Kikuo NoMOTO, Hiroyasu YAMADA, Shizuko MURAOKA, and Kenji IAKEYA Department of Bacteriology, School of Medicine, Kyushu University, Fukuoka 812

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1 Japan. J. Microbiol. Vol. 17 (1),1-5, 1973 Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains V. Anti-Hapten Antibody Production after Immunization with Hapten-Erythrocyte Conjugates Kikuo NoMOTO, Hiroyasu YAMADA, Shizuko MURAOKA, and Kenji IAKEYA Department of Bacteriology, School of Medicine, Kyushu University, Fukuoka 812 (Received for publication, April 7, 1972) ABSTRACT The production of anti-hapten antibody after immunization with trinitrophenylated (TNP) hamster erythrocytes (HRBC) or sheep erythrocytes (SRBC) was determined in high- and low-responder mouse strains against HRBC antigen. 1) Anti-TNP antibody was detected in sera of high-responder DDD and CF1 mice after primary immunization with TNP-HRBC, but not in those of low-responder C57BL/6 mice. 2) Anti-TNP antibody was detectable in sera of all the strains after primary immunization with TNP-SRBC. 3) Production of anti-tnp antibody was elicited after a booster injection of TNP-HRBC in low-responder C57BL/6 mice pre-sensitized with HRBC in Freund's complete adjuvant, These results suggest that functions of thymus-derived cells specific for HRBC antigen are deficient in low-responder mice. Strain differences in immune response against various kinds of antigens have been reported in several species of animals and many of these differences appear to be under genetic control [8]. In our previous papers, strain differences in immune response against hamster erythrocytes (HRBC) were reported on various mouse strains [14-16]. Magnitudes of antibody production of the strains after a primary intravenous injection 16]. The latent periods required for development of delayed hypersensitivity after sensitization with HRBC in Freund's complete adjuvant (FCA) were correlated inversely with the magnitudes of antibody production [15]. Requirement of the cooperation between Please address requests of reprints to Dr. Kikuo Nomoto, Department of Bacteriology, School of Medicine, Kyushu University, 1276 Katakasu, Higashi-ku, Fukuoka 812, Japan. thymus-derived cells (T cells) and bonemarrow-derived antibody-forming cell precursors (B cells) in antibody production has been reported in various combinations of antigens and hosts [1-4, 10]. The requirement of cell cooperation was also reported regarding the production of anti-hapten antibody, where hapten-specific antibody producers and carrier-specific helper cells contributed to antibody production [5, 6, 17]. These results suggested that the precursor T cells specific for carrier antigens were stimulated by treatment with the carrier alone and that these stimulated T cells facilitated the antibody production against hapten conjugated with the homologous carrier after their interaction with carrier sites of the conjugated antigen. The present investigation had been undertaken to resolve whether or not the functions of precursor cells for HRBC-specific helper cells were deficient in the low-responder C57BL/6 mice.

2 2 K. NOMOTO, H. YAMADA, S. MURAOKA AND K. TAKEYA MATERIALS AND METHODS Animals. Inbred mice of C57BL/6 and DDD strains, and outbred CF1 mice were obtained from the breeding unit of Kyushu University School of Medicine [16]. Eight to 10-weekold male mice were used for the experiments. Antigen. HRBC were obtained by cardiac puncture from adult golden hamsters and stored in Alsever's solution at 4C. Sheep erythrocytes (SRBC) in Alsever's solution were obtained commercially. Human erythrocytes were obtained from a volunteer possessing type O blood (ORBC). Preparation of hapten-conjugated RBC. TNP- RBC were prepared by the method of Rittenberg and Pratt [18]. A 60mg portion of 2, 4, 6-trinitrophenylbenzene sulfonate was dissolved in 21.0ml of cacodylate buffer in a 50ml flask. Three milliliters of packed RBC was added dropwise with stirring. The mixture was stirred slowly at room temperature for 10min, then centrifuged at 1000rpm for 8min. The supernatant was decanted and the sediment was resuspended in 35ml of barbital buffer (ph 7.3) containing 0.022g of glycylglycine. The suspension was stirred, centrifuged and the supernatant was decanted as above. This washing procedure was repeated 3 times. Finally, the treated RBC were adjusted to the desirable concentrations with phosphate-buffered saline (PBS). Immuniziation. Two-tenths milliliters of a 25% (v/v) HRBC or TNP-HRBC, or a 10% (v/v) TNP-SRBC was injected into the tail vein of the test mice. One-tenth milliliter of an emulsion of HRBC or TNP-HRBC (25%) in PBS and Freund's complete adjuvant (FCA) was injected subcutaneously into both sides of the flanks [16]. Titration of antibodies. Hemagglutinin (HA) titration was performed with a microtiter set as described previously [16]. TNP- ORBC were used as the antigen in order to titrate anti-tnp antibody. Titers were recorded in log2. Titers of the sera in which any of the first wells of the microplates did not exhibit a positive reaction were given the value of "0." RESULTS Production of Anti-TNP Antibody after a Primacy Immunization with TNP-RBC C57BL/6, DDD and CF1 mice were injected intravenously with TNP-HRBC or TNP-SRBC (Table 1). C57BL/6, DDD and CF1 mice produced anti-tnp antibodies of almost identical titers after a primary intravenous injection with TNP-SRBC. After a primary injection with TNP-HRBC, anti- TNP antibodies were detected in the sera of DDD and CFl mice, but not in C57BL/6 mice. Titers of anti TNP antibody waned from day 4 to day 14 in any combination of the immunizing antigens and hosts. The Effect of Pre-Sensitization of C57BL/6 Mice with HRBC in FCA on the Production of Anti-TNP Antibody after TNP- HRBC Injection After a booster injection of TNP-HRBC in saline, the production of anti-tnp antibody of high titer was detected in the C57BL/6 mice which had been pre-sensitized with HRBC in FCA 7 days in advance (Table 2). On the other hand, no anti-tnp antibody was detected in sera from non-pretreated Table 1. Production of anti-hapten antibody after a primary injection with TNP-RBC in saline a) 0.2ml of 25% (v/v) in the case of TNP-HRBC and 10% (v/v) in the case of TNP-SRBC. b) Average of 8 to 12 animals.

