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Evaluation of T helper function in lines of mice selected for high or low antibody production: quantitative inhibition of immune responses by anti-L3T4+ monoclonal antibody
Immunology Letters, 23 (1989/1990) 21-26 Elsevier
IMLET 01301
Evaluation of T helper function in lines of mice selected for high or low antibody production: quantitative inhibition of immune responses by anti-L3T4 ÷ monoclonal antibody Jacques Couderc, Yolande Bouthillier, Jean-Claude Mevel and Denise M o u t o n Service d'Immunogdndtique, Section de Biologie, Institut Curie, Paris, France
(Received 19 June 1989; revision received 1 July 1989; accepted 25 July 1989)
I. Summary The potentialities of T helper cell compartment were compared in lines of mice selected for high or low Ab production against heterologous erythrocytes (H1 and LI mice) by investigating the "in vivo" and "in vitro" modulation of immune responses by GK 1-5 anti-L3T4 + mAb. FACS analysis showed a frequency of L3T4+ cells similar in non-immunized mice of both lines. However, L I mice were more susceptible to inhibition of the IgG Ab response to SE when injected GK 1-5 prior to immunization. The "in vitro" proliferation of T cells specific for HEL was higher in H I than in L I LN cultures, and a higher GK 1-5 mAb concentration had to be applied to HI cultures in order to produce the inhibitory effect. In contrast, during MLR, the capacity for T proliferation was similar in H I and LI cultures stimulated by a common F1 target. Key words." High-antibody responder mouse; Low-antibody responder mouse; T helper cell; Macrophage Abbreviations: Ab, antibody; Ag, antigen; CFA, complete Freund's adjuvant; DTH, delayed-type hypersensitivity; FACS, fluorescent activating cell sorter; FITC, fluorescein isothiocyanate; GVH, graft-versus-host reaction; H I, high antibody producer line; HEL, hen egg lysozyme; LI, low antibody producer line; LN, lymph node; mAb, monoclonal antibody; MLR, mixed lymphocytic reaction; OVA, ovalbumin; SE, sheep erythrocytes. Correspondence to: Dr. Jacques Couderc, CNRS UPR A0305, Service d'Immunog6n6tique, Section de Biologie, Institut Curie, 26 rue d'Ulm, 75231 Paris Cedex 05, France.
This allo-Ag-induced proliferation was inhibited at similar GK 1-5 mAb concentrations in the two lines. These results demonstrate that there is no intrinsic LaT4+ cell deficiency in LI mice. Differences in GK 1-5 mAb required for inhibition of responses to Ag (other than allo-Ag) in HI and LI mice are mainly due to different macrophage T cell triggering. 2. Introduction The lines of mice obtained by selective breeding for high and low Ab production to heterologous erythrocytes [1] (HI and LI, Selection 1) differ greatly in immunoresponsiveness, not only to Ag used for selection, but also to most unrelated Ag investigated so far (multispecific effect). The interline difference in Ab production has been ascribed mainly to macrophage accessory function: the data converge in demonstrating that an increase in Ag stimulation, either by means of repeated injections "in vivo", or by the use of high Ag concentrations for macrophage pulse "in vitro", partially compensates the deficient L I mice's macrophage Ag presentation [2]. The regulatory function ofT cell subsets, though deserving prime attention, could not be clearly estimated in either line. Recently, a monoclonal Ab recognizing the L3T4 molecules on T cells (GK 1-5 rat mAb) was shown to inhibit the helper function of LaT4+ T cell subsets "in vitro" [3-5], as well as in "in vivo" T-dependent immune responses [6-9]. We were thus given the opportunity to quantify the helper subsets in the two lines, and to compare the "in vivo" inhibitory effects of GK 1-5. The ef-
0165-2478 / 89 / $ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
21
Male and female High (HI, H-2 q haplotype) or Low (LI, H-2 s haplotype) Ab responder mice (Biozzi Selection I) were bred at our animal unit, and used at 2-4 months old. (HI×LI) F1 hybrids were obtained by reciprocal crosses.
cells/well) were cultivated in microtitre plates (Falcon) in 200/A modified Click medium: RPMI-1640, supplemented with 1 mM ~.-Glutamine, 2.25 mM sodium pyruvate, 1°70 N.E. Amino acids 100×, 50 t~g streptomycin, 50 U penicillin/ml (Gibco), 5 10-5 M 2 ME (Sigma, St. Louis, MO U.S.A.)and 2% normal mouse serum (HI and LI serum, V/v). The cultures were incubated for 5 days with or without H E L and, when required, serial dilutions of mAb GK 1-5, in a i r + 5 % CO2 at 37°C. On day 4, cultures were pulsed with 0.2 ttCi tritiated thymidine (specific Activity 2 Ci/mmol.), and harvested 24 h later with a Skatron automated cell harvester (Flow Labs, Irvine, Scotland, U.K.) Filter discs were counted in a 1221 minibeta LKB counter. Results are expressed as arithmetic means of cpm from triplicate cultures.
