in vitro induction of immunological tolerance

CELLULAR IMMUNOLOGY 124,187-201(1989)

In Vitro Induction of Immunological Tolerance

GIOVANNI BIAS, MARIA MAZZOCCHI, PAOLA ZANOVELLO, DINO COLLAVO, AND HILLIARD FESTENSTEIN*

Institute of Oncology, Inter-University Centerfor Cancer Research, University ofpadua, Padua, Italy, and *Department of Immunology, The London Hospital Medical College, London, United Kingdom

Received November 2,1988; accepted August 6, 1989

IL-2 was previously shown to induce cytotoxic effecters with a broad spectrum of target speci- ficities in thymus and spleen cell cultures. This study was designed to show whether T cells activated by H-2 allogeneic cells in MLC or by syngeneic tumor cells in MLTC are also potential targets for these cytotoxic effecters. We found that thymocytes activated in vitro for 5 days by rIL-2 were capable of killing tumor cells as well as activated T cells. Thymocytes activated by IL-2 were accordingly utilized as a means of effecting clonal deletion of T cells activated by H-2 allogeneic target cells in MLC. To establish whether the unresponsiveness is specific, IL-2-a&- vated thymocytes were added as third party cells to MLC and MLTC. The results showed that both T cells, proliferating in response to H-2 allogeneic cells, and CTL, reactive against syngeneic tumors or H-2 allogeneic cells, are eliminated from the T cell pool. Only alloreactive T cells are specifically eliminated in MLC by IL-2-activated thymocytes, as the remaining T cells are capable of proliferating and generating CTL in response to antigenically unrelated third party allogeneic cells. The possibility that unresponsiveness might be due to soluble factors was ruled out by studies performed with a diffusable “chamber insert” culture system. The results provide evidence that IL-2-activated thymocytes induce in vitro T cell tolerance. o 1989 Academic

Press, Inc.

INTRODUCTION

The precise manner in which the T cell repertoire develops and MHC restriction to self-antigens comes about is controversial ( l-5). A key question in this context is how immune tolerance to self occurs. It has long been postulated that several different processes, including clonal deletion and active T cell suppression, are involved in immune tolerance induction (6-8). Numerous reports indicate that specific anti-allogeneic MHC (9, lo), anti-haptenic (1 I), and anti-Moloney murine leukemia virus (M-MuLV) ( 12) T cell clones are missing following tolerization. However, unequivo- cal evidence showing that T cell clonal deletion is the way in which immune tolerance to self-MHC and self-Mls” occurs has been provided only recently (13-15); it was seen that immature T cells have the relevant rearranged T cell receptor genes while mature T cells emerging from the thymus lack them and consequently fail to express the specific autoreactive receptor(s). Tolerance therefore is most likely initiated and maintained in the thymus as previously shown in different experimental systems ( 16- 18), even though some studies indicate that T cell precursors are deleted in the pre- thymic compartment ( 19,20) or differentiated T cell clones are suppressed after leav-

187

0008-8749/89$3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

188 BIAS1 ET AL.

ing the thymus (2 1, 22). What is not yet established is the precise mechanism by which self-recognizing T cell clones are eliminated in the thymus.

To ascertain whether specific unresponsiveness could be induced in vitro by IL- 2-activated thymocytes, we evaluated whether T cell clones, potentially reactive to syngeneic leukemic or H-2 allogeneic target cells, could be specifically killed by these effectors themselves. The in vitro IL-Zactivated thymocytes used were either syngeneic or allogeneic to the responder cells. Both T cell proliferative and cytolytic assays were employed in these studies and provide evidence that IL-2-activated thymocytes induce in vitro T cell tolerance.

MATERIALS AND METHODS

Cell donors. Inbred C57BL/6 (B6, H-2b), BALB/c (H-2d), BALB.K(H-2k), BALB.B (H-2b), and AKR (H-2k, Thy 1.1) mice, originally purchased from the Jackson Labo- ratory (Bar Harbor, ME), were maintained by brother/sister matings and served as thymus and spleen cell donors at 1 week and 2 months of age, respectively. B6 mice, inoculated im with 0.05 ml of cell-free preparation of the Moloney murine sarcoma virus/murine leukemia virus (M-MSV/M-MuLV) with an in vitro titer of 5 X lo6 focus-forming units/ml on 3T3/FL cells, were used as “M-MSV-immune” spleen cell donors (23).

Cell suspensions. Thymus lobes, carefully cleaned to avoid mediastinal lymph node or blood contamination, and spleens were gently pressed through a nylon sieve and suspended in Dulbecco’s modified MEM (Flow Lab, Irvine, UK) supplemented with L-glutamine (2 X lop3 M), antibiotics, Hepes (3 X low2 M), 2-mercaptoethanol (3 X lop5 M), and 10% heat-inactivated fetal calf serum (Flow Lab);

Cell lines. The MBL-2 (H-2b) leukemic cell line originally induced by Moloney murine leukemia virus in B6 mice and the P8 15 (H-2d) mastocytoma cell line obtained in DBA/2 mice were maintained in ascitic form by ip passage in syngeneic recipients or by continuous in vitro cell culture.

Antibodies and C treatment. BALB.K anti-BALB.B (anti-H-2b) and BALB.B anti- BALB.K (anti-H-2k) sera, obtained from hyperimmunized mice as previously described (23), were used in a C (Cederlane, Hornby, Canada)-mediated cytotoxicity test for selective cell elimination at a final dilution of 1:80. Monoclonal anti-Thy 1.2 antibodies (NEN, Dreieich, Germany) were used for T cell depletion at a final dilution of 1:50,000. C-mediated cytotoxicity was carried out using a two-step proce- dure (23).

Cell cultures. IL-2-activated thymocytes (1.5 to 2 X lo6 cells/ml) were cultured in 50 ml of complete medium supplemented with 300 U/ml of recombinant interleu- kin-2 (IL-2, Glaxo Institute for Molecular Biology S.A., Geneva, Switzerland) for 5 days in a water-saturated atmosphere containing 5% CO;!. Cell viability, determined by the eosine dye exclusion method in different experiments, ranged from 27 to 36% of the initial cell number in culture.

