CUTTING EDGE
IMMUNOLOGY
E OF
JOURNAL
Cutting Edge: Transitional T3 B Cells Do Not Give Rise to Mature B Cells, Have Undergone Selection, and Are
Reduced in Murine Lupus
1Brittany N. Teague,* Yujun Pan,* Philip A. Mudd,* Britt Nakken,* Qingzhao Zhang,
†Peter Szodoray,
†Xana Kim-Howard,* Patrick C. Wilson,
†and A. Darise Farris
2*
As the immediate precursors to mature follicular B cells in splenic development, immature transitional cells are an essential component for understanding late B cell differ- entiation. It has been shown that T2 cells can give rise to mature B cells; however, whether T3 B cells represent a normal stage of B cell development, which has been widely assumed, has not been fully resolved. In this study, we demonstrate both in vitro and in vivo that T3 B cells do not give rise to mature B cells and are instead selected away from the T13 T23mature B cell developmental pathway and are hyporesponsive to stimulation through the BCR. Sig- nificantly reduced numbers of T3 B cells in young lupus- prone mice further suggest that the specificity of this subset holds clues to understanding autoimmunity. The Journal of Immunology, 2007, 178: 7511–7515.
N
ewly formed B cells that have passed through bone marrow (BM)3tolerance checkpoints and express a functional BCR migrate to the spleen to complete development. These recent emigrants are known as transi- tional B cells. Of the⬃2 ⫻ 107surface (s) IgM⫹cells pro- duced in murine BM each day, only 10% enter the spleen and, furthermore, only 1–3% reach maturity (1). Transi- tional populations of B cells have been described previously (2, 3) and can be divided into three populations designated as T1 (CD93⫹sIgMhighCD23⫺), T2 (CD93⫹IgMhighCD23⫹), and T3 (CD93⫹sIgMlowCD23⫹) (4).Continuous in vivo BrdU labeling further supports a devel- opmental sequence in which T1 B cells give rise to T2 B cells, and T2 cells give rise to T3 B cells (4). DNA content analysis revealed that the transitional subsets do not undergo significant proliferation, suggesting that peripheral development is not as- sociated with a proliferative burst (4). Adoptive transfer studies have shown that T2 cells can give rise to mature follicular B cells (MB) in wild-type recipient mice and marginal zone (MZ) B
cells in lymphopenic recipients (5). CD21int/highCD24high cells, which likely encompass T2 cells, T3 cells, and MZ precursors, undergo maturation in vitro when given soluble B cell-activating factor (BAFF) in the presence of anti-IgM BCR stimulation (6). However, details concerning the role of the T3 subset in B cell development and tolerance are still unclear.
The T1 stage has been shown to be an important deletion checkpoint, as supported by studies showing that BCR cross- linking induces exaggerated apoptosis at this stage (2, 7, 8). T2 cells are also susceptible to anti-IgM-mediated apoptosis, con- sistent with susceptibility of T2 cells to deletional tolerance (4).
Mice overexpressing or underexpressing BAFF, an important B cell survival factor (6), have also revealed the importance of transitional B cell tolerance checkpoints. BAFF-deficient mice have a developmental block at the T1 stage. In contrast, BAFF–
transgenic mice have enhanced transitional and MZ B cell pop- ulations and develop a lupus-like disorder (9, 10). It has been demonstrated that excess soluble BAFF can rescue the lower af- finity hen egg lysozyme (HEL)-reactive CD21int/highCD23high population, which may include T2 and T3 B cells in HEL- transgenic mice. These self-reactive B cells were allowed to enter forbidden splenic microenvironments from which they are nor- mally excluded (10). BAFF did not rescue higher affinity HEL- reactive B cells deleted in the BM; thus, excess BAFF specifically perturbs transitional B cell tolerance (10).
Finally, a significant increase in the number of autoreactive B cells in the peripheral naive mature compartment compared with newly emigrated peripheral B cells in human systemic lu- pus erythematosus (SLE) further suggests that transitional B cell tolerance is faulty in human SLE (11). Together, these stud- ies highlight the importance of transitional B cell tolerance in regulating autoimmunity. The lack of knowledge concerning the relative role that T3 B cells play in B cell development and tolerance led us to further characterize this population and to determine whether it can give rise to MB cells.
*Arthritis and Immunology Research Program and†Molecular Immunogenetics Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 Received for publication October 16, 2006. Accepted for publication April 24, 2007.
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants P50 AR 48940, KO2 AI51647, and R01 AI48097.
