suppressor lyt 2+ t-cells demonstrated in mice late after thermal injury

Immunology Letters, 19 (1988) 33-40 Elsevier

IMI_. 01089

Suppressor Lyt 2 + T-cells demonstrated in mice late after thermal injury

Fuj io Suzuki and Richard B. Pollard Division o f Infectious Diseases, Department o f Internal Medicine, University o f Texas Medical Branch and Shriners Burns

Institute, Galveston, Texas 77550, I~]S.A.

(Received I1 April 1988; revision received 13 June 1988; accepted 15 June 1988)

1. Summary

Biological properties of suppressor cells mediating second suppressive state (S-SupS) of IFN-7 responsiveness in mice following thermal injury were investigated by their ability to inhibit [3H]thymidine incorporation by allogeneic cell-stimulated mononuclear cells (MNC) prepared from spleens of unburned normal mice (N-mice). Kinetic studies indicated that the suppressor cells appeared at approximately 15 days post-burn and persisted until 40 days after thermal injury. This cell population from the spleens of thermally injured mice (TI-mice) was inactivated by anti-Thy 1.2 monoclonai antibody (mAb) plus complement (C). Treatment with anti- mouse immunoglobulin and anti-Lyt 1.2 mAb followed by C failed to abrogate the activity. Hoewever, it could be eliminated by treatment with anti-Lyt 2.2 mAb and C. In addition, when Thy 1 + or l_yt 2 ~ T cells were depleted from spleen cells containing suppressor cell activities by treatment with mAbs and C, the suppressor activity for IFN-7 production by N- mice splenic MNC stimulated with concanavalin A was also eliminated. However, no alteration in the suppression of IFN production was observed when this splenic cell population from TI-mice was treated with anti-Lyt 1.2 mAb and anti-mouse Ig followed by C. Since it has been reported [1] that the suppres-

Key words: Thermal injury; Suppressor T-cells; MLR; Second suppressive state; IFN-3, production

Correspondence to: Dr. E Suzuki, University of Texas Medical Branch, Division of Infectious Diseases, Department of Internal Medicine, Clay Hall, Rt. H-82 Galveston, TX 77550, U.S.A.

sor cell in spleens of Tl-mice could neither be re- moved by plastic adherence nor by carbonyl-iron treatment, this secondarily-generated suppressor cell was concluded to be a T cell possessing the Lyt 1-2" phenotype. The present report describes the demonstration, following a generation of suppressor macrophages [2], of suppressor Lyt 2- T cells spleen of mice late after thermal injury.

2. Introduction

Gamma interferon (IFN-y) appears to be a media- tor of cellular immunity that can be induced by various immunopotentiators [3], after stimulation of lymphocytes with mitogens [4, 5], anti-lymphocyte sera [6], bacterial and viral antigens [7, 8], and al- Iogeneic lymphocytes [9]. The production of IFN-'), in thermally injured mice (TI-mice) has been reported [10] to be selectively suppressed from 2-7 days post-burn and this suppression was mediated by splenic macrophages [2]. Recently, a second suppressive state (S-SupS) of IFN-7 responsiveness was described in mice 3-5 weeks after thermal injury [1]. In addition, spleen ceils obtained during S-SupS were shown to possess a defect in the production of IFN-7 in vitro, and they inhibited IFN-7 production when co-cultured with mononuclear cells (MNC) prepared from spleens of unburned normal mice (N- mice) in the presence of concanavalin A (Con A). When phagocytic ceils were depleted from spleen cells obtained from thermally injured mice (TI- mice), suppressor activity could still be demonstrated, which indicated that the suppressor ceils were not of the macrophage lineage [1]. The present paper

0165-2478 / 88 / $ 3.50 ~ 1988 Elsevier Science Publishers B.V. (Biomedical Division) 33

describes further biological properties of the suppressor cells detected in spleens of TI-mice during S-SupS of IFN-7 production and the obtained results suggesting that, following the generation of suppressor macrophages, suppressor 1 - 2 " T cells were demonstrated in spleens of TI-mice 15-40 days after burn injury.

