Masayasu Kitagawa

Induction of anti-hapten antibody response by hapten-isologous carrier conjugate: I. Development of hapten-reactive helper cells by hapten-isologous carrier

Abstract Anti-hapten antibody production was elicited by the immunization of hapten-isologous carrier conjugate (DNP-MγG) in mice. The spleen and lymph node cells taken from those primed mice were effectively stimulated with hapten-heterologous carrier conjugates (DNP-KLH and DNP-BαA) as well as hapten-homologous carrier conjugate (DNP-MγG) when transferred into X-irradiated recipient mice. The reactivity of DNP-MγG-primed cells to DNP-heterologous carrier conjugates was not due to the mutual crossreactivity of the carrier with MγG on cellular level, since the spleen and lymph node cells primed with DNP-KLH or DNP-BαA could only be stimulated with corresponding hapten-homologous carrier conjugate. The responsiveness of DNP-MγG-primed cells to hapten-heterologous carrier conjugates was due to the result that hapten-reactive helper cells were developed by the immunization of hapten-isologous carrier and these cells cooperated with hapten-specific B cells. The helper activity of the hapten-isologous carrier-primed cells was resistant to 600-R X-irradiation in vitro and sensitive to in vivo ATS treatment. This suggests that the helper activity induced by hapten-isologous carrier is of T cell origin. The helper activity of hapten-isologous carrier-primed cells was also developed by the immunization of PAB-MγG, and clear cooperative interaction between PAB-MγG-primed cells and DNP-specific B cells was demonstrated through DNP-MγG-PAB. The possible mechanism of helper cell development induced by the immunization of hapten-isologous carrier conjugate was discussed in light of the hapten specificity of helper activity.

Enhancing factor on anti-hapten antibody response released from PPDs-stimulated tubercle bacilli-sensitized cells

Abstract There is a considerable body of evidence that cellular interaction is required for the induction of antibody response to various antigens (Claman and Chaperon, 1969); Miller and Mitchell, 1969; Taylor, 1969). It is now well established in hapten-carrier system that a good anti-hapten antibody response is obtained when hapten-committed antibody forming cell precursor (AFCP) cooperates with carrier-committed helper cells through hapten-carrier conjugate. The former cells are derived from bone marrow and have the immunoglobulin-like receptor specific for the haptenic group on their cell surface. The latter cells are thymus-derived and have a receptor specific for the carrier moleculr and help anti-hapten AFCP to produce anti-hapten antibody. We reported previously that tubercle bacilli-sensitized cells have helper activity on the secondary anti-hapten antibody formation in the presence of hapten-conjugated PPDs in vivo culture system (Takatsu et al., 1972a). The helper activity of tubercle bacilli-sensitized cells were diminished when the cells were treated with θ-antibody and guinea-pig complement prior to cell transfer (Takatsu et al., 1972b). It is however, still obscure how both cells of the tubercle bacilli-sensitized cells and hapten-specific AFCP interact to produce anti-hapten antibody in the presence of hapten-tuberculin protein conjugate. In the present study, we found that a soluble factor is released from tubercle bacilli-sensitized cells in vivo and invitro, and enhances effectively anti-hapten antibody response of a hapten-carrier-primed cells in the presence of a heterologous carrier conjugated with the hapten.

Induction of anti-hapten antibody response by hapten-isologous carrier conjugate: II. Specificity of hapten-reactive helper T cells

