Immunology Letters, 27 (1991) 1 - 6 Elsevier IMLET 01508
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Prothymocyte seeding in the thymus H e l e n C. O'Neill Developmental Haematology Group, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia (Received 16 March 1990; revision received 2 July 1990; accepted 19 September 1990)
1. Summary A major gap in our understanding of T lymphocyte development is the process of stem cell differentiation into T lymphocyte precursors. An important question is whether bone marrow-derived stem cells become committed to T lymphoid lineage within the bone marrow, or whether this occurs once cells have entered the microenvironment of the thymus. Attempts to identify a haemapoietic precursor of thymocytes in mice, a "prothymocyte", have involved cell transfer experiments involving isolated and selected populations of bone marrow stem cells, as well as transformed or continuous cell lines representing early stage in mouse T cell development. Current information on the properties of stem cells which can seed the thumus is reviewed in this paper, and the possibility that progenitor T cells may be identified by their expression o f receptor(s) which localise them into the thymus is considered.
2. Introduction The thymus is the central organ for differentiation of T lineage cells, yet the adult mouse thymus contains no endogenous stem cell population (reviewed in ref. 1). After initial development during embryogenesis, the thymus receives only slow traffic of cells from bone marrow to replenish its proliferating cell pool. After irradiation, stress or disease, it becomes Key words: Thymus; Stem cell; T cell progenitor; Cell adhesion; Lymphocyte migration Correspondence to." Helen C. O'Neill, Developmental Haematology Group, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
repopulated by a low level influx of stem ceils [1], and the irradiated animal model has been used widely to study stem cell reconstitution of the thymus [1 - 14]. In adult mice, most of these ceils come from bone marrow, but cells which can colonise the irradiated thymus have also been found in spleen [3, 6, 11] and thymus [5, 6, 11, 12]. The thymus operates primarily for T cell differentiation and, unlike lymph node cells, is not geared for entry of large numbers of cells, nor for expansion of antigen-activated cells. The medullary compartment of the thymus is however connected to the lymphoid circulation, and mature T lymphocytes have been shown to enter the thymus from the periphery [15, 16], and to localise within the medulla [17]. It is thought that progenitor T cells enter the thymus through a set of blood vessels in the cortico-medullary region and thence localise to the subcapsular cortical region which is the location of large immature blast cells [18]. In 1976, Basch and Kadish [3] reported that bone marrow stem cells entering the thymus of an irradiated host underwent a resting phase estimated to be between 8 and 12 days, and that only a small number, e.g., 10-100 stem cells, were needed to reconstitute the T cell compartment of an irradiated mouse thymus [3, 4]. More recent studies have confirmed the restricted number of. progenitors required to generate the T cell repertoire [1, 9, 11, 14]. Weissman and co-workers [9, 14] have used limiting numbers of bone marrow ceils to reconstitute the irradiated mouse thymus and have estimated that just a single stem cell, termed the T colony forming unit, CFU-T, is needed to repopulate thymic lobes. In these studies, individual thymic colonies expressing allele-specific cell surface markers of donor bone marrow cells have been identified by antibody staining. Analysis of thymic colonies
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[9], and of peripheral T cells derived from these colonies [14], have confirmed that a single CFU-T in bone marrow can give rise to thymic progeny representing each of the major T cell subsets of thymus [10, 14]. The major functional classes of T cells have also been identified in the periphery [14]. Recolonisation of deoxyguanosine-treated or alymphoid embryonic thymus lobes in organ culture has also been reported by Kingston et al. [19] using a single fetal thymic blast cell. Cell surface marker analysis of the progeny of a single intrathymic precursor T cell has confirmed the development of several major thymic subclasses amongst the progeny. Once stem cells have entered the thymus, they proliferate and develop into T cells with antigen specificity and immune function (reviewed in refs. 20 and 21). The process of T cell maturation is now well understood from studies of cell surface antigen expression using monoclonal antibodies (reviewed in refs. 22-24), and from analyses of T cell receptor rearrangements on selected T cell subpopulations [25, 26]. T lymphocytes become committed to the expression of one T cell receptor and one function, either cytotoxic, helper or suppressor. These cells can be broadly grouped into CD8-expressing cytotoxic cells or CD4-expressing helper cells which recognise antigen in the context of Class I or Class II MHC antigens, respectively. Rearrangement and expression of the T cell antigen-specific receptor occurs within the thymus in an ordered and defined fashion [23, 25, 26], and selection of the T cell repertoire occurs before cells leave the thymus. Thymic epithelial elements [27] and bone marrow-derived stromal cells expressing self MHC antigens [28] play a role in selection/elimination of cells expressing T cell receptors of low/high affinity for self determinants. Thymic stromal elements also function to drive proliferation and activation required for differentiation of cells, but how this process operates or in what order is not yet known. One interaction already described involves the CD2 molecule on immature T cells interacting with its ligand, the LFA-3 molecule on isolated thymic epithelial cells [29], and the activation and proliferation of human immature thymocytes by supernatants of human thymic epithelial cells containing IL-1, IL-3 and GM-CSF has been measured [30]. Monoclonal antibodies have now been derived which can divide the epithelial compartment into several distinct regions and it
is possible that different epithelial/stromal elements all have distinct functions during the maturation process [31]. Despite a wealth of information about differentiation of the major lineages of T cells within the thymus, very little is known about the properties of the haemapoietic precursor of thymus, or about the exact mechanism by which these cells seed the thymus. The early stages of stem cell differentiation within the thymus and the regulatory elements which control these processes are stil unknown. A paucity of antibodies specific for cell surface markers unique to T cell progenitors has made identification of these cells difficult. One anticipates that markers for T cell progenitors should reflect acquisition of T cell specific functions unique to early T cell development. Expression of CD3 and the T cell receptor, or known T cell specific growth factor receptors, correlates only with thymic development of mature T cells. Markers such as CD7 in humans [32] and Thy-1 in mouse [33] are expressed early during T cell development, but are also expressed by all T-lineage cells. Because of the absence of specific markers, very little is understood about the lineage relationship of stem cells and T cell progenitors, both within the thymus and in other lymphoid compartments. A detailed study of early T cell development, and the isolation of progenitor T cells prior to induction of self tolerance is central to therapies involving bone marrow transplants, immunotherapy in T cell deficient diseases and radiation therapy. Cells which are part way committed to the T cell lineage may also be the most appropriate target cells for gene therapy involving T cell specific genes.
