Anti-hapten Antibody Response Research Articles

In vitro modulation of antigen-primed T cells by a glycosylation-inhibiting factor that regulates the formation of antigen-specific suppressive factors.

BDF1 [(C57BL/6 X DBA/2)F1] mice were primed with alum-absorbed ovalbumin, and their spleen cells were cultured with ovalbumin to activate antigen-primed T cells. The activated T cells were then propagated in interleukin 2-containing medium in the presence or absence of affinity-purified glycosylation-inhibiting factor (GIF). Upon incubation with ovalbumin-pulsed macrophages, T cells propagated in the absence of GIF produced IgE-potentiating factor and glycosylation-enhancing factor. In contrast, T cells propagated in the presence of GIF produced IgE-suppressive factor and GIF. The ovalbumin-primed T cells propagated in the presence of GIF constitutively produced the 13-kDa GIF that lacked affinity for ovalbumin. However, stimulation of the same T cells with ovalbumin-pulsed macrophages resulted in the production of 80- and 35-kDa GIF that had affinity for ovalbumin. Both the antigen-specific GIF and nonspecific GIF from the activated BDF1 T cells had I-Jb determinants. Since the ovalbumin-specific GIF is derived from Lyt-2+, I-J+ ovalbumin-specific suppressor T cells and suppresses the anti-hapten antibody response to dinitrophenyl-coupled ovalbumin, the results strongly suggest that the presence of GIF during the propagation of antigen-primed T cells facilitates the generation of antigen-specific suppressor T cells.

Cloned, Ia-restricted T cells that do not produce interleukin 4(IL 4)/B cell stimulatory factor 1(BSF-1) fail to help antigen-specific B cells.

T cells can be subdivided based on cell surface markers, MHC restriction, function, and production of soluble factors. Analysis of the ability of cloned, Ia-restricted, L3T4+ T cells to induce an in vitro anti-hapten antibody response to hapten-carrier conjugates allowed the definition of three functional subtypes. To examine whether these functional subtypes also differed in the production of soluble mediators, supernatants of the cloned lines were examined for the production of T cell growth factors and factors inducing increased expression of Ia glycoproteins on small resting B cells. All of the cloned lines produced T cell growth factors that could be further differentiated by inhibition with monoclonal antibodies. None of the Ia-restricted, L3T4+ cloned T cell lines that failed to produce IL 4/BSF-1 could provide helper function. Thus, the activation of antigen-specific B cells by helper T cells appears to require IL 4/BSF-1 as a necessary but not sufficient signal for differentiation into antibody-forming cells.

Carrier-specific suppression of antibody responses by antigen-specific glycosylation-inhibiting factors.

We previously established an ovalbumin (OA)-specific T cell clone from spleen cells of BDF1 mice, which had been treated by i.v. injections of OA, and constructed antigen-specific T cell hybridomas from the T cell clone. One of the hybridomas constitutively released glycosylation-inhibiting factor (GIF) which lacked affinity for OA, and was called non-specific GIF. Incubation of the same hybridoma cells with OA-pulsed syngeneic macrophages or OA-pulsed B lymphoblastoid cells of BALB/c origin resulted in the formation of GIF molecules that had affinity for OA but not for bovine serum albumin or keyhole limpet hemocyanin. Both the OA-specific GIF and nonspecific GIF bound to monoclonal anti-lipocortin and possessed I-Jb determinants. The OA-specific GIF consisted of two species of molecules, of m.w. 80,000 and 30,000 to 40,000, respectively, whereas the nonspecific GIF from unstimulated cells had an m.w. of 15,000. Intravenous injections of OA-specific GIF or nonspecific GIF into BDF1 mice suppressed both the IgE and IgG1 anti-hapten antibody responses of the animals to dinitrophenyl derivatives of OA (DNP-OA), but OA-specific GIF was much more effective than nonspecific GIF in suppressing the antibody responses. When the same preparations of GIF were injected into DNP-KHL-primed mice, OA-specific GIF and nonspecific GIF were comparable in suppressing the anti-DNP antibody response. In contrast to the 40,000 m.w. species of OA-specific GIF, the 80,000 m.w. OA-specific GIF had carrier-specific suppressive effects. The similarities of antigen-specific GIF to antigen-specific TsF suggest that the phospholipase-inhibiting activity of the molecules may be involved in the immunosuppressive effects of some antigen-specific TsF.

