.....
[
TRENDS Korsmeyer,S.J. (1991) Nature 353, 71-73 15 Strasser, A., Whittingham, S., Vaux, D.L. et al. (1991) Proc. Natl Acad. Sci.
USA 88, 8661-8665 16 McDonnell,T.J. and Korsmeyer, S.J. (1991) Nature 349, 254-256 17 Strasser, A., Harris, A.W., Bath, M.L. and Cory, S. (1990) Nature 348, 331-334 18 Blackman,M., Kappler,J. and
Marrack, P. (1990) Science 248, 1335-1341 19 Sentman, C.L., Shutter, J.R.,
Hockenbery, D., Kanagawa, O. and Korsmeyer,S.J. (1991) Cell 67, 879-888 20 Strasser,A., Harris, A.W. and Cory, S. (1991) Cell 67, 889-899 21 Guidos, C.J., Danska, J.S., Fathman, C.G. and Weissman,I.L. (1990) J. Exp. Med. 172, 835-845
22 Shortman, K., Vremec, D. and Egerton, M. (1991)J. Exp. Med. 173, 323-332 23 Ellis, H.M. and Horvitz, H.R.
(1986) Cell44, 817-829 24 Hengartner, M.O., Ellis, R.E. and Horvitz, H.R. (1992) Nature 356, 494-499 25 Yonish-Rouach,E., Resnitzky,D., Lotem, J. et al. (1991) Nature 352, 345-347
Taking the thymus to pieces Bruno Kyewski and Thomas Hiinig
Since the initiation of the thymus workshops at Rolduc in 1988, thymus research has made rapid progress, highlighted by the definitive demonstration of positive and negative selection. One may wonder whether the thymus still holds major secrets and surprises. On second thoughts, however, it becomes clear that we still lack a comprehensive understanding of the molecular mechanisms and signals that determine the developmental fate of the different T-cell lineages during intrathymic differentiation.
Selection and lineage commitment Transgenic mice have proven invaluable in deciphering the principles of T-cell repertoire selection and it is obvious that the complementary 'knockout' technique (in which a gene of choice is inactivated by homologous recombination) will become an equally powerful tool. Moreover, the combination of the two approaches offers hitherto unforeseen possibilities: the crossing of knockout mice with corresponding transgenic mice, in which the transgene itself or its expression pattern is specifically altered, allows the function of molecules to be assessed in the intact in vivo environment. This is exemplified by the crossing of major histocompatibility complex (MHC) class-II-negative mice with transgenic mice expressing MHC class II on either cortical epithelial
Complex in vitro and in vivo techniques are being combined to unlock the remainingsecrets of the thymus. In this report from a recent thymus workshop*, Bruno Kyewski and Thomas Hiinig describe the genetic manipulations aimed at clarifyingthe mechanisms of T-cell selection and lineage commitment, and the use of organ culture and immunohistology to identify the thymic microenvironments in which these events take place.
tic cells and immature thymocytes, as indicated by the MHC restriction of the CD8 ÷ T cells by the haplotype of the bone marrow donor. This result argues against the idea that epithelial cell-specific MHC-peptide complexes are essential ligands for positive selection (as proposed by the so-called peptide hypothesis) 3. The combination of knockout cells or hemopoietic antigen- mice with transgenic technology has presenting cellst, thus restoring also been applied to the question MHC class II expression in particu- of lineage commitment; that is, lar microenvironments. Not unex- whether commitment to the CD4 + pectedly, the expression of MHC and CD8 + lineages follows stochasclass lI on cortical epithelial cells tic or instructional rules. Instruction, restores the differentiation of the as originally proposed by von CD4 + T-cell lineage, which is de- Boehmer4, implies that corecognition ficient in the absence of MHC class I1 by immature double positive expression; however, expression of (CD4+CD8 +) thymocytes of thymic MHC class II on bone marrow de- MHC class-I complexes by the T-cell rived cells does not (M. Mercken- receptor (TCR) plus CD8, delivers a schlager, Strasbourg). In contrast, specific signal to shut down the CD4 infusion of MHC class-I-positive expression; and that conversely cobone marrow cells into irradiated recognition of MHC class II com132-microglobulin ([32-m)-deficient, plexes by TCR plus CD4 turns off MHC class-I-negative mice2 does CD8 expression. The stochastic restore the differentiation of func- model assumes that CD4 and CD8 tional CD8 + T cells, albeit at 5-10 expression is turned off randomly in times lower efficacy than in wild- double positive thymocytes, and that type mice (D. Raulet, Berkeley). The subsequent engagement of the TCR selection of CD8 + T cells in these and the appropriate coreceptor reschimeras is the result of direct cell- cues the thymocytes. MHC class-I-negative mice that cell interactions between hemopoielack CD8 + T cells were bred with *The Fourth Rolduc Thymus Workshop, transgenic mice expressing CD4 organized by Thomas Hfinig and Bruno under the p56 I"k promotor (D. LittKyewski was held in Rolduc, The man, San Francisco). According to the instructional model, these mice Netherlands on 26-29 May 1992.
