differentiation of thymic epithelial progenitors

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1 Proc. Natl. Acad. Sci. USA Vol. 93, pp , June 1996 Immunology The nu gene acts cell-autonomously and is required for differentiation of thymic epithelial progenitors (nude mice/thymus) C. C. BLACKBURN*t, C. L. AUGUSTINE*, R. LI*, R. P. HARVEY*, M. A. MALINt, R. L. BOYDt, J. F. A. P. MILLER*, AND G. MORAHAN* *The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Parkville 3050, Australia; and tdepartment of Pathology and Immunology, Monash University Medical Centre, Commercial Road, Prahran 3181, Australia. Contributed by J. F. A. P. Miller, February 5, 1996 ABSTRACT The nude mutation (nu) causes athymia and hairlessness, but the molecular mechanisms by which it acts have not been determined. To address the role of nu in thymogenesis, we investigated whether all or part of the nude thymic epithelium could be rescued by the presence of wildtype cells in nude <-> wild-type chimeric mice. Detailed immunohistochemical analyses revealed that nude-derived cells could persist in the chimeric thymus but could not contribute to cortical or medullary epithelial networks. Nude-derived cells, present in few clusters in the medulla, expressed markers of a rare subpopulation of adult medullary epithelium. The thymic epithelial rudiment of nude mice strongly expressed these same markers, which may therefore define committed immature thymic epithelial precursor cells. To our knowledge, these data provide the first evidence that the nu gene product acts cell-autonomously and is necessary for the development of all major subpopulations of mature thymic epithelium. We propose that nu acts to regulate growth and/or differentiation, but not determination, of thymic epithelial progenitors. The thymus is the obligatory site of T-cell maturation and is therefore central to the development of a fully competent immune system; athymia results in profound immunodeficiency (1). The thymic epithelium not only supplies a framework in which T-cell development occurs, it also shapes the T-cell repertoire by mediating positive and negative selection of developing thymocytes. Consequently, its organization is quite complex (2). The nude mutation (nu) has a profound effect on thymic development (3, 4) but does not affect the lymphoid compartment (4, 5). During development of the nude thymic anlage, the pharyngeal ectoderm of the third cleft fails to proliferate at embryonic day 11.5 (6) and thymogenesis does not progress beyond this point; the anlage is devoid of lymphocytes and never proceeds to a lymphoid stage (4). Adult nude mice retain a nonfunctional cystic thymic rudiment (7). Two hypotheses have previously been offered to explain the nu defect (6, 8). One states that the primary lesion affects ectoderm (6). Because studies have clearly shown that avian thymic epithelium is derived principally from endoderm (9), this hypothesis suggests that nu does not affect the developmental potential of thymic epithelial precursors. The absence of thymic epithelial networks in nude mice would instead result from the failure of ectoderm to induce differentiation of thymic endoderm. Alternatively, the primary defect in nude mice might be the inability of the thymic epithelial anlage to attract and retain thymocyte precursors; loss of epithelial differentiation could be secondary to the absence of thymic lymphocytes (8). This suggests that the nu gene affects thymus development, because The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact inductive interactions cannot be initiated between the thymic epithelial rudiment and thymocyte progenitors. In this case, the primary defect need not be in the inductive pathway. A third hypothesis is that nu is a cell-autonomous gene product that is necessary for differentiation of all or part of the thymic epithelium itself. This hypothesis predicts that nu expression is minimally required in the endodermal germ layer. In this case, the observed ectodermal defect in nude mice could be explained by an additional requirement for nu expression in ectoderm per se, or by nu control of ectodermal proliferation via induction from endodermal cells. Recently, whn, a homologue of a Drosophila transcription factor gene, was identified as a candidate nu gene; expression of whn was detected in thymus and skin by reverse transcriptase-pcr, and mutations were found in alleles of whn carried by strains of nude mice and rats (10). However, its precise role in thymus development and hair formation remains unknown. All three of the above hypotheses could be fulfilled by this candidate nu gene, which could act to affect differentiation of any of the thymic epithelial subpopulations, or could control the expression of an inductive pathway in either the endodermal or ectodermal germ layers. To discriminate between these possibilities, the role of the nu gene was investigated directly by determining whether the nu thymic defect could be rescued in the presence of wild-type cells. Neither of the first two hypotheses postulates that nu acts autonomously to affect thymic epithelial differentiation; both predict that the nu defect should be rescued by wild-type cells. A chimeric thymus would be composed of both nude and wild-type cells under both models. Only if the third hypothesis was true would no rescue of the nu defect occur; in that case, thymic epithelial networks of chimeric mice should comprise only wild-type cells. Others have previously used nude < wild-type allophenic chimeras to investigate the nature of the 11; ref. nu defect [see reference to Matsunaga (1973) in ref. 12). However, no immunohistological analyses were conducted in those studies and the results obtained were inconclusive (12). This study therefore had three aims: (i) to determine whether nude-derived cells could contribute to the thymic epithelium in nude -> wild-type chimeras; (ii) to determine the phenotype of any such cells in the chimeric thymus; and (iii) to use this information to determine the likely mode of action of the nu gene. MATERIALS AND METHODS Animals. All mice were from specific pathogen-free colonies of the Walter and Eliza Hall Institute of Medical Research. Abbreviations: MHC, major histocompatibility complex; MTS, mouse thymic stroma. tto whom reprint requests should be sent at the present address: Molecular Immunology Group, Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom.

