Intravenous injection of allogeneic cells can prime T-lymphocytes with cytotoxic activity

Immunology Letters, 35 (1993) 13-18

0165

2478 / 93 / $ 6.00 4") 1993 Elsevier Science Publishers B.V. All rights reserved

IMLET 01886

Intravenous injection of allogeneic cells can prime T-lymphocytes with cytotoxic activity V a d i m V. K r o n i n , N a t a l y G. A n o s o v a , I g o r A. P o p o v , A n n N. P o l e s s k a y a , Boris D. B r o n d z a n d Y u r y S. K r i v o s h e i n Institute of Carcinogenesis, Cancer Research Center, Moscow, Russia

(Received 23 September 1992; accepted I October 1992)

I.

Summary

The possibility of specific cytotoxic T-lymphocyte (CTL) 2 induction was shown upon intravenous (i.v.) immunization of mice with 9 x 107 irradiated (2000 tad) allogeneic splenocytes. The induced CTL express the cell surface markers Thyl.2 +, L3T4- and Lyt2 +. No correlation between the level of cytotoxic activity and the ability to inhibit proliferation was shown in populations of lymphocytes, primed both by i.v. immunization and in mixed lymphocyte culture (MLC). The possible role of cytotoxic activity in downregulation of the immune response is discussed. 2.

Introduction

Intravenous immunization of mice with high doses of irradiated allogeneic splenocytes is the

Key words: Cytotoxic T lymphocyte; Down-regulatory activity; Alloantigen; Intravenous immunization; Mixed lymphocyte culture Correspondence to: V.V. Kronin, Laboratory of Immunity Regulation Mechanisms, Institute of Carcinogenesis, Cancer Research Center, Kashirskoye Shosse, 24, Moscow 115478, Russia. Abbreviations." CTL, cytotoxic T-lymphocytes; i.v., intravenous; MLC, mixed lymphocyte culture; T~, suppressor T cells; mAb, monoclonal antibody; C1, cytotoxic index; MC, mitomicin C; II, inhibition index; C', complement.

traditional system for inducing the specific suppressor activity of T-lymphocytes [1-5]. Without giving a strict definition of the suppression phenomenon [6], we shall mention that the existence of suppression as a separate function of T-lymphocytes implicates utilization of the unique mechanisms and mediators typical for this phenomenon. The existence of foreign functional activities in the studied cellular population can, in a number of systems, lead to inhibition of the immune response and therefore makes it difficult to reveal the effects of suppression [7]. In particular, the immune response could be decreased because of stimulator's alloantigen-specific CTL activity [8]. The necessity of paying attention to this occurrence has also been noted [9 12]. However, the answer to the question whether the specific cytotoxic activity of T lymphocytes can be induced under the above-mentioned i.v. immunization system is not yet clear. Although a number of groups have not taken this question into consideration [2,3,5], no specific cytotoxic activity has been revealed in the work [1]. However the principal possibility of CTL induction was shown in this work as far as the enrichment of induced upon i.v. immunization specific T-lymphocytes on donor macrophage monolayers resulted in bringing to light a high level of T-cell mediated cytotoxicity. The aim of our work was to investigate the given question more thoroughly.

13

3. 3.1.

Materials and Methods Mice

The inbred mouse strains C57B1/6 (B6:KbIbDb), C57B1/I0 (B10:KbIbDb), BALB/c (KdldDa), B10.BR (BR:KkIkDk), B10.M (KfIrDf), B10.D2(R101) (R101:KdIdDb), B10.MBR (MBR: KbIkD q) and B6.C-H-2 bml2 (bml2: KbIbml2Db) were provided by the nursery of Cancer Research Center. Both male and female mice aged 2 5 months were used in the experiments. The combinations were chosen in order to exclude a response to Y-antigen. 3.2.

L-transfectants

L t k - L-cells (H-2 k) and their transfected descendants 2-5 and 1-4, which express the D b and K b molecules, respectively, were generously provided by H. Allen [13]. L-cells DAP (H-2 k) and their transfected descendants FT 7.1 Cb and FT 7.2 C1H which express the I-A b and I-A bml2 molecules, respectively, were generously provided by R. Germain [14]. 3.3.

Monoclonal antibodies.

