B Lymphopoiesis in the Thymus Koichi Akashi, Lauren I

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B Lymphopoiesis in the Thymus Koichi Akashi, Lauren I. Richie, Toshihiro Miyamoto, William H. Carr and Irving L. Weissman This information is current as J Immunol 2000; 164:5221-5226; ; of September 29, 2021. doi: 10.4049/jimmunol.164.10.5221 http://www.jimmunol.org/content/164/10/5221 Downloaded from References This article cites 40 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/164/10/5221.full#ref-list-1

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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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. B Lymphopoiesis in the Thymus1

Koichi Akashi,2,3 Lauren I. Richie,3 Toshihiro Miyamoto, William H. Carr, and Irving L. Weissman

The thymus has been regarded as the major site of T cell differentiation. We find that in addition to ␣␤ and ␥␦ T cells, a significant number (ϳ3 ؋ 104 per day) of B220؉IgM؉ mature B cells are exported from the thymus of C57BL/6 mice. Of these emigrating B cells, we estimate that at least ϳ2 ؋ 104 per day are cells which developed intrathymically, whereas a maximum of ϳ0.8 ؋ 104 per day are cells which circulated through the thymus from the periphery. The thymus possesses a significant number of pro-B and pre-B cells that express CD19, VpreB, ␭5, and pax-5. These B cell progenitors were found in the thymic cortex, whereas increasingly mature B cells were found in the corticomedullar and medullary regions. Other lymphoid cells, including NK cells and lymphoid dendritic cells, are not exported from the thymus at detectable levels. Thus, the thymus contributes to the formation of peripheral pools of B cells as well as of ␣␤ and ␥␦ T cells. The Journal of Immunology, 2000, 164: 5221–5226. Downloaded from hymocyte progenitors migrate from hematopoietic tis- cells, B cells, and lymphoid dendritic cells at low frequencies (10– sues, such as the bone marrow, to the thymus where they 12). Therefore, one might expect to find some B cells, NK cells, T proliferate, mature, and undergo a stringent selection pro- and dendritic cells maturing within and emigrating from the adult cess resulting in the export of self-tolerant TCR␣␤ T cells and thymus. TCR␥␦ T cells. During their tenure in the thymus, maturing thy- To test this hypothesis, we analyzed recent thymic emigrants mocytes interact with the thymic microenvironment, which (RTEs) to peripheral lymphoid organs. FITC was injected intra- http://www.jimmunol.org/ provides the appropriate cellular and/or soluble factors to enable thymically to label cells in the thymus, and at various time points thymocyte proliferation, development, and selection. This micro- after labeling, the spleen and lymph nodes were analyzed for the environment includes several types of thymic epithelial cells, bone presence of RTE. We found that, in addition to ␣␤ and ␥␦ T cells, marrow-derived dendritic cells, macrophages, bone marrow de- a significant number (3 ϫ 104 per day) of non-T cells were ex- rived B cells, and several mesenchymal elements. Thymic epithe- ported from the thymus; these were B220ϩIgMϩ B cells. We also lial cells produce a number of cytokines, including steel factor and demonstrated that the majority of these cells were generated from IL-7, both of which play important roles in progenitor prolifera- precursors in the thymus, whereas a minority could have been tion, survival, and commitment to the T cell lineages (1, 2). Both derived from circulating B cells that had entered the thymus. steel factor and IL-7 are also critical for the maturation of B cells by guest on September 29, 2021 in the bone marrow (3–5). In early mouse fetal life, the first seeded thymic progenitors Materials and Methods collectively include cells capable of T or B cell maturation (6–8). Mouse strains In adults, the thymus is continuously seeded at a low rate. These The congenic strains of mice, C57BL6-Ly5.2 or C57BL6-Ly5.1 mice, bone marrow-derived precursors have not yet been fully charac- were used. The C57BL6-Ly5.1/Ly5.2 mice were made by crossing C57BL6-Ly5.2 with C57BL6-Ly5.1 (F ). The strains differed only at the terized as to their phenotype or function. One candidate is the 1 4 Ly5 allele, and this difference made it possible to detect donor-derived recently identified common lymphoid progenitor (CLP), which cells. C57BL6-TCR␤-deficient mice (13) were purchased from The Jack- has lymphoid-restricted differentiation potential into T, B, and NK son Laboratory (Bar Harbor, ME). Mice were bred and maintained in the cells (9), found in adult bone marrow. The CD4lowCD44highc-Kitϩ animal care facility at Stanford University School of Medicine and were earliest thymic precursor population (10) has been shown to be used at 4–8 wk of age. capable of differentiating not only into T cells but also into NK Labeling of thymocytes and flow cytometric analysis