2Address correspondence and reprint requests to Dr. A. Darise Farris, Oklahoma Medical Research Foundation, 825 North East 13th Street, Oklahoma City, OK 73104. E-mail address: [email protected]
3Abbreviations used in this paper: BM, bone marrow; MB, mature follicular B cell; MZ, marginal zone; BAFF, B cell-activating factor; SLE, systemic lupus erythematosus; s, sol- uble; SA, streptavidin; int, intermediate; HEL, hen egg lysozyme.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
Materials and Methods
Mice
Four- to 5-wk-old C57BL/6 (B6) female mice were used as a source for splenic B cell populations for maturation, Ca2⫹flux, and H chain usage studies. For quantitation of B cell subsets, 8- to 10-wk-old lupus-prone (BxSB/MpJ, NZM2410, NZBWF1, and MRL/MpJ, MRL/lpr/lpr) and control strains (BALB/c, B6, AKR/J, and 129/SvJ) were used. B6.SJL-PtprcaPepcb/BoyJ (B6.Ly5SJL) were used at the age of 4 –5 wk. All mice were purchased from The Jackson Laboratory). All studies were approved by the Oklahoma Medical Re- search Foundation Institutional Animal Care and Use Committee.
Abs and flow cytometric analyses
Abs used were as follows: anti-mouse IgM F(ab⬘)2-FITC or -Cy5 (Caltag Lab- oratories), anti-mouse CD23-PE (B3B4), anti-mouse B220 (RA3-6B2)-PerCP or PE-Cy5, and anti-mouse CD93 (493)-biotin. Biotinylated Abs were de- tected with secondary streptavidin (SA)-allophycocyanin, SA-allophycocyanin- Cy7, or SA-allophycocyanin-Cy5.5 (Molecular Probes). All Abs were pur- chased from BD Pharmingen, with the exceptions noted above. Anti-CD16/32 was used to block FcRs before staining 2⫻ 106cells, unless otherwise noted.
Events were collected on FACSCalibur or LSRII instruments (BD Biosciences).
Cell sorting
Splenic B cell subsets were isolated on a MoFlo cell sorter (DakoCytomation) or FACSAria (BD Biosciences). Purity of FACSAria-sorted cells ranged from 89 –98%, and purity of MoFlo-sorted cells ranged between 50 and 70%.
Gating strategy is shown in Fig. 1. An anti-IgM F(ab⬘)2was used during puri- fication to avoid BCR cross-linking.
In vitro maturation assay
Maturation assays were performed as previously described (6). Sorted cells were incubated at 3⫻ 106cells/ml in 96-well microtiter plates in DMEM supple- mented with 10% FCS, 100 U/ml penicillin, 100g/ml streptomycin, 2 mM glutamine, 2 mM sodium pyruvate, 0.1 mM Nonessential amino acids solu- tion, and 50M 2-ME. Cells were stimulated for 72 h with 1.5 g/ml recom- binant soluble human myc-tagged BAFF, provided by Dr. S. Kalled (Biogen Idec, Cambridge, MA) (12). This treatment was shown to eliminate any con- tamination of MB cells by the 50-h time point (6). F(ab⬘)2rabbit anti-mouse Ig (Zymed Laboratories) was added to cultures at a final concentration of 10
g/ml for the last 24 h of culture. At 72 h, cells were stained with mAbs to IgM, B220, and CD93 and analyzed by flow cytometry.
In vivo maturation assay
Sorted cells from B6 (CD45.2) donors were resuspended at 250,000 cells/250
l of PBS containing 0.5% FCS per recipient. Cells were adoptively transferred into B6.Ly5SJL(CD45.1) hosts by way of retro-orbital plexus. Ninety-six hours after transfer, B6.Ly5SJL hosts were euthanized and the phenotype of CD45.1⫺CD45.2⫹or CD45.1⫺donor B cells was assessed using mAbs to IgM, B220, and CD93.
Ca2⫹mobilization assay
Sorted cells (1⫻ 106) from each population (T1, T2, T3, and MB) were re- suspended in 1 ml of HBSS with Ca2⫹(Sigma-Aldrich) and loaded with 5M Indo-1 acetoxymethyl dye in the presence of 2% pluronic F-127 (Molecular Probes). Cells were washed twice, resuspended, and stimulated with 50g/ml F(ab⬘)2rabbit anti-mouse Ig (Zymed Laboratories). Ratiometric measurements of free intracellular Ca2⫹concentrations were obtained by a Photon Technol-
ogy International QuantaMaster spectrofluorometer equipped with an excita- tion monochromator set at 350 nm and two emission monochromators set at 405 and 485 nm.