2 Immunology: Blackbum et al. Allophenic, or chimeric, mice (11) were produced as follows. Three-day embryos were collected from BALB/cnu/+ x CBA.u/nu and (C57BL/6 x C57BL/10)Fl matings. The zona pellucida was removed from each of the embryos by incubation in acid Tyrode's solution, after which embryos were placed in pairs in a small volume of M16 medium (13), covered with equilibrated mineral oil, and incubated overnight at 37 C. Aggregated embryos were transferred into uteri of pseudopregnant recipients. Two types of chimera were generated: (BALB/c x CBA).nu/nu <- (C57BL/6 x C57BL/10)F2-i.e., nude <> wild-type (nu/nu <- +/+)-and (BALB/c CBA)nu/+ (C57BL/6 x <- x C57BL/10)F2--i.e., nu/+ +/+. Genotypes of chimeric mice were determined by typing < tail DNA for microsatellite markers DllMit8 (Whitehead/ MIT, Center for Genome Research, Genetic Map of the Mouse, data base release no. 3, 1993) and DD228 (14), which are closely linked to nu (15). Immunological Analyses. Six-week-old mice were irradiated (900 rad) and reconstituted by intravenous injection with 105 C57BL/6 bone marrow cells. The following day they received 0.2 ml of rat anti-thy 1 antibody T-24 (16) intraperitoneally. After irradiation, mice were maintained on antibiotic water (25,xg of neomycin per ml and 13,g of polymyxin per ml) for 6 weeks and then sacrificed. Thymi were prepared for sectioning and staining as described (17, 18). Briefly, binding of biotinylated monoclonal antibodies to H-2Kk (A.11.2.F; ref. 19) and anti-i-ak (39J; ref. 20) was detected after sequential incubation with horseradish peroxidase-streptavidin and diaminobenzidine. Three-color immunofluorescence was performed with monoclonal antibodies of the mouse thymic stroma (MTS) series specific for thymic stroma subsets (18) as follows. Sections were incubated in MTS antibody, washed, and incubated in fluorescein isothiocyanate-coupled anti-rat immunoglobulin (Silenus, Melbourne, Australia). They were then incubated sequentially in normal rat serum (1:10) and a mixture of biotinylated-a.11.2.f and rabbit anti-keratin, washed, incubated in tetramethylrhodamine B isothiocyanate-anti-rabbit immunoglobulin (Silenus) and amino methyl commarin acetic acid-avidin (The Jackson Laboratory), washed, and then visualized and photographed under appropriate excitation conditions. Flow cytometric analyses (performed as in ref. 21) used biotinylated antibodies to H-2Kk (A.11.2.F), H-2Kb (K9-178; ref. 22), Thy 1 (30H12; ref. 23), and CD3 (KT3.2; ref. 24). RESULTS Phenotype of Cells in the Nude Thymic Rudiment. Before examining epithelial cells in the chimeric thymus, it was necessary to define the phenotype of the thymic remnant from nude mice. Nude thymic remnants were therefore stained with a panel of MTS antibodies to defined thymic stromal elements (18). Two-color fluorescence staining was used to assess the reactivity of cells with each MTS marker and with an anticytokeratin antibody. Cytokeratins are expressed by all epithelial cells, distinguishing them from bone marrow-derived thymic components. Anti-keratin staining demonstrated that the nude rudiment consisted of clusters, or linear aggregates, of regular-shaped epithelial cells, some of which appeared to form cyst-like structures. Approximately 90% of these epithelial cells were positive for the epithelial marker MTS-44, and -15% expressed the medullary epithelial marker MTS-10. Three-color labeling for keratin, MTS-10, and MTS-44 confirmed that the majority of epithelial cells were either MTS44+ or MTS10+, but a distinct population expressed both MTS markers (data not shown). As previously reported (25), with the exception of isolated macrophages and occasional epithelial cells, the nude rudiment was major histocompatibility complex (MHC) class II negative/low. A striking finding was that MTS-20, MTS-24, and MTS-33 all reacted with the Proc. Natl. Acad. Sci. USA 93 (1996) 5743 nude epithelial rudiment (Fig. 1; Table 1) despite their recognition in the normal adult thymus of only infrequent single cells or small clusters of medullary epithelium (18). As