© 1992, Hsevier Science Publishers Ltd, UK.
lmmu,ology Today 2 8 8
vol 13 No. 8 1992
TRENDS should not harbor any mature CD8 + T cells, since all immature thymocytes expressing MHC class-Irestricted TCR fail to be selected and MHC class-ll-restricted cells should shut off CD8. The stochastic model predicts that cells which randomly turn off endogenous CD4, but retain CD8 expression, could still be rescued upon corecognition of MHC class II molecules by an MHC class-lI-restricted TCR and the transgenic CD4; hence, these would differentiate into CD4+CD8 + mature T cells. Indeed such cells were found in significant numbers, together with CD4 single positive cells, in peripheral lymphoid organs in these mice. In contrast to earlier reports s, these findings support the stochastic model, although a caveat to this interpretation is that the CD4 transgene is expressed at levels tenfold higher than normal. The role of CD8 in negative selection has been studied by two groups using double transgenic mice. In both cases, transgenic mice were made which expressed (1) an MHC class-I-specific TCR, derived from peripheral T cells, which strictly depends on CD8 for antigen recognition and (2) the respective MHC class I protein, structurally modified (either by a point mutation at residue 227 or by replacing the third domain of the heavy chain (u3) by a human homologue) to abolish or drastically reduce the CD8-MHC class 1molecule interaction. Depending on the model system studied, the process of negative selection of double positive thymocytes was unaffected (in the case of an anti-H-2K b TCR transgene; B. Arnold, Heidelberg) or was abolished (in the case of an anti-H-Y, H-2Db-restricted TCR transgene, D. Littman). Taken together, these findings imply that CD8-MHC class I molecule interaction is not an essential requirement for negative selection. They also provide further evidence for a lower triggering threshold in negative selection as compared to peripheral activation6. A series of knockout mice generated in the laboratory of S. Tonegawa (Cambridge, USA), and presented by J. Lafaille, shed light on important signal and switch points during thymic T-cell differentiation. Interestingly, inactivation of the
genes encoding the TCR (x and [3 chains results in different phenotypes. In mice deficient in the TCR c~ chain, the size of the thymus and its double positive thymocyte component are normal, whereas mice deficient in the TCR [3 chain have a poorly developed thymus containing mostly double negative thymocytes and a few double positive thymocytes. Mature, single positive ~13 T cells are missing in both mutants. These distinct phenotypes imply a decisive role for the TCR [3 chain rearrangement (and, probably, for subsequent surface expression of 13 chain multimers) in the generation of double positive thymocytes; expression of TCR o~chains seems limiting only for surface expression of the TCR heterodimer and subsequent selection events. Inactivation of e~[3 TCR differentiation does not affect y8 T-cell development, and vice versa, lending support to the notion that differentiation and pool size of both T-cell lineages are independently regulated. As an encore, mice in which the peptide transporter gene HAM-1 was inactivated were briefly mentioned. These mice completely lack surface expression of MHC class I antigens; this is a remarkable result given the recent description of 'leakage pathways' for MHC class-Ipeptide loading in RMA-S cellsr, which represent the in vitro equivalent of the HAM- l knockout mice. Microenvironments The thymic microenvironment, that is, thymic stromal cells, thymocyte subsets and their mutual interactions, was the connecting theme throughout the conference. The complexity of these cellular interactions is reflected by the distribution patterns of cell adhesion molecules. The very restricted expression of some of these, for example, vascular cell adhesion molecule 1 (VCAM-1) on cortical macrophages, points to highly selective cell-cell contacts in T-cell development (N. Reza, London; J. Lannes-Vieira, Rio de Janeiro). It is commonly accepted that thymocytes receive signals from stromal cells during their passage through the thymus; for example, epithelial cells signal positive selection. Thus, targeting of T cells bearing particu-
