Neonatal tolerance induction in the thymus to MHC-class II-associated antigens

CELLULAR

IMMUNOLOGY

108,162- 174 ( 1987)

Neonatal Tolerance Induction in the Thymus to MHC-Class II-Associated Antigens III. Significance of Hemopoietic Stem Cells for Induction and Maintenance of MIS Tolerance by Continuous Supply of Tolerance-Inducing Nonlymphocytes

MASAMICHI HOSONO, TOMOHIDE HOSOKAWA,* TATSUO KINA, AND YOSHIMOTO KATSURA Department of Bacteriology and Serology, Chest Disease Research Institute, Kyoto University, and *Department ofPreventive Medicine, Kyoto Prefectural University ofMedicine, Kyoto 606, Japan Received February 2, 1987; accepted March 18. 1987 The role of hemopoietic stem cells and of other cell types in the induction and maintenance of immunologic tolerance in the thymus was investigated by intravenous injection of Mls-semiallogeneic cells into newborn mice less than 24 hr after birth. Mls-specific tolerance was induced by inoculation of peritoneal cells and thymus cells, and the tolerant state was compared with that induced by bone marrow cells which had hemopoietic stem cell activity and were able to create a stable chimera in both central and peripheral lymphoid organs. When peritoneal or thymus cells were injected, the level of tolerance attained was proportional to the number of cells injected, though peritoneal cells were 20 times as effective as thymus cells. In vivo functions of tolerance-inducing cells and their immediate precursors were radiosensitive and belonged to a Thy-l-, nylon-wool-nonadherent (probably non-B), weakly Sephadex G-lo-adherent cell population. Tolerance induced by peritoneal cell injections was transient, starting to terminate within the first 2 weeks of life, while tolerance caused by bone marrow cell injections persisted through more than 6 weeks. Such transient tolerance induced by the former became long-lasting when followed by an additional injection of bone marrow cells, which did not cause thymic lymphocyte chimerism. All data indicated that bone marrow stem cells were engaged in tolerance induction and maintenance by continuously supplying tolerance-inducing nonlymphocytes. 0 1987 Academic Press, Inc.

INTRODUCTION Antigen-specific immunological tolerance to allogeneic major histocompatibility complex (MHC) and non-MHC antigens, induced by the injection of semiallogeneic hemopoietic cells into newborn mice, has long been studied as a model for natural tolerance to self-antigens (l-23). Tolerance in peripheral lymphoid tissues has been thought to be mediated by multiple mechanisms, such as irreversible inactivation of antigen-specific lymphocytes (10, 12, 14, 15, 1S), generation of suppressor cells (6-9, 11, 13) and/or reversible suppression of lymphocyte functions by serum-borne factors (3), depending upon the experimental system used (5, 20). Regardless of the precise mechanism involved in each case of neonatal tolerance, maintenance of the tolerant state was found to be closely related to chimerism in hematolymphoid cells 162 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved

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(2,4). However, since most these experimental resuls were obtained using peripheral lymphoid organs of adult animals, the cellular events in a tolerance induction phase, especially in the thymus where negative selection of T cells reactive to self-antigens must take place, seem to be clarified for further understanding self-tolerance. Recently it was demonstrated that injection of semiallogeneic hemopoietic cells into newborn mice also induced tolerance in the thymus at the level of MHC class Iand class II-reactive T-cell subsets within the first week of life, e.g., tolerance to alloMHC antigens in cytotoxic T-cell precursors ( 10, 16) and in proliferative T cells ( 12, 22) and also to non-MHC, MIS antigens of graft-versus-host reaction-inducing cells (20, 23). This central tolerance was not mediated by suppressor cell generation, suggesting that clonal inactivation was the mechanism, yet significantly chimerism in thymic lymphocytes at this early stage of life was not demonstrated. Central tolerance without demonstrable chimerism, in contrast to peripheral tolerance with apparent chimerism, would be an attractive subject in the study of cellular events of tolerance induction in developing, differentiating thymus T cells. Thus, Morrissey et al. (24) using a highly sensitive method has extensively analyzed the relationship between the degree of tolerance and chimerism in the adult thymus of mice in which tolerance had been induced neonatally. They showed that there was a close correlation between the two, though the percentage of chimerism was very low, being between 0.2 and 2.8%. This showed that even only a small fraction ofthe donorderived cells, probably at a level almost undetectable by usual techniques, could be sufficient for induction and maintenance of tolerance in the thymus. To identify the cells involved in the early stages of tolerance induction in the newborn thymus, and to assess the biological significance of pluripotent bone marrow stem cells in the maintenance of intrathymic tolerance, we injected neonates with tolerogens from several types of cells which did or did not show hemopoietic stem cell activity and with fractionated cells from Mls-semiallogeneic hybrid mice. Mls antigens used here as tolerogenic determinants were known to be recognized only by class II-reactive T-cells in association with self-Ia antigens (23, 25-29) so that the resulting data might illuminate the role of negative selection mechanisms in the development and repertoire generation of thymus helper-T-cell subsets (30-34). MATERIALS AND METHODS Mice. BALB/c (H-2d, Mlsb), DBA/2 (H-2d, MIS”), C3H/He (H-2k, MIS’), and BlO.BR (H-2k, Mlsb) mice were purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka). AKR/J (H-2k, Mls”) and CBA/ J (H-2k, Mlsd) mice were donated by Dr. S. Muramatsu, Faculty of Science, Kyoto University. Fl hybrid mice were produced in our animal facility, and used when 3 to 6 months of age, unless otherwise stated. Cef/ preparation. Cell suspensions from the thymus, spleen, mesenteric lymph nodes, and bone marrow were prepared in Eagle’s minimum essential medium (MEM, Nissui Seiyaku Co., Tokyo) without serum supplement, as described previously (20). Red blood cells, when necessary, were removed by suspending lymphoid cells in Gey’s hemolytic solution. Blood cells were collected by heart puncture and resident peritoneal cells by washing the peritoneal cavity with IO ml MEM containing 10% normal BALB/c mouse serum, following anesthetization with sodium pentobarbital (Somnopentil, Pitman-Moore, Inc., NJ). Cells were washed three times in MEM before use.

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Cellfractionation. T cells were removed from whole cell suspensions by incubation at room temperature with appropriately diluted anti-Thy- 1.2 monoclonal antibody (Meiji Institute of Health Science, Tokyo) for 40 min, followed by incubation at 37°C for 30 min in the presence of 10% normal guinea pig serum as a source ofcomplement (35). In some experiments, thymus cells were passed through nylon-wool columns in order to remove strongly adherent as well as B cells (36). For further removal of more weekly adherent cells, the nylon-wool-nonadherent thymus cells were passed over a column of Sephadex G- 10 beads (Pharmacia, Uppsala, Sweden) (37). The medium used for these cell fractionations was warm MEM supplemented with 5% normal BALB/c serum (35). Tolerance induction. BALB/c mice, which were less than 24 hr old, were injected via the anterior facial veins with 50 ~1 MEM containing appropriate numbers of cells from Fl hybrid mice, as previously described (20). Some littermates or age-matched neonates that were not injected served as positive controls. Between one and several weeks after injection, the mice and their thymus cells were tested for responsiveness to antigens of tolerance-inducing cells. Cells from Mls-semiallogeneic and/or H-2semiallogeneic mice were used as tolerogens: the differences in the Mls and H-2 haplotypes from BALB/c mice are indicated in circles. Cell surface antigen analysis by flow cytofluorometry. Conjugation of fluorescein isothiocyanate (FITC) with anti-Thy- 1.1 monoclonal antibody (T 11D7) was carried out, as described elsewhere (38). Samples of 5 X lo5 cells in 50 ~1of MEM were mixed with 10 ~1 fluorescein isothiocyanate (FITC) -anti-Thy- 1.1 (50 pg/ml), incubated for 30 min in an ice bath, and then washed twice. The medium used for thymocyte staining was MEM containing 2% fetal calf serum. After being resuspended in 1 ml medium, cells were analyzed by flow cytofluorometry (Spectrum III, Ortho Diagnostic Systems, MA). Host-versus-graft and graft-versus-host reactions. The tolerance states of individual mice were tested by a local host-versus-graft reaction (HVGR), as previously described (20). Each mouse received in one footpad, 2000-rad-irradiated spleen cells (1 X lo’/25 ~1) from Fl mice which had been used as the tolerance-inducing cell donors. Four days later, the mice were killed and their popliteal lymph nodes (PLN) were carefully removed and weighed to within kO.0 1 mg. As control, medium was injected into the contralateral footpad. To assess the anti-Mls activity of BALB/c lymphoid cells, the PLN-swelling assay for the local graft-versus-host reaction (GVHR) was employed, as reported elsewhere (20). Twenty five microliters of MEM and 2 X lo7 thymus cells from tolerance-induced mice were injected into the footpads of adult F 1 hybrid recipients. Cells prepared from untreated BALB/c mice and MEM were used as positive and negative controls, respectively. PLN-swelling assay. The degree of PLN enlargement was expressed as a lymph node-swelling index (LN index = PLN weight of the cell-injected group/PLN weight of the medium-injected group). The percentage of tolerance was calculated as follows: percentage tolerance = [ 1 - (mean PLN weight of tolerant group - mean PLN weight of medium control)/(mean PLN weight of positive control - mean PLN weight of medium control)] X 100. Statistical analysis. The significance of differences between experimental groups was examined by the two-tailed Mann-Whitney U test. Experimental groups with probability values larger than 0.05 were not significantly different from controls.

