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Differences in transformation of adult and newborn lymphocytes stimulated by antigen, antibody, and antigen-antibody complexes
CELLULAR
IMMUSOLOGY
Differences
1, 46&175
(1971)
in Transformation
Lymphocytes
Stimulated
of Adult by Antigen,
Antigen-Antibody
and
Newborn
Antibody,
Complexes
and
’
SANFORD LEIKIN George Washington University School of Medicine, Children’s Hospital of the District of Colzlmbia, alzd Research Foundation of the Children’s Hospital, Washington, D.C. 20009 AND National
JOOST J. OPPENHEIM
Institute
of Dental
Received
Research,
Bethesda,
Md.
May 11, 1970
stimulation of lymphocytes by It has been proposed that i?t vitro antigen-antibody complexes is due to an immunologically nonspecific action. The lymphocytes of newborn infants and adults were tested for their ability to respond to tetanus and diphtheria toxoid, the respective antitoxinIs and antigen-antibody complexes. Cord blood lymphocytes failed to transform with these stimulants whereas the lymphocytes of previously immunized adults were transformed by these antigens and the antigen-antibody complexes. In addition, BSA-anti-BSA complex stimulated lymphocytes from ostensibly unsensitized adults but not the cord blood cells. It is concluded from these results that lymphocyte transformation by antigen-antibody complexes is due to a specific acquired immunologic response and that adult individuals may have unknowingly become sensitized to ubiquitous antigens such as BSA.
INTRODUCTION
The stimulants used to produce in vitro lymphocyte transformation include such specific antigens as tetanus and diphtheria toxoids to which adults react only if previously sensitized. “Nonspecific” stimulants, such as phytohemagglutinin (PHA), are mitogens to which lymphocytes react in the absense of prior sensitization. However, when such antigens as bovine serum albumin (BSA) are complexed with antibody they stimulate lymphocytes from “unimmunized” adult donors (1). Moreover, Miiller (2) found that antibodies complexed to ubiquitous antigens, such as sheep erythrocytes, Sall~onella bacteria, E. coli endotoxin, bovine serum albumin (BSA), and mouse gamma globulin, also stimulate ostensibly unsensitized adult human lymphocytes. It has been suggested that these antigen-antibody complexes, therefore, act in a nonspecific fashion by irritating the lymphocyte 1 Supported
by NIH
Grant HD-01273. 468
STIMULATION
OF
NEWBORN
LYMPHOCYTES
469
membrane and that this may be the basic mechanism for the lymphocyte response to all nonspecific stimulants (1). It is known that cord blood lymphocytes are capable of responding vigorously to immunologically specific stimulation with homologous cells in mixed leukocyte cultures and to nonspecific stimulants such as PHA, pokeweed mitogen, and exotoxin of staphylococcal filtrate (3). If antigen-antibody complexes do react in a nonspecific manner, it would be expected that they would stimulate cord blood lymphocytes. In this study we have, therefore, compared the reaction of lymphocytes from normal adults with those from randomly chosen cord bloods to antigen-antibody complexes. The cord blood lymphocytes did not react, which suggests that transformation of adults’ lymphocytes by antigen-antibody complexes is due to a specific immunologically acquired sensitization rather than a nonspecific process. MATERIAL AND METHODS
Horse antidiptheria serum was kindly provided by Parke Davis Co. Its titer was 2600u/ml. Rabbits were immunized with 0.5 ml of alum-precipitated (Parke Davis Co.) diphtheria-tetanus toxoid subcutaneously and into the foot pads administered every other week on four occasions. Two weeks after the last injection the animal was bled and the antiserum was obtained. The anti-BSA was prepared in rabbits by three weekly intraperitoneal injections of 50 mg BSA in complete Freund’s adjuvant. The antiserum was obtained 12 weeks after the last injection. The antigens used for preparation of the antigen-antibody complexes were diphtheria toxoid (Pittman-Moore Co.), tetanus toxoid (kindly supplied by Dr. L. Levine, Massachussetts Department of Health), and BSA (Mann Research Laboratories) . Precipitated antigen-antibody complexes were prepared at equivalence by incubating BSA with rabbit anti-BSA, diphtheria toxoid with horse anti-diphtheria, and tetanus toxoid with rabbit anti-tetanus at 37” for 2 hr and then at 4” for 48 hr (4). These were then washed three times with ice-cold isotonic saline, and resuspended with the antigen concentrations as indicated. Fresh antisera were used in all cases for preparation of the complexes. Cord blood specimens were obtained by venous aspiration from the fetal side of the placenta after uncomplicated delivery of mothers who were free of illness and who had received diphtheria and tetanus immunization prior to but not during their pregnancies. The adult donors had all received previous diphtheria and tetanus immunizations. The leukocytes from the adult donors and cord specimens were obtained from heparinized blood by differential sedimentation. Three-milliliter leukocyte cultures were prepared using 3 X lo6 leukocytes, 20% fresh antologous plasma, NCTC 199 culture media, and appropriate stimulant. The concentrations of antigens and antigen-antibody complexes used in the cultures were equivalent to 1 LF/ml of diphtheria toxoid, 1 LF/ml of tetanus toxoid or BSA in various concentrations as mentioned below. The antibody used was 1 U/ml horse diphtheria antiserum, 1 U/ml of rabbit tetanus antiserum or 0.1 ml/culture of rabbit anti-BSA in a concentration equivalent to that used to prepare the Ag-AB complex. The leukocyte cultures were incubated in triplicate at 37” for 5 days (3), and the degree of lymphocyte transformation was measured by the uptake of tritiated thymidine (TdR3H) in trichloracetic acid-precipitated material by liquid-scintillation counting (5).
