168

F. Y. Liew and W. L. Chan-Liew

F. Y . Liew and W. L. Chan-Liew Department of Genetics and Cellular Biology, University of Malaya, Kuala Lumpur

Eur. J. Immunol. 1978.8: 168-171

Regulation of delayed-type hypersensitivity I I. Specific suppressor factor for delayed-type hypersensitivit to sheep erythrocytes in mice An antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is described. Lymph node cells and spleen cells from mice injected intravenously with 1 x lo9 SRBC 4 days previously were incubated in vitro for 48 h in culture medium. Supernatant obtained from the culture inhibited the induction of DTH t o SRBC in normal mice. It also suppressed the expression of DTH in presensitized mice. The suppression is specific as the suppressor factor had no effect on the DTH to a noncross-reacting antigen, chicken red blood cells. Treatment of the spleen cells with anti-theta serum and complement prevented the production of the suppressor factor, whereas treatment with anti-Ig serum and complement had no effect. Suppressor factor produced by H-2k mice suppressed the DTH in H-2b mice. The factor thus seems to act across the H-2 barrier. The suppressor factor was not removed by adsorption with goat anti-mouse immunoglobulin immunoadsorbent, but could be adsorbed by SRBC. It was stable at 5 6 OC for 1 h, but was partially inactivated by freezing and thawing. The factor has a molecular weight of less than 35 000 daltons.

1 Introduction

2 Materials and methods

Cell-cell interaction which involves diffusible cell products, has an obvious advantage over interaction involving direct cellular contact in that more cells can be affected rapidly. There are now a number of reports on suppressor factors released by T cells which are active in vivo and in vitro. Specific T cell suppressor factors for antibody responses [ 1-41, allotype suppression [ 5, 61 and homocytotropic antibody [ 7, 81 have been well documented. Specific T suppressor factor for a tumor system has also been reported [9]. Recently, Zembala et al. [ 101 produced T cell suppressor factor in vitro by incubating lymph node and spleen cells from mice injected with picryl sulfonic acid. This factor was antigen-specific and inhibited the passive transfer of contact sensitivity to picryl chloride when the cells transferred were incubated in it.

2.1 Mice

In the first paper [ 111 of this series, specific T suppressor cells for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice were described. The suppressor cells in mice were induced by a single high intravenous (i.v.) dose of SRBC. In the present paper, a specific suppressor factor produced by incubating the suppressor cells in vitro is described. This suppressor factor is T cell-dependent and antigen-specific. It inhibits the induction as well as the expression of DTH in vivo. The factor appears t o have a molecular weight of less than 35 000 daltons and appears to act across the H-2 barrier. It is adsorbable by antigen but not removed by anti-mouse immunoglobulin (MIg) immunoadsorbent. [I 19101 Correspondence: F. Y. Liew, Department of Experimental Immunobiology, The Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, GB. Abbreviations: DTH: Delayed-type hypersensitivity SRBC: Sheep red blood cells CRBC: Chicken red blood cells CY: Cyclophosphamide MIg: Mouse immunoglobulin

Inbred CBA and B10.129(5M) mice, 6-8 weeks old of both sexes, raised and maintained in our laboratory by brother and sister mating, were used. In all experiments, except those described in Sect. 3.5, CBA mice were used.

2.2 Previously described methods These include the induction of DTH to SRBC, the induction of suppressor cells to DTH, the measurement of DTH by the footpad swelling method, and the anti-@and anti-Ig treatments of the suppressor cells [ 111.

2.3 Harvesting and testing of suppressor factor Spleen cells or lymph node cells of mice injected i.v. with 1 x lo9 SRBC 4 days previously were obtained, and a single cell suspension was made in washing medium (RPMI 1640 with HEPES). The cells were spun gently and resuspended t o 2 x l o 7 viable cells/ml in complete medium (RPMI 1640 with 10 % fetal calf serum, sodium hydrogencarbonate, gentamycin and glutamine, pH adjusted t o 7.2 with C02). The cell suspension was dispensed into plastic petri dishes (2.0 ml/ 5.5 cm petri dish) and incubated for 48 h a t 37 O C in a humidifed atmosphere of 5 % C 0 2 in air. After incubation, the cell viability was normally reduced from 90 % to 50-60 %. The incubated cells were then resuspended and pelleted by centrifugation at 1 500 x g for 10 min at room temperature. The supernatant was collected and concentrated 5 x with Aquacide I1 (Calbiochem, San Diego, CA). To test for suppressive activity, normal mice were injected i.v. with 0.5 ml of the supernatant which was equivalent to the amount derived from 5 x lo7 viable cells. Control mice were injected with a supernatant derived from incubating normal spleen cells. Immediately after the injection, mice were simultaneously challenged in the left footpad with 1 x lo8 SRBC and intraperitoneally (i-p.) with 200 mg/kg body weight cyclophos-