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B

I

231561

6

8

IO

Weeks

1

after

2

3

5

6

1

6

g

IO

birth

FIG. 1. Chimerism of central and peripheral T lymphocytes in tolerant mice neonatally induced by semiallogeneic bone marrow cells. BALB/c (Thy- 1.2) neonates, less than 24 hr after birth, were iv injected with 2 X 10’ bone marrow cells of (BALB/c X AKR)Fl (Thy-1.1/2) mice. One to ten weeks after birth they were killed, and the percentage of donor-derived Thy-l. I+ cells in the thymus (panel A) and in the spleen (panel B) were assessed by means of cytofluorometry. Mean values were indicated by open circles, with numerals indicating the number of mice tested, and with -+ 1 standard errors (vertical bars): standard errors were omitted when smaller than the size of the symbol. Closed circles represent the development of Thy- 1.2’ cells in normal BALB/c mice.

RESULTS

Kinetics of Chimer&m in the Thymus and Spleen of Mice Injected Neonatally Semiallogeneic Bone Marrow Cells

with

Newborn BALB/c (H-2d, Mlsb, Thy- 1.2) mice were injected with 2 X 10’ (BALB/ c X AKR)Fl (H-2 d’k, M1sbla Thy- 1.2/ 1) (CAKFI ) bone marrow cells. At various ages, these mice were killed for assessment of chimerism in the thymic lymphocyte populations and calculation of the percentage of Thy- 1. 1+, CAKF 1-derived thymocytes. As shown in panel A in Fig. 1, Thy-l. If cells became appreciable just after 2 weeks of age, and continued to increase, following the increase in thymus weight, until 4 weeks after birth; the peak percentage at that time was about 27%. This then gradually decreased as the thymus weight also decreased; by 10 weeks of age the percentage of donor-derived cells had decreased to about 7%. On the other hand, during the first 7 weeks of life, the proportion of host T cells in the spleen increased gradually from 0.1% to around one-fourth of total spleen cells in this experiment (panel B in Fig. 1). However, T-cell chimerism in tolerance-induced spleens was apparently not observed: Although T cells of CAKE1 bone marrow cell origin became detectable at around 7 weeks of age, the level was quite low, and fell to undetectable levels soon thereafter. In another experiment, the formation of chimerism in the periphery of 4-week-old BALB/c recipients was assessed by a functional analysis. Spleen cells of individual mice which had received CAKFl bone marrow cells while they were neonates were

O 0 0 0 0 r 0

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10 9 8

0

s7 $8 L .ol

5

$4 z g

0 0

0

3

0

I 2 c

I .125

I .25

1 .5

B I 1 wo-‘)

No. of spleen cells injected FIG. 2. Chimerism in HVGR-inducing cells in the spleen of 4-week-old mice in which tolerance was neonatally induced with semiallogeneic bone marrow cells. Graded numbers of spleen cells of normal (BALB/c X AKR)Fl mice (0) and 10’ spleen cells either from normal BALB/c mice (Cl) or from BALB/c mice transfused neonatally with 2 X 10’ (BALB/c X AKR)Fl bone marrow cells(O), after 2000 rad irradiation, were injected into footpads of normal BALB/c recipients in order to assessthe level of chimerism in the HVGR-inducing spleen cells. Four days later, the recipients were killed and their popliteal lymph nodes (PLN) were weighed. In the tolerance-induced mouse group, each symbol indicates the mean of two PLN for each spleen cell donor. In others each symbol represents the weight of one PLN after injection with various number of Fl spleen cells.