470
LEIKIN
AND
OPPENHEIM
The Student t test was used for statistical analysis of paired comparisons of unstimulated and stimulated cultures. The analysis evaluated data after it was normalized by logarithmic transformation of the counts per minute. Analysis of variance of the response of triplicate cultures was also performed and revealed that a 3 :1 or greater ratio of TdR”H incorporation by stimulated to unstimulated cultures was statistically significant (p < .Ol). RESULTS
The TdR3H incorporation by leukocyte cultures from cord blood and immunized adults stimulated with diphtheria and tetanus toxoids, their antitoxins and toxoidantibody complexes was determined (Fig. 1 and Tables 1 and 2). When analyzed as a group no significant difference could be found between the unstimulated cultures of cord cells and those stimulated by diphtheria toxoid, horse antidiphtheria, and its antigen-antibody complex. The lymphocytes from one cord blood specimen responded to all three of these stimulants. A second cord blood specimen was stimulated only by antigen and a third only by complex. In contrast, the adults’ lymphocyte cultures were significantly stimulated by diphtheria toxoid (p < .OZS) w h en compared to their control cultures. The diphtheria toxoid-antibody complexes also stimulated adults’ lymphocytes (p < .Ol ) . Adults’ lymphocytes cultured with antidiphtheria antibody did not show a response. Cord blood leukocyte cultures were also generally not stimulated significantly by tetanus toxoid or tetanus toxoid-antitoxin complexes when compared to their unstimulated controls. In nine cord blood samples, responses were produced by tetanus toxoid in only two, and by tetanus toxoid-antibody complexes in only one of the subjects. Rabbit anti-tetanus antibody stimulated five of the nine cord blood specimens. Adults’ lymphocytes were stimulated by tetanus toxoid (p < .OOS). The rabbit anti-tetanus antiserum also stimulated adult leukocyte cultures (p = .OS). The tetanus toxoid antigen-antibody complexes stimulated adult cultures maximally (p < .OOl). Although both the tetanus-anti-tetanus and diphtheria-anti-diphtheria complexes stimulated the adults’ lymphocytes to a greater extent than did the uncomplexed antigens, the increments were not statistically different. In view of the positive response of immunized adults and usually negative reaction of newborn lymphocytes to the above complexes, we tested the lymphocyte response of both unimmunized newborn and unimmunized adults with BS,4, its antibody, and BSA-antibody complexes Figs. 2 and 3). Tenfold increases in concentrations ranging from 0.006 pg/ml to 60 pg/ml of l3SA had no effect on newborn or adult leukocyte cultures. High concentrations of heat-aggregated BSA of 2 and/or 20 mg/ml significantly stimulated only two of seven adults’ lymphocyte cultures and one of five cord blood cultures (Tables 3 and 4). However, BS.4 at a concentration of 60 pg/ml when complexed to anti-BSA produced optimal stimulation in the adults’ lymphocyte cultures p < .OOl) but not in cord blood cultures (p = .5). Furthermore, lo-fold increases in concentrations of complexes ranging from ,006 pg/ml to 600 /Jg of BSA did not have a significant effect on newborn leukocyte cultures. It was found that adults’ lymphocytes also responded as well to anti-BSL% as to BSA-anti-BSA complex. L5Tealso tested to see if the response to BSA-anti-USA
STIMULATION
OF
NEWBORN
TABLE TRITIATED
THYMIDINE STIMULATED
471
LYMPHOCYTES
1
INCORPORATIOX BY ADULT AND CORD BLOOD WITH DIPHTHERIA TOXOID, ANTIBODI’, AND TOXOID-ANTIBODY COMPLESES (cpm)
Antibody
Control
LEUKOCYTE DIPHTHERIA
CULTURI’S
Ahtigen-nntibodq complex
Adults 1 2 3 4 5 6 7 Geometric
mean (cpm)
t test p value
77 439 90 226 33.5 171 567 216 -