Suppressor factor for DTH

Eur. J. Immunol. 1978.8: 168-171 phamide (CY). DTH reactions were elicited in t h e right footpad 5 days after challenge. To test f o r the suppression of DTH expression by the supernatant, 0.5 ml of concentrated supernatant was injected i.v. into mice which were sensitized 6 days previously with 1 x lo8 SRBC in t h e left hind footpad and i.p. with CY. DTH was elicited in these mice immediately after the injection of supernatant. 2.4 Adsorption with antigen Spleen cell supernatant was incubated with a n equal volume of packed SRBC o r chicken red blood cells (CRBC) for I h at room temperature. The adsorbed supernatant was harvested by centrifugation at 1500 x g a t room temperature. 2.5 Adsorption with anti-Ig immuoadsorbent

169

Table 1. Effect of suppressor factor on the induction and expression of DTH to S R B C ~ ) Source of supernatant

Recipients Challenge

Primed lymph node cells Normal Primed spleen cells Normal Normal spleen cells Normal Normal Primed lymph node cells Sensitized Primed spleen cells Sensitized Normal spleen cells Sensitized Sensitized Normal

+ + + + -

-

24-h footpad inaease (%) 9.8 * 1.8 10.6 2.5 21.6 f 2.2 +_

26.1 i: 1.6 9.1 2 2.3 10.2 k 2.1 31.3 i: 1.9 32.5 t 2.3 6.5 i: 1.1

a) For experimental details see Sect. 2.3.

Goat anti-MIg coupled t o CNBr-activated Sepharose 4B was a gift from Dr. U. Krawinkel. The immunoadsorbent was prepared according t o t h e method of Axen et al. [ 121, and its binding capacity and specificity have been described [ 131. 2.6 Fractionation of suppressor factor Ascending flow gel filtration chromatography was performed on a 2.6 x 89 cm column (Pharmacia, Uppsala) of Sephadex G-200 (Pharmacia) equilibrated with phosphate-buffered saline, pH 7.2, Flow rates of 20 ml/h were maintained. Supernatant of spleen cells from mice primed i.v. 4 days previously with 1 x 109 SRBC, was concentrated 5 x and applied in a volume of 7.5 ml. Fractions were collected in 3.2 m l volumes with an LKB fraction collector (LKB Produkter AB, Bromma, Sweden). Elution profile was traced by measuring t h e absorbance of alternate fractions a t 280 nm in a Beckman spectrophotomerer (Beckman Instruments Inc., Fullerton, CA). Three distinct peaks were obtained. Each peak fraction was pooled, concentrated and tested for suppressor activity. F o r details of the test see legend to Fig. 2.

3 Results 3.1 Suppression of DTH b y cell-free supernatant

Two separate experiments show that the supernatant of spleen or lymph node cells from primed donors completely suppressed either t h e induction o r the expression of DTH in t h e recipients, while the supernatant from unprimed cells had n o effect (Table 1). 3.2 Titration of suppressor factor

In the experiments reported above, each recipient received 0.5 ml of supernatant harvested from 5 x 10' spleen cells. This amount completely suppressed both the induction and the expression of DTH in the recipients. Fig. 1 demonstrates that as t h e supernatant was diluted, t h e suppressive effect linearly decreased.

4

1

0

I

10-2

DILUTION

OF SUPPRESSIVE

I

10-1

1

10

SUPERNATANT

Figure I. Dose response of suppressor factor on the induction of DTH. Supernatant of spleen cells from mice injected i.v. with 1 x lo9 SRBC 4 days previously, was injected (0.5 ml/mouse) i.v. in various

dilutions (in phosphate-buffered saline) into syngeneic recipients. Recipients were challenged immediately with 1 x lo8 SRBC in the right footpad and i.p. with CY. Five days later, DTH was elicited in the left footpad with 1 x 10s SRBC. Control mice (0)were not challenged. Vertical bars show 1 S.E.M. of 5 mice. MIg treatment 40.5 % and normal serum treatment 8.7 % of spleen cells. The cells were tested as before for their ability t o produce a factor which could suppress expression of DTH. Table 2 shows that supernatants from spleen cells treated with anti-Ig and C, o r normal serum plus C, were as suppressive as supernatant from untreated cells. In contrast, supernatant from spleen cells treated with anti-@ plus C contained no suppressive activity. The results clearly indicate that the production of the suppressor factor is a function of T cells, or at least, is T cell-dependent. 3.4 Specificity of suppressor factor