3 ANTI-HAPTEN ANTIBODY IN LOW-RESPONDERS 3 Table 2. Production of anti-hapten antibody after TNP-HRBC injection in C57BL/6 mice pre-sensitized with HRBC in FCA a) 0.2ml of 25% (v/v) TNP-HRBC suspension. b) Average of 8 to 12 animals. Table 3. The effect of pretreatment of C57BL/6 mice with HRBC or TNP-HRBC on their antibody production against TNP after TNP-HRBC injection a) 0.2ml of 25% (v/v) TNP-HRBC. b) Average of 8 to 12 animals. Table 4. Antibody production against carrier RBC in mice a) The same animals described in Tables 1 and 2. b) 0.2ml of 25% (v/v) in the case of TNP-HRBC and 10% (v/v) in the case of TNP-SRBC.c ) Average of 8 to 10 animals. mice or mice pretreated with saline in FCA. The Effect of Pretreatment with HRBC or TNP- HRBC in FCA or in Saline on the Production of Anti-TNP Antibody in C57BL/6 Mice C57BL/6 mice were subcutaneously inoculated with HRBC or TNP-HRBC in FCA, or intravenously with HRBC or TNP-HRBC in saline. Seven days later, these mice were intravenously injected with TNP-HRBC and the production of anti- TNP antibody was examined (Table 3). The production of anti-tnp antibody was elicited in all mice pretreated as described above. Efficiency of the enhancing effect was found to be better with pretreatment using HRBC than with TNP-HRBC. The enhancing effects of antigens in FCA were superior to those in saline.

4 4 K. NOMOTO, H. YAMADA, S. MURAOKA AND K. TAKEYA An tibody Production against Carrier RBC sensitivity Relationships among these T Antibody to HRBC was not detectable in cells will be discussed in a following paper the sera of C57BL/6 mice after a primary [13]. The cell types (T or B) under genetic intravenous injection with TNP-HRBC control might he different in each combination of antigen and host, as suggested by (Table 4). Antibodies to HRBC were detected in very low titers in DDD and CF1 several reports [7, 9, 11, 12, 19]. mice after a primary injection of TNP- The production of anti-hapten antibody HRBC. Antibodies to HRBC of high-titers after a booster with TNP-HRBC was enhanced by pre-sensitization with HRBC in were produced by the C57BL/6 mice after an intravenous is injection of TNP-HRBC, if FCA (Tables 2 and 3). Treatment with they had been pretreated with HRBC in HRBC in FCA appeared to stimulate the FCA (Table 4). After a primary immunization with TNP-SRBC, antibodies to SRBC in terms of helper cells in antibody pro- proliferation of T cells of different functions, were detectable in all mice of the C57BL/6, duction (Tables 2 and 3) and effector cells DDD and CF1 strains (Table 4). Cross in delayed hypersensitivity [15]. reaction was not detected between HRBC Pretreatment with TNP-HRBC exhibited and SRBC. Antibody production against a lower enhancing effect on the production carrier HRBC after an intravenous injection of anti-tnp antibody after a booster with of TNP-HRBC was better in C57BL/6 mice TNP-HRBC in saline than pretreatment pretreated with HRBC than in those pretreated with TNP-HRBC (Table 3). gens for pretreatments were given in FCA or with unconjugated HRBC, whether the anti- in saline (Table 3). This appeared to be due DISCUSSION to a decreased stimulation of T cells specific Several reports indicated that antibody for HRBC that might be brought by a production against haptenic antigens required the cooperation between T cells decrease in immunogenicity of HRBC after conjugation with TNP (Tables 3 and 4). specific for carrier antigens and B cells specific for hapteris [5, 6, 17]. Anti-hapten REFERENCES antibodies were produced after a primary intravenous injection with INP-HRBC in DDD and CF1 mice, the high-responder strains in antibody production against HRBC, but not in C57BL/6 mice which belonged to the low-responder strain against HRBC antigen (Table 1). On the other hand, anti-hapten antibodies were produced by all three strains alter a primary injection with TNP-SRBC (Table 1). SRBC were equally immunogenie for all mouse strains [16]. Therefore, the low-responder C57BL/6 mice appeared to be deficient in their functions of the precursor T cells which should differentiate into HRBC-specific helper cells. Results in the present work, together with those in previous papers [14-16], suggested that the low-responder C57BL/6 mice had some deficiencies in their functions of the T cells specific for HRBC antigen, in terms of helper function in antibody production against HRBC antigen itself and against haptenic antigen conjugated with HRBC, and also development of sensitized lymphocytes as effector cells in delayed hyper- [1] Brody, T Identification of two cell populations required for mouse immunocompetence. J. Immunol. 105: [2] Chiller J. M., Habicht, G. S., and Weigle, W. O Cellular sites of immunologic unresponsiveness. Proc. Nat. Acad. Sci. U. S. 65: [ 3] Claman, H. N., Chaperon, E. A., and Tripett, R. F Immunocompetence of transferred thymus-marrow cell combinations. J. Immunol. 97: [4 ] Davies, A. J. S., Leuchars, E., Wallis, V., Maichant, R., and Elliott, E. V The failure of thymus-derived cells to produce antibody. Transplantation 5: [5] Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B Carrier function in antihapten immune responses. I. Enhancement of primary and secondary antihapten antibody responses by carrier preimmunization. J. Exp. Med. 132: [6] Kettman. J., and Dutton, R. W An in vitro primary immune response to TNP-snbstituted erythrocytes. Response against carrier and hapten. J. Immunol. 104: [7] Levin, H. A., Levine, H., and Schlossman, S. F Antigen recognition and antigen specificity. Carrier specificity and genetic control of anti-dinitrophenyl-oligolysinc andibody. J. Exp. Med. 133:

