- Email: [email protected]
The ontogeny of T lymphocytes
Ann. Immunol. (Inst. Pasteur) 1983, 134 D, 115-122
THE
ONTOGENY
OF
T
LYMPHOCYTES
by J. J. T. Owen, E. J. Jenkinson and R. Kingston
Department o] Anatomy, University o/Birmingham, Birmingham (United Kingdom)
SUMMARY We have provided a speculative overview of some of the questions concerning the nature of thymic stem cells and the role of the t h y m u s in their maturation, and have suggested experimental approaches which might provide solutions to some of the questions. Throughout, we have stressed the complexity of the cellular constitution of the t h y m u s and have suggested roles for particular cell types which seem plausible based on present evidence. KEY-WORDS: T lymphocyte, Thymus, Ontogeny; Stem cell, Maturation.
INTRODUCTION.
Almost two decades have passed since (to use Jacques Miller's phrase [25]) the ~cgolden age of thymology ~ was ushered in by experiments demonstrating the adverse consequences of neonatal t h y m e c t o m y on lymphopoiesis and immunity. During the intervening years, considerable progress has been made in demonstrating the importance of the t h y m u s in T-lymphocyte maturation; in identifying the need for stem cell inflow to the t h y m u s in the embryo and adult; and in analysing the heterogeneous populations of T lymphocytes in t h y m u s and in peripheral tissues. Hgwever, a number of important issues remain unresolved. We will try to focus on t h e m and to offer some experimental approaches which might provide solutions in the future. THYMIG STEM CELLS (PRE-T CELLS).
Stem cells, which are precursors of all t h y m u s lymphocytes, migrate to t h y m u s from sites of blood cell formation - - foetal liver in the embryo and Manuscrit regu le 15 ffvrier 1983.
116
J . J . T . OWEN, E. J. JENKINSON AND B. KINGSTON
bone marrow in the adult [7, 18, 26]. Migration takes place via the vascular system, although in early embryogenesis, before the thymic primordium is vascularized, the final pathway involves extravascular m o v e m e n t of stem cells from local vessels through the basement membrane which envelopes the t h y m u s and into the primordium. It is likely t h a t the rate of migration of stem cells into the t h y m u s is slow. This idea is supported by studies in which repopulation of the t h y m u s was examined in mice after a potentially lethal dose of irradiation and injection of two syngeneic but chromosomally distinguishable bone marrow populations [32]. In comparing individual thymic lobes, considerable variations in the proportion of cells derived from each marrow type were noted and, using a probability analysis, the authors concluded t h a t as few as ten stem cells were responsible for t h y m i c repopulation. If t h e rate of inflow is slow, then thymic stem cells must have considerable proliferative capabilities in order to produce large numbers of t h y m i c lymphocytes, most of which are renewed every 3-4 days [24]. The fact t h a t stem cells within a 14-day mouse embryo t h y m i c graft can produce all of the t h y m i c lymphocytes during a period of considerable thymic growth after transplantation supports the idea that t h y m i c stem cells have extensive proliferative and perhaps some self-renewal capabilities [29]. An important group of questions centres on the nature of thymic stem ceils. Are they already partially differentiated along the T-cell p a t h w a y before t h e y enter the t h y m u s and, if so, what level of maturation is achieved? In particular, do stem cells acquire antigen recognition receptors pre-thymically? In considering these issues, it m u s t be noted t h a t T cells recognise antigen in the context of self molecules encoded within the major histocompatibility complex (MHC) and hence they may have either a single receptor for a combined antigenic determinant (self and foreign) or two receptors, one for self and one for foreign antigens [6]. At the moment, there is no general agreement as to which is the case, but if T cells have two receptors, then either one or both might be acquired at a pre-thymic level. Recent studies have argued for the view t h a t thymic stem cells are partially differentiated and, indeed, may express antigen recognition receptors at a prethymic level. In one study, parental strain A t h y m u s was transplanted into (A x B)F1 mice which were then irradiated and reconstituted with strain A bone marrow [27]. Cells repopulating the t h y m i c graft were shown to be of donor A marrow origin but they were found to be specifically tolerant to allogeneic host strain B determinants. Since the authors could not detect strain B MHC determinants in t h y m u s cell suspensions, they argued t h a t pre-T cells must have been exposed to these determinants pre-thymically and hence t h a t they possess antiallogeneic MHC receptors through which they can be rendered tolerant. If alloantigens are recognised by the same population of T-cell receptors which recognize
CTL-P = cytotoxic T - l y m p h o c y t e precursor. MHC = m a j o r h i s t o c o m p a t i b i l i t y complex.