Influence of immunoglobulin-dependent T cells on antibody class switching.

Normal and B cell-deficient, carrier-primed mice were irradiated and were adoptively transferred with B cells to evaluate the role of putative Ig- and B cell-dependent T cells in anti-hapten antibody responses. The response was analyzed by using the splenic focus assay, which allowed us to examine the frequency of responding B cells and the production of multiple isotypes by single precursor B cells. This analysis revealed that both primary and secondary B cells were activated at higher frequency in the spleens of normal recipients, and production of isotypes other than IgM and IgG1 was enhanced in normal recipients as compared with anti-mu-treated recipients. Both changes could be restored to control levels by co-transfer of T cells from normal donors primed with an unrelated carrier, provided the free carrier was added to the assay culture. These results are consistent with a role for Ig or B cell-dependent helper T cells in the optimal activation and the resulting isotype expression of both primary and secondary B cells.

Immunosuppressive effects of glycosylation inhibiting factor on the IgE and IgG antibody response.

Glycosylation inhibiting factor (GIF) was purified from culture filtrates of a T cell hybridoma, 23A4, by affinity chromatography on anti-lipomodulin Sepharose. The factor exhibited phospholipase inhibitory activity upon dephosphorylation. Immunization of BDF1 mice with aluminum hydroxide gel (alum)-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) resulted in persistent IgE and IgG antibody formation. However, repeated injections of the affinity-purified GIF into the DNP-OA-primed mice beginning on the day of priming prevented the primary anti-hapten antibody responses of both the IgE and the IgG1 isotypes. Treatment with GIF also diminished on-going IgE antibody formation in the DNP-OA-primed mice. The treatment changed the nature of IgE-binding factors formed by BDF1 spleen cells. Incubation of spleen cells from OA + alum-primed mice with OA resulted in the formation of IgE-potentiating factor, whereas spleen cells of OA-primed, GIF-treated mice formed IgE-suppressive factor upon antigenic stimulation. It was also found that Lyt-2+ T cells in the OA-primed, GIF-treated mouse spleen cells released GIF, which had affinity for OA and bore I-Jb determinant(s). Transfer of a Lyt-1+ cell-depleted fraction of the OA-primed, GIF-treated mouse spleen cells into naive syngeneic animals resulted in suppression of the primary anti-DNP IgE antibody response of the recipients to alum-absorbed DNP-OA, but failed to affect the anti-DNP antibody response to DNP-keyhole limpet hemocyanin. The results indicate that GIF treatment during the primary response to OA facilitated the generation of antigen-specific suppressor T cells.

Use of particulated antigen to enhance primary in vitro antibody responses to trinitrophenyl-keyhole limpet hemocyanin

Methods are described for the efficient induction of primary anti-hapten antibody responses to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) in vitro. Mouse leukocytes stimulated in vitro with particulated (bentonite-adsorbed) TNP-KLH demonstrated 10-fold more IgM-secreting, plaque-forming cells (PFC) as measured by a hemolytic plaque assay than did leukocytes stimulated with soluble TNP-KLH. The T dependency of the enhanced antibody response was confirmed using highly enriched B and T lymphocyte populations. These methods should greatly facilitate studies regarding primary in vitro antibody responsiveness to T-dependent antigens.

Primary antibody responses to a well-defined and unique hapten are not enhanced by preimmunization with carrier: analysis in a viral model.