3.3 Effect of anti-@ treatment Spleen cells from mice primed with 1 x lo9 SRBC were treated with a n t i 4 and complement (C), anti-MIg and C o r normal mouse serum and C. Anti-@ treatment lysed 31.2 %, anti-

Supernatants of spleen cells from mice primed with 1 x 1O9 SRBC were injected i.v. into normal syngeneic mice which were treated with CY and injected into t h e footpad with either 1 x lo8 SRBC o r 1 x lo8 CRBC. Five days later, DTH

F. Y.Liew and W. L. Chan-Liew

170

Eur. J. Immunol. 1978.8.168-171

Table 2. Effect of anti-@treatment on the production of suppressor factor by spleen cell& Supernatant from cells treated with Anti-O + C Anti-Ig + C Normal serum Untreated

f

Studies were carried out to determine some of t h e biological properties of t h e suppressor factor. The suppressive supernatant was adsorbed with equal volumes of packed SRBC, CRBC or goat-anti-MIg immunoadsorbent at room temperature f o r 1 h with gentle agitation. The adsorbed supernatants were then tested f o r suppressive activity and for thermostability. Table 5 summarizes the data from a number of experiments. T h e suppressor factor was removed by adsorbing with SRBC, but n o t with CRBC or anti-MIg immunoadsorbent. It was resistant to heating a t 56 "C f o r 1 h but was partially inactivated b y freezing and thawing once.

24-11 footpad increase

(%I 32.5 i 3.1 12.6 f 0.5 11.9 f 2.1 9.3 * 2.7 33.0 i 3.5

C

3.6 Some biological properties of t h e suppressor factor

a) For experimental details see Sect. 3.3. reactions were elicited with either SRBC or CRBC. Results shown in Table 3 demonstrated that while t h e supernatant completely suppressed t h e induction of DTH t o SRBC, it had no effect o n t h e induction of DTH t o CRBC. T h e same specificity also held for t h e suppression of t h e expression of DTH (results not shown).

Table 5. Characterization of suppressor factord Supernatant

(70) Adsorbed with SRBC Adsorbed with CRBC Adsorbed with anti-MIg Freeze and thaw 56 "C, 1 h Untreated -

Table 3. Specificity of suppressor factoral Supernatant

Challenge

Elicit

24-h footpad

increase

a)

SRBC-primed SRBC-primed ~

-

CY CY CY CY

+ SRBC + CRBC + SRBC

+ CRBC -

SRBC CRBC SRBC CRBC SRBC CRBC

10.0 f 0.8 43.7 f 2.5 36.7 t 2.1 45.3 f 2.8 10.2 f 1.3 8.5 t 0.9

The suppressive supernatant was fractionated with Sephadex (3-200 column chromatography. The protein elution profile revealed 3 distinct peaks which were pooled separately, concentrated and tested for their ability to suppress the induction of DTH t o SRBC. Fig. 2 shows that Fraction 111 (peak 3 ) conMID BGG MN A PEPSIN RIBONUCLEASE

3.5 Suppressor factor acts across H-2 barrier

20

Supernatant prepared as in Sect. 2.3, was injected into normal syngeneic or allogeneic recipients which were challenged immediately with 1 x 1O8 SRBC and CY. DTH was elicited 5 days later. In a separate experiment, supernatant was also injected into recipients which had been sensitized 5 days previously with 1 x 1O8 SRBC and CY. Table 4 shows that t h e suppressor factor from CBA mice (H-2k) suppressed t h e induction and t h e manifestation of DTH i n b o t h t h e syngeneic recipients as well as t h e allogeneic B 10.129( 5M) mice (H-2b).