Immunology Today
289
lar TCRs to cortical epithelial cells by hybrid antibodies in situ results in selective survival and subsequent phenotypic and functional maturation of immature thymocytes (K. Miiller, Heidelberg). It is less well appreciated that normal development of the thymic non-lymphoid architecture, in turn, depends on unperturbed T-cell development. This is well illustrated by the lack of the thymic medulla in severe combined immunodeficiency (SCID) mices and in mice deficient for p56 lea',and by the appearance of thymic medullary epithelial cells upon restoration of normal T-cell development (W. van Ewijk, Rotterdam)9. Moreover, a thymocyte to stromal cell signalling pathway was directly demonstrated by the rapid autophosphorylation of a 90 kDa membrane glycoprotein on a medullary epithelial cell line upon binding of double positive thymocytes in vitro. This 90kDa protein is part of a complex comprising three polypeptides, the in vivo function of which is unknown (E. Potworowski, Quebec). Attempts to mimic the thymic microenvironment using less complex in vitro suspension cultures have been reported by several groups. Certain cultures seem to support the process of negative selection (Y. Tanaka, London) but efforts to induce phenotypic T-cell maturation using cytokines and anti-TCR antibodies, although partially successful, have fallen short of mimicking MHC-specific positive selection of the major T-cell subsets (S. Saiagh, Lyon; M. Small, Tel Aviv; J. Park, Wiirzburg; C. Uittenbogaart, Los Angeles). Due to the disruption of the microenvironment, suspension cultures cannot reveal subtle differences among distinct compartments, such as differential cell turnover or antigen accessibility. As an example, medullary dendritic cells are far more efficient in presenting circulating antigens in situ than cortical epithelial cells, but this difference is not apparent when antigen is added to these thymic antigenpresenting cells in vitro (B. Kyewski, Heidelberg). Apart from signals mediated by cell interaction molecules, cytokines have been implicated in thymopoiesis, notably interleukin 2 (1L-2), IL-4, IL-7 and 1L-10. In situ
Vol. 13 No. 8 1992
TRENDS hybridization studies reveal a strictly controlled expression pattern of different cytokines in the fetal thymus (J. Deman, Liege). The putative functional role of a cytokine is usually assessed by addition of the recombinant cytokine or neutralizing anti-cytokine antibodies to fetal thymic organ cultures (FTOC), or by injection of antibodies in vivo. These studies have yielded conflicting resuits, particularly in the case of IL-2, but the advent of gene inactivation by homologous recombination promises to provide a clearer view of whether a given cytokine is indispensable for thymic function. Surprisingly, none of the cytokine knockout mutants studied so far, namely IL-2 (A. Schimpl, Wtirzburg) l°, IL-4 and 1L-10 loss mutants (W. Mfiller, K61n)11, show striking alterations of thymic T-cell differentiation in vivo. Although the two experimental situations (sudden withdrawal of a cytokine by an antibody versus absence of the cytokine throughout ontogeny) may not be equivalent, the mutant mice clearly show that each of these cytokines, despite being operational in wild-type mice, can be functionally replaced. The obvious next step, crossing two or more cytokine-deficient mice, may lead to a state of deprivation which cannot be compensated for. FTOC provides a unique system to study early thymopoiesis in vitro, since it is easily accessible and sensitive to experimentation by, for example, the addition of antibodies to surface receptors or soluble factors. Due to this sensitivity, however, results require careful control and interpretation. This was well illustrated by the observations that (1) the ratio of ~J3 to y8 T cells is profoundly altered by addition of certain cytokines, such as IL-7 (de Smedt, Gent); (2) culture of FTOC at the air-medium interface or submerged in culture medium produce very different results (R. Ceredig, Strasbourg), and (3) raising the percentage of oxygen in the medium favors ~!B T-cell development (Y. Watanabe, Tokyo).
