Intrathymic and extrathymic T cell maturation.

Immunological Rev. (1978), Vol. 42 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

Intrathymic and Extrathymic T Cell Maturation OSIAS STUTMAN

PTP: NTx: DC: MHC:

ABBREVIATIONS Postthymic precursor cell Neonatally thymectomized Diffusion chambers Major histocompatibility complex

TABLE OF CONTENTS

I. II.

Introduction Traffic A. A brief description of methods B. Hemopoietic cells migrate to thymus C. Yolk sac cells need an additional "step" before thymus migration D. Migration to thymus prefers H-2 identity E. H-2 preference of thymus migration is radiosensitive F. Time of intrathymic residence G. Emigration from the thymus H. Emigration of postthymic cells is unidirectional I. Short- and long-lived emigrant lymphocytes J. Steroid resistance and sensitivity K. Generation of competent T cells after thymus traffic L. Conclusions

Memorial Sloan-Kettering Cancer Center New York, N.Y. 10021, U.S.A.

INTRATHYMIC AND EXTRATHYMIC T CELL MATURATION

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III. Postthymic precursor (PTP) cells A. Characteristics of PTP cells in mice B. Direct demonstration that PTP cells give rise to competent T cells C. Lyt 123 PTP cells give rise both to Lyt 1 and Lyt 23 subclasses of T cells D. Conclusions IV. Development of T cells A. Models for T cell differentiation B. In vitro induction and T cells in nude mice C. Nonfimctional differentiation D. Cell-cell interaction and T cell development E. T cell development and H-2 restriction F. Summary V. Tolerance and Thymus A. Tolerance induction with thymus grafts B. Interpretation VL Epilogue

I. INTRODUCTION

The possibility of an instructional role of the thymus in imparting and directing immunological reactivity to T cells has received new impetus from the recent work af Zinkernagel et al. (1978a, b, c). In previous articles we indicated that two mechanisms seemed critical for the development and renewal of the T cell pool: 1) the traffic of hemopoietic cells to and from the thymus and 2) the evidence for further maturation of postthymic precursor (PTP) cells in extrathymic lymphoid sites (Stutman, 1970, 1972, 1975a, b, c, 1977). "Postthymic" is understood in our model of T cell development as indicating a cell that has been processed by the thymus (through traffic) and is presently in an extrathymic location in the periphery (i. e. any of the mature T subclasses as well as the PTP cells woxild qualify as "postthymic"). The concept of extrathymic maturation also implies that what is being exported by the thymus is not necessarily a competent T cell and that the PTP cells actually behave as precursors in the periphery for the immimologically functional T lineage (Stutman, 1975a, b. c. 1977). Using the terminology applied to other cell renewal systems of the hemopoietic tissues, the PTP cell would probably qualify as a "recognizable" precursor, as opposed to the restricted stem cell which are committed for one major line of differentiation (Lajtha, 1975). In the present review I will discuss these two main features of T cell development and attempt to correlate the intrathymic and

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extrathymic maturational steps for T cell development with the H-2 restriction phenomena of the effector T cells. In addition, I will discuss some of our older data on "tolerance induction with thymus grafts" (Stutman et al., 1967, 1969c) in light of the possible re-interpretation of the role of the thymus in dictating T cell reactivity (Zinkernagel et al., 1978a, b, c).

IL TRAFFIC

The term "traffic" will be used to indicate the movement of hemopoietic cells to the thymus and the subsequent export of such cells or their descendants to the periphery, as part of a maturational pathway (Ford, 1966, Stutman, 1972, 1977). By the use of cells with chromosome markers in irradiated mice, a migration of cells of marrow origin via thymus to the lymph nodes was demonstrated (Ford & Micklem, 1963, Micklem et al., 1966). This journey has a probable duration of weeks and the thymus seems to play an "instructional" role upon the migrating cells either in irradiated, thymectomized (and thymus grafted) or even normal hosts (Harris & Ford, 1963, 1964, Ford et al., 1966, Ford, 1966, Stutman, 1970, 1972, Doenhoff et al., 1970). Most of the early observations on thymus traffic dealt with the finding of cells with chromosome markers in different anatomical sites after appearance in the thymus (Ford & Micklem, 1963, Harris & Ford, 1963, 1964, Micklem et al., 1966, Ford, 1966) or as dividing cells in the periphery derived from thymus grafts and showing some quantitative changes in response to immune stimuli (Davies et al., 1966, 1971, Davies, 1969). Later studies showed that cells responding to phytohemagglutinin (PKL\) in the peripheral tissues of reconstituted mice were derived from the thymus grafts (Davies et al., 1968, 1971, Davies, 1969, Doenhoff et al., 1970). Subsequently, it was demonstrated that the cells responding to PHA in the peripheral lymphoid tissues were actually of hemopoietic origin but processed through the thymus (Stutman, 1970). Thus, the following step in the clarification of this peculiar biological traffic was the direct demonstration that precursors from adult marrow or from embryonic tissues (liver, blood, yolk sac) could become immimologically reactive T cells capable of responding to mitogens and allogeneic cells after traffic through the thymus and subsequent export to the periphery (Stutman & Good, 1969, 1971 a & b, Stutman, 1970, 1972, 1976, 1977). This last set of findings, as well as some interesting pecularities of the traffic will be discussed briefly in this section, especially in relation to the possible role of the thymus in determining the range of specificities of T cells (Zinkernagel et al., 1978a, b, c). As was indicated by Ford (1966), " . . . it seems unlikely that so specific a


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property could be entirely an experimental artifact," the property alluded to being that of natural movement of cells irom the thymus to the periphery (as well as the movement of cells into the thymus). Thus, traffic through the thymus seems to be a critical step for T cell differentiation and fits well with the theoretical (Jerne, 1971) as well as the experimental evidence (Bevan, 1977a, Zinkernagel et al., 1977, 1978a, b, c) suggestive of a role of the thymic stroma not only in the induction of T cell differentiation but also in the regulation of the spectrum of T cell reactivity. However, the complex traffic patterns described in this section are not biologically isolated events since extensive movement of either single cells or groups of cells, as well as highly selective migration and differentiation patterns are some of the major features of embryonic development in vertebrates (see Weston, 1970, and LeDourain, 1976, to mention just a few examples). The term "traffic" has also been used to describe the rapid recirculation of small mature lymphocytes (mostly T) from blood into lymphoid tissues and back to blood, which takes place within hours (Ford & Gowans, 1969). As will be seen, this second type of "traffic" is a property of mature T lymphocytes and is indeed one of the consequences of the first type of traffic (i. e. the traffic of hemopoietic cells through the thymus, Stutman, 1977). A. A Brief Description of Methods The use of CBA/H and CBA/HT6T6 mice and their F^ hybrids allows the detection of three different cell types in mitosis, characterized by their chromosome patterns: no markers (CBA/H), two small T6 chromosomes (CBA/HT6T6) and one single small T6 chromosome (the Fj hybrids, CBA/HT6). The two basic models used are: Model 1—Fifty to 60-days-old neonatally thymectomized (NTx) CBA/H grafted intraperitoneally or under the kidney capsule with a thymus lobe from newborn CBA/HT6 or CBA/H donors and injected intraperitoneally with hemopoietic cells of CBA/HT6T6 origin. The T6T6 cells are derived from adult marrow, newborn liver or spleen and embryonic liver, blood or yolk sac (from 10-17-days-old embryos, depending on cell type, see Stutman, 1976). Ten to 90 days after treatment, chromosome preparations are made of thymus grafts and lympho-hemopoietic tissues of the host, with or without mitogenic stimulation. This model was used to study traffic of hemopoietic cells to the thymus proper (Stutman, 1970, 1972, 1976 & 1977, Stutman & Good, 1969, 1971a, b). The injected cells serve only as a functional probe and are in competition with the host's own bone marrow cells. Model 2—Fifty to 60-days-old NTx CBA/H hosts are used as secondary hosts for thymus grafts from the Model 1 animals.

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Such thymus grafts, 20-30 days after injection of hemopoietic cells in the primary host, contain migrating T6T6 cells in transit (Stutman, 1970, 1972, 1976 & 1977, Stutman & Good, 1971a, b). Twenty to 90 days after thymus grafting, chromosome preparations are made of lymph nodes or thoracic duct lymphocytes, as well as other lymphoid tissues, of the secondary hosts. These cells are studied either unstimulated or after stimulation with mitogens or allogeneic cells. In this model the only source of T6T6 cells is the thymus grafts itself, and permits the study of export of such "in transit" cells from thymus to the periphery. Although there is good evidence that hemopoietic cells migrate and repopulate the tissues of irradiated hosts (Micklem et al., 1966), the proportion of migrating cells is much lower when hemopoietic cells are injected into normal hosts (Micklem et al., 1968). On the other hand, the rate of entry of hemopoietic cells into thymus grafts is quite comparable for normal or thymectomized hosts (Leuchars et al., 1967), probably as a consequence af the lymphoid depletion observed after thymus grafting (Gottesman & Jaffe, 1926, Law & Miller, 1950) and the subsequent repopulation of the graft by host cells (Metcalf & WakonigVaartaja, 1964). One advantage of our model is that by using 50-60-daysold NTx hosts, which are also deprived of PTP cells in the periphery (Stutman, 1977), both the traffic of cells to the graft as well as the export of such cells to the periphery appear to be increased, or at least, reach levels where workable numbers of mitoses can be scored. B. Hemopoietic Cells Migrate to Thymus Twenty days after cell injection, lO-25*/o of the dividing cells within the thymus grafts are of CBA/HT6T6 origin in Model 1 animals, that is, derived from the injected probe of hemopoietic precursors of embryonic or adult origin (Stutman, 1970, 1972, 1976, 1977, Stutman & Good, 1969, 1971a, b). Donor T6T6 bone marrow or liver cells can be detected within thymus graft as early as 10 days after injection (Stutman, 1976). The T6T6 dividing cells are also found in marrow (usually 3-6''/o of the unstimulated dividing cells) but are absent from lymph nodes or thoracic duct (Stutman, 1972, 1977, Stutman & Good, 1971a, b). The T6T6 cells are detected in nodes and thoracic duct only when tested 30 days or more after treatment (Stutman, 1972, 1976, 1977), which fits with their timing of appearance in lymph nodes in radiation chimeras (Micklem et al., 1966, Ford et al., 1966). The ability of hemopoietic cells to migrate to nodes or appear in thoracic duct (i. e. recirculate) was not detected in thymectomized mice that were injected with cells alone and had not received a thymus graft or that were grafted wih a thymus in a diffusion chamber (Stutman & Good, 1971a, b). In these animals the T6T6 cells were detected almost exclusively in marrow