MAb G K 1.5, L3T4-specific [15], and mAb 3.168, Lyt2-specific [16] were generously provided by Dr. A. Mitchison. Thyl.2-specific mAbs G4 was produced in our laboratory and generously provided by Dr. A. Chervonsky. All mAbs were used as cultural supernatants. 3.4.

Intravenous immunization

Recipients were immunized by single i.v. injection of 9 x 10 7 2000 rad-irradiated allogeneic splenocytes. Immunization was conducted in BALB/c anti-B6, R101 anti-B6 and B6 anti-bml2 systems. The splenocytes of immune mice were tested on the 4th day after i.v. immunization. 3.5.

In vitro priming

5 x 106 responder cells (intact BALB/c splenocytes) were incubated in MLC with 10 6 irradiated (2000 rad) stimulators (intact B6 splenocytes). 14

Mixtures were incubated at 37°C in a 5% CO2 atmosphere in wells of 24-well plates (Linbro) in 2 ml of RPMI-1640 medium, supplemented with 10% of heat-treated fetal calf serum (all from Flow Laboratories), 2 mM L-glutamine/100 U/ml gentamycin/10 mM Hepes buffer (Flow). Mixtures were incubated for 120 h, after which the viable cells were counted and used in tests. 3.6.

Cytotoxicity assay

51Cr-labeled target cells (6 x 104 peritoneal macrophages o r l 0 4 L-cells per well) were settled in 96-well flat-bottomed plates (Linbro). Targets were washed twice and then incubated for 16 h with lymphocytes tested for cytotoxic activity [17]. The cytotoxic index (CI) was calculated according to the formula: CI = [ ( e - s ) / ( m - s ) ] x 100%, where e, s and m are cpm for 51Cr released, respectively, in the experimental variant, spontaneously and maximally (in the presence of 1% Triton X-100 (Calbiochem)). 3.7.

Assay on down-regulation o[prolf/bration

3 x 105 lymph node cells from BALB/c mice (responder cells) were incubated in MLC with 106 irradiated (2000 rad) spleen cells (stimulators) of B6-donor, B10.M-third-party and BALB/c-syngeneic strains. Mixtures were incubated for 120 h in 150/~1 RPMI-1640 medium, supplemented with 5% of heat-treated human serum, 5 x 10 5 M 2mercaptoethanol (Serva) and other ingredients. 16 h before termination of the culture [3H]thymidine was added (1 #Ci per well). To determine the level of inhibitory activity, 1 2 × 105 mitomycin C (MC) (Sigma)-treated (25/~g/ml, 30 min) immune (intact in control) BALB/c lymphocytes were added. The inhibition index (II) was calculated according to the formula: II = [1 (A-B)/(C D)] x 100%, where A and B denote [3H]thymidine incorporation (in cpm) in response to allogeneic and syngeneic stimulators, respectively in mixtures with addition of immune lymphocytes; and C and D the same indexes in mixtures with addition of intact lymphocytes.

3.8.

Assessment expression

of C T L

surface marker

I m m u n e splenocytes (107/ml) were treated for 40 min at 4°C with cultural supernatants from hybridomas, washed once and incubated with nontoxic rabbit c o m p l e m e n t (C') at 1/20 dilution for 50 rain at 37°C and finally washed three times. After counting the viable cells were used in the C T L assay.

TABLE 1 Cytotoxic activity of splenocytes, induced by i.v. immunization. The priming was conducted in BALB/c (H-2d) anti-B6 (H-2 b) combination. (A) Experiment Target cell (haplotype) (L-fibroblasts)

3.9. Statistics The statistical significance of the data was determined by the Student's t-test [18]. A P value o f less than 0.05 was taken as significant. 4.

4.1.

Results

Specific cytotoxic activity can be induced upon i.v. immunization of mice with high dose of allogeneic irradiated splenocytes

4

Cytotoxic index (%) Effector:target ratio 90:1

30:l

10:1

3:1

2-5 (H-2 k, D b) DAP (H-2 k)

12" 0

6

3

3

0

0

n.d.

1-4 (H-2 k, K b) DAP (H-2 k)

63* 34*

36* 20

23* 3

5 0

1-4 (H-2 k, K h) Ltk- (H-2 k)

22* 9*

5 0

n.d. n.d.

n.d. n.d.

1-4 (H-2k, K b)

27*

10"

n.d.

n.d.

