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Visualization of bispecific antigen-binding in immunized splenic lymphocytes of the newt, Triturus viridescens
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Printed in the United States
Vol. 2, pp. 175-180, 1978 Pergamon Press, Inc.
VISUALIZATION OF BISPECIFIC ANTIGEN-BINDING IN IMMUNIZED SPLENIC LYMPHOCYTES OF THE NEWT, TRITURUS VIRIDESCENS.
Laurens N. Ruben and Benjamin F. Edwards Department of Biology Reed College Portland, Oregon 97202, U.S.A.
Antigen-binding splenic lymphocytes of hapten-carrier immunized adult newts, Triturus viridescens, may be bispecific as shown by selective blocking experiments (i). Carrier-specific enhancement of an anti-hapten response, monitored by antigen-binding (immunocytoadherence), was first reported in 1973 in this species (2). Since then, optimal immunization protocols have been defined (3). Newts injected !-~- with a high dose (10%) of trinitrophenyl (TNP)-conjugated heterlogous erythrocytes; e.g., chicken (CRBC), generate maximum antigen-binding cell (ABC) responses 8 days thereafter. Maximum responses are detected if newts are injected 2-4 days prior to hapten presentation with a low dose (0.0025%) of the carrier erythrocyte. A priming injection of a different erythrocyte or a single injection of 10% TNP-CRBC will not initiate an anti-hapten response. Antigen binding was monitored after incubating aliquots of dissociated sensitized splenic lymphocytes with a different erythrocyte; e.g., horse (HRBC) or its TNP-conjugate. Preferential binding by splenic lymphocytes of TNP-HRBC as compared with HRBC measured carrier-specific helper activity and the intensity of anti-hapten responses. That excess numbers of TNP-RBC binding cells were specific for TNP-binding was determined by prior incubation of sensitized splenocytes with either TNP-human serum albumin (2) or monovalent TNP-glycine (i), both of which eliminated this excess without affecting HRBC binding. Control experiments with TNP-glycine showed that antigen binding by splenic lymphocytes from newts immunized with HRBC, but not TNP, was unaffected. If either TNP-HRBC or TNP-CRBC sensitized splenic lymphocytes were incubated with the other conjugate, subsequent binding to immunogenic carrier erythrocytes was blocked (i), suggesting that at least some of the newt spleen cells could bind either the hapten o__[radeterminant on the carrier erythrocyte membrane. As additional confirmation, when the erythrocyte homologous with the immunogenic carrier was used to test antigen binding, TNP-RBC immunized splenic lymphocytes showed no binding preference for the TNP-conjugate. Furthermore, preincubation with TNPglycine blocked binding equally well between the carrier and the TNP-carrier, suggestimg bispecific binding capacities. There is probably no tendency of newt splenic lymphocytes to recognize either the TNP-lysine-erythrocyte link or of cross reactivity between TNP and an erythrocyte membrane determinant, since reversal of the order of presentation of carrier and TNP-RBC fails to generate bispecific binding cells. Thus different immunocyte 175
176
ANTIGEN BINDING CELLS IN NEWTS
Vol. 2, No. 1
populations may be selected by the same immunogens depending upon the order of presentation (4). The present experiments were designed to test bispecific antigen-binding by newt immunocytes using a technique allowing direct visualization of bispecific cells. Adult newts (l-3g body weight) were obtained from Connecticut Valley Biological Supply (Massachusetts, U.S.A.). They were stored in distilled water at 4°C and fed chopped beef liver at weekly intervals; the water was changed on the day following feeding. After removal from storage, they were maintained at 25°C and fed twice daily. All newts were injected initially !.£. with 0.2 ml of 0.0025% HRBC in Alsever's solution and two days later with 0.2 ml of 10% TNP-HRBC. Our method of TNP-RBC conjugation has been noted previously (2). Eight days after hapten presentation, spleens were mechanically dissociated into a medium of 7 parts Leibowitz (L-15, GIBCO) to 2 parts twice glass distilled water with i00 ug/ml streptomycin and I00 units/ml penicillin as described previously (3). Prior to preparation of spleen cell suspension, bovine serum albumin (BSA) was coupled to polacrylamide beads ("Bio-gel P-10", Bio-Rad, 200-400 mesh) according to the methods of Weston and Avrameas (5). Five ml of settled hydrated beads were suspended in 12.5 ml distilled water and 2.5 ml, 1.0 M phosphate buffer solution (PBS) at pH 7.4. Dropwise addition of 5 ml of 25% glutaraldehyde was followed by slow stirring for 18 hours at 25°C. Excess glutaraldehyde was removed by washing with PBS at pH 7.7. The washed glutaraldehyde-beads were then combined with 50 mg BSA in 5 ml PBS, pH 7.7 and gently stirred for 18 hours at 4°C. The BSA-beads were washed with PBS (pH 7.7), incubated in 0.i M lysine at pH 7.4 for 2 hours, treated with HCL-glycine at pH 7.8 for 15 minutes and washed finally in PBS at pH 7.7. The amount of BSA bound to the beads was determined by monitoring its removal from the supernatant fluid at 280 nm with a spectrophotometer. At this stage, half of the preparation was set aside