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and spleen (3 to 9''/o of the dividing cells in unstimulated cultures). However, re-transplantation of such bone marrow containing T6T6 cells of embryonic origin showed that the embryonic cells had retained their ability to migrate to thymus (Stutman & Good, 1971a). These results indicate that a viable and accessible thymus stroma is a requirement for the production of lymph node migrating and/or recirculating T cells from the injected hemopoietic precursor cells. C. Yolk Sac Cells Need An Additional "Step" Before Thymus Migration Our initial observations did not show any differences in thymus migration among the different hemopoietic cells tested, including yolk sac, when the animals were studied at 20-30 days after cell injection (Stutman & Good, 1971a). However, when the grafts were studied earlier after cell injection, some marked differences became apparent: while adult marrow and embryonic liver cells were readily detected 10 days after injection, no yolk sac cells could be found in the thymus grafts at that period (Stutman, 1976). This time gap suggested that yolk sac cells required an additional step before being able to migrate to thymus, possibly a sojourn in a secondary hemopoietic site such as liver in the physiological situation and marrow in the Model 1 animals. To test this hypothesis we used the effects of 89Sr, a boneseeking isotope that produces a functional abrogation of marrow activity, with retention of spleen hemopoiesis (Fried et al., 1966). While marrow or 14-17-days-old embryonic liver cells could migrate to thymus in the 89Srtreated animals, yolk sac cells were incapable of such migration (studied up to 75 days after cell injection) and the spleen, which was the main site where the yolk sac cells could be found, could not replace the marrow function in providing thymic migration capacity to the injected yolk sac cells (Stutman, 1976, 1977). When spleen cells from the 89Sr-treated animals containing T6T6 yolk sac cells were re-transplanted to Model 1 animals, T6T6 dividing cells could be detected in the thymus 20 days after cell injection (Stutman, unpublished). We also showed that in the 89Sr-treated animals, competent T cells capable of responding to PHA and recirculating in thoracic duct can be found in the animals injected with marrow or liver but not with yolk sac (Stutman, 1976). On the other hand, yolk sac cells can indeed become competent T cells after thymus traffic, provided that there is an intact marrow in the host (Stutman, 1976, 1977) or when transferred as intrathymic "in transit" cells (Stutman & Good, 1971a). D. Migration to Thymus Prefers H-2 Identity The migration of hemopoietic precursors to thymus grafts in animals of a modified Model 1 was highly sensitive to histocompatibility differences

STUTMAN

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TABLE I Syngeneic Preference of Migration of Hemopoietic Cells to Thymus Grafts and Abrogation of Preference by Thymus Irradiation Thymus'' ] . CBA/HT6T6 2. CBA/HT6T6 3. CBA/HT6T6 4. CBA/HT6T6

HT6T6 HT6T6 HT6T6 HT6T6

5. CBA/HT6T6 6. CBA/HT6T6

HT6T6 (450R) HT6T6 (750R)

Donor cellse

Percent donor-host metaphasesd Unstimulated Phytohemagglutinin Donor Host Donor Host

CBA/H 20 CBA/J 9 C3H/He 5 (CBA/H X 2 C57BL/6) Fi C3H/He 2 C3H/He 15

80 91 95 98

43 18 10 9

57 82 90 91

98 85

17 33

83 67

Sixty-days-old neonatally thymectomized hosts. Thymus from 5-15-days-old donor, implanted intraperitoneally at 60 days of age. In groups 5 and 6, thymus was irradiated in vitro with 450R and 750R, respectively. Thymus grafting and retrieval as in Stutman et al., 1972. Cells from 15-17-days-old embryonic livers of the indicated strains, injected intraperitoneally (5 X10' / mouse), 3 days after thymus grafting. Percent metaphases scored per thymus, 30 days after cell injection. "Donor" indicates the normal chromosome type of the injected liver cells; "host" indicates the T6T6 chromosome type of the host and thymus graft. Pooled results from three animals per group, 225 to 670 metaphases per mouse scored.

between cells and thymus grafts, even weak non-H-2 differences (Stutman & Good, 1969). When 45-days-old NTx C3H mice were grafted with CBA/H, C3H or Fj hybrid thymuses, and injected with CBA/HT6T6 embryonic liver cells, the percent T6T6 dividing in the thymus graft 15 days after cell injection was approximately IS^Io for the syngeneic combination (i. e. CBA/H grafts and CBA/HT6T6 cells) versus O-50/o when thymus graft and cells were incompatible (Stutman & Good, 1969). Table I shows a repeat of these experiments using a reversed Model 1: CBA/HT6T6 hosts and thymus grafts injected with cells that have normal chromosomes. While 2O»/o of tlie dividing cells and 43"/o of the Con A stimulated cells from the thymus graft are of the injected type, in the fully syngeneic combination, only 2-9''/o (which increased to 9-180/0 in the Con A-stimulated cells) are of the injected type in the allogeneic combinations. These experiments indicate that in the presence of competing syngeneic hemopoietic precursors from the host's own marrow (indicated in Table I as "host"), the allogeneic cells are at a disadvantage in repopulating the thymus graft in this experimental model. This "restriction" contrasts with most of the published literature of thymic repopulation in radiation chimeras which shows that


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mouse thymus can be repopulated by allogeneic cells and even by xenogeneic (rat) cells (Gengozian et al., 1957, Ford et al., 1957, Roller et al., 1961). However, it is in agreement with data on the fate of allogeneic thymus grafts in thymectomized hosts, which show poor repopulation (Dukor et al., 1965, Miller, 1966, Stutman et al., 1967). E. H-2 Preference of Thymus Migration is Radiosensitive The discrepancy indicated in the previous section between the irradiation models and those in which the thymus is not irradiated (the former showing minimal H-2 restriction for repopulation by hemopoietic cells while the latter shows a relative degree of H-2 restriction favoring syngeneic combinations) could be explained by a radiosensitive "trap" in the thymus which favors the proliferation of syngeneic hemopoietic precursors. Table I (groups 5 and 6) shows the effect of pre-irradiation of the thymus graft with 450 or 750R in their capacity to accept allogeneic (albeit H-2 identical) hemopoietic migrants. It is apparent that while the thymus irradiated with 450R behaves like the non-irradiated thymus (compare group 5 with group 3), the 750Rirradiated thymus allowed a proportion of the allogeneic hemopoietic cells to divide, giving values comparable to the syngeneic combination (compare group 6 with group 1). This abrogation of H-2 preference by irradiation of the thymus is probably important in explaining the successful functional chimeras obtained by whole body irradiation (von Boehmer et al., 1975, Zinkernagel, 1976, Bevan, 1977a) or with thymus grafts irradiated with 850 -900R implanted in thymectomized irradiated recipients (Zinkernagel et al., 1978a, b, c). F. Time of Intrathymic Residence By measuring the timing of the dilution of migrant T6T6 cells by host hemopoietic cells in the thymus, an estimate of the time of intrathymic residence can be made (Stutman & Good, 1969, Stutman, 1977). Our results indicate that most of the injected cells that migrated to thymus have disappeared by 45-50 days after cell injection, and are replaced by migrants of host origin (Stutman, 1977). However, a small population of dividing cells of donor origin remained for as long as 180 days, and can be associated with resident population of steroid resistant cells in the thymus (Stutman, 1977). G. Emigration from the Thymus When thymus grafts containing migrating hemopoietic cells of CBA/HT6T6 origin (including yolk sac cells) were transplanted to secondary NTx CBA/H hosts (Model 2), lO-SO^/o of the dividing cells in lymph nodes and thoracic duct were of T6T6 origin, 30 days after intraperitoneal grafting (Stutman, Immunological Rev. (1978), Vol. 42

lo

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STUTMAN

1970, 1972, 1977, Stutman & Good, 1971a, b). In addition 5-lO»/o of the dividing cells in spleen and Peyer's patches were of T6T6 origin, and in about half of the animals, T6T6 cells were also fotmd in bone marrow (Stutman & Good, 1971a, b). Using isotopic labeling procedures, most evidence indicates that there is export of thymus-derived cells to the periphery and such export is higher during early perinatal stages (Joel et al., 1972, Bryant et al., 1975, Laiusse et al., 1976); however, a substantial number of the labeled thymocytes die within the thymus or have a very short life in the periphery. Our data, although not quantitative, suggest that a proportion of the dividing cells, especially in nodes and thoracic duct, are derived from cells processed and exported by the thymus. These cells could be generated by the expansion in the periphery of a relatively small pool of postthymic precursors (PTP, see Section III. B) and/or by the daily constant output of relatively small numbers of short-lived lymphocytes which accumulate in the periphery. The extent of intrathymic cell death compared to emigration is still an unresolved question, as well as the magnitude of export at different ages (Joel et al., 1972, 1974, Bryant, 1972, Bryant et aL, 1975, Shortman et al., 1975, Laiusse et al., 1976). Based on labeling indices as well as generation times, it has been calculated that the mouse thymus produces approximately 1X106 thymocytes per mg per day (Joel et al., 1974). The daily replacement values for mice (i. e. the number of times/day that the thymic migrants can replace the circulating blood lymphocyte pool) has been given as 5 (Bryant, 1972) which compares with the values defined for other species (Joel et 1., 1974). In addition, our own data presented in Section III B & C indicate that most of the T cells in 50-days-old neonatally thymectomized mice injected with relatively low numbers of PTP cells (10«-10^ cells) plus a thymus graft in a diffusion chamber are derived from the expansion of the injected cells (Stutman, 1975d, 1977). H. Emigration of Postthymic Cells is Unidirectional To study whether cells exported by thymus can return to the thymus or if traffic is unidirectional. Model 2 animals were modified and either had a normal CBA/H thymus grafted before the thymus from Model 1 animals was implanted (Model 2. A in Table II) or were grafted simultaneously with a CHA/H normal thymus and a thymus from Model 1 donors (Model 2. B in Table II). In both instances, no T6T6 cells could be detected in the second CBA/H thymus, while they could be detected in nodes and thoracic duct, i. e. were exported to the periphery but did not return to the thymus (Table n , also Stutman, 1972, 1977).