3.2. Monoclonal anti-L3T4 + mAb
3.6. Mixed lymphocyte reaction (MLR)
Rat GK 1-5 monoclonal Ab was partly purified from ascitic fluid by ammonium sulfate precipitation, followed by an extensive dialysis. The same batch was used in all experiments, and its protein content evaluated by measuring absorption at 280 nm.
Inguinal, periaortic and popliteal LN from normal HI or LI mice were used as reactive cells. Macrophages from (H I × El) F l normal hybrids were purified by plastic adherence [11] and used as stimulator cells. 2×105-106 macrophages were mixed with 106LN cells in 200 #l Click modified medium. Cultures, thymidine pulse, harvest and counting were performed as described above.
fect of GK 1-5 mAb on two "in vitro" immune responses, T-specific proliferation assay and oneway MLR, was also compared in H I and L I mice. The rationale for all these experiments was that the mAb GK 1-5 dose requirement in H I and L l mice could serve as a measure of T cell subset potentialities. 3. Materials and Methods
3.1. Mice
3.3. Antigen and immunization 108SE (Pasteur Institute) were injected i.v. in 0.2 ml saline. H E L was a gift from Societa Prodotti Antibiotici (Milan, Italy). 10/zg H E L emulsified in CFA (Difco) were injected at the base of the tail in a 0.1 ml volume. 3.4. Antibody titration Direct SE agglutination was made by micromethods on individual mouse sera. 2 MEresistant Ab titres were determined by incubating a 1:4 serum dilution in an equal volume of 0.1 M. 2 ME at 37 °C for 30 min, just before agglutinin titration. These Ab are referred to as IgG Ab [10]. 3.5. T cell proliferation assay Mice were killed by cervical dislocation 8 days after H E L priming, and inguinal LN cells pooled (3 mice/group). Single cell suspensions (106 viable 22
3.7. FACS analysis L3T4 + T-cell numbers were evaluated by indirect fluorescence staining on spleen cell suspensions from a pool of 3 mice either pretreated or not with GK 1-5 mAb, 24 h before sacrifice. Cells were analyzed in a flow cytometer (FACSTAR; Becton Dickinson, Mountain View, CA, U.S.A.) and data collected from 10000 cells/sample. The results were expressed in percentages of stained cells and fluorescence modes. 4. Results
4.1. Evaluation of L3T4 + T cells in the spleen of normal and GK 1-5 mAb-pretreated Hy and L t mice FACS analysis was carried out in order to determine the proportion of the L 3 T 4 + cell population in
H I and L[ mice, and to verify that complete depletion does occur after injection o f an optimal GK 1-5 mAb dose. The percentages o f LaT4 + cells are not significantly different in control HI or L I mice (21.5_+6.9 and 16.2_+3.6, respectively). The expression o f surface markers is also similar in both lines, as evidenced by the equivalent values of the fluorescence mode: 120_+54 in H I, and 118+52 in L[ mice. The administration o f the GK 1-5 mAb doses, chosen to elicit a full inhibition o f Ab response: 1 mg in H I mice and 0.3 mg in L I mice (c.f. Fig. 1 and 2) leads to complete depletion of the LaT4 + T cell subsets in either line. 4.2. Modulation o f A b production "'in vivo'" after treatment with GK 1-5 m A b Increasing doses of GK 1-5 were injected i.v. in groups of HI and LI mice, 8 h before primary or secondary i.v. immunization by SE. The booster injection was done 14 days after primary immunization. Total and IgG Ab were titrated. Those IgG titres that were most illustrative of the T helper effect were selected, and appear in Figs. 1 and 2). The results shown in Fig. 1 mark a clear dosedependent inhibition o f IgG Ab production in H I whenever GK 1-5 mAb is administered before primary immunization (Fig. IA). The inhibition, which is transient at the lowest mAb doses, never persists when a booster injection is given (data not shown). Likewise, even the highest GK 1-5 mAb dose remains uneffective when given before the A 15" .iO.lmg
eJ =o~ :.® =~"I0
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Fig. 1. Effect o f "in vivo" administration of increasing GK 1-5 mAb doses on the kinetics o f IgG Ab responses to SE in H I mice. (A) GK 1-5 given 8 hours before first SE injection; (B) GK 1-5 given 8 hours before second SE injection.