ExtrathymicT-cell development Although the thymus provides a most efficient microenvironment for T-cell differentiation and selection, an extrathymic route for T-cell
maturation exists in nude mice and rats as a salvage pathway, and in euthymic rodents as a physiological pathway for certain T-cell subsets. Gut-associated intra-epithelial lymphocytes (IELs) in rat and mice deviate in phenotype (a large proportion express CD8 (~c~homodimers rather than ~x[3heterodimers) and in repertoire composition from splenic and lymph node T cells. Using a panel of anti-rat TCR V region antibodies, N. Torres-Nagel (Wfirzburg) found that gut-associated T cells displayed a 'patchy' (oligoclonal) distribution of distinct V regions with great variations among individual animals, in contrast to the precisely controlled frequencies of different V regions among T cells from spleen and lymph nodes. A well-characterized transgenic model expressing an MHC classI-restricted TCR specific for the male antigen H-Y was used to show that development of IELs does not follow the selection rules that apply to thymus-derived otJ3T cells; IELs are not positively selected in the presence of the appropriate MHC (H-2D b) and the absence of the antigen, and are not deleted in the presence of antigen and H-2D b, but they do require corecognition of the appropriate antigen-MHC complex for development (B. Rocha, Paris). These distinct recognition requirements speak strongly in favor of an extrathymic route of development of IELs 12. For the second time, the Rolduc Workshop devoted a special session to the classification of thymic epithelial cell antibodies in different species 13, and the exchange of reagents which not only included antibodies of interest but also stromal cell lines, transgenic and knockout mice.
Big strides have been made in thymus research in recent years and we now have firm knowledge of the principles, but not yet of the details, of thymic function. Will they yield as rapidly under the weight of the growing number of thymologists and new tools?
Bruno Kyewski is at the Forschungsschwerpunkt Tumor-Immunologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg I, FRG; and Thomas Hiinig is at the Institut fiir Virologie u nd Immunobiologie, Versbacher Strasse 7, 8700 Wiirzburg, FRG.
References 1 van Ewijk, W., Ron, Y., Monaco, J. et al. (1988) Cell 53,357-370 2 Zijlstra, M., Bix, M., Simister,N. et al. (1990) Nature 344, 742-746 3 Marrack, P, and Kappler,J. (1987) Science 238, 1073-1079 4 von Boehmer,H. (1986) Immunol. Today 7, 333-336 5 Robey, F.A., Fowlkes,B.J., Gordon, J.W. et al. (1991) Cell 64, 99-107 6 Pircher,H., Rohrer, K.H., Moskophidis, D. et al. (1991) Nature 351,482-486 7 Wei, M.L. and Cresswell,P. (1992) Nature 356, 443-446 8 Shores,E.W., van Ewijk, W. and Singer, A. (1991) Eur.J. Immunol. 21, 1657-1661 9 Molina, T.J., Kishihara, K., Siderovski, D.P. et al. (1992) Nature 357, 161-164 10 Schorle,H., Holtschke, T., Hfinig, T. et al. (1991) Nature 352, 621-624 11 Kfihn,R., Rajewsky,K. and Miiller, W. (1991) Science 254, 707-710 12 Rocha, B., von Boehmer,H. and Guy-Grand,D. Proc. Natl Acad. Sci. USA (in press) 13 Kampinga,J., Berges,S., Boy&R.L. et al. (1989) Thymus 13, 165-173
Debate in
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~mm.notogy Today
290
Vol 13 No. 8
•992
[
TRENDS Korsmeyer,S.J. (1991) Nature 353, 71-73 15 Strasser, A., Whittingham, S., Vaux, D.L. et al. (1991) Proc. Natl Acad. Sci.