62 2099 257 5438
189 713 69
811
1214 20.54 864 <.02.5
328 374 891 28.5 > .2
624 7205 97 9925 1280 1217 1655 1406 < .Ol
1202 429 32 196 2559 3752 77 539 443 35 161 90 695 >.2
1098 920 122 1497 6445 4271 472 350 726 35 52 101 468 > ..5
65 34 31 41 7237 4562 1630 363 988 2474 367 182 502 >.5
154
Cord blood 1 i 4 5 6 7 8 9 10 11 12
Geometric mean (cpm) t test p value
427 668 249 361 135 3014 1405 166 1204 197 339 278 441
was due to a cross-reaction between anti-BSA and human serum albumin (HSA), but found that our preparations of HSA-anti-BSA complexes failed to stimulate adults’ lymphocytes. DISCUSSION
Both diphtheria and tetanus antigen and antigen-antibody complexes stimulated the lymphocytes of previously immunized adults but usually had no effect on lymphocytes from unimmunized newborns. These results strongly suggest that the responses in these instances are immunologically acquired rather than nonspecific. This is in agreement with the report of the lack of response of unsensitized guinea pig lymphocytes to diphtheria and tetanus antigen complexed by antibodies (6). BSA did not stimulate “unsensitized” cord blood and adults’ lymphocytes in concentrations of 60 pg/ml or less. However, the stimulation of a minority of adults’ and cord blood lymphocyte cultures with high concentration (2 and 20 ~g/ml) of heat-aggregated or unaggregated BSA suggests that a state of detectable low-grade prior sensitization may indeed exist in some individuals. Furthermore, the absence of a response of cord blood cells to uncomplexed and complexed BSA at a concen-
472
LEIKIN
AND
TABLE TDR~H
INCORPORATION BY ADULT BY TETANUS TOXOID, ANTIBODY,
Adults 1
805 360 326 123 606 121 264 323 304
Geometric mean (cpm) t test p value
2
AND CORD BLOOD LEUKOCYTE CULTURES STIMULATED AND TETANUS TOXOID-AXTIBODY CONPLEX (cpm)
Control
2 3 4 5 6 7 8
OPPENHEIM
Antigen-antibody complex
Antigen
Antibody
5476 57.56 12,057 2468 4109 12,772 321 1488 3519
872 1684 5600 579 6173 1355 49 597 1019
4356 4.523 14,422 3939 4244 4657 11,483 686 4520
< ,005
.05
< .OOl
Cord blood 1
1902
1980
2123
114
2 3 4 5 6 7 8 9
1043 1616 109 2578 629 871 690 943
1807 2179 4085 1934 2252 33.5 967 1928
881
1642 NS
8167 3882 520 13,295 405 1461 3172 3326 2425
1033 2248 104 4.521 644 3347 93.5 2070 930
Geometric mean (cpm) t test p value
NS
<.Ol
.
.
D T Cord Blood Antigen
Adults
D T D T Cord Blood Adults Antibody
1. Diphtheria toxoid (D) and tetanus toxoid (AgAb) complex stimulation of cord blood and adult represents the mean of triplicate cultures from a subject. FIG.
Ag-Ab
:
m
: .
:
D Adult: Complex
(T) antibody and antigen-antibody blood leukocyte cultures. Each point
STIMULATION
OF
NEWBORN
TABLE
LYMPHOCYTES
473
3
RATIO OF TRITIATED THYMIDINE INCORPORATION (STIMULATED/UNSTIMULATED) BY ADULTS’ LYMPHOCYTE CULTURES STIMULATED WITH HIGH CONCENTRATIONS OF AGGREGATED AND UNACGREGATED BS.4 Ratio No. 20 mg
2w
.14 1.8 .90
1 4x. Unagg. 2 kg. Unagg. 3 Aa. Unagg. 4 Am. Unagg. 5 Am. Unagg. 6 Aa. Unagg. 7 &a. Unagg.
.39 2.3 1.3 1.4 1.1 2.4 5.1 2.4 1.4 1.6 15.7 6.6 1.8 .68
0.7
.05 1.0 8.1 1.0 1.9 1.1 2.1 2.6 .24 .34
tration of 60 &ml, and the stimulation of adults’ lymphocytes by complexed BSA observed by us as well as others (1,Z) strongly suggests that adult donors have unknowingly been sensitized. The in vitro-stimulating effects of antigens may be enhanced by aggregation with antibody (1,2,6,). It has been shown by Uhr and Dose BSA
--
Response
Curve
Anti
Complex
BSA
\ \
0 6 Antigen
60 Concentration
.g;>, 1.6 600 ( fig/ml)
FIG. 2. Dose-response curve of adults’ lymphocyte cultures with BSA-anti-BSA complex at varying concentrations of antigen. The solid and interrupted lines show the results of two separate experiments. Each point represents the mean of triplicate cultures from a subject.