5 ANTI-HAPTEN ANTIBODY IN LOW-RESPONDERS 5 [8] McDevitt, H. O., and Benacerraf, B Genetic control of the specific immune response. Advances Immunol. 11: [9] Mitchell, F. G., Grumet, F. C., and McDevitt, H. O Genetic control of the immune response. The effect of thymectomy on the primary and secondary antibody response of mice to poly-l-(tyr, Glu)-poly-D, L-Ala- -poly-l-lys. J. Exp. Med. 135: [10] Mitchell, G. F., and Miller, J. F. A. P Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. J. Exp. Med. 128: [11] Mozes, E., and Shearer, G. M Contribution of bone marrow cells and lack of expression of thymocytes in genetic controls of immune responses for two immunopotent regions within poly-(phe, Glu)-poly-Pro- -poly-lys in inbred mouse strains. J. Exp. Med. 134: [12] Mozes, E., Shearer, G. M., and Sela, M Celluar basis of the genetic control of immune response to synthetic polypeptides. I. Differences in frequency of splenic precursor cells specific for a synthetic polypeptide derived from multichain polypeptide ([T, G]-Pro- -L) in high- and low-responder inbred mouse strains. J. Exp. Med. 132: [13] Nomoto, K., Kuroiwa, A., Mashiba, H., and Takeya, K Immune response against hamster erythrocytes in the low-responder mouse strains. VII. Competitive suppression of antibody production against hamster erythrocytes by simultaneous development of delayed hypersensitivity. Japan. J. Microbiol. 17 (3) in press. [14] Nomoto, K., Kuroiwa, A., and Takeya, K. in the low-responder mouse strains. II. Effects of pre-sensitization with hamster lymphoma cells on antibody production against hamster erythrocytes. Japan. J. Microbiol. 16: [15] Nomoto, K., Makidono, R., and Takeya, K. in the low-responder mouse strains. IV. Delayed hypersensitivity against solubilized hamster erythrocytes in mice. Japan. J. Microbiol. 16: [16] Nomoto, K., Mashiba, H., and Takeya, K. in the low-responder mouse strains. I. Strain difference in the antibody response to primary antigenic stimulation and its disappearance after pre-sensitization with the antigen in Freund's complete adjuvant. Japan. J. Microbiol. 16: [17] Rejewsky, K., Schirrmacher, V., Nase, S., and Nordin, N. K The requirement of more than one antigenic determinant for immunogenicity. J. Exp. Med_ 129: [18] Rittenberg, M. B., and Pratt, K. L Antitrinitrophenyl (TNP) plaque assay. Primary response of Balb/c mice to soluble and particulate immunogen. Proc. Soc. Exp. Biol. Med. 132: [19] Shearer, G. M., Mozes, E., and Seta, M Cellular basis of the genetic control of immune responses to synthetic polypeptides. II. Frequency of immunocompetent precursors specific for two distinct regions within (Phe, G)-Pro- -L, a synthetic polypeptide derived from multichain polyproline, in inbred mouse strains. J. Exp. Med. 133: