Clin. exp. Immunol. (1976) 23, 525-535.
Characterization of the antibody response of the marmoset to sheep red blood cells N. GENGOZIAN, BRENDA L. SALTER, NANCY L. BASFORD & J. R. KATELEY Marmoset Research Center, Medical and Health Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee, U.S.A.
(Received 7 July 1975) SUMMARY
The immune competence of two species of marmosets, S. fuscicollis and S. oedipus, was evaluated by the intravenous (i.v.) and intramuscular (i.m.) injection of sheep red blood cells (SRBC). In S. fuscicollls marmosets, 1 ml of a 50% suspension yielded titres of haemolysin and agglutinating antibodies equal to or greater than 1 ml of a 10% dose of antigen. In both species, the i.v. route, while resulting in formation of 19S and 7S agglutinins, yielded only 19S haemolysins, even after multiple antigen injections. Repeated i.v. injections resulted in a progressive decrease in peak titres, in contrast to the i.m. route, where booster inoculations gave a typical anamnestic response. Jerne plaque-forming cells (PFC) in the spleens of S. oedipus marmosets showed predominately 19S plaques after a primary i.v. challenge; only 19S PFC were detected in the spleen of an animal that had been given multiple inoculations, the type of antibody produced reflecting that found in the serum. 19S but not 7S haemolysins of both species were sensitive to heating at 560C for 1/2 hr. The serum titres and splenic PFC data from the marmosets suggest these animals, particularly S. oedipus, respond poorly to SRBC when a comparison is made to similar studies in mice and rats.
INTRODUCTION The marmoset, a South American primate, is well suited for laboratory investigation due to its small size, ease of maintenance, and breeding in captivity (Gengozian, 1969). Its most unique feature, however, is its high frequency of fraternal twinning, which in the laboratory occurs in approximately 80% of all conceptions; further, placental vascular anastomoses between the foetuses result in haemopoietic chimerism in the twins (Benirschke, Anderson & Brownhill, 1962; Gengozian, Batson & Eide, 1964). Properties of chimerism that have been studied are frequency of occurrence; degree; haemopoietic tissues involved; stability; tolerance to co-twin antigens (Gengozian et al., 1969; Porter & Gengozian, 1969, 1973; Gengozian, 1971). The immunological capability of marmosets has been demonstrated in skin allograft studies; specific sensitization is indicated by the accelerated rejection of second- and thirdset grafts (Gengozian & Porter, 1971). The reactivity of blood lymphocytes in mixed lymphocyte cultures parallels the allograft studies in revealing histocompatibility differences (Barnhart & Gengozian, 1975). Very little is known, however, about the ability of the marmoset to form humoral antibodies to parenterally introduced antigens. In this study we report the response of two marmoset species, S. oedipus and S. fuscicollis, to multiple Correspondence: Dr N. Gengozian, Oak Ridge Associated Universities, P.O. Box 117, Oak Ridge, Tennessee 37830, U.S.A.
