA Abs (all Bethyl Laboratories) in PBS (pH 7.4), washed with PBS/Tween, and incubated with blocking solution (0.2% BSA PBMC, enriched B cells, and postculture B and subsets were in PBS, pH 7.4) for3hatroom temperature. B cell culture supernatants stained with various mAb combinations for 20 min on ice in staining and standards were diluted in blocking solution and incubated on coated buffer (PBS with 1% BSA). The directly conjugated mAb used were plates overnight at 4°C. Normal human IgG, IgM (both Caltag Laborato- anti-IgD-FITC, anti-CD27-FITC, anti-CD10-PE, anti-CD95-PE, anti- ries/Invitrogen) and human reference serum (Bethyl Laboratories) for IgA CD4-PerCP-Cy5.5, anti-CD38-PE-Cy7, anti-CD20-allophycocyanin- ELISA were used as standards. Culture supernatants and standards were Cy7, anti-CD11b-allophycocyanin-Cy7, anti-CD69-allophycocyanin-Cy7 (all removed by vacuum aspiration and plates were washed three times with BD Biosciences), anti-CD21-PE, anti-CD5-PE, anti-HLA-DR-PE, anti- PBS/Tween. Alkaline phosphatase-conjugated affinity-purified goat anti- CD19-PE-Texas Red, anti-CD19-PE-Cy5.5 (all Caltag Laboratories). Anti- human IgG, IgM, or IgA (all Bethyl Laboratories) in blocking solution CD44-Alexa Fluor 647, anti-BAFF-R-Alexa Fluor 647, anti-CD9-Alexa (200 ng/ml, 50 ␮l/well) were added as secondary Abs and incubated over- Fluor 647, anti-CD38-Alexa Fluor 647, and anti-CD27-Alexa Fluor 647 night at 4°C. SigmaFast (Sigma-Aldrich) was used as alkaline phosphatase were labeled from purified mAbs (BD Biosciences) using the Alexa Fluor substrate and specific absorbance measured at 410 nm using a PowerWave 647 mAb labeling kit (Molecular Probes/Invitrogen). Anti-IgM-Pacific X 96-well plate reader (Bio-Tek Instruments) and KC4 version 3.1 soft- Blue, anti-CD27-Pacific Blue, anti-IgD-Pacific Blue, and anti-CD19- ware (Bio-Tek Instruments). Pacific Blue were prepared from purified mAbs (BD Biosciences) using the Downloaded from Pacific Blue 647 mAb labeling kit (Molecular Probes/Invitrogen). An- Statistics nexin-V-allophycocyanin (Caltag Laboratories) and the Live/Dead fixable Statistical analysis was performed using JMP 7. Comparison between two violet dead cell staining kit (Invitrogen) were used to assess apoptosis and groups was performed using unpaired Student’s t test, and comparison cell death. Stained cells were washed and fixed in 1% paraformaldehyde, between different conditions was performed using one-way analysis (error and data were collected by performing eight-color flow cytometry using the bars represent SEM values). The Mann-Whitney U nonparametric test was CyAn multiparameter flow cytometer (Dako). Data were analyzed using used for comparisons between patients and controls (data are expressed as FlowJo software (Tree Star). B cell populations were sorted using the Dako means Ϯ SD). For analysis of correlation between variables, a linear re- http://www.jimmunol.org/ MoFlo cell sorter. gression analysis was performed. Values of p Ͻ 0.05 were considered .(p Ͻ 0.005 ,ء ;p Ͻ 0.05 ,ء) Cell culture statistically significant B cell populations were cultured in 96-well plates at 3 ϫ 104 cells/ml in Results 100-␮l volumes of culture medium (RPMI 1640 with 10% FCS, Gluta- CD27ϪCD38intIgDϩ intermediate B cells comprise a distinct MAX, and gentamicin) at 37°C alone or supplemented with various cyto- kines and stimuli: IL-21 (200 ng/ml; R&D Systems), IL-4 (100 ng/ml; B cell subset R&D Systems), IL-10 (200 ng/ml; R&D Systems), BAFF (200 ng/ml; We examined in detail the human CD27ϪIgDϩ B cell population R&D Systems), anti-CD40 (10 ␮g/ml), and goat F(abЈ) anti-IgM (10 ␮g/ ϩ 2 in the peripheral blood to search for a CD5 B cell population

ml; Jackson ImmunoResearch Laboratories). To induce in vitro B cell dif- by guest on September 23, 2021 ferentiation, B cells were cocultured with human CD4ϩ T cells activated other than transitional B cells. Previous studies revealed that tran- by immobilized anti-CD3 as described previously (21). In brief, 96-well sitional cells and naive B cells in humans could be distinguished flat bottom plates were coated with 10 ␮g/ml anti-CD3 clone UCHT1 (BD by expression of CD38, in that transitional B cells expressed high ϩ Biosciences) overnight at 37°C in Tris buffer (50 mM (pH 7.4)). CD4 T levels of CD38 compared with low levels in naive B cells (20). cells were purified as described above and treated with a final