ONTOGENY OF T LYMPHOCYTES
117
self MHC determinants plus foreign antigen, then these results suggest t h a t the T-cell repertoire is generated at a pre-thymic level. However, the possibility that s o m e F1 cells were present within the engrafted parental t h y m u s could not be rigorously excluded in these experiments. For example, it is known that in t h y m i c chimeras, some thymie medullary cells which express Ia antigen, are of host origin [9.]. These cells, which might be thymie dendritic cells [3], could have populated the thymic graft during the period of regeneration following transplantation and so might provide a source of strain B antigen with which the donor strain A cells could be rendered tolerant intrathymically. A further counterargument to the notion t h a t there is substantial T-cell repertoire diversity at a prethymic level is provided by the evidence reviewed earlier, namely, that relatively few stem cells migrate into the organ and that those which do are responsible for large numbers of progeny. If the repertoire is generated prethymically without further diversification intrathymically, the products of thymic maturation, i. e. mature T cells, would have a repertoire of responsiveness limited to the few stem cells from which they are derived. Although none of the evidence is decisive, it seems reasonable to continue to think of the extensive production of cells within the t h y m u s as being of importance in the generation of new specificities [30], although prethymieally created diversity, perhaps in foetal liver or adult bone marrow, would have the virtue of economy if it occurred in a common T- and B-cell precursor pool. Regardless of the question of repertoire generation, evidence that t h y m i e stem cells are partially differentiated along the T-cell pathway is far from convincing. Some cells in foetal liver and bone marrow can be ~ induced 5~ (by t r e a t m e n t with agents which elevate intraeellular cyclic AMP) to express cell membrane antigens which are characteristic of mature T cells [16, 17]. However, expression of T-cell antigens is insufficient evidence to justify the claim that all (c inducible 5~cells are pre-T cells in the absence of data showing t h a t these cells migrate to thymus. Similarly, the presence of (c inducible ~5 cells and, indeed, the presence of precursors of cytotoxic T cells (CTL-P) in Nude mice [1, 5, 9,-11, 20,-22] is as much an argument for the possibility of some T-cell maturation in the absence of a lymphoid t h y m u s as for the notion t h a t t h y m i c stem cells are partially differentiated before entry to the thymus. A~ major difficulty is the fact that t h y m i c stem cells cannot be studied directly. Within marrow and foetal liver, they form a small proportion of a highly heterogeneous population and, as yet, have not been isolated in purified form. Assays for thymie stem cells using irradiated animals [15] suffer from the complexity of the system - - the presence of surviving host lymphoid cells within the t h y m u s and the seeding of haemopoietic stem cells to spleen and marrow from which t h y m i c colonization may secondarily occur. In order to circumvent some of these difficulties, we have tried to establish an in vitro assay for thymic stem cells. In part, the assay depends upon a method we have described for the production of c( e m p t y 55thymic rudiments free of indigenous lymphoid cells [131. Deoxyguanosine, which is
118
J . J . T . OWEN, E. J. JENKINSON AND R. KINGSTON
toxic for T lymphocytes, is used during a short initial period of organ culture to deplete embryonic thymic lobes of lymphocytes and stem cells, leaving an epithelial thymus which can be repopulated by added stem cells. In the absence of exogenous stem cells, the treated thymic rudiments remain small and alymphoid [13]. We have now developed this system further so as to allow repopulation of (( e m p t y )) thymic rudiments with known numbers of cells. The general scheme of the experiments is as follows: Stage 2
Stage 1
Thymic lobes removed from 14-day mouse embryos ~-
Culturein hanging drops for two days
Organ culture with deoxyguanosine for 5 days
Stage 3 ~
--
Recolonized thymiclobes transferred to organ culture
(( E m p t y ))thymic lobes placed in hanging drop cultures with stem cells
>
Organ culture for 7 to 10 days
Stage 4
Whole lobes pulsed for 18 h with 11.5 UdR for measurement of DNA synthesis or lymphocytes harvested for analysis of phenotype and function
Stage 2 is carried out in (( hanging )) drops in microwell plates (Terasaki) so as to allow (( empty )~thymic lobes to come into direct contact with small numbers of stem cells at the point of each drop. Sources of stem cells may be suspensions of foetal liver, bone marrow cells or of the (( blasts )) present within another early thymic rudiment (13-15 day's gestation). We have found that the latter are a particularly rich source of stem cells - - as few as 60 cells will produce recolonisation, and we are now investigating t h e use of even lower cell numbers. Stage 3 of the procedure is important because the period of organ culture after recolonization allows expansion of the lymphoid population. In stage 4, the overall growth of the t h y m u s may be measured, or the lymphocytes generated in a chimeric t h y m u s may be tested for functional activity, as discussed later. Experiments of this type should be capable of providing assays of cells able to migrate into the t h y m u s and, in addition, should provide a means of examining the effects of various agents, including antibodies acting on the cell surface, on the migration process and upon the proliferation of cells within the thymus. THE
INTERACTION
BETWEEN
STEM
CELLS
AND
THYMIC
STROMA.
In addition to any hormonal role which the thymic stroma may have in lymphocyte maturation, it is implicit in many theories of T-cell receptor
ONTOGENY OF T LYMPHOCYTES
119