3. Materials and Methods

3.1. Media, mitogens, IFN, cells and reagents

RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 5 × 10 -5 M 2-ME, 2 mM L-glutamine, penicillin G (100 U/ml) and streptomycin (100 ~g/ml), was used as mixed lymphocyte response (MLR) medium. Monolayer cultures of murine L cells were grown in 150-cm 2 tissue culture plastic flasks in Eagle's modified minimal essential medium (MEM) supplemented with 10070 FBS and antibiotics, and used as target cells for IFN titration. [3H]thymidine (specific activity = 6 Ci /mM) was acquired from Schwarz/Mann, Orangeburg, NY. Monoclonal antibodies (mAb) and antisera utilized included: anti- Thy 1.2 mAb (Accurate Chemical and Scientific Corporation, Westbury, NY), goat anti-mouse Ig antiserum (Cappel Laboratories, Inc., Cochraneville, PA), anti-Lyt 1.2 mAb, (New England Nuclear, Boston, MA), and anti-Lyt 2.2 mAb, (Cedarlane Laboratories, Ltd., Hornby, Ontario, Canada). Low-tox-M rabbit C was also obtained from Cedarlane Laboratories. The reference standard of murine IFN (G-002-904-511) was obtained from the Antiviral Substance Program of the Na- tional Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda [10]. Con A was used as IFN-3, inducer; and the reference IFN was used to standardize the IFN assay [10].

3.2. Mice and thermal injury

Eight-week-old male and female inbred BALB/c and CBA/J mice, propagated from breeders obtained from The Jackson Laboratory, Bar Harbor, ME, were used in experiments. A thermal injury was produced in mice which resulted in a third degree burn over approximately 30°7o of the total body surface area as described previously [10].

34

3.3 Preparation of responder and stimulator cells for MLR

Spleens obtained from normal BALB/c (responders) or CBA/J (stimulators) mice were teased onto steel mesh as previously described [2, 10]. The MNC were separated from spleen cell sus- pensions by Ficoll-Hypaque (density: 1.074 g/ml) centrifugation as described previously [2]. The cells in the interface were collected and resuspended in MLR medium at a cell density of 3 x 10 7 cells/ml. Stimulator cells were incubated in a 37 °C waterbath with 20 ~g/ml of mitomycin C (MMC, Mutamycin, Bristol Laboratories, Syracuse, NY) tor 20 min, washed three times and resuspended in MLR medium.

3.4. Preparation of splenic MNC containing suppressor cell activity

Five to 10× 106 MNC/spleen were obtained from TI-mice on day 20 after burn injury, and were pretreated with MNC in the same fashion as were the stimulator cells. In order to determine the time course of the appearance of suppressor cell activity after thermal injury, splenic MNC were obtained from mice at various intervals (0-50 days) postburn.

3.5. Assay of suppressor cell activity in MLR

Five × 104 responder cells in 0.05 ml and the same amount of MMC-treated stimulator cells were co-cultured in a 96-well round-bottomed microtiter plate (Flow Laboratories, Hamden, CT). Various concentrations of MMC-treated splenic MNC (3× 104 to 2.5× 105 cells/well) from TI-mice were added to produce a final volume of 0.15 ml/well for 6 days at 37 °C in a 5°/0 CO 2 incubator with a hu- midified atmosphere. The same volume of media was added to control wells. One ~Ci of [-aH]thymidine in 0.05 ml was added for the last 24 h of culture (7 days total incubation time), and the incorporation of [3H]thymidine into DNA was measured in a liq- uid scintillation counter (Beckman kS 6800). The inhibition of the proliferative response in MLR was ex- pressed as the percent suppression using the following formula: 1- (cpm in the presence of suppressor cells)/(cpm in the absence of suppressor cells) × 100.

3.6 IFN induction and assay

In the presence or absence of splenic MNC from Tl-mice which were pretreated with various block- ers, cultures of MNC prepared from spleens of N- mice were stimulated by 2 #g/ml of Con A, as described previously [2]. The IFN titer was calculated as the reciprocal of the greatest dilution of the test sample that reduced virus plaques by 50°7o and compared to murine standard IFN [1].

3.7. Treatment of splenic MNC containing suppressor cell activity with monoclonal antibodies" and antisera

Six groups of MMC-treated splenic suppressor cells obtained from BALB/c mice 20 days after burn were prepared at a concentration of 3 x 10 7 cells/ml in MLR medium. Four groups were treated with a 1:500 dilution of anti-Thy 1.2 mAb, a 1:10 dilution of anti-mouse immunoglobulin (anti-mouse Ig), a 1:500 dilution of anti-Lyt 1.2 mAb, or a 1:20 dilution of anti-Lyt 2.2 mAb. Following incubation at 4°C for 1 h, the blocker-treated groups and one other group were centrifuged (450xg for 10 min), resuspended in 2 ml of a 1:10 dilution of C in cytotoxicity medium (RPMI 1640 medium supplemented with 0.3% BSA, 25 mM Hepes buffer, 2 mM L-glutamine and antibiotics) at a concentration of 3x107 cells/ml, and incubated for 1 h in a 37 °C waterbath. All 6 groups were then washed with the media, centrifuged three times (450xg for 10 min) and resuspended in MLR medium for MLR or MEM supplemented 2°70 FBS and antibiotics for IFN induction at concentration of 1 - 2 x 10 6 cells/ml.