concentration of 30 ␮g/ml mitomycin C (Sigma-Aldrich) for 30 min at 37°C. A total of This suggested that the CD38 expression profile might serve as a 1 ϫ 105 mitomycin C-treated CD4ϩ B cells and 3 ϫ 104 sorted B cell marker of peripheral B cell maturation. We therefore performed populations were cultured together in duplicates or triplicates in 200 ␮lon multicolor flow cytometric analysis of peripheral blood B cells the anti-CD3-precoated microtiter wells. including staining for CD19, CD27, CD38, and IgD expression. As ϩ Ϫ Cell proliferation and functional assays shown in Fig. 1A, CD19 CD27 B cells can be subdivided by low 7 CD38 expression into previously described CD38 (naive) and To determine proliferation, purified B cells (2–5 ϫ 10 cells/ml) were high labeled with CFSE (Molecular Probes) at a final concentration of 2.5 mM CD38 (transitional) B cell populations. Additionally, we ob- for 8 min and cultured in medium supplied with various stimuli: anti-CD40 served another B cell subpopulation that expresses intermediate ␮ Ј ␮ (100 g/ml) and goat F(ab )2 anti-IgM (10 g/ml) for 48 or 72 h. Prolif- levels of CD38. When differential expression of CD5 was assessed eration was analyzed by dilution of CFSE. CFSE-labeled B cells cocultured Ϫ int ϩ ϩ on these populations, CD27 CD38 IgD (intermediate) B cells with human CD4 T cells activated by immobilized anti-CD3 as described expressed CD5, as did CD27ϪCD38highIgDϩ transitional B cells, above were cultured for 96–144 h and analyzed for CFSE dilution. To and accounted for the majority of circulating CD5ϩ B cells (87% determine B cell-induced allogeneic T cell proliferation, CFSE-labeled ϩ negatively selected CD4 T cells (1 ϫ 105) were cultured alone or with an of total CD5 B cells). Of the total 12 normal donors examined, equal number of purified B cells with or without anti-CD40 (10 ␮g/ml) for the mean (ϮSE) frequency of naive B cells was 63 Ϯ 2.8% (range, 96 h, and the percentage of divided T cells was assessed. 44–82%), intermediate B cells 7 Ϯ 0.9% (range, 3–16%), and For analysis of calcium signaling, purified B cells were stained with ϩ transitional B cells 2 Ϯ 2.9% (range, 1–4%) of the total CD19 B anti-CD20-allophycocyanin-Cy7, anti-CD38-PECy-7, and anti-CD27- Ϫ int ϩ Pacific Blue, resuspended in HBSS containing 1 mM Ca and Mg and 1% cells. CD27 CD38 IgD intermediate B cells accounted for a ϩ Ϫ FCS at a concentration of 2 ϫ 106 cells/ml, and loaded with Fluo-3 AM mean (ϮSE) of 10 Ϯ 1% of the total circulating CD19 CD27 B (Molecular Probes; final concentration 4 ␮g/ml) for 30 min at 37°C in the cells. presence of probenecid (Sigma-Aldrich; final concentration 4 mM). ϩ Ϫ high ϩ Ϫ ϩ Using eight-color flow cytometry, we determined phenotypic CD20 CD27 CD38 and CD20 CD27 CD38 B cells were gated and Ϫ int ϩ calcium responses were measured using a CyAn flow cytometer (Dako) by characteristics of the CD27 CD38 IgD intermediate B cells Fluo-3 fluorescence at 515–535 nm using linear amplification over 4–5 (Fig. 1B). When expression of a variety of B cell markers was ␮ Ј min. After 30 s, 10 g/ml F(ab )2 goat anti-human IgM was added for BCR analyzed, intermediate B cells displayed similar levels of expres- crosslinking. Intensity of Fluo-3 fluorescence was analyzed using FlowJo sion of most B cell markers including CD23, CD21, CD24, CD44, software (TreeStar). IgM, CD95, and CD11b as naive B cells. Both naive B cells and To determine ABCB1 transporter activity, PBMCs were prestained with Ϫ int ϩ anti-CD20-allophycocyanin-Cy7, anti-CD38-PECy-7, and anti-CD27- CD27 CD38 IgD intermediate B cells could be distinguished Pacific Blue and stained with R123 (Invitrogen) at 12.5 ␮M for 10 min at from transitional B cells by expression of CD23, CD21, CD24, 4120 HUMAN CD5ϩ PRE-NAIVE B CELL POPULATION IDENTIFIED Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Intermediate B cells are functionally distinct. A, The CD27ϪCD38low B cell population represents the naive B cell subset, and the CD27ϪCD38int B cell population represents the intermediate B cell population. CD5 and IgD expression by each B cell population is shown. B, Proliferative responses of purified B cell subsets after incubation for 48 h and 72 h in medium alone, or with anti-IgM or anti-CD40. Proliferation was analyzed by dilution of CFSE and is representative of results from three experiments. C,Ca2ϩ mobilization of intermediate and naive B cells after