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TABLE II Cells Exported by the Thymus Do Not Return to Thymus Unless the Thymus is Irradiated Tissued Studied^ • •

CBA/HT6T6 metaphases per total metaphasesb Model 2.A Model 2.B Model 2.C

CBA/H thymus graft 0/976 CBA/H thymus graft (PHA) 0/699 Lymph nodes 98/899 (llVo) Lymph nodes (PHA) 168/902 (19»/o)

0/1233 0/873 76/801 ( 9»/o) 159/863 (I8V0)

49/630 ( 8°/o) 68/510 (130/0) 73/752 (lOVo) 83/540 (15»/o)

» Results from four experiments (5-9 animals) 30 days after intraperitoneal transplantation of the thymus from a Model 1 animal (injected with hemopoietic T6T6 cells, see section II. a). PHA indicates the results after incubation of the cells with phytohemagglutinin. "Thymus graft" indicates the second thymus, derived from a normal CBA/H, implanted under the kidney capsule, either 20 days before intraperitoneal implantation of the Model 1 thymus (Model 2.A) or simultaneously with the Model 1 thymus (Model 2.B). Model 2.C consisted of a CBA/H thymus irradiated in vitro with 750R and implanted under the kidney capsule simultaneously with the intraperitoneal implantation of the Model 1 thymus. It should be noted that the only source of T6T6 cells in all these experiments is the Model 1 thymus proper. b Pooled results. Numbers in parentheses indicate percent T6T6 metaphases scored.

Two exceptions to the unidirectional traffic should be mentioned. The first one is the capacity of leukemic T cells to return to the thymus after injection as a cell suspension (Stutman, 1972). The second exception is presented as Model 2. C in Table II. Using a modified Model 2. B system (both a Model 1 thymus and a normal CBA/H thymus grafted simultaneously intraperitoneally and under the kidney capsule, respectively) it can be seen that a significant population of T6T6 cells can be detected in the second thymus, provided that such thymus was previouly irradiated with 750R. Thus, irradiation of the thymus appears to alter some of the selectivity (see Section II C & D) as well as the characteristics of thymus traffic. The present observation may also explain the description of a subpopulation of thymocytes that can come back to the thymus in irradiated hosts (Kadish & Basch, 1977). I. Short- and Long-lived Emigrant Lymphocytes Using Model 2 animals and subsequent injections of 3H-thymidine in schedules that will preferentially label short- and long-lived cells (Everett et al., 1964), we tried to determine if both types of lymphocytes were derived from hemopoietic precursors after thymic traffic and subsequent export (Stutman, 1977). The association of radioactive grains with T6T6 chromosome markers

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in lymph node lymphocytes would determine, depending on the laheling schedules, if short- and/or long-lived cells were generated after traffic. Both cell types were generated, although most of the cells hearing hoth markers were short-lived (i. e. had divided at least once within a 6-day period). However, as was indicated in Section II. F, thymus emigrants can he detected in lymph nodes and thoracic duct of Model 2 recipients for long periods of time, even 600-700 days after thymus transfer (Stutman, 1977), suggesting that long-lived recirculating lymphocytes are also generated after thymus traffic. One alternative possihility is that the short-lived population may give rise in the periphery to the long-lived recirculating population. And indeed, as will he discussed in Section III, the PTP cells are a rapidly dividing population (Stutman, 1975, 1977). By temporary exposure of NTx mice to a thymus graft, we have heen ahle to determine that a relatively long-lived pool of competent cells is developed, which in the ahsence of thymus begins to decUne at approximately 6 months of age (Stutman et al., 1972). This pool is probably produced by traffic as well as expansion of PTP cells in the periphery (see Section III). These results fit well with the data on replacement kinetics of T cells determined by the recovery from tolerance induction to alloantigens (Leech & Mitchison, 1976). J. Steroid Resistance and Sensitivity In previous studies we showed that the PTP cell was sensitive to high doses of corticosteroids (Stutman, 1977, see also Section III). In the traffic model we observed that: 1) the population of corticosteroid resistant thymocytes within thymus grafts was also derived from hemopoietic immigrants and 2) that when thymus grafts of Model 1 animals pre-treated with high doses of hydrocortisone were transferred to secondary thymectomized hosts (Model 2), there was no evidence of export of the T6T6 corticosteroid resistant intrathymic population (Stutman, 1977). By stimulation with PHA, it was possible to show that this resident population was long-lasting and could still be detected 180 days after grafting when most of the donor T6T6 cells in the unstimulated thymus have been diluted by host-derived cells (Stutman, 1977). It was also observed that no detectable exported cells derived from the steroid-treated thymus grafts in Model 2 animals could be found in nodes or thoracic duct at any thne from 30 to 180 days after grafthig, either in imstimvilated or PHA-stimulated culture, indicating that the steroid resistant cells do not belong to the pool that is exported (Stutman, 1977). Experiments with in situ label of the thymic cortex indicate that the steroid-resistant medullary thymocytes appear to be derived from cortical thymocytes (Weissman, 1972, Weissman et al., 1975). On the other hand.


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mature T cells in the periphery show both sensitivity and resistance to high dosages of steroids (Claman & Moorhead, 1972). Our studies indicate the intrathytnic steroid-resistant poptilation of thymocytes is also derived from hemopoietic precursors, as are probably all lymphoid cells in the thymus (Stutman, 1977). The two other conclusions from our work are: 1) that the bulk of the PTP cells exported by the thymus are steroid-sensitive cells, which apparently can produce a subpopulation of steroid-resistant cells in the periphery (Stutman, 1977) and 2) that there is no detectable evidence for export of the intrathymic steroid resistant population to the periphery (Stutman, 1977) supporting the observations by Elliott (Elliott et aL, 1971, Elliott, 1973, 1977). The presence of the mattire T-like lymphocytes within the thymus has been interpreted as representing the compartment of cells ready for export and as the probable source of the peripheral T lymphocytes (Raff, 1971, Leckband & Boyse, 1971, Dyminsky & Smith, 1974, Shortman et al., 197). However, Elliott et al (1971) showed that the cortisone resistant PHAresponsive population in thymus grafts was of donor origin for at least 28 days after transplantation, i. e. was not renewed during that period by host cells. In subsequent works it was demonstrated that the "mature" subpopulation is not tmdergoing rapid replacement for relatively long periods of time (Elliott, 1973, 1977) nor any detectable export to the periphery for up to 7 months after grafting (Stutman, 1977). When the steroid-resistant thymocytes were made into cell suspension and injected intravenously into irradiated hosts, the cells appeared to circulate in blood but were unable to penetrate to the lymphoid organs and were undetectable in nodes and spleen (Elliott, 1977). Thus, it appears that the steroid-resistant mature T cells in thymus represent a special resident population of undetermined function (see Section FV A). In irradiated animals (Sharp & Thomas, 1975, Kadish & Basch, 1975) and in animals with severe protein deficiency (Bell & Hazel, 1977), there is an intrathytnic pool of cells resistant to such maneuvers which is capable of partial thymic repopulation. There is no evidence that the pool of competent T cells is derived from such cells, and in both instances, cells of hemopoietic origin seem to mediate the complete repopulation of the thymus as well as the reconstitution of the peripheral pool of T cells. It is apparent that these types of intrathymic resident populations deserve further study, especially in their possible role as regulators of T cells specificity, proposed by the theories of positive or negative selection of reactive T cells within thymus (Bumet, 1962, Jeme, 1971, Langman, 1978, Schwartz, 1978, Blanden & Ada, 1978).