booster SE injection (Fig. 1B). Corresponding data for L I mice are shown in Fig. 2. No IgG Ab production can be detected after a single SE injection in immunized control L~ mice. However, the inhibitory effect of GK 1-5 mAb can be observed when a booster injection is effected (without repeating the GK 1-5 mAb injection) (Fig. 2A). This effect is also dose-dependent. Compared to the results shown in Fig. 1, the effective dose is more than 10 times lower in L~ than in HI immunized mice. When GK 1-5 mAb is injected into L I mice prior to the booster injection (Fig. 2B), inhibition of IgG Ab production still occurs, but with a higher mAb dose requirement (approx. 3 times higher). Compared to each other, Figs. 1 and 2 bring forth the demonstration of a quantitative variation of GK 1-5 mAb effect in function of the intensity of the Ab response (primary vs. secondary challenge, and the immunogenetic status of HI vs. L]). 4.3. Effect of GK 1-5 on the "'in vitro" T cell proliferative response of Hi or L t mice to HEL LN lymphocytes from primed animals were stimulated with an increasing amount o f H E L in the classical T cell proliferation assay (see Materials and Methods). As shown in Fig. 3A, H I displayed a dose-dependent T cell proliferation with H E L in culture, and no proliferation at all against the nonrelated Ag OVA. In contrast, a poor proliferation occurred in LN of L I mice unless stimulated with a high dose of Ag (500 #g/ml). To estimate T helper cell capacity of the two lines "in vitro", inhibition experiments were started at a constant H E L concentration (500 #g/ml) and with increasing doses of GK 1-5 mAb (Fig. 3B). Complete inhibition o f the proliferation was observed in both strains of mice with 12.5 #g/ml of GK 1-5 added at day 0 for the 5 days of culture. For LI mice, however, a 50°7o inhibition occurred at 1/zg/ml, while 3.1 #g/ml were required to inhibit by 50o7o the proliferation in H I LN cells. 4.4. Effect o f GK 1-5 m A b on one-way M L R In order to investigate another type o f Ag-specific T cell proliferation directed towards class II allo-Ag, we performed a one-way MLR. LN cells (1 × 106) of H I or L 1 mice were co-cultured with 2 × 105 2000-r23
A
B
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Fig. 2. Effect of "in vivo" administration of increasing GK 1-5 mAb doses on the kinetics of IgG Ab responses to SE in Lt mice. (A) GK 1-5 mAb given 8 h before first injection; (B) GK 1-5 mAb given 8 h before second injection.
irradiated purified resident peritoneal macrophages from ( H I × L D F 1 hybrids. Fig. 4A shows that H I and L I LN cells proliferate with a similar magnitude (9252___3684 for HI, 11202+_5305 for LI). The higher proliferation for L l is reproducible but not significant (P> 0.9). GK 1-5 added at day 0 until the end of culture has a dose-dependent inhibitory effect. The mAb dose required for 50% MLR inhibition, calculated from a regression curve, is 63 ng for H Tand 48 ng/ml for L I. If the ratio of stimulatory cells/reacting cells is increased to 0.5 and 1 (Fig. 4B), the 50% inhibiting mAb concentration increases
linearly. The regression slopes are similar in both lines (b=2.17 for HI and b=2 for L 0. Moreover, the HI/LI ratio, in terms of proliferation (cpm) remains < 1, whatever the effector/target ratio.
B
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,'5 ~,
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Fig. 3. "In vitro" Ag-dependent T cell proliferation assay. Cultures of inguinal LN cells (llYVwell) from H 1 and L I immunized 7 days previously with 10 #g HEL in CFA at the base of the tail. (A) Counts (cpm) for increasing Ag concentration in five-day cultures pulsed 24 h before harvest with 0.2 #Ci tritiated thymidine; (B) dose-dependent GK 1-5 mAb inhibition of optimal T proliferative response (induced by 500 tzg/ml HEL), GK 1-5 being added at culture onset.
24
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Fig. 4. T proliferation induced by one-way MLR in H I and L t LN cultures stimulated by 2000-r-irradiated resident peritoneal macrophages from (H I x LI) F 1 hybrids. Cells (1 x 106) were cultivated for 5 days and pulsed 24 h before harvest with 0.2 #Ci tritiated thymidine. (A) Counts (cpm) in cultures stimulated by 2 x 105 target cells without and with increasing concentration of GK 1-5 mAb (added at day 0 of culture); (B) relationship between the 50% mAb inhibitory concentration and the number of target cells added to the cultures: 2x105, 5xlO 5, and 106 F l macrophages/well, corresponding to effector/target ratios of 0.2, 0.5 and 1.00 respectively. Values in parentheses are the ratios: H x cpm/L I cpm.