(B)

Splenocytes of i.v. immunized recipients were assayed on cytotoxic activity on the fourth day after immunization. Layers of StCr-labeled macrophages or murine L-fibroblasts transfected with M H C genes ( H - 2 K b, H - 2 D b, I-A b, I-A bml2) were used as targets in cytotoxic assay. In some experiments the bulk populations of i m m u n e splenocytes were treated with M C before C T L assay. In total, 17 experiments were performed: 13 tests in BALB/c anti-B6, 2 tests in R101 anti-B6, and 2 in B6 a n t i - b m l 2 donor/recipient combinations. Specific cytotoxicity (CI > 10%, P <0.05) was observed in 7 experiments (BALB/c anti-B6 combination) and in one out o f two experiments in both other c o m b i n a t i o n s (Table 1, Fig, 1). Preliminary treatment of i m m u n e splenocytes with M C m a k e s the levels of cytotoxic activity lower but does not abolish it completely (Fig. 1).

4.2.

CTL induced following i.v. immunization display Thyl.2 +, L3T4 , Lyt2 + T cell surface markers

I m m u n i z a t i o n was p e r f o r m e d in BALB/c antiB6 system. It follows from Fig. 2 that the main p o p u l a t i o n of CTL, induced by i.v. immunization

Experiment Target cell (haplotype) (macrophages)

Cytotoxic index (%) Effector:target ratio 15:1

5:1

1.7:1 0.5:1

B6 (H-2 b) B10.M (H-2 r) BALB/c (H-2a)

58* 46 47*

100" 47 0

97 33 4

42* 32 8

B10 (H-2b) BR (H-2 k) BALB/c (H-2d)

19" 9 5

12" 0 9

n.d. n.d. n.d.

n.d. n.d. n.d.

MBR (KblkDq) BR (H-2k) BALB/c (H-2a)

37" 15 5

28* 12 2

n.d. 0 0

n.d. n.d. n.d.

n.d., not determined. *Significant difference between control and experimental variants (P <0.05). display T h y l +, L 3 T 4 - , Lyt2 + cell surface markers since cytotoxic activity almost disappears after the elimination of T h y l + or Lyt2 + cells (by 72% and 61%, respectively) and only slightly decreases after the elimination of L3T4 + cells (by 11%). Naturally, the h y b r i d o m a s ' supernatants were non-toxic (data not shown). 15

g SPECIFIC Cr-51

SPECIFIC Cr-51

REI~ASE

g SPECIFIC Cr-5t

RELEASE

70 , +

l

d i

RELEASE

:

k b~I ~_ D,P(a-A[

FTT.2CIH(H-2, I-k )

f

6O

25"

5O

40

D

30

I0 Z0 d

5 I0

-°-'-.'-~a 0

-° ~,~-~

. . . [. . . . .

^

3:1

10:I

30:1

90:1

10:1

b --[ ' 30:1

- 0

EFFECTOR:TARGET RATIO

EFFECTOR:TARGET RATIO

~,~

10:1

30:1

Q0:1

EFFECTOR:TARGET RATIO

Fig. 1. The influence of preliminary treatment with MC on the magnitudes of cytotoxic activity, induced in splenocytes of i.v. immunized recipients. Experiments were performed in BALB/c anti-B6 (A), RI01 anti-B6 (B), and B6 anti-bml2 (C) donor/recipient combinations. Cytotoxic activity of MC-treated (. . . . . . ) or untreated ( - - - ) splenocytes was studied. 5~Cr-labeled murine L-cells (see insets) were used as targets. SPECIFIC Cr-51 RELEASE

4.3.

The cytotoxic activity of immune splenocytes does not correlate with their ability to inhibit the in vitro proliferative response

25

Populations of immune lymphocytes, induced in BALB/c anti-B6 system by i.v. immunization and those obtained during MLC were both simultaneously tested in assays on cytotoxicity and down-regulation. In total, 5 experiments were carried out. No correlation between CI and II magnitudes was revealed (Table 2). The existence of strong cytotoxic activity in population of lymphocytes (Expt. 5) is not sufficient for inhibition of proliferation in MLC. On the contrary, the high level of inhibition can be observed in spite of the absence of any cytotoxic activity (Expts. 2 and 3).