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K. Generation of Competent T Cells After Thymus Traffic The final test to prove that this peculiar traffic implies a true differentiative process towards immunological competence was to show that the immimologically competent T cells in nodes, thoracic duct or blood of Model 2 animals were indeed descendants of the T6T6 hemopoietic precursors in transit within the grafted thymuses. Our results indicated that a substantial fraction of the responding cells (capable of responding in vitro to PHA, Con A or allogeneic cells in mixed lymphocyte cultures, using C57BL/6 cells as stimulators) were of the T6T6 type, thus, derived from the embryonic or adult hemopoietic precursors after thymus traffic and export (Stutman, 1970, 1972, 1976, 1977, Stutman & Good, 1971a, b). Table III shows that a proportion of T6T6 cells exported from the thymus graft, ranging from 10 to 27"/o, is responding in nodes and thoracic duct of the treated animals to PHA and allogeneic cells, while no detectable T6T6 mitoses can be observed responding to LPS, a B cell mitogen. As was indicated previously, our evaluations of these results were purely qualitative, without any attempts at quantification. We were satisfied that progeny of the T6T6 cells in transit within the thymus graft were detected in the periphery as a recirculating TABLE III Chromosome Analysis of Mitogenic Responses to PHA, Allogeneic Cells and LPS on Lymph Nodes and Thoracic Duct Cells from Neonatally Thymectmized CBA/H Secondary Hosts Grafted with Thymus Grafts of CBA/H Origin Containing in Transit CBA/HT6T6 of Various Hemopoietic Sources Origin of T6T6 cells in thymus graft* Yolk Sac (10-13 Yolk Sac (10-13 Embryonic liver Embryonic liver Bone marrow Bone marrow

days) days) (13-19 days) (13-19 days)

Tissue testedb LN TD LN TD LN TD

(4) (4) (9) (6) (5) (5)

Number of T6T6 Metaphases per total": MLC LPS PHA 39/233 (16«/o) 63/289 (22«/o) 664/2466 (27»/o) 198/893 (22»/o) 120/890 (14»/o) 163/885 (18»/o)

36/340 (lOVo) 76/489 (15°/o) 90/899 (ll»/o) 62/578 (ll»/o) 77/569 (13»/o) 88/620 (14»/o)

0/483 0/450 0/973 0/830 0/1002 0/889

Model 1 thymuses (see Section IL a) were used for grafting 60-days-old neonatally thymectomized CBA/H mice. In parentheses the embryonic age in days. Thymuses were implanted intraperitoneally. LN: pooled peripheral lymph nodes; TD: thoracic duct cells from 26-h drainages. Numbers in parentheses indicate number of animals per group. All animals were tested 60 days after thymus grafting. Number of T6T6 metaphases per total scored, pooled results. PHA: response to phytohemagglutinin measured at 72 h of culture; MLC: mixed lymphocyte cultures using C57BL/6 cells as stimulators in 3- and 4-days cultures; LPS: response to lypopolysaccharide from Salmonella Typhosa (Difco Labs, Detroit, Mich.) at 20 ^g concentration and 24-48 h after stimulation.


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and immunologically competent T cell, and considered it as sufficient proof that the thymus traffic process is indeed a maturational pathway involved in the generation of competent T cells in the periphery (Stutman, 1972, 1977). L. Conclusions The following conclusions can be drawn from the information discussed in this section: 1. Cells in hemopoietic tissues af adults and embryos migrate to thymus grafts. 2. Yolk sac hemopoietic cells (but not hemopoietic cells from adult marrow or embryonic liver) need an additional step before thymus migration, apparently a sojourn in a secondary hemopoietic organ such as liver in the embryo and marrow in the adult experimental model used. 3. Migration of hemopoietic cells to thymus prefers H-2 identity with thymus. 4. H-2 preference of thymus migration (or of proliferation within thymus) is regulated by the thymus and is radiosensitive (can be abrogated by 750R). 5. The time of intrathymic residence of the hemopoietic immigrants is difficult to determine, but by 45 days after entry, most of the injected cells have been diluted by cells derived from the host. 6. A proportion of cells emigrate from the thymus and appear in lymph nodes and thoracic duct lymph. 7. Emigration of these postthymic cells is unidirectional to nodes and thoracic duct but not back into thymus. However, irradiated thymus grafts (750R) permit the return of postthymic exported cells. 8. The exported postthymic cells are mostly short-lived lymphocytes, although some long-lived lymphocytes of exported origin are also generated (there is the possibility that long-lived lymphocytes are derived in the periphery from short-lived postthytnic precursors). 9. The steroid-resistant intrathymic pool of cells is also derived from hemopoietic precursors. 10. The steroid-resistant intrathymic pool is a resident population which is not exported to the periphery. 11. Competent T cells in nodes and thoracic duct capable of responding to mitogens and allogeneic cdls (but not to LPS) are produced from thymus-processed exported postthymic cells, derived from the original hemopoietic precursors of embryonic or adult origin. 12. This mattirational pathway requires a viable and accessible thymic stroma and cannot be replaced by thymus within cell impermeable dif-

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fusion chambers, especially when truly prethymic hemopoietic precursors such as yolk sac or early embryonic liver are used (prethymic is used here in the sense of hemopoietic tissues that have appeared in ontogeny before the development of the thymus). 13. Thus, the idea of an "instructional" role of the intrathymic environment had important heuristic value and progressed from: a) the demonstration that the instruction allowed migration of the exported cells to nodes (Harris & Ford, 1964), to b) the demonstration that the emigrants processed by the thymus were competent T cells (Stutman, 1970, Stutman & Good, 1971a, b) and finally to c) the demonstration that specificity of H-2 restriction may actually be the consequence of the thymic environment in which the T cells differentiate from their precursors (Zinkemagel et al., 1978a, b, c).

in. POSTTHYMIC PRECURSOR CELLS

During our studies on immunological restoration of neonatally thymectomized mice, especially with thymus in diffusion chambers (DC), we observed that the animals become refractory when treatment was delayed for 40 days or more after thymectomy (Stutman et al., 1967, 1969a). We interpreted these results as indicative of a population of cells in the peripheral Iymphoid tissues of these mice which declines in the absence of a thymus, was generated probably in late embryonation, before thymectomy was performed, and was sensitive to thymic humoral influence (Stutman et al., 1969 a, b). We termed this population "postthymic" due to its thymus dependency for renewal and the probability that it represented a pool of cells which was processed by the thymus (Stutman et al., 1969a, b). It should be noted that the refractoriness observed with delay of treatment after thymectomy was found even when free thymus grafts were used, suggesting that the peripheral effects of the thymus on this postthymic compartment, as opposed to the intrathymic and import-export functions, were quite critical for the rapid achievement of immimological restoration (Stutman et al., 1967, 1969a, b). When we studied the effects of various cell supplements in augmenting the capacity of thymus in DC to restore immune functions in 50-days-old neonatally thymectomized mice, we observed that: 1) Iymphoid and hemopoietic cells from adult or newbom animals, at cell dosages in which they were ineffective by themselves, acted synergically with thymus in DC in restoration of immune functions (Stutman et al., 1969a, b, 1970a, b); 2) the actual content of immunologically competent cells in the different cell supplements was irrelevant since tissues with low or absent competent T cells.


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such as newborn liver or spleen, were as effective as adult spleen in cooperative restoration (Stutman et al., 1970 a, b) and 3) embryonic hemopoietic cells such as yolk sac or liver, especially if obtained from embryos before day 14-15 of gestation, cooperated only with free thymus grafts and not with thymus in DC in restoring immune functions (Stutman et al., 1969b, 1970b). It should be noted that those same embryonic tissues contain "prethymic" precursors that can generate competent T cells via thymus traffic (see Section II). In a strict sense, the only true prethymic cells would be those in yolk sac (and perhaps in 11-days-old embryonic liver) which appear before lymphoid cells can be detected in thymus, and even before the development of the epithelial thymus (see Stutman, 1977, for details on the timing of these events). However, "prethymic" was used as an operational term, meaning that the cells have not yet been processed by the thymus (Stutman et al., 1970a, b). Based on these findings, two types of precursor cells for the T lineage could be defined: 1) Prethymic precursors which require contact with the thymic stroma through traffic for further differentiation and 2) Postthymic precursors (PTP) which are immunologically incompetent thymus-processed precursor cells exported from the thymus to the periphery (Stutman et a., 1969b, 1970a, b, Stutman 1975a, b, c). It is difficult to determine the magnitude of these two differentiation steps (i. e. intrathymic via traffic and exrathymic via PTP cells), especially the differences in early versus adult life (Stutman, 1977). However, both mechanisms are essential for maintaining homeostasis of T cell production and carmot be separated. As a matter of fact, PTP cells in the periphery are derived from prethymic hemopoietic precursors processed by the thymus after migration (Stutman, 1975, 1977). By temporary exposure to thymic function early in life, neonatally thymectomized mice can generate a pool of immunologically competent T cells within a relatively short period after exposure, but this pool of cells is eventually depleted with time in absence of the thymus, i. e. is incapable of self-renewal (Stutman et al., 1972). Based on this data we feel that most of the differentiation of precursor cells into competent T cells in adult life, occurs in the periphery from the PTP pool under thymic influence. These interpretations of intrathymic as well as extrathymic steps for T cell development agree well with the proposed role of the thymic stroma in imparting the range of reactivity for T cells, as well as the role of the peripheral "lymphoreticular tissues" in further refining such reactivity (Zinkernagel et al., 1978a, b, c).

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A. Characteristics of FTP cells in Mice Thus, the postthymic pool in the periphery includes PTP as well as the different functional subsets of T cells (Stutman, 1977). The fact that tissues such as marrow and newborn spleen have relatively low levels of competent T cells, prompted studies to characterize the PTP cells in the peripheral TABLE IV Some Characteristics of Postthymic Precursor (PTP) Cells in the Mouse, Compared to Prethymic Precursors and Competent T cells Biological characteristics

Prethymic

marrow liver yolk sac, liver, blood 9 (embryo) Time of appearance (days) Yes Migration to thymus No Recirculation spleen-seeking Trafic patterns No Immunological competence^ Restoration of Tx animals: Alone No Yes with thymus graft No with thymus in DC None? Effect of neonatal thymectomy None Effect of adult thymectomy Yes Presence in nude mice None Effect of short-term ALS None Sensitivity to steroids No? 3H-thymidine suicide ? Life span, probable Low Density in BSA gradients Sedimentation ratesb Adult spleen 5.0 Bone marrow 4.6 Newborn spleen 4.8 7.7 & 9.5 Embryonic liver No Adherence to nylon wool No Surface markers: Thy. 1 TL No No Lyt 1, 2, 3 Fc No

Tissue distribution: Adult Newborn Embryo

PTP

T cell

spleen, marrow lymphoid tissues spleen, liver liver 15 (embryo) No No spleen-seeking No No Yes Yes Depletion Depletion No None Depletion Yes Short Low

1-15 No Yes node-seeking Yes Yes Yes Yes Depletion None, mostly No Depletion Mostly none

5.9 5.4 5.8 5.9 Yes Yes, high No 123+ No

3.7 3.6 3.5 No, mostly Yes, low No 1, 23, 123+ No, usually

No Short & long High

Measured as response to mitogens (PHA, Con A), graft-versus-host reactivity, capacity to react in mixed leukocyte cultures (MLC) against major histocompatibility differences, capacity to generate cytotoxic cells after MLC and helper activity (primary antibody response to sheep red cells). See Stutman, 1975a, b, c, 1977. Velocity sedimentation at unit gravity in fetal calf serum or ficoll gradients expressed in mm/h.