Single-cell RT-PCR
Presorted cells were resorted into 96-well plates, subjected to RT-PCR, and sequenced using recently described primers and procedures (13).
Results and Discussion
BAFF and BCR stimulation leads to maturation of T2 cells but not T3 cells
In vitro studies have shown that immature B cells give rise to mature B cell populations; however, reports using similar strat- egies to determine the developmental fate of T3 B cells are not available. It was recently reported that BAFF delivered in com- bination with BCR stimulation enabled the maturation of CD21int/highCD24highimmature B cells into a MB phenotype (6). We used a similar approach to determine whether the T2 and T3 populations can give rise to MB cells. As expected, BCR stimulation in the presence of BAFF led to maturation of a sub- set of the immature T2 population (Fig. 2). However, no mat- uration of the immature T3 population was observed as assessed by cell surface retention of the immature marker CD93 under
FIGURE 1. Separation of splenic B cell subsets. Representative gating strategy of a B6 mouse. Plots were first gated for live lymphocytes based on forward vs side scatter profiles. Using the Allman classification scheme (4), B220⫹CD93⫹cells were subdivided based on CD23 and IgM expression. MB cells were identified within the B220⫹CD93⫺subset as IgM⫹CD23⫹.
FIGURE 2. T2 but not T3 B cells undergo maturation in vitro in response to BCR cross-linking in the presence of BAFF. A, Representative results from one of three in vitro maturation assays. Cultures initiated with sorted T2 (upper panels) or T3 cells (lower panels) were gated to removed dead cells, and each sample was restained for CD93, IgM, and B220 expression. Unstained controls are shown to verify that no residual staining from the sort procedure remains cell associated. IgM and CD23 levels could not be reliably assessed due to the complicating presence of cross-linking anti-IgM in the assay and due to a sort- ing-induced down-regulation of CD23 in all cultures regardless of treatment or cell origin. B, Results of three independent experiments presented in graphical form. Induced loss (⌬%) of the immature marker CD93 on B cells cultured in BAFF alone (left panels) compared with B cells cultured in BAFF plus anti-IgM F(ab⬘)2(right panels) is reported. One experiment included two separate cul- tures. The Student’s t test indicates that the results are significant at p⫽ 0.007.
similar conditions (Fig. 2). Although the purity (see Materials and Methods) and overall viability of isolated T2 and T3 cells varied with the sorter used, the degree of purity of the two sub- sets within a single experiment was always similar. Moreover, similar results were obtained in each experiment performed, as shown in Fig. 2B, which depicts results from one experiment using lower purity/higher viability MoFlo-sorted cells and from two experiments using higher purity/lower viability FACSAria- sorted cells. Even though BAFF receptor levels were marginally lower in T3 cells compared with T2 B cell (data not shown), there was no significant difference in the number of live-gated cells recovered between T2 and T3 cultures under conditions of BAFF alone vs BAFF plus anti-sIgM (p⫽ 0.127 for three in- dependent experiments, Kruskal-Wallis test). This indicated that increases in the fraction of mature cells in T2 cultures re- flected phenotypic changes and not differential cell death.
T2 B cells adoptively transferred into wild-type mice have been shown to acquire a MB phenotype within 4.5 days after transfer (5). We predicted that under similar conditions, T3 B cells would remain phenotypically immature. To address this question, we performed adoptive transfer experiments (5). Two hundred fifty thousand T2 and T3 B cells were sorted from B6 (CD45.2) donors and transferred into B6.Ly5SJL (CD45.1) hosts. Four days after transfer, the hosts were euthanized and the phenotype of donor cells was assessed. Quantitation of the CD45.1⫺CD45.2⫹ donor cells positive for B220 expression revealed that a portion of donor T2 cells assume a mature phe- notype as evidenced by loss of CD93 expression and down-reg- ulation of cell surface IgM, whereas essentially all donor T3 cells remain CD93⫹. These results demonstrate that T2 cells but not T3 cells acquire a mature phenotype in vivo (Fig. 3). These re-
sults lead us to conclude that the T3 population makes at most a limited contribution to the mature B cell pool and does not represent a normal stage B cell development.