expected, MTS-12+ cells (vascular endothelium) were present in the rudiments, indicating that nu did not affect these cells. Phenotypic Analysis of Nude <-> Wild-Type Chimeric Mice. Two types of chimeras were generated: nu/nu <- +/+ and nu/+ +/+, <- the latter serving as controls because nu/+ mice are physiologically normal. The (BALB/c x CBA)F1 embryo is referred to hereafter as the donor, and the (C57BL/6 x C57BL/10)F2 wild-type embryo as the host. Donors and hosts were mismatched at the MHC, being H-2k/d and H-2b, respectively. This allowed immunohistochemical discrimination between donor and host cells at single-cell resolution. As described for similar aggregations (11, 12), some chimeric animals showed nude skin patches (Fig. 2), the extent of which varied greatly between chimeras. Although the presence of nude patches on chimeric skin appeared to be a good indicator of donor genotype, it was important to confirm this by direct methods. Therefore, tail DNA was analyzed by PCR at two loci closely linked to nu, DllMit8 and D11DD228 (14, 15). Because the BALB/c.nu, CBA.nu, C57BL/6, and C57BL/10 alleles were identical at these loci, a single PCR product was generated in nu/nu <-> +/+ chimeras, whereas nu/+ <-> +/+ mice had an additional product corresponding to the BALB/c or CBA allele (15). Genotyping in this manner confirmed that all nu/nu <-> +/+ mice also exhibited patches of nude skin. To determine whether all of the nu/nu - + / + chimeras had developed a functional thymus, peripheral blood was analyzed for the presence of T cells by flow cytometry. CD3+Thy 1+ FIG. 1. Phenotype of nude stromal cells in the nude thymic remnant: double-color fluorescence using antibodies to MTS-20 (A), MTS-24 (C), and MTS-33 (E; green fluorescence). The same sections were stained with antibodies to keratin (B, D, and F; red fluorescence). Scale bar = 24 um.

3 5744 Immunology: Blackburn et al. Table 1. Summary of expression of MTS markers Antibody Target nu/+ nu/nu nu thymic rudiment H2-Kk MHC class I Many Few + Cytokeratin Epithelium MTS-6 MHC class II + -/low epithelium negative MTS-7 Epithelial and non-epithelial stroma + - MTS-10 Supcapsular and medullary epithelial + (+) (+) MTS-20 Medullary epithelial subset +* + +t +t MTS-24 Medullary epithelial subset +* +t +t MTS-33t Medullary epithelial subset; MTS-44 cortical thymocytes (ThB) +* +t +t Cortical epithelium; medullary epithelial subset MTS-12t Vascular endothelium MTS-16 Extracellular matrix MTS-37t Macrophage (heat stable antigen) *Isolated epithelial cells in medulla. tall nude epithelial cells. talso occurs on lymphocytes. cells were found in the peripheral blood of all chimeras (data not shown). They therefore had circulating T cells and a functional thymus. Contribution of Nude-Derived Cells to the Chimeric Thymus. To determine whether the nude defect in thymus development could be rescued in the presence of wild-type cells, thymi from nu/nu <-> +/+ chimeras were analyzed for the presence of nude-derived (i.e., H-2k/d) cells. To discriminate between expression of MHC class I antigens on thymic epithelial cells and on thymocytes and other bone marrow-derived cells, it was necessary to deplete the chimeras of donor embryo-derived lymphoid cells that could complicate analysis. Mice were therefore irradiated and reconstituted with C57BL/6 (H-2b) bone marrow. After 6 weeks, mice were sacrificed and their thymi were removed for sectioning and staining with donor-specific anti-h-2kk antibody. No H-2Kk-positive cells were observed in the thymic cortical epithelium of the five nu/nu /- +/+ chimeras tested. In two out of five nu/nu <- +/+ chimeras, no H-2Kk-positive cells were observed in the thymic medulla, whereas in the other three, occasional small, isolated clusters of H-2Kk-positive cells were seen in an otherwise negative medulla (Fig. 3 A and B). In contrast, thymi from the nine nu/+ -> +/+ control chimeras tested showed heavy anti-h-2kk staining throughout both cortical and medullary regions (Fig. 3 C and D). H-2Kkpositive cells also contributed to the vasculature in all chimeras ṀHC class I