Unless otherwise indicated, M-MSV-specific CTL were generated in vitro in a 5- day mixed leukocyte tumor cell culture (MLTC) system as previously reported (23). Briefly, cultures were carried out in 50-ml flasks (Falcon) with 25 X lo6 responder spleen cells from M-MSV-immune B6 mice and 5 X lo6 mitomycin C-blocked (m) MBL-2 stimulator cells in 15 ml of complete medium or in 35-mmdiameter wells (Costar 3406, Cambridge, MA) utilizing 7.5 X 1 O6 responders and 1.5 X 1 O6 stimula-

IN VZTRO INDUCTION OF T CELL TOLERANCE 189

tors in 4 ml of complete medium. Cell viability ranged from 39 to 46 and 20 to 28% of the initial cell number in culture on Days 3 and 5, respectively, of culture.

Bulk unidirectional standard allogeneic primary mixed leukocyte cultures (l- MLC) were set up with 2 X 10’ thymus or spleen responder cells and 3 X 10’ (m) spleen cells as stimulators in 15 ml of complete medium or with 5 X 1 O6 responder cells and lo7 stimulators in 35-mm Costar wells. Viable cell recovery of thymocytes cultured in MLC on Days 3 and 6 ranged from 27 to 32 and 13 to 19%, respectively, of the initial cell number in culture; viable spleen cell recovery ranged from 38 to 46 and 3 1 to 37% on Days 3 and 6, respectively. MLR detection was achieved in MLC made up of 6 X lo4 thymus or spleen responder cells stimulated with lo6 (m) spleen cells in a final volume of 0.2 ml in round-bottom 96-well microplates (Costar, 3799).

In third party cultures 7 X lo6 to lo7 thymocytes in bulk cultures and 2.5 X lo6 thymocytes in 35-mm-diameter wells were added. In some runs, 10’ thymocytes were isolated in a porous cell culture insert (24 mm) with 0.4-pm-diameter pores; the insert was placed in Costar dishes (Transwell, 3408), where MLTC or MLC were carried out in a total volume of 4 ml complete medium. The third party cells in MLTC and MLC consisted of unirradiated or irradiated (4000 rad) fresh cells or 5day IL-2- activated cells.

Secondary allogeneic MLCs (2-MLC) were carried out with the 1 -MLC responder cells that had been set up in 15 ml of complete medium. The third party unirradiated IL-Zactivated thymocytes added in the 1 -MLCs were eliminated by antibody and C treatments. After repeated washings, the recovered cells were adjusted to the desired viable cell concentrations and used in unidirectional 2-MLC set up with 5 X lo6 responders and 10’ stimulators (4 ml final volume) in 35-mm-diameter wells (Costar, 3406) or 6 X lo4 responders and lo6 (m) spleen cells as stimulators in 96-well microplates (0.2 ml final volume).

Mitogen-stimulated blast cells were obtained by culturing 25 X lo6 spleen cells in 10 ml of complete medium (2 X 1 06/cm2) containing 5 &ml of Con A (Pharmacia, Uppsala, Sweden) for 2 or 3 days in 50-ml flasks. Microcultures, carried out in 96-well microplates with 6 X lo4 spleen cells/well in 0.2 ml of complete medium containing 1 pg of Con A, served to detect mitogen stimulation.

Prolijkution assay. Eighteen Bequerels of [methy/-3H]thymidine ([3H]TdR, sp act 74 GBq/mM; New England Nuclear, Boston, MA) was added to MLC and Con A- stimulated microcultures 16 hr before harvesting, which was accomplished using a semiautomatic cell harvester (Skatron, Lyerbyen, Norway); [3H]TdR uptake was determined by liquid scintillation counting. The data are expressed as the average cpm in triplicate or in terms of stimulation index, SI = cpm (A + Bm) - (Bm)/cpm(A + Am) - (Am), where (A + Bm) and (A + Am) are experimental and control combinations of responder (A) and stimulator (Am, Bm) cells, respectively.

“0 release assay. Tumor cells and Con A-stimulated blast cells, as well as cells recovered on Day 3 from MLTC and MLC, served as target cells, which were labeled with 51Cr by incubating 3 X lo6 cells with 0.1 ml Na2”Cr04 (NEN) for 60 min at 37°C. Dead cells were removed by a lymphocyte separation medium (Plow Lab) and the residual labeled cells were washed three times. Cytotoxic activity of IL-Zactivated thymocytes and CTL generated in MLTC and MLC was determined by a 4-hr incubation “Cr release assay in which lo4 “Cr-labeled target cells and different numbers of effector cells were employed (23). Spontaneous release in tumor cells, Con A-activated blasts, and cells from MLTC or MLC was calculated by incubating target cells

190 BIAS1 ET AL.

in medium alone and ranged from 9 to 14, 16 to 23, and 17 to 22%, respectively, of the total S’Cr release obtained in Triton X-treated targets. Cytotoxicity was expressed as the percentage of specific lysis calculated as 100 X (experimental release - spontaneous release)/(total (Triton X) release - spontaneous release).

DNA release/fragmentation assay. A modification of the DNA fragmentation assay described by Duke and Cohen was used (24). Cells from cultures labeled with [3H]TdR (45 Bq/ml) during the last 24 hr of culture served as targets. Effector cell lytic activity was evaluated in a 4-hr incubation assay utilizing 2 X 1 O4 labeled targets mixed with different numbers of effecters in 0.2 ml of medium (Costar, 3799). The release of radiolabeled DNA for spontaneous and experimental conditions was mea- sured in 0.1 ml of microculture supematant using a liquid scintillation counter (cultures were centrifuged at 200g for 10 min). Total fragmented DNA, including the portion induced in target cells that did not yet undergo lysis during the incubation period, was also determined (24). Pelleted cells were lysed by adding hypotonic buffer (0.2 ml total volume/well) and the lysates were transferred in microfuge tubes and centrifuged at high speed ( 13,OOOg for 10 min) in order to measure radioactivity in 0.1 ml of ( 13,OOOg) supernatant, which contained only fragmented DNA, as well as in the remaining 0.1 ml, which included the pellet with intact DNA.

Maximum DNA release was evaluated in target cells as the total radioactivity present (incubation medium + 13,000g supematant and pellet). The percentage of released and fragmented DNA was calculated as follows: percentage DNA released = 100 (cpm incubation medium/cpm maximum release); percentage fragmented DNA = 100 (cpm incubation medium + 13,000g supematant/cpm maximum release). In the absence of effector cells, spontaneous DNA release in target cells ranged from 9 to 12%, while the values of spontaneous total fragmented DNA ranged from 18 to 26% of the maximum release values.

The percentage of specific released and fragmented DNA was calculated according to the expression 100 (percentage experimental release - percentage spontaneous release)/( 100 - percentage spontaneous release).