We used a viral model to reexamine classical experiments showing that mice previously primed with a "carrier" molecule alone and then challenged with the carrier-hapten conjugate exhibited an enhanced antihapten antibody response. Mice were primed with live or UV-inactivated vesicular stomatitis virus (VSV) Indiana (Ind) serotype with or without complete Freund's adjuvant. After challenge with VSV New Jersey (NJ), these mice developed a secondary-type IgG response, measured by antibody binding in an ELISA, against both VSV-Ind and VSV-NJ. The same result was found for the reciprocal experiments where mice were primed with VSV-NJ. Similarly, when mice were primed with live VSV, UV-inactivated VSV, or purified VSV glycoprotein G of Ind or NJ serotype and later were challenged with dinitrophenyl (N2ph)-conjugated, UV-inactivated VSV or with N2ph-conjugated G protein of either serotype, they exhibited a secondary-type anti-N2ph antibody response as demonstrated by the binding of IgG to dinitrophenylated bovine serum albumin measured by ELISA. In contrast, when neutralizing antibody responses were monitored, VSV-Ind-primed mice challenged with VSV-NJ developed a strictly primary type of anti-VSV-NJ response and vice versa. We conclude that preexistent helper T cells specific for shared carrier determinants do not improve virgin B-cell responses specific for "new," unique determinants that are the target for the biologically relevant neutralizing antibodies. These findings suggest that priming of B cells rather than of helper T cells may be of importance to induce protective immunity mediated by antibodies.

Suppression of the anti-hapten IgE antibody response with hapten-modified spleen cells.

Spleen cells of BALB/c mice were chemically modified with phosphorylcholine or benzylpenicilloyl hapten. The i.v. administration of such cells into syngeneic animals suppressed the formation of specific IgE antibodies against the respective hapten. The IgE antibody response against ovalbumin, which was used as an immunogenic carrier for the haptens, was not affected and the anti-hapten IgG or IgG1 response remained at the levels of the controls. The suppression could be transferred into X-irradiated mice by T cells from tolerized animals. Moreover, it was demonstrated that not only the induction of IgE, but also an established anti-hapten IgE antibody response is accessible to suppression by treatment with hapten-modified spleen cells from syngeneic animals. The results indicate that the i.v. administration of antigen coupled to syngeneic spleen cells induces T cells which suppress the formation of specific IgE antibodies in the primary and the secondary response without significantly affecting the formation of IgG antibodies.

Hapten-specific unresponsiveness in mice: IV.—Significance of anti-hapten antibody responses elicited by E. coli lipopolysaccharides in tolerant mice

The antibody response to lipopolysaccharide (LPS) and trinitrophenol-LPS (TNP-LPS) was investigated in mice after tolerance induction to chemically reactive hapten 2-4-6-trinitrobenzene sulphonic acid. The depression of the anti-TNP IgM response elicided by LPS was moderate and lasted 1 week. Depression of the response to TNP-LPS was severe and lasted 5 weeks. Tolerant mice given LPS 7 days after tolerance induction, at a time when it elicited a normal response, and challenged with TNP-LPS 2 weeks later, exhibited the same depression of their anti-TNP response as tolerant mice not given LPS. Furthermore, the anti-TNP IgM elicided by TNP-LPS in normal mice was of higher avidity than that observed with LPS. These results suggest that the anti-TNP response elicided by LPS cannot account for the overall immune status of tolerant mice.

Antigen non-specific activities in culture supernatant of ovalbumin-specific, cloned helper T lymphoma

Antigen non-specific activities in culture supernatants from clones of a radiation leukemia virus transformed, ovalbumin-specific helper T lymphoma were studied. After stimulation with ovalbumin-pulsed macrophages, these clones release factor(s) capable of providing carrier-specific help for anti-hapten antibody responses. It was found that in addition to IL 2-likeactivity, one clone constitutively released a suppressive factor that could inhibit polyclonal growth and differentiation of B cells stimulated with LPS. The factor inhibited activation of both syngeneic and allogeneic B cells directly, rather than via activation of accessory suppressor T lymphocytes. However, upon dilution of the supernatant, B cell growth and differentiation activities were detected. A role for a polyclonal B cell suppressor factor in the overall helper activity of the clone is proposed.

Novel mechanisms of specific suppression of anti-hapten antibody response mediated by monoclonal anti-carrier antibody.