3.8. Statistical analysis

In inhibition studies, groups tested for suppressor activity were compared with the positive control in each experiment by Student 's t-test, with a p value <0.05 considered significant.

4. Results

4.1. Suppressor cell activity of splenic MNC from Tl-mice in MLR

As described previously [1, 10], the suppression of

IFN- 7 production appeared initially at day 3 and persisted until day 7 after thermal injury and a second decrease in IFN production was demonstrated in Tl-mice 3 weeks after burn injury. Therefore, the suppressor cell activity found in the S-SupS was ex- amined in MLR systems. First, splenic MNC from BALB/c N-mice (responders) were co-cultured with MMC-treated splenic MNC (stimulators) from CBA/J mice and MMC-treated splenic MNC from BALB/c mice 20 days after thermal injury (suppressors) in MLR. As shown in Table 1, MLR with responder and stimulator concentrations of 5 x 104 cells/well, and splenic MNC containing suppressor cell activity at a concentration of 1 × 105 cells/well, resulted in a maximum suppression of 84°/0, as compared to responder and stimulator alone 7 days after cultivation. A similar phenomenon occurred in MLR with responder and stimulator cell concentrations of 1xl05 and 2.5x104 cells/well, and 2x105 and 5 × 10 4 cells/well of suppressor ceils. The suppression of MLR induced by splenic MNC from TI- mice was statistically significant (p < 0.005).

4.2. Kinetics of the appearance of suppressor cell activity

To determine the time course of the appearance of suppressor cell activity after thermal injury, splenic MNC of Tl-mice werc obtained from BALB/c micc at 10, 15, 20, 30, 40, and 50 days after thermal injury and l x 105 MMC-treated putative suppressor cells were co-cultured with 5 x 104 responder and 5 × 104 MMC-treated stimulator cells in MLR. Suppressor cell activity was first observed at 15 days after thermal injury and persisted beyond 40 days postburn but was not detected at 50 days after thermal t rauma (Fig. 1). Subsequent experiments focused on suppressor cells at 20 days post thermal injury and on determining the nature of this activity.

4.3. Inhibitory effect of various concentrations of splenic suppressor cells on MLR

Since suppressor cell activity was described at a suppressor cell to responder cell ratio of 2:1, the effect of varying the concentrations of splenic MNC with suppressor cell activity was determined in MLR. Increasing numbers of splenic MNC from TI- mice (0.6× 104 to 1.7× 10 6 cells/well) were added to

35

TABI .E 1

Suppress ion of MI.R by splenic M N C from mice 20 days post thermal injury, a

M L R System b Responder S t imula to r M N C from cpm c Suppress ion ~' p (ce l l s /wel l l (cel ls /wel l ) T l -mice (%)

(cel ls /wel l )

Resc and Stit 1 x 10 :~ I x 105 8478 + 385 Res, S t i a n d M N C f r o m T l - m i c e 1 x 105 1 x 1(15 2 x 1() ~ 3 7 8 4 = 314 57 < 0 . 0 0 3

Rcs and Sti 5 x 104 5 x 104 23437 :. 1640 - Res, Sti and MNC from Tl -micc 5 × 104 5 x 104 1 x 105 3887 _+ 1821 84 < 0.001

Res and Sti 2.5 x 1(1 ~ 2.5 × 104 - 3721 _+ 1366 Res, Sti and M N C from Tl -mice 2.5 x 104 2.5 x 10 a 5 × 104 2657 & 551 45 <0.01

a Mice were subjected to a f l ame-burn resul t ing in a third degree burn over app rox ima te ly 30°7.,0 of the total body surface area. b A total vo lume of 0.15 ml of cell suspens ion /we l l was used in MLR. Responder cells were prepared f rom BAI .B /c N-mice. S t imula tor cells prepared f rom C B A / J N-mice were pre t rea ted with M M C (20 p.g/ml for 20 min) before in t roduc t ion into MI.R. MNC from TI-micc

20 days pos t -burn were pre t rea ted with M M C in the same fashion as were the s t imula to r cells.

