CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
7, l-9 (1977)
Studies of Tetanus Toxoid-Induced Blast Transformation of Lymphocytes of Immunized Healthy Donors A. EYQUEM Service
d’lmmunothkrapie Institut
AND C. BONA
ExpPrimentale and Pasteur and C.N.R.S.
Centre de Transfusion Paris, France
Sanguine,
Received December 29, 1975 Tetanus toxoid induced-blast transformation of peripheral blood lymphocytes of 15 immunized healthy subjects was studied. According to the magnitude of the [3H]thymidine incorporation, the subjects were classified into two groups: high and low blast specific responses. The high or low blast response induced by tetanus toxoid did not correlate with the stimulation induced by PHA and PPD nor with the antitetanusantibody titer of these subjects. T cells were shown to be involved in this specific blast response. The differences in the magnitude of the blast response could be easily explained by the existence of B cells with suppressor activity on T cells functions rather than by inhibition of the tetanus toxoid induced stimulation by an antigen-antibody complex. Relationship with HL-A system will be investigated.
INTRODUCTION
Various soluble antigens are able to induce blast transformation of lymphocytes in vitro after active in vivu immunization of animals. Antigen-induced proliferation in vitro is a correlate of in vivo cellular immunity (1.2) and generally T cells are involved in the blast response (3 -6). It has been reported that the lymphocytes of guinea pigs actively immunized with tetanus toxoid in Freund’s complete adjuvant were transformed specifically by toxoid and that the blast response required the presence of macrophages in the system (7). In this work, we have investigated various aspects of in vitro blast transformation of lymphocytes of human healthy subjects hyperimmunized with tetanus toxoid as well as the relationship of this phenomenon to various parameters, i.e., nonspecific blast response, specific blast response with a nonrelated antigen, titer of antitetanus antibody, and the schedule of immunization. MATERIALS
AND METHODS
Blood lymphocytes were recovered from healthy human blood donors, vaccinated several times with tetanus toxoid vaccine (I.P.A.D.T. Institut Pasteur). The lymphocytes were separated according to the Boyum technique (8). Separation of T and B-derived lymphocytes. Purified T- or B-derived lymphocytes were obtained by the passage of ficoll separated lymphocytes through a nylon column according to a previously described technique (32). The degree of purification of lymphocytes was controlled by rosetting the lymphocyte purified populations with SRBC or by staining with FAB fragments of goat IgG anti-human Ig labeled with peroxydase (Pasteur Institute), according to the method of Graham and Karnovsky (9). Determination of Ig positive cells with Lymphocytes.
Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved
7
EYQUEM
.-\Nl>
BOh.4
Fab fragments of goat IgG anti-human IgG antibodies exclude the possibility of staining cells which bind heterologous IgG by their Fc receptors. Moreover, staining was performed after incubation of cells at 37°C according to the method of Lobo et al. (10) which is more selective than after incubation at 4°C. Blast transformatiotz. Lymphocytes (106) were cultured for 4 days in I ml of Eagles medium (General Island Biological Co., Grand Island. N. Y.) supplemented with nonessential aminoacids and 15% of AB homologous serum previously inactived for 30 min at 56°C. The cultures were performed in an incubator model IH-100 (Gallenkamp) under a continuous flow of a mixture of 5% CO, and 95% air. Antigen was added at various concentrations in 0. I ml of MEM medium. I &i r3H]thymidine (1 ciimmol specific activity. Saclay. France) was added to each culture tube 12 hr before harvesting the cells. 13H]thymidine incorporation was measured by liquid scintillation counting according to a previously described technique (I I). Antigens. Purified protein derivative (PPD-neutre pour test de transformation blastique. Institut Pasteur), tetanus toxoid containing 30 Lf/ml and 2.8 mg protein/ml (lnstitut Pasteur) were the antigens employed. Other reagents. Human Ig antitetanus antibody (15.5 g% and 125 IU/ml) (Pasteur Institute) PHA-P (Difco). Antibody assays. Titers of antitetanus antibody were measured either in vitro by means of agglutination of tetanus toxoid coated latex particles according to a previously described technique (12) or in ~ivo by protection of mice according to described techniques ( 13). RESULTS
Blast Transformation of Lymphocytes [3H]thymidine incorporation of lymphocytes of human hyperimmunized subjects with tetanus toxoid was estimated after incubation with various amounts of tetanus toxoid (0.03-3 Lf) as well as with 10 pg PPD and 2 pg PHA. The level of specific antibody was determined simultaneously with thymidine incorporation. According to the level of specific tetanus toxoid blast responses of lymphocytes. the 15 subjects could be classified into two groups: Eight subjects with a stimulation index higher than five and seven subjects with a stimulation index lower than four. High responder subjects. In this group, the peak of blast response induced by various doses of tetanus toxoid ranged between 5.5-31. In seven out of eight cases, the peak of [3H]thymidine incorporation was obtained with doses smaller than 1 Lff 106 cells; doses varied between 0.03-0.3 Lf. The lymphocytes of these eight subjects could be stimulated by a nonspecific mitogen such as PHA as well as by another specific mitogen such as PPD. As can be seen in Fig. 1, there was no correlation between the blast responses induced by tetanus toxoid and the level of specific antibody or the level of [3H]thymidine incorporation induced by PHA and PPD. Low, responder subjects. The peak of blast response induced by various doses of tetanus toxoid was smaller than 4 in the case of seven subjects. As can be seen in Fig. 2, the dose-response relationship curves of these subjects shows very clearly that the highest stimulation indices were obtained in all seven cases with 1 Lf
TETANUS
CELLULAR
IMMUNITY
IN
MAN
FIG. 1. Dose-response relationship of stimulation of human peripheral lymphocytes by tetanus toxoid (high responder group with stimulation index > 5).