RESULTS

Cytotoxic Activity of IL-2-Activated Thymocytes

IL-Z-containing supematants in thymus and spleen cell cultures were previously shown to induce cytotoxic effecters that reacted with a broad spectrum of target speci- ficities (25). We studied whether activated T cells also constitute potential targets for these cytotoxic effecters.

Cytotoxic effecters, induced by culturing B6 thymocytes in the presence of high concentrations (300 U/ml) of recombinant IL-2 for 5 days, were assayed against a wide pattern of target cells including syngeneic (MBL-2) and allogeneic (P8 15) tumor cells, spleen cells obtained from 2-day Con A-stimulated cultures, splenocytes from M-MSV-immune mice restimulated in MLTC, and splenocytes and thymocytes activated in 3day MLC. The “Cr release assay results of one representative experiment (Table 1) show that IL-Zactivated thymocytes are capable of killing syngeneic and allogeneic tumor and normal target cells. In 12 repetitive experiments utilizing various target cell combinations, no significant differences were observed in the lytic activity exerted against syngeneic and allogeneic target cells, but Con A blasts and cells

IN VITRO INDUCTION OF T CELL TOLERANCE 191

TABLE 1

Cytotoxic Activity of IL-2-Activated B6 Thymocytes against Different Target Cells Evaluated by 5’Cr Release Assay

% Specific “Cr release”

Target cells 45:l 15:l 5:l

MBL-2 40 29 18 P815 38 24 17 B6 Con A bla.stsh 27 20 14 BALB/C Con A blastsb 26 19 11 B6 splenocytes anti-(MBL-2),’ 18 14 9 B6 splenocytes anti-(BALB/c),d 20 14 7 B6 thymocytes anti-(BALB/c),d 19 I4 6 BALB/c thymocytes anti-(B6),” 21 15 9

Note. m, Mitomycin C-treated. ’ “Cr release assay at different effector:target cell ratios. b Two-day Con A-stimulated splenocytes. ’ M-MSV immune B6 responder spleen cells (25 x 106) cultured in MLTC for 3 days with 5 X lo6 (MBL-

2), syngeneic leukemic cells. ‘Spleen or thymus responder cells (2 X 10’) cultured in MLC for 3 days with 3 X IO’ H-2 allogeneic

(m) spleen cells.

from MLTC or MLC were invariably less efficiently lysed (specific “Cr release rang- ing from 13 to 29% at 45: 1 effector to target cell ratio) than tumor cells (37-48%).

To evaluate if this lytic activity was directed against proliferating cells, a modified radiolabeled DNA release/fragmentation assay (24) was employed. MBL-2 leukemic cells, Con A-stimulated B6 splenocytes, and cells from H-2 incompatible MLC were labeled with [3H]TdR during the last 24 hr of culture, or with “Cr at the end of culture, and then used as target cells. Three runs were performed, and similar findings were obtained. The results of one representative experiment are reported in Table 2

TABLE 2

Cytotoxic Activity of IL-2-Activated B6 Thymocytes against Proliferating Target Cells Evaluated by DNA Release/Fragmentation Assay

Target cells”

% Specific DNA release’

15:l 5:l

% Specific fragmented

DNA’

15:l 5:I

% Specific “Cr release ’

15:l 5:I

MBL-2 26 18 82 64 33 21 B6 Con A blasts 27 19 70 52 17 12 B6 anti-(BALB/c), 25 16 64 49 13 7

Note. m, Mitomycin C-treated. a MBL-2 leukemic cells, 2-day Con A-stimulated splenocytes, and B6 splenocytes activated for 3 days

in MLC by (BALB/c), splenocytes were prelaheled with [‘H]TdR 24 hr before testing, or labeled with “Cr at the end ofculture, and were utilized for DNA release/fragmentation and “Cr release assays, respectively.

b DNA release, DNA fragmentation, and “Cr release assays at different effector:target cell ratios.

192 BIAS1 ET AL.

TABLE 3

The Effect of IL-2-Activated Cells on M-MuLV-Specific CTL Generation in MLTC”

Third party cells b Third party cells eliminated by’

% Specific 5’Cr released MBL-2 target cells

15:l 5:1 1.5:l

None B6 thymocytes IL-2-activated B6 thymocytes BALB.K thymocytes IL-2-activated BALB.K

thymocytes

None 43 26 18 None 41 23 16 None 0 1 0

Anti-H-Z’ + C 37 24 11

Anti-H-Z’ + C 2 2 1

’ M-MSV-immune B6 responder spleen cells (25 X 106) were cultured with 5 X lo6 (MBL-Z), for 5 days in MLTC.

b Fresh or IL-2-activated lymphoid cells (7 X 106) were added to MLTC at Time 0. ’ Third party cells were eliminated by anti-H-2k serum + C treatment prior to 5’Cr release assay. d 5’Cr release assay at different effector:target cell ratios.

and show that similar amounts of [3H]TdR are released by the different target cells into the medium (specific DNA release) during the 4-hr incubation test. Moreover, the effecters caused DNA fragmentation in all target cells, as shown by the high percentage of specific fragmentation detected at various effector to target cell ratios.

These findings indicate that IL-Zactivated thymocytes induce the nuclear damage (apoptosis) described by others in anti-H-2-specific CTL-mediated killing (24) and suggest that lytic activity against normal T cells is preferentially exerted against the cell subset that responds to antigens by proliferating.

Inhibition of CTL Generation and T Cell Proliferation Induced by IL-2-Activated Thymocytes

The effect of adding 5-day IL-Zactivated cells as third party cells to MLTC, in which B6 splenic T cells from M-MSV-immune mice were restimulated in vitro with the M-MuLV-induced (MBL-2), leukemic cells, is shown in Table 3. Five-day activated cytotoxic MLTCs specifically lysed the MBL-2 targets, and no inhibition of virus specific CTL generation occurred when fresh B6 thymocytes (third party control) were added to the MLTCs. Third party IL-2-activated syngeneic H-2b or allogeneic H-2k thymocytes, however, blocked CTL generation when added on Day 0 in MLTC. The third party allogeneic thymocytes were specifically eliminated after 5 days of culture with anti-H-2k sera and C prior to the 5’Cr release assay to avoid anti- H-2b cytotoxicity. In five out of five repetitive experiments, exposure to IL 2-activated syngeneic cells completely prevented virus-specific CTL generation in MLTC.