Twcn ty - four h before harvest , 1 ,uCi of [~H]TdR was added to each well. d Percent suppress ion was ca lcu la ted by c o m p a r i n g cul tures with M N C from T l -micc to controls .

¢ Responders , splenic M N C from B A L B / c N-mice. S t imula to r s , M M C t rea ted splenic M N C from C B A / J N-mice.

19

• g

ao_

o-

0 I i I l I i I L L i

0 10 20 30 ~0 50

Days P0st Thermal Inlury

l:ig. I. Appearance of suppressor cell activity in mouse spleens after thermal injury. One x ]05 splenic mononuclear cells (MNC) obtained from BALB/c thermally injured mice at 10-50 days postburn were co-cultured ( o - ) with 5x lO 4 MMC- treated splenic MNC from CBA/J normal mice (stimulators) and the same number of splenic MNC from BALB/c normal mice (responders) in MLR. The % suppression (- • -) was calculated by comparing [3H]thymidine incorporation between cultures with putative suppressor cells and controls. The illustrations are mean values (circle) and deviations (bar) of [3H]thymidine incor-

poration.

5x10 4 responder and 5x104 stimulator cells. As shown in Fig. 2, increasing ratios of suppressor to responder cells resulted in an augmented suppression of blastogenesis of splenic MNC from BALB/c N-mice. While 0.6x 104 cells/well of splenic MNC of Tl-mice failed to inhibit blastogenesis of responder cells, 5.3x104 cells/well (suppressor:responder = 1:1) resulted in approximately 50°7o inhibition. At a ratio of 4.3:1 or higher, maximum suppression of this reaction was observed (more than 80°70 inhibition).

4.4. Monoclonal antibody and antiserum t r e a t m e n t s

The next experiments focused on the type o f lymphocyte responsible for the suppression. Anti-Thy 1.2 mAb, anti-Lyt 1.2 mAb, and anti-Lyt 2.2 mAb for the determination of T cell subpopulations and anti- mouse Ig for B lymphocytess, followed by C treatment were utilized. Splenic MNC containing suppressor cell activity were pretreated with mAbs or antisera at 4 °C for 1 h, followed by C treatment at 37 °C for 1 h before being introduced into MLR. As shown in Table 2, pre-treatment of splenic MNC obtained from Tl-mice with anti-Thy 1.2 mAb eliminated suppressor activity as compared to controls.

36

24- - - I00

I 20- - I • o I - -80 I

:~ 12- -

03 8-

4 t 200 d 6 , 's ' ' ' ' ' " IO.G 213 42.5 85.0 1700(x lO cells/well)

(O.hl) (O.$f} (0.5"1) (I.l:l) (2.1:1) (4.$'t) (8.5:1) ( l? ' l ) {$4:1)

Concentrotions Of Suppressor Cells (SUPPRESSOR CELLS:RESPONDER CELLS)

Fig. 2. Effect of various suppressor to responder cell ratios on the suppression of MLR. Five x 104 responder and 5 x 104 stimulator cells were co-cultured with graded numbers of suppressor cells ( - • - ) as indicated in MLR. Twenty-four h before harvest, 1 ~Ci/well of

[]H]thymidine was added to each sample and the percent suppression (- o -) was calculated as described.

A n t i - m o u s e Ig d id n o t dec r ea se the s u p p r e s s i o n sig-

n i f i c a n t l y (68°7o vs. 7107o in con t ro l ) . Th i s sugges t ed

t h a t t he s u p p r e s s o r cell was a T cell. To f u r t h e r

c h a r a c t e r i z e t h e s u p p r e s s o r ceils, an t i -Ly t m A b s

were e m p l o y e d . W h i l e t r e a t m e n t o f sp l en i c M N C

f r o m T I - m i c e w i t h an t i -L y t 1.2 m A b a n d C fa i led to

a b r o g a t e t he s u p p r e s s o r ac t iv i ty in M L R , an t i -Ly t

2.2 m A b plus C e l i m i n a t e d the s u p p r e s s i o n o f t he

M L R o f sp l en i c M N C o f N - m i c e (Table 2). Next ,

t he se s u p p r e s s o r ceils t r e a t e d wi th v a r i o u s b l o ck e r s

TABLE 2

Effect on various monoclonal antibodies and antisera treatments on suppressor cell activity of splenic MNC from TI-mice in MLR.