tetanus toxoid406 cells. Three out of seven subjects had a strong blast response to PHA and only one subject did not respond to tuberculin. The low blast responses to tetanus toxoid in these seven subjects could not be related to the titer of specific antibody measured simultaneously with rH]thymidine incorporation.
M .a c-i)
LnDt
done
**,
“./ml
FIG. 2. Dose-response relationship of stimulation of human peripheral lymphocytes by tetanus toxoid (low responder group with stimulation index < 4).
EYQUEM
.!
l FIG.
Kinetics
.,,..
+ f , ,‘, 420123456789
3. Kinetics
of Speci’c
to blast
,
!.....,’
,
response
and Nonspecific
,
AND
,
,
of two
BON.\
,
,
, . loweeks
subjects
with
stimulation
index
‘> 5
Blast Response
The kinetics of specific and nonspecific blast responses were studied in two cases (No. 10207 and 10486) during immunization and in another two cases (No. 10208 and 10211) 40 and 90 days after the last booster (see the schedules of immunization of these last two subjects in Fig. I). Doses of tetanus toxoid corresponding to the peak of blast responses established previously (see Figs. 1 and 2) were used in the kinetics study. As can be seen in Fig. 3, a time dependent increase of specific blast response was obtained in the case of subject No. 10207 whereas a plateau was observed in
I
FIG.
4. Kinetics
1111,,1111,,,11 123456789
of blast
response
to
of two subjects
11
12
with
13
14
15 1r3ICCkI
stimulation
*
index
c 5
TETANUS
CELLULAR
IMMUNITY
IN
5
MAN
the case of subject No. 10486. PHA blast response paralleled the specific response. In the case of subjects studied 40 days (No. 10208) and 90 days (No. 10211) after their last booster, a decrease of the specific blast response was observed during the first 8 or 9 weeks as compared with the nonspecific blast response induced by PHA (see Fig. 4). Evidence of T-Derived Lymphocytes Involvement in the Specific Blast Response Induced by Tetanus Toxoid In three cases, the blast response induced by tetanus toxoid was tested on nonseparated lymphocytes as well as T- and B-derived purified lymphocytes. As can be seen in Table 1, a stimulation of rH]thymidine incorporation was obtained in the case of nonseparated lymphocytes as well as T- and B-derived purified lymphocytes. As can be seen in Table 1, a stimulation of [3H]thymidine incorporation was obtained in the case of nonseparated lymphocytes and purified T-derived lymphocytes whereas the B lymphocytes did not respond. Influence of Human ZgG Antitetanus Antibody or Tetanus Toxoid Induced PHfthymidine Incorporation by T Lymphocytes It is well documented that antibody regulates functions of T lymphocytes (14- 17) and could influence the expression of delayed hypersensitivity (18). We have tested the influence of various amounts of human IgG antitetanus Ab. on the [3H]thymidine incorporation induced by an unique dose of tetanus toxoid by highly purified T lymphocytes. As can be seen in Fig. 5, high concentrations of Ab (0.5- 15 mg corresponding to 1.25- 12.5 IU) incubated with 10s T lymphocytes in 1 ml of medium strongly inhibited the rH]thymidine incorporation in control cultures whereas the smaller amounts (0.005-O. 15 mg) had no effect. Incubation of lymphocytes with 0.3 Lf induced a strong stimulation which was not affected by the small amounts of antitetanus Ab (0.0015-0.015 mg) whereas the higher TABLE TETANUS
TOXOID
[3H]T~~~~~~~~
INCORPORATION
1 BY HUMAN
LYMPHOCYTE
POPULATIONS
Lymphocyte populations
Number of subject
Nonseparated
T
B
10478
RFC (%) Ig positive cells (%) Stimulation index
62 18 2.0
87 0 4.6
0.3 76 0.6
10747
RFC (%) Ig positive cells (%) Stimulation index
60 21 6.8
84 1.9 6.4
0.6 74
RFC (%) Ig positive cells (%) Stimulation index
33
87 0 13.7
10211
11
5.6
1.0
0 85
0.8
EYQUEM
AND
BONA
FIG. 5. Dose-response relationship of stimulation of highly purified T-lymphocytes by 0.3 L tetanus toxoid in presence of various amounts of human IgG antitetanus antibody.
amounts (0.5-15 mg) strongly inhibited the [3H]thymidine similar to that which was observed with control cultures. Similar results were obtained in three individual experiments. Influence of B-Derived Lymphocytes on Tetunus Toxoid Induced pH]thymidine Incorporation by T Lymphocytes The influence of autologous B cells on the blast response of T-derived lymphocytes induced by tetanus toxoid was studied by the addition of lo5 or 3 x 105 B cells to 106 T lymphocytes. As can be seen in Table 2, in four cases studied 40-96 days after the last booster and with different schedules of immunization, the addition of B cells on T cells inhibited the [3H]thymidine incorporation (expressed as stimulation index) induced by tetanus toxoid. The inhibition was dependent on the number of B cells but was not related to the number of Ig-bearing cells in nonseparated lymphocytes. DISCUSSION
As expected, T cells were shown to be involved in the specific blast responses of ‘human lymphocytes induced by tetanus toxoid in human subjects hyperimmunized with this antigen. The present study shows that the magnitude of specific blast responses varied in a group of 15 human healthy donors. We classified these subjects into two groups according to their stimulation index. In the case of subjects with a high response (stimulation index > 5) the peak of [3H]thymidine incorporation was obtained with
106 days 45 days 19 months 28 months
Period of immunization
ON TETANUS
40 96 90 75
Time of assay after last booster (days)
TOXOID-INDUCED
D T-cell population contained 0.5 + 0.4% Ig-bearing cells. b B-cell population contained 78 -C 7% Ig-bearing cells. C Stimulation index = (cpm culture + TT)/cpm control culture.
3 3 4 9
CELLS
10201 10891 10211 10750
B
Number of subject
OF
Number of boosters before assay
INFLUENCE
TABLE
2
18 20 13 10
BY
T
2.w 1.0 6.8 5.6
106 nonseparated
INCORPORATION
Ig-bearing cells in nonseparated lymphocytes (%)
[‘HITHYMIDINE
OF HYPERIMMUNIZED
4.6 2.6 15.8 5.0
106 Ta 0.6 0.3 0.9 1.2
lo6 Bb
2.9 2.1 6.7 4.2
1.8 0.9 1.2 3.6
lo6 T 3 x 1O+5 B
SUBJECTS
IO6 T 105+ B
Lymphocyte population
CELLS
2
3 9
$
z f
n ? c’
2 cn
3
8
EYQUEM
AND
BONA
0.03-0.3 Lf in seven out of eight subjects, whereas all the subjects with a low response (stimulation index < 4) reached their peak at I Lf. These findings clearly suggest that the affmity of the receptor of T lymphocytes of high responders for tetanus toxoid was greater as compared with the affinity of low responders. The differences in the response of these two groups cannot be attributed to a general defect in cell mediated immunity of low responder subjects since the blast response induced by a nonspecific mitogen such as PHA or by another antigen such as PPD was quite similar (lymphocytes provided by French subjects are in fact sensitized to tuberculin as a consequence of the compulsory BCG vaccination of French infants). Furthermore, no striking differences between the levels of specific antibody were observed between the two groups. Kinetic studies of blast response during immunization showed a slight increase or a plateau of responses. However. the blast response decreased after the last booster. Three hypothesis could be entertained for explanation of differences in the blast response and its regulation. (i) Relationship bent)een magnitude of tetanus toxoid induced blast response and HLA system. In humans a significant contribution of genetic factors in the immune responses to measles and diphtheria toxoid were shown (19). This is in contrast to the immune response to poliomyelitis vaccina, rubella, influenza virus ( 19). PPD response and anti-Rhesus antibody (20), as well as terpolymer glutamic acid and lysine-thyrosine (21), where no significant deviation from the controls was found for any of the HLA antigens tested for. Studies are in progress in order to test the HLA antigens of our group of subjects with high or low blast response induced by tetanus toxoid. (ii) Injluence ofspecific antibodies. It has