expression is relatively low in thymic cortical stroma and can be difficult to detect. However, MHC class II l ~ l I '' FIG. 2. nu/nu <-> +/+ chimeric mouse. Note patches of nudederived skin around eye and on flank. Proc. Natl. Acad. Sci. USA 93 (1996) is expressed at high levels on cortical stroma (2). The analysis was therefore repeated using a monoclonal anti-i-ak antibody, 39J (20). No I-Ak-positive cells were seen in the thymic cortex of nu/nu + / + chimeras, while nu/+ + +/+ cortex stained heavily with anti-i-ak (data not shown). The pattern of anti-i-ak staining in the medulla was similar to that observed with anti-h-2kk: two out of five nu/nu +- +/+ chimeras had no I-Ak-positive cells in the thymic medulla, whereas in the other three mice, infrequent isolated clusters of I-Ak weakly positive cells were seen in an otherwise negative medulla (data not shown). The I-Ak-positive nu/nu cells in these clusters were far fewer than the H-2Kk-positive cells and stained less intensely than cells in the control, nu/+ <- +/+ chimeras, suggesting that MHC class II expression was markedly reduced on nu/nu cells (see below). Phenotype of nu/nu Cells within Chimeric Thymi. To determine whether the clusters of nu/nu cells were a normal thymic subpopulation, the chimeric thymi were stained with a panel of MTS antibodies to defined thymic stromal elements (18). Two- and three-color fluorescence staining was used to assess the reactivity of H-2Kk cells with each MTS marker and with an anti-cytokeratin antibody. Radio-resistant, bone marrow-derived cells from the nude donor were detected by counterstaining with MTS-37, which recognizes heat-stable antigen. Nude-derived vascular endothelium showed a normal pattern by staining with MTS-12 (data not shown). Despite developing in a normal thymic environment, the nude-derived epithelial cells within the chimeric thymi were very similar morphologically and phenotypically to those seen in the thymic rudiment. They remained as regular-shaped clusters or linear aggregates and appeared to form cyst-like structures. These cells were negative/low for MTS-10, which recognizes subcapsular and medullary epithelial cells (Fig. 4A). They were negative for MTS-16 (Fig. 4B), expressed on the extracellular matrix, and also for MTS-7, which recognizes reticular fibroblasts and isolated epithelial cells (data not shown). In addition, as predicted by the anti-i-ak data presented above, they were negative, or stained only weakly, for the nonpolymorphic MHC class II determinant recognized by MTS-6 (Fig. 4C). Most of the nude-derived epithelial cells within nu/nuu +/+ chimeric thymi expressed MTS-44 or MTS-10 in the proportion observed in the nude rudiment; -10% expressed both markers. Again as for the nude rudiment, they stained strongly positive with MTS-20, MTS-24, and MTS-33 (illustrated by MTS-24 and MTS-33 in Fig. 4 D-K), which identify infrequent epithelial cells in normal adult thymus (18). Nudederived cells positive for these markers occurred in higher

4 Immunology: Blackbumn et al. Proc. Natl. Acad. Sci. USA 93 (1996) 5745 FIG. 3. Contribution of nude-derived cells to chimeric thymus stroma. (A and B) nu/nu *- +/+ thymus stained with anti-h-2kk. (C and D) control nu/+ + +/+ thymus stained with anti-h-2kk. Scale bar = 72.Lm (A and C); scale bar = 30 pum (B and D). numbers than the equivalent population in a normal thymus. H-2Kk-positive, cytokeratin-positive cells in the control, nu/+ <- +/+ thymi showed a normal pattern of reactivity for all markers (data not shown; Table 1). DISCUSSION Cell-Autonomous Role of nude in Thymogenesis. Normal cells could not rescue the nu defect, as nude-derived cells could not form mature thymic epithelial networks in nude -> wildtype chimeras, although they could form mesoderm-derived (endothelial) and bone marrow-derived stromal elements. Therefore, the nu gene product acts cell-autonomously and is necessary for the development of most thymic epithelial subpopulations. These data have major implications regarding the role of the nu gene in thymus development. First, as thymic epithelium