injected into footpads of syngeneic mice (BALB/c) and tested for lymph node-swelling inducibility caused by the HVGR. The degree of lymph node swelling indicated the degree of chimerism, probably mainly due to radioresistant nonlymphocytic cells. Results are shown in Fig. 2. The HVGR inducibility of 1 X 10’ spleen cells of tolerance-induced mice was almost equal to that achieved by - 1O6spleen cells of CAKF 1 mice, suggesting approximately 10% chime&m. Relationship of the Degree of Tolerance to the Chimer&m of Thymic Lymphocytes in Mice at 4-Weeks ofAge and the Number of Bone Marrow Cells Injected Neonatally Graded numbers of CAKF 1 bone marrow cells were injected into neonatal BALB/ c mice. Four weeks later, the chimerism of thymic lymphocytes was analyzed, and simultaneously the degree of anti-Ml9 tolerance in the thymus was assessed. As shown in Table 1, when the inoculum of bone marrow cells was less than 1 X 10’ cells per neonate, there was a good correlation among these parameters, an increasing number of bone marrow cells resulting in a higher degree of chimerism and a higher level of tolerance. Comparison

of Tolerance Induction

by Diferent

Types of Tolerogen Cells

Up to this point anti-Ml9 tolerance had been investigated using only bone marrow cells as tolerogens, containing a high proportion of pluripotent stem cells. In the next experiments, tolerance induction by other types of cells, including erythrocytes, peritoneal cells, and thymus cells, was investigated. Graded numbers of cells from (BALB/c X DBA/2)Fl (H-2d, M~s~‘~) (CDFl) mice were injected into newborn

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Relationship

between the Degree of Tolerance BALB/c Mice Neonatally Transfused

No. of bone marrow cells injected into neonates None 2.5 x 5x 10 x 20 x

(positive IO6 lo6 IO6 IO6

cont.)

INTRATHYMIC 1

and Chimerism with (BALB/c

% Chimerism (% Thy-l. I+ cells)b 0.0 0.0, 0.1 0.1,O.l 1.5,3.4 17.9,27.1,34.7

167

TOLERANCE

of T Cells in the Thymus X AKR)FI Bone Marrow

GVHR activity (LN index +- SE) (n) 2.15 2.16 1.89 0.83

kO.1 I(4) f 0.04 (3) * 0.25 (2) f 0.20 (3) ND=

of 4-Week-Old Cells”

% Reduction

2 26 100 ND

a Newborn BALB/c mice (124 hr) were injected iv with various numbers of (BALB/c X AKR)Fl bone marrow cells. Four weeks later, thymuses were taken out, and the percentage chimerism was determined by a fluorocytometry technique. Twenty million thymus cells from each group of mice were tested by routine methods. ’ Percentage of Thy-l. I+ cells in (BALB/c X AKR)Fl thymus cells was 93.5%. ’ Not done.

BALB/c mice. One week later, the GVH activity of thymus cells was tested in CDFl recipients; results are shown in Fig. 3. One marked difference among these tolerogen cells was the effect of cell dose. Clearly the degree of tolerance was directly proportional to the number of inoculated peritoneal or thymus cells, both of which probably contained few hemopoietic stem cells. On the other hand, tolerance induction by bone marrow cells was much less affected by a logarithmic decrease in the number of cells inoculated. Erythrocytes were ineffective even at doses as high as 5 X 10’. Cells Participating

in Tolerance Induction

Cells from different organs showed varying degrees of potency in tolerance induction. Thus, the cell numbers required for 50% tolerance, as shown in Fig. 3, were 25 X 106, 1.25 X 106, and around 0.5 X lo6 for thymus, peritoneal, and bone marrow cells, respectively. As shown in Fig. 3, irradiation of peritoneal cells prior to injection completely abrogated their ability to induce tolerance in the thymus, indicating that either the cells’ functions or their normal physiological behavior in newborn recipients were radiosensitive. Removal of thymic lymphocytes by pretreatment of inoculum cells with anti-Thy1.2 + complement (C) did not affect tolerance inducibility (Fig. 3), and again it was not affected when thymus cells were passed through a nylon-wool column (see the closed triangle with asterisk in Fig. 3), a treatment which removes strongly adherent and B cells (37). This also prevented death of neonates caused by embolism following injection of a high cell dose. Futher passage of these fractionated cells through a Sephadex G- 10 column reduced, through only partially, tolerance inducibility (see open triangles in Fig, 3). All these results indicated that the tolerance-inducing cells were generally distributed throughout all the hematolymphoid tissues, though to different degrees, and also in a Thy- 1-, non-B, weakly Sephadex G- 1O-adherent cell population.