crosslinking of BCR with 15 ␮g/ml anti-IgM. Ca2ϩ flux was measured by flow cytometry after loading cells with Fluo-3. D, Spontaneous apoptosis and cell death were analyzed in intermediate and naive B cells immediately ex vivo and after 48 h of culture. Apoptosis was measured by the frequency of annexin V-positive cells, excluding Live/Dead-binding cells. Cell death p Ͻ 0.01 between naive and intermediate ,ءء .was measured by the frequency of Live/Dead-binding cells. Data shown are the means Ϯ SEM of three experiments B cells. E, Intermediate and naive cells were cultured in medium alone or with either the addition of anti-IgM or anti-CD40. After 48 h, apoptosis and cell death p Ͻ ,ءء p Ͻ 0.05 and ,ء .were assessed by staining the cells with annexin V and Live/Dead as described in D. Data are the means Ϯ SE of three experiments 0.01 between culture medium alone and anti-IgM or anti-CD40. F, Sorted cells were cultured with medium alone or with various cytokines, including BAFF, IL-4, p Ͻ 0.01 between ,ءء .IL-21, and IL-10. After 48 h, apoptosis and cell death were assessed as described in D. Data are the means Ϯ SEM of three experiments cultures with medium alone and various cytokines. The Journal of Immunology 4121 Downloaded from http://www.jimmunol.org/

FIGURE 4. Intermediate B cells can develop into naive B cells. A, Sorted CD27ϪCD38int B cells were cultured with mitomycin C-treated CD4ϩ T cells in anti-CD3-precoated microtiter wells and assessed for CD5 expression and CFSE dilution following 4 days and 6 days of culture. Data are representative of three experiments with similar results. B, CD5ϩCD27ϪCD38low B cells were sorted and cultured with anti-CD40 and IL-21. CD5 expression and CFSE dilution were assessed after 4 days and 6 days of culture. Data are representative of three experiments with similar results. by guest on September 23, 2021

BAFF-R, CD44, IgM, CD95, CD11b, and CD86. However, the population of CD20ϩCD38intCD27Ϫ B cells comparable to the expression of B cell maturation markers CD9 and CD10 by CD19ϩCD38intCD27ϪIgDϩ B cells previously analyzed. As also CD27ϪCD38intIgDϩ B cells was distinct from naive and transi- shown in Fig. 3A, these cells could be easily distinguished from tional B cells, with intermediate levels of these markers noted. CD20ϩCD27ϪCD38low naive B cells. These two populations were Additionally, ABCB1 transporter expression was assessed by both IgDϩ, but only the CD20ϩCD38intCD27Ϫ population ex- R123 dye extrusion. CD27ϪCD38intIgDϩ intermediate B cells pressed CD5 (Fig. 3A). Naive B cells started to proliferate to both also displayed an intermediate degree of R123 extrusion, whereas anti-BCR and anti-CD40 stimulation after 48 h of culture, and they transitional B cells showed poor extrusion, and naive B cells proliferated robustly to both stimuli by 72 h of culture, whereas showed full extrusion (Fig. 1C). intermediate B cells showed no proliferative responses to either

Ϫ int ϩ stimulus after 48 h, and by 72 h of culture, they exhibited a min- CD27 CD38 IgD intermediate B cells are increased in cord imal proliferative response, but only to anti-CD40 stimulation blood, but not in normal bone marrow (Fig. 3B). To characterize responsiveness of intermediate B cells, We next examined the distribution of intermediate B cells in hu- Ca2ϩ mobilization was measured after crosslinking of BCR with man cord blood. As previously reported, transitional B cells were anti-IgM. The increase in intracellular Ca2ϩ of intermediate B markedly increased in cord blood (20). Additionally, intermediate cells in response to BCR crosslinking was significantly less than B cells were also significantly increased in cord blood (Fig. 2A). In that of the naive B cells (Fig. 3C). To evaluate apoptosis and cell contrast, intermediate B cells were infrequent in normal bone mar- death, the frequencies of annexin V and Live/Dead violet stain- row (Fig. 2B). As in the blood, bone marrow intermediate B cells positive cells were measured in both populations immediately ex expressed CD5, as did transitional and immature B cells, but not vivo and after 48 h of culture. Intermediate B cells contained a pro- or pre-B cells. significantly higher percentage of apoptotic cells and dead cells compared with the naive B cells (Fig. 3D). Both intermediate and Ϫ int ϩ Functional activity of CD27 CD38 IgD intermediate B cells naive B cells were saved from