525
526
N. Gengozian et al.
inoculations of sheep red blood cells (SRBC). Examination of the humoral (serum antibody) and cellular (Jerne plaque assay) responses has revealed that the marmoset can show an anamnestic response with 7S antibody formation; this, however, is dependent upon the dose of antigen, route of injection, and the marmoset species immunized. A primary deficiency of the marmoset, regardless of species and antigen dose, is failure to synthesize 7S haemolysins following multiple intravenous infusions of SRBC; the antigen given intramuscularly elicits an anamnestic 7S response, the magnitude of which is dependent upon the species. MATERIALS AND METHODS Animals. Two species of marmosets were used, Saguinus oedipus and Saguinus fuscicollis ssp. Imported and laboratory-bred marmosets were used randomly, with no significant effects in any experiment being attributable to the source. Ages of the imported animals were not known; however, all had been in our colony 2 yr and therefore, based on our breeding studies, were considered to be sexually mature. Laboratory bred marmosets ranged in age from 2 to 44 yr. Antigen and immunizations. SRBC were maintained in Alsever's solution for week at 40C prior to use. The cells were washed with saline and suspended to the desired concentration. Two different concentrations were used, ml of a10O% or 50%Y suspension of cells, given either intravenously (i.v.) in the femoral vein or intramuscularly (i.m.) in the outer aspects of the thigh (0 5 ml per thigh). Multiple injections were given, each antigen inoculum (dose and route) being the same as the initial challenge. Animals were bled at regular intervals and the antisera stored at-70C until tested. Assay for serum haemolysins and agglutinins. To detect haemolysins, two-fold serial dilutions of individual serum samples were made in 0 1 ml of modified barbital buffer, pH 7 3-74. The sera were either heated (560C for hr) or not prior to each titration as indicated in the text. The source of complement (C') was fresh human or guinea-pig serum since preliminary tests showed no significant difference between the two with a 50%4 haemolysis endpoint. Serum dilutions were incubated at 370C for 30 min after addition of 01 ml SRBC (1%Y) and 0 1 ml C'(1:10) absorbed with SRBC. To test for agglutinins, 01 ml of diluted antiserum was incubated with 1 ml of SRBC at 370C for 30 min, after which the tubes were centrifuged for 15 sec at approximately 1500 g. Titres have been expressed aslog2 of the reciprocal of the highest dilution showing agglutination or haemolysis. Significant differences between the mean peak titres of the various groups were determined by one-way analysis of variation and the F test. Distinction between 1 9S and7S antibodies Three procedures were used to identify the molecular class of serum antibodies. (1) 2-Mercaptoethanol (2-ME) treatment of serum (Deutsch & Morton, 1957). This permitted determination of 2-ME-sensitive or 2-ME-resistant antibodies, hereafter referred to as 19S and 7S antibodies, respectively. (2) Sucrose density gradient ultracentrifugation. Serum diluted 1:2 with PBS was which was centrifuged at layered on a sucrose gradient (10% to 5°C at 35,000 rev/min for 16 hr in a Spinco L-2 ultracentrifuge with an SW 39 swinging bucket rotor. Fifteen fractions of 03 ml each were collected. (3) Sephadex gel filtration. Sera with similar composition of 19S and 7S antibodies (as revealed by sucrose gradients and 2-ME) taken from identical and/or adjacent blood sample days were pooled and applied to columns of Sephadex G-200 and eluted at room temperature with0-02 phosphate-buffered saline. Jerne plaque technique. Ten marmosets were splenectomized at various intervals after ani.v. injection of SRBC. Splenectomy was performed following anaesthesia with Sernylan (0 025 mg/100 g body weight). The individual spleens were weighed and cell suspensions prepared, washed with Eisen's medium and resuspended to the desired concentration. A slide modification of the Jerne plaque technique was used to assay for haemolytic plaque-forming cells (PFC) (Jerne & Nordin, 1963; Sell, Park & Nordin, 1970). Prior to performing this assay with cells, however, several 'haemolytic spot' tests with immune (to SRBC) marmoset serum were made to determine the optimal conditions for detection of antibody released by spleen cells while in the agarose gel (Hubner & Gengozian, 1969). Variables considered were the C' source (human, rabbit and guinea-pig serum absorbed with SRBC), dilution of C' (1:4, 1:7, 1:10), and concentration of Ca2+ and Mg2 + in the agarose and Eisen's medium used to dilute the C' and to suspend spleen cells. Testing of several marmoset sera containing either 19S or 7S antibody revealed that maximum sensitivity for detection of 19S haemolysins was with human C' (AB, Rh-positive serum) diluted 1: 10, using Eisen's medium containing 493 x 10-4M Ca2 + and x 10-4M Mg 2. For detection of 7S antibody, slightly greater sensitivity was obtained with guinea-pig C' diluted 1:10, using Eisen's medium containing 2-46 x 104M Ca 2 and 1-25 x 104M Mg2+ Facilitation of 7S 'haemolytic spots' was obtained with a goat anti-marmoset IgG antiserum (1:80). Verification that these conditions were optimal for the PFC assay was obtained during the course of this study with spleen cells from four sensitized marmosets.
37%/)
2-50
.