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tissues of mice (Stutman, 1975a, b, c, 1977, Stutman & Shen, 1977). Although we still have not been able to isolate pure PTP, considerable enrichment can be obtained by separation procedures such as velocity sedimentation at unit gravity permitting some of the studies indicated in Sections III b & c. Table IV presents a summary of the properties of the PTP cells studied so far, and compares such properties with the prethymic precursor (i. e. the cell in hemopoietic tissues that can migrate to the thymus) and the compartment of immunologically competent T cells (which for the sake of space will be considered as a single entity). A rather extensive discussion of most of the properties of the PTP cells described in Table IV can be found in Stutman (1977) and will not be repeated here. However, three points deserve comment: 1) the PTP cell is sensitive to corticosteroids, thus indicating that what the thymus exports is not necessarily a steroid-resistant cell and that steroid-resistance may be acquired after maturation in the periphery (see also Section II, J); 2) the PTP cell is retained by nylon wool columns (Stutman, 1975a), a property recently reported for several classes of T cells TABLE V PTP Cells in Newborn Spleen Have the Lyt 123 Phenotype

Treatment of cells a None C control a Lyt 1.1 + a Lyt 2.1 -ta Lyt 3.2 + a Lyt 2.2 +

C C C C

Restoration of immune functions'* With thymus in DC With empty DC 8/12 (67»/o) 7/10 (7O«/o) 0/10 0/11 0/8 6/9 (67»/o)

0/11 0/11 0/6 0/6 0/6 0/6

« This work was done in collaboration with F. W. Shen (see Stutman & Shen, 1977). Sixty-days-old neonatally thymectomized CBA/H mice were injected intraperitoneally with 2X10' viable newborn spleen cells and implanted IP with a CBA/H thymus within a DC or an empty DC (for details on preparation of DC see Stutman, 1975a). The Lyt phenotype of CBA/H mice is 1.1, 2.1 and 3.2. The percent of spleen cells lysed by Lyt 1 or Lyt 2, 3 ranged from 27 to 35, which is comparable with the values obtained by Cantor & Boyse (1975). Due to the fact that the newborn lymphocytes in spleen appear to be mostly Ly 123, experiments with mixtures of Lyt 1 and Lyt 23 cells could not be performed. The nonappropriate Lyt 2.2 serum was used as control. •> Restoration was measured 30 days after cell injection in peripheral lymph nodes and in some animals with thoracic duct cells obtained after 26-h drainages. Animals were considered restored when they showed normal PHA and mixed lymphocyte responses. In some animals, cells were also tested for capacity to produce graftversus-host reactions when injected into 8-days-old (CBA/H X C57BL/6) Fj hybrids.

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with regulatory functions (Cohen & Livnat, 1976, Tada et al., 1977, Cantor et al., 1978, R. K. Gershon, personal communication) and 3) the PTP belongs to the group of postthymic cells which express the Lyt 123 phenotype (i. e. are Lyt 1+, Lyt 2,3+, see Cantor & Boyse, 1977, and Boyse et al., 1977, for nomenclature), which represents an important and poorly studied compartment of the peripheral T cell pool (5O''/o of adult spleen cells and lOO^/o of 1-week-old spleen lymphocytes. Cantor & Boyse, 1975). A compartment that also includes different T cells with complex regulatory functions (Tada et al, 1977, Cantor et al., 1968). Table V shows some of our studies on the Lyt phenotype of the PTP cell (Stutman & Shen, 1977). B. Direct Demonstration that PTP cells Give Rise to Competent T Cells The basic model used for these studies consists of 50-60-days-old NTx CBA/H or other strains, which are injected intraperitoneally with syngeneic cells from either newborn spleen or adult marrow in most of the experiments (in some instances the cells have been treated in vitro with alloantisera and C, etc., or are derived from donors treated in vivo by different procedures) and subsequently are implanted intraperitoneally with a thymus in a DC (control chambers were empty or contained a spleen fragment). At different times after this treatment, usually 30 days later, the lymph nodes or thoracic duct cells are tested for immune functions. When purified PTP cells obtained by velocity sedimentation from newborn CBA/HT6T6 spleens were injected into 60-days-oId neonatally thymectomized mice and then implanted with a CBA/H thymus in DC, we could show that the PTP cells differentiated into competent T cells in the peripheral tissues of the host (Stutman, 1975b). When tested 30 days after treatment, 86-98''/o of the cells in nodes and thoracic duct which were responding to PHA or allogeneic cells had the T6T6 marker of the PTP cell (Stutman, 1975b). Thus, these results indicate that as a consequence of a maturation event, probably regulated by humoral thymic factors, and which is time dependent (the earliest detectable responding population was observed at 7-8 days after teatment), cells with the chromosome marker of the injected PTP showed recirculating capacities and could respond to PHA and allogenetic cells. No such responding nor recirculating cells could be detected in control animals receiving either an empty DC or a DC containing spleen (Stutman, 1975b, c). C. Lyt 123 PTP cells Give Rise Both to Lyt 1 and Lyt 23 Subclasses of T Cells Table VI shows results indicating that by the use of relatively pure PTP cells from newborn CBA/HT6T6 spleen, it can be shown that 40 days after


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TABLE VI PTP Lyt 123 Cells Give Rise Both To Lytl and Lyt23 Subsets of T Cells

Treatment of cells* 1. 2. 3. 4. 5.

Untreated 1 (Lyt 1, 23, 123) C Contro I (Lyt 1, 23, 123) a Lyt 1.1 + C (Lyt 2) a Lyt 2 1 + C (Lyt 1) a Lyt 2.2 + C (Lyt 1, 23, 123)

Percent T6T6 metaphases'' Spleen Lymph nodes ]PHA MLC PHA MLC 68 66 16 33 69

(560) (420) (666) (690) (445)

ND 89 (462) 20 (689) 63 (502) 85 (599)

86 88 23 45 88

(430) (399) (430) (492) (386)

ND 95 (396) 15 (491) 52 (309) 88 (302)

"

Spleen and lymph node cells were obtained from CBA/H animals that were neonatally thymectomized and treated at 60 days of age with the intraperitoneal injection of 5X10' PTP cells separated by velocity sedimentation at unit gravity from CBA/HT6T6 newborn spleens and with a CBA/H thymus enclosed in a DC. The spleen and lymph node cells were obtained from these animals 40 days after treatment. Before testing for responses to PHA and MLC, cells were treated with C, appropriate Lyt antisera and an inappropriate Lyt control. The dead cells were removed by passage on a ficoU gradient. b Percent T6T6 metaphases per total number of metaphases scored (in parentheses). Results are pooled from three animals per point. PHA measured after 72 h in culture; MLC using C57BL/6 cells as stimulators in 3-4-days cultures.

treatment (the injection of 5X108 PTP cells plus grafting of a CBA/H thymus in DC to 60-days-old NTx CBA/H hosts) a substantial proportion of the cells in lymph nodes and spleen responding to PHA or allogeneic cells belongs to either the Lyt 23 (group 3, Table VI) or to the Lyt 1 (group 4, Table VI) subclass of mature T cells. It has been shown that both subclasses can respond to mitogens (Cantor & Boyse, 1977) and that both subclasses respond to allogeneic cells, albeit recognizing different regions of the major histocompatibility complex (MHC) (Nagy et al., 1976). On the other hand, it is also well established that both subclasses, once established, behave as if they belong to independent differentiative pathways (Huber et al., 1976). The present data show that they may have a common Lyt 123 precursor. One of the problems with the Lyt serological analysis of T cells is that, although clean preparations of either Lyt 1 or Lyt 23 cells can be obtained, in each case, the Lyt 123 cells are also eliminated and estimates of this compartment have to be made by arithmetical accounting of the serological effects with both sets of Lyt reagents (Cantor & Boyse, 1975, 1977). Thus, from the results presented in Table VI, it can be assumed that Lyt 123 cells are also generated from the PTP cell. When the proportion of dividing T cells in groups 3 and 4 is added (i. e. the response of the Lyt 23 and Lyt 1