generation t h a t the t h y m i c stroma plays an i m p o r t a n t part t h r o u g h contact with the differentiating lymphoid cells [30]. Irrespective of w h e t h e r receptor diversity is generated prethymieally or intrathymically, t h e stroma might have a crucial role in receptor selection. One hypothesis has proposed t h a t the stroma might drive the diversification process [14]. The cellular composition of t h e stroma and the expression of MHC antigens on stromal cells, which might influence self MHC recognition and tolerance induction, are therefore matters of considerable interest. The epithelial cells are of mixed ectodermal/endodermal origin, b u t only endodermal cells probably contribute to the alymphoid t h y m i c remn a n t of the Nude mouse [4, 28]. The t h y m i c stroma also contains cells of mesenchymal origin, some of which form the connective tissue strands which separate lobules and surround vessels; others, however, form t h e (( dendritic ~, network of macrophages which is most obvious in t h e t h y m i c medulla [2]. Class II (I region) MHC antigens are a b u n d a n t on t h y m i c epithelial cells (probably on those of ectodermal rather t h a n endodermal origin - - the Nude t h y m u s is Ia-negative [12]). Class I (K and D region) antigens are also expressed on t h y m i c epithelial cells, although probably at lower levels. The expression of class lI antigens is particularly notew o r t h y because these antigens play a central role in the induction of proliferative responses by T cells and ace not widely distributed on other tissues, especially during embryogenesis. Some confusion has arisen as to whether the cells expressing class II antigens are of intrinsic or extrinsic t h y m i c origin. The m a t t e r has been clarified with the demonstration of Ia antigens on both (( intrinsic ,, epithelial cells and c( extrinsic ~., dendritic cells [2]. It would appear t h a t both cell types synthesize and express class II antigens independently, and t h a t antigen is not acquired by one t y p e from the other. Dendritic cells also strongly express class I antigens [8]. The functional significance of MHC antigen expression on t h y m i c stromal cells can be analysed in t h y m i c chimeras in which stromal cells and lymphoid cells are of different but known genetic origins. In this way, t h e influence of t h e stroma on the generation of the T-cell repertoire m a y be assessed. T h y m i c chimeras constructed in irradiated or Nude mice in vivo have been used to demonstrate a t h y m i c influence on MHC restriction of responses to exogenous antigen [33]. However, the results of these experiments were not clear-eric, and the role of t h e t h y m u s in restriction remains controversial, due in part to the complex nature of the in vivo chimera model. The construction of t h y m i c chimeras entirely in vitro in the m a n n e r we have already described provides an alternative approach in which the influence of the t h y m i c stroma can be separated from the effects of peripheral lymphoid tissues. Thus, stem cells introduced into an allogeneie stroma can be used to generate lymphoid cells whose functional capabilities in terms of MHC restriction and tolerance to MHC antigens can be examined using in vilro assays. F u r t h e r m o r e , the e x t e n t of repertoire generation t o alloantigens can be measured in clonal assays [31] and the size of the repertoire can therefore be studied in relation to the n u m b e r
120
J . J . T . OWEN, E. J. JENKINSON AND R. KINGSTON
of stem cells used for t h y m i c colonisation. In particular, the following question m a y be asked: does the use of limited numbers of stem cells restrict the size of the T-cell repertoire in the resulting lymphoid population? If not, the experiment would favour the idea t h a t most repertoire generation occurs intrathymically; conversely, if it does, the results would argue for pre-thymic generation of diversity. One final point is worthy of note. We have found t h a t dendritic cells within our chimeric t h y m u s express Ia antigen correspopding to the genotype of the donor stem cells. It seems likely t h a t these cells are therefore of extrinsic origin (as t h e y are in vivo) and so are derived from precursors intermixed with the lymphoid stem cell population. The relative importance of class II MHC antigen expression on epithelial cells as opposed to dendritic cells is unclear, but our experimental system might provide an answer. It is t e m p t i n g to speculate t h a t (1) because the major proliferative zone of lymphoid cells is in the outer thymic cortex and (2) because, during the formation of noncycling small thymocytes, there is a progressive migration of cells through the cortical region to the cortico-medullary junction where functional maturity is t h o u g h t to be achieved, the expression of MHC antigens on cortical epithelial cells might act as a selective influence on the transit population. Since substantial cell production is t h o u g h t to be matched by considerable cell loss within the t h y m u s [23], it is unlikely that MHC antigen expression on stromal cells acts in a positive way to drive proliferation of cells with matching specificies, but rather t h a t the products of cortical proliferation might need to match receptor specificity with stromal cell antigens during their passage through the cortex in order to achieve final maturation at the cortico-medullary junction This view argues for a role of cortical epithelial cells in selecting for MHC restriction. Again, it is tempting to speculate that medullary dendritic cells, by presenting antigen to cortical lymphocytes at their point of maturation at the cortico-medullary junction, might be responsible for intra-thymic tolerance induction. This pattern of tolerance induction in T cells would closely parallel the situation in B cells where newly formed B cells in foetal liver and adult marrow are t h o u g h t to be especially sensitive to tolerogenic signals [19]. Also, this scheme would provide a plausible explanation for long-term tolerance induced in T cells by neonatal injection of allogeneic cell suspensions, if this procedure resulted in the establishment of long-lived allogeneic dendritic cell populations within the t h y m i c medulla. RI~SUMI~ ONTOGENIESE DES LYMPHOCYTES
T
Nous avons discut~ des probl~mes concernant la nature des cellules souches thymiques ainsi que le r61e du t h y m u s dans leur maturation. Nous sugg~rons l'utilisation d'approches exp6rimentales qui devraient donner
ONTOGENY OF T LYMPHOCYTES
121
des solutions A certains de ces probl~mes. Nous avons t o u t particuli6rement port6 l'accent sur la complexit6 de la structure cellulaire du t h y m u s et avons sugg6r6 des r61es particuliers pour certains t y p e s cellulaires. MOTS-CL~S : L y m p h o c y t e T, Thymus, Ontogen~se; Cellule souche, Maturation.