In addition, in six comparable experiments we observed that CTL generation in different H-2 incompatible allogeneic MLCs can also be blocked by IL-Zactivated thymocytes; the results of three such runs are reported in Table 4. When allogeneic IL-2-activated cells were used, irradiation or elimination with specific antibodies and C before testing precluded target cell lysis overlapping in the 5’Cr release assay. IL-2-


TABLE 4

The Effect of IL-2-Activated Thymocytes on Allospecilic CTL Generation in MLC

Effecters

MLC”

Stimulators

% Specific “Cr Third release*

party cells Third party cells b eliminated by’ 15:l 5:l l.S:l

Experiment 1 AKR thymocytes

AKR thymocytes

AKR splenocytes

AKR splenocytes

Experiment 2 BALB.K

splenocytes BALB.K

splenocytes

Experiment 3 B6 thymocytes

B6 thymocytes

B6 splenocytes

B6 splenocytes

(B6), thymocytes

(B6), thymocytes

(B6), thymocytes

(B6), thymocytes

(B6), thymocytes B6 thymocytes

(B6), thymocytes IL2-activated B6 thymocytes

WWchn splenocytes

(BALB/c)m splenocytes

@ALB/c)m splenocytes

@ALB/c), splenocytes

B6 thymocytes

IL-2-activated B6 thymocytes

B6 thymocytes

IL-Zactivated B6 thymocytes

None

(Rx)IL-2-activated B6 thymocytes

None

(Rx)IL-2-activated B6 thymocytes

Anti-Thy 1.2+c

Anti-Thy 1.2 +c

Anti-Thy 1.2+c

Anti-Thy 1.2 + c

MBL-2 target cells

20 14 8

0 0 1

24 17 11

0 0 0

Anti-H-2b + C 54 36 20

Anti-H-2b + C 0 I 0

None

None

None

None

P815 target cells

23 13 7

0 0 0

54 25 9

0 0 0

Note. m, Mitomycin C-treated. ’ Allogeneic primary MLC set up with 2 X 10’ thymus or spleen responder cells and 3 X 10’ (m) spleen

cells as stimulators. ’ Fresh or IL-2-activated (7 X 106), or 10’ 4000-rad-irradiated (Rx) thymocytes were added to MLC at

Time 0. ’ Third party cells were eliminated by anti-Thy 1.2 or anti-H-2b antibodies + C treatment prior to 5’Cr

release test. * 5’Cr release assay at different effector:target cell ratios.

activated thymocytes, but not fresh thymocytes, were able to abrogate allospecific CTL generation in the thymocyte or splenocyte responder cell populations in MLC (Table 4).

In the light of these findings, we explored the possibility that the observed lack of CTL generation might be due to the production of diffusible factors. We examined the potential inhibitory properties of soluble factors released by IL-2-activated thymocytes by means of a diffusible “chamber insert” culture system. As shown by a representative experiment reported in Table 5, no inhibitory effects on CTL generation were observed either in MLTC or in MLC. The same results were observed in

194 BIAS1 ET AL.

TABLE 5

Soluble Factors Released by IL-Z-Activated Tbymocytes: Effect on CTL Generation in MLTC and MLC

Cell cultures

Mixed cultures

Responders Stimulators Third

party cells

% Specific “Cr release b

Insert chamber’ 15:l 5:l 1.5:1

MLTC’ Anti-M-MuLV

B6 splenocytes Anti-M-MuLV

B6 splenocytes Anti-M-MuLV

B6 splenocytes

MLCd B6 splenocytes B6 splenocytes

WBI-4,

(MBL-Z),

(MBI-4,

MBL-2 target cells B6 thymocytes + 66 53 32

IL-2-activated + 64 56 40 B6 thymocytes

IL-2-activated - 3 2 2 B6 thymocytes

P8 15 target cells (BALB/c), splenocytes B6 thymocytes + 48 29 10 (BALB/c), splenocytes IL2-activated + 50 30 13

B6 thymocytes

Note. m, Mitomycin C-treated. B Fresh or IL-Z-activated thymocytes (10’) were isolated in a culture chamber insert with 0.4~pm-

diameter pores (+), or mixed in MLTC (-). b -Wr release assay at different effkctor:target cell ratios. ’ MLTC carried out with 7.5 X lo6 M-MSV-immune B6 responder spleen cells and I.5 X lo6

(MBL-2), . d MLC carried out with 5 X lo6 responder and 10’ (m) stimulator cells.

four different MLTC runs using doses up to 2 X lo7 of IL-2-activated thymocytes; thus, direct contact with the responder cell population is absolutely necessary to prevent effector cell generation.

The next set of experiments was carried out to test whether IL-Zactivated thymocytes were also capable of inhibiting T cell proliferation (Table 6). Primary MLCs with thymocytes and splenocytes as responding cells were set up together with H- 2 incompatible (m)-treated splenocytes: the combinations were B6 thymocytes or splenocytes stimulated by (BALB/c), splenocytes (experiment 1) and vice versa (experiment 2). B6 thymocytes were cultured for 5 days with IL-2, irradiated with 4000 rad, and then added as third party cells to the MLCs at Time 0; irradiation prevented [3H]TdR uptake without interfering with cytotoxic activity for the following 2-3 days only (data not shown). Control cultures consisted of (a) a set of primary autologous MLCs with no third party cells and(b) H-2 incompatible MLCs set up in the presence of third party freshly irradiated thymocytes, while the above-described IGZactivated and irradiated thymocytes served as third party cells in the experimental cultures (c).

The two experiments reported in Table 6 were repeated twice. Baseline uptake in the thymocyte control cultures (a) was invariably much lower than in the correspond- ing splenocyte control cultures (32 1 f 3 1 vs 1148 + 207 mean cpm f SD). In H-2 incompatible MLC (b) the mean f SD stimulation indexes (SI) were 6.7 + 2.1 and 8.8 f 1.8, respectively. However, when the IL-Zactivated cells were the third party instead of fresh thymocytes (c), uptake fell to levels seen in the autologous controls (a) (means +- SD Sl, 1.1 f 0.2 and 1.2 + 0.2, respectively).