undergoing apoptosis by anti-CD40 We next determined the in vitro functional responses of interme- stimulation, but apoptosis of intermediate B cells was significantly diate B cells to BCR ligation, anti-CD40, and various cytokines. increased by anti-IgM, whereas this was not observed in the naive For functional studies, we developed a strategy to sort naive and B cells. When total dead cells were evaluated, intermediate B cells intermediate B cells without engaging receptors that could activate were rescued from cell death only by the anti-CD40 signal, them. A combination of mAbs to CD20, CD38, and CD27 was whereas naive B cells were saved by both anti-CD40 stimulation employed that, as shown in Fig. 3A, could identify a unique and by anti-IgM to a lesser degree (Fig. 3E). Various cytokines 4122 HUMAN CD5ϩ PRE-NAIVE B CELL POPULATION IDENTIFIED Downloaded from

FIGURE 5. Intermediate B cells can undergo terminal differentiation similarly to naive B cells. CD27ϪCD38int intermediate B cells and CD27ϪCD38low http://www.jimmunol.org/ naive B cells were sorted and cultured in the presence of anti-CD40 and IL-21 and assessed for IgD and CD38 expression after 3 days, 6 days, and 9 days of culture. Boxed regions represent plasma cells, and numbers represent percentage of plasma cells. Data are representative of three experiments with similar results. were examined to determine the effect on survival of intermediate and present Ag. Stimulation of both intermediate and naive B cells and naive B cells. BAFF had no significant effect on the survival with anti-CD40 and IL-21 induced down-modulation of surface of intermediate B cells, in contrast to its ability to rescue naive B IgD and the differentiation of CD38highIgDϪ plasma cells (Fig. 5). cells from both apoptosis and cell death ( p Ͻ 0.01). Of note, IL-4, Similar results were noted when intermediate B cells were stimu- ϩ IL-21, and IL-10 increased survival of both intermediate and naive lated by culturing them with activated CD4 T cells. As can be by guest on September 23, 2021 B cells significantly ( p Ͻ 0.01), with IL-10 having the least con- seen, by day 10 of culture, a large population of IgDϪ B cells was sistent effect (Fig. 3F). generated (Fig. 6A). All of these had proliferated, as gauged by dilution of CFSE (data not shown). Of these, a fraction was plasma CD20ϩCD38intCD27Ϫ intermediate B cells can develop into ϩ cells. Plasma cells derived from intermediate B cells secreted naive B cells via transition to CD5 naive B cells mainly IgM (4.2–5.6 ␮g/ml), and less IgG (0.06–0.08 ␮g/ml) and Levels of expression of CD38, CD10, CD9, and the ABCB1 IgA (0.24–0.53 ␮g/ml) than those from naive B cells, but the total transporter by CD20ϩCD38intCD27Ϫ B cells that were between amount of secreted Abs was 2- to 6-fold less than plasma cells those displayed by transitional and naive B cells suggested derived from naive B cells (Fig. 6B). that these B cells might be developmental intermediates The capacity of intermediate B cells to function as APCs was between these two B cell populations. When we examined ex- also examined. First, CD86 expression was assessed after stimu- pression of CD5 on naive B cells, we noticed a small fraction of lating intermediate and naive B cells with anti-CD40. As shown in naive B cells that expressed CD5 (Fig. 3A). This suggested the Fig. 7A, intermediate B cells increased CD86 expression when possibility that CD20ϩCD38intCD27Ϫ B cells might mature stimulated with anti-CD40. In additional experiments, intermedi- into naive B cells and gradually lose CD5. To test this hypoth- ate or naive B cells were cultured with allogeneic T cells to assess esis, we attempted to induce differentiation of intermediate B their capacity to stimulate the MLR. As shown in Fig. 7B, both cells and CD5ϩ naive B cells into CD5Ϫ naive B cells by pro- populations induced comparable degrees of allogeneic T cell pro- viding Ag nonspecific bystander T cell help conferred by acti- liferation with or without stimulation by anti-CD40. vated T cells. As can be seen in Fig. 4A, activated CD4ϩ T cells induced CD38intIgDϩ intermediate B cells to proliferate, and Increased frequencies of intermediate B cells are found in they differentiated into CD38lowIgDϩ naive B cells, and this peripheral blood of SLE patients population subsequently lost expression of CD5. To ensure that Since SLE is associated with a loss of B