REFERENCES
[1] ANDO, I. & HURME, M., Self-MHCorestricted cytotoxic T-cell response without thymic influence. Nature (Lond.), 1981, 289, 494-495. [2] BARCLAY,A. N. & MAYROHOFER,G., Bone marrow origin of Ia-positive cells in the medulla of rat thymus. J. exp. Med., 1981, 153, 1660-1671. [3] BELLER, D. I. & UNANNE, E. R., I-A antigens and antigen-presenting function of thymic macrophages. J. Immunol., 1980, 124, 1443-1450. [4] CORDIER,A. C. & HEREMANS,J. F., Nude mouse embryo. Ectodermal nature of the primordial thymic defect. Scand. J. Immunol., 1975, 4, 193-196. [5] DENNERT, G. & HAYMAN, R., Functional Thy-1 + cells in cultures of spleen cells from nu/nu mice. Europ. J. Immunol., 1980, 10, 583-589. [6] DOUGHERTY, P.C. & ZINKERNAGEL,]~. M., H-2 compatibility is required for T-cell-mediated lysis of target cells of mice injected with lymphocyte choriomeningitis virus. J. exp. Med., 1975, 141, 502-507. [7] FORD, C. E., MICKLEM, H. S., EVANS, E. P., GRAY, J. S. & OGDEN, D. A., The inflow of bone marrow cells to the thymus: studies with part-body irradiated mice injected with chromosome marked bone marrow and subjected to antigenic stimulation. Ann. N. Y. Acad. Sci., 1966, 129, 283-296. [8] FUKUMOTO, T., McMASTER, W . R . & WILLIAMS, A. S., Mouse monoclonal antibodies against rat major histocompatibility antigens. Two Ia antigens and expression of Ia and class I antigens in rat thymus. Europ. J. Immunol., 1982, 12, 237-243. [9] GILLIS, S., UNION, N. A., BAKER, P. E. & SMITH, K. h., In vitro generation and sustained culture of nude mouse cytolytic T-lymphocytes. J. exp. Med., 1979, 149, 1460-t476. [10] GILLIS, S. & WATSON, J., Interleukin-2 induction of hapten-specific cytolytic T cells in nude mice. J. Immunol., 1981, 126, 1245-1248. [11] HUNIG, T. & BEVAN, M. ft., Specificity of cytotoxic T cells from athymic mice. J. exp. Med., 1980, 152, 688-702. [12] JENKINSON,E. J., VAN EWIJK, ~V. & OWEN, J. J. T., Major histocompatibility antigen expression on the epithelium of the developing thymus in normal and (~nude )) mice. J. exp. Med., 1981, 153, 280-293. [13] JENKINSON, E. J., FRANCHI, L. L., KINGSTON, R. & OWEN, J. J. T., Effect of deoxyguanosine on lymphopoiesis in the developing thymic rudiment in vitro: application in the production of chimeric thymus rudiments. Europ. J. Immunol., 1982, 12, 583-587. [14] JERNE, N. K., The somatic generation of immune recognition. Europ. J. Immunol., 1971, 1, 1-9. [15] KADISH, J . L . & BASCH, R. S., Haematopoietic thymocyte precursors. --I. Assay and kinetics of the appearance of progeny. J. exp. Med., 1976, 143, 1082-1099. [16] KoMuno, K. & BOYSE, E.A., Induction of T-lymphocytes from precursor cells in vitro by a product of the thymus. J. exp. Med., 1973, 138, 479-482.
122
J.J.T.
0WEN, E. J. JENKINSON AND R. KINGSTON
[17] KOMURO,K., GOLDSTEIN,G. & BOYSE, E. A., Thymus re-populating capacity of cells that can be induced to differentiate to T cells in vitro. J. Immunol., 1975, 115, 195-198. [18] LE DOUARIN, N. M. & JOTEREAU, F., Tracing of cells of the avian thymus through embryonic life in interspecific chimeras. J. exp. Med., 1975, 142, 17-40. [19] LEVITT,D. & COOPER,M. D., Mouse pre-B cells synthesize and secrete 9 heavy chains but not light chains. Cell, 1980, 19, 617-625. [20] LIPSICK, J. S., SERUNIAN, L., SATO, V. L. & KAPLAN, N. 0., Differentiation and activation of nu/nu splenic T cell precursors by mature peripheral T cells in the absence of thymus. J. Immunol., 1982, 129, 40-45. [21] Loon, F. & KINDRED, B., Differentiation of T-cell precursors in nude mice demonstrated by immunofluorescence of T-cell membrane markers. J. exp. Med., 1973, 138, 1044-1055. [22] Loon, F. & ROELANTS, G. F., High frequency of T-lineage lymphocytes in nude mouse spleen. Nature (Lond.), 1974, 251, 229-230. [23] McPHEE, D., PYE, J. & SHORTMAN,K., The differentiation of T lymphocyte. - V. Evidence for intrathymic death of most thymocytes. Thymus, 1979, 1, 151-156. [24] METCALF,D. & WIADROWSKI, M., Autoradiographic analysis of lymphocyte proliferation in the thymus and in thymic lymphoma tissue. Cancer Res., 1966, 26, 483-491. [25] MILLER, J. F. A. P., The thymus. Yesterday, today and tomorrow. Lancet, 1967, II, 1299-1302. [26] MOORE,M. A. S. & OWEN, J. J. T., Experimental studies on the development of the thymus. J. exp. Med., 1967, 126, 715-726. [27] MORRISSEY, P. J., KRUISBEEK, A. M., SHARROW, S. O. & SINGER, A., Tolerance of thymic cytotoxic T lymphocytes to allogeneic H-2 determinants encountered pre-thymically: evidence of expression of anti-H-2 receptors prior to entry to the thymus. Proc. nat. Acad. Sci. (Wash.), 1982, 79, 2003-2007. [281 OWEN, J. J. T. & JENKINSON, E. J., Embryology of the lymphoid system. Progr. Allergy, 1981, 29, 1-34. [29] OwEs, J. J. T. & I~AFF, M. C., Studies on the differentiation of thymusderived lymphocytes. J. exp. Med., 1970, 132, 1216-1232. [30] SCHRADER,J. W., A single T-cell receptor: a speculative review of the intrathymic generation and modulation of the repertoire. Thymus, 1982, 4, 181-207. [31] VON BOEHMER, H. & HAAG, W., H-2 restricted cytolytic and non-cytolytic T cell clones: isolation, specificity and functional analysis. Immunol. Rev., 1981, 54, 27-55. [32] WALLIS,V., LEUCHARS,E., CHWALINSKI,S. & DAVIES,A. J. S., On the sparse seeding of bone marrow and thymus in radiation chimaeras. Transplant., 1975, 19, 2-11. [33] ZINKERNAGEL, Ft. M., Thymus and lymphohemopoietic cells: their role in T-cell maturation in selection of T cells' H-2 restriction specificity and in H-2 linked in gene control. Immunol. Rev., 1978, 42, 224-270.