as "control" BSA-beads, whereas the remainder were conjugated with TNP. BSA-beads (in 2.5 ml) were washed in 0.5 M NaHC03, pH 8, mixed with 200 mg trinitrobenzene sulfonic acid (TNBS) in 2.5 ml 0.5 M NaHC03 and incubated for 30 minutes in the dark at 25°C. TNP-BSA-beads were then washed once in 0.i M glycylglycine, pH 8.0, several times in PBS, pH 7.7 and stored at 4°C. Both types of beads were washed and resuspended in the same L-15 medium used to prepare spleen cell suspensions, before the binding assays. Binding was first effected with the beads and the immunized spleen cells. Fifty ul each of the suspension of beads and of splenocyte suspensions were placed in a small glass tube. Depending upon whether competition with haptenbinding was provided, 25 ul of 10 -7 M glycine was added. Dosages of glycine or TNP-glycine were chosen from earlier experiments (i), which tested the effects of various concentrations of TNP-glycine as a selective blocking agent. Splenocytes, beads and appropriate medium were then placed on a slowly turning roller drum apparatus for 4-5 hours at 4°C, a time determined previously as the minimum time required to maximize antigen-cell binding. Suspensions were then removed from the drum, diluted with 1 ml of L-15 medium and 50 ul of 10% HRBC, the original immunogenic carrier, were added. This dilution provided background cell populations for microscopic examination. The mixture was gentle swirled and placed upright for 16 hours at 4°C. After gentle resuspension with a Pasteur pipette, each tube was sampled 3 times by streaking a glass slide. Although most macrophages are removed during spleen dissociation, those which remain readily flatten onto the bead surface and can be visually excluded because they are large and granular. To remove more of the initial macrophage population, cells can be cultured for 30 minutes in a plastic (Falcon) tissue culture dish. Only round lymphocytes which remain and are clearly attached to the bead surface were counted. Thin streaks of 5 cm can be scanned at a magnification of x250 before they dry. A variety of depressionslide arrangements, as well as Cunningham-Szenberg chambers were tried, but
Vol. 2, No. 1
ANTIGEN BINDING CELLS IN NEWTS
177
streaking with a Pasteur pipette produced the best condition for viewing. Binding trials with erythrocytes and spleen cells mixed together with beads gave less satisfactory results, possibly because of competition for available binding sites. Only upper hemispheres of beads were scrutinized to ensure that the counted cells are actually attached to and are not simply in contact with the beads. Three samples from each incubation mixture were streaked individually on a slide and the first i00 beads counted, including those with attached lymphocytes and numbers of those attached lymphocytes that also bound HRBC. Beads which showed any abnormal appearance were excluded, such as severely fractured beads which tended to aggregate HRBC and lymphocytes. Since each incubation was normally made in triplicate, 900 beads were counted for each experimental group per trial. Our experiments were performed 4 times with different groups of immunized newts providing spleen cell pools. Thus, 3600 beads were counted to supply each value in Table I. Standard deviations desscribe experimental variations among non-isogenic newts collected from the wild. An additional control to test for binding specificity was provided by utilizing newt spleen cells immunized with HRBC, but not TNP. After a single injection of 25% HRBC, spleen cells were harvested 8 days later, at the time of maximum antigen-binding cell activity (3). The same protocols as those described above were used except that they were only incubated with TNP-BSA beads. When TNP-HRBC immunocytes were incubated with TNP-BSA beads in the presence of 10 -7 M glycine, at least one of every 5 beads bound a lymphocyte (Table i). After subsequent incubation with HRBC, the immunogenic carrier, approximately one of every fourth bead-attached-lymphocyte also bound HRBC. BSA-beads were remarkably free of attached lymphocytes, suggesting that lymphocytes bound to beads were TNP-specific. Figure IA shows a low power (xl00) view of a BSA-bead preparation with fractured beads visible at (f); splenic lymphocytes and erythrocytes are on a different plane of focus and are barely visible. Figures IB and IC illustrate bead-attached lymphocytes which also bind smaller HRBC. Incubation of cells and TNP-BSA beads with TNP-glycine substantially reduced the number of bound lymphocytes and none of those still attached were erythrocyte-binding cells. This second result supported our contention that binding of these lymphocytes to beads involved TNP-binding sites. TABLE 1 The Visualization of Bispecific Splenocytes from Newts Immunized in vivo against TNP-HRBC Biogel Bead Conjugate
BSA TNP-BSA TNP-BSA aTNP-BSA
Pre-incubation Treatment
-
10 -7 M glycine (25 ~i) 10 -7 M TNP-glycine (2S ~i) -
Percent Bound Lymphocytes 0 18 4 7
± ± ± ±
0 5 3 3
Percent Bound ABC 0 5 0 0
± ± ± ±
0 5 0 0
a The spleen cells used for these data were pooled from 4-6 newts/group which had been immunzied only against HRBC by a single 25% injection, 8 days prior to assay.