474
LEIKIN
AND
OPPENHEIM
.
.
. . . . .
.
Ai .
d
. A &. 4
Adults d lib0 dy
tigc
FIG. 3. Bovine serum albumin (BSA), anti-BSA, and BSA-anti-BSA (AgAb) complex stimulation of cord blood and adult blood leukocyte cultures. Each point represents the mean of triplicate cultures from a subject.
TABLE RATIO
4
OF RADIOACTIVE COUNTS (STIMULATED/UNSTIMULATED) OF CORD LYMPHOCYTES CONTAINING HIGH CONCENTRATIONS OF AGGREGATED AND UNAGGREGATED BSA
Ratio No. 1 && Unagg.
2 kg.
___ 20 mg/ml
2 mg/ml
1.1 9.0
6.1 15.8
.53 1.7
1.2 1.7
3 Am. Unagg.
0.4 .79
1.4 1.0
4 Am. Unagg.
.87 .45
.76 .97
5 Am. Unagg.
.76 .41
.14 .45
Unagg.
BLOOD
STIMULATION
OF
NEWBORN
LYMPHOCYTES
475
Phillips (7) that antigen-antibody complexes manifest increased macrophage and lymphocyte sensitization. This mechanism might, therefore, facilitate macrophage opsonization and “processing” of the antig-en and thus promote lymphocyte transformation (S) . These findings in conjunction with our observations suggest that in zlitro lymphocyte transformation becomes sufficiently sensitive to detect postnatal sensitization by a wide variety of ubiquitous antigens only when one increases the immunogenicity of these antigens by complexing them with antibodies. In agreement with others (1, 2) we also found that adults’ lymphocytes responded significantly to rabbit anti-BSA, and adult and cord blood cells to rabbit anti-tetanus. Additional studies by us (9) have shown that the stimulating effect of rabbit anti-BSA was equalled by that of normal rabbit serum. Therefore, lymphocytes may be responding to some stimulant in the rabbit serum other than the anti-B&4 antibody ( 10). Furthermore, the precipitated BSIZ-anti-BSA complexes were washed free of most of the free rabbit serum. Hence the stimulating effect of antigen-antibody complexes is probably due to the antigenic component rather than the antibody. It has also been reported that the stimulating effect of rabbit antiBSA could be absorbed out with lymphocytes, without blocking subsequent BSXanti-13SA stimulation (1). Since both newborn and adults’ lymphocytes responded to rabbit serum, but only the latter to complexes of rabbit BSA-anti-B54 the rahbit antibody probably does not participate in this phenomenon. Although as a group the cord blood cells were not stimulated by diphtheria, tetanus, or BSA antigens, or their antibodies or complexes, isolated responses were seen in several of the cord blood cultures. It is possible that this could be due to the passage of maternal cells across the placenta into the fetal circulation. However, karyotypic analyses of cord blood cells by Turner et a.Z.(11) and by us (12) indicate that this occurs only rarely. Alternatively, it is possible that these responses represent evidence of prenatal sensitization by these antigens or detection of genetic differences in lymphocyte reactivity at the time of birth. Studies are in progress to examine this problem. ACKNOWLEDGMENT We thank Mrs. Carole Harris for excellent technical assistance. We are also deeply indebted to Dr. C. MacLean for assistance in the statistical analysis. We gratefully acknowledge the helpful suggestions of Dr. Jacqueline Whang-Peng.
REFERENCES 1. 2. 3. 4.
Block-Shtacher, N., Hirschhorn, K., and Uhr, J., Cl&z. Exfi. Z~~zmmol. 3, 889, 1968. MGller, G., Ch. Exfi. I~mtzclcol. 4, 65, 1969. Leikin, S., Mochir-Fatemi, F., Park, K., J. Pcdiat. 72, 510, 1968. Kabat, E., Mayer, M., 1~ “Experimental Innnunology” C. E. Thomas, Springfield, Ill. (1963).
5. Oppenheim,J., Wohlstencroft, R., and Gel!, P. G. H., Inzmz~zology12,89, 1967. 6. 7. 8. 9. 10.
11. 12.
Oppenheim, J., Fed. Proc. 28, 565, 1969. Uhr, J., and Phillips, J., Amz. N. Y. Acad. Sci. 129, 793, 1966. Hersh, E., and Harris, J., /. Immzu~ol. 100, 1184, 1968. Leikin, S., and Oppenheim, J., Unpubli,shed results. Elrod, M., and Schrek, K., La~zcet 2, 595, 1964. Turner, J., Wald, N., and Quinlivan, W., Amr. J. Obsfet. Gynecol. 95, 831, 1966. Leikin, S., Whang, J., and Oppenheim,J., Proc. Sot. Pcdiat. Rcs., p. 21. 1970.