THE
ONTOGENY
OF
T
LYMPHOCYTES
by J. J. T. Owen, E. J. Jenkinson and R. Kingston
Department o] Anatomy, University o/Birmingham, Birmingham (United Kingdom)
SUMMARY We have provided a speculative overview of some of the questions concerning the nature of thymic stem cells and the role of the t h y m u s in their maturation, and have suggested experimental approaches which might provide solutions to some of the questions. Throughout, we have stressed the complexity of the cellular constitution of the t h y m u s and have suggested roles for particular cell types which seem plausible based on present evidence. KEY-WORDS: T lymphocyte, Thymus, Ontogeny; Stem cell, Maturation.
INTRODUCTION.
Almost two decades have passed since (to use Jacques Miller's phrase [25]) the ~cgolden age of thymology ~ was ushered in by experiments demonstrating the adverse consequences of neonatal t h y m e c t o m y on lymphopoiesis and immunity. During the intervening years, considerable progress has been made in demonstrating the importance of the t h y m u s in T-lymphocyte maturation; in identifying the need for stem cell inflow to the t h y m u s in the embryo and adult; and in analysing the heterogeneous populations of T lymphocytes in t h y m u s and in peripheral tissues. Hgwever, a number of important issues remain unresolved. We will try to focus on t h e m and to offer some experimental approaches which might provide solutions in the future. THYMIG STEM CELLS (PRE-T CELLS).
Stem cells, which are precursors of all t h y m u s lymphocytes, migrate to t h y m u s from sites of blood cell formation - - foetal liver in the embryo and Manuscrit regu le 15 ffvrier 1983.
116
J . J . T . OWEN, E. J. JENKINSON AND B. KINGSTON
bone marrow in the adult [7, 18, 26]. Migration takes place via the vascular system, although in early embryogenesis, before the thymic primordium is vascularized, the final pathway involves extravascular m o v e m e n t of stem cells from local vessels through the basement membrane which envelopes the t h y m u s and into the primordium. It is likely t h a t the rate of migration of stem cells into the t h y m u s is slow. This idea is supported by studies in which repopulation of the t h y m u s was examined in mice after a potentially lethal dose of irradiation and injection of two syngeneic but chromosomally distinguishable bone marrow populations [32]. In comparing individual thymic lobes, considerable variations in the proportion of cells derived from each marrow type were noted and, using a probability analysis, the authors concluded t h a t as few as ten stem cells were responsible for t h y m i c repopulation. If t h e rate of inflow is slow, then thymic stem cells must have considerable proliferative capabilities in order to produce large numbers of t h y m i c lymphocytes, most of which are renewed every 3-4 days [24]. The fact t h a t stem cells within a 14-day mouse embryo t h y m i c graft can produce all of the t h y m i c lymphocytes during a period of considerable thymic growth after transplantation supports the idea that t h y m i c stem cells have extensive proliferative and perhaps some self-renewal capabilities [29]. An important group of questions centres on the nature of thymic stem ceils. Are they already partially differentiated along the T-cell p a t h w a y before t h e y enter the t h y m u s and, if so, what level of maturation is achieved? In particular, do stem cells acquire antigen recognition receptors pre-thymically? In considering these issues, it m u s t be noted t h a t T cells recognise antigen in the context of self molecules encoded within the major histocompatibility complex (MHC) and hence they may have either a single receptor for a combined antigenic determinant (self and foreign) or two receptors, one for self and one for foreign antigens [6]. At the moment, there is no general agreement as to which is the case, but if T cells have two receptors, then either one or both might be acquired at a pre-thymic level. Recent studies have argued for the view t h a t thymic stem cells are partially differentiated and, indeed, may express antigen recognition receptors at a prethymic level. In one study, parental strain A t h y m u s was transplanted into (A x B)F1 mice which were then irradiated and reconstituted with strain A bone marrow [27]. Cells repopulating the t h y m i c graft were shown to be of donor A marrow origin but they were found to be specifically tolerant to allogeneic host strain B determinants. Since the authors could not detect strain B MHC determinants in t h y m u s cell suspensions, they argued t h a t pre-T cells must have been exposed to these determinants pre-thymically and hence t h a t they possess antiallogeneic MHC receptors through which they can be rendered tolerant. If alloantigens are recognised by the same population of T-cell receptors which recognize
CTL-P = cytotoxic T - l y m p h o c y t e precursor. MHC = m a j o r h i s t o c o m p a t i b i l i t y complex.