5. Discussion The L3T4 marker is broadly represented in the mouse T helper cell compartment, and plays a role in Ag recognition by T cells in association with class II molecules [12]. The inhibition pattern o f "in vivo" Ab production to SE by pretreatment with GK 1-5 mAb differs in H I and L I mice. The mAb dose required for complete inhibition o f the primary response is much lower in L I than in H I mice, and moreover, suboptimal mAb doses give only a transient reduction o f IgG Ab titres in H I mice. This effect is abolished if a booster injection is given, even when mAb is administered just beforehand (Fig. 1). In contrast, in L I mice, full inhibition can be obtained after a booster, even though the mAb dose required is higher for primed than for normal mice (Fig. 2). Although the proportion of L3T4 + T cells and the expression of L3T4 molecules/cell were found to be similar in non-immunized H I and LI mice, a clear and constant difference between the two lines in the GK 1-5 mAb requirement for Ab response inhibition was observed after primary and secondary immunizations. The results o f the "in vitro" experiments are in agreement with the "in vivo" data. The background of unstimulated cultures is similar for both lines, with a slight advantage to L I (618_+37 cpm vs, 242_+ 32 for HI). This fits the similar proportion o f L3T4 + T ceils found in the spleen by FACS analysis. In the Ag-specific T proliferation assay, the Ag concentration required for T cell stimulation is much higher for L I than for H I (60 ktg H E L in H I to 500/zg in L l for a similar response). This finding is in agreement with previous results o f Adorini [13]. When GK 1-5 mAb is added to the cultures, the more proliferation obtained in control cultures, the more mAb concentration is needed in order to get a 50% inhibition o f the response. This is an incitement to interpret the "in vivo" dose effect as a quantitative evaluation of the T helper proliferation capacity. In contrast to the results for T proliferation in response to HEL, H I and L I LN cells proliferate equally when stimulated, in an M L R assay, with normal (H I ×Li) F 1 hybrid irradiated macrophages. Taking into account that adherent spleen cells expressing Ia molecules are potent stimulators of MLR [14, 15], Falo et al. demonstrated that no further
processing of allo-antigens was required for T cell activation [16]. This is also documented by data showing that allo-reactive T cells bind directly to the surface o f the target [17], and can be stimulated by means of purified class II molecules and synthetic membrane systems [18]. On these grounds, our results demonstrate that when macrophages are not involved in T helper cell functions as Ag-presenting cells, there is no difference in T proliferation. Thus, H l or L I MLR can be inhibited by similar concentrations of GK 1-5 mAb. The mAb dose required for 50% inhibition increases with the ratio of target/effector cells in a linear relationship for both lines (Fig. 4B), with very similar regression slopes, showing that the F 1 target delivers an equivalent stimulus for H I and L t cells. These findings are in agreement with the results obtained by Milon et al. [19]: studying the kinetics of T cells capable to transfer locally the delayed-type hypersensitivity, the authors point out that expansion of this T cell population occurs faster in H I than in L I mice, due to a more efficient macrophage accessory function. Thus, our results lead to the conclusion that there is no intrinsic difference in frequency or basic activity of L3T4 + cell population between H I and L I normal mice. It is the divergent macrophage Ag processing/presenting capacity that accounts for the faster proliferation o f T helper cells after T-dependent Ag stimulation.
Acknowledgements The authors would like to thank Dr. E Fitch of the University of Chicago for providing hybridoma GK 1-5, and Professor P. Debr6, URA A 86 CNRS, for offering us access to FACSTAR apparatus.
References [1] Biozzi, G., Siqueira, M., Stiffel, C., Ibanez, P. M., Mouton, D. and Ferreira, V. C. A. (1980)in: Immunology 80: Progress in Immunology IV (M. Fougereau and J. Dausset, Eds.) pp. 432-457, Academic Press, London, New York, Toronto, Sydney, San Francisco. [2] Biozzi, G., Mouton, D., Stiffel, C. and Bouthillier, Y. (1984) Adv. Immunol. 36, 189. [3] Wilde, D. B., Marrack, E, Kappler, J., Dyalinas, D. E and Fitch, E W. (1983) J. ImmunoL 131, 2178. [4] Watts, T. H., Brian, A. A., Kappler, J. W., Marrack, P. and McConnell, H. M. (1984) Proc. Natl. Acad. Sci. USA 81, 7574.
25
[5] Ranges, G. E., Cooper, S. M. and Sriram, S. (1987) Cell. Immunol. 106, 163. [6] Cobbold, S. P., Jayasuriya, A., Nash, A., Prospero, T. D. and Waldmann, H. (1984) Nature 312, 548. [7] Coulie, P. G., Coutelier, J. P., Uyttenhove, C., Lambotte, P. and Van Snick, J. (1985) Eur. J. Immunol. 15, 638. [8] Wofsy, D., Mayes, D. C., Woodcock, J. and Seaman, W. E. (1985) J. Immunol. 135, 1698. [9] Goronzy, J., Weyand, C. M. and Fathman, C. G. (1986) J. Exp. Med. 164, 911. [10] Silver, D. M., McKenzie, I. F. C. and Winn, A. J. (1972) J. Exp. Med. 136, 1063. [11] Mosier, D. E. (1967) Science 158, 1573. [12] Carteron, N. L., Wofsy, D. and Seaman, W. E. (1988) J. Immunol. 140, 713.
26
[13] Adorini, L. and Doria, G. (1981) Eur. J. Immunol. 11, 984. [14] Minami, M., Shreffler, D. C. and Cowing, C. (1980) J. Immunol. 124, 1314. [15] Taira, S. and Nariuchi, H. (1988) J. Immunol. 141, 440. [16] Falo, Jr., L. D., Benacerraf, B. and Rock, K. L. (1986) Proc. Natl. Acad. Sci. USA 83, 6994. [17] Golstein, P., Svedmyr, E. A. J. and Wigzell, H. (1971) J. Exp. Med. 134, 1385. [18] Coeshott, C. M., Chesnut, R. W., Kubo, R. T., Grammer, S. F., Jenis, D. M. and Grey, H. M. (1986) J. Immunol. 136, 2832. [19] Milon, G., Marchal, G., Mouton, D. and Biozzi, G. (1985) in: Genetic control of Host Resistance to Infection and Malignancy (E. Skamene, Ed.) pp. 623-628, Alan R. Liss, New York.