USA 88, 8661-8665 16 McDonnell,T.J. and Korsmeyer, S.J. (1991) Nature 349, 254-256 17 Strasser, A., Harris, A.W., Bath, M.L. and Cory, S. (1990) Nature 348, 331-334 18 Blackman,M., Kappler,J. and
Marrack, P. (1990) Science 248, 1335-1341 19 Sentman, C.L., Shutter, J.R.,
Hockenbery, D., Kanagawa, O. and Korsmeyer,S.J. (1991) Cell 67, 879-888 20 Strasser,A., Harris, A.W. and Cory, S. (1991) Cell 67, 889-899 21 Guidos, C.J., Danska, J.S., Fathman, C.G. and Weissman,I.L. (1990) J. Exp. Med. 172, 835-845
22 Shortman, K., Vremec, D. and Egerton, M. (1991)J. Exp. Med. 173, 323-332 23 Ellis, H.M. and Horvitz, H.R.
(1986) Cell44, 817-829 24 Hengartner, M.O., Ellis, R.E. and Horvitz, H.R. (1992) Nature 356, 494-499 25 Yonish-Rouach,E., Resnitzky,D., Lotem, J. et al. (1991) Nature 352, 345-347
Taking the thymus to pieces Bruno Kyewski and Thomas Hiinig
Since the initiation of the thymus workshops at Rolduc in 1988, thymus research has made rapid progress, highlighted by the definitive demonstration of positive and negative selection. One may wonder whether the thymus still holds major secrets and surprises. On second thoughts, however, it becomes clear that we still lack a comprehensive understanding of the molecular mechanisms and signals that determine the developmental fate of the different T-cell lineages during intrathymic differentiation.
Selection and lineage commitment Transgenic mice have proven invaluable in deciphering the principles of T-cell repertoire selection and it is obvious that the complementary 'knockout' technique (in which a gene of choice is inactivated by homologous recombination) will become an equally powerful tool. Moreover, the combination of the two approaches offers hitherto unforeseen possibilities: the crossing of knockout mice with corresponding transgenic mice, in which the transgene itself or its expression pattern is specifically altered, allows the function of molecules to be assessed in the intact in vivo environment. This is exemplified by the crossing of major histocompatibility complex (MHC) class-II-negative mice with transgenic mice expressing MHC class II on either cortical epithelial
Complex in vitro and in vivo techniques are being combined to unlock the remainingsecrets of the thymus. In this report from a recent thymus workshop*, Bruno Kyewski and Thomas Hiinig describe the genetic manipulations aimed at clarifyingthe mechanisms of T-cell selection and lineage commitment, and the use of organ culture and immunohistology to identify the thymic microenvironments in which these events take place.
tic cells and immature thymocytes, as indicated by the MHC restriction of the CD8 ÷ T cells by the haplotype of the bone marrow donor. This result argues against the idea that epithelial cell-specific MHC-peptide complexes are essential ligands for positive selection (as proposed by the so-called peptide hypothesis) 3. The combination of knockout cells or hemopoietic antigen- mice with transgenic technology has presenting cellst, thus restoring also been applied to the question MHC class II expression in particu- of lineage commitment; that is, lar microenvironments. Not unex- whether commitment to the CD4 + pectedly, the expression of MHC and CD8 + lineages follows stochasclass lI on cortical epithelial cells tic or instructional rules. Instruction, restores the differentiation of the as originally proposed by von CD4 + T-cell lineage, which is de- Boehmer4, implies that corecognition ficient in the absence of MHC class I1 by immature double positive expression; however, expression of (CD4+CD8 +) thymocytes of thymic MHC class II on bone marrow de- MHC class-I complexes by the T-cell rived cells does not (M. Mercken- receptor (TCR) plus CD8, delivers a schlager, Strasbourg). In contrast, specific signal to shut down the CD4 infusion of MHC class-I-positive expression; and that conversely cobone marrow cells into irradiated recognition of MHC class II com132-microglobulin ([32-m)-deficient, plexes by TCR plus CD4 turns off MHC class-I-negative mice2 does CD8 expression. The stochastic restore the differentiation of func- model assumes that CD4 and CD8 tional CD8 + T cells, albeit at 5-10 expression is turned off randomly in times lower efficacy than in wild- double positive thymocytes, and that type mice (D. Raulet, Berkeley). The subsequent engagement of the TCR selection of CD8 + T cells in these and the appropriate coreceptor reschimeras is the result of direct cell- cues the thymocytes. MHC class-I-negative mice that cell interactions between hemopoielack CD8 + T cells were bred with *The Fourth Rolduc Thymus Workshop, transgenic mice expressing CD4 organized by Thomas Hfinig and Bruno under the p56 I"k promotor (D. LittKyewski was held in Rolduc, The man, San Francisco). According to the instructional model, these mice Netherlands on 26-29 May 1992.