Response to SRBC in the marmoset
527
RESULTS Intramuscular immunizations of S. fuscicollis Eight S.fuscicollis marmosets were given i.m. injections of SRBC, four receiving 1 ml of a 10% suspension and four receiving 1 ml of a 500 suspension. There was no significant difference (P = 0 05) between the two SRBC doses with respect to the qualitative character
:--
-0 0
cacl To
10 9 8 76 54 3
2
10
20
30
40
50
60
70
80
90
100
110
120 130 140 150 160
Days FIG. 1. Mean agglutinin titres to sheep RBC following intramuscular immunizations in S. fuscicois marmosets. Data from two groups of four animals each, one receiving 1 ml of a 10% and the other 1 ml of a 50% suspension, were combined, since no significant differences were noted. (0-0) Total antibody activity; (o - - *) antibody activity after 2-ME treatment of sera. (T) Antigen injection. 10 9
8 7
6 A>
'8 l_
r_
5 4 3
24
Days
FIG. 2. Mean haemolysin titres to SRBC following intramuscular immunizations in S.fuscicollis marmosets. Serum samples from same animals depicted in Fig. 1. (0-0) Total antibody activity; (e - - e) antibody activity remaining after heat (560C for i hr) treatment of sera.
(T) Antigen injection. of the responses or the peak titres following each injection. Therefore, the results were pooled and the mean titres for the eight marmosets are shown in Fig. 1. The agglutinins detected on day 7 were almost all 19S as determined by sucrose density gradients and 2-ME tests. The 7S antibodies reached peak activity on day 14 and remained at a high level throughout. Each booster inoculation resulted in a peak titre greater than that observed after the previous injection. Titrations for haemolysins were performed on the same sera. Two methods were used: in method 1 the sera were unheated before titration and in method 2 the sera had been heated for i hr at 560C. Unheated sera contained low titres (log2 1-3) of 'natural' haemolysins, but I
N. Gengozian
528
et
al.
after heating these could not be detected (Fig. 2). Similarly, although unheated sera showed a titre of 7 (log2) on day 7 after antigen injection, significant haemolysin activity could not be detected following heat treatment. By method 2, peak titres in the primary response were quite low, but became progressively higher following the second, third and fourth injection TABLE 1. Localization and heat sensitivity of 7S and 19S haemolysins in sucrose density gradient fractions Day serum obtained after antigen
injection* 7
14
49
Treatment of fraction for titrationt Fraction numbert 1-8 9 10 11 12 13 1-8 9 10 11 12 13 5
6 7 8 9 10 11
154
12 3 4 5 6 7 8 9 10 11 12
A
B
0
0
3
0
5
0
6
0
5
0 0
I
0
0 0
3
0
5
0
5
0
3
0
3
3
5
5
4 4 2 3 5 3 2 6 7 8 6 5 3 3 3 2
4 3 0
0 0 0 2 6 7
8 6
5 0 0 0 0
* See Fig. 2 for immunizations and serum sample days. t Fractions 1-8 represent the light (7S antibody) or upper portions of the gradient, and fractions 9-13 represent the heavy (19S antibody) or lower portions of the gradient. t A, fraction incubated with SRBC and complement at 370C for i hr; B, fraction heated at 560C for i hr. then incubated with SRBC and complement at 370C for i hr. The results are expressed as 10g2 titres.
of antigen. These results are to be contrasted to the titres detectable by method 1. Thus, peak titres of the primary and secondary response by method 1 were significantly higher than by titration method 2, but the third and fourth response titres were the same for both. As with the agglutinins, there was no significant difference between the dose groups (P = 005).
Response to SRBC in the marmoset
529
The haemolytic tests suggested that some of the antibody during the primary and secondary response was due to heat-sensitive 19S molecules, while the increased titres observed with time represented heat-resistant 7S haemolysins. To verify this, sucrose density gradients of primary and hyperimmune sera were examined. The results (Table 1) show: (1) haemolysins in the lower gradient fractions (19S) of primary serum (days 7 and 14) were heatlabile, since addition of C' did not restore haemolytic activity. 1 9S Haemolysins after booster inoculations (days 49 and 154) were also inactivated by the same conditions. (2) Haemolysins in the upper gradient fractions (7S) were not affected by heat. Further verification that the heat-sensitive haemolysins were 19S molecules was obtained through treatment of gradient samples with 2-ME. Complete loss of activity was obtained 9
(a)
8 _ 765
-
3
-
.01
2IIV~~~~~~~~~~~~~~~~~~~~' 4
-t
Characterization of the antibody response of the marmoset to sheep red blood cells N. GENGOZIAN, BRENDA L. SALTER, NANCY L. BASFORD & J. R. KATELEY Marmoset Research Center, Medical and Health Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee, U.S.A.