cell tolerance and result- ϩ T cell signals could also induce CD5 naive B cells to become ant disturbances in many B cell populations (22–25), we examined Ϫ ϩ CD5 , CD5 naive B cells were cultured with a combination of the frequency of intermediate B cells in the peripheral blood of anti-CD40 and IL-21. As can be seen in Fig. 4B, such stimu- SLE patients. The relative frequency of this B cell population in ϩ Ϫ lation induced CD5 naive B cells to become CD5 . the peripheral blood of 10 healthy adult donors was compared with that of 6 active SLE patients. Intermediate B cells were Intermediate B cells can manifest some functions of mature found in increased proportions in patients with active SLE. Of naive B cells note, the frequency of intermediate B cells was inversely pro- To determine whether intermediate B cells can perform normal portional to the frequency of total B cells, suggesting that the mature B cell functions, we assessed their ability to differentiate expansion of the intermediate B cell population was related to The Journal of Immunology 4123 Downloaded from http://www.jimmunol.org/ FIGURE 6. Intermediate B cells can differentiate into IgDϪ B cells and plasma cells by stimulation with anti-CD3-activated T cell help. A, Sorted CD27ϪCD38int B cells were cultured with mitomycin C-treated CD4ϩ T cells in anti-CD3-precoated microtiter wells and, after various days of culture, were gated into CD38ϩIgDlow/Ϫ cells, which were then assessed for expression of CD27 to identify plasma cells (PC). Numbers represent percentage of CD38ϩIgD low/Ϫ cells. B, IgM, IgA, and IgG concentrations were determined in intermediate and naive B cells after 10 days of culture, and data are the means Ϯ SEM from three independent experiments. lymphopenia. Additionally, naive B cells of SLE patients also Discussion expressed CD5, suggesting a B cell population shift toward im- We have defined a pre-naive B cell population that stands between mature B cells (Fig. 8). transitional and naive B cells, comprising an additional definable by guest on September 23, 2021 subset during normal human B cell development in the periphery. This new subset accounted for the majority of the circulating CD5ϩ B cell population, and it was defined by both its phenotypic and functional characteristics. Although pre-naive B cells exhibit some capabilities of normal mature naive B cells, their main func- tional role under physiologic conditions appears to be as a devel- opmental intermediate in maturation of human naive B cells, as shown for transitional B cells of both mice and humans (26, 27). However, their functional maturity suggests that under certain cir- cumstances they could be inappropriately activated and contribute to autoimmune disease. In mice, transitional B cells, which bridge immature B cells in the bone marrow and mature naive B cells in the periphery, can be subdivided into several developmental subsets. Initially, murine transitional B cells were separated into two maturational subsets, T1 and T2, based on expression of CD21 and IgD. Thus, CD24high transitional B cells could be subdivided into CD21lowIgDlow T1 and CD21highIgDhigh T2 cells. These two populations appear to have different population dynamics and require different matura- tion signals (28). Others have subdivided murine transitional B cells into three populations, that is, T1 (CD23Ϫ, IgMhigh), T2 (CD23ϩ, IgMhigh), and T3 (CD23ϩ, IgMlow), based on CD23 and FIGURE 7. Intermediate B cells can express CD86 and induce T cell IgM expression (29). It is unclear how these subsets relate to stages proliferation as efficiently as naive B cells. A, Intermediate and naive B of human B cell maturation. However, it is clear in both mice and cells were compared for CD86 expression after 24 h of culture in the humans, B cell development during the transition from immature presence of medium or anti-CD40. Numbers represent mean fluorescence to mature naive B cells is a multistep process, and at each step B intensity of each population. Data are representative of three experiments with similar results. B, CFSE-labeled T cells were cultured alone or with cells are influenced by selection pressure to ensure tolerance be- purified intermediate B cells or naive B cells with or without anti-CD40 (C) fore full maturation (29–31). It is notable that these populations addition in the wells, and the percentages of divided T cells were assessed. reside in the murine and are usually not found in other sites. Data are the means Ϯ SEM of three experiments. A similar multistep developmental process is presumed to exist in 4124 HUMAN CD5ϩ PRE-NAIVE B CELL POPULATION IDENTIFIED Downloaded from http://www.jimmunol.org/

FIGURE 8. Intermediate B cells are found in increased proportions in patients with SLE. A, B cells from peripheral blood of SLE patients and healthy adult donors were stained with anti-CD19, anti-CD27, anti-CD38, and anti-IgD to examine the frequencies of CD27ϪCD38lowIgDϩ naive B cells, CD27ϪCD38intIgDϩ intermediate B cells, and CD27ϪCD38highIgDϩ transitional B cells, and the differential expression of CD5 was assessed. Data are representative results from 6 SLE patients and 10 healthy adult donors. The average mean fluorescence intensity (ϮSD) of CD38 on naive, intermediate, and transitional B cells in SLE was 160 Ϯ 17, 846 Ϯ 74, 2541 Ϯ 25, respectively (compared with the mean fluorescence intensity Ϯ SD of naive, intermediate, and transitional B cells in healthy adult donors: 135 Ϯ 15, 855 Ϯ 29, 2659 Ϯ 91, respectively). B, The relative frequency of intermediate B cells in peripheral blood of healthy adult donors (n ϭ 10) and in patients with active SLE (n ϭ 6) was determined. Mann-Whitney U test was used to p Ͻ 0.01). C, Frequency of intermediate B cells is inversely proportional to the frequency of total B cells shown in a linear ,ءء) detect significant differences by guest on September 23, 2021 regression plot (r ϭϪ0.6363, p ϭ 0.0048). Data include results from 6 SLE patients and 10 normal donors. humans, but intermediate cell subsets during human peripheral B of the ABCB1 transporter activity, which has been described as a cell development have not been fully characterized. Importantly, definitive method to discriminate resting naive B cells from tran- intermediates in human B cell development can be found in the sitional B cells (32), also distinguished these cells from transitional peripheral blood (20), raising questions about the site(s) of matu- and naive B cells. ABCB1 transporter expression, confirmed by ration of these cells in humans. R123 dye extrusion, showed that CD38int B cells have the ability One of the difficulties in identifying human peripheral B cell to extrude the dye, but to a much lesser extent than do naive B subsets is that there are multiple phenotypic changes during de- cells, consistent with the conclusion that they are developmental velopment, and current understanding of the pattern of expression intermediates. In summary, the phenotypic analysis indicated this of these markers is remarkably limited. Previous studies revealed newly identified subset is a pre-naive B cell population, which that transitional cells and naive B cells in humans could be dis- appears to be a developmental stage between transitional and naive tinguished by expression of CD38, among other phenotypic mark- B cells. ers, in which transitional B cells express high levels compared with Fundamental functional differences between immature and ma- low levels in naive B cells (20). We found an additional B cell ture B cells in the periphery have been described previously (33). population that expressed an intermediate level of CD38 within the Ϫ In this regard, immature B cells have a shorter life span (34–36), CD27 Ag inexperienced B cells, which were neither CD38low and they undergo apoptosis rather than proliferation in response to naive nor CD38high transitional B cells. Further analysis revealed BCR ligation (20, 28, 35–37). These functional characteristics of that these cells were phenotypically more similar to the naive B cells and not to transitional B cells. In this regard, CD38intCD27Ϫ immature B cells were also found in the pre-naive B cell popula- intermediate B cells differed from transitional B cells by expres- tion, but to a lesser extent than manifested in transitional B cells. sion of CD23, CD21, CD24, CD44, IgM, CD95, and CD11b. Pre-naive B cells were more prone to both spontaneous apoptosis However, these cells also differed in phenotype from naive B cells and cell death than were naive B cells, but less so than transitional by expression of the immature B cell markers CD9 and CD10, and B cells (20). Additionally, BCR stimulation induced increased ap- they were intermediate between transitional and naive B cells, dis- optosis compared with