IMLET 01301
Evaluation of T helper function in lines of mice selected for high or low antibody production: quantitative inhibition of immune responses by anti-L3T4 ÷ monoclonal antibody Jacques Couderc, Yolande Bouthillier, Jean-Claude Mevel and Denise M o u t o n Service d'Immunogdndtique, Section de Biologie, Institut Curie, Paris, France
(Received 19 June 1989; revision received 1 July 1989; accepted 25 July 1989)
I. Summary The potentialities of T helper cell compartment were compared in lines of mice selected for high or low Ab production against heterologous erythrocytes (H1 and LI mice) by investigating the "in vivo" and "in vitro" modulation of immune responses by GK 1-5 anti-L3T4 + mAb. FACS analysis showed a frequency of L3T4+ cells similar in non-immunized mice of both lines. However, L I mice were more susceptible to inhibition of the IgG Ab response to SE when injected GK 1-5 prior to immunization. The "in vitro" proliferation of T cells specific for HEL was higher in H I than in L I LN cultures, and a higher GK 1-5 mAb concentration had to be applied to HI cultures in order to produce the inhibitory effect. In contrast, during MLR, the capacity for T proliferation was similar in H I and LI cultures stimulated by a common F1 target. Key words." High-antibody responder mouse; Low-antibody responder mouse; T helper cell; Macrophage Abbreviations: Ab, antibody; Ag, antigen; CFA, complete Freund's adjuvant; DTH, delayed-type hypersensitivity; FACS, fluorescent activating cell sorter; FITC, fluorescein isothiocyanate; GVH, graft-versus-host reaction; H I, high antibody producer line; HEL, hen egg lysozyme; LI, low antibody producer line; LN, lymph node; mAb, monoclonal antibody; MLR, mixed lymphocytic reaction; OVA, ovalbumin; SE, sheep erythrocytes. Correspondence to: Dr. Jacques Couderc, CNRS UPR A0305, Service d'Immunog6n6tique, Section de Biologie, Institut Curie, 26 rue d'Ulm, 75231 Paris Cedex 05, France.
This allo-Ag-induced proliferation was inhibited at similar GK 1-5 mAb concentrations in the two lines. These results demonstrate that there is no intrinsic LaT4+ cell deficiency in LI mice. Differences in GK 1-5 mAb required for inhibition of responses to Ag (other than allo-Ag) in HI and LI mice are mainly due to different macrophage T cell triggering. 2. Introduction The lines of mice obtained by selective breeding for high and low Ab production to heterologous erythrocytes [1] (HI and LI, Selection 1) differ greatly in immunoresponsiveness, not only to Ag used for selection, but also to most unrelated Ag investigated so far (multispecific effect). The interline difference in Ab production has been ascribed mainly to macrophage accessory function: the data converge in demonstrating that an increase in Ag stimulation, either by means of repeated injections "in vivo", or by the use of high Ag concentrations for macrophage pulse "in vitro", partially compensates the deficient L I mice's macrophage Ag presentation [2]. The regulatory function ofT cell subsets, though deserving prime attention, could not be clearly estimated in either line. Recently, a monoclonal Ab recognizing the L3T4 molecules on T cells (GK 1-5 rat mAb) was shown to inhibit the helper function of LaT4+ T cell subsets "in vitro" [3-5], as well as in "in vivo" T-dependent immune responses [6-9]. We were thus given the opportunity to quantify the helper subsets in the two lines, and to compare the "in vivo" inhibitory effects of GK 1-5. The ef-
0165-2478 / 89 / $ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
21
Male and female High (HI, H-2 q haplotype) or Low (LI, H-2 s haplotype) Ab responder mice (Biozzi Selection I) were bred at our animal unit, and used at 2-4 months old. (HI×LI) F1 hybrids were obtained by reciprocal crosses.
cells/well) were cultivated in microtitre plates (Falcon) in 200/A modified Click medium: RPMI-1640, supplemented with 1 mM ~.-Glutamine, 2.25 mM sodium pyruvate, 1°70 N.E. Amino acids 100×, 50 t~g streptomycin, 50 U penicillin/ml (Gibco), 5 10-5 M 2 ME (Sigma, St. Louis, MO U.S.A.)and 2% normal mouse serum (HI and LI serum, V/v). The cultures were incubated for 5 days with or without H E L and, when required, serial dilutions of mAb GK 1-5, in a i r + 5 % CO2 at 37°C. On day 4, cultures were pulsed with 0.2 ttCi tritiated thymidine (specific Activity 2 Ci/mmol.), and harvested 24 h later with a Skatron automated cell harvester (Flow Labs, Irvine, Scotland, U.K.) Filter discs were counted in a 1221 minibeta LKB counter. Results are expressed as arithmetic means of cpm from triplicate cultures.
3.2. Monoclonal anti-L3T4 + mAb
3.6. Mixed lymphocyte reaction (MLR)
Rat GK 1-5 monoclonal Ab was partly purified from ascitic fluid by ammonium sulfate precipitation, followed by an extensive dialysis. The same batch was used in all experiments, and its protein content evaluated by measuring absorption at 280 nm.