IMMUSOLOGY
Differences
1, 46&175
(1971)
in Transformation
Lymphocytes
Stimulated
of Adult by Antigen,
Antigen-Antibody
and
Newborn
Antibody,
Complexes
and
’
SANFORD LEIKIN George Washington University School of Medicine, Children’s Hospital of the District of Colzlmbia, alzd Research Foundation of the Children’s Hospital, Washington, D.C. 20009 AND National
JOOST J. OPPENHEIM
Institute
of Dental
Received
Research,
Bethesda,
Md.
May 11, 1970
stimulation of lymphocytes by It has been proposed that i?t vitro antigen-antibody complexes is due to an immunologically nonspecific action. The lymphocytes of newborn infants and adults were tested for their ability to respond to tetanus and diphtheria toxoid, the respective antitoxinIs and antigen-antibody complexes. Cord blood lymphocytes failed to transform with these stimulants whereas the lymphocytes of previously immunized adults were transformed by these antigens and the antigen-antibody complexes. In addition, BSA-anti-BSA complex stimulated lymphocytes from ostensibly unsensitized adults but not the cord blood cells. It is concluded from these results that lymphocyte transformation by antigen-antibody complexes is due to a specific acquired immunologic response and that adult individuals may have unknowingly become sensitized to ubiquitous antigens such as BSA.
INTRODUCTION
The stimulants used to produce in vitro lymphocyte transformation include such specific antigens as tetanus and diphtheria toxoids to which adults react only if previously sensitized. “Nonspecific” stimulants, such as phytohemagglutinin (PHA), are mitogens to which lymphocytes react in the absense of prior sensitization. However, when such antigens as bovine serum albumin (BSA) are complexed with antibody they stimulate lymphocytes from “unimmunized” adult donors (1). Moreover, Miiller (2) found that antibodies complexed to ubiquitous antigens, such as sheep erythrocytes, Sall~onella bacteria, E. coli endotoxin, bovine serum albumin (BSA), and mouse gamma globulin, also stimulate ostensibly unsensitized adult human lymphocytes. It has been suggested that these antigen-antibody complexes, therefore, act in a nonspecific fashion by irritating the lymphocyte 1 Supported
by NIH
Grant HD-01273. 468
STIMULATION
OF
NEWBORN
LYMPHOCYTES
469
membrane and that this may be the basic mechanism for the lymphocyte response to all nonspecific stimulants (1). It is known that cord blood lymphocytes are capable of responding vigorously to immunologically specific stimulation with homologous cells in mixed leukocyte cultures and to nonspecific stimulants such as PHA, pokeweed mitogen, and exotoxin of staphylococcal filtrate (3). If antigen-antibody complexes do react in a nonspecific manner, it would be expected that they would stimulate cord blood lymphocytes. In this study we have, therefore, compared the reaction of lymphocytes from normal adults with those from randomly chosen cord bloods to antigen-antibody complexes. The cord blood lymphocytes did not react, which suggests that transformation of adults’ lymphocytes by antigen-antibody complexes is due to a specific immunologically acquired sensitization rather than a nonspecific process. MATERIAL AND METHODS
Horse antidiptheria serum was kindly provided by Parke Davis Co. Its titer was 2600u/ml. Rabbits were immunized with 0.5 ml of alum-precipitated (Parke Davis Co.) diphtheria-tetanus toxoid subcutaneously and into the foot pads administered every other week on four occasions. Two weeks after the last injection the animal was bled and the antiserum was obtained. The anti-BSA was prepared in rabbits by three weekly intraperitoneal injections of 50 mg BSA in complete Freund’s adjuvant. The antiserum was obtained 12 weeks after the last injection. The antigens used for preparation of the antigen-antibody complexes were diphtheria toxoid (Pittman-Moore Co.), tetanus toxoid (kindly supplied by Dr. L. Levine, Massachussetts Department of Health), and BSA (Mann Research Laboratories) . Precipitated antigen-antibody complexes were prepared at equivalence by incubating BSA with rabbit anti-BSA, diphtheria toxoid with horse anti-diphtheria, and tetanus toxoid with rabbit anti-tetanus at 37” for 2 hr and then at 4” for 48 hr (4). These were then washed three times with ice-cold isotonic saline, and resuspended with the antigen concentrations as indicated. Fresh antisera were used in all cases for preparation of the complexes. Cord blood specimens were obtained by venous aspiration from the fetal side of the placenta after uncomplicated delivery of mothers who were free of illness and who had received diphtheria and tetanus immunization prior to but not during their pregnancies. The adult donors had all received previous diphtheria and tetanus immunizations. The leukocytes from the adult donors and cord specimens were obtained from heparinized blood by differential sedimentation. Three-milliliter leukocyte cultures were prepared using 3 X lo6 leukocytes, 20% fresh antologous plasma, NCTC 199 culture media, and appropriate stimulant. The concentrations of antigens and antigen-antibody complexes used in the cultures were equivalent to 1 LF/ml of diphtheria toxoid, 1 LF/ml of tetanus toxoid or BSA in various concentrations as mentioned below. The antibody used was 1 U/ml horse diphtheria antiserum, 1 U/ml of rabbit tetanus antiserum or 0.1 ml/culture of rabbit anti-BSA in a concentration equivalent to that used to prepare the Ag-AB complex. The leukocyte cultures were incubated in triplicate at 37” for 5 days (3), and the degree of lymphocyte transformation was measured by the uptake of tritiated thymidine (TdR3H) in trichloracetic acid-precipitated material by liquid-scintillation counting (5).