MLR system a Suppressor cells pretreated with: b cpm c Suppression d p (%)

Res e, Sti f, and Sup¢ Media 4781 _+ 677 71 < 0.001 Res, Sti, and Sup C alone 5451 +_ 541 67 < 0.001 Res, Sti, and Sup Anti-Thy 1.2 mAb + C 16067 ~ 2747 2 Res, Sti, and Sup Anti-mouse lg + C 5190 _+ 1405 68 < 0.001 Res, Sti, and Sup Anti-Lyt 1.2 mAb + C 6323 +_ 2619 61 < 0.002 Res, Sti, and Sup Anti-Lyt 2.2 mAb + C 17516 _+ 3390 0 Res and Sti - 16316 + 3586

a Cell concentrations used in MLR (0.15 ml/well) were: responder, 5 x 104 cells; stimulator, 5 × 10 a cells; suppressor, I × 105 cells. t, Splenic MNC from TI-mice were pretreated with various mAbs or anti-mouse Ig and C before introduction into MLR, as described

in text. c [3H]Td R (1 ,uCi/well) was added 24 h before harvest.

d Percent suppression was calculated by comparing cultures with MNC from Tl-mice to controls. e Responders, splenic MNC from BALB/c N-mice.

f Stimulators, MMC-treated splenic MNC from CBA/J N-mice. Suppressors, MMC-treated splenic MNC from BALB/c Tl-mice 20 days post thermal injury.

37

T A B I . t 3

S u p p r e s s o r cell ac t iv i ty o f T l -mice splenic M N C on IFN p r o d u c t i o n by N-mice M N C s t imula t ed wi th C o n A.

N-mice M N C were c o - c u l t u r e d wi th T l - m i c e M N C p re t r ea t ed wi th ''

C o n A IFN liter h S u p p r e s s i o n (2 #g / rn l ) ( U / m l ) (%,)

Med ia 84 C , 89 A n t i - T h y 1 . 2 m A b 4 C ~ 396

A n t i - M o u s e Ig + C ~ 81 A n t i - L y t 1.2 m A b 4- C ÷ 92

A n t i - L y t 2 . 2 m A b ~ C * 413 C o n t r o l d 427

Med ia < 6 C - < 6 A n t i - T h y 1.2 m A b + C < 6

A n t i - M o u s e lg + C - < 6 A n t i - L y t 1.2 m A b 4 (" < 6

A n t i - L y t 2.2 m A b + C < 6 C o n t r o l d < 6

80 ---. 0 .00 l 79 < 0.()01

6 N . S y 81 < 0.001

78 < 0.(X)I

3 0

a Tile N-mice M N C (I x 106 ce l l s /ml ) were c o - c u l t u r e d wi th 4 x 106 ce l l s /ml o f T l - m i c e M N C t r ea t ed wi th va r i ous m A b s or an t i s e r a fo l lowed by C , in the p resence or ab sence o f 2 /~g /ml o f C o n A. S u p p r e s s o r cells were p r e p a r e d f r o m TI -mice 20 days pos t t h e r m a l in ju ry .

b C u l t u r e f lu ids ha rves t ed 24 h a f t e r cu l t i va t ion were a s sayed for IFN act ivi t ies . c S t u d e n t ' s t - test .

d As a c o n t r o l , N-mice M N C (4 × lf~' ce l l s /ml ) were a d d e d .

c Not s ign i f ican t .

were co-cultured with N-mice splenic MNC in the presence of Con A. Twenty-four h after cultivation at 37 °C in CO2, culture fluids were harvested and as- sayed for IFN. As shown in Table 3, pretreatment of splenic MNC from Tl-mice with anti-Thy 1.2 and Lyt 2.2 mAbs followed by C eliminated suppressor cell activity. However, the treatment with anti-mouse lg antiserum and anti-Lyt 1.2 mAb followed by C did not alter the suppression of IFN production. There- fore, the suppressor cell demonstrated in the spleen of mice late after thermal injury appeared to be a T cell of the Lyt 1-2 + phenotype.