The cellular mechanisms of the antibody-induced suppression of immune responses were analyzed in the keyhole limpet hemocyanin (KLH) system. Some of the monoclonal anti-KLH antibodies, like KLH-specific suppressor T cell factor (KLH-TsF), were demonstrated to suppress the anti-2,4-dinitrophenyl IgG but not IgM plaque-forming cell responses in a KLH-specific and H-2-restricted manner. The anti-KLH antibodies with suppressive activity reacted with, and in turn, stimulated the suppressor hybridoma (34S-281) with the anti-idiotypic receptor complementary to the idiotypic KLH-TsF of the inducer type. Moreover, because the suppressive activity of the anti-KLH antibody was completely abolished by the treatment of responding spleen cells with anti-Lyt-2 and complement, it was apparent that the suppressive antibody activated suppressor T cell pathways. The isotype or affinity of antibodies is not related to the suppressive activity, because suppressive and nonsuppressive antibodies possess a similar affinity belonging to the same Ig isotypes. It also has been demonstrated that the Fc portion is not the functional site, because the F(ab')2 fragment still has the activity. The antibody specificity is found to be important for determining whether the antibody is suppressive or not. In fact, anti-KLH 26, but not other antibodies without activity, recognizes the particular KLH epitope seen by KLH-TsF, and exclusively interacts with the anti-idiotypic suppressor T cells. Thus, the anti-idiotypic suppressor T cell receives signals both from the suppressive anti-KLH antibody and from KLH-TsF, and transmits the antibody-induced suppressor signals to the effector-suppressor pathway. The size of the repertoire of anti-idiotypic suppressor T cells involved in the suppression seems to be very limited, because only four out of 120 monoclonal anti-KLH antibodies were found to have suppressor activity. The possible mechanisms of the cell interaction mediated by the suppressive antibody are discussed.

23] Antigen-specific suppressor T cells and their soluble products

Several types of suppressor T cells and their factors are described. In this chapter, the focus is mainly on the preparation, separation, and characterization of the antigen-specific suppressor T cell with Thy-I, Lyt-2, and I-J phenotypes and on those of their soluble suppressor factor composed of two polypeptide chains. It is well known that an interaction between carrier protein-primed helper T cells and hapten-primed B cells in the presence of hapten-carrier protein conjugates is required in order to elicit an anti-hapten antibody response. Moreover, the anti-hapten response largely depends upon carrier specificity, since animals that have been immunized with hapten carrier X do not respond when boosted with hapten carrier Y. This is also the case of suppressor T cells. Thus, suppressor T cells are primed with carrier antigens and possess the specificity for the determinants on the carrier protein (carrier specificity). The chapter further explains establishment of antigen-specific suppressor T Cell hybridomas.

Evaluation of accessory cell heterogeneity. I. Differential accessory cell requirement for T helper cell activation and for T-B cooperation.

Several Ia+ tumor cell lines and peritoneal exudate macrophages were tested as accessory cells (AC) for the activation of antigen-specific T cells and for T-B cooperation. The macrophages and all the Ia+ tumor lines tested induced the release of lymphokines from T cells in a major histocompatibility complex (MHC)-restricted fashion and reconstituted the antibody responses of AC-depleted spleen cells or of purified T and B cells. However, only the normal macrophages but none of the tumor lines induced carrier-specific T helper (Th) cells which help B cells for specific antihapten antibody responses by linked recognition. For T-B cooperation accessory cells were also required, but in contrast to Th cell activation any type of Ia+ AC (e.g. macrophage or tumor line) was effective. Strong MHC-restriction between the lymphocytes and the AC was seen if antigen-pulsed AC were added into the AC-depleted T-B cooperation cultures. If the AC and antigen were concomitantly added to the AC-depleted T-B cultures, MHC-restriction was less obvious. Concanavalin A supernatant reconstituted the response of AC-depleted T-B cultures provided antigen-specific Th cells and the hapten-carrier conjugate were present. If, however, tumor line-activated T cells were added instead of macrophage-induced Th cells, no cooperation with B cells took place even in the presence of Con A supernatant. The results obtained demonstrate a differential AC requirement for the induction of Th cells depending on the differentiation stage of the Th cells.

Regulation of anti-hapten antibody responses in vivo: memory, carrier-specific Lyt-2+ T cells can terminate antibody production without altering the peak of response.