been shown that antibodies regulate functions of T lymphocytes (14- 18). In our cases, the magnitude of the humoral response did not correlate with the high or low specific blast responses. Furthermore, the blast transformation assays were performed in our experiments after six washings of the cells and incubation in an AB pool serum without detectable antitetanus antibodies. In addition, specific antibodies present during blast transformation assays in doses between 0.0015-0.05 mg did not alter the level of [3H]thymidine incorporation induced by tetanus toxoid by T lymphocytes. An inhibition was observed only with high amounts (0.15-S mg) of antibody both in the control cultures as well as in the stimulated cultures by tetanus toxoid. This observation is in agreement with that reported by Benezra et al. (22) who showed that the addition of specific antibodies did not affect the blast response indiced by antigens in rabbits. (iii) Suppressor cells. It is well known that there exist general homeostatic cellular phenomenon which control the afferent limbs of B- or T-mediated immune responses (23-31). In a previous work, we demonstrated that in the peripheral blood lymphocytes of BCG vaccinated subjects there were B cells which inhibited the PPD induced [3H]thymidine incorporation by T lymphocytes (32). The data presented here clearly show that the removal of lymphocytes which are not rosetting SRBC also increased the [3H]thymidine incorporation induced by tetanus toxoid in T-derived lymphocytes. In these cases. the addition of 105 or 3 x
TETANUS
CELLULAR
IMMUNITY
IN MAN
9
1V B-derived lymphocytes to 106 T cells strongly and significantly inhibited the blast response. In conclusion, these observations show that among a group of 15 human healthy subjects hyperimmunized with tetanus toxoid according to various schedules, important differences were observed in the magnitude of the specific blast response of T cells. These differences could be easily explained by the existence of B cells with suppressor activity on T functions. Nevertheless, either an inhibitory antibody or a correlation with a particular HLA antigen can not be excluded. Studies are in progress in order to explore the mechanism of this inhibitory activity mediated by B cells as well as the HLA typing of our groups of subjects. ACKNOWLEDGMENTS We are grateful to Dr. Sheldon Woolf and A. Faucci for helpful advice. We thank Mrs. Edith Bourgeois for expert technical assistance, donors for their gift of blood, and nurses for their help, and Mrs. Robba and Mrs. Petit for tetanus antibody titration.
REFERENCES 1. Mills, J. A., J. Immunol. 37, 239, 1966. 2. Oppenheim, J. J., Fed. Proc. 27, 21, 1968. 3. Chess, L., Mac Dermott, R. P., and Schlossman, S. S., J. Immunol. 113, 1122, 1974. 4. Rosenthal, A. S., and Shevach. E. M., J. Exp. Med. 138, 1194, 1973. 5. Cohen, B. E.. Rosentahl. A. S., and Paul, W. E., J. Zmmunol. 111, 811, 1973. 6. Gatien, J. G., Merler, E., and Colten, H. R., C/in. Immune/. zmmunopafh. 4, 32, 1975. 7. Seeger, R. C., and Oppenheim, J. J., J. Exp. Med. 132, 44. 1970. 8. Biiyum, A., Stand. J. Clin. Lab. Invest. 97, 77, 1968. 9. Graham. R. C., Karnovsky, M. J.. J. Histochem. Cytochem. 14, 191, 1966. 10. Lobo. P. I., Westervelt, F. B., and Horowitz, 0. A., J. Immunol. 114, 116, 1975. 11. Bona, C., Chedid, L., Damais, C., Ciorbaru, R., Shek, P. N., Dubiski, S., and Cinader. B., J. Immunol. 114, 348, 1975. 12. de Saint Martin, J., Eyquem, A., Turpin, A., and Bizzini, B., VOX Sanguinis. 15, 39, 1974. 13. Turpin. A., Bizzini, B., and Raynaud, M., Med. Mal. Infect. 2, 65, 1973. 14. Playfair. J. H. L.. Clin. Exp. Zmmunol. 17, 1. 1974. 15. Haugton, G., and MLkelB, O., J. Exp. Med. 138, 103, 1973. 16. Hamaoka, T., Takatsu, K.. Kitgawa, N., Immunology 21, 259, 1971. 17. Tada, T., and Okumura, K., J. Immunol. 107, 1137, 1971. 18. Neta. R., Slavin, S. B., Cc/l. Zmmunol. 9, 242, 1973. 19. Haverkorn, M. J., Hoffman, B., Masurel, N., and Van Rood, J. J., Transpl. Rev. 22, 120, 1975. 