470
LEIKIN
AND
OPPENHEIM
The Student t test was used for statistical analysis of paired comparisons of unstimulated and stimulated cultures. The analysis evaluated data after it was normalized by logarithmic transformation of the counts per minute. Analysis of variance of the response of triplicate cultures was also performed and revealed that a 3 :1 or greater ratio of TdR”H incorporation by stimulated to unstimulated cultures was statistically significant (p < .Ol). RESULTS
The TdR3H incorporation by leukocyte cultures from cord blood and immunized adults stimulated with diphtheria and tetanus toxoids, their antitoxins and toxoidantibody complexes was determined (Fig. 1 and Tables 1 and 2). When analyzed as a group no significant difference could be found between the unstimulated cultures of cord cells and those stimulated by diphtheria toxoid, horse antidiphtheria, and its antigen-antibody complex. The lymphocytes from one cord blood specimen responded to all three of these stimulants. A second cord blood specimen was stimulated only by antigen and a third only by complex. In contrast, the adults’ lymphocyte cultures were significantly stimulated by diphtheria toxoid (p < .OZS) w h en compared to their control cultures. The diphtheria toxoid-antibody complexes also stimulated adults’ lymphocytes (p < .Ol ) . Adults’ lymphocytes cultured with antidiphtheria antibody did not show a response. Cord blood leukocyte cultures were also generally not stimulated significantly by tetanus toxoid or tetanus toxoid-antitoxin complexes when compared to their unstimulated controls. In nine cord blood samples, responses were produced by tetanus toxoid in only two, and by tetanus toxoid-antibody complexes in only one of the subjects. Rabbit anti-tetanus antibody stimulated five of the nine cord blood specimens. Adults’ lymphocytes were stimulated by tetanus toxoid (p < .OOS). The rabbit anti-tetanus antiserum also stimulated adult leukocyte cultures (p = .OS). The tetanus toxoid antigen-antibody complexes stimulated adult cultures maximally (p < .OOl). Although both the tetanus-anti-tetanus and diphtheria-anti-diphtheria complexes stimulated the adults’ lymphocytes to a greater extent than did the uncomplexed antigens, the increments were not statistically different. In view of the positive response of immunized adults and usually negative reaction of newborn lymphocytes to the above complexes, we tested the lymphocyte response of both unimmunized newborn and unimmunized adults with BS,4, its antibody, and BSA-antibody complexes Figs. 2 and 3). Tenfold increases in concentrations ranging from 0.006 pg/ml to 60 pg/ml of l3SA had no effect on newborn or adult leukocyte cultures. High concentrations of heat-aggregated BSA of 2 and/or 20 mg/ml significantly stimulated only two of seven adults’ lymphocyte cultures and one of five cord blood cultures (Tables 3 and 4). However, BS.4 at a concentration of 60 pg/ml when complexed to anti-BSA produced optimal stimulation in the adults’ lymphocyte cultures p < .OOl) but not in cord blood cultures (p = .5). Furthermore, lo-fold increases in concentrations of complexes ranging from ,006 pg/ml to 600 /Jg of BSA did not have a significant effect on newborn leukocyte cultures. It was found that adults’ lymphocytes also responded as well to anti-BSL% as to BSA-anti-BSA complex. L5Tealso tested to see if the response to BSA-anti-USA
STIMULATION
OF
NEWBORN
TABLE TRITIATED
THYMIDINE STIMULATED
471
LYMPHOCYTES
1
INCORPORATIOX BY ADULT AND CORD BLOOD WITH DIPHTHERIA TOXOID, ANTIBODI’, AND TOXOID-ANTIBODY COMPLESES (cpm)
Antibody
Control
LEUKOCYTE DIPHTHERIA
CULTURI’S
Ahtigen-nntibodq complex
Adults 1 2 3 4 5 6 7 Geometric
mean (cpm)
t test p value
77 439 90 226 33.5 171 567 216 -