that found with naive B cells, suggesting tinguishing this CD38int B cell population as a distinct subset. that BCR engagement might be involved in negative selection at Importantly, these cells expressed CD5, which is also characteris- this developmental stage. It is notable that anti-CD40 and T cell- tic of human transitional B cells and immature B cells in the bone derived cytokines, including IL-4, IL-21, and IL-10, were able to marrow, but not of naive B cells, suggesting that CD5 might be an enhance survival and prevent apoptosis of pre-naive B cells, but important development marker for human B cells. The expression the known B cell survival and growth factor BAFF/BLyS was The Journal of Immunology 4125 unable to enhance survival or prevent these cells from undergoing Human pre-naive B cells can be distinguished from the murine apoptosis (20, 37). T2 B cells by several distinctive observations that may be associ- Previously, it has been shown that human transitional B cells ated with their functional role. In contrast to the murine T2 cells, also did not respond to BAFF by increasing survival, although they which are found exclusively in the spleen, human pre-naive B cells expressed BAFF-R, but at a lesser density than that of mature were observed in the peripheral blood, suggesting that their mat- naive B cells (20). Thus, during human B cell development, neither uration occurs at multiple sites. It is possible that human pre-naive transitional B cells nor pre-naive CD38intCD27ϪCD5ϩ B cells re- B cells matured at different sites may give rise to different subsets spond to BAFF with increased survival. Since both populations of pre-naive B cells with different functions, as exemplified by express BAFF receptors, failure of BAFF to promote survival of marginal zone B cells and B-1 B cells in the murine system (14). these populations cannot be explained by absence of these recep- However, phenotypic markers are not currently available that tors, although downstream signaling events may be defective. would permit examination of this question. Human pre-naive cells Moreover, it is clear that a number of T cell-derived cytokines, also seem to be phenotypically more mature than murine T2 cells, such as IL-4 and IL-21, can promote survival of pre-naive B cells, with most markers other than CD9, CD10, and the ABCB1 trans- whereas IL-4 can also promote survival of transitional B cells (20). porter assuming naive B cell density, whereas murine T2 B cells These results indicate that different signaling pathways than those continue to express a more immature pattern of B cell markers, activated by BAFF are involved in promoting survival of human with the exception of IgD and CD21 (28). The expression of CD5 pre-naive and transitional B cells. Finally, the lack of responsive- and the lack of responsiveness to BAFF are also characteristic of ness of transitional and pre-naive human B cells to BAFF differs human pre-naive B cells but not murine T2 B cells, and suggest from findings in the mouse, in which B cell developmental stages additional mechanisms to avoid responsiveness to autoantigens. Downloaded from from the T2 stage on are thought to be BAFF responsive (34). These differences predict unique developmental functions for pre- Pre-naive B cells proliferated minimally to anti-CD40 or anti- naive B cells in humans that are different from the role of T2 B IgM stimuli, and mIgM-induced Ca2ϩ mobilization was much cells in the murine system. lower compared with that manifested by naive B cells. This may We found that subjects with SLE have increased proportions of relate to the fact that CD5 is expressed by human pre-naive B cells. pre-naive B cells, and this expansion correlated with the degree of CD5 expression appeared to be characteristic of human immature B cell lymphopenia. Previously, it had been observed in patients http://www.jimmunol.org/ with SLE and Sjo¨gren’s syndrome who had been treated with the B cells, being expressed on immature B cells, transitional B cells, ϩϩ ϩ ϩ and pre-naive B cells. In this regard, the most notable difference in B cell-depleting therapy rituximab that CD38 IgD CD5 im- phenotype between murine and human T1 B cells was reported to mature B cells repopulated during the recovery phase of B cell be the expression of CD5 (20). CD5 expression has been shown to depletion, and this correlated with an increase in serum