20. Van Rood, J. J., Van Hooff. J. P., and Keuning, J. J., Trunspl. Rev. 22, 1975. 21. Sher, I., Ahmed, D., and Green, I., 1975 European Immunology meeting Abstracts, Amsterdam, 1975. 22. Benezra, D., Gery, I., and Davies, A. M.. Proc. SOC. Exp. Rio/. Med. 125, 1305, 1967, 23. Gershon, R. K., and Kondo, K., Immunology 18, 723, 1970. 24. Gershon, R. K., Cohen, P., Hencin, R., and Liebhaker, S. A., J. Zmmuno/. 108, 586, 1972. 25. Gershon, R. K., Orbach-Arbouys, S., and Calkins, C.. In “Immunology II,” (L. Brent and J. Holborow, Eds.) Vol. 2, p. 123, North-Holland Publishing, Amsterdam, 1974. 26. Droege, W., Eur. J. Immunol. 3, 804, 1973. 27. Herzenberg. L. A., Chan, E. L., Ravitch, M. M.. Riblet, R. J., and Herzenberg, L. A., J. Exp. Med. 137, 1311, 1973. 28. Allison, A. C., Denman, A. M., and Barnes, R. D., Lancer II, 135. 1971. 29. Katz. S. I.. Parker. D.. and Turk, J. L., Nature (London) 251, 550. 1974. 30. Gorczinski. R. M.. J. Immunol. 112, 1926. 1974. 31. Russell, A. S.. Liburd, E. M., and Dinner, E., Nature (London) 249, 43, 1974. 32. Bona, C., and Chedid, L., J. Infect. Dis. 133, 465, 1976.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
7, l-9 (1977)
Studies of Tetanus Toxoid-Induced Blast Transformation of Lymphocytes of Immunized Healthy Donors A. EYQUEM Service
d’lmmunothkrapie Institut
AND C. BONA
ExpPrimentale and Pasteur and C.N.R.S.
Centre de Transfusion Paris, France
Sanguine,
Received December 29, 1975 Tetanus toxoid induced-blast transformation of peripheral blood lymphocytes of 15 immunized healthy subjects was studied. According to the magnitude of the [3H]thymidine incorporation, the subjects were classified into two groups: high and low blast specific responses. The high or low blast response induced by tetanus toxoid did not correlate with the stimulation induced by PHA and PPD nor with the antitetanusantibody titer of these subjects. T cells were shown to be involved in this specific blast response. The differences in the magnitude of the blast response could be easily explained by the existence of B cells with suppressor activity on T cells functions rather than by inhibition of the tetanus toxoid induced stimulation by an antigen-antibody complex. Relationship with HL-A system will be investigated.
INTRODUCTION
Various soluble antigens are able to induce blast transformation of lymphocytes in vitro after active in vivu immunization of animals. Antigen-induced proliferation in vitro is a correlate of in vivo cellular immunity (1.2) and generally T cells are involved in the blast response (3 -6). It has been reported that the lymphocytes of guinea pigs actively immunized with tetanus toxoid in Freund’s complete adjuvant were transformed specifically by toxoid and that the blast response required the presence of macrophages in the system (7). In this work, we have investigated various aspects of in vitro blast transformation of lymphocytes of human healthy subjects hyperimmunized with tetanus toxoid as well as the relationship of this phenomenon to various parameters, i.e., nonspecific blast response, specific blast response with a nonrelated antigen, titer of antitetanus antibody, and the schedule of immunization. MATERIALS
AND METHODS
Blood lymphocytes were recovered from healthy human blood donors, vaccinated several times with tetanus toxoid vaccine (I.P.A.D.T. Institut Pasteur). The lymphocytes were separated according to the Boyum technique (8). Separation of T and B-derived lymphocytes. Purified T- or B-derived lymphocytes were obtained by the passage of ficoll separated lymphocytes through a nylon column according to a previously described technique (32). The degree of purification of lymphocytes was controlled by rosetting the lymphocyte purified populations with SRBC or by staining with FAB fragments of goat IgG anti-human Ig labeled with peroxydase (Pasteur Institute), according to the method of Graham and Karnovsky (9). Determination of Ig positive cells with Lymphocytes.
Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved
7
EYQUEM
.-\Nl>
BOh.4
Fab fragments of goat IgG anti-human IgG antibodies exclude the possibility of staining cells which bind heterologous IgG by their Fc receptors. Moreover, staining was performed after incubation of cells at 37°C according to the method of Lobo et al. (10) which is more selective than after incubation at 4°C. Blast transformatiotz. Lymphocytes (106) were cultured for 4 days in I ml of Eagles medium (General Island Biological Co., Grand Island. N. Y.) supplemented with nonessential aminoacids and 15% of AB homologous serum previously inactived for 30 min at 56°C. The cultures were performed in an incubator model IH-100 (Gallenkamp) under a continuous flow of a mixture of 5% CO, and 95% air. Antigen was added at various concentrations in 0. I ml of MEM medium. I &i r3H]thymidine (1 ciimmol specific activity. Saclay. France) was added to each culture tube 12 hr before harvesting the cells. 13H]thymidine incorporation was measured by liquid scintillation counting according to a previously described technique (I I). Antigens. Purified protein derivative (PPD-neutre pour test de transformation blastique. Institut Pasteur), tetanus toxoid containing 30 Lf/ml and 2.8 mg protein/ml (lnstitut Pasteur) were the antigens employed. Other reagents. Human Ig antitetanus antibody (15.5 g% and 125 IU/ml) (Pasteur Institute) PHA-P (Difco). Antibody assays. Titers of antitetanus antibody were measured either in vitro by means of agglutination of tetanus toxoid coated latex particles according to a previously described technique (12) or in ~ivo by protection of mice according to described techniques ( 13). RESULTS
Blast Transformation of Lymphocytes [3H]thymidine incorporation of lymphocytes of human hyperimmunized subjects with tetanus toxoid was estimated after incubation with various amounts of tetanus toxoid (0.03-3 Lf) as well as with 10 pg PPD and 2 pg PHA. The level of specific antibody was determined simultaneously with thymidine incorporation. According to the level of specific tetanus toxoid blast responses of lymphocytes. the 15 subjects could be classified into two groups: Eight subjects with a stimulation index higher than five and seven subjects with a stimulation index lower than four. High responder subjects. In this group, the peak of blast response induced by various doses of tetanus toxoid ranged between 5.5-31. In seven out of eight cases, the peak of [3H]thymidine incorporation was obtained with doses smaller than 1 Lff 106 cells; doses varied between 0.03-0.3 Lf. The lymphocytes of these eight subjects could be stimulated by a nonspecific mitogen such as PHA as well as by another specific mitogen such as PPD. As can be seen in Fig. 1, there was no correlation between the blast responses induced by tetanus toxoid and the level of specific antibody or the level of [3H]thymidine incorporation induced by PHA and PPD. Low, responder subjects. The peak of blast response induced by various doses of tetanus toxoid was smaller than 4 in the case of seven subjects. As can be seen in Fig. 2, the dose-response relationship curves of these subjects shows very clearly that the highest stimulation indices were obtained in all seven cases with 1 Lf
TETANUS
CELLULAR
IMMUNITY
IN
MAN
FIG. 1. Dose-response relationship of stimulation of human peripheral lymphocytes by tetanus toxoid (high responder group with stimulation index > 5).
tetanus toxoid406 cells. Three out of seven subjects had a strong blast response to PHA and only one subject did not respond to tuberculin. The low blast responses to tetanus toxoid in these seven subjects could not be related to the titer of specific antibody measured simultaneously with rH]thymidine incorporation.
M .a c-i)
LnDt
done
**,
“./ml
FIG. 2. Dose-response relationship of stimulation of human peripheral lymphocytes by tetanus toxoid (low responder group with stimulation index < 4).
EYQUEM
.!
l FIG.
Kinetics
.,,..
+ f , ,‘, 420123456789
3. Kinetics
of Speci’c
to blast
,
!.....,’
,
response
and Nonspecific
,
AND
,
,
of two
BON.\
,
,
, . loweeks
subjects
with
stimulation
index
‘> 5
Blast Response
The kinetics of specific and nonspecific blast responses were studied in two cases (No. 10207 and 10486) during immunization and in another two cases (No. 10208 and 10211) 40 and 90 days after the last booster (see the schedules of immunization of these last two subjects in Fig. I). Doses of tetanus toxoid corresponding to the peak of blast responses established previously (see Figs. 1 and 2) were used in the kinetics study. As can be seen in Fig. 3, a time dependent increase of specific blast response was obtained in the case of subject No. 10207 whereas a plateau was observed in
I
FIG.