(Received 7 July 1975) SUMMARY
The immune competence of two species of marmosets, S. fuscicollis and S. oedipus, was evaluated by the intravenous (i.v.) and intramuscular (i.m.) injection of sheep red blood cells (SRBC). In S. fuscicollls marmosets, 1 ml of a 50% suspension yielded titres of haemolysin and agglutinating antibodies equal to or greater than 1 ml of a 10% dose of antigen. In both species, the i.v. route, while resulting in formation of 19S and 7S agglutinins, yielded only 19S haemolysins, even after multiple antigen injections. Repeated i.v. injections resulted in a progressive decrease in peak titres, in contrast to the i.m. route, where booster inoculations gave a typical anamnestic response. Jerne plaque-forming cells (PFC) in the spleens of S. oedipus marmosets showed predominately 19S plaques after a primary i.v. challenge; only 19S PFC were detected in the spleen of an animal that had been given multiple inoculations, the type of antibody produced reflecting that found in the serum. 19S but not 7S haemolysins of both species were sensitive to heating at 560C for 1/2 hr. The serum titres and splenic PFC data from the marmosets suggest these animals, particularly S. oedipus, respond poorly to SRBC when a comparison is made to similar studies in mice and rats.
INTRODUCTION The marmoset, a South American primate, is well suited for laboratory investigation due to its small size, ease of maintenance, and breeding in captivity (Gengozian, 1969). Its most unique feature, however, is its high frequency of fraternal twinning, which in the laboratory occurs in approximately 80% of all conceptions; further, placental vascular anastomoses between the foetuses result in haemopoietic chimerism in the twins (Benirschke, Anderson & Brownhill, 1962; Gengozian, Batson & Eide, 1964). Properties of chimerism that have been studied are frequency of occurrence; degree; haemopoietic tissues involved; stability; tolerance to co-twin antigens (Gengozian et al., 1969; Porter & Gengozian, 1969, 1973; Gengozian, 1971). The immunological capability of marmosets has been demonstrated in skin allograft studies; specific sensitization is indicated by the accelerated rejection of second- and thirdset grafts (Gengozian & Porter, 1971). The reactivity of blood lymphocytes in mixed lymphocyte cultures parallels the allograft studies in revealing histocompatibility differences (Barnhart & Gengozian, 1975). Very little is known, however, about the ability of the marmoset to form humoral antibodies to parenterally introduced antigens. In this study we report the response of two marmoset species, S. oedipus and S. fuscicollis, to multiple Correspondence: Dr N. Gengozian, Oak Ridge Associated Universities, P.O. Box 117, Oak Ridge, Tennessee 37830, U.S.A.
525
526
N. Gengozian et al.
inoculations of sheep red blood cells (SRBC). Examination of the humoral (serum antibody) and cellular (Jerne plaque assay) responses has revealed that the marmoset can show an anamnestic response with 7S antibody formation; this, however, is dependent upon the dose of antigen, route of injection, and the marmoset species immunized. A primary deficiency of the marmoset, regardless of species and antigen dose, is failure to synthesize 7S haemolysins following multiple intravenous infusions of SRBC; the antigen given intramuscularly elicits an anamnestic 7S response, the magnitude of which is dependent upon the species. MATERIALS AND METHODS Animals. Two species of marmosets were used, Saguinus oedipus and Saguinus fuscicollis ssp. Imported and laboratory-bred marmosets were used randomly, with no significant effects in any experiment being attributable to the source. Ages of the imported animals were not known; however, all had been in our colony 2 yr and therefore, based on our breeding studies, were considered to be sexually mature. Laboratory bred marmosets ranged in age from 2 to 44 yr. Antigen and immunizations. SRBC were maintained in Alsever's solution for week at 40C prior to use. The cells were washed with saline and suspended to the desired concentration. Two