Inguinal, periaortic and popliteal LN from normal HI or LI mice were used as reactive cells. Macrophages from (H I × El) F l normal hybrids were purified by plastic adherence [11] and used as stimulator cells. 2×105-106 macrophages were mixed with 106LN cells in 200 #l Click modified medium. Cultures, thymidine pulse, harvest and counting were performed as described above.
fect of GK 1-5 mAb on two "in vitro" immune responses, T-specific proliferation assay and oneway MLR, was also compared in H I and L I mice. The rationale for all these experiments was that the mAb GK 1-5 dose requirement in H I and L l mice could serve as a measure of T cell subset potentialities. 3. Materials and Methods
3.1. Mice
3.3. Antigen and immunization 108SE (Pasteur Institute) were injected i.v. in 0.2 ml saline. H E L was a gift from Societa Prodotti Antibiotici (Milan, Italy). 10/zg H E L emulsified in CFA (Difco) were injected at the base of the tail in a 0.1 ml volume. 3.4. Antibody titration Direct SE agglutination was made by micromethods on individual mouse sera. 2 MEresistant Ab titres were determined by incubating a 1:4 serum dilution in an equal volume of 0.1 M. 2 ME at 37 °C for 30 min, just before agglutinin titration. These Ab are referred to as IgG Ab [10]. 3.5. T cell proliferation assay Mice were killed by cervical dislocation 8 days after H E L priming, and inguinal LN cells pooled (3 mice/group). Single cell suspensions (106 viable 22
3.7. FACS analysis L3T4 + T-cell numbers were evaluated by indirect fluorescence staining on spleen cell suspensions from a pool of 3 mice either pretreated or not with GK 1-5 mAb, 24 h before sacrifice. Cells were analyzed in a flow cytometer (FACSTAR; Becton Dickinson, Mountain View, CA, U.S.A.) and data collected from 10000 cells/sample. The results were expressed in percentages of stained cells and fluorescence modes. 4. Results
4.1. Evaluation of L3T4 + T cells in the spleen of normal and GK 1-5 mAb-pretreated Hy and L t mice FACS analysis was carried out in order to determine the proportion of the L 3 T 4 + cell population in
H I and L[ mice, and to verify that complete depletion does occur after injection o f an optimal GK 1-5 mAb dose. The percentages o f LaT4 + cells are not significantly different in control HI or L I mice (21.5_+6.9 and 16.2_+3.6, respectively). The expression o f surface markers is also similar in both lines, as evidenced by the equivalent values of the fluorescence mode: 120_+54 in H I, and 118+52 in L[ mice. The administration o f the GK 1-5 mAb doses, chosen to elicit a full inhibition o f Ab response: 1 mg in H I mice and 0.3 mg in L I mice (c.f. Fig. 1 and 2) leads to complete depletion of the LaT4 + T cell subsets in either line. 4.2. Modulation o f A b production "'in vivo'" after treatment with GK 1-5 m A b Increasing doses of GK 1-5 were injected i.v. in groups of HI and LI mice, 8 h before primary or secondary i.v. immunization by SE. The booster injection was done 14 days after primary immunization. Total and IgG Ab were titrated. Those IgG titres that were most illustrative of the T helper effect were selected, and appear in Figs. 1 and 2). The results shown in Fig. 1 mark a clear dosedependent inhibition o f IgG Ab production in H I whenever GK 1-5 mAb is administered before primary immunization (Fig. IA). The inhibition, which is transient at the lowest mAb doses, never persists when a booster injection is given (data not shown). Likewise, even the highest GK 1-5 mAb dose remains uneffective when given before the A 15" .iO.lmg
eJ =o~ :.® =~"I0
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///
~, <.-zs-
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/
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,~
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IO Atl,~ ISE GK 1.5
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~0
Fig. 1. Effect o f "in vivo" administration of increasing GK 1-5 mAb doses on the kinetics o f IgG Ab responses to SE in H I mice. (A) GK 1-5 given 8 hours before first SE injection; (B) GK 1-5 given 8 hours before second SE injection.