Abstract Anti-hapten antibody production was elicited by the immunization of hapten-isologous carrier conjugate (PAB-MGG) in mice. Spleen and lymph node cells taken from these primed mice could demonstrate their helper activity for anti-DNP antibody production when transferred intravenously into 600R X-irradiated recipient mice along with DNP-primed B cells and the double hapten conjugated carrier, DNP-MGG-PAB. Isologous carrier (MGG)-primed cells could not demonstrate this helper activity. Accordingly, helper cells reactive for a haptenic group are considered to develop by the immunization of hapten-isologous carrier conjugate. Hapten-reactive helper activity was also induced by the immunization of other hapten-isologous carrier conjugates, e.g., MAB-MGG, PABS-MGG or PAB-MSA. These hapten-reactive helper cells were T lymphocytes, as the helper activity of PAB-MGG-primed cells was completely abolished by in vivo ATS-treatment. Helper activity of PAB-MGG-primed cells for DNP-primed B cells was also demonstrated through the double hapten conjugated heterologous carrier DNP-HGG-PAB to be the same as with DNP-MGG-PAB, but weakly through DNP-KLH-PAB. As HGG but not KLH resembles MGG in composition, almost all hapten-reactive helper T cells can be considered to recognize not only haptenic groups but also physicochemical properties of the hapten-conjugated carrier site. However, these helper T cells could discriminate structural differences among related haptenic groups, because PAB-MGG-primed cells clearly responded to DNP-MGG-PAB to demonstrate their helper activity for DNP-primed B cells, but responded only weakly to DNP-MGG-PABS or DNP-MGG-MAB. When the specificity restrictions of T and B cells to the same haptenic group were compared by responsiveness measured after the antigenic stimulation (B cell function by anti-hapten antibody production and T cell function by helper activity), differences were noted, as PAB-MGG-primed T cells could respond not only to DNP-MGG-PAB but also fairly well to DNP-MGG-MAB to demonstrate their helper activity, but PAB-MGG-primed B cells responded to only PAB-MGG. Thus, hapten specificity appears to be much more restricted for B cells than T cells. The difference of this responsivity between B cells and helper T cells was thought to derive from the specificity difference of B cell and helper T cell receptors rather than from any sensitivity differences of the experimental procedure. The differences in the specificity restrictions of receptors of B and helper T cells were discussed in the light of hapten-specificity.

Immune maturation of T lymphocytes: Sequential changes in the functional specificity and apparent affinity of hapten-reactive helper T cells during an immune response

Abstract The maturation of helper T lymphocytes during an immune response was studied with respect to sequential changes in the functional specificity and affinity toward certain antigens. Protein-carrier (BαA)-reactive helper T cells obtained after a relatively long priming period were effectively stimulated by relatively lower doses of antigen than shortly primed helper T lymphocytes. When hapten (PAB)-reactive helper T lymphocytes were utilized as a model of helper T cells, reactivity also increased progressively to smaller concentrations of PAB-conjugates at successive intervals after primary immunization. Concomitantly, the cross-reactivity of PAB-reactive helper T cells to structurally related MAB- or OAB-determinants also decreased. Moreover, the PAB-reactive helper T cells of the relatively longer priming period were very susceptible to tolerance induction upon treatment with PAB- d -GL, whereas the reactivity of those helper T cells of the relatively shorter priming period was not abolished by this treatment. These results clearly indicate that there are qualitative changes in the helper T lymphocyte population during an immune response, and that this represents the sequential development or selection of helper T lymphocytes of higher specificity and apparent affinity to a corresponding antigenic determinant.

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice: I. Establishment of the experimental conditions for the termination of immunological tolerance to human gamma globulin☆