© 1992, Hsevier Science Publishers Ltd, UK.
lmmu,ology Today 2 8 8
vol 13 No. 8 1992
TRENDS should not harbor any mature CD8 + T cells, since all immature thymocytes expressing MHC class-Irestricted TCR fail to be selected and MHC class-ll-restricted cells should shut off CD8. The stochastic model predicts that cells which randomly turn off endogenous CD4, but retain CD8 expression, could still be rescued upon corecognition of MHC class II molecules by an MHC class-lI-restricted TCR and the transgenic CD4; hence, these would differentiate into CD4+CD8 + mature T cells. Indeed such cells were found in significant numbers, together with CD4 single positive cells, in peripheral lymphoid organs in these mice. In contrast to earlier reports s, these findings support the stochastic model, although a caveat to this interpretation is that the CD4 transgene is expressed at levels tenfold higher than normal. The role of CD8 in negative selection has been studied by two groups using double transgenic mice. In both cases, transgenic mice were made which expressed (1) an MHC class-I-specific TCR, derived from peripheral T cells, which strictly depends on CD8 for antigen recognition and (2) the respective MHC class I protein, structurally modified (either by a point mutation at residue 227 or by replacing the third domain of the heavy chain (u3) by a human homologue) to abolish or drastically reduce the CD8-MHC class 1molecule interaction. Depending on the model system studied, the process of negative selection of double positive thymocytes was unaffected (in the case of an anti-H-2K b TCR transgene; B. Arnold, Heidelberg) or was abolished (in the case of an anti-H-Y, H-2Db-restricted TCR transgene, D. Littman). Taken together, these findings imply that CD8-MHC class I molecule interaction is not an essential requirement for negative selection. They also provide further evidence for a lower triggering threshold in negative selection as compared to peripheral activation6. A series of knockout mice generated in the laboratory of S. Tonegawa (Cambridge, USA), and presented by J. Lafaille, shed light on important signal and switch points during thymic T-cell differentiation. Interestingly, inactivation of the
genes encoding the TCR (x and [3 chains results in different phenotypes. In mice deficient in the TCR c~ chain, the size of the thymus and its double positive thymocyte component are normal, whereas mice deficient in the TCR [3 chain have a poorly developed thymus containing mostly double negative thymocytes and a few double positive thymocytes. Mature, single positive ~13 T cells are missing in both mutants. These distinct phenotypes imply a decisive role for the TCR [3 chain rearrangement (and, probably, for subsequent surface expression of 13 chain multimers) in the generation of double positive thymocytes; expression of TCR o~chains seems limiting only for surface expression of the TCR heterodimer and subsequent selection events. Inactivation of e~[3 TCR differentiation does not affect y8 T-cell development, and vice versa, lending support to the notion that differentiation and pool size of both T-cell lineages are independently regulated. As an encore, mice in which the peptide transporter gene HAM-1 was inactivated were briefly mentioned. These mice completely lack surface expression of MHC class I antigens; this is a remarkable result given the recent description of 'leakage pathways' for MHC class-Ipeptide loading in RMA-S cellsr, which represent the in vitro equivalent of the HAM- l knockout mice. Microenvironments The thymic microenvironment, that is, thymic stromal cells, thymocyte subsets and their mutual interactions, was the connecting theme throughout the conference. The complexity of these cellular interactions is reflected by the distribution patterns of cell adhesion molecules. The very restricted expression of some of these, for example, vascular cell adhesion molecule 1 (VCAM-1) on cortical macrophages, points to highly selective cell-cell contacts in T-cell development (N. Reza, London; J. Lannes-Vieira, Rio de Janeiro). It is commonly accepted that thymocytes receive signals from stromal cells during their passage through the thymus; for example, epithelial cells signal positive selection. Thus, targeting of T cells bearing particu-