Departments of Pathology and Developmental Biology, Stanford University School of The technique for in vivo intrathymic labeling of thymocytes with FITC Medicine, Stanford, CA has been previously described (14). Briefly, 10 ␮l of FITC (1 mg/ml; Sigma, St. Louis, MO) was injected into both thymic lobes of 5- to 8-wk- Received for publication August 30, 1999. Accepted for publication February 25, 2000. old C57BL/6 mice. Single-cell suspensions were made from the spleen and the lymph nodes, including cervical, axillary, submandibular, inguinal, bra- The costs of publication of this article were defrayed in part by the payment of page chial, and mesenteric lymph nodes. Cells were stained with PE or allophy- charges. This article must therefore be hereby marked advertisement in accordance cocyanin-conjugated anti-IgM (clone 331; obtained from Dr. L. A. Her- with 18 U.S.C. Section 1734 solely to indicate this fact. zenberg, Stanford University, Stanford, CA), Mac-1 (M1/70), Gr-1 (8C5), 1 This work was supported by U.S. Public Health Service Grant CA42551 (to I.L.W.) anti-CD3 (KT31.1), anti-CD4 (GK1.5), anti-CD5 (53-7.8), and/or anti- and by a grant from the Jose Carreras International Leukemia Society (to K.A.). L.I.R. CD8 (53-6.7) Abs. Anti-IAb, anti-CD11c, anti-␣␤TCR, and anti-␥␦TCR is a Howard Hughes Medical Institute predoctoral fellow, and a Stanford Graduate Abs were purchased from PharMingen (San Diego, CA). In analyzing Fellow. CD4ϪCD8Ϫ RTEs, cells were additionally stained with Texas Red-conju- 2 Address correspondence and reprint requests to Dr. Koichi Akashi at the current gated anti-CD4 and anti-CD8 Abs. For thymic B cell progenitor analysis, address: Department of Cancer Immunology and AIDS, Dana-Farber Cancer thymocytes were stained with FITC-conjugated anti-CD43 (S7) and anti- Institute, Sm 770B, 44 Binney Street, Boston, MA 02115. E-mail address: mouse IgD (clone 11-26; obtained from Dr. L. A. Herzenberg); PE; or [email protected] Cy5-PE-conjugated anti-mouse IgM, PE-conjugated anti-CD19, allophy- 3 K.A. and L.I.R. contributed equally to the research described in this paper. cocyanin-conjugated B220, and Texas Red-conjugated anti-CD4 and anti- 4 Abbreviations used in this paper: CLP, common lymphoid progenitor; RTE, recent CD8 Abs (PharMingen). Cells were analyzed by a highly modified five- thymic emigrants. color FACS Vantage (Becton Dickinson, Mountain View, CA) (9). Dead

cation of 36, 32, or 32 cycles, respectively. The following primers were used: pax-5-forward, CTA CAG GCT CCG TGA CGC AG; pax-5-reverse,

TCT CGG CCT GTG ACA ATA GG (Tanneal, 65°C; expected length, 439 bp; Ref. 17); VpreB-forward, GTC TGA ATT CCT CCA GAG CCT AAG ATC CC; VpreB-reverse, CAG GTC TAG AGC CAT GGC CTG GAC ␭ GTC TG (Tanneal, 60°C; expected length, 400 bp; Ref. 18); 5-forward, GGG TCT AGT GGA TGG TGT CC; and ␭5-reverse, CAA AAC TGG

GGC TTA GAT GG (Tanneal, 60°C; expected length, 205 bp; Ref. 19). Results Recent thymic emigrants include B cells as well as ␣␤ and ␥␦ T cells FITC was injected intrathymically to label cells in the thymus, and the spleen and lymph nodes were later analyzed for the presence of RTE. After intrathymic FITC injection, the percentage of labeled cells in the thymus was followed over time (Fig. 1A). Six hours FIGURE 1. Changes in the FITC level of thymocytes after intrathymic ϩ ϩ postinjection, 95% of the thymocytes were FITC (Fig. 1A)in injection of FITC. A, Decrease in the number of FITC thymocytes after intrathymic injection of FITC. The vertical bars represent SD in each time comparison with noninjected controls. From 6 to 48 h postinjec- point of analysis (each point represents ﬁve mice). B, Levels of FITC in tion, the percentage of FITC-labeled cells decreased in a nearly

thymocytes 6 h after FITC injection. ƒ, Limit of FITC intensity detectable linear fashion. One of the main causes of the loss of FITC might Downloaded from on FACS. Each vertical broken line represents a scale for a two-fold dif- be cell division, resulting in a 50% decrease in surface labeling per ference in FITC intensity. Therefore, the number on top of each vertical cell division (20), although it is possible that the loss of FITC label line indicates the time of cell division that allows the cells on the lines to could be due to normal turnover of membrane proteins in the ab- remain detectable by FACS. sence of division (21). As shown in Fig. 1B, the intensity of FITC signals at 6 h postinjection was strong enough to detect more than

cells were excluded by positive staining with propidium iodide and were 85% of FITC-labeled cells after one or two cell divisions and http://www.jimmunol.org/ detected in a Cy5-PE channel. ϳ50% of cells after four cell divisions. Because the estimated cycling time for thymocytes is ϳ8–12 h (22), the majority of prog- Estimation of mature T and B cell immigrants into the thymus eny from cycling FITC-labeled thymocytes might maintain detect- Single spleen cell suspension was obtained from C57BL6-Ly5.1/Ly5.2 able levels of FITC at least 24 h after injection. At 24 h postin- mice. Myeloid spleen cells were removed by incubation with Gr-1, Mac-1, jection, ϳ84% of thymocytes retained detectable levels of FITC, and Ter119 Abs before negative selection using anti-rat IgG-conjugated indicating that the bulk of thymocytes can be estimated to turn immunomagnetic beads (Dynal, Oslo, Norway). The purified 1 ϫ 108 ϫ over in ϳ6 days. This estimate is in accordance with turnover rates splenic lymphocytes from adult (Ly5.1 Ly5.2)F1 mice were injected into 1.5- to 3-wk-old Ly5.1 mice. The percentages of donor-derived mature ␣␤ of thymocytes in previous reports (23). Because only ϳ1% of thy- T cells and B cells in the secondary lymphoid organs were analyzed 7, 18, mocytes emigrate per day (14), the major loss of labeled cells is by guest on September 29, 2021 and 24 days postinjection, when the injected Ly5.1 mice reached the age of likely due to apoptosis of thymocytes that either failed positive 4–5 wks. selection or were deleted by negative selection (22, 24). Immunohistochemical staining The phenotype of FITC-labeled thymic emigrants in the spleen is shown in Fig. 2. Approximately 95% of FITCϩ recent thymic Sections of the thymus were cut from frozen samples at 4 microns and were ϩ Ϫ Ϫ ϩ emigrants were CD4 CD8 or CD4 CD8 single-positive ␣␤T fixed with acetone for 10 min (15). Samples were treated with the Vector ϩ Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, CA) and cells (Fig. 2A). The remaining ϳ5% of FITC RTEs were Ϫ Ϫ stained with biotinylated anti-IgM, B220, or MD2 Abs (16). They were CD4 CD8 double-negative cells that were composed of incubated with Streptavidin HRP (Caltag, South San Francisco, CA), vi- CD3ϩTCR␥␦ϩ T cells, B220ϩIgMϩ mature B cells, and some sualized with 3-amino-9-ethylcarbazole as the chromagen for 20 min, and CD3ϩTCR␥␦Ϫ T cells (Fig. 2C). The majority of B220ϩIgMϩ counterstained with Gill’s hematoxylin (Medical Chemical, Fairfield, NJ). ϩ Isotype-matched rat IgG was used as a negative control. mature B cells expressed IgD, but only a minority were CD5 (Fig. 2C). The rate of appearance of FITC-labeled RTEs is almost RT-PCR analysis linear in the periphery up to 24 h postinjection (Fig. 3). The pro- Total RNA was isolated from 1000 purified thymic pro-B and pre-B cells. files of RTE in the lymph nodes were similar to those in the spleen cDNA was analyzed for the presence of pax-5, VpreB, or ␭5 by amplifi- (data not shown). Because most lymphocytes collect in the spleen