1 1 1 1

15

E

=

Fr I

0

10

u w

3 m m 0

205

-

Table 4. Suppressor factor acts across H-2 bamera) 0

Recipient

Challenge 24-h footpad increase (%)

Primed CBA spleen Normal CBA Primed CBA spleen Normal B10.129(5M) Normal CBA spleen Normal B10.129(5M) Normal B10.129(5M) Primed CBA spleen Sensitized CBA Primed CBA spleen Sensitized B10.129 (5M) Normal CBA spleen Sensitized B10.129(5M) Sensitized B10.129 (5M) __ Normal CBA Normal B10.129 (5M) a) For experimental details see Sect. 3.5.

25.5 i 1.5 10.6 i 0.7 9.6 f 1.0 25.1 f 1.5 10.5 f 0.6 8.2 f 0.5 35.6i 2.3

a) For experimental details see Sect. 3.6.

a) For experimental details see Sect. 3.4.

Source of supernatant

24-h footpad increase

+ f + t -

-

__

7.8 i 3.0 5.8 i 1.2 21.0i 2.3 22.2 f 0.2 8.9 2 2.5 7.6* 1.6 26.0+ 2.5 27.2f 2.2 6.4 * 1.1 5.5 i 0.8

0

50

100 1% FRACTION NUMBER

200

250

Figure 2. Fractionation of suppressive supernatant with Sephadex G-200 column. The following fractions were pooled: I (no. 60-73); I1 (no.90-105); I11 (no. 150-1 73). The pooled fractions were concentrated to 2.5 ml each. 0.5 ml of each fraction was injected i.v. into normal syngeneicrecipients which was immediately challenged subcutaneously with 1 x 108 SRBC. DTH was elicited 5 days after challenge. Histograms show the effect of various fractions on the induction of DTH. Whole = unfractionated supernatant; +ve C. = control mice receivedpo supernatant; -ve C. = control mice received no supernatant and were not challenged. Vertical bars show 1 S.E.M. of 5 mice. Arrows indicate the positions of chromatographicruns of standard proteins. BGG = bovine IgC (moL wt. 170 000); Con A = concanavalin A (mol. wt. 71 000); oeosin = hog stomach mucosa pepsin (mol. wt.'34000);ribonu&a& (moL G. 15 000).

Eur. J . Immunol. 1978.8: 168--171 tained all the suppressive activity of the supernatant, there being n o detectable activity i n Fractions I o r 11. Thus, the suppressor factor appears to be of small mol. wt. and may be rather homogenous. By comparing it with chromatographic runs of standard proteins, the mol. wt. of t h e suppressor factor was estimated t o be less than 35 000 daltons and close t o that of rihonuclease. 4 Discussion

The present paper describes a specific suppressor factor for DTH t o SRBC produced by spleen and lymph node cells of mice, which inhibits b o t h t h e induction and expression of DTH to SRBC. It is antigen-specific since (a) it inhibits DTH to SRBC but not DTH t o a noncross-reacting antigen CRBC, and (b) it is removable b y adsorption with SRBC but not with CRBC. Failure tcb remove t h e suppressor factor from the supernatant by anti-Ig immunoadsorbent indicates that t h e factor is nonIg in nature. This is in keeping with t h e earlier observation [ 111 that the suppression of DTH t o SRBC in the present system is not due t o t h e antibody which had been reported by Crowle and his colleagues [ 14, 151 and Mackaness et al. [ 161. It is also consistent with the report of Ramshaw et al. [ 171 who found that t h e suppression of DTH t o horse red blood cells was not mediated b y antibody, and that of Morikawa et al. [ 181 who showed that the suppression of DTH t o methylated human serum albumin was also not mediated by humoral antibody. Experiments using Sephadex gel filtration suggest that the suppressor factor has a relatively low mol. wt. I t should be pointed out that t h e mol. wt. estimation is only an approximate value. More rigorous determination will depend o n further purification. T h e separation was clear-cut, indicating that the factor may be rather homogenous. It should b e noted that due t o the large amount of material and number of animals needed for t h e in vivo test, it was not possible t o test the suppressive activity for each fraction. T h e total protein recovered for the pooled fractions accounted for approximately 65 % of the protein input. The pooled fractions were concentr,ited t o approximately the amount of protein that was used for injection with the unfractionated whole supernatant. This, however, should not seriously influence t h e results obtained, since even a 10-fold dilution of the whole supernatant still contained sufficient suppressive activity to suppress about 50 % of control DTH (see Fig. 1). A biochemical characterization of t h e suppressor factor is currently in progress. Suppressor factor prepared from spleen cells of SRBC-primed CBA mice specifically suppressed t h e DTH response t o SRBC in CBA and B10.129(5M) mice. These data suggest that t h e suppressor factor is effective across the H-2 barrier. This contrasts wil h t h e observations of Tada and Taniguchi [ 191 who found that a keyhole limpet hemocyanin-specific factor can only effectively suppress antibody responses of H-2-compatible strains. The suppressor factor for DTH t o SRBC in the present system is reminiscent of that of t h e GAT-specific suppressor factor of Kapp et al. [4] which could suppress antibody responses b y H-2-incompatible cells, t h e only restriction being that they must be nonresponder cells. The suppressor factor