Immunology Today
289
lar TCRs to cortical epithelial cells by hybrid antibodies in situ results in selective survival and subsequent phenotypic and functional maturation of immature thymocytes (K. Miiller, Heidelberg). It is less well appreciated that normal development of the thymic non-lymphoid architecture, in turn, depends on unperturbed T-cell development. This is well illustrated by the lack of the thymic medulla in severe combined immunodeficiency (SCID) mices and in mice deficient for p56 lea',and by the appearance of thymic medullary epithelial cells upon restoration of normal T-cell development (W. van Ewijk, Rotterdam)9. Moreover, a thymocyte to stromal cell signalling pathway was directly demonstrated by the rapid autophosphorylation of a 90 kDa membrane glycoprotein on a medullary epithelial cell line upon binding of double positive thymocytes in vitro. This 90kDa protein is part of a complex comprising three polypeptides, the in vivo function of which is unknown (E. Potworowski, Quebec). Attempts to mimic the thymic microenvironment using less complex in vitro suspension cultures have been reported by several groups. Certain cultures seem to support the process of negative selection (Y. Tanaka, London) but efforts to induce phenotypic T-cell maturation using cytokines and anti-TCR antibodies, although partially successful, have fallen short of mimicking MHC-specific positive selection of the major T-cell subsets (S. Saiagh, Lyon; M. Small, Tel Aviv; J. Park, Wiirzburg; C. Uittenbogaart, Los Angeles). Due to the disruption of the microenvironment, suspension cultures cannot reveal subtle differences among distinct compartments, such as differential cell turnover or antigen accessibility. As an example, medullary dendritic cells are far more efficient in presenting circulating antigens in situ than cortical epithelial cells, but this difference is not apparent when antigen is added to these thymic antigenpresenting cells in vitro (B. Kyewski, Heidelberg). Apart from signals mediated by cell interaction molecules, cytokines have been implicated in thymopoiesis, notably interleukin 2 (1L-2), IL-4, IL-7 and 1L-10. In situ
Vol. 13 No. 8 1992
TRENDS hybridization studies reveal a strictly controlled expression pattern of different cytokines in the fetal thymus (J. Deman, Liege). The putative functional role of a cytokine is usually assessed by addition of the recombinant cytokine or neutralizing anti-cytokine antibodies to fetal thymic organ cultures (FTOC), or by injection of antibodies in vivo. These studies have yielded conflicting resuits, particularly in the case of IL-2, but the advent of gene inactivation by homologous recombination promises to provide a clearer view of whether a given cytokine is indispensable for thymic function. Surprisingly, none of the cytokine knockout mutants studied so far, namely IL-2 (A. Schimpl, Wtirzburg) l°, IL-4 and 1L-10 loss mutants (W. Mfiller, K61n)11, show striking alterations of thymic T-cell differentiation in vivo. Although the two experimental situations (sudden withdrawal of a cytokine by an antibody versus absence of the cytokine throughout ontogeny) may not be equivalent, the mutant mice clearly show that each of these cytokines, despite being operational in wild-type mice, can be functionally replaced. The obvious next step, crossing two or more cytokine-deficient mice, may lead to a state of deprivation which cannot be compensated for. FTOC provides a unique system to study early thymopoiesis in vitro, since it is easily accessible and sensitive to experimentation by, for example, the addition of antibodies to surface receptors or soluble factors. Due to this sensitivity, however, results require careful control and interpretation. This was well illustrated by the observations that (1) the ratio of ~J3 to y8 T cells is profoundly altered by addition of certain cytokines, such as IL-7 (de Smedt, Gent); (2) culture of FTOC at the air-medium interface or submerged in culture medium produce very different results (R. Ceredig, Strasbourg), and (3) raising the percentage of oxygen in the medium favors ~!B T-cell development (Y. Watanabe, Tokyo).