TABLE 6

Inhibition of T Cell Proliferation by Third Party IL-Z-Activated Thymocytes

MLC”

Responders Stimulators Third party cells6

in culture t3H]TdR cpm SI’

Experiment 1 [a] B6 thymocytes [b] B6 thymocytes [c] B6 thymocytes

[a] B6 splenocytes [b] B6 splenocytes [c] B6 splenocytes

None (Rx)B6 thymocytes (Rx)IGZ-activated

B6 thymocytes None (Rx)B6 thymocytes (Rx)IL-2-activated

B6 thymocytes

324 - 1175 4.1 353 1.1

1324 - 9097 7.1 1835 1.4

Experiment 2 [a] BALB/c thymocytes [b] BALB/c thymocytes [c] BALB/c thymocytes

[a] BALB/c splenocytes [b] BALB/c splenocytes [c] BALB/c splenocytes

None (Rx)B6 thymocytes (Rx)IL2-activated

B6 thymocytes None (Rx)B6 thymocytes (Rx)IL-Zactivated

B6 thymocytes

286 - 2024 8.2

265 0.9

936 - 8136 10.2 1028 1.1

Note. m, Mitomycin C-treat&, Rx, irradiated 4000 rad. a MLC carried out with 6 X lo4 thymus or spleen responder cells and IO6 (m) stimulator spleen cells. ’ Fresh or IL-2-activated 4000~rad-irradiated (Rx) thymocytes ( 105) were added to MLC at Time 0. ‘Stimulation index as cpm (experimental - (m))/cpm (control - (m)).

Specijic Inhibition Exerted by IL-2-Activated Thymocytes

Primed lymphocyte culture experiments were set up with fresh thymocytes or splenocytes responding to H-2 allogeneic (m)-treated thymocytes or splenocytes in the presence of a third party cell (Tables 7 and 8). This last was an unirradiated/ irradiated fresh or 5day IGZstimulated suspension of B6 thymocytes. 2-MLC and mitogen stimulation were carried out following third party cell elimination with anti- Thy 1.2 or anti-H-2b antibody and C treatment. The (m)-treated splenocyte stimulators in 2-MLC were (a) autologous cells, (b) specific allogeneic cells (the same used as stimulators in 1 -MLC), and (c) H-2 unrelated ahogeneic cells.

The results of five representative experiments (in a total of nine) are reported in Tables 7 and 8 and show that the IL-2-activated thymocytes apparently eliminated only specific anti-target proliferating T cells in I-MLC. In fact, the response of thymocytes (Table 7) and splenocytes (Table 8) to specific allogeneic target cells in 2-MLC (b) was strongly reduced (overall mean SI -I- SD of four experiments, 2.3 k 1.1 and 2.3 f 1.3, respectively), while their response to H-2 unrelated cells (c) was not inhibited (12.6 + 5.9 and 14.1 -t 4.0, respectively). The partial SI inhibition (SI > 3) we observed in two out of eight different 2-MLCs (one reported in Table 8, experiment 2) in response to specific ahogeneic target cells might be explained by a residual response of the cells not activated and consequently not eliminated during I-MLC.

196 BIAS1 ET AL.

TABLE 7

Specific Inhibition Exerted by &Z-Activated Thymocytes on the Proliferative Responses of Thymocytes Activated in I-MLC

Responders

I-MLC’

Stimulators Third party cells

in culture?

2-MLC’

Third party cells Stimulator [‘H]TdR eliminated byd splenocytes CPd SI’

Experiment I AKR thymocytes

AKR tbymocytes

AKR thymocytes

AKR thymocytes

AKR thymocytes

AKR thymocytes

Experiment 2 B6 thymocytes

B6 thymocytes

86 thymocytes

B6 thymocytes

B6 thymocytes

B6 thymocytes

NW, tbmocytff

W), thymocytff

(B6A, thymocytes

(W, tbmocytes

(B6X. thymocytes

(B6x. thymocytes

(BALB/c). W.W splenocytes thymcqtes

(BALWc). (Rx)B6 splenocytes thymccytes

(BALB/c)m (Rx)B6 splenocytes thymocytes

@ALB/c), (Rx)ILZ-activated SpltTVXyt~S B6 thymocytes

(BALWc), (Rx)lLZ-activated spknocytes B6 thymocytes

(BALB/c). (Rx)IL-2-activated splenocytes B6 thymocytes

B6 thymocytes

B6 thymocytes

B6 thymocytes

IL-Z-activated B6 thymocytes

IL-Z-activated B6 thymocytes

IL-2-activated B6 thymwytes

[a] Anti-Thy 1.2+c

[b] Anti-Thy 1.2+c

[cl Anti-Thy 1.2+c

[a] Anti-Thy 1.2+c

[b] Anti-Thy 1.2+c

[c] Anti-Thy 1.2+c

(AKRLo 144

Pal 1157

(BAWc), 1032

W‘Wm 219

W), 333

(BAWc), 1946

[a] None Pal 319

[b] None (BALB/ch 2930

[cl None (BALB.K), 2151

[a] None Pa, 361

[b] None (BALB/c), 837

[cl None (BALB.K), 1683

-

14.0

12.4

1.8

12.3

-

Il.6

8.7

-

2.8

5.7

Note. m, Mitomycin C-treated. y Allogeneic primary MLC set up with 2 X IO’ thymus responder cells and 3 X IO’ (m) thymus or spleen

cells as stimulators. ’ Fresh or IL-2-activated (7 X 106), or IO’ 4000~rad-irradiated (Rx) thymocytes were added to MLTC at

Time 0. ’ 2-MLC carried out with 6 X IO4 thymus responder cells and IO6 (m) spleen stimulator cells. d Third party cells were eliminated by anti-Thy 1.2 antibodies + C treatment prior to use in 2-MLC. ’ Average cpm in triplicates at Day 3. ‘Stimulation index as cpm (experimental - (m))/cpm (control - (m)).

Moreover, in two experiments we observed that mitogen activation of the splenocytes recovered from I-MLC was not inhibited (Table 8); this further confirms that the splenocytes were deprived of only a relatively small subset of specifically activated T cells.

In the experiments shown in Table 9, B6 thymocytes and splenocytes or BALBK splenocytes were used as the responder cells. Irradiated and unirradiated IL-Zacti- vated thymocytes were once again employed as third party cells in l-MLC to elimi- nate the specifically activated T cells during the l-MLCs in which (BALB/c), or (BALB.K), cells stimulated B6 cells, and (BS),,, cells stimulated BALB.K cells. In 2- MLC, the responders were cocultured with the same stimulators used in I-MLC, or an unrelated H-2 allogeneic target cell. After 4 more days of secondary stimulation in MLC, CTL generation was evaluated using the following targets: P815 cells for

IN VITRO INDUCTION OF T CELL TOLERANCE

TABLE 8

Specific Inhibition Exerted by IL-2-Activated Thymocytes on the Proliferative Responses of Splenocytes Activated in I-MLC

197

Responders

I-ML?