FIGURE 2. Analysis of FITCϩ recent thymic emigrants in the spleen 24 h after intrathymic injection of FITC. A, FITCϩ spleen cells contain CD4ϪCD8Ϫ cells as well as CD4ϩCD8Ϫ and CD4ϪCD8ϩ single-positive mature T cells. B, Gate for the FITCϩCD4ϪCD8Ϫ cells. C, Surface phenotypes of the FITCϩCD4ϪCD8Ϫ cells. The Journal of Immunology 5223

FIGURE 3. Numbers of FITCϩ ␣␤ T(A), ␥␦ T(B), and B cell (C) emigrants in the spleen and lymph nodes. The vertical bars represent SD in each time point of analysis (each point represents ﬁve mice). Downloaded from and lymph nodes, it can be estimated that the thymus exports at of donor origin (Table I and Fig. 4). If 100% of peripheral B cells least 1.2 ϫ 106 ␣␤ T cells, ϳ3 ϫ 104 ␥␦ T cells, and ϳ3 ϫ 104 could be replaced by donor-derived B cells, then 0.6 ϫ (100/ B cells per day to the periphery (Fig. 3). The estimated numbers of 5ϳ7) ϭ 12ϳ15% of thymic B cells could be replaced by B cells ␣␤ and ␥␦ T cell emigrants are compatible with previous reports of donor origin. Therefore, a maximum of (12ϳ15%) ϫ (ϳ5 ϫ ϩ ϩ 4 ϭ ϳ ϫ 4

(14, 25). On the other hand, Mac-1 Gr-1 granulocytes/macro- 10 ) 0.6 0.8 10 thymic B cells could have migrated from http://www.jimmunol.org/ phages, CD3ϪNK1.1ϩ NK cells, and CD8␣ϩ/ϪCD11cϩMHC- the periphery. Even if all such B cell immigrants could leave the class IIϩ dendritic cells were not detectable in the RTE (FITCϩ) thymus in 1 day, a maximum of 3 ϫ 104 Ϫ 0.8 ϫ 104 ϭϳ2 ϫ 104 fractions throughout these experiments (Fig. 2C). The lack of thymic B cell emigrants that developed intrathymically would be FITCϩ granulocytes/macrophages indicates that the FITC label exported each day. did not leak into the circulation, and therefore, unincorporated FITC does not persist in a form that would be available for labeling B cell progenitors in the thymus newly immigrating or circulating cells. It has been reported that B cell progenitors are present in the thymus and that isolated thymic B cell progenitors can intrathymically

A significant fraction of B cells that are exported from the differentiate into mature B cells after reinjection into the thymus by guest on September 29, 2021 thymus developed in the thymus (28). The phenotype of thymic B cell progenitors is similar to that It is important to know whether the export of B cells from thymus of bone marrow B cell progenitors (29). In the C57BL/6 strain, the thy- reflects B cell production in the thymus or results from FITC la- mus contains significant numbers of immature B220ϩCD43ϪIgMϪ beling of B cells that have migrated into the thymus from the pre-B cells (ϳ1.8 ϫ 104/thymus) and B220ϩCD43ϩIgMϪ pro-B cells periphery. We tested whether or not peripheral B cells could re- (ϳ1.2 ϫ 104/thymus) (Fig. 5A and Table II). These thymic B220ϩ enter the thymus and contribute to the thymic B cell pool. We B cell progenitors coexpressed CD19 but not NK1.1 (Fig. 5B). The injected i.v. 1 ϫ 108 splenic lymphocytes from adult (Ly5.1 ϫ thymic IgMϩ B cells were composed of IgDϪ and IgDϩ B cells as

Ly5.2)F1 mice into Ly5.1 mice and evaluated the percentages of in bone marrow B cells. Although thymic B cells are reported to donor-derived mature ␣␤ T cells and B cells in the secondary express a broad range of CD5 in the C3H mouse strain (28, 30, 31), lymphoid organs 7, 18, and 24 days postinjection. The peak chimerism for donor-derived cells was seen at 18 days postinjection. Approximately 4% of ␣␤ T cells in peripheral organs were of donor origin, whereas only 0.01% of mature ␣␤ T cells in the thymus were of donor origin (Table I). The rare reentry of these T cells into the thymus is compatible with previous reports (26, 27). On the other hand, ϳ5–7% of B cells in peripheral organs were of donor origin, whereas only ϳ0.6% of thymic mature B cells were