We have been able to directly visualize bispecific antigen-binding spleen cells in the newt, Triturus viridescens. Previous data (I) using TNP-glycine
178
ANTIGEN BINDING CELLS IN NEWTS
Vol. 2, No. 1
as a selective blocking agent showed that generalized utilization of TNPbinding sites on splenic lymphocytes prevented subsequent binding to carrier erythrocyte membrane determinants. However, local TNP-binding to the TNPBSA-bead clearly does not eliminate the possibility of demonstrating the dualbinding capacity of immunized splenic lymphocytes of newts. These findings are significant with respect to the evolution and development of individual lymphocyte specificity (6,7).
ACKNOWLEDGEMENTS We are grateful for the technical assistance of Judith M. Ruben. This research was partially supported by a grant (AI-12846) from the National Institutes of Health, Bethesda, Maryland, U.S.A.
REFERENCES (i) RUBEN, L. N. and SELKER, E. U. Polyfunctional antigen-binding in haptencarrier responses of the newt, Triturus viridescens. Adv. Exp. Med. Biol. 64, 387, 1975. (2) RUBEN, L. N., V ~ DER HOVEN, A. and DUTTON, R. W. Cellular cooperation in hapten-carrier responses in the newt, Triturus viridescens. Cell. Immunol. 6, 300, 1973. (3) RUBEN, L. N. Ontogeny, phylogeny and cellular cooperation. Amer. Zool. 15, 93, 1975. (4) RUBEN, L. N. and EDWARDS, B. F. The inference of lymphoid cell heterogeneity in the newt, Triturus viridescens from hapten-carrier antigenbinding studies. In: Phylogeny of T and B Cells. R. K. Wright and E. L. Cooper (Eds.) Amsterdam, No. Holland Publ. Co. 1977, P. 161. (5) WESTON, P. D. and AVRAMEAS, S. Proteins coupled to polyacrylamide beads using glutaraldehyde. Biochem. Biophys. Res. Commun. 45, 1574, 1971. (6) RUBEN, L. N., WARR, G. W., DECKER, J. M. and MARCHALONIS, J. J. Phylogenetic origins of immune recognition: Lymphoid heterogeneity and the hapten-carrier effect in the goldfish, Carrasuis auratus. Cell Immunol. 31, 266, 1977. (7) RUBEN, L. N. and EDWARDS, B. F. Phenotypic restriction of antigen-binding specificity on immunized amphibian spleen cells. Cell Immunol. 33, 437, 1977.
Vol. 2, No. 1
ANTIGEN BINDING CELLS IN NEWTS
179
FIGURE 1 (A) View of "control" polyacrylamide beads conjugated with bovine serum albumin showing the variety of bead sizes and fractured beads (f). Lymphocytes and erythrocytes are barely visible out of the plane of focus - xl00. (B) Horse erythrocyte (HRBC)-binding splenic lymphocyte attached to a TNP-BSA conjugated bead. The animal had been immunized in vivo with HRBC and TNP-HRBCx240. (C) HRBC-binding splenic lymphocyte attached to a TNP-BSA conjugated bead. This animal had also been immunized in vivo with TNP-HRBC - x300.