3. Attempts to identify progenitor T cells amongst stem cell populations The main question under consideration here is whether stem cells become committed to T cell lineage within the bone marrow environment or whether this occurs once stem cells have localised within the thymus. The former possibility is consistent with the presence of a T-committed "prothymocyte" or even a lymphoid-committed stem cell present in bone marrow. The latter is consistent with the model that multipotential stem cells can seed the thymus. Most attempts to define the haemapoietic precursor of thymocytes have involved selectively depleted bone
marrow populations and a test of their capacity to reconstitute irradiated mouse thymus. In the early experiments of Basch et al. [34], procedures for depletion of B cells, T cells, and macrophages from bone marrow, were found to give enrichment of prothymocytes as judged by their content of the enzyme terminal deoxyribonucleotide transferase. Recently, more rigorous depletion of bone marrow by sorting out lineage-negative cells using a fluorescence activated cell sorter and antibodies specific for macrophages, granulocytes, T and B cells have confirmed the presence of a bone marrow stem cell subset expressing low levels of the Thy-1 antigen [35]. This population of cells has been shown to contain precursors of all lymphomyeloid lineages, reconstituting irradiated animals with T, B and macrophage/monocyte lineage cells [35, 36]. Cells which can repopulate the thymus of an irradiated animal have now been identified amongst those bone marrow cells which appear to represent the pluripotential subset [13]. These cells have been identified as lineage-negative bone marrow cells which bind antibody specific for the Thy-1 and Ly-6 molecules (the Sca-1 antibody) [13] and represent a 0.05% population of non-dividing bone marrow cells. A comparison of the number of precursors in whole bone marrow versus this highly purified stem cell population has indicated a 1000-fold enrichment of precursors capable of reconstituting the thymus of an irradiated animal. The number of these cells capable of generating a thymic clone was found to be as low as 20, and was reduced by 5-fold if cells were injected directly into the thymus rather than the bloodstream [13]. These increased estimates could, however, reflect the higher proliferative potential of stem cells, when introduced directly into the thymic microenvironment. This data suggests several possibilities. Firstly, pluripotential stem cells may become committed to the lymphoid lineage once they are located within the thymus, and secondly, pluripotential stem cells may be heterogenous with respect to their capacity to migrate to and localise in the thymic environment. Thymic reconsfitution capacity of whole adult bone marrow has also been shown to be greater after intrathymic versus intravenous transfer [11, 36]. Capacity of stem cells to traverse endothelial cell barriers and to enter the thymus from bloodstream may represent a characteristic unique to T cell progenitors, but may not reflect unique commitment
of these cells to the T cell lineage. Consistent with this hypothesis is a report on human thymocytes, which shows that the most immature thymocyte population does contain cells not irreversibly committed to T cell lineage [37]. Some of these cells have been induced to differentiate into cells of the myeloerythroid lineages when cultured in the presence of appropriate growth factors.
4. Isolation and properties of cell lines representing progenitor T cells Many attempts have been made to culture T cell progenitors from hemapoietic tissues, in the hope of generating continuous or transformed progenitor T cell lines [38-43]. Most of these have been derived from either bone marrow or spleen cultured in vitro in the presence of various T cell growth factors and splenic cell feeder layers [38- 43]. Others have been derived by induction of thymomas in mice using Abelson virus pseudotyped with a murine leukaemia virus, A-MuLV [44, 45]. Most cell lines are thought to represent early T cells. They show absence of T lineage specific markers, but do express low levels of the Thy-1 and Ly5 markers common to bone marrow and early T cells [38-40, 42]. The most definive criterion for the progenitor T cell nature of these cell lines has been their capacity to replicate and differentiate within the thymus. The pT-D8 cell line described by Goodwin et al. [38] is a Thy-1 l°, Ly5 + , CD4 + cell line derived from spleen and has shown limited replication capacity within the thymus of an irradiated host and can generate thymic progeny expressing mature T cell markers. While this information is consistent with the T-like nature of the cells, it is not yet clear whether this cell line only generates thymic progeny or whether it has capacity to differentiate into B cells, or even myeloid lineage cells. Similar T-like cells have now been isolated from spleen induced to transform in vitro with a Radiation Leukemia Virus (RadLV) [43]. These cells express low levels of the CD3-e molecule, and have the phenotype of a Thy-1 - Ly-5 + C D 4 - C D 8 - cell. All clones isolated have been found to carry germ line T cell receptor 7, 6 and/3 genes as well as a germ line IgH gene. All cell lines carry a common Va gene rearrangement and it is not yet known whether this is related to the transformation process, or whether these particular cells have aberrantly rearranged
their ~ gene first (H.C. O'N., unpublished data). One of these cell lines has been shown to have specific capacity to migrate to the thymus in just a 3-h homing assay, suggesting that it may express a receptor which can localise cells within the thymus [42]. The relationship of these spleen-derived null, T-like cells and the pT-D8 cell line to T cell progenitors, is not yet known. One possibility which cannot be excluded is that they represent an early thymic subset which has spilled into the periphery for some unknown reason, prior to full maturation within the thymus [43]. Another interpretation of the origin of progenitor T cell lines is that they are derived from stem ceils by athymic T cell differentiation which may be possible in vitro if stem cells are given appropriate growth factors and stromal cells to support the replication of T cells. This interpretation could apply for the spleen derived cells described above and for cell lines derived by Hurwitz [41] from bone marrow, and by Palacios et al. [39] from nude mouse bone marrow. In vitro athymic T cell differentiation has been demonstrated since T cell receptor gene rearrangements can proceed in bone marrow derived T cell cultures [46]. Any attempt to interpret the lineage relationship of early T cells cultured in vitro therefore becomes difficult, and the distinction between a Tcommitted stem cell and a multipotential stem cell as the originator of these celllines becomes blurred. The pro-T cell lines described by Palacios et al. [39] have been aligned with progenitor T cells because they carry all T cell receptor genes in germ line configuration, and because they have capacity to migrate to and differentiate within the thymus of sublethally irradiated mice. These were found not to generate B cell progeny in sublethally irradiated SCID mice, but did develop in the thymus of SCID mice [39]. These cell lines reflect properties of committed T cell progenitors, but is is still not clear whether this T commitment pre-existed in in vitro culture. Monoclonal antibody to cell surface markers on these cells may be valuable in studies of early T cell differentiation and in defining T-committed stem cells in bone marrow [401. An interesting contrast can be drawn between in vitro generated progenitor T cell lines and the thym o m a s induced by A-MuLV which represent early T cells in thymus [44, 45]. These are Thy-1 - cell lines which upon transformation have been found to carry rearrangements at both IgH chain genes as well
as T cell receptor 3' chain genes [44, 45]. Comparative studies on A-MuLV induced bone marrow derived pre-B and thymus derived pre-T cell lines have shown that both Ig and T cell receptor 3' chain genes are accessible for rearrangement in both types of cell lines [45]. All of this data confirms that a multipotential lymphoid stem cell is present in bone marrow and thymus which can rearrange and express both IgH and T cell receptor 3' chain genes. Both 3' and ix chain genes are available for rearrangement and can be activated in the same early haemapoietic cells. With evidence supporting the existence of a lymphoid-committed stem cell present in both bone marrow and thymus, the challenge is now to determine what prethymic event, if any, can constitute commitment to the T cell lineage in stem cells.
5. Attempts to identify T cell progenitors which seed the thymus Information on the multipotential nature of cells in thymus raises questions about the nature of cells which can localise in the thymic environment and the specific means by which they enter. Several considerations are necessary. If multipotential stem cells are recirculating, localisation into thymus could be a chance phenomenon, and stem cells may be induced to differentiate under the hormonal influence of the environment in which they localise. However, in just a 3 or 4 hour assay of bone marrow reconstitution of irradiated mice, fluorochrome-labelled cells have been found to enter thymus [6, 7], and to differentiate there showing expression of the T cell specific Thy-1 antigen within 24 h. This result confirms the existence of CFU-T in bone marrow, and in such a short term assay, precludes the requirement for colonisation of bone marrow in the irradiated host, prior to thymic entry of stem cells [6]. Bone marrow stem cells committed to T cell lineage may carry specific receptors which bind these cells to endothelial cell surfaces allowing entry to the thymus by crossing this cell barrier. Expression of a specific "thymus-homing" receptor would not necessarily indicate irreversible commitment to T lineage, and, in fact, stem cells which enter thymus could still be quite heterogenous with respect to any lineage commitment. Spangrude and Weissm a n [14] have compared the seeding of genetically marked bone marrow cells into the thymus of irradi-
ated animals followed by a chase of syngeneic bone marrow or lymph node cells. They report specific blockade of thymic colony formation by excess bone marrow cells, and not by lymph node cells, which do not have the capacity to home to thymus [6]. This suggests that CFU-T in bone marrow may use a specific homing mechanism to enter thymus. The capacity of bone marrow stem cells to express receptors for endothelial ceils in thymic sites could reflect a minimal commitment to T cell lineage. A transformed cell line has now been described which has specific capacity to localise within thymus and not other lymphoid organs [43, 47]. These ceils develop primarily as thymomas when injected intravenously into mice, and preliminary analyses have revealed that lesions initiate as a clonal proliferation in the subcapsular region of the cortex, known to be the location of immature lymphoblasts in normal thymic ontogeny [18]. This 16C1 cell line and not "sister" clones of the same immature T cell phenotype have been isolated from thymus within 3 h after i.v. inoculation. Only about 3000 16C1 cells enter thymus over a 3-h assay following i.v. injection into an irradiated host. This could mean that there is only a limited number of sites within the thymus to which incoming cells can bind (H.C. O'N., unpublished data). The restricted number of cells which can enter thymus makes analysis of the seeding mechanism, and of any receptors involved, very difficult. The 16C1 cell line did not adhere to thin sections of thymus (or control tissue) when tested in the Stamper and Woodruff [48] assay for lymphocyte adherence as an in vitro assay for "homing specificity" (H.C. O'N., unpublished data). This cell line has been used for investigating cellular interactions and receptors apparent in thymus entry with a view to definition of any receptors involved. Antibodies specific for the Pgp-1 molecule have been tested on this cell line since they were previously shown to inhibit reconstitution of the thymus of lethally irradiated mice using both C D 4 - C D 8 thymocytes and bone marrow [12, 49]. Precursor thymocytes which can home back to the thymus have also been identified as the Pgp-1 ÷ (phagocytic glycoprotein-1) subpopulation of C D - C D 8 thymocytes [12]. Antibodies specific for Pgp-I were found to inhibit localisation of both 16C1 cells and of bone marrow into thymus in a short term homing assay [47]. Anti-Pgp-1 antibodies do not appear to
have any effect on 16C1 cell function, i.e. inhibition of proliferation, cell death or modulation of Pgp-1 from the cell surface and the simplest prediction is that the mechanism for inhibition of thymic entry is blockade of receptor-ligand interactions [47]. Expression of the Pgp-1/CD44 molecule is not uniquely related to thymus homing capacity, since other T cell lines, phenotypically identical to 16C1, and expressing Pgp-1, are known to localise in spleen and not thymus in a 3-h homing assay [47]. The possibility that 16C1 expresses a second receptor besides Pgp1, or a variant form of Pgp-1, which confers "thymic homing" specificity needs investigation. The Pgp-1 molecule appears to operate as a receptor involved in general leukocyte adhesion, with no known specificity for any particular organ site [50]. A role for CD44/Pgp-1 has been described in both adhesion and activation events, but since it interacts with the cytoskeleton, presumably via its long cytoplasmic tail [50], expression of this marker could also reflect the "migratory" capacity of a cell. It is expressed on many tumour cell lines, a high percentage of bone marrow cells and a low percentage of spleen and thymus cells [51]. Its identity with the human CD44 molecule has now been confirmed since genes expressing each of these molecules have been cloned and shown to have identity [51, 52]. Antibodies to the CD44 molecule, e.g., Hermes-l, have been shown to inhibit adhesion of lymphocytes to high endothelial venules present in lymph nodes and mucosal sites [54], and the Hermes/CD44 group of adhesion molecules represents a distinct class of receptors broadly distributed and implicated in lymphocyte-endothelial cell adhesion in multiple tissues [50]. The CD44 molecule now contrasts with a second family of cell-cell adhesion molecules, including the lymph node homing receptor defined by the MEL-14 antibody [55, 56], and the endothelial leukocyte adhesion molecule, ELAM-1 [57]. In contrast to Pgp-1, expression of these molecules reflects tissue specific adhesive properties of a cell. It now seems clear that adhesion events during lymphocyte recirculation may involve several types of receptors and that some of these may act together. The requirement for a site-specific receptor appears to be warranted for the entry of 16C1 into the thymus and a role for Pgp-1/CD44 in thymic colonisation is now suggested. At this point, analysis of progenitor T cell lines such as 16C1 and the expression of adhesive
m o l e c u l e s / h o m i n g receptors may represent an opportunity to search for similar receptors on Tcommitted stem cells in bone marrow. Receptors involved in thymic localisation may be the most appropriate markers for defining those stem cells which can enter thymus. References [1] Scollay, R., Smith, J. and Stauffer, V. (1986) lmmunol. Rev. 91, 129. [2] Ford, C., Micklem, H., Evans, E., Gray, J. and Ogden, D. (1966) Ann. NY Acad. Sci. 129, 283. [3] Kadish, J. and Basch, R. (1976) J. Exp. Med. 143, 1082. [4] Basch, R. and Kadish, J. (1977) J. Exp. Med. 145,405. [5] Kadish, J. and Basch, R. (1977) Cell. lmmunol. 30, 12. [6] Lepault, F. and Weissman, I. (1981) Nature 293, 151. [7] Lepault, E, Coffman, R. and Weissman, I. (1983) J. lmmunol. 131, 64. [8] Boersma, W., Betel, I., Daculsi, R. and van der Westen (1981) Cell. Tissue Kinet. 14, 179. [9] Ezine, S., Weissman, 1. and Rouse, R. (1984) Nature 309, 629. [10] Ezine, S., Jerabek, L. and Weissman, I. (1987) J. lmmunol. 139, 2195. [11] Katsura, Y., Kina, T., Amagai, T., Tsubata, T., Hirayoshi, K., Takaoki, Y., Sado, T. and Nishikawa, S-I. (1986) J. Immunol. 137, 2434. [12] Lesley, J., Hyman, R. and Schulte, R. (1985) Cell. Immunol. 91, 397. [13] Spangrude, J., Heimfeld, S. and Weissman, 1. (1988) Science 241, 58. [14] Spangrude, G. and Weissman, I. (1988) J. lmmunol. 141, 1877. [15] Fink, P., Bevan, M. and Weissman, I. (1984) J. Exp. Med. 159, 436. [16] Naparstek, Y., Holoshitz, J., Eisenstein, S., Reshef, T., Rappaport, S., Chemke, J., Ben-Nun, A. and Cohen, 1. (1982) Nature 300, 262. [17] Mitchie, S. and Rouse, R. (1988) Transplantation 46, 98. [18] Weissman, I. (1967) J. Exp. Med. 126, 291. [19] Kingston, R., Jenkinson, E. and Owen, J. (1985) Nature 317, 811. [20] Adkins, B., Mueller, C., Okada, C., Reichert, R., Weissman, I. and Spangrude, G. (1987) Annu. Rev. lmmunol. 5, 325. [21] Von Boehmer, H. (1988) Annu. Rev. Immunol. 6, 309. [22] Scollay, R., Bartlett, P. and Shortman, K. (1984) Immunol. Rev. 82, 79. [23] McDonald, R., Howe, R., Pedrazinni, T., Lees, R., Budd, R., Schneider, R., Liao, N., Zinkernagel, R., Louis, J., Raulet, D., Hentgartner, H. and Miescher, G. (1988) Immunol. Rev. 104, 157. [24] Scollay, R., Wilson, A., D'Amico, A., Kelly, K., Egerton, M., Pearse, M., Wu, L. and Shortman, K. (1988) Immunol. Rev. 104, 81.
[25] Wilson, R., Lai, E., Concannon, P., Barth, R. and Hood, L. (1988) Immunol. Rev. 101, 149. [26] Strominger, J. (1989) Science 244, 943. [27] Bevan, M. and Fink, P. (1978) lmmunol. Rev. 42, 3. [28] Marrack, P., Lo, D., Brinster, R., Palmiter, R., Burkly, L., Flavell, R. and Kappler, J. (1988) Cell 53, 627. [29] Vollger, W., Tuck, D., Springer, T., Haynes, B. and Singer, K. (1987) J. Immunol. 138, 358. [30] Denning, S., Kurtzberg, J., Le, P., Tuck, D., Singer, K. and Haynes, B. (1988) Proc. Natl. Acad. Sci. USA 85, 3125. [31] Boyd, R., Izon, D., Godfrey, D., Wilson, T. and Tucek, C. (1989) Adv. Exp. Med. Biol. 237, 263. [32] Haynes, B., Denning, S., Singer, K. and Kurtzberg, J. (1989) Immunol. Today 10, 87. [33] Basch, R. and Berman, J. (1982) Eur. J. lmmunol. 12, 359. [34] Basch, R., Kadish, J. and Goldstein, G. (1978) J. Exp. Med. 147, 1843. [35] Muller-Sieberg, C., Whitlock, C. and Weissman, I. (1986) Cell 44, 653. [36] Spangrude, G., Muller-Sieberg, C., Heimfeld, S. and Weissman, I. (1988) J. Exp. Med. 167, 1671. [37] Kurtzberg, J., Denning, S., Nycum, L., Singer, K. and Haynes, B. (1989) Proc. Natl. Acad. Sci. USA 86, 7575. [38] Goodwin, L., Rocha, A. and Basch, R. (1986) Nature 323, 166. [39] Palacios, R., Kiefer, M., Brockhaus, M., Kajalainen, K., Dembic, Z., Kisielow, P. and von Boehmer, H. (1987) J. Exp. Med. 166, 12. [40] Palacios, R. and Pelkonen, J. (1988) lmmunol. Rev. 104, 158. [41] Hurwitz, J. (1987) Eur. J. lmmunol. 17, 751. [42] O'Neill, H. (1987) Cell. Immunol. 109, 222. [43] O'Neill, H. Submitted ms. [44] Cook, W. (1985) Mol. Cell. Biol. 5, 390. [45] Cook, W. and Balaton, A. (1987) Mol. Cell. Biol. 7, 266. [46] Hurwitz, J., Samaridis, J. and Pelkonen, J. (1988) Cell 52, 821. [47] O'Neill, H. (1989) Immunology 68, 59. [48] Stamper, H. and Woodruff, J. (1976) J. Exp. Med. 144, 828. [49] Trowbridge, I., Lesley, J., Schulte, R., Hyman, R. and Trotter, J. (1982) lmmunogenetics 15, 299. [50] Stoolman, L. (1989) Cell 56, 907. [51] Haynes, B., Telen, M., Hale, L. and Denning, S. (1989) Immunol. Today 10, 423. [52] Goldstein, L., Zhou, D., Picker, L., Minty, C., Bargatze, R., Ding, J. and Butcher, E. (1989) Cell 56, 1063. [53] Stamenkovic, I., Amiot, M., Pesando, J. M. and Seed, B. (1988) Cell 56, 1057. [54] Jalkanen, S., Reichert, R., Gallatin, W., Bargatze, R., Weissman, i. and Butcher, E. (1986) Immunol. Rev. 91, 39. [55] Siegelman, M., van de Rijn, M. and Weissman, I. (1989) Science 243, 1166. [56] Lasky, L., Singer, M., Yednock, T., Oowbenko, D., Fennie, C., Rodriguez, R., Nguyen, T., Stachel, S. and Rosen, D. (1989) Cell 56, 1045. [57] Bevilacqua, M., Stengelin, S., Gimbrone, M. and Seed, B. (1989) Science 243, 1160.