is mainly derived from the endoderm (9), these data show that the nu mutation acts autonomously in epithelial lineages derived from the endoderm, contradicting the hypothesis that nu principally affects cells of the ectodermal layer (6). Although it remains possible that nu may also act in cells of ectodermal origin, it is not clear that any thymic epithelial subpopulations are ectoderm-derived. Secondly, the chimeric thymi were all fully lymphoid and (before irradiation and reconstitution) could support development of T cells of both host and donor type. Therefore, attracting hemopoietic cells into the thymus and thereby facilitating epithelial-lymphocyte crosstalk was insufficient to rescue the nu defect, refuting the second hypothesis regarding the role of the nu gene (8). Although it is still possible that nu/nu epithelial cells are unable to participate in thymocyte crosstalk, due to, for instance, an inability to receive or respond to lymphoid inductive signals, these data show that the defect is not restricted to an inability of nude epithelial cells to draw bone marrow-derived cells into the thymic anlage. Identification of a Putative Thymic Epithelial Progenitor Cell. Despite the inability of cells carrying the nu mutation to contribute to normal thymic epithelial networks, there were some nude-derived cells present within the medulla of the chimeric thymi. These cells were positive for the immature thymic epithelial markers MTS-20, MTS-24, and MTS-33, and they were also positive for either MTS-44 or the medullary marker MTS-10, or both. These nude-derived, MTS-20+, 24+, 33+, H-2Kk+, I-Ak-no cells appeared in clusters or linear aggregates and had no apparent equivalent in the normal thymus, in which the rare subpopulation of medullary epithelial cells that express these antigens are usually found as single cells. MTS-20 and MTS-24 are highly expressed, pan-epithelial markers early in thymic ontogeny (day 13, the earliest stage tested, to day 17; ref. 18). In addition, MTS-33 is expressed strongly at times when thymic epithelium is regenerating, for instance, postirradiation (26). Our data show that the nude thymic rudiment also consists largely of poorly differentiated epithelial cells that express MTS-20, MTS-24, and MTS-33 (although it was not determined whether these markers were coexpressed) and do not express MHC class II. This suggests that in nude mice, the thymic rudiment contains cells that have been committed to thymic epithelial lineages but that are developmentally arrested. It also suggests that the nudederived cells observed in the chimeric thymus are equivalent to epithelial cells in the nude thymic rudiment. We therefore postulate that the MTS-20+, 24+, 33+, H-2 Kk+, I-Ak-1 phenotype describes thymic epithelial precursors. This is consistent with the observation that determination of thymic epithelial precursors occurs before branchial pouch formation in chicken embryos (9). The data suggest that thymic epithelial precursor population or populations may reside in the medulla, rather than the cortex, of the adult thymus. whn as the nu Gene. It was recently proposed that whn may be the nu gene (10). This gene is a member of the winged-helix class of transcription factors, and may play a role in transcriptional regulation. Such a role, in principle, may or may not be cell-autonomous, because transcription factors could control expression of, for instance, cell-signaling molecules that act in tissue induction (reviewed in ref. 27). The data we present are consistent with the nu gene encoding a cell-autonomous transcription factor. Taken together, our results suggest that nu directly affects the growth and differentiation, but not the determination, of thymic epithelial progenitors and that failure of inductive interactions is not central to the nude defect. Other genes of the forkhead family (28) may have a similar mode of action, as may other transcription factors shown to be essential for the development of particular organs, such as Hoxll in the spleen (29). The nude mouse may therefore provide a model to study the role of individual transcription factors in the interdependent relationships of growth and differentiation during organogenesis. Finally, the identification of a putative epithelial progenitor cell population in the adult thymus suggests that nu is not only required in development of the embryonic thymus, but also in the neonatal and