Table I. Chimerism of mature T and B cells 18 days after injection of mature lymphocytesa

Percentage of Donor-Derived Cells

Spleen LN Blood Thymus ϩ ϩ Mature B cells 5.3 Ϯ 1.2 6.7 Ϯ 1.6 5.1 Ϯ 1.8 0.6 Ϯ 0.2 FIGURE 4. Donor-derived mature B cells (Ly5.1 Ly5.2 ) in lymphoid Mature T cells 4.1 Ϯ 1.3 3.7 Ϯ 1.6 3.9 Ϯ 0.9 0.01 Ϯ 0.002 organs 18 days after injection of mature splenic lymphocytes into 2-wk-old mice (Ly5.1ϩLy5.2Ϫ). Percentages of donor-type B220ϩIgMϩ B cells a ϫ 8 Ϫ Ϫ Ϫ A total of 1 10 myeloid cell-depleted (Mac-1 Gr-1 Ter119 ) splenocytes (Ly5.1ϩLy5.2ϩ) are 5–7% in lymph nodes, blood, and spleen but only from C57BL/6 (Ly5.1/5.2) mice were injected i.v. into C57BL/6 (Ly5.2) mice. B220ϩIgMϩ B cells and TCR␣␤high T cells were analyzed. Data are shown as the 0.6% in the thymus. Data from a representative mouse are shown (see mean Ϯ SD in four chimeric mice. Table I). 5224 THYMIC B LYMPHOPOIESIS

FIGURE 5. Phenotypic analysis of thymic B cell compartments in either C57BL/6- or C57BL/ 6-TCR␤-deficient mice. A, B220/IgM profiles of CD4ϪCD8Ϫ thymocytes (upper panels) and B220/CD43 profiles of CD4ϪCD8ϪIgMϪ thymocytes (lower panels). B, Additional surface phenotypic analysis of thymic B cell progenitors: the CD19/CD43 (a) and CD5/CD43 (b) profiles of B220ϩIgMϪ cells, the B220/NK1.1 profile of CD4ϪCD8Ϫ cells, and the IgM/IgD profile of B220ϩ cells.

Table II. Numbers and frequencies of B cell compartments in the thymus Downloaded from

Absolute Numbers/Thymus (ϫ 104)a

Mouse Pro-B Pre-B Mature B

C57BL/6 1.20 Ϯ 0.10 (0.006)b 1.81 Ϯ 0.14 (0.010) 4.96 Ϯ 0.14 (0.028) TCR␤-deﬁcient C57BL/6 10.60 Ϯ 2.77 (0.530) 25.20 Ϯ 3.93 (1.260) 84.12 Ϯ 6.15 (4.206) http://www.jimmunol.org/ a Data are shown as the mean Ϯ SD in four mice in each strain. b Percentage of total cells.

CD5 expression was almost limited to the pro-B cell fraction in the The thymus possesses B cell progenitors of each stage that are C57BL/6 strain thymi (Fig. 5B). similar to those in bone marrow. This suggests that the thymic The thymic pro-B and pre-B cells were puriﬁed and analyzed for microenvironment can fully support B as well as T cell maturation. expression of early B cell-related molecules such as VpreB and ␭5 Adult thymi have been shown to be capable of generating B cells