Minireview
Prothymocyte seeding in the thymus H e l e n C. O'Neill Developmental Haematology Group, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia (Received 16 March 1990; revision received 2 July 1990; accepted 19 September 1990)
1. Summary A major gap in our understanding of T lymphocyte development is the process of stem cell differentiation into T lymphocyte precursors. An important question is whether bone marrow-derived stem cells become committed to T lymphoid lineage within the bone marrow, or whether this occurs once cells have entered the microenvironment of the thymus. Attempts to identify a haemapoietic precursor of thymocytes in mice, a "prothymocyte", have involved cell transfer experiments involving isolated and selected populations of bone marrow stem cells, as well as transformed or continuous cell lines representing early stage in mouse T cell development. Current information on the properties of stem cells which can seed the thumus is reviewed in this paper, and the possibility that progenitor T cells may be identified by their expression o f receptor(s) which localise them into the thymus is considered.
2. Introduction The thymus is the central organ for differentiation of T lineage cells, yet the adult mouse thymus contains no endogenous stem cell population (reviewed in ref. 1). After initial development during embryogenesis, the thymus receives only slow traffic of cells from bone marrow to replenish its proliferating cell pool. After irradiation, stress or disease, it becomes Key words: Thymus; Stem cell; T cell progenitor; Cell adhesion; Lymphocyte migration Correspondence to." Helen C. O'Neill, Developmental Haematology Group, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
repopulated by a low level influx of stem ceils [1], and the irradiated animal model has been used widely to study stem cell reconstitution of the thymus [1 - 14]. In adult mice, most of these ceils come from bone marrow, but cells which can colonise the irradiated thymus have also been found in spleen [3, 6, 11] and thymus [5, 6, 11, 12]. The thymus operates primarily for T cell differentiation and, unlike lymph node cells, is not geared for entry of large numbers of cells, nor for expansion of antigen-activated cells. The medullary compartment of the thymus is however connected to the lymphoid circulation, and mature T lymphocytes have been shown to enter the thymus from the periphery [15, 16], and to localise within the medulla [17]. It is thought that progenitor T cells enter the thymus through a set of blood vessels in the cortico-medullary region and thence localise to the subcapsular cortical region which is the location of large immature blast cells [18]. In 1976, Basch and Kadish [3] reported that bone marrow stem cells entering the thymus of an irradiated host underwent a resting phase estimated to be between 8 and 12 days, and that only a small number, e.g., 10-100 stem cells, were needed to reconstitute the T cell compartment of an irradiated mouse thymus [3, 4]. More recent studies have confirmed the restricted number of. progenitors required to generate the T cell repertoire [1, 9, 11, 14]. Weissman and co-workers [9, 14] have used limiting numbers of bone marrow ceils to reconstitute the irradiated mouse thymus and have estimated that just a single stem cell, termed the T colony forming unit, CFU-T, is needed to repopulate thymic lobes. In these studies, individual thymic colonies expressing allele-specific cell surface markers of donor bone marrow cells have been identified by antibody staining. Analysis of thymic colonies
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[9], and of peripheral T cells derived from these colonies [14], have confirmed that a single CFU-T in bone marrow can give rise to thymic progeny representing each of the major T cell subsets of thymus [10, 14]. The major functional classes of T cells have also been identified in the periphery [14]. Recolonisation of deoxyguanosine-treated or alymphoid embryonic thymus lobes in organ culture has also been reported by Kingston et al. [19] using a single fetal thymic blast cell. Cell surface marker analysis of the progeny of a single intrathymic precursor T cell has confirmed the development of several major thymic subclasses amongst the progeny. Once stem cells have entered the thymus, they proliferate and develop into T cells with antigen specificity and immune function (reviewed in refs. 20 and 21). The process of T cell maturation is now well understood from studies of cell surface antigen expression using monoclonal antibodies (reviewed in refs. 22-24), and from analyses of T cell receptor rearrangements on selected T cell subpopulations [25, 26]. T lymphocytes become committed to the expression of one T cell receptor and one function, either cytotoxic, helper or suppressor. These cells can be broadly grouped into CD8-expressing cytotoxic cells or CD4-expressing helper cells which recognise antigen in the context of Class I or Class II MHC antigens, respectively. Rearrangement and expression of the T cell antigen-specific receptor occurs within the thymus in an ordered and defined fashion [23, 25, 26], and selection of the T cell repertoire occurs before cells leave the thymus. Thymic epithelial elements [27] and bone marrow-derived stromal cells expressing self MHC antigens [28] play a role in selection/elimination of cells expressing T cell receptors of low/high affinity for self determinants. Thymic stromal elements also function to drive proliferation and activation required for differentiation of cells, but how this process operates or in what order is not yet known. One interaction already described involves the CD2 molecule on immature T cells interacting with its ligand, the LFA-3 molecule on isolated thymic epithelial cells [29], and the activation and proliferation of human immature thymocytes by supernatants of human thymic epithelial cells containing IL-1, IL-3 and GM-CSF has been measured [30]. Monoclonal antibodies have now been derived which can divide the epithelial compartment into several distinct regions and it
is possible that different epithelial/stromal elements all have distinct functions during the maturation process [31]. Despite a wealth of information about differentiation of the major lineages of T cells within the thymus, very little is known about the properties of the haemapoietic precursor of thymus, or about the exact mechanism by which these cells seed the thymus. The early stages of stem cell differentiation within the thymus and the regulatory elements which control these processes are stil unknown. A paucity of antibodies specific for cell surface markers unique to T cell progenitors has made identification of these cells difficult. One anticipates that markers for T cell progenitors should reflect acquisition of T cell specific functions unique to early T cell development. Expression of CD3 and the T cell receptor, or known T cell specific growth factor receptors, correlates only with thymic development of mature T cells. Markers such as CD7 in humans [32] and Thy-1 in mouse [33] are expressed early during T cell development, but are also expressed by all T-lineage cells. Because of the absence of specific markers, very little is understood about the lineage relationship of stem cells and T cell progenitors, both within the thymus and in other lymphoid compartments. A detailed study of early T cell development, and the isolation of progenitor T cells prior to induction of self tolerance is central to therapies involving bone marrow transplants, immunotherapy in T cell deficient diseases and radiation therapy. Cells which are part way committed to the T cell lineage may also be the most appropriate target cells for gene therapy involving T cell specific genes.