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D-

)-

)-

No. of cells injected(&) FIG. 3. Tolerance induction by different organ-derived cells. BALB/c (Mlsb, H-2d) neonates less than 24 hr after birth were iv injected with various numbers of (BALB/c X DBA/2)Fl (Ml&““, H-2d) cells from different organs. One week later their thymus cells were tested for GVHR inducibility in the routine PLNswelling assayin (BALB/c X DBA/2)Fl recipients. Cells inoculated into the neonates were resident peritoneal cells, following (0) or without (0) 2000 rad irradiation, bone marrow cells (m), thymus cells (A), and red blood cells (+). In one group (A with a), thymus cells were pretreated with anti-Thy- 1.2 + complement to kill T cells: this treatment killed more than 98% of thymocytes as judged by trypan blue dye exclusion. In another group (A with +), a thymus cell suspension was passed through a nylon-wool column to remove strongly adherent cells as well as B cells. Thymus cell populations nonadherent to both nylon-wool fibers and Sephadex G-10 beads (A) were also tested for their tolerance inducibility. Open circles represent antiMIS=-H-2” tolerance inducibility of peritoneal cells from (BALB/c X AKR)Fl (M1sbla, H-2““) mice with hybrid Mls” and MHC haplotypes.

Antigen-Specijic Intrathymic ated by Suppressor Cells

Tolerance Induced by Peritoneal

Cells Was Not Medi-

Experiments were done to test whether anti-Mls tolerance induced by injection of peritoneal cells was Mls-determinant specific but not mediated by suppressor cells, as was so in tolerance induction by bone marrow cells (20,23). As shown in Table 2, thymus cells of l-week-old BALB/c mice, which were tolerance induced by CDFl peritoneal cells, were unresponsive to the corresponding M~s”-H-~~ antigens of CDFl mice, but were normally responsive to unrelated M~s~-H-~~ antigens of (BALB/c X CBA/J)F 1 mice and to M~s~-H-~~ antigens, which had different MHC haplotypes,

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Specificity

Expt

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2

of Anti-Mls”-H-2d Tolerance Induced in the Thymus with Mls-Semiallogeneic Peritoneal Cells”

Recipients for GVHR (H-2, Mls) (BALB/c X DBA/Z)Fl (4 b/C9 (BALB/c X CBA/J)F

State of BALB/c

1

W@> MiD (BALB/c X DBA/Z)FI (4 b/@) (BALB/c X AKR)Fl WB, b/B)

LN index

f

P

SE(n)

2.73 f 0.16(5) l.lO~O.ll(5) 2.51 f 0.45 (4)

Nor. Tol. Nor. Tol.

2.43

Nor. Tol. Nor. Tol.

f

0.01

0.20(4)

2.14 f 0.21 (4) 1.28 f 0.06 (4) 1.721rO.17(3) 1.81

f

93

-

-

0.48

Nontol.h

0.02

-

0.26(2)

% Tolerance

NT‘

75

-

Nontol.

’ Newborn BALB/c mice (~24 hr) were injected iv with (BALB/c X DBA/2)Fl (H-2d, MIS”“) peritoneal cells at a dose of 5 X 106. One week later, the newborn thymus cells (Tol.) or thymus cells of untreated, age-matched BALB/c mice (Nor.) were tested for GVHR inducibility in Fl hybrid mice. b Nontolerant because of nonsignificant difference (P > 0.05) from or larger value of LN index than normal control groups. ’ Not tested.

of (BALB/c X AKR)Fl mice. On the other hand, neonatal tolerance induced by peritoneal cells was probably not mediated by suppressor cells, as judged by the cellmixture experiments shown in Table 3. Efect of MHC Antigen Dosage on Intrathymic, neal Cell Tolerogens

MIS Tolerance Inducibility

by Perito-

Comparison of tolerance induced in BALB/c neonates to M~s~-H-~~ antigens by inoculation of (DBA/2 X B lO.BR)Fl (Mls a’b, H-2d’k) peritoneal cells with that induced by (BALB/c X DBA/2)Fl (Mls b’a, H-2d) peritoneal cells suggested that the degree of anti-Mls tolerance induced was dependent on the MHC antigen dosage (open and closed circles, Fig. 3). Thus, almost twice as many cells of the former geno-

TABLE Neonatal

Group 1 2 3

Tolerance

Thymus Nor. Tol. Nor.