as well as a transcription factor, pax-5. All of these were detectable after intrathymic injection of hematopoietic stem cells or CLPs (9, by guest on September 29, 2021 in the thymic pro-B cells and were increasingly expressed in pre-B 35). Although it has not been shown that the CLPs themselves cells (Fig. 6). The expression pattern of these molecules was con- migrate from the bone marrow to the thymus, it is possible that sistent with that in bone marrow pro-B and pre-B cells (data not CLPs or one of their immediate offspring could migrate to and seed shown). the thymus. We have previously reported that IL-7, which is pre- Thymic B lymphopoiesis was significantly enhanced in C57BL/ sumably secreted from thymic epithelial cells, promotes survival 6-TCR␤-deficient mice in which thymic T cell development is of thymocytes undergoing positive selection through the up-regu- impaired. The TCR␤-deficient thymi had more than a 100-fold lation of Bcl-2 (1, 4). IL-7 is essential also for Ig gene rearrange- increase in the frequencies (Fig. 5A) and more than a 10-fold in- ment in developing B cells (5). Because IL-7 is known to exist in crease in absolute numbers of both mature B cells and B cell pro- the thymic milieu, IL-7 would be available for the intrathymic genitors in comparison to wild-type thymi (Table II). development of B cells as well as T cells (36). Therefore, the In immunohistochemical stainings of normal and TCR␤-defi- thymic microenvironment should be able to physiologically sup- cient thymi, IgMϩ B cells reside mainly in the corticomedullar port B lymphopoiesis as well as T lymphopoiesis. junction and in the medulla, whereas B220ϩ cells were found Recent studies have shown that, in CD3-⑀ transgenic mice (37) throughout the thymus, indicating that immature B220ϩIgMϪ B and Notch1-deficient mice (38), the number of thymic B cells sig- cells mainly reside in the cortex through the corticomedullar junc- nificantly increases in association with severe impairment of thy- tion (Fig. 7). Accordingly, thymic B lymphopoiesis might occur mic T cell development. It is possible that alterations of these concomitantly with B cell progenitor migration from the cortex to genes could skew the commitment of immature thymic progenitors the medulla of the thymus, mirroring T lymphopoiesis (32). toward the B cell lineage (37, 38). However, we demonstrate in

Discussion We demonstrate that the adult C57BL mouse thymus physiologically generates and exports a significant number of B cells into the peripheral B cell pool. The findings of B cell development in the thymus and the recently described ␣␤ T cell development in the bone marrow (33, 34) challenge the paradigm of lymphocyte classification into bone marrow-derived B cells and thymus-derived T cells. Our study also suggests that the thymus may not actively export NK cells and dendritic cells into the periphery or FIGURE 6. RT-PCR analysis of sorted thymic B cell progenitors. The that the level of export may be below the level of detection in this sorted CD4ϪCD8ϪIgMϪB220ϩCD43ϩ pro-B cells and CD4ϪCD8ϪIgMϪ- study. B220ϩCD43Ϫ pre-B cells increasingly expressed pax-5, VpreB, and ␭5. The Journal of Immunology 5225

FIGURE 7. Immunohistochemi- cal staining of a TCR␤-deficient thymus. The thymus from a TCR␤- deficient mouse was stained with MD-2 Abs that react with medullar epithelial cells (A), B220 Abs (B), and anti-IgM Abs (C). Cx, Cortex; M, medulla. Downloaded from this study that the disruption of T cell development simply by a 10. Wu, L., M. Antica, G. R. Johnson, R. Scollay, and K. Shortman. 1991. Devel- TCR␤ gene knockout also results in a relative and absolute in- opmental potential of the earliest precursor cells from the adult mouse thymus. J. Exp. Med. 174:1617. crease in thymic B cell compartments. Accordingly, these data 11. Matsuzaki, Y., J. Gyotoku, M. Ogawa, S. Nishikawa, Y. Katsura, G. Gachelin, collectively suggest that in normal thymi, rapid expansion of T and H. Nakauchi. 1993. Characterization of c-kit positive intrathymic stem cells cells might take up most of the microenvironmental niches, pre- that are restricted to lymphoid differentiation. J. Exp. 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This may allow a more diverse set of Ig recep- quantitative aspects of cellular traffic from the thymus to the periphery in mice. tors in the periphery. It is also possible that the thymic B cells may Eur. J. Immunol. 10:210. contribute to the cellular interactions that mediate positive and 15. Gutman, G. A., and I. L. Weissman. 1972. Lymphoid tissue architecture: exper- imental analysis of the origin and distribution of T-cells and B-cells. Immunology by guest on September 29, 2021 negative selection of maturing T cells (39), including Ig isotype 23:465. and idiotype as selecting elements (40). Thus, it is important to 16. Small, M., W. Van Ewijk, A. M. Gown, and R. V. Rouse. 1989. Identification of clarify the role of these ectopically but physiologically developed subpopulations of mouse thymic epithelial cells in culture. Immunology 68:371. thymic B cells in the immune system. 17. Adams, B., P. Dorfler, A. Aguzzi, Z. Kozmik, P. Urbanek, I. Maurer-Fogy, and M. Busslinger. 1992. 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