T3 cells exhibit reduced mobilization of Ca2⫹in response to BCR cross- linking and a skewed JH3 gene usage
Studies have shown that T1 and T2 immature B cells mobilize Ca2⫹ to a greater extent than mature B cells following BCR cross-linking (14), whereas anergic B cells have a reduced mo- bilization of Ca2⫹(15, 16). To examine the relative capacity of T3 B cells to respond to BCR cross-linking, we sorted the im- mature transitional cells into T1, T2, T3, and MB phenotypes, then measured Ca2⫹ mobilization following loading of cells with Indo-1. In response to BCR cross-linking, we found a re- producibly reduced Ca2⫹flux to anti-IgM BCR stimulation in the T3 population when compared to T1, T2, and MB B cell populations (Fig. 4A). T1 and T2 cells both mobilized Ca2⫹
more efficiently than mature B cells as predicted. All popula- tions fluxed Ca2⫹to similar extents with the addition of iono- mycin, demonstrating efficient Indo-1 loading in all popula- tions (Fig. 4A). Similar results were observed in Fluo-4-labeled
FIGURE 3. Portions of T2 but not T3 cells assume a mature phenotype in vivo following adoptive transfer into B6.Ly5SJLrecipients as assessed by loss of CD93 expression. T2 or T3 populations from CD45.2⫹mice were transferred into CD45.1⫹recipient mice, retrieved after 96 h, and analyzed for CD93 and IgM expression. Representative results of one of four in vivo maturation assays.
Quantitation of CD93 expression on the CD45.1⫺CD45.2⫹donor popula- tion shows that portions of T2 donor cells lose the CD93 marker in vivo (middle panel), whereas T3 cells remain CD93-positive in vivo (upper panel). Cells within the gated populations were B220⫹. No cells with a donor phenotype were identified in mock recipients (lower left panel). IgM levels down-regulate in the CD93⫺T2 donor cells, consistent with a follicular MB cell phenotype (lower right panel). Results are based on the collection of 3.0 – 4.5⫻ 106total events. Donor cells represented 0.01– 0.3% of recipient splenocytes
FIGURE 4. T3 B cells exhibit reduced mobilization of Ca2⫹and altered JH
gene usage. A, T3 B cells exhibit reduced mobilization of Ca2⫹when stimu- lated through BCR cross-linking. T1, T2, and T3 immature transitional and MB cells were sorted as previously described. Fifty micrograms of anti-IgM F(ab⬘)2was added at the 60-s (denoted by arrow) or 80-s (MB only) time points, and ratiometric quantitation of Indo-1 was measured for⬃200 s. Iono- mycin added at a later time point verified that each subset was efficiently loaded with Indo-1. B, Single-cell RT-PCR used to analyze murine Ig VH/JHgene us- age by the T1, T2, T3, and MB populations revealed a significant bias toward JH3 gene usage in T3 B cells. Results from three independent experiments are shown. Statistical differences between JHusage of the T3 subset compared with the T1, T2, or MB subsets were judged using the t test. The average number of single cells examined for T1, T2, T3, and MB cells per experiment was 27, 32, 25, and 36, respectively.
cells by flow cytometry (data not shown). Our results are con- sistent with the contention that the T3 population exhibits an anergic phenotype in response to BCR cross-linking (17).
To determine whether T3 cells show any evidence of selec- tion away from the T13 T23 MB pathway, we performed sin- gle-cell RT-PCR on the T1, T2, T3, and MB populations to analyze their Ig VH/JHgene usage. Interestingly, we observed a significant enhancement of JH3 segment usage in the T3 pop- ulation (46 ⫾ 13%), compared with T2 (17 ⫾ 10%; p ⫽ 0.022) and mature cells having a follicular phenotype (17⫾ 5%; p⫽ 0.027; Fig. 4B). A predominance of JH6 usage has been observed within an edited autoreactive B cell subset in humans (18); however, no association between particular JHsegments and potentially autoreactive B cell subsets has been described in the mouse. To determine whether JH3 might demonstrate an association with autoantibodies in the mouse, the Immunoge- netics database was exhaustively searched for murine Abs di- rected to self-Ags or nonrodent Ags. Significantly more autoan- tibodies (38 of 110; 35%) than Abs to nonrodent Ags (64 of 262, 24%) used the JH3 segment (p ⫽ 0.014; Fisher’s exact test), suggesting an association of JH3 with autoreactivity (Sup- plemental Tables Ia and Ib).4Thus, biased JH3 usage within the T3 subset might be explained by enrichment of this subset with autoreactive clones. These data are consistent with T3 B cells being anergic and autoreactive.
Lupus-prone mice have reduced numbers of T3 B cells
Given the impaired BCR responsiveness of the T3 population, reports of reversal of B cell anergy (19) led us to hypothesize that a tolerance defect at this selection point would most likely man- ifest itself as a significant reduction in the T3 population in au- toimmune strains of mice. To explore this possibility, we enu- merated T1, T2, T3, and MB cells in lupus-prone (BxSB/MpJ, NZBWF1NZM2410, MRL/lprlpr, and MRL/MpJ) and normal inbred (BALB/c, B6, AKR/J, and 129/SvJ) strains of mice.