3. Attempts to identify progenitor T cells amongst stem cell populations The main question under consideration here is whether stem cells become committed to T cell lineage within the bone marrow environment or whether this occurs once stem cells have localised within the thymus. The former possibility is consistent with the presence of a T-committed "prothymocyte" or even a lymphoid-committed stem cell present in bone marrow. The latter is consistent with the model that multipotential stem cells can seed the thymus. Most attempts to define the haemapoietic precursor of thymocytes have involved selectively depleted bone
marrow populations and a test of their capacity to reconstitute irradiated mouse thymus. In the early experiments of Basch et al. [34], procedures for depletion of B cells, T cells, and macrophages from bone marrow, were found to give enrichment of prothymocytes as judged by their content of the enzyme terminal deoxyribonucleotide transferase. Recently, more rigorous depletion of bone marrow by sorting out lineage-negative cells using a fluorescence activated cell sorter and antibodies specific for macrophages, granulocytes, T and B cells have confirmed the presence of a bone marrow stem cell subset expressing low levels of the Thy-1 antigen [35]. This population of cells has been shown to contain precursors of all lymphomyeloid lineages, reconstituting irradiated animals with T, B and macrophage/monocyte lineage cells [35, 36]. Cells which can repopulate the thymus of an irradiated animal have now been identified amongst those bone marrow cells which appear to represent the pluripotential subset [13]. These cells have been identified as lineage-negative bone marrow cells which bind antibody specific for the Thy-1 and Ly-6 molecules (the Sca-1 antibody) [13] and represent a 0.05% population of non-dividing bone marrow cells. A comparison of the number of precursors in whole bone marrow versus this highly purified stem cell population has indicated a 1000-fold enrichment of precursors capable of reconstituting the thymus of an irradiated animal. The number of these cells capable of generating a thymic clone was found to be as low as 20, and was reduced by 5-fold if cells were injected directly into the thymus rather than the bloodstream [13]. These increased estimates could, however, reflect the higher proliferative potential of stem cells, when introduced directly into the thymic microenvironment. This data suggests several possibilities. Firstly, pluripotential stem cells may become committed to the lymphoid lineage once they are located within the thymus, and secondly, pluripotential stem cells may be heterogenous with respect to their capacity to migrate to and localise in the thymic environment. Thymic reconsfitution capacity of whole adult bone marrow has also been shown to be greater after intrathymic versus intravenous transfer [11, 36]. Capacity of stem cells to traverse endothelial cell barriers and to enter the thymus from bloodstream may represent a characteristic unique to T cell progenitors, but may not reflect unique commitment
of these cells to the T cell lineage. Consistent with this hypothesis is a report on human thymocytes, which shows that the most immature thymocyte population does contain cells not irreversibly committed to T cell lineage [37]. Some of these cells have been induced to differentiate into cells of the myeloerythroid lineages when cultured in the presence of appropriate growth factors.
4. Isolation and properties of cell lines representing progenitor T cells Many attempts have been made to culture T cell progenitors from hemapoietic tissues, in the hope of generating continuous or transformed progenitor T cell lines [38-43]. Most of these have been derived from either bone marrow or spleen cultured in vitro in the presence of various T cell growth factors and splenic cell feeder layers [38- 43]. Others have been derived by induction of thymomas in mice using Abelson virus pseudotyped with a murine leukaemia virus, A-MuLV [44, 45]. Most cell lines are thought to represent early T cells. They show absence of T lineage specific markers, but do express low levels of the Thy-1 and Ly5 markers common to bone marrow and early T cells [38-40, 42]. The most definive criterion for the progenitor T cell nature of these cell lines has been their capacity to replicate and differentiate within the thymus. The pT-D8 cell line described by Goodwin et al. [38] is a Thy-1 l°, Ly5 + , CD4 + cell line derived from spleen and has shown limited replication capacity within the thymus of an irradiated host and can generate thymic progeny expressing mature T cell markers. While this information is consistent with the T-like nature of the cells, it is not yet clear whether this cell line only generates thymic progeny or whether it has capacity to differentiate into B cells, or even myeloid lineage cells. Similar T-like cells have now been isolated from spleen induced to transform in vitro with a Radiation Leukemia Virus (RadLV) [43]. These cells express low levels of the CD3-e molecule, and have the phenotype of a Thy-1 - Ly-5 + C D 4 - C D 8 - cell. All clones isolated have been found to carry germ line T cell receptor 7, 6 and/3 genes as well as a germ line IgH gene. All cell lines carry a common Va gene rearrangement and it is not yet known whether this is related to the transformation process, or whether these particular cells have aberrantly rearranged
their ~ gene first (H.C. O'N., unpublished data). One of these cell lines has been shown to have specific capacity to migrate to the thymus in just a 3-h homing assay, suggesting that it may express a receptor which can localise cells within the thymus [42]. The relationship of these spleen-derived null, T-like cells and the pT-D8 cell line to T cell progenitors, is not yet known. One possibility which cannot be excluded is that they represent an early thymic subset which has spilled into the periphery for some unknown reason, prior to full maturation within the thymus [43]. Another interpretation of the origin of progenitor T cell lines is that they are derived from stem ceils by athymic T cell differentiation which may be possible in vitro if stem cells are given appropriate growth factors and stromal cells to support the replication of T cells. This interpretation could apply for the spleen derived cells described above and for cell lines derived by Hurwitz [41] from bone marrow, and by Palacios et al. [39] from nude mouse bone marrow. In vitro athymic T cell differentiation has been demonstrated since T cell receptor gene rearrangements can proceed in bone marrow derived T cell cultures [46]. Any attempt to interpret the lineage relationship of early T cells cultured in vitro therefore becomes difficult, and the distinction between a Tcommitted stem cell and a multipotential stem cell as the originator of these celllines becomes blurred. The pro-T cell lines described by Palacios et al. [39] have been aligned with progenitor T cells because they carry all T cell receptor genes in germ line configuration, and because they have capacity to migrate to and differentiate within the thymus of sublethally irradiated mice. These were found not to generate B cell progeny in sublethally irradiated SCID mice, but did develop in the thymus of SCID mice [39]. These cell lines reflect properties of committed T cell progenitors, but is is still not clear whether this T commitment pre-existed in in vitro culture. Monoclonal antibody to cell surface markers on these cells may be valuable in studies of early T cell differentiation and in defining T-committed stem cells in bone marrow [401. An interesting contrast can be drawn between in vitro generated progenitor T cell lines and the thym o m a s induced by A-MuLV which represent early T cells in thymus [44, 45]. These are Thy-1 - cell lines which upon transformation have been found to carry rearrangements at both IgH chain genes as well
as T cell receptor 3' chain genes [44, 45]. Comparative studies on A-MuLV induced bone marrow derived pre-B and thymus derived pre-T cell lines have shown that both Ig and T cell receptor 3' chain genes are accessible for rearrangement in both types of cell lines [45]. All of this data confirms that a multipotential lymphoid stem cell is present in bone marrow and thymus which can rearrange and express both IgH and T cell receptor 3' chain genes. Both 3' and ix chain genes are available for rearrangement and can be activated in the same early haemapoietic cells. With evidence supporting the existence of a lymphoid-committed stem cell present in both bone marrow and thymus, the challenge is now to determine what prethymic event, if any, can constitute commitment to the T cell lineage in stem cells.