Abstract In order to study the role of hapten-reactive helper T cells in the induction of autoimmunity in mice, an attempt was made to establish an experimental model for the development of hapten-reactive helper T cells and the termination of immunological tolerance against heterologous proteins. Spleen cells taken from mice which were immunized with hapten-isologous protein conjugates (PAB-MGG) demonstrated helper activity for the anti-DNP antibody response of DNP-primed B cells responding to DNP and PAB-conjugated protein, but spleen cells from hapten-heterologous protein conjugate (PAB-HGG)-primed mice could not respond to PAB-determinant. Thus, hapten-reactive helper T cells can develop in mice by the immunization with hapten-isologous protein conjugate, but not with hapten-heterologous protein conjugate. However, spleen cells from mice which had been rendered tolerant by treatment with 2.5 or 0.2 mg of DHGG and then immunized with PAB-HGG could demonstrate helper activity responding to PAB-determinant. This helper activity was PAB-specific, because these spleen cells did not demonstrate helper activity if PAB-determinant was omitted in the primary and the secondary antigen. This helper activity was abrogated by the treatment of spleen cells with anti-θ serum and complement. Thus, hapten-reactive helper T cells were successfully induced by the challenge with hapten-heterologous protein conjugate in carrier-protein tolerant mice. When mice were treated with 2.5 or 0.2 mg of DHGG, no anti-HGG antibody response was induced by the challenge with HGG or PAB-HGG. However, the termination of HGG-tolerance was demonstrated only when the mice were preimmunized with PAB-MGG to raise PAB-rcactive helper T cells, treated with 0.2 mg of DHGG, and then challenged with PAB-HGG. This termination of immunological tolerance was not observed when the mice were preimmunized with PAB-BαA to raise PAB-specific B cells and anti-PAB antibody, or when the mice were treated with 2.5 mg of DHGG. Thus, if HGG-specific B cells remain intact in mice such as treated with low dose of DHGG, these B cells can be activated by some bypass mechanisms in the presence of PAB-reactive helper T cells through the PAB-determinant even in the absence of HGG-reactive helper T cells. These data clearly showed the role of hapten-reactive helper T cells in the termination of immunological tolerance and provide experimental supports to the hypothesis on the termination mechanism proposed by Weigle. The cellular mechanism for the development of hapten-reactive helper T cells in tolerant animals and the cellular mechanism of autoantibody production were discussed on the basis of T-B cell collaboration.

Naturally occurring rabbit antibodies against the tryptic 3β5S and 5S fragments of rabbit IgG—II

Abstract (1) The homoreactants directed to the tryptic 3β5S fragments, Fab″ and 5S fragments, F(ab″)2 from rabbit IgG could be demonstrated in normal rabbit IgG by agglutination of Fab″ or F(ab″)2-conjugated erythrocytes. (2) These homoreactants could be distinguished from those directed to the papain fragments, Fab and the peptic fragment, Fab′ by inhibition test of hemagglutination with each fragment. The homoreactant directed to Fab″ seems to be different from that directed to F(ab″)2. (3) Immunoadsorbents were prepared by conjugation of Fab, Fabt or Fab″ with insoluble polymer of rabbit serum albumin. The homoreactants directed to Fab and Fab″ could be purified by adsorption on, elution with 1 M propionic acid from the homologous immunoadsorbents and by absorption with the heterologous immunoadsorbents. (4) These purified homoreactants (radiolageled) showed preferential binding activities with the homologous immunoadsorbents. The binding activity was inhibited strongly by the homologous fragments but weakly by the heterologous fragments. These results indicate that the antigenic determinants exposed by tryptic digestion are different from those done by papain or peptic digestion.

An abnormal ratio of cytochromes in the respiratory chain of mouse and human myelomas

Mouse myeloma cells and mitochondria had the same kinds of cytochrome components in the respiratory chain as the normal ones. Their constitution, however, was abnormally different from that found in normal cells and mitochondria. The cytochrome aa3 concentration was especially low in the myeloma as compared with cytochrome c concentration, and the resulting cytochrome aa3/c ratio was 0.25, which was the lowest ever reported in animal mitochondria. Normal lymph node cells, producing the immunoglobulin similar to the myeloma cells, had a ratio of 1.1. Human myeloma mitochondria had the same characteristics as the mouse myeloma. Ascite form myeloma originated from mouse solid from myeloma grew faster, and yet aa3/c of 0.5 in the ascites myeloma was found to be quite similar to that observed in various ascites tumor cells such as hepatomas, Ehrlich and sarcoma 180. A significant part of the cytochromes in the respiratory chain of the mouse myeloma remained in the oxidized form in the cyanide-inhibited or anaerobic states, and was reduced only by the addition of dithionite. The properties of the b cytochromes in mouse myeloma mitochondria are also described and discussed in the context of multiple forms of the b cytochromes in the respiratory chain.