ONTOGENY OF T LYMPHOCYTES
117
self MHC determinants plus foreign antigen, then these results suggest t h a t the T-cell repertoire is generated at a pre-thymic level. However, the possibility that s o m e F1 cells were present within the engrafted parental t h y m u s could not be rigorously excluded in these experiments. For example, it is known that in t h y m i c chimeras, some thymie medullary cells which express Ia antigen, are of host origin [9.]. These cells, which might be thymie dendritic cells [3], could have populated the thymic graft during the period of regeneration following transplantation and so might provide a source of strain B antigen with which the donor strain A cells could be rendered tolerant intrathymically. A further counterargument to the notion t h a t there is substantial T-cell repertoire diversity at a prethymic level is provided by the evidence reviewed earlier, namely, that relatively few stem cells migrate into the organ and that those which do are responsible for large numbers of progeny. If the repertoire is generated prethymically without further diversification intrathymically, the products of thymic maturation, i. e. mature T cells, would have a repertoire of responsiveness limited to the few stem cells from which they are derived. Although none of the evidence is decisive, it seems reasonable to continue to think of the extensive production of cells within the t h y m u s as being of importance in the generation of new specificities [30], although prethymieally created diversity, perhaps in foetal liver or adult bone marrow, would have the virtue of economy if it occurred in a common T- and B-cell precursor pool. Regardless of the question of repertoire generation, evidence that t h y m i e stem cells are partially differentiated along the T-cell pathway is far from convincing. Some cells in foetal liver and bone marrow can be ~ induced 5~ (by t r e a t m e n t with agents which elevate intraeellular cyclic AMP) to express cell membrane antigens which are characteristic of mature T cells [16, 17]. However, expression of T-cell antigens is insufficient evidence to justify the claim that all (c inducible 5~cells are pre-T cells in the absence of data showing t h a t these cells migrate to thymus. Similarly, the presence of (c inducible ~5 cells and, indeed, the presence of precursors of cytotoxic T cells (CTL-P) in Nude mice [1, 5, 9,-11, 20,-22] is as much an argument for the possibility of some T-cell maturation in the absence of a lymphoid t h y m u s as for the notion t h a t t h y m i c stem cells are partially differentiated before entry to the thymus. A~ major difficulty is the fact that t h y m i c stem cells cannot be studied directly. Within marrow and foetal liver, they form a small proportion of a highly heterogeneous population and, as yet, have not been isolated in purified form. Assays for thymie stem cells using irradiated animals [15] suffer from the complexity of the system - - the presence of surviving host lymphoid cells within the t h y m u s and the seeding of haemopoietic stem cells to spleen and marrow from which t h y m i c colonization may secondarily occur. In order to circumvent some of these difficulties, we have tried to establish an in vitro assay for thymic stem cells. In part, the assay depends upon a method we have described for the production of c( e m p t y 55thymic rudiments free of indigenous lymphoid cells [131. Deoxyguanosine, which is
118
J . J . T . OWEN, E. J. JENKINSON AND R. KINGSTON
toxic for T lymphocytes, is used during a short initial period of organ culture to deplete embryonic thymic lobes of lymphocytes and stem cells, leaving an epithelial thymus which can be repopulated by added stem cells. In the absence of exogenous stem cells, the treated thymic rudiments remain small and alymphoid [13]. We have now developed this system further so as to allow repopulation of (( e m p t y )) thymic rudiments with known numbers of cells. The general scheme of the experiments is as follows: Stage 2
Stage 1
Thymic lobes removed from 14-day mouse embryos ~-
Culturein hanging drops for two days
Organ culture with deoxyguanosine for 5 days
Stage 3 ~
--
Recolonized thymiclobes transferred to organ culture
(( E m p t y ))thymic lobes placed in hanging drop cultures with stem cells
>
Organ culture for 7 to 10 days
Stage 4
Whole lobes pulsed for 18 h with 11.5 UdR for measurement of DNA synthesis or lymphocytes harvested for analysis of phenotype and function
Stage 2 is carried out in (( hanging )) drops in microwell plates (Terasaki) so as to allow (( empty )~thymic lobes to come into direct contact with small numbers of stem cells at the point of each drop. Sources of stem cells may be suspensions of foetal liver, bone marrow cells or of the (( blasts )) present within another early thymic rudiment (13-15 day's gestation). We have found that the latter are a particularly rich source of stem cells - - as few as 60 cells will produce recolonisation, and we are now investigating t h e use of even lower cell numbers. Stage 3 of the procedure is important because the period of organ culture after recolonization allows expansion of the lymphoid population. In stage 4, the overall growth of the t h y m u s may be measured, or the lymphocytes generated in a chimeric t h y m u s may be tested for functional activity, as discussed later. Experiments of this type should be capable of providing assays of cells able to migrate into the t h y m u s and, in addition, should provide a means of examining the effects of various agents, including antibodies acting on the cell surface, on the migration process and upon the proliferation of cells within the thymus. THE
INTERACTION
BETWEEN
STEM
CELLS
AND
THYMIC
STROMA.
In addition to any hormonal role which the thymic stroma may have in lymphocyte maturation, it is implicit in many theories of T-cell receptor
ONTOGENY OF T LYMPHOCYTES
119