By using the classical hapten-carrier antigenic system (dinitrophenyl-keyhole limpet hemocyanin) (DNP-KLH), and positive immunoselective techniques for purification of lymphocyte subsets, we studied the kinetics and role of KLH-primed T cells in the termination of a secondary in vivo plaque-forming cell (PFC) response against DNP in the mouse. Separation of T cells by Lyt-2 phenotype and recombination of T cell subsets demonstrated that a single injection of KLH in complete Freund's adjuvant (CFA) concomitantly generated both helper and suppressor T cells in the spleens of primed mice. When these KLH-primed, purified T cells were transferred into irradiated recipients along with DNP-primed, purified B cells and DNP-KLH, helper activity was demonstrable first, with the early production of a high anti-DNP PFC response. Suppression of the PFC response was observed only after the response had fully developed; the PFC response was then "terminated" within a few days. Helper activity was obtained with the carrier-primed Lyt-2- T cell population, whereas suppression required the addition of Lyt-2+ T cells. Both activities were generated over a wide dose range, were specific for the priming antigen KLH, and could be detected as early as 8 days or as late as 9 mo after a single priming injection of KLH. Lyt-2+ T cells from unprimed donors or from animals primed with a different carrier did not have the capacity to terminate the response when added with the primed Lyt-2- helper T cells. Thus, we have now demonstrated the existence of carrier-specific, long-lived, "memory" suppressor T cells, which are initially generated in parallel with specific helper T cells. Whereas these memory suppressor T cells do not inhibit the induction of a secondary antibody response, they can effectively terminate it once it has developed, and may therefore account for the normal termination of antibody responses.

Studies on the immune response in chickens IV. Generation of hapten-specific memory and absence of increase in carrier-specific helper memory in antibody response to sheep red blood cell antigen and its hapten-conjugate

The role of carrier recognition in the hapten-specific antibody response of chickens to hapten (TNP) conjugate of sheep red blood cells (SRBC) was studied. The anti-TNP antibody response to the challenge with either TNP-SRBC or DNP conjugate of Salmonella enteritidis (DNP-Sal) of chickens previously primed with TNP-SRBC was definitely higher than that of unprimed chickens. Injection of TNP-chicken red blood cells, which did not provoke any significant primary anti-TNP response, also induced the memory for secondary anti-TNP response to the challenge with TNP-SRBC or DNP-Sal. The TNP-specific memory generated by stimulation with TNP-SRBC was adoptively transferable by spleen cells into cyclophosphamide (CY)-treated X-irradiated syngeneic recipients. On the other hand, the SRBC-specific memory for augmentation of anti-TNP response to the challenge with TNP-SRBC was never detected in chickens previously primed with either any of a wide range of doses (10 4–10 8) of SRBC, solubilized SRBC or SRBC fixed with formaldehyde, nor in CY-treated X-irradiated chickens receiving SRBC-primed spleen cells. The anti-TNP response of chickens to the challenge with TNP-SRBC was rather suppressed by the SRBC preimmunization. The passively administered anti-SRBC chicken serum also suppressed the response to TNP-SRBC. The carrier-specific suppressor activity generated by SRBC stimulation was not adoptively transferable by spleen cells. It is suggested from these results that the mechanism of amplification of the anti-hapten antibody response of chickens to hapten-SRBC conjugate is primarily mediated by the hapten-recognition, and carrier-recognition is more important for suppression rather than for amplification of the anti-hapten response, although the inductive mechanism for anti-hapten response itself should somehow involve the carrier recognition.

Prevention of cerebral malaria by adoptive transfer of malaria-specific cultured T cells into mice infected with Plasmodium berghei.

Murine T cell populations specific for Plasmodium berghei parasites were generated in vitro from BALB/c immune lymph node cells. The malaria-specific T lymphocytes were shown: a) to proliferate specifically in vitro in response to stimulation with P. berghei-infected red blood cells; b) to exhibit the Thy-1+, Lyt-1+2- cell surface phenotype; c) to provide specific helper activity for an in vitro anti-hapten (TNP) plaque-forming cell antibody response; and d) to protect P. berghei-infected mice from early mortality due to cerebral malaria.