62 2099 257 5438
189 713 69
811
1214 20.54 864 <.02.5
328 374 891 28.5 > .2
624 7205 97 9925 1280 1217 1655 1406 < .Ol
1202 429 32 196 2559 3752 77 539 443 35 161 90 695 >.2
1098 920 122 1497 6445 4271 472 350 726 35 52 101 468 > ..5
65 34 31 41 7237 4562 1630 363 988 2474 367 182 502 >.5
154
Cord blood 1 i 4 5 6 7 8 9 10 11 12
Geometric mean (cpm) t test p value
427 668 249 361 135 3014 1405 166 1204 197 339 278 441
was due to a cross-reaction between anti-BSA and human serum albumin (HSA), but found that our preparations of HSA-anti-BSA complexes failed to stimulate adults’ lymphocytes. DISCUSSION
Both diphtheria and tetanus antigen and antigen-antibody complexes stimulated the lymphocytes of previously immunized adults but usually had no effect on lymphocytes from unimmunized newborns. These results strongly suggest that the responses in these instances are immunologically acquired rather than nonspecific. This is in agreement with the report of the lack of response of unsensitized guinea pig lymphocytes to diphtheria and tetanus antigen complexed by antibodies (6). BSA did not stimulate “unsensitized” cord blood and adults’ lymphocytes in concentrations of 60 pg/ml or less. However, the stimulation of a minority of adults’ and cord blood lymphocyte cultures with high concentration (2 and 20 ~g/ml) of heat-aggregated or unaggregated BSA suggests that a state of detectable low-grade prior sensitization may indeed exist in some individuals. Furthermore, the absence of a response of cord blood cells to uncomplexed and complexed BSA at a concen-
472
LEIKIN
AND
TABLE TDR~H
INCORPORATION BY ADULT BY TETANUS TOXOID, ANTIBODY,
Adults 1
805 360 326 123 606 121 264 323 304
Geometric mean (cpm) t test p value
2
AND CORD BLOOD LEUKOCYTE CULTURES STIMULATED AND TETANUS TOXOID-AXTIBODY CONPLEX (cpm)
Control
2 3 4 5 6 7 8
OPPENHEIM
Antigen-antibody complex
Antigen
Antibody
5476 57.56 12,057 2468 4109 12,772 321 1488 3519
872 1684 5600 579 6173 1355 49 597 1019
4356 4.523 14,422 3939 4244 4657 11,483 686 4520
< ,005
.05
< .OOl
Cord blood 1
1902
1980
2123
114
2 3 4 5 6 7 8 9
1043 1616 109 2578 629 871 690 943
1807 2179 4085 1934 2252 33.5 967 1928
881
1642 NS
8167 3882 520 13,295 405 1461 3172 3326 2425
1033 2248 104 4.521 644 3347 93.5 2070 930
Geometric mean (cpm) t test p value
NS
<.Ol
.
.
D T Cord Blood Antigen
Adults
D T D T Cord Blood Adults Antibody
1. Diphtheria toxoid (D) and tetanus toxoid (AgAb) complex stimulation of cord blood and adult represents the mean of triplicate cultures from a subject. FIG.
Ag-Ab
:
m
: .
:
D Adult: Complex
(T) antibody and antigen-antibody blood leukocyte cultures. Each point
STIMULATION
OF
NEWBORN
TABLE
LYMPHOCYTES
473
3
RATIO OF TRITIATED THYMIDINE INCORPORATION (STIMULATED/UNSTIMULATED) BY ADULTS’ LYMPHOCYTE CULTURES STIMULATED WITH HIGH CONCENTRATIONS OF AGGREGATED AND UNACGREGATED BS.4 Ratio No. 20 mg
2w
.14 1.8 .90
1 4x. Unagg. 2 kg. Unagg. 3 Aa. Unagg. 4 Am. Unagg. 5 Am. Unagg. 6 Aa. Unagg. 7 &a. Unagg.
.39 2.3 1.3 1.4 1.1 2.4 5.1 2.4 1.4 1.6 15.7 6.6 1.8 .68
0.7
.05 1.0 8.1 1.0 1.9 1.1 2.1 2.6 .24 .34
tration of 60 &ml, and the stimulation of adults’ lymphocytes by complexed BSA observed by us as well as others (1,Z) strongly suggests that adult donors have unknowingly been sensitized. The in vitro-stimulating effects of antigens may be enhanced by aggregation with antibody (1,2,6,). It has been shown by Uhr and Dose BSA
--
Response
Curve
Anti
Complex
BSA
\ \
0 6 Antigen
60 Concentration
.g;>, 1.6 600 ( fig/ml)
FIG. 2. Dose-response curve of adults’ lymphocyte cultures with BSA-anti-BSA complex at varying concentrations of antigen. The solid and interrupted lines show the results of two separate experiments. Each point represents the mean of triplicate cultures from a subject.