Vol. 2, pp. 175-180, 1978 Pergamon Press, Inc.
VISUALIZATION OF BISPECIFIC ANTIGEN-BINDING IN IMMUNIZED SPLENIC LYMPHOCYTES OF THE NEWT, TRITURUS VIRIDESCENS.
Laurens N. Ruben and Benjamin F. Edwards Department of Biology Reed College Portland, Oregon 97202, U.S.A.
Antigen-binding splenic lymphocytes of hapten-carrier immunized adult newts, Triturus viridescens, may be bispecific as shown by selective blocking experiments (i). Carrier-specific enhancement of an anti-hapten response, monitored by antigen-binding (immunocytoadherence), was first reported in 1973 in this species (2). Since then, optimal immunization protocols have been defined (3). Newts injected !-~- with a high dose (10%) of trinitrophenyl (TNP)-conjugated heterlogous erythrocytes; e.g., chicken (CRBC), generate maximum antigen-binding cell (ABC) responses 8 days thereafter. Maximum responses are detected if newts are injected 2-4 days prior to hapten presentation with a low dose (0.0025%) of the carrier erythrocyte. A priming injection of a different erythrocyte or a single injection of 10% TNP-CRBC will not initiate an anti-hapten response. Antigen binding was monitored after incubating aliquots of dissociated sensitized splenic lymphocytes with a different erythrocyte; e.g., horse (HRBC) or its TNP-conjugate. Preferential binding by splenic lymphocytes of TNP-HRBC as compared with HRBC measured carrier-specific helper activity and the intensity of anti-hapten responses. That excess numbers of TNP-RBC binding cells were specific for TNP-binding was determined by prior incubation of sensitized splenocytes with either TNP-human serum albumin (2) or monovalent TNP-glycine (i), both of which eliminated this excess without affecting HRBC binding. Control experiments with TNP-glycine showed that antigen binding by splenic lymphocytes from newts immunized with HRBC, but not TNP, was unaffected. If either TNP-HRBC or TNP-CRBC sensitized splenic lymphocytes were incubated with the other conjugate, subsequent binding to immunogenic carrier erythrocytes was blocked (i), suggesting that at least some of the newt spleen cells could bind either the hapten o__[radeterminant on the carrier erythrocyte membrane. As additional confirmation, when the erythrocyte homologous with the immunogenic carrier was used to test antigen binding, TNP-RBC immunized splenic lymphocytes showed no binding preference for the TNP-conjugate. Furthermore, preincubation with TNPglycine blocked binding equally well between the carrier and the TNP-carrier, suggestimg bispecific binding capacities. There is probably no tendency of newt splenic lymphocytes to recognize either the TNP-lysine-erythrocyte link or of cross reactivity between TNP and an erythrocyte membrane determinant, since reversal of the order of presentation of carrier and TNP-RBC fails to generate bispecific binding cells. Thus different immunocyte 175
176
ANTIGEN BINDING CELLS IN NEWTS
Vol. 2, No. 1
populations may be selected by the same immunogens depending upon the order of presentation (4). The present experiments were designed to test bispecific antigen-binding by newt immunocytes using a technique allowing direct visualization of bispecific cells. Adult newts (l-3g body weight) were obtained from Connecticut Valley Biological Supply (Massachusetts, U.S.A.). They were stored in distilled water at 4°C and fed chopped beef liver at weekly intervals; the water was changed on the day following feeding. After removal from storage, they were maintained at 25°C and fed twice daily. All newts were injected initially !.£. with 0.2 ml of 0.0025% HRBC in Alsever's solution and two days later with 0.2 ml of 10% TNP-HRBC. Our method of TNP-RBC conjugation has been noted previously (2). Eight days after hapten presentation, spleens were mechanically dissociated into a medium of 7 parts Leibowitz (L-15, GIBCO) to 2 parts twice glass distilled water with i00 ug/ml streptomycin and I00 units/ml penicillin as described previously (3). Prior to preparation of spleen cell suspension, bovine serum albumin (BSA) was coupled to polacrylamide beads ("Bio-gel P-10", Bio-Rad, 200-400 mesh) according to the methods of Weston and Avrameas (5). Five ml of settled hydrated beads were suspended in 12.5 ml distilled water and 2.5 ml, 1.0 M phosphate buffer solution (PBS) at pH 7.4. Dropwise addition of 5 ml of 25% glutaraldehyde was followed by slow stirring for 18 hours at 25°C. Excess glutaraldehyde was removed by washing with PBS at pH 7.7. The washed glutaraldehyde-beads were then combined with 50 mg BSA in 5 ml PBS, pH 7.7 and gently stirred for 18 hours at 4°C. The BSA-beads were washed with PBS (pH 7.7), incubated in 0.i M lysine at pH 7.4 for 2 hours, treated with HCL-glycine at pH 7.8 for 15 minutes and washed finally in PBS at pH 7.7. The amount of BSA bound to the beads was determined by monitoring its removal from the supernatant fluid at 280 nm with a spectrophotometer. At this stage, half of the preparation was set aside as "control" BSA-beads, whereas the remainder were conjugated with TNP. BSA-beads (in 2.5 ml) were washed in 0.5 M NaHC03, pH 