20

15

10

5. 1

NOr TItFa~D

1

ANT[U~T4+C' ~

~

~g.r~+c'

~

~

~rl'I 1"n~q.a+C'

~+C'

Fig. 2. C T L induced following i.v. immunization display T h y l . 2 + , L 3 T 4 - , Lyt2 + cell surface markers. BALB/c anti-B6 spIenocytes were treated with mAbs plus C' and tested for cytotoxic activity. The figure presents the data (means and standard errors) of 4 experiments in which StCr-labeled B6 (H-2 b) and. BI0 (H-2 ~') macrophages and 1-4 (K b) L-cells were used as targets and equal doses of effector cells (9 x l0 s per well) were used.

16

Discussion

The results show that specific CTL are induced following i.v. immunization of mice with 9 × 10 7 2000 rad irradiated allogeneic splenocytes. Several donor/recipient mouse strain combinations were used: BALB/c anti-B6, R101 anti-B6 and B6 anti-bml2. Most of the induced CTL express the Thyl.2 ~, L3T4- and Lyt2 + markers. Exactly the same surface molecules were found on so-called

TABLE 2 The comparison of cytotoxic and down-regulatory activities in populations of immune splenocytes. Priming was conducted in BALB/c (H-2 a) anti-B6 (H-2b) combination. Immune splenocytes were studied simultaneously in assays on cytotoxicity and on down-regulation of proliferation (II magnitudes of specific activity are given). Expt.

Cytotoxic index (%) Target cell origin

Effector: target ratio

Inhibition index (%) Way of priming

Ts:responder cell ratio

i.v.

MLC

Way of priming i.v.

MLC

1

2-5(D b)

90:1

12"

83"

1:2

0

100"

2

2-5(D b)

90:1

0

64"

1:2

37*

87

3

1-4(K b)

90:1

4

25*

1: 1.5 1:3

78* 51"

92* 73"

4

1-4(K b)

90:1

63*

80*

1: 1.5 1:3

43* 14

100" 100"

5

B6(H-2 b)

15:1

58"

100"

1: 1.5 1:3

10 28"

0 0

*Significant difference between control and experimental variants (P <0.05).

specific suppressor T-cells (Ts), primed using the same immunization protocol [19,20]. The slight decrease in cytotoxicity after the treatment of immune splenocytes with anti-L3T4 mAb plus C' could be attributed to the presence of some L3T4 ÷ CTL in this population. The remaining cytotoxicity observed following the treatment of immune splenocytes with anti-Thyl.2 mAb plus C' is probably connected with cytotoxicity mediated by macrophages [1]. Cytotoxic activity of splenocytes of i.v.-immunized mice was observed in approximately half of our experiments and the levels of cytotoxicity varied considerably. One can suggest different reasons for such variability. The possible influence of latent infection can not be excluded either. Intravenous immunization with irradiated allogeneic splenocytes is known to induce down-regulatory (suppressor) activity of Tlymphocytes. However, cytotoxic activity was detected in our experiments. Thus, in view of the fact that the treatment of immune splenocytes with MC does not abolish the CTL function, it could be supposed that the down-regulatory activity of immune splenocytes in M L C could be attributed to the killing of allogeneic stimulators.

To address this question, the splenocytes of the i.v. immunized recipients were studied simultaneously for their cytotoxic activity and ability to inhibit proliferation in M L C in an antigen-specific manner. The splenocytes primed in vitro (MLC) were also tested in this way. We observed no correlation between the levels of cytotoxicity and down-regulation. These results support the existing data concerning the possibility of true Tcell suppressor activity induction in these systems [1-5,11,21-25]. Considering the possible role of CTL in down-regulation of immune response in our particular test-system, it becomes apparent that Expt. 5 (Table 2) provides evidence against this role. Nevertheless we can not completely rule out the involvement of CTL in inhibition of proliferation as far as the down-regulatory effect mediated by cytotoxic activity in the given experiment could have been recompensed by the activity of helper T cells in the same population of immune splenocytes [26]. The possible role of CTL in down-regulation of immune response could explain the controversial results, described in the literature, in the case that the responder cells and Ts induced by i.v. immunization were stimulated in 17

vitro by different antigens. It has been shown [27] that for the realization of inhibition of immune response to the third-party (for Ts) stimulators, the presence of both antigens in the system will be enough, while other reports [28] show the necessity for both antigens to be localized on the surface of the same stimulator. In view of the above data it seems more reliable to investigate the down-regulatory activity of T-lymphocytes with the help of a test system which allows this to be separated from cytotoxic activity. Work to establish such a test system is now in progress in our laboratory.