different concentrations were used, ml of a10O% or 50%Y suspension of cells, given either intravenously (i.v.) in the femoral vein or intramuscularly (i.m.) in the outer aspects of the thigh (0 5 ml per thigh). Multiple injections were given, each antigen inoculum (dose and route) being the same as the initial challenge. Animals were bled at regular intervals and the antisera stored at-70C until tested. Assay for serum haemolysins and agglutinins. To detect haemolysins, two-fold serial dilutions of individual serum samples were made in 0 1 ml of modified barbital buffer, pH 7 3-74. The sera were either heated (560C for hr) or not prior to each titration as indicated in the text. The source of complement (C') was fresh human or guinea-pig serum since preliminary tests showed no significant difference between the two with a 50%4 haemolysis endpoint. Serum dilutions were incubated at 370C for 30 min after addition of 01 ml SRBC (1%Y) and 0 1 ml C'(1:10) absorbed with SRBC. To test for agglutinins, 01 ml of diluted antiserum was incubated with 1 ml of SRBC at 370C for 30 min, after which the tubes were centrifuged for 15 sec at approximately 1500 g. Titres have been expressed aslog2 of the reciprocal of the highest dilution showing agglutination or haemolysis. Significant differences between the mean peak titres of the various groups were determined by one-way analysis of variation and the F test. Distinction between 1 9S and7S antibodies Three procedures were used to identify the molecular class of serum antibodies. (1) 2-Mercaptoethanol (2-ME) treatment of serum (Deutsch & Morton, 1957). This permitted determination of 2-ME-sensitive or 2-ME-resistant antibodies, hereafter referred to as 19S and 7S antibodies, respectively. (2) Sucrose density gradient ultracentrifugation. Serum diluted 1:2 with PBS was which was centrifuged at layered on a sucrose gradient (10% to 5°C at 35,000 rev/min for 16 hr in a Spinco L-2 ultracentrifuge with an SW 39 swinging bucket rotor. Fifteen fractions of 03 ml each were collected. (3) Sephadex gel filtration. Sera with similar composition of 19S and 7S antibodies (as revealed by sucrose gradients and 2-ME) taken from identical and/or adjacent blood sample days were pooled and applied to columns of Sephadex G-200 and eluted at room temperature with0-02 phosphate-buffered saline. Jerne plaque technique. Ten marmosets were splenectomized at various intervals after ani.v. injection of SRBC. Splenectomy was performed following anaesthesia with Sernylan (0 025 mg/100 g body weight). The individual spleens were weighed and cell suspensions prepared, washed with Eisen's medium and resuspended to the desired concentration. A slide modification of the Jerne plaque technique was used to assay for haemolytic plaque-forming cells (PFC) (Jerne & Nordin, 1963; Sell, Park & Nordin, 1970). Prior to performing this assay with cells, however, several 'haemolytic spot' tests with immune (to SRBC) marmoset serum were made to determine the optimal conditions for detection of antibody released by spleen cells while in the agarose gel (Hubner & Gengozian, 1969). Variables considered were the C' source (human, rabbit and guinea-pig serum absorbed with SRBC), dilution of C' (1:4, 1:7, 1:10), and concentration of Ca2+ and Mg2 + in the agarose and Eisen's medium used to dilute the C' and to suspend spleen cells. Testing of several marmoset sera containing either 19S or 7S antibody revealed that maximum sensitivity for detection of 19S haemolysins was with human C' (AB, Rh-positive serum) diluted 1: 10, using Eisen's medium containing 493 x 10-4M Ca2 + and x 10-4M Mg 2. For detection of 7S antibody, slightly greater sensitivity was obtained with guinea-pig C' diluted 1:10, using Eisen's medium containing 2-46 x 104M Ca 2 and 1-25 x 104M Mg2+ Facilitation of 7S 'haemolytic spots' was obtained with a goat anti-marmoset IgG antiserum (1:80). Verification that these conditions were optimal for the PFC assay was obtained during the course of this study with spleen cells from four sensitized marmosets.
37%/)
2-50
.