booster SE injection (Fig. 1B). Corresponding data for L I mice are shown in Fig. 2. No IgG Ab production can be detected after a single SE injection in immunized control L~ mice. However, the inhibitory effect of GK 1-5 mAb can be observed when a booster injection is effected (without repeating the GK 1-5 mAb injection) (Fig. 2A). This effect is also dose-dependent. Compared to the results shown in Fig. 1, the effective dose is more than 10 times lower in L~ than in HI immunized mice. When GK 1-5 mAb is injected into L I mice prior to the booster injection (Fig. 2B), inhibition of IgG Ab production still occurs, but with a higher mAb dose requirement (approx. 3 times higher). Compared to each other, Figs. 1 and 2 bring forth the demonstration of a quantitative variation of GK 1-5 mAb effect in function of the intensity of the Ab response (primary vs. secondary challenge, and the immunogenetic status of HI vs. L]). 4.3. Effect of GK 1-5 on the "'in vitro" T cell proliferative response of Hi or L t mice to HEL LN lymphocytes from primed animals were stimulated with an increasing amount o f H E L in the classical T cell proliferation assay (see Materials and Methods). As shown in Fig. 3A, H I displayed a dose-dependent T cell proliferation with H E L in culture, and no proliferation at all against the nonrelated Ag OVA. In contrast, a poor proliferation occurred in LN of L I mice unless stimulated with a high dose of Ag (500 #g/ml). To estimate T helper cell capacity of the two lines "in vitro", inhibition experiments were started at a constant H E L concentration (500 #g/ml) and with increasing doses of GK 1-5 mAb (Fig. 3B). Complete inhibition o f the proliferation was observed in both strains of mice with 12.5 #g/ml of GK 1-5 added at day 0 for the 5 days of culture. For LI mice, however, a 50°7o inhibition occurred at 1/zg/ml, while 3.1 #g/ml were required to inhibit by 50o7o the proliferation in H I LN cells. 4.4. Effect o f GK 1-5 m A b on one-way M L R In order to investigate another type o f Ag-specific T cell proliferation directed towards class II allo-Ag, we performed a one-way MLR. LN cells (1 × 106) of H I or L 1 mice were co-cultured with 2 × 105 2000-r23
A
B
15-
,~ I0-
o
~"'~"--~
4""~' " " - - - ~ control 0.05 mg
control
1
o=5.
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(..9
O. I mg
o,o0
~<2.5-
...........
ling
0.3 m g
E GK 1.5
SE
days
SE
~ SE GK 1.5
days
Fig. 2. Effect of "in vivo" administration of increasing GK 1-5 mAb doses on the kinetics of IgG Ab responses to SE in Lt mice. (A) GK 1-5 mAb given 8 h before first injection; (B) GK 1-5 mAb given 8 h before second injection.
irradiated purified resident peritoneal macrophages from ( H I × L D F 1 hybrids. Fig. 4A shows that H I and L I LN cells proliferate with a similar magnitude (9252___3684 for HI, 11202+_5305 for LI). The higher proliferation for L l is reproducible but not significant (P> 0.9). GK 1-5 added at day 0 until the end of culture has a dose-dependent inhibitory effect. The mAb dose required for 50% MLR inhibition, calculated from a regression curve, is 63 ng for H Tand 48 ng/ml for L I. If the ratio of stimulatory cells/reacting cells is increased to 0.5 and 1 (Fig. 4B), the 50% inhibiting mAb concentration increases
linearly. The regression slopes are similar in both lines (b=2.17 for HI and b=2 for L 0. Moreover, the HI/LI ratio, in terms of proliferation (cpm) remains < 1, whatever the effector/target ratio.
B
A 150-
c4, .9
~-IO0x
LO
E o_ tO
A 40-
o 30.
HEL . . . . OVA
--
o
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/
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•
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",, 15
62
250 I000 HEL p g / m l
,'5 ~,
' 6.2 12.5 GK 1_5 p g / r n l
Fig. 3. "In vitro" Ag-dependent T cell proliferation assay. Cultures of inguinal LN cells (llYVwell) from H 1 and L I immunized 7 days previously with 10 #g HEL in CFA at the base of the tail. (A) Counts (cpm) for increasing Ag concentration in five-day cultures pulsed 24 h before harvest with 0.2 #Ci tritiated thymidine; (B) dose-dependent GK 1-5 mAb inhibition of optimal T proliferative response (induced by 500 tzg/ml HEL), GK 1-5 being added at culture onset.
24
o:o~
0.12
o:zs' GKI.5
I0.
LI
r = 0.98
~2.
'
u
J
i
0.5 pg/ml
LI
,o
HI
o_
~0.821 c0.771
-
d.2 0.5 effector/target
10.4~1 ratio
Fig. 4. T proliferation induced by one-way MLR in H I and L t LN cultures stimulated by 2000-r-irradiated resident peritoneal macrophages from (H I x LI) F 1 hybrids. Cells (1 x 106) were cultivated for 5 days and pulsed 24 h before harvest with 0.2 #Ci tritiated thymidine. (A) Counts (cpm) in cultures stimulated by 2 x 105 target cells without and with increasing concentration of GK 1-5 mAb (added at day 0 of culture); (B) relationship between the 50% mAb inhibitory concentration and the number of target cells added to the cultures: 2x105, 5xlO 5, and 106 F l macrophages/well, corresponding to effector/target ratios of 0.2, 0.5 and 1.00 respectively. Values in parentheses are the ratios: H x cpm/L I cpm.