BAFF level (42, 43). Furthermore, an expanded population of be associated with negative regulation of BCR and TCR signaling, CD38ϩϩIgDϩCD5ϩ B cells within the naive B cell compartment as well as maintenance of tolerance in anergic B cells (38–40). was found in peripheral blood of active SLE patients (44). These These findings suggest that during development from the immature previously described B cell populations appear to fall within the through the pre-naive stages, human B cells may be inhibited from by guest on September 23, 2021 pre-naive B cell subset characterized in our study. Collectively, responding to BCR stimulation by expression of CD5. Presum- these observations strongly imply that pre-naive B cells can be ably, this could contribute to the culling of autoreactive B cells specifically amplified under certain conditions that can alter B cell during these stages of development, since a considerable percent- homeostasis, including those characteristic of patients with active age of immature B cells is known to be autoreactive (9). Interest- SLE and those recovering from B cell depletion. Characterization ingly, we have observed that activated T cell help caused down- of this B cell population as pre-naive and demonstration that it has regulation of CD5 on pre-naive B cells and induced proliferation the capacity to respond to bystander T cell signals with differen- and differentiation into naive B cells. These findings are consistent tiation into Ab-secreting plasma cells suggest that these cells may, with a role for CD5 in limiting BCR stimulation and autoantigen- under specific circumstances, contribute to autoimmunity. induced activation during B cell development. However, noncog- In summary, human peripheral B cell development is a multistep nate T cell help may permit autoreactive pre-naive B cells to ex- process involving at least three developmental subsets: transi- pand and differentiate, thus bypassing this checkpoint. tional, pre-naïve, and naive B cells, and likely subsets within each As experimentally defined in our study, the pre-naive B cells stage. Pre-naive B cells possess a phenotype and function that are comprise a population with the capacity to differentiate into plasma intermediate between transitional and naive B cells in that they cells, and also the capacity to function as APCs. When these cells have some of the characteristics of the more immature transitional were stimulated with anti-CD40 in the presence of IL-21, a potent B cells, but, at the same time, they have some of the functional set of signals capable of inducing differentiation, pre- capacity of the mature B cells to differentiate and present Ag. naive B cells exhibited the potential to undergo terminal differen- These immature features of the human pre-naive B cells may pro- tiation into plasma cells, as previously shown for human peripheral vide the basis for normal selection mechanism in the periphery at blood naive B cells (41). Similarly, activated T cells induced pre- this stage of differentiation. However, the finding that human pre- naive B cells to proliferate and differentiate into plasma cells that naive B cells can proliferate and differentiate in response to non- mainly secreted IgM. We also found that anti-CD40 stimulation cognate T cell signals suggests that under certain circumstances enhanced expression of CD86 by pre-naive B cells and that pre- tolerance may be bypassed at this stage of development and auto- naive B cells could stimulate proliferation of allogeneic T cells in immunity could result. an MLR. These results clearly indicate that pre-naive B cells have acquired some of the functions associated with mature naive B cells. These findings also suggest the possibility that under certain Acknowledgments circumstances, activation of pre-naive B cells, perhaps during non- We thank James Simone and Rafael Villasmil of the National Institute of cognate bystander interactions with activated T cells, could con- Arthritis and Musculoskeletal and Skin Diseases Flow Cytometry Facility tribute to the functional activation and differentiation of these cells for expert technical assistance with cell sorting, and Ho-Youn Kim (Cath- and the emergence of autoimmunity. olic Univerisity) for providing some of the SLE patient samples. 4126 HUMAN CD5ϩ PRE-NAIVE B CELL POPULATION IDENTIFIED

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