ExtrathymicT-cell development Although the thymus provides a most efficient microenvironment for T-cell differentiation and selection, an extrathymic route for T-cell
maturation exists in nude mice and rats as a salvage pathway, and in euthymic rodents as a physiological pathway for certain T-cell subsets. Gut-associated intra-epithelial lymphocytes (IELs) in rat and mice deviate in phenotype (a large proportion express CD8 (~c~homodimers rather than ~x[3heterodimers) and in repertoire composition from splenic and lymph node T cells. Using a panel of anti-rat TCR V region antibodies, N. Torres-Nagel (Wfirzburg) found that gut-associated T cells displayed a 'patchy' (oligoclonal) distribution of distinct V regions with great variations among individual animals, in contrast to the precisely controlled frequencies of different V regions among T cells from spleen and lymph nodes. A well-characterized transgenic model expressing an MHC classI-restricted TCR specific for the male antigen H-Y was used to show that development of IELs does not follow the selection rules that apply to thymus-derived otJ3T cells; IELs are not positively selected in the presence of the appropriate MHC (H-2D b) and the absence of the antigen, and are not deleted in the presence of antigen and H-2D b, but they do require corecognition of the appropriate antigen-MHC complex for development (B. Rocha, Paris). These distinct recognition requirements speak strongly in favor of an extrathymic route of development of IELs 12. For the second time, the Rolduc Workshop devoted a special session to the classification of thymic epithelial cell antibodies in different species 13, and the exchange of reagents which not only included antibodies of interest but also stromal cell lines, transgenic and knockout mice.
Big strides have been made in thymus research in recent years and we now have firm knowledge of the principles, but not yet of the details, of thymic function. Will they yield as rapidly under the weight of the growing number of thymologists and new tools?
Bruno Kyewski is at the Forschungsschwerpunkt Tumor-Immunologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg I, FRG; and Thomas Hiinig is at the Institut fiir Virologie u nd Immunobiologie, Versbacher Strasse 7, 8700 Wiirzburg, FRG.
References 1 van Ewijk, W., Ron, Y., Monaco, J. et al. (1988) Cell 53,357-370 2 Zijlstra, M., Bix, M., Simister,N. et al. (1990) Nature 344, 742-746 3 Marrack, P, and Kappler,J. (1987) Science 238, 1073-1079 4 von Boehmer,H. (1986) Immunol. Today 7, 333-336 5 Robey, F.A., Fowlkes,B.J., Gordon, J.W. et al. (1991) Cell 64, 99-107 6 Pircher,H., Rohrer, K.H., Moskophidis, D. et al. (1991) Nature 351,482-486 7 Wei, M.L. and Cresswell,P. (1992) Nature 356, 443-446 8 Shores,E.W., van Ewijk, W. and Singer, A. (1991) Eur.J. Immunol. 21, 1657-1661 9 Molina, T.J., Kishihara, K., Siderovski, D.P. et al. (1992) Nature 357, 161-164 10 Schorle,H., Holtschke, T., Hfinig, T. et al. (1991) Nature 352, 621-624 11 Kfihn,R., Rajewsky,K. and Miiller, W. (1991) Science 254, 707-710 12 Rocha, B., von Boehmer,H. and Guy-Grand,D. Proc. Natl Acad. Sci. USA (in press) 13 Kampinga,J., Berges,S., Boy&R.L. et al. (1989) Thymus 13, 165-173
Debate in
ImmunologyToday If there is an issue that you would like to see aired in the Debate section of Immunology Today please contact the Editor.
~mm.notogy Today
290
Vol 13 No. 8
•992