Acknowledgements The authors are profoundly grateful to Dr. H. Allen and Dr. R. Germain for providing L-transfectants, to Dr. A. Mitchison and Dr. A. Chervonsky for providing mAbs, to Dr. E. Fedoseeva and Dr. G. Benichou for critical reading of the manuscript.

References [1] Brondz, B.D., Karaulov, A.V., Abronina, I.F. and Blandova, Z.K. (1981) Scand. J. Immunol. 13, 517. [2] Van der Kwast, T.H., Bianchi, A.T.J., Bril, H. and Benner, R. (1981) Transplantation 31, 79. [3] Liew, F.Y. (1981) Eur. J. lmmunol. 11,883. [4] Crispe, I.N., Gascoigne, N.R.J., Lai, P.K., Mitchison, N.A. and Owens, T. (1983) Transplant. Proc. 15, 685. [5] Aoki, I., Ishii, N., Minami, M., Misugi, K., Doff, M.E. and Okuda, K. (1987) Transplantation 43, 888. [6] Moller, G. (1988) Scand. J. Immunol. 27, 247. [7] Mitchison, N.A. (1989) lmmunol. Today 10, 392. [8] Sugarbaker, P.H. and Matthews, W. (1981) Cell. Im-

18

munoI. 57, 124. [9] Sinclair, N.R.StC.~ Lees, R.K., Wheeler, M.E., Vichos, E.E. and Fung, F.Y. (1976) Cell. lmmunol. 27, 153. [10] Fitch, F.W., Engers, H.D., Cerottini, J.C. and Brunner, K.T. (1976) J. Immunol. 116, 716. [11] Holan, V. and Mitchison, N.A. (1983) Eur. J. lmmunol. 13, 652. [12] Brondz, B.D. (1988) T Lymphocytes and Their Receptors in Immunologic Recognition. Harwood, London. [13] Allen, H., Wraith, D., Pala, P., Askonas, B. and Flavell, R.A. (1984) Nature 309~ 279. [14] Ronchese, F., Brown, M.A. and Germain, R.N. (1987) J. lmmunol. 139, 629. [15] Dialynas, D.P., Quan, Z.S., Wall, K.A., Pierres, A., Quantans, J., Loken, M.R., Pierres, M. and Fitch, F.W. (1983) J. lmmunol. 131, 2445. [16] Sarmiento. M., Glasebrook, A.L. and Fitch, F.W. (1980) J. lmmunol. 125, 2665. [17] Drizlich, G.I,, Andreev, A.V., Brondz, B.D. and Kotomiha, I.F. (1975) J. Immunol. Methods 8, 383. [18] Urbach, V.Yu. (1964) Biometric Methods, Science Publishing Co., Moscow (in Russian). [19] Fedoseeva, E.V., Apasov, S.G. and Chervonsky, A.V. (1990) Immunogenetics, 32, 129. [20] Molendijk, A., Van Gurp, R.H.L.M., Knulst, A.C. and Benner, R. (1989) Immunobiol. 178, 56. [21] Sinclair, N.R,StC., Lees, R.K., Missiuna, P.C. and Vichos, E.E. (1976) Cell. Immunol. 27, 163. [22] Al-Adra, A.R. and Pilarski, L.M, (1978) Eur. J. Immunol. 8, 504. [23] Orosz, C.G. and Bach, F.H. (1979) J. lmmunol. 123, 1419. [24] Schwartz, A., Sutton, S.L. and Gershon, R.K. (1982) Eur. J. Immunol. 12, 380. [25] Zaitzeva, M.B. and Brondz, B.D. (1990) Immunology 70, 372. [26] Perry, L.L., Williams, I.R. and Dirusso, S. (1984) J. Immunol. 133, 16. [27] Bianchi, A.T.J., Bril, H. and Benner, R. (1983) Nature 301, 614. [28] Brondz, B.D., Karaulov, A.V., Chervonsky, A.V. and Blandova, Z.K. (1982) lmmunogenetics 15, 167.