generation t h a t the t h y m i c stroma plays an i m p o r t a n t part t h r o u g h contact with the differentiating lymphoid cells [30]. Irrespective of w h e t h e r receptor diversity is generated prethymieally or intrathymically, t h e stroma might have a crucial role in receptor selection. One hypothesis has proposed t h a t the stroma might drive the diversification process [14]. The cellular composition of t h e stroma and the expression of MHC antigens on stromal cells, which might influence self MHC recognition and tolerance induction, are therefore matters of considerable interest. The epithelial cells are of mixed ectodermal/endodermal origin, b u t only endodermal cells probably contribute to the alymphoid t h y m i c remn a n t of the Nude mouse [4, 28]. The t h y m i c stroma also contains cells of mesenchymal origin, some of which form the connective tissue strands which separate lobules and surround vessels; others, however, form t h e (( dendritic ~, network of macrophages which is most obvious in t h e t h y m i c medulla [2]. Class II (I region) MHC antigens are a b u n d a n t on t h y m i c epithelial cells (probably on those of ectodermal rather t h a n endodermal origin - - the Nude t h y m u s is Ia-negative [12]). Class I (K and D region) antigens are also expressed on t h y m i c epithelial cells, although probably at lower levels. The expression of class lI antigens is particularly notew o r t h y because these antigens play a central role in the induction of proliferative responses by T cells and ace not widely distributed on other tissues, especially during embryogenesis. Some confusion has arisen as to whether the cells expressing class II antigens are of intrinsic or extrinsic t h y m i c origin. The m a t t e r has been clarified with the demonstration of Ia antigens on both (( intrinsic ,, epithelial cells and c( extrinsic ~., dendritic cells [2]. It would appear t h a t both cell types synthesize and express class II antigens independently, and t h a t antigen is not acquired by one t y p e from the other. Dendritic cells also strongly express class I antigens [8]. The functional significance of MHC antigen expression on t h y m i c stromal cells can be analysed in t h y m i c chimeras in which stromal cells and lymphoid cells are of different but known genetic origins. In this way, t h e influence of t h e stroma on the generation of the T-cell repertoire m a y be assessed. T h y m i c chimeras constructed in irradiated or Nude mice in vivo have been used to demonstrate a t h y m i c influence on MHC restriction of responses to exogenous antigen [33]. However, the results of these experiments were not clear-eric, and the role of t h e t h y m u s in restriction remains controversial, due in part to the complex nature of the in vivo chimera model. The construction of t h y m i c chimeras entirely in vitro in the m a n n e r we have already described provides an alternative approach in which the influence of the t h y m i c stroma can be separated from the effects of peripheral lymphoid tissues. Thus, stem cells introduced into an allogeneie stroma can be used to generate lymphoid cells whose functional capabilities in terms of MHC restriction and tolerance to MHC antigens can be examined using in vilro assays. F u r t h e r m o r e , the e x t e n t of repertoire generation t o alloantigens can be measured in clonal assays [31] and the size of the repertoire can therefore be studied in relation to the n u m b e r
120
J . J . T . OWEN, E. J. JENKINSON AND R. KINGSTON
of stem cells used for t h y m i c colonisation. In particular, the following question m a y be asked: does the use of limited numbers of stem cells restrict the size of the T-cell repertoire in the resulting lymphoid population? If not, the experiment would favour the idea t h a t most repertoire generation occurs intrathymically; conversely, if it does, the results would argue for pre-thymic generation of diversity. One final point is worthy of note. We have found t h a t dendritic cells within our chimeric t h y m u s express Ia antigen correspopding to the genotype of the donor stem cells. It seems likely t h a t these cells are therefore of extrinsic origin (as t h e y are in vivo) and so are derived from precursors intermixed with the lymphoid stem cell population. The relative importance of class II MHC antigen expression on epithelial cells as opposed to dendritic cells is unclear, but our experimental system might provide an answer. It is t e m p t i n g to speculate t h a t (1) because the major proliferative zone of lymphoid cells is in the outer thymic cortex and (2) because, during the formation of noncycling small thymocytes, there is a progressive migration of cells through the cortical region to the cortico-medullary junction where functional maturity is t h o u g h t to be achieved, the expression of MHC antigens on cortical epithelial cells might act as a selective influence on the transit population. Since substantial cell production is t h o u g h t to be matched by considerable cell loss within the t h y m u s [23], it is unlikely that MHC antigen expression on stromal cells acts in a positive way to drive proliferation of cells with matching specificies, but rather t h a t the products of cortical proliferation might need to match receptor specificity with stromal cell antigens during their passage through the cortex in order to achieve final maturation at the cortico-medullary junction This view argues for a role of cortical epithelial cells in selecting for MHC restriction. Again, it is tempting to speculate that medullary dendritic cells, by presenting antigen to cortical lymphocytes at their point of maturation at the cortico-medullary junction, might be responsible for intra-thymic tolerance induction. This pattern of tolerance induction in T cells would closely parallel the situation in B cells where newly formed B cells in foetal liver and adult marrow are t h o u g h t to be especially sensitive to tolerogenic signals [19]. Also, this scheme would provide a plausible explanation for long-term tolerance induced in T cells by neonatal injection of allogeneic cell suspensions, if this procedure resulted in the establishment of long-lived allogeneic dendritic cell populations within the t h y m i c medulla. RI~SUMI~ ONTOGENIESE DES LYMPHOCYTES
T
Nous avons discut~ des probl~mes concernant la nature des cellules souches thymiques ainsi que le r61e du t h y m u s dans leur maturation. Nous sugg~rons l'utilisation d'approches exp6rimentales qui devraient donner
ONTOGENY OF T LYMPHOCYTES
121
des solutions A certains de ces probl~mes. Nous avons t o u t particuli6rement port6 l'accent sur la complexit6 de la structure cellulaire du t h y m u s et avons sugg6r6 des r61es particuliers pour certains t y p e s cellulaires. MOTS-CL~S : L y m p h o c y t e T, Thymus, Ontogen~se; Cellule souche, Maturation.