Stimulators Third party cells

in cultur@

2-MLC’

Third party cells Stimulator [‘H]TdR eliminated byd ?&tlocyt~ CPIII' SI’

Experiment I AKR splenwytes

AKR splenocytes

AKR splenocytes

AKR splenocytes

AKR splenocytes

AKR splenocytes

Experiment 2 BALB/c splenocytes

BALB/c splenocytes

BALB/c splenocytes

BALB/c splenocytes

BALB/c splenocytes

BALB/c splenocytes

BALB/c splenocytes

BALBlc splenocytes

Experiment 3 BALB.K

SpletlGCyteS BALB.K

SplWXyteS

(B6), thymocytes

(B6), thymcqtes

(B6), thymocytes

Wk., thnocytff

(B6X. thymocytes

(B6), tbymocytes

(B6), splenocytes

(B6), splenocytes

(B6), splenocytes

(B6), splenacytes

(B6), splenocytes

(B6), splenocytes

(B6), splenocytes

(B6), splenocytes

(B6), splenocytes

(B6), splenocytes

B6 thymocytes

B6 thymocytes

B6 thymocytes

112-activated 86 thymocytes



(Rx)B6 thymocytes

(Rx)B6 thymocytes

(Rx)B6 thymocytes

(Rx)lL-Z-activated B6 thymocytes

(Rx)IL-2-activated 86 thymocytes


(Rx)B6 tbymocyies


B6 thymocytes Anti-H-2b + C Con A 15.004


Anti-H-2” + C Con A 48,245

[a] Anti-Thy 1.2+c

[b] Anti-Thy l.2fC

[c] Anti-Thy 1.2+c

[a] Anti-Thy 1.2+c

[b] Anti-Thy 1.2+c

[c] Anti-Thy 1.2fC

(AKRX.

@6),

(BALB/c)m

(AK%

Km,

(BALB/ch

1,143

16,285

12,521

1.396 -

2,486 I.8

17,414 12.8

[a] None

(b] None

[c] None

[a] None

[b] None

[c] None

(BALB/c)m

W),

WKR),

(BALB/c)m

@6),

(AKR),

1,248

26,236

16,930

2,464

9,864

46,63 I

Mitogen stimulation*

NOtIe Con A 18,090

N0ll.Z Con A 22,324

-

14.7

11.3

-

22.2

13.5

-

4.1

19.4

-

-

-

Note. m, Mitomycin C-treated. ” Allogeneic primary MLC set up with 2 X 10’ spleen responder cells and 3 X lo7 (m) cells as stimulators. ’ Fresh or IL-2-activated (7 X 106), or lo7 4000-rad-irradiated (Rx) thymocytes were added to MLC at

Time 0. ’ 2-MLC carried out with 6 X IO“ spleen responder cells and IO6 (m) spleen stimulator cells. d Third party cells were eliminated by anti-Thy 1.2 or anti-H-2 b antibodies + C treatment prior to use

in 2-MLC. ’ Average cpm in triplicates at Day 3. ’ Stimulation index as cpm (experimental - (m))/cpm (control - (m)). I Microcultures carried out with 6 X lo4 spleen cells plus 5 &ml of Con A for 3 days.

TABL

E 9

Spec

ific

Inhi

bitio

n Ex

erte

d by

IL-Z

-Act

ivat

ed

Thym

ocyt

es o

n th

e C

TL G

ener

atio

n in

I-M

LC

Effe

ctor

cel

ls fr

om 2

-MLC

Res

pond

ers

I-MLC

”

Stim

ulat

ors

Third

par

ty c

ells

in

cultu

reb

2-M

LCC

%

Spe

cific

5’C

r-rel

ease

r Th

ird p

arty

cel

ls

Stim

ulat

or

elim

inat

ed b

yd

sple

nocy

tes

Targ

et c

ells

’ 15

1 5:

l IS

:1

Expe

rimen

t 1

B6 th

ymoc

ytes

B6

thym

ocyt

es

(BA

LB/c

), sp

leno

cyte

s (B

ALB

/c),

sple

nocy

tes

(Rx)

B6 t

hym

ocyt

es

(Rx)

IGZa

ctiv

ated

B6

thym

ocyt

es

Non

e N

one

P81

5 21

13

10

P81

5 0

0 0

Expe

rimen

t 2

B6 s

plen

ocyt

es

B6 s

plen

ocyt

es

B6 s

plen

ocyt

es

25

17

6 E 9

39

27

14

Ki

(BAL

B.K)

, sp

leno

cyte

s (B

ALB.

K),

sple

nocy

tes

(BAL

B.K)

, sp

leno

cyte

s

Non

e N

one

(Rx)

IL-2

-act

ivat

ed

B6 th

ymoc

ytes

(R

x)IG

2-ac

tivat

ed

B6 th

ymoc

ytes

B6

thym

ocyt

es

B6 th

ymoc

ytes

(R

x)IL

-2-a

ctiv

ated

B6

thym

ocyt

es

(Rx)

IL-2

-act

ivat

ed

B6 th

ymoc

ytes

Non

e (B

ALB.

K),

Non

e (B

AW

dm

Non

e (B

ALB.

K),

H-2

’ C

on A

bla

sts

P81

5

1 1

2 2 F

H-2

k C

on A

bla

sts

B6 s

plen

ocyt

es

(BAL

B.K)

, sp

leno

cyte

s N

one

P81

5 M

BG

2 P

815

. 31

17

9

40

35

25

49

31

13

BALB

.K s

plen

ocyt

es

(B6)

m sp

leno

cyte

s BA

LB.K

spl

enoc

ytes

(B

6)m

sple

nocy

tes

BALB

.K

sple

nocy

tes

(B6)

, sp

leno

cyte

s

Ant

i-H-2

b +

C

Ant

i-H-2

b +

C

Non

e M

BL-

2 5

5 4

BALB

.K s

plen

ocyt

es

(B6)

, sp

leno

cyte

s N

one

@A

Wc)

m

P81

5 54

40

21

Not

e. m

, Mito

myc

in

C-tr

eate

d; R

x, I

rradi

ated

400

0 ra

d.

a Allo

gene

ic p

rimar

y M

LC s

et u

p w

ith 2

X 1

0’ th

ymus

or s

plee

n re

spon

der c

ells

and

3 X

10’

(m) s

plee

n ce

lls a

s stim

ulat

ors.

b F

resh

or I

L-2-

activ

ated

(7

X 10

6), o

r 10

’ 400

0~ra

d-irr

adia

ted

(Rx)

thy

moc

ytes

wer

e ad

ded

to M

LC a

t Tim

e 0.

c 2

-MLC

car

ried

out w

ith 5

X lo

6 re

spon

der c

ells

and

10’

(m)

sple

en s

timul

ator

cel

ls.

d Thi

rd p

arty

cel

ls w

ere

elim

inat

ed b

y an

ti-H

-2 b

seru

m +

C tr

eatm

ent

prio

r to

use

in 2

-MLC

. ’ T

umor

cel

ls o

r mito

gen-

stim

ulat

ed B

ALB.