Groups (n⫽ 10) of mice were examined at 8–10 wk of age to reduce the influence of chronic inflammation on B cell num- bers and to enhance the likelihood of detecting primary defects in B cell development, selection, or homeostasis. Examination of total spleen cellularity among the strains tested revealed two statistically separable groups (Fig. 5A) that were consistent with strain-to-strain variation in spleen size. We analyzed the two groups separately and compared the lupus prone strains to the non-autoimmune control strain harboring the closest number of T3 B cells. In the large spleen group, the T3 population in MRL/MpJ and MRL/lprlprmice was significantly reduced com- pared to the T3 population in the AKR/J strain (closest normal control) (Fig. 5B). In the small spleen group, significantly re- duced numbers of T3 B cells were also observed in both NZM2410 and NZBWF1strains compared with 129/SvJ mice (Fig. 5B). No reduction in T3 B cell numbers was observed in BXSB males.
To detect potential selection defects at earlier developmental stages, the numbers of T1 and T2 B cells among lupus-prone and control mice were also examined (Fig. 5, C and D). Both MRL/MpJ and MRL/lprlprmice exhibited reductions in the T1 and T2 populations in addition to the T3 population. Interest- ingly, there were no significant differences in the sizes of the T1 and T2 populations in NZBWF1and NZM2410 strains com-
pared with normal strains, indicating specific reductions in the T3 compartment in these two genetically related strains. These data suggest a possible genetic etiology of reduced T3 B cell numbers in NZB/W-related strains consistent with either im- paired T3 cell development or anergy. No significant differ- ences in the numbers of MB cells were detected among the strains tested and no significant differences in MZ B cell num- bers were observed in NZBWF1or NZM2410 strains com- pared with controls (data not shown). However, it is likely that the MB compartments of the autoimmune-prone strains har- bor enhanced numbers of autoreactive cells.
Cumulatively, these data strongly suggest that the T3 transi- tional B cell population is not a normal stage of B cell develop- ment and is unlikely to give rise to mature B cells under normal conditions. The BAFF in vitro maturation assays and in vivo adoptive transfer assays demonstrate that T3 B cells, contrary to the T2 population, remain phenotypically immature when given survival factors plus BCR stimulation or when placed into a normal peripheral environment in the mouse. Furthermore,
4The online version of this article contains supplemental material.
FIGURE 5. T3 B cells are reduced in lupus-prone mouse strains. A, Total spleen cell counts revealed two statistically separable groups that each contained representative control strains. Absolute numbers of B cell populations were compared within each group. For each statistical comparison, lupus-prone strains were compared with the group of control mice giving the closest number of cell counts. B, In the large spleen group, the T3 population in MRL/MpJ and MRL/lpr/lprstrains was significantly reduced compared with nonautoimmune strains. In the small spleen group, T3 B cells in NZM2410 and NZBWF1
strains were significantly reduced compared with T3 cells in control nonauto- immune strains. C and D, To determine whether the reduction in T3 cells in the lupus-prone mice is specific for this subset, the T1 (C) and T2 (D) immature subsets were quantitated. In the large spleen group, MRL/MpJ mice had re- duced numbers of T1 cells, and MRL/lpr/lprmice had reduced numbers of T2 cells. No significant differences in the T1 or T2 subsets were noted in the NZM2410 or NZBWF1autoimmune-prone strains compared with controls.
Thus, the NZB/NZW-related strains exhibited a selective reduction in the T3 compartment. E, No significant differences were found in MB numbers in the large or small spleen groups.ⴱ, p ⬍ 0.05 and ⴱⴱ, p ⬍ 0.005 by ANOVA with contrast.
defective mobilization of Ca2⫹in response to BCR stimulation and a biased JHgene usage provide evidence that the T3 popu- lation harbors cells of low responsiveness that have been selected away from the T23 MB pathway. The observations that JH3 us- age is associated with autoreactivity and T3 B cells are selectively reduced in two genetically related lupus-prone models, along with recent data highlighting defective peripheral tolerance checkpoints in human SLE patients, suggest that T3 B cells are likely to be im- portant in the regulation of autoimmunity.
Acknowledgments
We thank Dr. Susan Kalled for providing BAFF, the staff of the Oklahoma Medical Research Foundation Flow Cytometry Core, Andy Duty for support with calcium signaling, and Karen Schwarz for mouse husbandry. We also thank Drs. Mark Coggeshall, Carol Webb, and Shannon Maier for helpful comments on this manuscript.
Disclosures
The authors have no financial conflict of interest.
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