3

in the Thymus Induced without Thymic Lymphocyte Is Not Mediated by Suppressor Cells” cells

+ Tol.

LN index

f SE (n)

2.73 f 0.16(5) l.lO+-0.11(5) 3.65 k 0.37(3)

P 0.008 0.07

Chimerism

7%Tolerance

93 Nontol.’

’ Twenty million thymocytes from I-week-old BALB/c mice (Mlsb) neonatally tolerized with (BALB/c X DBA/2)Fl (Mlsbla) resident peritoneal cells (5 X 106) (Tol.) or those from normal, age-matched BALB/ c mice (Nor.) were tested for their GVHR inducibility in (BALB/c X DBA/2)Fl recipients. In the last group, a mixture of thymocytes of Tol. and Nor. was assayed. b Nontolerant due to no reduction of the response.

HOSONO ET AL. TABLE 4 Transient Induction of Intrathymic Tolerance by Injection of Semiallogeneic Peritoneal Cells without Thymic Lymphocyte Chimerism” Cells transfused into neonates

Expt I. Tolerance in the thymus (GVH activity) II. Tolerance in the mouse (HVG activity)

Weeks after birth 1 2 3 3 6

Peritoneal cells (5 % Suppression 83.8 f 49.0 rt 12.5 f 39.0 f 22.0 f

5.6 (6)b 24.5 (3) 9.6 (6) 15.0 (2) 11 .o (3)

X

106)

Bone marrow cells (2 X 10’)

% Chimerism

% Suppression

% Chimerism

0.0,0.1,0.1 ND’ 0.0,o.o

84.2+6.2(g) 90.0 + 4.0 (4) 85.2 f 8.5 (4) ND 100.0 + 0.0 (3)

0.0,0.0,0.1,0.1 0.5,0.6,0.6 6.7, 14.5,20.0

a Newborn BALB/c mice (Mlsb, Thy-l .2) were injected either with resident peritoneal cells or with bone marrow cells from (BALB/c X AKR)Fl (Mls b’a, Thy-1.1/2) mice. At various weeks after injection, the recipients were tested for degree of tolerance and chimerism in the thymus by means of GVHR inducibility and fluorocytometry techniques (Experiment I). Some individual mice were also tested for their HVGR activity to corresponding antigens (Experiment II). b Figures in parentheses indicate the number of PLNs tested. ’ Not done.

type were required as those of the latter to induce the same level of tolerance, as is the case of bone marrow cell tolerogens (20). Transient Induction of Intrathymic Tolerance by Peritoneal Cells without Development of Thymic Lymphocyte Chimer&m Neonatal BALB/c mice (Thy- 1.2) were injected either with bone marrow cells or with peritoneal cells from (BALB/c X AKR)Fl mice (Mlsbla, Thy-1.2/l). With both types of tolerogen cells, almost complete tolerance was reproducibly induced as early as 1 week after birth, without demonstrable chimerism in thymic lymphocytes (Table 4). However, a significant difference between the effects of the two cell types became apparent after 2 weeks of age. The tolerant state in the peritoneal cell-induced group gradually declined and disappeared by 3 weeks of age. During this period, no chimerism of Thy- 1. It cells was observed. On the other hand, in the bone marrow cellinjected group complete tolerance lasted for the period of observation and was accompanied by a gradual increase in the number of Thy- 1. l+ cells in the thymus (Table 4, Fig. 1). This was also so when the tolerant state was measured in individual BALB/c mice by local HVGR to the tolerance-inducing antigens. Thus, complete tolerance was observed in 6-week-old BALB/c mice injected with bone marrow cells, while in the peritoneal cell-injected group termination of tolerance had started by 3 weeks of age, and was completely abolished by 6 weeks of age (Experiment II in Table 4). Comparison between the Degree of Lymphocyte Chimerism and of Tolerance in the Thymus Although the tolerant state in bone marrow cell-transfused groups seemed to be correlated with thymic lymphocyte chimerism (Table l), it was possible that a unique

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Percent of donor-derived Thyl’ thymus cells FIG. 4. Dissociation of lymphocyte chimerism and the degree of tolerance in the thymus. BALB/c (MIs!, Thy-l .2) neonates were injected once with 5 X lo6 peritoneal cells of (BALB/c X AKR)FI (MIsbla, Thy1.1/2) mice to induce transient unresponsiveness in the thymus (see Table 4) and then reinjected iv with body weight-equivalent numbers of bone marrow cells from the same mouse hybrid strain. At 4 weeks of age, mice were killed, and GVHR inducibility and thymus cell chimerism were assessed by routine techniques.

cell type of nonlymphocyte lineage, participating in tolerance induction, was generated from the inoculated pluripotent bone marrow stem cells, and was continuously supplied into the newborn thymus, thus keeping thymocytes unresponsive to the tolerogens. If so, chimerism of thymic lymphocytes would not always be correlated with the degree of tolerance. To test this possibility, we induced tolerance in BALB/c neonates with peritoneal cells, causing no thymic lymphocyte chimerism (Table 4) and then 1 week later reinjetted them intravenously with bone marrow cells to maintain the tolerant state. At 4 weeks of age, the mice were killed and their thymus cells were tested for chimerism and degree of tolerance. As shown in Fig. 4, Thy- 1.1+ cell chimerism in four mice was0.1,4.4,5.1, and 9.5%, respectively, thus showing a loo-fold variation. However, all thymuses were completely tolerant. Thus, the tolerant state can be maintained by addition of pluripotent bone marrow stem cells without thymic lymphocyte chimerism. DISCUSSION In the negative selection of T cells active against self-antigens as well as against exogenous transplantation antigens, the thymus is thought to be a central and critical organ, in which T-cell development, differentiation, and repertoire generation, especially of MHC class II-reactive T cells, takes place (30-34), and it was shown that neonatal tolerance in this T-cell subset was easily induced as early as 1 week after birth (12, 18, 20, 22, 23). In this report, cells engaged in tolerance induction in the thymus were studied for Mls determinants, to which classII-reactive T cells preferentially responded in association with self-Ia antigens (25-29). Although we could not

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ET AL.

completely exclude an involvement of minor histocompatibility antigens other than Mls antigens in our experimental system in which Mls-semiallogeneic cells of ordinary strains of mice were used as antigens, this seems unlikely when judged by strain combinations among the neonatal recipients, tolerance-inducing cell donors, and cellular antigens used for assays (20,23). Cells participating in intrathymic tolerance in our system, referred to as toleranceinducing cells (TIC), probably existed throughout the body, though their presence in each organ probably varied. TIC and/or their immediate precursors were Thy- lnegative (non-T), nylon-wool-nonadherent (probably non-B), partially Sephadex Glo-adherent (weakly adherent) cells. Their functions were radiosensitive (Fig. 3) though they probably did not proliferate rapidly, as discussed later. The mechanism of intrathymic tolerance induced by peritoneal cells and by thymic nonlymphocytes seemed to be the same as that of bone marrow cells (20,23), in terms of specificity, absence of mediation by suppressor cells, and dependence on the MHC-gene dosage (Tables 2 and 3, Fig. 3). Thus, TIC, irrespective of their in vivo localization, probably belonged to the same cell population. A varying ability for different spleen cell types to induce neonatal tolerance was first reported by Weissman et al. ( 17), who indicated that the most effective cells for tolerance induction in splenic cytotoxic T-cell precursors were T cells; this has recently been confirmed by Ishizaka et al. (21), though the data may not be extended to cytotoxic T-cell precursors in the thymus ( 16). In contrast to this, nonlymphocytic adherent cells have been reported to be possibily the most effective spleen cells in neonatal tolerance induction in splenic interleukin-2 (IL-2)-producing, MHC class II-reactive T cells (21). These data are similar to those presented in this report on thymic GVHR-inducing cells, though the precise mechanism remains to be clarified. Using Mlsb-thymus-grafted Mlsa recipients, which had been irradiated and reconstituted with Mlsb bone marrow cells, Morrissey et al. (39) demonstrated that tolerance to Mls” determinants in the thymus was induced only intrathymically. We speculate that within a few days after iv injection TIC migrated into the newborn thymus and encountered thymocytes in which they induced tolerance. When tolerogens were peritoneal cells, the tolerant state induced was transient and abrogated within the following 2 weeks, indicating at least that the TIC were limited in their capacity for self-renewal. Early termination of tolerance may have been due to dilution of the TIC in the developing, growing thymus, since the number of cells per thymus generally increases about twofold between 1 and 2 weeks after birth or due to short-life span of TIC. The long-term intrathymic tolerance induced by a bone marrow cell injection, or by a peritoneal cell injection followed by a bone marrow cell injection, provides good evidence for bone marrow origin of the TIC. This is further supported by the similar mechanisms of tolerance induction shown by both peritoneal and bone marrow cells, as described above. TIC may be of a nonlymphocyte lineage, since tolerance induction was not always accompanied by chimerism in thymic lymphocytes (Table 4 and Fig. 4), and because there was a difference in kinetic profiles of the degrees of tolerance and T-lymphocyte chimerism in the thymus; TIC seemed to generate from bone marrow stem cells within a week, while it took at least 11 days for donor-derived thymic T cells to be detected in the host thymus (Fig. 1) (40,41). TIC remained in the nylon-wool- and G- lo-nonadherent fractions of thymus cell preparations, of which 0.5% were Thy- 1- cells as judged by trypan blue dye exclusion

INDUCTION

AND

MAINTENANCE

OF

INTRATHYMIC

173

TOLERANCE

(data not shown). A half-maximal tolerance seemed to be induced by 7 X lo7 cells of this preparation (Fig. 3), so that the largest number of TIC in each inoculum would have been 3.5 X lo5 (7 X lo7 X 0.005) cells per neonate. If we consider a dilution factor depending on an organs’ blood supply of -0.005 for soluble materials entering the thymus after iv injection into adult mice (42) and a ratio of thymus to whole body weight of 0.003 in neonates (data not shown), which have not yet developed a complete thymus-blood barrier (43), the number of TIC initially entering the thymus may be - 2 X 103. The number of donor-derived cells necessary to induce complete tolerance in the thymus of the newborn recipients can be calculated as -0.02% of total thymocytes (there being - lo7 cells/thymus at birth). This value does not seem insignificant when compared with the lowest chimeric percentage of about 0.2% donor-derived cells in bone marrow cell tolerance-induced thymus, reported by Morrissey et al. (24), since in the latter situation chimera should have been created in all bone marrow-derived thymocyte populations. Recent reports indicate that Ia-positive, bone marrow-derived nonlymphocytes but not thymic epitheiial cells are possibly the cells that participate in negative selection of self- and allo-class II-reactive T lymphocytes (33, 44, 45). This may also be the case for thymic anti-Mls tolerance in our system, since BALB/c (Mlsb) thymocytes developing in a (BALB/c X DBA/2)Fl (M~s~‘~) thymus grafted under the kidney capsule of thymectomized, lethally irradiated, syngeneic bone marrow cell-reconstituted BALB/c mice were normally responsive to the Mls” determinant 4 weeks after thymus grafting (data not shown). There may be at least two well-known types of nonlymphocytes of bone marrow cell origin in the thymus: dendritic cells and macrophages. Since anti-Mls tolerance is restricted to MHC, probably Ia haplotypes of tolerogen cells (23) TIC at the functional stage must be Ia positive. Together with the previously mentioned characteristics of TIC, one may consider dendritic cells as the most likely candidate, as Kyewski et ul. (46) have speculated on the basis of experiments with mice that received bone marrow cell injections as neonates. In these experiments, dendritic cells (Ia’) readily appeared in the medullary area of the thymus. However, the identity of the TIC cell type cannot be finally resolved until TIC activity has been identified by a clearcut demonstration of tolerance induction with a single cell type. For long-term tolerance, continuous generation of TIC regardless of the cell type, and presentation by them of nominal antigens as “self-antigens” to the thymic lymphocytes in which tolerance is to be induced (45), should be certified by the presence of their stem cells. ACKNOWLEDGMENT We thank

Mr. S. Araya,

Kansai

Medical

Laboratory

Center,

Kyoto,

for flow cytofluorometry

analysis.

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