4. Kinetics
1111,,1111,,,11 123456789
of blast
response
to
of two subjects
11
12
with
13
14
15 1r3ICCkI
stimulation
*
index
c 5
TETANUS
CELLULAR
IMMUNITY
IN
5
MAN
the case of subject No. 10486. PHA blast response paralleled the specific response. In the case of subjects studied 40 days (No. 10208) and 90 days (No. 10211) after their last booster, a decrease of the specific blast response was observed during the first 8 or 9 weeks as compared with the nonspecific blast response induced by PHA (see Fig. 4). Evidence of T-Derived Lymphocytes Involvement in the Specific Blast Response Induced by Tetanus Toxoid In three cases, the blast response induced by tetanus toxoid was tested on nonseparated lymphocytes as well as T- and B-derived purified lymphocytes. As can be seen in Table 1, a stimulation of rH]thymidine incorporation was obtained in the case of nonseparated lymphocytes as well as T- and B-derived purified lymphocytes. As can be seen in Table 1, a stimulation of [3H]thymidine incorporation was obtained in the case of nonseparated lymphocytes and purified T-derived lymphocytes whereas the B lymphocytes did not respond. Influence of Human ZgG Antitetanus Antibody or Tetanus Toxoid Induced PHfthymidine Incorporation by T Lymphocytes It is well documented that antibody regulates functions of T lymphocytes (14- 17) and could influence the expression of delayed hypersensitivity (18). We have tested the influence of various amounts of human IgG antitetanus Ab. on the [3H]thymidine incorporation induced by an unique dose of tetanus toxoid by highly purified T lymphocytes. As can be seen in Fig. 5, high concentrations of Ab (0.5- 15 mg corresponding to 1.25- 12.5 IU) incubated with 10s T lymphocytes in 1 ml of medium strongly inhibited the rH]thymidine incorporation in control cultures whereas the smaller amounts (0.005-O. 15 mg) had no effect. Incubation of lymphocytes with 0.3 Lf induced a strong stimulation which was not affected by the small amounts of antitetanus Ab (0.0015-0.015 mg) whereas the higher TABLE TETANUS
TOXOID
[3H]T~~~~~~~~
INCORPORATION
1 BY HUMAN
LYMPHOCYTE
POPULATIONS
Lymphocyte populations
Number of subject
Nonseparated
T
B
10478
RFC (%) Ig positive cells (%) Stimulation index
62 18 2.0
87 0 4.6
0.3 76 0.6
10747
RFC (%) Ig positive cells (%) Stimulation index
60 21 6.8
84 1.9 6.4
0.6 74
RFC (%) Ig positive cells (%) Stimulation index
33
87 0 13.7
10211
11
5.6
1.0
0 85
0.8
EYQUEM
AND
BONA
FIG. 5. Dose-response relationship of stimulation of highly purified T-lymphocytes by 0.3 L tetanus toxoid in presence of various amounts of human IgG antitetanus antibody.
amounts (0.5-15 mg) strongly inhibited the [3H]thymidine similar to that which was observed with control cultures. Similar results were obtained in three individual experiments. Influence of B-Derived Lymphocytes on Tetunus Toxoid Induced pH]thymidine Incorporation by T Lymphocytes The influence of autologous B cells on the blast response of T-derived lymphocytes induced by tetanus toxoid was studied by the addition of lo5 or 3 x 105 B cells to 106 T lymphocytes. As can be seen in Table 2, in four cases studied 40-96 days after the last booster and with different schedules of immunization, the addition of B cells on T cells inhibited the [3H]thymidine incorporation (expressed as stimulation index) induced by tetanus toxoid. The inhibition was dependent on the number of B cells but was not related to the number of Ig-bearing cells in nonseparated lymphocytes. DISCUSSION
As expected, T cells were shown to be involved in the specific blast responses of ‘human lymphocytes induced by tetanus toxoid in human subjects hyperimmunized with this antigen. The present study shows that the magnitude of specific blast responses varied in a group of 15 human healthy donors. We classified these subjects into two groups according to their stimulation index. In the case of subjects with a high response (stimulation index > 5) the peak of [3H]thymidine incorporation was obtained with
106 days 45 days 19 months 28 months
Period of immunization
ON TETANUS
40 96 90 75
Time of assay after last booster (days)
TOXOID-INDUCED
D T-cell population contained 0.5 + 0.4% Ig-bearing cells. b B-cell population contained 78 -C 7% Ig-bearing cells. C Stimulation index = (cpm culture + TT)/cpm control culture.