K sp

leno

cyte

s at t

hird

day

of c

ultu

re.

J5’C

r rel

ease

assa

y at d

iffer

ent e

ffect

or:ta

rget

cel

l rat

ios.


anti-BALB/c sensitization, H-2k Con A blasts for anti-BALB.K sensitization, and MBL-2 cells for B6 sensitization. The results of two representative experiments (out of four) showed that only the T cell clones specifically responsive to the target antigens had been eliminated by the IL-Zactivated thymocytes in 1 -MLC, since the remaining T cells were unable to generate CTL specific for the relevant allogeneic target cells.

DISCUSSION

Our findings show that thymocytes cultured in the presence of IL-2 generate LAK- like cytotoxic effecters capable of killing syngeneic and allogeneic tumor cells, as well as lymphocytes stimulated by Con A mitogen, syngeneic tumors, or H-2 allogeneic determinants.

That IL-2-cultured thymocytes acquire a non-MHC-restricted cytotoxic activity has been widely demonstrated for murine and human systems (25-29), and in some cases they also lyse normal, lectin-stimulated, syngeneic, and allogeneic lymphocytes (25,27). These last observations and the results reported here suggest that LAK cells, in contrast with other reports (30, 3 l), do not spare normal target cells, since cell membrane changes in response to various signals seem to render the lymphocytes susceptible to LAK lysis. Interestingly enough, those T cell subpopulations that pro- liferate in response to antigens are efficiently lysed by the IL-2-activated thymocytes.

Furthermore, the addition of these effecters to mixed cultures abrogates the proliferative response to H-2 alloantigens as well as the generation of CTL reactive against syngeneic tumor or H-2 incompatible cells. Thus, it appears that the same populations that are killed in the cytotoxic assay are also eliminated during culture. In fact, IL-Zactivated effecters did not lyse the B6 splenocytes when these were used as targets in a “Cr release assay following their activation in MLC in the presence of (Rx) IL-Zactivated thymocytes (data not shown). Taken together, these findings indicate that the functional unresponsiveness we observed in MLTC and MLC is due to the elimination of antigen-reactive cell subsets by the IL-Zactivated thymocytes.

Numerous in vivo and in vitro findings suggest that T cell tolerance might be mediated by suppressor T cells (32-34), or by veto cells which inactivate CTL directed against antigens expressed on themselves (35, 36). Unlike veto cells, however, H-2b IL-2-activated thymocytes also abrogate the (H-2b) T cell response to the not shared H-2d and H-2k alloantigens. Moreover, suppressor and veto cells lack cytotoxic activity, while IL-2-activated thymocytes induce in vitro T cell unresponsiveness thanks to their lytic activity. Soluble factors, possibly released by IL-Zactivated cells, are not implicated in this phenomenon, since cell-to-cell contact is required to prevent antigen-specific cell differentiation. Therefore, in agreement with the recent demon- stration that spontaneous ( 13- 15) or induced (37) tolerance to Mls” antigens is due to a clonal deletion, the unresponsiveness we observed is also achieved by in vitro elimination of antigen-reactive T cell clones.

In our experimental model, alloreactive T cells in MLC are specifically deleted by IL-2-activated thymocytes, as the potential reactivity of the remaining T cells to unrelated antigens is not affected. An in vitro elimination of specific immunoreactive cells in MLC was also obtained by Zoschke and Bach (38) using a 5-bromodeoxyuri- dine photolysis method. In both cases, nonactivated cells remain unphased and therefore are spared, but the interesting aspect of our findings is that specific antigen unresponsiveness is induced by activated thymocytes possessing LAK-like activity.

BIAS ET AL.

Following in vitro exposure to IL-2, thymocytes with LAK characteristics have been described in both CDV and CD4-8- subpopulations (25-29). The identifica- tion of the relevant subpopulation responsible for the elimination of the alloacti- vated T cells in our experiments is currently under study.

The main point that remains to be clarified is whether our in vitro findings are relevant to the in vivo situation. If LAK-like cells have a role in in vivo tolerance induction, this implies that IL-2 must be available within the thymus. Indeed, cells competent for varying amounts of IL-2 production are present in the thymus (29, 39), but high, not physiological IL-2 doses are required to generate cytotoxic thymocytes in vitro.

On the other hand, even in the early stages of thymocyte development, an IL-2- dependent autocrine pathway seems to sustain intrathymic precursor cell proliferation (39). It has also been observed that anti-IL-2 receptor antibodies inhibit T cell proliferation and differentiation when added to fetal organ cultures (40), despite a recent report to the contrary (4 1). Moreover, by adding a high amount of IL-2 to the organ cultures, cell proliferation and the appearance of CD4+8+ and CD4+ thymocytes were inhibited, thus suggesting that IL-Zactivated thymocytes become cytotoxic and involved in thymic cell selection (41, 42). These results seem to indicate that while IL-2 is utilized in the thymus for proliferation and differentiation, the presence of surplus IL-2 restricts the aliquot of proliferating cells and CD4+8+/CD4+ emerging cells that might be targets of clonal elimination in the self-recognition process. Therefore, if IL-2/IL-2 receptor interaction plays a role in both the proliferation and the elimination taking place in the thymus, it is conceivable that different amounts of IL-2 might be present in some microenvironmental sites, and that IL-2 could reach a sufficient concentration for cytotoxic cell generation when or where autoreactive cells are activated by accessory cells carrying the relevant autoantigens.