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populations) there is still a 20-300/0 unaccounted population, when compared to the controls (i. e. groups 1, 2 & 5) which probably represents the Lyt 123 population, especially in nodes where most of the responding cells are derived from the injected PTP cells. Cantor & Boyse (1975) proposed two possibilities for the generation of the Lyt 1 and Lyt 23 subclasses of T cells: 1) different pathways with different precursors for each subset and 2) a common precursor for both lineages. In a later article (Cantor & Boyse, 1977), they proposed a modification of the second possibility, based on the fact that the two subclasses are stable and do not give rise to one another (Huber et al., 1975). The modified hypothesis called for a TL+ Lyt 123 precursor either giving rise directly to Lyt 1 and Lyt 23 subclasses (as well as Lyt 123 cells) or alternatively, giving rise to a TL— Lyt 123 cell which acted as precursor for the other sets (Cantor & Boyse, 1977). Cantor & Boyse (1977) supported the latter possibility since in one type of cytotoxic response the evidence suggested that Lyt 123 cells acted as precursors for the Lyt 23 cells. Our present results show direct evidence that Lyt 123 cells can give rise to both Lyt 1 and Lyt 23 subclasses (and probably also to Lyt 123 cells). In addition, we propose that the differentiation takes place in the periphery, probably xmder thymic control via humoral factors, but also influenced by "inducer" cells with the appropriate MHC display for the generation of subclass-committed precursors (see Section IV, D & E), and that further differentiation into the functionl subclasses is driven by inducer cells with the appropriate matching determinants as well as by antigen and soluble factors produced by different cell types such as macrophages or other cells (Beller et al., 1978). The fact that the Lyt 123 compartment of the T cells in the periphery contains both the PTP cells as well as a complex set of cells with a wide range of regulatory fimctions (Cantor & Boyse, 1977, Cantor et al., 1978), complicates the studies aimed at characterization of the different cell populations. In addition, if our interpretations are correct, the Lyt 123 PTP cell may actually give rise to all of the other T subclasses, including the regulatory cells in the Lyt 123 compartment. Thus, two possible hypotheses for the T-T regulatory interactions can be proposed (Cantor & Asofsky, 1972): 1) the interacting T lymphocytes in any given system belong to a single line of differentiation but differ in degree of maturation or 2) that there are at least two separate differentiated T lines which interact with each other. Probably both interpretations are correct, in the sense that the first applies to the PTP-differentiated T cell model discussed, while the second, which is supported by the Lyt data, would apply to some of the T-T fimctional


159

interactions resulting in a defined immune response (Jerne, 1974, Gershon, 1974, Gershon et al., 1977, Tada et al., 1977). D. Conclusions A precursor population of immunologically incompetent cells committed to differentiation into immunologically competent T cells of the different subclasses defined by Lyt antisera in mice, can be characterized by multiple biological criteria (see Table IV), as being of postthymic origin and substantially different from both the immunologically competent T lymphocytes as well as the prethymic hemopoietic stem cells. Some of the critical and still unanswered questions concerning specialization of T cell subclasses are: Where does subclass segregation take place? What are the regulatory stimuli which determine subclass segregation? The evidence that Lyt 123 cells can give rise to other Lyt subsets in the periphery is progressively growing (see Section III C, and Cantor & Boyse, 1977). However, we still ignore what the mechanisms are by which T cells express their predetermined program and, for example, within the different Lyt subsets, what mechanisms actually regulate the expression of particular I region determinants (McDevitt et al., 1976, Shreffler, 1977, Murphy et al., 1977, Tada et al., 1977). The demonstration that the T helper cells for H-2restricted cytotoxic responses apparently react with I region determinants and that such reactivity can be regulated by the appropriate expression of such determinants in the periphery (Zinkernagel et ala., 1978c) supports our views of further differentiation and specialization of T cells in the periphery. Thus, the PTP compartment would not only allow an economic means for T cell renewal but would assure the further refinement of T cell function. Indeed, it is probable that PTP cells which can "specifically' interact with "inducer" cells in the periphery may generate the different T subclasses by way of the signaling produced by display of the appropriate MHC determinants on the surface of the inducer cell. In a way, this is a proposition of cell interactions comparable to that of Boyse & Cantor (1978), who suggest that in ontogeny, the unit of differentiation is not a single cell but a pair of cells that undergo complementary differentiative steps to prepare them for their collaborative tasks (see also Section IV, E, for further discussion of matching populations). Although we are still ignorant of the full range of these cell-interaction molecules, the fact that la antigens, as well as H-2, are expressed early during embryonic life (Delovitch et al., 1978), suggests that the appropriate inducer cells in the periphery may be available for the ontogenic programming of the T cell subclasses. In addition, it is probable that functional differentiation may require cell division for expression of the functional state (Goldstein et al., 1977, Cantor

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& Boyse, 1977, Boyse & Cantor, 1978), supporting our view of expansion and differentiation of the PTP pool in the periphery. Thus, fulfillment of a differentiation program for a cell may consist in premitotic expression of a set of genes for phenotype (which will permit the appropriate interaction" with the inducer cell) and postmitotic expression of another set of genes for function (after the successful interaction with the inducer cell). In addition, this differentiation step in the periphery, as described for T-T or T-inducer interactions fits well with the adaptive differentiation proposed for T-B interactions (Katz & Benacerraf, 1976, Katz, 1977).

IV. DEVELOPMENT OF T CELLS

A. Models for T Cell Generation From the experimental information in mice and most other species, as well as the human immimodeficiency data, it appears that the thymus is an obligatory step for the generation of competent T cells. The studies described in previous sections show that the thymus exports to the periphery a postthymic precursor (PTP) cell which is derived from prethymic hemopoietic precursors that migrated to thymus and that the competent T cells are generated from further differentiation of the PTP in the periphery. In section II we also discussed some of the intrathymic events and it appears that hemopoietic immigrants have probably three choices within thymus: 1) become part of the pool of cells which will be exported to the periphery, most probably as PTP cells; 2) enter the pool of thymocytes which are not exported and apparently die in the thymus or 3) become part of the intrathymic pool of resident mature T cells, which are also not exported. If the theoretical possibility of some form of clonal deletion or selection taking place within thymus is to be considered (Burnet, 1962, Jerne, 1971, Bevan, 1977b, Nagholz & Miggiano, 1977, Blanden & Ada, 1978, Langman, 1978, Schwartz, 1978), the first two (and perhaps all three) choices indicated above would be in reality a single process, and only the cells with the appropriate repertoire would be allowed to emigrate as PTP cells. However, these interpretations were (and to some extent still are) not the prevalent ones and the "conventional wisdom" indicated by Weissman et al. (1975) supports the view of a single lineage of differentiation from cortical thymocytes to medullary thymocytes and then to the periphery as competent T cells. On the other hand,, the increasing evidence for peripheral as well as intrathymic T cell heterogeneity brought forth ideas of more complex differentiation systems for T cells. Three schemes can be proposed for the


161

generation of T cells (Stutman, 1977): 1) several differentiation steps in a single developmental pathway taking place exclusively within the thymus (the "conventional wisdom" approach); 2) branched pathways leading to different specialized end points, although derived from a single lymphoid precursor for the different subclasses (it is still not dear where the branching takes place, i. e. intrathymically or in the periphery, our own data supporting the latter) and 3) specialized lymphocyte lineages without any common precursors (except perhaps at the hemopoietic stem cell level). The advantage of Model 2, especially the availability in the periphery of the PTP compartment that can be driven into further differentiation by thymic influence (and perhaps by other influences), is that it seems the most economic way for maintaining homeostasis of the T cell pool (and is symmetrical with the other models of cell renewal and differentiation in the hemopoietic tissues). In addition, the proposed model for T cell development agrees with the requirements for H-2 regulation of immune reactivity as well as the instructional role of thymus in determining T cell specificity. Thus, the intrathymic step would permit the development of the appropriate recognition units for modified self-H-2 and non-self (Jerne, 1971, Zinkernagel, 1978a, b, c). On the other hand, the postthymic maturation in the periphery would permit the functional diversification of T cell subclasses, including the T help required for x-plus-self specific cytotoxicity (Zinkernagel et al., 1978c) as well as the "adaptive differentiation" required for other types of cell interactions (Katz & Benacerraf, 1976, Katz, 1977). A corollary of this interpretation is that in the absence of the intrathymic step, T cell differentiation may be nonfunctional (see Sections IV b & c). B. In Vitro Induction and T Cells in Nude Mice The instructional role of the thymus, providing not only the maturational impulse but also the actual range of specificity for the effector T cells, permits some re-evaluation of the data on in vitro induction of T cells and the presence of T cells in nude mice. Hemopoietic cells of adult or embryonic origin from normal or nude mice can express an array of cell surface antigens which are characteristic of the T lineage (i. e. TL, Thy 1, Lyt, etc.) after short-term incubation in vitro with various thymus extracts (Komuro & Boyse, 1973). However, a variety of substances that have in common the ability to increase intracellular levels of cyclic AMP are also capable of producing similar changes, albeit at different concentrations than some of the thymic humoral factor (Scheid et al., 1973, 1975a, b, Goldstein et al., 1975, 1977). The target cell for this in vitro induction appears to be a predetermined T cell progenitor, probably Immunological Rev. (1978), Vol. 42

ii

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prethymic although this is still debatable (Stutman, 1977), termed a "prothymocyte" in most publications (Komuro & Boyse, 1973, Komuro et al., 1975, Scheid et al., 1973, 1975a, b, Goldstein et al., 1975, 1977). It has been shown that the inducible prothymocyte has the ability to migrate and repopulate the irradiated thymus (Komuro et al., 1975). The in vitro induction assays have two interesting qualities: 1) they mimic to a large extent the intrathymic step of differentiation, including the appearance of TL antigen, which is an accepted marker for intrathymic thymocytes and 2) the induced cells are mostly devoid of immunological competence (Scheid et al., 1973, 1975a, b) or have a modest reactivity to T cell mitogens, especially Con A (Basch & Goldstein, 1975). Thus, it appears that even with the most purified thymic humoral factors such as thymopoietin (Goldstein et al., 1975, 1977), it is not possible to drive a restricted stem cell or progenitor to become a competent T cell in vitro (i. e. in the absence of the thymic stroma). The incubation of prothymocytes from nude mice on monolayers of thymic reticuloepithelial cells also generated cells endowed only with a moderate response to Con A, and minimal response to allogeneic cells (Sato et al., 1976), suggesting that the interaction of the prothymocyte with the thymic stroma may be quite complex. These results contrast somewhat with the type of differentiation obtained by incubation of thymocytes with macrophages in which, besides the antigenic changes suggesting peripheral type T cells (i. e. decrease TL, increase HI 2, etc.), a relatively good response to allogeneic cells is generated (Beller et al., 1978). Using organ cultures of fetal thymus, it was observed that while that antigenic changes are rapid, 4 days in culture are required for recovery of cells with reactivity to allogeneic cells in MLC (Robinson & Owen, 1977), again showing the complexities of the intrathymic step of differentiation. Some experiments with nude mice point in the same direction as the in vitro induction, suggesting a strict requirement for the intrathymic step for functional differentiation of T cells. Thy 1+ cells are known to occur in nude mice (Raff, 1973, Loor & Kindred, 1973, Scheid et al., 1975a, Loor et al., 1976a, Roelants et al., 1976, Ropke,. 1977), although their functional significance and origin are still imdefined. The putative role of the rudimentary thymic anlage in the nudes, as well as maternal influences in nudes bred from heterozygote pairs have been invoked to explain their origin (see Pritchard & Micklem, 1974, Stutman, 1977, for reviews), while others think that these cells belong to a prethymic "preprogrammed" precursor pool (Scheid et al., 1975a, b, Loor et al., 1976a, Roelants et al., 1976). Injection of nude mice with thymic humoral factors, as well as other cell products such as ubiquitin (Goldstein et al, 1975) has been shown to induce the