REFERENCES
[1] ANDO, I. & HURME, M., Self-MHCorestricted cytotoxic T-cell response without thymic influence. Nature (Lond.), 1981, 289, 494-495. [2] BARCLAY,A. N. & MAYROHOFER,G., Bone marrow origin of Ia-positive cells in the medulla of rat thymus. J. exp. Med., 1981, 153, 1660-1671. [3] BELLER, D. I. & UNANNE, E. R., I-A antigens and antigen-presenting function of thymic macrophages. J. Immunol., 1980, 124, 1443-1450. [4] CORDIER,A. C. & HEREMANS,J. F., Nude mouse embryo. Ectodermal nature of the primordial thymic defect. Scand. J. Immunol., 1975, 4, 193-196. [5] DENNERT, G. & HAYMAN, R., Functional Thy-1 + cells in cultures of spleen cells from nu/nu mice. Europ. J. Immunol., 1980, 10, 583-589. [6] DOUGHERTY, P.C. & ZINKERNAGEL,]~. M., H-2 compatibility is required for T-cell-mediated lysis of target cells of mice injected with lymphocyte choriomeningitis virus. J. exp. Med., 1975, 141, 502-507. [7] FORD, C. E., MICKLEM, H. S., EVANS, E. P., GRAY, J. S. & OGDEN, D. A., The inflow of bone marrow cells to the thymus: studies with part-body irradiated mice injected with chromosome marked bone marrow and subjected to antigenic stimulation. Ann. N. Y. Acad. Sci., 1966, 129, 283-296. [8] FUKUMOTO, T., McMASTER, W . R . & WILLIAMS, A. S., Mouse monoclonal antibodies against rat major histocompatibility antigens. Two Ia antigens and expression of Ia and class I antigens in rat thymus. Europ. J. Immunol., 1982, 12, 237-243. [9] GILLIS, S., UNION, N. A., BAKER, P. E. & SMITH, K. h., In vitro generation and sustained culture of nude mouse cytolytic T-lymphocytes. J. exp. Med., 1979, 149, 1460-t476. [10] GILLIS, S. & WATSON, J., Interleukin-2 induction of hapten-specific cytolytic T cells in nude mice. J. Immunol., 1981, 126, 1245-1248. [11] HUNIG, T. & BEVAN, M. ft., Specificity of cytotoxic T cells from athymic mice. J. exp. Med., 1980, 152, 688-702. [12] JENKINSON,E. J., VAN EWIJK, ~V. & OWEN, J. J. T., Major histocompatibility antigen expression on the epithelium of the developing thymus in normal and (~nude )) mice. J. exp. Med., 1981, 153, 280-293. [13] JENKINSON, E. J., FRANCHI, L. L., KINGSTON, R. & OWEN, J. J. T., Effect of deoxyguanosine on lymphopoiesis in the developing thymic rudiment in vitro: application in the production of chimeric thymus rudiments. Europ. J. Immunol., 1982, 12, 583-587. [14] JERNE, N. K., The somatic generation of immune recognition. Europ. J. Immunol., 1971, 1, 1-9. [15] KADISH, J . L . & BASCH, R. S., Haematopoietic thymocyte precursors. --I. Assay and kinetics of the appearance of progeny. J. exp. Med., 1976, 143, 1082-1099. [16] KoMuno, K. & BOYSE, E.A., Induction of T-lymphocytes from precursor cells in vitro by a product of the thymus. J. exp. Med., 1973, 138, 479-482.
122
J.J.T.
0WEN, E. J. JENKINSON AND R. KINGSTON
[17] KOMURO,K., GOLDSTEIN,G. & BOYSE, E. A., Thymus re-populating capacity of cells that can be induced to differentiate to T cells in vitro. J. Immunol., 1975, 115, 195-198. [18] LE DOUARIN, N. M. & JOTEREAU, F., Tracing of cells of the avian thymus through embryonic life in interspecific chimeras. J. exp. Med., 1975, 142, 17-40. [19] LEVITT,D. & COOPER,M. D., Mouse pre-B cells synthesize and secrete 9 heavy chains but not light chains. Cell, 1980, 19, 617-625. [20] LIPSICK, J. S., SERUNIAN, L., SATO, V. L. & KAPLAN, N. 0., Differentiation and activation of nu/nu splenic T cell precursors by mature peripheral T cells in the absence of thymus. J. Immunol., 1982, 129, 40-45. [21] Loon, F. & KINDRED, B., Differentiation of T-cell precursors in nude mice demonstrated by immunofluorescence of T-cell membrane markers. J. exp. Med., 1973, 138, 1044-1055. [22] Loon, F. & ROELANTS, G. F., High frequency of T-lineage lymphocytes in nude mouse spleen. Nature (Lond.), 1974, 251, 229-230. [23] McPHEE, D., PYE, J. & SHORTMAN,K., The differentiation of T lymphocyte. - V. Evidence for intrathymic death of most thymocytes. Thymus, 1979, 1, 151-156. [24] METCALF,D. & WIADROWSKI, M., Autoradiographic analysis of lymphocyte proliferation in the thymus and in thymic lymphoma tissue. Cancer Res., 1966, 26, 483-491. [25] MILLER, J. F. A. P., The thymus. Yesterday, today and tomorrow. Lancet, 1967, II, 1299-1302. [26] MOORE,M. A. S. & OWEN, J. J. T., Experimental studies on the development of the thymus. J. exp. Med., 1967, 126, 715-726. [27] MORRISSEY, P. J., KRUISBEEK, A. M., SHARROW, S. O. & SINGER, A., Tolerance of thymic cytotoxic T lymphocytes to allogeneic H-2 determinants encountered pre-thymically: evidence of expression of anti-H-2 receptors prior to entry to the thymus. Proc. nat. Acad. Sci. (Wash.), 1982, 79, 2003-2007. [281 OWEN, J. J. T. & JENKINSON, E. J., Embryology of the lymphoid system. Progr. Allergy, 1981, 29, 1-34. [29] OwEs, J. J. T. & I~AFF, M. C., Studies on the differentiation of thymusderived lymphocytes. J. exp. Med., 1970, 132, 1216-1232. [30] SCHRADER,J. W., A single T-cell receptor: a speculative review of the intrathymic generation and modulation of the repertoire. Thymus, 1982, 4, 181-207. [31] VON BOEHMER, H. & HAAG, W., H-2 restricted cytolytic and non-cytolytic T cell clones: isolation, specificity and functional analysis. Immunol. Rev., 1981, 54, 27-55. [32] WALLIS,V., LEUCHARS,E., CHWALINSKI,S. & DAVIES,A. J. S., On the sparse seeding of bone marrow and thymus in radiation chimaeras. Transplant., 1975, 19, 2-11. [33] ZINKERNAGEL, Ft. M., Thymus and lymphohemopoietic cells: their role in T-cell maturation in selection of T cells' H-2 restriction specificity and in H-2 linked in gene control. Immunol. Rev., 1978, 42, 224-270.