The existence of IL-2-activated thymocytes within the thymus under physiological conditions has not yet been demonstrated, and we did not observe inhibition of antigen responses by mature lymphocytes following addition of fresh thymus cell suspensions to MLTC or MLC. However, putative LAK-like cells might possibly be diluted out when the thymic cell suspensions are prepared. In conclusion, even if the mechanism described in this study has not yet been shown to occur in vivo, our findings delineate a novel approach to study the process underlying antigen-reactive T cell clone elimination during tolerance induction.

ACKNOWLEDGMENTS

This work was supported by grants from the Consiglio Nazionale delle Ricerche, PFO No. 87.01189.44, 87.01239.44, from the Associazione Italiana per la Promozione delle Ricerche sul Cancro, and from the Medical Research Council of Great Britain. We are grateful for excellent technical assistance provided by S. Mezzalira, and to Denny Williams for preparing the manuscript. Maria Mazzocchi is recipient of a fellowship of AIRC.

REFERENCES

1. Jeme, N. K., Eur. J. Immunol. 1, 1, 1971. 2. Fink, P. J., and Bevan, M. J., J. Exp. Med. 8,766, 1978. 3. Zinkemagel, R. M., Callahan, G. N., Althage, A., Cooper, S., Klein, P. A., and Klein, J., J. Exp. Med.

147,882,1978. 4. Kruisbeek, A. M., and Longo, D. L., Suw. Zmmunol. Rex 4, 110, 1985.


5. Schwartz, R. H., In “Advances in Immunology” (F. J. Dixon, Ed.), Vol. 38, p. 3 1. Academic Press, San Diego, 1986.

6. Bumet, F. M., “The Clonal Selection Theory of Acquired Immunity.” Cambridge Univ. Press, New York, 1959.

7. Nachtigal, D., Zan-Bar, I., and Feldman, M., Transplant. Rev. 26,87, 1975. 8. Weigle, W. O., Sieckmann, D. G., Doyle, M. V., and Chiller, J. M., Transplant. Rev. 26, 186, 1975. 9. Nossal, G. J. V., and Pike, B. L., Proc. Natl. Acad. Sci. USA 78,3844, 1981.

10. Feng, H. M., Glasebrook, A. L., Engers, H. D., and Louis, J. A., J. Zmmunol. 131,2 165, 1983. 11. Good, M. F., and Nossal, G. J. V., J. Zmmunoi. 130,78, 1983. 12. Collavo, D., Ronchese, F., Zanovello, P., Biasi, G., and Chieco-Bianchi, L., J. Zmmunol. 126, 774,

1982. 13. Kappler, J. W., Roehm, N., and Marrack, P., CeZl49,273, 1987. 14. Kappler, J. W., Staerz, U., White, J., and Marrack, P. C., Nature (London) 332,35, 1988. 15. McDonald, R. H., Schneider, R., Lees, R. K., Howe, R. C., Acha-Orbea, H., Festenstein, H., Zinkema-

gel, R. M., and Hengartner, H., Nature (London) 332,40,1988. 16. Chieco-Bianchi, L., Collavo, D., and Biasi, G., Adv. Cuncer Rex 51,277, 1988. 17. Morrissey, P. J., Bradley, D., Sharrow, S. O., and Singer, A., J. Exp. Med. 158,365, 1983. 18. von Boehmer, H., and Hafen, K., Nature (London) 320,626, 1986. 19. Bradley, S. M., Morrissey, P. J., Sharrow, S. O., and Singer, A., J. Exp. Med. 155, 1638, 1982. 20. Mot-r&y, P. J., Kruisbcek, A. M., Sharrow, S. G., and Singer, A., Proc. Natl. Acad. Sci. USA 79,

2003, 1982. 21. Benjamin, D. C., J. Exp. Med. 141,635, 1975. 22. Miller, S. D., Sy, M. S., and Claman, H. N., Eur. J. Zmmunol. 7, 165, 1987. 23. Biasi, G., Saggin, L., Dar& F., De Rossi, A., and Collavo, D., J. Zmmunol. 130, 1447, 1983. 24. Duke, R. C., and Cohen, J. J., Zn “Cytolytic Lymphocytes and Complement as Eff’tors of the Im-

mune System” (E. R. Podack, Ed.), pp. 35-68. CRC Press, Bcca Raton, FL, 1986. 25. Biasi, G., Rivarollo, R., Colmagro, R., Ronchese, F., and Collavo, D., J. Biol. Homeostatic Agents 1,

I19,1987. 26. Budd, R., Cerottini, J. C., and McDonald, H. R., J. Zmmunof. 137,3734, 1986. 27. Ballas, Z. K., and Rasmussen, W., J. Zmmunol. 139,3542, 1987. 28. Phillips, J. H., and Lanier, L. L., J. Zmmunol. 139,683, 1987. 29. Toribio, M. L., Alonso, J. M., B&cena, A., Gultierrez, J. C., de la Hera, A., Marcos, M. A. R., Mirquez,

C., and Martinez-A, C., Zmmunol. Rev. 104,55, 1988. 30. Grimm, E. A., and Rosemberg, S. A., Lymphokines 9,279,1984. 3 1. Rosemberg, S. A., J. Natl. Cancer Inst. 75,595, 1985. 32. Dorsch, S., and Roser, B., J. Exp. Med. 145, 1144, 1987. 33. Bitter-Suermann, H., and Shevach, E. M., Transplant. Proc. 15, 1851, 1983. 34. Tutschka, P. J., Belanger, R., Hess, A. D., Beschomer, W. E., and Santos, G. W., Transplant. Proc.

15,853, 1983. 35. Miller, R. G., Nature(London) 287,544, 1980. 36. Rammensee, H. G., Bevan, M. J., and Fink, P. J., Zmmunol. Today6,41, 1985. 37. MacDonald, H. R., Pedrazzini, T., Schneider, R., Louis, J. A., Zinkemagel, R. M., and Hengartner,

H., J. Exp. Med. 167,2005, 1988. 38. Zoschke, D. C., and Bach, F. H., J. Zmmunol. Methods 1,55, 197 1. 39. Rothenberg, E. V., McGuire, K. L., and Boyer, P. D., Zmmunol. Rev. 104,29, 1988. 40. Jenkinson, E. J., Kingston, R., and Owen, J. J. T., Nature (London) 329, 160, 1987. 41. Plum, J., and De Smedt, M., Eur. J. Zmmunol. 18,795, 1988. 42. Skinner, M., Legros, G., Marbrook, J., and Watson, J., J. Exp. Med. 165, 148 1, 1987.

in vitro induction of immunological tolerance

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