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appearance of Thy 1+ TL+ cells in spleens of nude mice (Scheid et al., 1975a), prompting the interpretation that such cells have been triggered to differentiate from Thy 1— precursors by products of bacterial infection or tissue breakdown (i. e. ubiquitin or similar). It should be indicated that the precursor cell described by Roelants et al. (1976) is also Thy 1+ TL+. However, it appears that such cells, although bearing markers of the T cell lineage, have only limited immunological capacity, i. e. only moderate response to polyclonal activators for the Thy 1+ cells in nude marrow (Ropke, 1977). Similarly, nude mice do not show signs of immunological restoration when treated with thymus enclosed in cell impermeable diffusion chambers or thymic humoral factors (Stutman, 1974, Pierpaoli & Besedovsky, 1975, Stutman, 1977), or show only some restoration of Con A reactivity (Thurman et al., 1975). C. Nonfunctional Differentiation Based on the above findings, it is tempting to postulate that in the absence of direct contact with the thymic reticulo-epithelial stroma (and perhaps with intrathymic thymocytes), the T cell precursors go into a trivial alternate pathway of differentiation which generates nonfunctional T cells (or cells only endowed with some response to polyclonal activators), perhaps of the type which are destined to intrathymic death. One possible mechanism of this nonfunctional differentiation could be due to the lack of selection within the thymus for the appropriate "cell-interaction molecules" (Katz, 1977), related to the MHC which are critical for the interactions of the antigen specific T cells. This interpretation will fit with either positive or negative intrathymic selection models (Bumet, 1962, Jerne, 1971, Bevan, 1977b, Blanden & Ada, 1978; Langman, 1978, Zinkemagel et al, 1978a, b, c). D. Cell-Cell Interactions and T cell Development Although the MHC regions involved in different functional cell to cell interactions are known and in some instances the effector and regulatory cells involved are also known, the question of soluble factors versus cell-cell contacts, as well as the direction of the signals given in such interactions, are still undefined (Katz & Benacerraf, 1976, McDevitt et al., 1976, Shreffler, 1977, Gershon et al., 1977, Katz, 1977, Benacerraf & Germain, 1978). If we design a symmetrical cell-interaction system not for function but for development of T cells, which is also regulated by MHC regions and their products, it may be postulated that: 1) different MHC regions in the "inducer" cells, as well as specific and nonspecific humoral factors, will regulate the intrathymic and extrathymic steps of T cell development, and that such signals are necessary for functional differentiation of T cells and 2) the

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"inducer" cells will impart at least three types of different signals: one for irreversible commitment to the T lineage, the other for the elaboration of the appropriate dictionary of cell-interaction molecules and the third one for specialization into a determined subclass of T cell. It is probable that the first two signals are intrathymic events, while the third may be a postthymic one. It is also probable that the first type of signal is independent of MHC and that some overlap between the last two signals may occur (see Section III, D). E. T Cell Development and H-2 Restriction A key question raised by the phenomenon of H-2 restriction of T effector cells, concern the range of specificity of the restriction (Zinkemagel & Doherty, 1977, Shearer et al., 1977, Bevan, 1977b). Is the specificity established during the induction of the antigen-reactive T cell and defined simply by the H-2 specificity on the antigen-presenting cell or is H-2 restriction limited only to the self-H-2- specificity? Most of the recent results favored the antigen-presentation theory (Zinkemagel & Doherty, 1977, Shearer et al., 1977, Bevan, 1977a, b, Paul et al., 1977, Schmitt-Verhulst et al., 1978). However, most of the above mentioned experiments could also be interpreted as predetermination of self-H-2 specificity (i. e. the results of Schmitt-Vedhulst et al., 1978, showing that H-2 restricted cytotoxic cells can be produced by immunization with hapten-modified soluble proteins). On the other hand, the experimental evidence that the specificity of H-2 restriction may actually be the consequence of the environment in which T cells differentiate from their precursors and that such a critical environment was the thymus (Zinkemagel et al., 1978a, b c), strongly suggests that functional T cell development and restrictions to self-H-2 specificities may be intimately related processes. Thus, the intrathymic step described in Section II would represent not only a necessary requirement for commitment towards T cell differentiation, but a critical step for the programming of the cellinteraction molecules required for function and regulation of the effector T cells. The extensive intrathymic lymphopoiesis was considered as a mechanism to generate random genetic variation which would provide the lymphoid cells with the different immune reactivity pattems, while the extensive intrathymic cell death may reflect the operation of mechanisms that destroy autoaggressive cells (Bumet, 1962). A more restricted and defined version of this idea was proposed by Jeme (1971). Jeme's hypothesis proposes that self-H-2 antigens drive the generation of receptor diversity. The immatiu-e T cells first express anti-self-H-2 activity in the thymus, leading to proliferation and accumulation of V gene mutations until there is no reaction


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against self, and it is such a population which is eventually exported to the periphery. Some modified versions of the theory ascribe either positive or negative selection within thymus to explain as well, such phenomena as H-2 restriction and MHC requirements for cell-cell interaction (Bevan, 1977b, Nabholz & Miggiano, 1977, Langman, 1978, Blanden & Ada, 1978, Schwartz, 1978). The large amount of intrathymic cell death discussed in Section II. G, would support a priori the idea of intrathymic selection (Burnet, 1962, Jeme, 1971). However, it is not easy to define at present writing the nature of the selective process. The long-term persistence of abnormal mutant cells in the thymus (Joneja & Stich, 1963, Session & Stich, 1965, Nowell & Cole, 1967) as well as the development of lymphoid leukemias first within the thymus (Miller, 1971) would suggest that "surveillance" by cell deletion is not an intrathymic event and that the high rate of cell death within thymus is probably not directly related to some kind of censorship function as proposed by Bumet (1962). Thus, one would be inclined to consider the process of excess cell production within thymus as a selective process essential for functional development of T cells, and which may parallel the selection of appropriate matching populations proposed for the development of the central nervous system in vertebrates (Katz & Lasek, 1978). It has been observed that during development, more neuroblasts are generated than survive to become members of the mature functional units (Hamburger, 1975, Clarke & Cowan, 1976, HoUyday & Hamburger, 1976). This excess cell production cannot be attributed to imprecise programming of the number of mitotic cycles because the total number of excess neuroblasts produced in similar animals is quite constant (Hambuger, 1975, Katz & Lasek, 1978). So far, the same can be said for the excess intrathymic cell production (see Joel et al., 1974, and Section II. G). This surplus production must be of some use to the animal, and it has been proposed that they may represent a safety factor that permits the population to accomodate to the range of variation within its innervation field (Hamburger, 1975, Clarke & Cowan, 1976). These authors also indicate that the process of forming appropriate synapses may be probabilistic and that the surplus neuroblasts may be necessary to achieve the correct final connection. Thus, the excess cells may be needed to compensate for developmental errors (Clarke & Cowan, 1976) or other trophic interactions required for normal growth (HoUyday & Hamburger, 1976). In addition, the rate of cell death within the excess population is influenced by changes in the periphery (Hollyday & Hamburger, 1976). Whatever the precise role of these excess cells during ontogeny, they are probably required for the successful matching of populations, a matching of exquisite sensitivity, as exemplified also by the studies on nerve regeneration (Sperry, 1976). It is not difficult to use the same terminology indicated

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above to describe the intrathymic steps of T cell functional development, as well as the generation of functional T cells in the periphery. Katz & Lasek (1978) propose an interesting theory for neural development that can be translated with ease into immunological terms. They indicate that the nervous tissue accomodates populations of cells that are connected in a highly predictable manner, and call the interconnected groups a pair of "matching populations." Genetic changes that affect a pair of matching populations can be evolutionary only when the matching quality is not disruped and call them "concordant heritable changes." The "nonconcordant" are those changes that do not autonomously preserve the match (Katz & Lasek, 1978). This interpretation bears some resemblance to the theory proposed by Boyse & Cantor (1978) on complementary differentiation of pairs of cells. However, the idea proposed by Katz & Lasek (1978) also included the need for surplus cell production for selection of the appropriate matching structures. Thus, intrathymic selection may be mostly for concordant changes that preserve appropriate matching between precursor and inducer cells. Such concordant matchings would generate different cells capable also of concordant interaction with the appropriate matches in the periphery. The nonconcordant matches would produce nonfunctional differentiation, probably destined to intrathymic death (Section IV. C). This last idea is supported by the results of Zinkernagel et al. (1978b, c) showing that fully allogeneic combinations produce nonfunctional cells incapable of reacting with modified self from any of the members of the combination. Our interpretation also proposed that the matching populations, both intra and extrathymic, are selected via compatibility for MHC regions as well as other surface determinants. It has been suggested that one conceivable function for HLA (or H-2) antigens may be to serve as the constant region for other functionally important membrane antigens (Bodmer, 1972), an idea expanded by Ohno (1977), proposing MHC antigens as the main anchorage sites for organogenesis-directing proteins. Thus, it is possible that MHC associated regions (such as I or the newly described Qa antigens, Stanton & Boyse, 1976, Flaherty, 1976) may be critical factors both at intrathymic and extrathymic levels for the induction of T cell specialization permitting the interaction of matchng pairs of precursor and inducer cells. The capacity of expansion in the periphery (including clonal expansion) of T cell development, in addition to the intrathymic step with selection by excess cell production, permits indeed, a wide range of diversification of function. Concerning the functional repertoire of the T cell obtained at intrathymic and extrathymic level, it appears that three major selections may take place at intrathymic level: 1) a selection leading to self tolerance (or to a mech-