474
LEIKIN
AND
OPPENHEIM
.
.
. . . . .
.
Ai .
d
. A &. 4
Adults d lib0 dy
tigc
FIG. 3. Bovine serum albumin (BSA), anti-BSA, and BSA-anti-BSA (AgAb) complex stimulation of cord blood and adult blood leukocyte cultures. Each point represents the mean of triplicate cultures from a subject.
TABLE RATIO
4
OF RADIOACTIVE COUNTS (STIMULATED/UNSTIMULATED) OF CORD LYMPHOCYTES CONTAINING HIGH CONCENTRATIONS OF AGGREGATED AND UNAGGREGATED BSA
Ratio No. 1 && Unagg.
2 kg.
___ 20 mg/ml
2 mg/ml
1.1 9.0
6.1 15.8
.53 1.7
1.2 1.7
3 Am. Unagg.
0.4 .79
1.4 1.0
4 Am. Unagg.
.87 .45
.76 .97
5 Am. Unagg.
.76 .41
.14 .45
Unagg.
BLOOD
STIMULATION
OF
NEWBORN
LYMPHOCYTES
475
Phillips (7) that antigen-antibody complexes manifest increased macrophage and lymphocyte sensitization. This mechanism might, therefore, facilitate macrophage opsonization and “processing” of the antig-en and thus promote lymphocyte transformation (S) . These findings in conjunction with our observations suggest that in zlitro lymphocyte transformation becomes sufficiently sensitive to detect postnatal sensitization by a wide variety of ubiquitous antigens only when one increases the immunogenicity of these antigens by complexing them with antibodies. In agreement with others (1, 2) we also found that adults’ lymphocytes responded significantly to rabbit anti-BSA, and adult and cord blood cells to rabbit anti-tetanus. Additional studies by us (9) have shown that the stimulating effect of rabbit anti-BSA was equalled by that of normal rabbit serum. Therefore, lymphocytes may be responding to some stimulant in the rabbit serum other than the anti-B&4 antibody ( 10). Furthermore, the precipitated BSIZ-anti-BSA complexes were washed free of most of the free rabbit serum. Hence the stimulating effect of antigen-antibody complexes is probably due to the antigenic component rather than the antibody. It has also been reported that the stimulating effect of rabbit antiBSA could be absorbed out with lymphocytes, without blocking subsequent BSXanti-13SA stimulation (1). Since both newborn and adults’ lymphocytes responded to rabbit serum, but only the latter to complexes of rabbit BSA-anti-B54 the rahbit antibody probably does not participate in this phenomenon. Although as a group the cord blood cells were not stimulated by diphtheria, tetanus, or BSA antigens, or their antibodies or complexes, isolated responses were seen in several of the cord blood cultures. It is possible that this could be due to the passage of maternal cells across the placenta into the fetal circulation. However, karyotypic analyses of cord blood cells by Turner et a.Z.(11) and by us (12) indicate that this occurs only rarely. Alternatively, it is possible that these responses represent evidence of prenatal sensitization by these antigens or detection of genetic differences in lymphocyte reactivity at the time of birth. Studies are in progress to examine this problem. ACKNOWLEDGMENT We thank Mrs. Carole Harris for excellent technical assistance. We are also deeply indebted to Dr. C. MacLean for assistance in the statistical analysis. We gratefully acknowledge the helpful suggestions of Dr. Jacqueline Whang-Peng.
REFERENCES 1. 2. 3. 4.
Block-Shtacher, N., Hirschhorn, K., and Uhr, J., Cl&z. Exfi. Z~~zmmol. 3, 889, 1968. MGller, G., Ch. Exfi. I~mtzclcol. 4, 65, 1969. Leikin, S., Mochir-Fatemi, F., Park, K., J. Pcdiat. 72, 510, 1968. Kabat, E., Mayer, M., 1~ “Experimental Innnunology” C. E. Thomas, Springfield, Ill. (1963).
5. Oppenheim,J., Wohlstencroft, R., and Gel!, P. G. H., Inzmz~zology12,89, 1967. 6. 7. 8. 9. 10.
11. 12.
Oppenheim, J., Fed. Proc. 28, 565, 1969. Uhr, J., and Phillips, J., Amz. N. Y. Acad. Sci. 129, 793, 1966. Hersh, E., and Harris, J., /. Immzu~ol. 100, 1184, 1968. Leikin, S., and Oppenheim, J., Unpubli,shed results. Elrod, M., and Schrek, K., La~zcet 2, 595, 1964. Turner, J., Wald, N., and Quinlivan, W., Amr. J. Obsfet. Gynecol. 95, 831, 1966. Leikin, S., Whang, J., and Oppenheim,J., Proc. Sot. Pcdiat. Rcs., p. 21. 1970.