Idiotypic determinants of immunoglobulins—I Antigenic homology between the heavy and light polypeptide chains from a γA myeloma protein

Abstract A λ-type γA myeloma protein (Hi) was studied in respect of individual-specific or ‘idiotypic’ antigenicity. Rabbit antiserum against γA(Hi) aws rendered specific to the homologous antigen, αA(Hi), by absorption with pooled normal human serum and showed the reaction specific for the idiotypic determinants of the γA(Hi). Absorption of the anti-γA(Hi) with increased amounts of pooled normal human serum resulted in decreased amounts of specific precipitation with the γA(Hi), suggesting that a small quantity of immunoglobulins in the pooled serum may have antigenic determinants similar to the idiotypic determinants of the γA(Hi). To analyze molecular localization of the idiotypic determinants, papain digest, heavy and light polypeptide chains of the γA(Hi) were prepared. The papain digest, heavy chain and intact γA(Hi) showed reactions of identity with the anti-idiotypic antiserum (anti-Hi-idio), but the light chain did not show any precipitate line with the anti-Hi-idio upon immunodiffusion. This seems to indicate that the idiotypic determinants of the γA(Hi) localized on the amino terminal portion of the heavy chain. However, the light chain was also found to possess the reactivity with the anti-Hi-idio as clearly demonstrated by pasive hemagglutination of rabbit red blood cells coated with the light chain and by inhibition to the precipitation reactions of the γA(Hi) and the heavy chain with the anti-Hi-idio. The facts that the heavy chain and the intact γA(Hi) showed completely identical reactions with the anti-Hi-idio upon immunodiffusion and that not only the reaction of the intact γA(Hi) but also of the heavy chain were inhibited by the presence of the light chain strongly suggest that, so far as the idiotypic determinants are concerned, there exists some homology in the antigenic structure between the heavy and light chains of γA(Hi). The significance of these findings was discussed.

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice: II. Termination of self-tolerance to erythrocytes by immunization with hapten-isologous erythrocytes☆

Abstract When mice which had been primed with hapten-isologous protein conjugate (PAG-MGG) were challenged with PAB-conjugated isologous mouse erythrocytes (MRBC), they developed Coombs positivity and anemia. However, when mice primed with hapten-heterologous protein conjugate (PAG-HGG) were challenged with PAB-MRBC, neither Coombs positivity nor anemia developed. Since it was demonstrated that PAB-reactive helper T cells were generated by immunization with PAB-MGG but not with PAB-HGG, PAB-reactive helper T cells were considered to play a very crucial role in the induction of autoantibody. These results, as a model for autoantibody production in mice, were discussed on the basis of cellular cooperation mediated by a hapten-mechanism, and are consistent with the hypothesis proposed by Weigle for the mechanism of termination of self-tolerance.

Regulatory Mechanism of Reagin Production in Mice at the T Cell Level

Helper T cell activities specific for purified protein derivative (PPD) generated by immunization with Mycobacterium tuberculosis (Tbc) or PPD were investigated concerning adoptive IgE and IgG antibody responses. It is interesting that preferential triggering activity of IgG antibody response was observed when PPD‐reactive cells from mice immunized with Tbc were used as a helper cell source. The selective triggering of IgG B cells by Tbc‐primed cells was consistently observed using DNP‐primed B cell populations from mice immunized with DNP‐carrier conjugate in either ICFA or alum. T cell dependency of helper activity was demonstrated by the fact that treatment of Tbc‐primed cells with anti‐Thy 1 antiserum plus complement abolished their helper activity. We also demonstrated that purified T cell populations selectively triggerred IgG B cells.