5. Attempts to identify T cell progenitors which seed the thymus Information on the multipotential nature of cells in thymus raises questions about the nature of cells which can localise in the thymic environment and the specific means by which they enter. Several considerations are necessary. If multipotential stem cells are recirculating, localisation into thymus could be a chance phenomenon, and stem cells may be induced to differentiate under the hormonal influence of the environment in which they localise. However, in just a 3 or 4 hour assay of bone marrow reconstitution of irradiated mice, fluorochrome-labelled cells have been found to enter thymus [6, 7], and to differentiate there showing expression of the T cell specific Thy-1 antigen within 24 h. This result confirms the existence of CFU-T in bone marrow, and in such a short term assay, precludes the requirement for colonisation of bone marrow in the irradiated host, prior to thymic entry of stem cells [6]. Bone marrow stem cells committed to T cell lineage may carry specific receptors which bind these cells to endothelial cell surfaces allowing entry to the thymus by crossing this cell barrier. Expression of a specific "thymus-homing" receptor would not necessarily indicate irreversible commitment to T lineage, and, in fact, stem cells which enter thymus could still be quite heterogenous with respect to any lineage commitment. Spangrude and Weissm a n [14] have compared the seeding of genetically marked bone marrow cells into the thymus of irradi-
ated animals followed by a chase of syngeneic bone marrow or lymph node cells. They report specific blockade of thymic colony formation by excess bone marrow cells, and not by lymph node cells, which do not have the capacity to home to thymus [6]. This suggests that CFU-T in bone marrow may use a specific homing mechanism to enter thymus. The capacity of bone marrow stem cells to express receptors for endothelial ceils in thymic sites could reflect a minimal commitment to T cell lineage. A transformed cell line has now been described which has specific capacity to localise within thymus and not other lymphoid organs [43, 47]. These ceils develop primarily as thymomas when injected intravenously into mice, and preliminary analyses have revealed that lesions initiate as a clonal proliferation in the subcapsular region of the cortex, known to be the location of immature lymphoblasts in normal thymic ontogeny [18]. This 16C1 cell line and not "sister" clones of the same immature T cell phenotype have been isolated from thymus within 3 h after i.v. inoculation. Only about 3000 16C1 cells enter thymus over a 3-h assay following i.v. injection into an irradiated host. This could mean that there is only a limited number of sites within the thymus to which incoming cells can bind (H.C. O'N., unpublished data). The restricted number of cells which can enter thymus makes analysis of the seeding mechanism, and of any receptors involved, very difficult. The 16C1 cell line did not adhere to thin sections of thymus (or control tissue) when tested in the Stamper and Woodruff [48] assay for lymphocyte adherence as an in vitro assay for "homing specificity" (H.C. O'N., unpublished data). This cell line has been used for investigating cellular interactions and receptors apparent in thymus entry with a view to definition of any receptors involved. Antibodies specific for the Pgp-1 molecule have been tested on this cell line since they were previously shown to inhibit reconstitution of the thymus of lethally irradiated mice using both C D 4 - C D 8 thymocytes and bone marrow [12, 49]. Precursor thymocytes which can home back to the thymus have also been identified as the Pgp-1 ÷ (phagocytic glycoprotein-1) subpopulation of C D - C D 8 thymocytes [12]. Antibodies specific for Pgp-I were found to inhibit localisation of both 16C1 cells and of bone marrow into thymus in a short term homing assay [47]. Anti-Pgp-1 antibodies do not appear to
have any effect on 16C1 cell function, i.e. inhibition of proliferation, cell death or modulation of Pgp-1 from the cell surface and the simplest prediction is that the mechanism for inhibition of thymic entry is blockade of receptor-ligand interactions [47]. Expression of the Pgp-1/CD44 molecule is not uniquely related to thymus homing capacity, since other T cell lines, phenotypically identical to 16C1, and expressing Pgp-1, are known to localise in spleen and not thymus in a 3-h homing assay [47]. The possibility that 16C1 expresses a second receptor besides Pgp1, or a variant form of Pgp-1, which confers "thymic homing" specificity needs investigation. The Pgp-1 molecule appears to operate as a receptor involved in general leukocyte adhesion, with no known specificity for any particular organ site [50]. A role for CD44/Pgp-1 has been described in both adhesion and activation events, but since it interacts with the cytoskeleton, presumably via its long cytoplasmic tail [50], expression of this marker could also reflect the "migratory" capacity of a cell. It is expressed on many tumour cell lines, a high percentage of bone marrow cells and a low percentage of spleen and thymus cells [51]. Its identity with the human CD44 molecule has now been confirmed since genes expressing each of these molecules have been cloned and shown to have identity [51, 52]. Antibodies to the CD44 molecule, e.g., Hermes-l, have been shown to inhibit adhesion of lymphocytes to high endothelial venules present in lymph nodes and mucosal sites [54], and the Hermes/CD44 group of adhesion molecules represents a distinct class of receptors broadly distributed and implicated in lymphocyte-endothelial cell adhesion in multiple tissues [50]. The CD44 molecule now contrasts with a second family of cell-cell adhesion molecules, including the lymph node homing receptor defined by the MEL-14 antibody [55, 56], and the endothelial leukocyte adhesion molecule, ELAM-1 [57]. In contrast to Pgp-1, expression of these molecules reflects tissue specific adhesive properties of a cell. It now seems clear that adhesion events during lymphocyte recirculation may involve several types of receptors and that some of these may act together. The requirement for a site-specific receptor appears to be warranted for the entry of 16C1 into the thymus and a role for Pgp-1/CD44 in thymic colonisation is now suggested. At this point, analysis of progenitor T cell lines such as 16C1 and the expression of adhesive