5. Discussion

Interferon-' , /production in mice 3-5 weeks post thermal injury has been reported as being decreased and phagocytic cell-depleted splenic MNC obtained from mice during this period inhibited IFN-3, production when co-cultured with N-mice splenic MNC stimulated with Con A [1]. In the present study some biological properties of the suppressor cells which mediated with the suppression of IFN-,,,,

responsiveness were investigated by their ability to inhibit blastogenesis of allogeneic cell-stimulated splenic MNC from N-mice and the IFN response by N-mice splenic MNC stimulated with Con A. Splen- ic MNC from mice 20 days after thermal injury could decrease the proliferation of syngeneic lymphocytes in a one-way MLR. The greatest suppression (84°'/o) was detected when 5 x 104 cells/well of responders (splenic MNC of BALB/c N-mice), the same number of stimulators (MMC-treated splenic MNC of CBA/J N-mice), and Ix 105 cells/well of MMC-treated splenic MNC obtained from BALB/c TI-mice were co-cultured. The suppression of the proliferation of responder cells was dependent upon the number of suppressor cells in the cultures, and 50% suppression of the proliferative response was observed when equal numbers of responder, stimulator, and suppressor cells were co-cultured. As previously described [1], the same suppressor cell preparation obtained from Tl-mice also inhibited IFN production when these cells were co-cultured with N-mice MNC in the presence of 2/xg/ml of Con A. Suppression of approximately 50% was observed

38

when N-mice MNC (I x 106 cells/ml) and Tl-mice MNC were co-cultured at a ratio of 1:1. Although the splenic suppressor cells during S-SupS of IFN-q, responsiveness were not of the macrophage lineage [1], they were sensitive to treatment with anti-Thy 1.2 mAb or anti-Lyt 2.2 mAb plus C, and insensitive to anti-mouse Ig or anti-Lyt 1.2 mAb plus C. This sug- gested that the cells mediating S-SupS were T cells with a Lyt I-2+ phenotypic classification.

Splenic suppressor T cells in burned mice which inhibited the generation of the primary antibody forming cell response by syngeneic normal cells had been reported [11]. Also, Kupper et al. [12] reported that Ly I ÷ 2 3 and Ly 1-23 ÷ T cells were involved in the suppression of immune responsiveness after thermal injury. Apparently, the suppressor cells appeared early and were of the Ly 1 ~ 23- phenotype or these cells acted as a suppressor-inducer of the Ly 1-23 ~ suppressor cell which subsequently mediated the suppression of the primary antibody response later in the post-burn period [11].

While the suppressor T cell described in this paper was detected approximately 15-40 days after thermal injury, the kinetics of the appearance of these suppressor cells differ. Miller and Claudy [11] reported suppressor cell activity was demonstrated between 4 to 7 days postburn, while Kupper et al. [12] observed suppressor activity on 3, 5, 7, and 15 days after thermal injury. In addition, Kupper et al. used

burned mouse splenic MNC as responder cells [12], while N-mice splenic MNC were used in the present study. Despite the possibility of two subpopulations of suppressor T cells being generated after thermal injury, the suppressor cells mediating the suppression of IFN-7 responsiveness 2-7 days postburn in mice were not T cells [2]. As well as the suppressor macrophage [2], suppressor T cells reported herein may play a role on immunological defects of burned patients and animals.

References

[I] Suzuki, F. and Pollard, R. B. (1987) J. Trauma 27, 379. [2] Suzuki, E and Pollard, R. B. (1982) J. Immunol. 129, 1811. [31 Matsubara, S., Suzuki, M., Suzuki, E and Ishida, N. (1980)

Microbiol. Immunol. 24, 87. [4] Johnson, H. M., Stanton, G. J. and Baron, S. (1977) Proc.

Soc. Exp. Biol. Med. 154, 138. [5] Reem. G. H., Cook, L. A., Henriksen, D. M. and Vilcek, J.

(1982) Infect. lmmun. 37, 216. [6] Falcoff, E., Falcoff, R., Catinot, L., Vomecourt, A. and San-

ceau, J. (1972) Re','. Eur. Etud. Clin. Biol. 17, 20. [7] Younger, J. S. and Salvin, S. B. (1973) J. Immunol. 11 I, 1914. 18] Ennis, E A. and Meager, A. (1981) J. Exp. Med. 154, 1279. [9] Ito, Y., Shimokata, K., and Maeno, K. (1982) Infect. lmmun.

37, 427. [101 Suzuki, E and Pollard, R. B. (1982) J. Immunol 129, 1806. [111 Miller, C. L. and Claudy, B. J. (1979) Cell. Immunol. 44,

102. [12] Kupper, T. S., Green, D. R., Chaudry, I. H., Clemens, M.

and Baue, A. E. (1981) Fed. Proc. 60, 1031.

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suppressor lyt 2+ t-cells demonstrated in mice late after thermal injury

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