8, mixed with 200 mg trinitrobenzene sulfonic acid (TNBS) in 2.5 ml 0.5 M NaHC03 and incubated for 30 minutes in the dark at 25°C. TNP-BSA-beads were then washed once in 0.i M glycylglycine, pH 8.0, several times in PBS, pH 7.7 and stored at 4°C. Both types of beads were washed and resuspended in the same L-15 medium used to prepare spleen cell suspensions, before the binding assays. Binding was first effected with the beads and the immunized spleen cells. Fifty ul each of the suspension of beads and of splenocyte suspensions were placed in a small glass tube. Depending upon whether competition with haptenbinding was provided, 25 ul of 10 -7 M glycine was added. Dosages of glycine or TNP-glycine were chosen from earlier experiments (i), which tested the effects of various concentrations of TNP-glycine as a selective blocking agent. Splenocytes, beads and appropriate medium were then placed on a slowly turning roller drum apparatus for 4-5 hours at 4°C, a time determined previously as the minimum time required to maximize antigen-cell binding. Suspensions were then removed from the drum, diluted with 1 ml of L-15 medium and 50 ul of 10% HRBC, the original immunogenic carrier, were added. This dilution provided background cell populations for microscopic examination. The mixture was gentle swirled and placed upright for 16 hours at 4°C. After gentle resuspension with a Pasteur pipette, each tube was sampled 3 times by streaking a glass slide. Although most macrophages are removed during spleen dissociation, those which remain readily flatten onto the bead surface and can be visually excluded because they are large and granular. To remove more of the initial macrophage population, cells can be cultured for 30 minutes in a plastic (Falcon) tissue culture dish. Only round lymphocytes which remain and are clearly attached to the bead surface were counted. Thin streaks of 5 cm can be scanned at a magnification of x250 before they dry. A variety of depressionslide arrangements, as well as Cunningham-Szenberg chambers were tried, but
Vol. 2, No. 1
ANTIGEN BINDING CELLS IN NEWTS
177
streaking with a Pasteur pipette produced the best condition for viewing. Binding trials with erythrocytes and spleen cells mixed together with beads gave less satisfactory results, possibly because of competition for available binding sites. Only upper hemispheres of beads were scrutinized to ensure that the counted cells are actually attached to and are not simply in contact with the beads. Three samples from each incubation mixture were streaked individually on a slide and the first i00 beads counted, including those with attached lymphocytes and numbers of those attached lymphocytes that also bound HRBC. Beads which showed any abnormal appearance were excluded, such as severely fractured beads which tended to aggregate HRBC and lymphocytes. Since each incubation was normally made in triplicate, 900 beads were counted for each experimental group per trial. Our experiments were performed 4 times with different groups of immunized newts providing spleen cell pools. Thus, 3600 beads were counted to supply each value in Table I. Standard deviations desscribe experimental variations among non-isogenic newts collected from the wild. An additional control to test for binding specificity was provided by utilizing newt spleen cells immunized with HRBC, but not TNP. After a single injection of 25% HRBC, spleen cells were harvested 8 days later, at the time of maximum antigen-binding cell activity (3). The same protocols as those described above were used except that they were only incubated with TNP-BSA beads. When TNP-HRBC immunocytes were incubated with TNP-BSA beads in the presence of 10 -7 M glycine, at least one of every 5 beads bound a lymphocyte (Table i). After subsequent incubation with HRBC, the immunogenic carrier, approximately one of every fourth bead-attached-lymphocyte also bound HRBC. BSA-beads were remarkably free of attached lymphocytes, suggesting that lymphocytes bound to beads were TNP-specific. Figure IA shows a low power (xl00) view of a BSA-bead preparation with fractured beads visible at (f); splenic lymphocytes and erythrocytes are on a different plane of focus and are barely visible. Figures IB and IC illustrate bead-attached lymphocytes which also bind smaller HRBC. Incubation of cells and TNP-BSA beads with TNP-glycine substantially reduced the number of bound lymphocytes and none of those still attached were erythrocyte-binding cells. This second result supported our contention that binding of these lymphocytes to beads involved TNP-binding sites. TABLE 1 The Visualization of Bispecific Splenocytes from Newts Immunized in vivo against TNP-HRBC Biogel Bead Conjugate
BSA TNP-BSA TNP-BSA aTNP-BSA
Pre-incubation Treatment
-
10 -7 M glycine (25 ~i) 10 -7 M TNP-glycine (2S ~i) -
Percent Bound Lymphocytes 0 18 4 7
± ± ± ±
0 5 3 3
Percent Bound ABC 0 5 0 0
± ± ± ±
0 5 0 0
a The spleen cells used for these data were pooled from 4-6 newts/group which had been immunzied only against HRBC by a single 25% injection, 8 days prior to assay.