m o l e c u l e s / h o m i n g receptors may represent an opportunity to search for similar receptors on Tcommitted stem cells in bone marrow. Receptors involved in thymic localisation may be the most appropriate markers for defining those stem cells which can enter thymus. References [1] Scollay, R., Smith, J. and Stauffer, V. (1986) lmmunol. Rev. 91, 129. [2] Ford, C., Micklem, H., Evans, E., Gray, J. and Ogden, D. (1966) Ann. NY Acad. Sci. 129, 283. [3] Kadish, J. and Basch, R. (1976) J. Exp. Med. 143, 1082. [4] Basch, R. and Kadish, J. (1977) J. Exp. Med. 145,405. [5] Kadish, J. and Basch, R. (1977) Cell. lmmunol. 30, 12. [6] Lepault, F. and Weissman, I. (1981) Nature 293, 151. [7] Lepault, E, Coffman, R. and Weissman, I. (1983) J. lmmunol. 131, 64. [8] Boersma, W., Betel, I., Daculsi, R. and van der Westen (1981) Cell. Tissue Kinet. 14, 179. [9] Ezine, S., Weissman, 1. and Rouse, R. (1984) Nature 309, 629. [10] Ezine, S., Jerabek, L. and Weissman, I. (1987) J. lmmunol. 139, 2195. [11] Katsura, Y., Kina, T., Amagai, T., Tsubata, T., Hirayoshi, K., Takaoki, Y., Sado, T. and Nishikawa, S-I. (1986) J. Immunol. 137, 2434. [12] Lesley, J., Hyman, R. and Schulte, R. (1985) Cell. Immunol. 91, 397. [13] Spangrude, J., Heimfeld, S. and Weissman, 1. (1988) Science 241, 58. [14] Spangrude, G. and Weissman, I. (1988) J. lmmunol. 141, 1877. [15] Fink, P., Bevan, M. and Weissman, I. (1984) J. Exp. Med. 159, 436. [16] Naparstek, Y., Holoshitz, J., Eisenstein, S., Reshef, T., Rappaport, S., Chemke, J., Ben-Nun, A. and Cohen, 1. (1982) Nature 300, 262. [17] Mitchie, S. and Rouse, R. (1988) Transplantation 46, 98. [18] Weissman, I. (1967) J. Exp. Med. 126, 291. [19] Kingston, R., Jenkinson, E. and Owen, J. (1985) Nature 317, 811. [20] Adkins, B., Mueller, C., Okada, C., Reichert, R., Weissman, I. and Spangrude, G. (1987) Annu. Rev. lmmunol. 5, 325. [21] Von Boehmer, H. (1988) Annu. Rev. Immunol. 6, 309. [22] Scollay, R., Bartlett, P. and Shortman, K. (1984) Immunol. Rev. 82, 79. [23] McDonald, R., Howe, R., Pedrazinni, T., Lees, R., Budd, R., Schneider, R., Liao, N., Zinkernagel, R., Louis, J., Raulet, D., Hentgartner, H. and Miescher, G. (1988) Immunol. Rev. 104, 157. [24] Scollay, R., Wilson, A., D'Amico, A., Kelly, K., Egerton, M., Pearse, M., Wu, L. and Shortman, K. (1988) Immunol. Rev. 104, 81.
[25] Wilson, R., Lai, E., Concannon, P., Barth, R. and Hood, L. (1988) Immunol. Rev. 101, 149. [26] Strominger, J. (1989) Science 244, 943. [27] Bevan, M. and Fink, P. (1978) lmmunol. Rev. 42, 3. [28] Marrack, P., Lo, D., Brinster, R., Palmiter, R., Burkly, L., Flavell, R. and Kappler, J. (1988) Cell 53, 627. [29] Vollger, W., Tuck, D., Springer, T., Haynes, B. and Singer, K. (1987) J. Immunol. 138, 358. [30] Denning, S., Kurtzberg, J., Le, P., Tuck, D., Singer, K. and Haynes, B. (1988) Proc. Natl. Acad. Sci. USA 85, 3125. [31] Boyd, R., Izon, D., Godfrey, D., Wilson, T. and Tucek, C. (1989) Adv. Exp. Med. Biol. 237, 263. [32] Haynes, B., Denning, S., Singer, K. and Kurtzberg, J. (1989) Immunol. Today 10, 87. [33] Basch, R. and Berman, J. (1982) Eur. J. lmmunol. 12, 359. [34] Basch, R., Kadish, J. and Goldstein, G. (1978) J. Exp. Med. 147, 1843. [35] Muller-Sieberg, C., Whitlock, C. and Weissman, I. (1986) Cell 44, 653. [36] Spangrude, G., Muller-Sieberg, C., Heimfeld, S. and Weissman, I. (1988) J. Exp. Med. 167, 1671. [37] Kurtzberg, J., Denning, S., Nycum, L., Singer, K. and Haynes, B. (1989) Proc. Natl. Acad. Sci. USA 86, 7575. [38] Goodwin, L., Rocha, A. and Basch, R. (1986) Nature 323, 166. [39] Palacios, R., Kiefer, M., Brockhaus, M., Kajalainen, K., Dembic, Z., Kisielow, P. and von Boehmer, H. (1987) J. Exp. Med. 166, 12. [40] Palacios, R. and Pelkonen, J. (1988) lmmunol. Rev. 104, 158. [41] Hurwitz, J. (1987) Eur. J. lmmunol. 17, 751. [42] O'Neill, H. (1987) Cell. Immunol. 109, 222. [43] O'Neill, H. Submitted ms. [44] Cook, W. (1985) Mol. Cell. Biol. 5, 390. [45] Cook, W. and Balaton, A. (1987) Mol. Cell. Biol. 7, 266. [46] Hurwitz, J., Samaridis, J. and Pelkonen, J. (1988) Cell 52, 821. [47] O'Neill, H. (1989) Immunology 68, 59. [48] Stamper, H. and Woodruff, J. (1976) J. Exp. Med. 144, 828. [49] Trowbridge, I., Lesley, J., Schulte, R., Hyman, R. and Trotter, J. (1982) lmmunogenetics 15, 299. [50] Stoolman, L. (1989) Cell 56, 907. [51] Haynes, B., Telen, M., Hale, L. and Denning, S. (1989) Immunol. Today 10, 423. [52] Goldstein, L., Zhou, D., Picker, L., Minty, C., Bargatze, R., Ding, J. and Butcher, E. (1989) Cell 56, 1063. [53] Stamenkovic, I., Amiot, M., Pesando, J. M. and Seed, B. (1988) Cell 56, 1057. [54] Jalkanen, S., Reichert, R., Gallatin, W., Bargatze, R., Weissman, i. and Butcher, E. (1986) Immunol. Rev. 91, 39. [55] Siegelman, M., van de Rijn, M. and Weissman, I. (1989) Science 243, 1166. [56] Lasky, L., Singer, M., Yednock, T., Oowbenko, D., Fennie, C., Rodriguez, R., Nguyen, T., Stachel, S. and Rosen, D. (1989) Cell 56, 1045. [57] Bevilacqua, M., Stengelin, S., Gimbrone, M. and Seed, B. (1989) Science 243, 1160.