5. Discussion The L3T4 marker is broadly represented in the mouse T helper cell compartment, and plays a role in Ag recognition by T cells in association with class II molecules [12]. The inhibition pattern o f "in vivo" Ab production to SE by pretreatment with GK 1-5 mAb differs in H I and L I mice. The mAb dose required for complete inhibition o f the primary response is much lower in L I than in H I mice, and moreover, suboptimal mAb doses give only a transient reduction o f IgG Ab titres in H I mice. This effect is abolished if a booster injection is given, even when mAb is administered just beforehand (Fig. 1). In contrast, in L I mice, full inhibition can be obtained after a booster, even though the mAb dose required is higher for primed than for normal mice (Fig. 2). Although the proportion of L3T4 + T cells and the expression of L3T4 molecules/cell were found to be similar in non-immunized H I and LI mice, a clear and constant difference between the two lines in the GK 1-5 mAb requirement for Ab response inhibition was observed after primary and secondary immunizations. The results o f the "in vitro" experiments are in agreement with the "in vivo" data. The background of unstimulated cultures is similar for both lines, with a slight advantage to L I (618_+37 cpm vs, 242_+ 32 for HI). This fits the similar proportion o f L3T4 + T ceils found in the spleen by FACS analysis. In the Ag-specific T proliferation assay, the Ag concentration required for T cell stimulation is much higher for L I than for H I (60 ktg H E L in H I to 500/zg in L l for a similar response). This finding is in agreement with previous results o f Adorini [13]. When GK 1-5 mAb is added to the cultures, the more proliferation obtained in control cultures, the more mAb concentration is needed in order to get a 50% inhibition o f the response. This is an incitement to interpret the "in vivo" dose effect as a quantitative evaluation of the T helper proliferation capacity. In contrast to the results for T proliferation in response to HEL, H I and L I LN cells proliferate equally when stimulated, in an M L R assay, with normal (H I ×Li) F 1 hybrid irradiated macrophages. Taking into account that adherent spleen cells expressing Ia molecules are potent stimulators of MLR [14, 15], Falo et al. demonstrated that no further
processing of allo-antigens was required for T cell activation [16]. This is also documented by data showing that allo-reactive T cells bind directly to the surface o f the target [17], and can be stimulated by means of purified class II molecules and synthetic membrane systems [18]. On these grounds, our results demonstrate that when macrophages are not involved in T helper cell functions as Ag-presenting cells, there is no difference in T proliferation. Thus, H l or L I MLR can be inhibited by similar concentrations of GK 1-5 mAb. The mAb dose required for 50% inhibition increases with the ratio of target/effector cells in a linear relationship for both lines (Fig. 4B), with very similar regression slopes, showing that the F 1 target delivers an equivalent stimulus for H I and L t cells. These findings are in agreement with the results obtained by Milon et al. [19]: studying the kinetics of T cells capable to transfer locally the delayed-type hypersensitivity, the authors point out that expansion of this T cell population occurs faster in H I than in L I mice, due to a more efficient macrophage accessory function. Thus, our results lead to the conclusion that there is no intrinsic difference in frequency or basic activity of L3T4 + cell population between H I and L I normal mice. It is the divergent macrophage Ag processing/presenting capacity that accounts for the faster proliferation o f T helper cells after T-dependent Ag stimulation.
Acknowledgements The authors would like to thank Dr. E Fitch of the University of Chicago for providing hybridoma GK 1-5, and Professor P. Debr6, URA A 86 CNRS, for offering us access to FACSTAR apparatus.
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[5] Ranges, G. E., Cooper, S. M. and Sriram, S. (1987) Cell. Immunol. 106, 163. [6] Cobbold, S. P., Jayasuriya, A., Nash, A., Prospero, T. D. and Waldmann, H. (1984) Nature 312, 548. [7] Coulie, P. G., Coutelier, J. P., Uyttenhove, C., Lambotte, P. and Van Snick, J. (1985) Eur. J. Immunol. 15, 638. [8] Wofsy, D., Mayes, D. C., Woodcock, J. and Seaman, W. E. (1985) J. Immunol. 135, 1698. [9] Goronzy, J., Weyand, C. M. and Fathman, C. G. (1986) J. Exp. Med. 164, 911. [10] Silver, D. M., McKenzie, I. F. C. and Winn, A. J. (1972) J. Exp. Med. 136, 1063. [11] Mosier, D. E. (1967) Science 158, 1573. [12] Carteron, N. L., Wofsy, D. and Seaman, W. E. (1988) J. Immunol. 140, 713.
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[13] Adorini, L. and Doria, G. (1981) Eur. J. Immunol. 11, 984. [14] Minami, M., Shreffler, D. C. and Cowing, C. (1980) J. Immunol. 124, 1314. [15] Taira, S. and Nariuchi, H. (1988) J. Immunol. 141, 440. [16] Falo, Jr., L. D., Benacerraf, B. and Rock, K. L. (1986) Proc. Natl. Acad. Sci. USA 83, 6994. [17] Golstein, P., Svedmyr, E. A. J. and Wigzell, H. (1971) J. Exp. Med. 134, 1385. [18] Coeshott, C. M., Chesnut, R. W., Kubo, R. T., Grammer, S. F., Jenis, D. M. and Grey, H. M. (1986) J. Immunol. 136, 2832. [19] Milon, G., Marchal, G., Mouton, D. and Biozzi, G. (1985) in: Genetic control of Host Resistance to Infection and Malignancy (E. Skamene, Ed.) pp. 623-628, Alan R. Liss, New York.