We have been able to directly visualize bispecific antigen-binding spleen cells in the newt, Triturus viridescens. Previous data (I) using TNP-glycine
178
ANTIGEN BINDING CELLS IN NEWTS
Vol. 2, No. 1
as a selective blocking agent showed that generalized utilization of TNPbinding sites on splenic lymphocytes prevented subsequent binding to carrier erythrocyte membrane determinants. However, local TNP-binding to the TNPBSA-bead clearly does not eliminate the possibility of demonstrating the dualbinding capacity of immunized splenic lymphocytes of newts. These findings are significant with respect to the evolution and development of individual lymphocyte specificity (6,7).
ACKNOWLEDGEMENTS We are grateful for the technical assistance of Judith M. Ruben. This research was partially supported by a grant (AI-12846) from the National Institutes of Health, Bethesda, Maryland, U.S.A.
REFERENCES (i) RUBEN, L. N. and SELKER, E. U. Polyfunctional antigen-binding in haptencarrier responses of the newt, Triturus viridescens. Adv. Exp. Med. Biol. 64, 387, 1975. (2) RUBEN, L. N., V ~ DER HOVEN, A. and DUTTON, R. W. Cellular cooperation in hapten-carrier responses in the newt, Triturus viridescens. Cell. Immunol. 6, 300, 1973. (3) RUBEN, L. N. Ontogeny, phylogeny and cellular cooperation. Amer. Zool. 15, 93, 1975. (4) RUBEN, L. N. and EDWARDS, B. F. The inference of lymphoid cell heterogeneity in the newt, Triturus viridescens from hapten-carrier antigenbinding studies. In: Phylogeny of T and B Cells. R. K. Wright and E. L. Cooper (Eds.) Amsterdam, No. Holland Publ. Co. 1977, P. 161. (5) WESTON, P. D. and AVRAMEAS, S. Proteins coupled to polyacrylamide beads using glutaraldehyde. Biochem. Biophys. Res. Commun. 45, 1574, 1971. (6) RUBEN, L. N., WARR, G. W., DECKER, J. M. and MARCHALONIS, J. J. Phylogenetic origins of immune recognition: Lymphoid heterogeneity and the hapten-carrier effect in the goldfish, Carrasuis auratus. Cell Immunol. 31, 266, 1977. (7) RUBEN, L. N. and EDWARDS, B. F. Phenotypic restriction of antigen-binding specificity on immunized amphibian spleen cells. Cell Immunol. 33, 437, 1977.
Vol. 2, No. 1
ANTIGEN BINDING CELLS IN NEWTS
179
FIGURE 1 (A) View of "control" polyacrylamide beads conjugated with bovine serum albumin showing the variety of bead sizes and fractured beads (f). Lymphocytes and erythrocytes are barely visible out of the plane of focus - xl00. (B) Horse erythrocyte (HRBC)-binding splenic lymphocyte attached to a TNP-BSA conjugated bead. The animal had been immunized in vivo with HRBC and TNP-HRBCx240. (C) HRBC-binding splenic lymphocyte attached to a TNP-BSA conjugated bead. This animal had also been immunized in vivo with TNP-HRBC - x300.