5 5746 Immunology: Blackburn et al. Proc. Natl. Acad. Sci. USA 93 (1996) FIG. 4. Phenotype of nude-derived stromal cells in chimeric thymus. (A-C) Double-color fluorescence of nu/nu <- +/+ thymus sections stained with anti-h-2kk (blue fluorescence) and counterstained (green fluorescence) with the following specific antibodies: MTS-10 (A), recognizing subcapsular and medullary epithelia; MTS-16 (B), specific for extracellular matrix; and MTS-6 (C), reacting with MHC class II molecules of all haplotypes. (D-K) Triple-color fluorescence of nu/nu * +/+ thymus sections stained with anti-h-2kk (blue fluorescence), anti-keratin (red fluorescence), and MTS-24 (D-G) and MTS-33 (H-K) antibodies (green fluorescence). G and K show all three colors. Scale bar = 12 tm. the adult thymus, perhaps as the thymic epithelium interacts with thymocytes and T cells (30, 31). The authors thank P. Crossley, I. Lyons, J. Allison, L. Hartley, and L. Parsons for advice and discussion, and J. Falso, F. Karamalis, J. Parvis, and A. Simos for technical assistance. C.C.B. was supported by the Wellcome Trust. This work was supported by the National Health and Medical Research Council of Australia, by the Rebecca L. Cooper and Ian Potter Foundations, and by National Institutes of Health Grant no. AI Miller, J. F. A. P. (1961) Lancet ii, Boyd, R. L., Tucek, C. L., Godfrey, D. I., Izon, D. J., Wilson, T. J., Davidson, N. J., Bean, A. G. D., Ladyman, H. M., Ritter, M. A. & Hugo, P. (1993) Immunol. Today 14, Flanagan, S. P. (1966) Genet. Res. 8, Pantelouris, E. M. (1973) Differentiation 1, Wortis, H. H., Nehlsen, S. & Owen, J. J. (1971)J. Exp. Med. 134, Cordier, A. C. & Heremans, J. F. (1975) Scand. J. Immunol. 4, Holub, M., Rossman, P., Tlaskalova, H. & Vidmarova, H. (1975) Nature (London) 256, Ritter, M. A. & Boyd, R. L. (1993) Immunol. Today 14, Le Douarin, N. M. & Jotereau, F. V. (1975) J. Exp. Med. 142, Nehls, M., Pfiefer, D., Schorpp, M., Hedrich, H. J. & Boehm, T. (1994) Nature (London) 372, Mintz, B. (1974) Annu. Rev. Genet. 8, Zinkernagel, R., Burki, IC, Cottier, F., Kossodo, Althage, A. & Illmensee, K. (1983) EMBO J. 2, Whittingham, D. G. (1971) J. Reprod. Fertil. Suppl. 14, Nehls, M., Luno, K., Schorpp, M., Krause, S., Matysiak-Scholze, U., Prokop, C.-M., Hedrich, H. J. & Boehm, T. (1994) Eur. J. Immunol. 24, Blackburn, C. C., Griffith, J. & Morahan, G. (1994) Genomics 26, Dennert, G., Hyman, R., Lesley, J. & Trowbridge, I. S. (1980) Cell. Immunol. 53, Allison, J., Campbell, I. L., Morahan, G., Mandel, T. E., Harrison, L. C. & Miller, J. F. A. P. (1988) Nature (London) 333, Godfrey, D. I., Izon, D. J., Tucek, C. L., Wilson, T. J. & Boyd, R. L. (1990) Immunology 70, Oi, V.T., Jones, P.P., Goding, J. W., Herzenberg, L.A. & Herzenberg, L. A. (1978) Curr. Topics Microbiol. Immunol. 81, Pierres, M., Devaux, C., Dosseto, M. & Marchetto, S. (1981) Immunogenetics 14, Morahan, G., Hoffman, M. & Miller, J. (1991) Proc. Natl. Acad. Sci. USA 88, Hammerling, G. J., Rusch, E., Tada, N., Kimura, S. & Hammerling, U. (1982) Proc. Natl. Acad. Sci. USA 79, Ledbetter, J. A., Rouse, R. V., Micklem, H. S. & Herzenberg, L. A. (1980) J. Exp. Med. 152, Tomonari, K. (1988) Immunogenetics 28, Jenkinson, E. J., van Ewijk, W. & Owen, J. J. T. (1981) J. Exp. Med. 153, Randle-Barrett, E. S. & Boyd, R. L. (1995) Dev. Immunol., in press. 27. Andrew, D. J. & Scott, M. P. (1992) The New Biologist 4, Pierrou S., Hellqvist M., Samuelsson L., Enerback S. & Carlsson P. (1994) EMBO J. 13, Roberts, C. W., Shutter, J. R. & Korsmeyer, S. J. (1994) Nature (London) 368, Surh, C. D., Ernst, B. & Sprent, J. (1992) J. Exp. Med. 176, Hollander, G., Wang, B., A., N., Platenburg, P. P., van Ewijk, W., Burakoff, S. J., Gutierrez-Ramos, J. C. & Terhost, C. (1995) Nature (London) 373,