3 3 4 9
CELLS
10201 10891 10211 10750
B
Number of subject
OF
Number of boosters before assay
INFLUENCE
TABLE
2
18 20 13 10
BY
T
2.w 1.0 6.8 5.6
106 nonseparated
INCORPORATION
Ig-bearing cells in nonseparated lymphocytes (%)
[‘HITHYMIDINE
OF HYPERIMMUNIZED
4.6 2.6 15.8 5.0
106 Ta 0.6 0.3 0.9 1.2
lo6 Bb
2.9 2.1 6.7 4.2
1.8 0.9 1.2 3.6
lo6 T 3 x 1O+5 B
SUBJECTS
IO6 T 105+ B
Lymphocyte population
CELLS
2
3 9
$
z f
n ? c’
2 cn
3
8
EYQUEM
AND
BONA
0.03-0.3 Lf in seven out of eight subjects, whereas all the subjects with a low response (stimulation index < 4) reached their peak at I Lf. These findings clearly suggest that the affmity of the receptor of T lymphocytes of high responders for tetanus toxoid was greater as compared with the affinity of low responders. The differences in the response of these two groups cannot be attributed to a general defect in cell mediated immunity of low responder subjects since the blast response induced by a nonspecific mitogen such as PHA or by another antigen such as PPD was quite similar (lymphocytes provided by French subjects are in fact sensitized to tuberculin as a consequence of the compulsory BCG vaccination of French infants). Furthermore, no striking differences between the levels of specific antibody were observed between the two groups. Kinetic studies of blast response during immunization showed a slight increase or a plateau of responses. However. the blast response decreased after the last booster. Three hypothesis could be entertained for explanation of differences in the blast response and its regulation. (i) Relationship bent)een magnitude of tetanus toxoid induced blast response and HLA system. In humans a significant contribution of genetic factors in the immune responses to measles and diphtheria toxoid were shown (19). This is in contrast to the immune response to poliomyelitis vaccina, rubella, influenza virus ( 19). PPD response and anti-Rhesus antibody (20), as well as terpolymer glutamic acid and lysine-thyrosine (21), where no significant deviation from the controls was found for any of the HLA antigens tested for. Studies are in progress in order to test the HLA antigens of our group of subjects with high or low blast response induced by tetanus toxoid. (ii) Injluence ofspecific antibodies. It has been shown that antibodies regulate functions of T lymphocytes (14- 18). In our cases, the magnitude of the humoral response did not correlate with the high or low specific blast responses. Furthermore, the blast transformation assays were performed in our experiments after six washings of the cells and incubation in an AB pool serum without detectable antitetanus antibodies. In addition, specific antibodies present during blast transformation assays in doses between 0.0015-0.05 mg did not alter the level of [3H]thymidine incorporation induced by tetanus toxoid by T lymphocytes. An inhibition was observed only with high amounts (0.15-S mg) of antibody both in the control cultures as well as in the stimulated cultures by tetanus toxoid. This observation is in agreement with that reported by Benezra et al. (22) who showed that the addition of specific antibodies did not affect the blast response indiced by antigens in rabbits. (iii) Suppressor cells. It is well known that there exist general homeostatic cellular phenomenon which control the afferent limbs of B- or T-mediated immune responses (23-31). In a previous work, we demonstrated that in the peripheral blood lymphocytes of BCG vaccinated subjects there were B cells which inhibited the PPD induced [3H]thymidine incorporation by T lymphocytes (32). The data presented here clearly show that the removal of lymphocytes which are not rosetting SRBC also increased the [3H]thymidine incorporation induced by tetanus toxoid in T-derived lymphocytes. In these cases. the addition of 105 or 3 x
TETANUS
CELLULAR
IMMUNITY
IN MAN
9
1V B-derived lymphocytes to 106 T cells strongly and significantly inhibited the blast response. In conclusion, these observations show that among a group of 15 human healthy subjects hyperimmunized with tetanus toxoid according to various schedules, important differences were observed in the magnitude of the specific blast response of T cells. These differences could be easily explained by the existence of B cells with suppressor activity on T functions. Nevertheless, either an inhibitory antibody or a correlation with a particular HLA antigen can not be excluded. Studies are in progress in order to explore the mechanism of this inhibitory activity mediated by B cells as well as the HLA typing of our groups of subjects. ACKNOWLEDGMENTS We are grateful to Dr. Sheldon Woolf and A. Faucci for helpful advice. We thank Mrs. Edith Bourgeois for expert technical assistance, donors for their gift of blood, and nurses for their help, and Mrs. Robba and Mrs. Petit for tetanus antibody titration.
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