Suppressor factor for DTH

171

for DTH t o SRBC also resembles the helper T cell-replacing factor described b y Mozes [20] and Taussig et al. [21] in that their manifestation was not H-2-restricted. Further serological and genetic analysis of t h e DTH suppressor factor will require congenic strains of mice. There seem t o b e certain common properties among t h e various soluble immunospecific factors described recently. They have similar mol. wts. (approximately 30 000-50 000), they lack Ig determinants but possess antigen binding sites and they have determinants recognized b y alloantisera specific for t h e I-region gene products. It thus seems likely that t h e antigen-specific suppressor factor for DTH described in t h e present report belongs t o this group of mediators. We wish to thank Mr. K. L. Teh for excellent technical assistance. We are grateful to Dr. U. Krawinkel for anti-MIg immunoadsorbent and Dr. B. A . Askonas for a n t t o serum. We are also indebted t o Professor Klaus Rajewsky and Dr. G. L. Asherson f o r helpful criticism.

Received October 5,1977; inrevised form December 23,1977.

5 References 1 Feldmann, M., Eur. J. Zmmunol. 1974.4: 667. 2 Tada, T., Taniguchi, M. and Takemori, T., Transplant. Rev. 1975. 26: 106. 3 Kishimoto, T. and Ishizaka, K., J. ZmmunoL 1974.112: 1685. 4 Kapp, J., Pierce, C. W. and Benacerraf, B., J. Exp. Med. 1977. 145: 828. 5 Jacobson, E.B., Eur. J. ZmmunoL 1973.3: 619. 6 Herzenberg, L. A., Okumura, K. and Herzenberg, L. A., in Singhal, S. K. and Sinclair, N. R. (Eds.), Suppressor Cells in Immunity, University of Western Ontario Press, 1975. p. 93. 7 Tada, T., Okumura, K. and Taniguchi, M., J. ImmunoL 1973. I l l : 952. 8 Okumura, K. and Tada, T., J. ZmmunoL 1974.112: 783. 9 Fujimoto, S., Greene, M. and Sehon, A. H., ImmunoL Commun. 1975.4: 201. 10 Zembala, M., Asherson, G. L., Mayhew, B. and Krejci, J . , Nature 1975.253: 72. 11 Liew, F. Y., Eur. J. ZmmunoL 1977. 7 : 714. 1 2 Ax&, R., Porath, J. and Ernback, S., Nature 1967. 214: 1302. 1 3 Krawinkel, U. and Rajewsky, K., Eur. J. ZmmunoL 1976.6: 529. 14 Crowle, A. J. and Hu, C. C., J. Allergy 1969. 43: 209. 15 Yonemasu, K. and Crowle, A. J.,Zmmunology 1973.25: 541. 16 Mackaness, G. B., Lagrange, P. H., Miller, T. E. and Ishibahi, T., J. Exp. Med. 1974.139: 543. 17 Ramshaw, I. A., Bretcher, P. A. and Parish, C. R., Eur. J. ZmmunoL 1976.6: 674. 18 Morikawa, S., Baba, M., Harada, T. and Mitsuoka, A., J. Exp. Med. 1977.145: 237. 19 Tada, T. and Taniguchi, M., in Katz, D. and Benacerraf, B. (Eds.), The Role of Products of the Histocompatibility Gene Complex in Immune Responses, Academic Press Inc., New York 1976. p. 513. 20 Mozes, E., in Katz, D. and Benacerraf, B. (Eds.), The Role of Products of the Histocompatibility Gene Complex in Immune Responses, Academic Press Inc., New York 1976, p. 485. 21 Taussig, M. J., MUNO, A. J., Campbell, R., David, C. S. and Stains, N. A., J. Exp. Med. 1975.142: 694.