Response to SRBC in the marmoset
527
RESULTS Intramuscular immunizations of S. fuscicollis Eight S.fuscicollis marmosets were given i.m. injections of SRBC, four receiving 1 ml of a 10% suspension and four receiving 1 ml of a 500 suspension. There was no significant difference (P = 0 05) between the two SRBC doses with respect to the qualitative character
:--
-0 0
cacl To
10 9 8 76 54 3
2
10
20
30
40
50
60
70
80
90
100
110
120 130 140 150 160
Days FIG. 1. Mean agglutinin titres to sheep RBC following intramuscular immunizations in S. fuscicois marmosets. Data from two groups of four animals each, one receiving 1 ml of a 10% and the other 1 ml of a 50% suspension, were combined, since no significant differences were noted. (0-0) Total antibody activity; (o - - *) antibody activity after 2-ME treatment of sera. (T) Antigen injection. 10 9
8 7
6 A>
'8 l_
r_
5 4 3
24
Days
FIG. 2. Mean haemolysin titres to SRBC following intramuscular immunizations in S.fuscicollis marmosets. Serum samples from same animals depicted in Fig. 1. (0-0) Total antibody activity; (e - - e) antibody activity remaining after heat (560C for i hr) treatment of sera.
(T) Antigen injection. of the responses or the peak titres following each injection. Therefore, the results were pooled and the mean titres for the eight marmosets are shown in Fig. 1. The agglutinins detected on day 7 were almost all 19S as determined by sucrose density gradients and 2-ME tests. The 7S antibodies reached peak activity on day 14 and remained at a high level throughout. Each booster inoculation resulted in a peak titre greater than that observed after the previous injection. Titrations for haemolysins were performed on the same sera. Two methods were used: in method 1 the sera were unheated before titration and in method 2 the sera had been heated for i hr at 560C. Unheated sera contained low titres (log2 1-3) of 'natural' haemolysins, but I
N. Gengozian
528
et
al.
after heating these could not be detected (Fig. 2). Similarly, although unheated sera showed a titre of 7 (log2) on day 7 after antigen injection, significant haemolysin activity could not be detected following heat treatment. By method 2, peak titres in the primary response were quite low, but became progressively higher following the second, third and fourth injection TABLE 1. Localization and heat sensitivity of 7S and 19S haemolysins in sucrose density gradient fractions Day serum obtained after antigen
injection* 7
14
49
Treatment of fraction for titrationt Fraction numbert 1-8 9 10 11 12 13 1-8 9 10 11 12 13 5
6 7 8 9 10 11
154
12 3 4 5 6 7 8 9 10 11 12
A
B
0
0
3
0
5
0
6
0
5
0 0
I
0
0 0
3
0
5
0
5
0
3
0
3
3
5
5
4 4 2 3 5 3 2 6 7 8 6 5 3 3 3 2
4 3 0
0 0 0 2 6 7
8 6
5 0 0 0 0
* See Fig. 2 for immunizations and serum sample days. t Fractions 1-8 represent the light (7S antibody) or upper portions of the gradient, and fractions 9-13 represent the heavy (19S antibody) or lower portions of the gradient. t A, fraction incubated with SRBC and complement at 370C for i hr; B, fraction heated at 560C for i hr. then incubated with SRBC and complement at 370C for i hr. The results are expressed as 10g2 titres.
of antigen. These results are to be contrasted to the titres detectable by method 1. Thus, peak titres of the primary and secondary response by method 1 were significantly higher than by titration method 2, but the third and fourth response titres were the same for both. As with the agglutinins, there was no significant difference between the dose groups (P = 005).
Response to SRBC in the marmoset
529
The haemolytic tests suggested that some of the antibody during the primary and secondary response was due to heat-sensitive 19S molecules, while the increased titres observed with time represented heat-resistant 7S haemolysins. To verify this, sucrose density gradients of primary and hyperimmune sera were examined. The results (Table 1) show: (1) haemolysins in the lower gradient fractions (19S) of primary serum (days 7 and 14) were heatlabile, since addition of C' did not restore haemolytic activity. 1 9S Haemolysins after booster inoculations (days 49 and 154) were also inactivated by the same conditions. (2) Haemolysins in the upper gradient fractions (7S) were not affected by heat. Further verification that the heat-sensitive haemolysins were 19S molecules was obtained through treatment of gradient samples with 2-ME. Complete loss of activity was obtained 9
(a)
8 _ 765
-
3
-
.01
2IIV~~~~~~~~~~~~~~~~~~~~' 4
-t