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nism to control anti-self reactivity); 2) a selection leading to self recognition which will restrict the host response preferentially towards modified self and 3) a selection for reactivity towards non-self (alloreactivity). However, if MHC products are considered as anchorage sites for other relevant determinants in cell differentiation and function (Ohno, 1977), the three processes may be a single event and related to the functional requirements imposed upon MHC products by endogenous (i. e. cell-differentiation or cell-interaction determinants) as well as exogenous (i. e. viruses arid other parasitic proteins) components. On the other hand, as was discussed in Section III and IV, the extrathymic step would assure further specialization towards T cell subclasses by reaction with the appropriate inducer cells. Some of the peculiarities of the chimeras studied by Zinkernagel et al. (1978a, b, c) suggest that self-recognition and self-tolerance occur as two different events (i. e. AXB cells in a syngeneic host that has an A thymus graft, behave like A cells, with the exception that they do not react to B antigens). Similarly, as will be discussed in Section V, tolerance to the antigens present in the thymus is not the absolute consequence of the processing of T cell precursors by such a thymus, suggesting that the interaction may require additional determinants or steps which may not be present in certain strain combinations used for the experiments. However, most of those experiments were done with other aims in mind, and need to be re-explored before they can be used as arguments in favor or against the role of the thymus in actual determination of the T cell repertoire. From a purely teleological standpoint, H-2 restriction appears as the most natural consequence of our fight with foreign invaders (Zinkemagel & Doherty, 1977). However, it is still possible that the association may be fortuitous and that the parasite (virus, etc.) may actually derive some advantage from its association with MHC products, perhaps in relation to their fimction as anchoring posts (Ohno, 1977). F. Summary T cells develop as a consequence of intrathymic and postthymic events, in which hemopoietic progenitors are differentiated into precursors and effector cells. We are proposing that such a process includes three integrated events: 1) T cell differentiation; 2) selection of the T cell repertoire and 3) specialization into functional subsets of T cells. Although there is evidence of specific and nonspecific humoral factors (i. e. thymic hormones, etc.) affecting T cell differentiation, it is also proposed that the most critical factors in this integrated process are a consequence of direct cell to cell interaction between precursor and inducer cells. The three processes are thus the consequence of the appropriate or concordant matching of precursor-inducer pop-

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ulations, both as intra- and extrathymic sites. It is also proposed that MHC detenninants are critical in permitting the appropriate matching. Thus, the model can account for nonfunctional differentiation when the appropriate matching is not available and with intrathymic selection by excess cell production favoring the appropriate matching.

V. TOLERANCE AND THYMUS

One of the predictions of the theory that invokes selection of the T cell repertoire within the thymus (Jerne, 1971) as well as its modernized versions (Nabholz & Miggiano, 1977, Blanden & Ada, 1978, Langman, 1978, Schwartz, 1978) is that the cells processed by an allogeneic thymus should be unable to react to the alloantigens displayed in that thymus. These theories are somewhat derived from ideas proposed by Bumet (1962) who ascribed to the thymus a dual role: a generative function in the production of clones with "definable immimological fimctions" and a censorship function with "elimination or inhibition of self-reactive clones." However, as will be seen from the following discussion, there is experimental evidence both from NTx as well as nude mouse studies indicating that such type of tolerance is not an invariable consequence of allogeneic thymus grafts and that it is apparently related to the type of donor-host combinations used. A. Tolerance Induction with Thymus Grafts Our initial observations with neonatally thymectomized mice showed that acceptance of an F^ thymus grafted into the parental host was variable, and depended on the strain combination used (Stutman et al., 1967, 1969c). In certain combinations such as C3H grafted with (C3H X A) Fj thymus, tolerance was observed in almost every instance, while in other combinations such as C3H grafted with (C3HXC57BL) Fj thymus, tolerance was not detected (Stutman et al., 1969c). Tolerance to transplantation antigens is indeed complex and its exact mechanisms are still not clearly defined (Brent et al., 1976). In our experiments, tolerance was defined as: 1) acceptance of skin of the same origin as the thymus graft for 60 days or more accompanied by normal rejection of a third party skin graft; 2) "permanent" acceptance of the thymus graft (usually studied at 160 days after grafting) and 3) no GVH reactivity of spleen cells of the C3H grafted animals when tested against the appropriate F^ hybrids (i. e. same as the thymus and skin donor) accompanied by reactively against an inappropriate C3H F^ hybrid (Stutman et al., 1968c). Thus, tolerance was defined by both the lack of reactivity against the appropriate antigens and by the presence of reactivity


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against a third party. The fact that both the acceptance of the thymus graft as well as the lack of GVH reactivity were considered besides the acceptance of skin grafts, precludes the possibility that the discrepancies in skin rejections could be related to skin-specific antigens not present in the toleranceinducing thymus (Sk antigens have been described in mice by Boyse et al., 1970). The reaction to third party alloantigens was also used as a requirement for definition of tolerance, since it was observed that fully allogeneic thymus grafts usually produced incomplete immune restoration (Miller et al., 1964, Miller, 1966, Stutman et al., 1967) and in some cases the apparent inconsistency of rejection of skin from the same origin as the thymus graft with retention of third party skin (Miller et al., 1964). The paradox that the host cells processed by the grafted hemi-allogeneic thymus could eventually reject the restoring thymus was interpreted at that time as supportive of the evidence for the thymus being an essential step for T cell differentiation via traffic of hemopoietic cells and not as an intrinsic prodcer of competent cells (Stutman et al., 1967, 1968c, 1972). Such results also supported the early observations that in thymectomized animals, immunological restoration was mediated niostly by host cells and not by thymus donor type cells (Dalmasso et al., 1963). We were also able to determine that in the animals that were restored, but actually rejected the restoring thymus, immunological capacity eventually declined with time in absence of the thymus (Stutman et al., 1972, Stutman & Good, 1974). Our results also disproved, to some extent, the concept that antigen presentation within the thymus was necessary and sufficient for induction of tolerance, either to transplantation antigens (Vojtiskova & Lengerova, 1965, 1968) or soluble proteins (Isakovic et al., 1965, Staples et al., 1966, see also Waksman, 1977, for an update on this problem). These experiments stemmed from the concept by Burnet (1962) that the thymus could be the ideal place for deletion of auto-reactive clones. Tables VII to IX will present some information, most of it unpublished and generated in 1969-1970, on tolerance induction by thymus grafts. It will become apparent that a re-investigation of the problem using modem technology seems justified at the present time, in view of the proposed complex instructional role ascribed to the thymus by Zinkernagel et al. (1978a b, c). Table VII shows some results in which C3Hf/Umc (C3H) NTx mice were grafted at 20 days of age with allogeneic H-2 kk compatible thymii (groups 1 to 6) or Fi hybrid thymuses of C3H with different allogeneic H-2 incompatible strains (groups 7 to 10). In the case of the H-2 kk thymus grafts, prolonged acceptance of the thymus graft and skin of the same origin as the graft was high in most instances, ranging from 50 to lOO^/o. However,

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STUTMAN TABLE VII Tolerance Induction to Skin Grafts of Same Origin as Thymus Grafts in Neonatally Thymectomized C3Hf/Umc Mice

Thymus donor^ 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

C3H/He Ce/J CBA/H AKR/J C58/J (C3H X C58) Fi (C3H X A) Fi (C3H X C57BL/1) Fi (C3H X C57BL/6) Fj (C3H X T6) Fi

H-2

Number of mice tolerantii

GVH activity

Presence of thyinus graft

Extrathymic T-cell selection.

The extrathymic T-cell development pathway.

PD-1 regulates extrathymic regulatory T-cell differentiation.

An overview of the intrathymic intricacies of T cell development.

Role of CD4 molecule in intrathymic T-cell development.

Ontogeny and development of extrathymic T cells in mouse liver.

CD5 instructs extrathymic regulatory T cell development in response to self and tolerizing antigens.

Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and unequal T-cell repertoire?

Isolation and Ex Vivo Culture of Vδ1+CD4+γδ T Cells, an Extrathymic αβT-cell Progenitor.

delta repertoire.

Extrathymic positive selection of alpha beta T-cell precursors in nude mice.

Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns.

Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction.

Positive selection of T-lymphocytes induced by intrathymic injection of a thymic epithelial cell line.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters intrathymic T-cell development in mice.

T-cell maturation and clonal deletion in cyclosporine-induced autoimmunity.

Ly-49 antigen defines an alpha beta TCR population in i-IEL with an extrathymic maturation.

Fibroblast and T cells conditioned media induce maturation dendritic cell and promote T helper immune response.

In Vitro Differentiation and Expansion of Intrathymic T Cell Progenitors from Human Umbilical Cord Blood-Derived CD34(+) Cells.

γδ T cells restrain extrathymic development of Foxp3+-inducible regulatory T cells via IFN-γ.

Programmed cell death and extrathymic reduction of Vbeta8+ CD4+ T cells in mice tolerant to Staphylococcus aureus enterotoxin B.

Molecular events in late stages of T-cell functional maturation.

beta + lymphocytes reveals a major extrathymic pathway of T cell differentiation.

Fluorochrome-based definition of naturally occurring Foxp3(+) regulatory T cells of intra- and extrathymic origin.