LPS greatly enhances the antibody response to hapten-polysaccharide conjugates, but not to hapten-protein conjugates

In confirmation of earlier findings, we observed that an injection of bacterial lipopolysaccharide (LPS) into mice caused a considerable increase in the serum concentrations of IgM and IgG (total Ig rose three- to four-fold in 7 days), and a corresponding increase in the concentrations of "natural" anti-(3-iodo-4-hydroxy-5-nitrophenyl) acetyl (NIP) and anti-trinitrophenol (TNP) antibodies. Our main purpose was to determine what effect LPS had on antigen-dependent responses. Hapten conjugates of a polysaccharide and of proteins were used as antigens. Hapten-protein conjugates induced a strong anti-hapten antibody response (up to 1 mg/ml of anti-hapten antibodies on day 7). Hapten-polysaccharide conjugates induced only a meagre increase in anti-hapten antibodies from the pre-immunization level (maximal concentration 65 micrograms/ml on day 7). LPS, when injected with the antigen, greatly enhanced the antibody response to the hapten-polysaccharide conjugates (up to 2.6 mg/ml of anti-hapten antibodies on day 7). It had little effect on antibody responses to hapten-protein conjugates. The combination treatment had the same effect on immunoglobulin concentrations as LPS alone.

Maturation of the lymphoid system IV. Ontogenetic compartmentalization of T cell function

Pretreatment of one day old but not eight day old or adult A/J mice with soluble ovalbumin (OVA) initiated specific T-cell unresponsiveness as reflected both in T-cell dependent cellular proliferation and in anti-hapten antibody responses to dinitrophenyl-OVA. In contrast, injection of soluble human gamma globulin into either neonatal or adult A/J mice resulted in unresponsiveness. The ability of lymph node T-cells to be sensitized by protein antigens occurred shortly after birth, since the degree of sensitization in 9 and 26 day old mice was similar. Finally, a striking ontogenetic difference was noted in the capacity of lymphocytes derived from the lymph nodes and spleens of young mice to respond to T-cell mitogens. Thus, while splenocytes obtained from 9 day old mice exhibited meager responses to PHA and Con A, lymph node cells from these animals responded at nearly adult levels. These observations are interpreted as reflecting an ontogenetic and tissue-specific division of T-cell function.

The donor cell type controls anti-hapten (fluorescein isothiocyanate) primary antibody response to hapten-modified syngeneic cells

Hapten (fluorescein isothiocyanate, FITC)-sensitized syngeneic red blood cells (FITC-RBC) are exceptionally active for induction of anti-hapten primary antibody response, and FITC-modified syngeneic spleen cells depleted of RBC (FITC-SSC) are not immunogenic [4]. The present study has demonstrated that FITC-SSC injected simultaneously with FITC-RBC inhibit partially the anti-FITC response to the latter. Either the immunogenicity of FITC-RBC or the response-inhibiting activity of FITC-SSC was increased as the concentration of hapten-sensitizing cells was raised from 0.005 mg/ml to 2 mg/ml. The inhibition of anti-FITC response by FITC-SSC strictly required live donor cells, but was not dependent on T-cell activity of either the donor or recipient. Neither FITC-thymocytes nor the FITC-T-cell-rich fraction of SSC showed a definite activity for inhibition, whereas the FITC-B-cell-rich fraction of SSC acted very effectively. These results suggest that the primary anti-hapten antibody response to hapten-modified syngeneic cells is primarily controlled by antigen-bearing live donor cells of different cell types.

Cell-to-cell interaction controlled by immunoglobulin genes. Role of Thy-1-, Lyt-1+, Ig+ (B') cell in allotype-restricted antibody production.

A novel lymphocyte subpopulation, designated "B' cell" because of its characteristic dual expression of Ig and Lyt-1 antigen, is described in relation to its ability to augment the in vitro secondary antibody response. The cells are found in the spleens of normal unprimed mice as well as those of athymic nude mice and represent a small of normal unprimed mice as well as those of athymic nude mice and represent a small number (2-3%) of immunoglobulin-positive cells. No other distinguishing surface markers of conventional T and B cells, such as Thy-1, Lyt-2, Ia, and ThB antigens, are detected on the B' cell. In the in vitro anti-hapten secondary antibody response, the addition of a small number of B' cells from unprimed mice to the mixture of T and B cells greatly augmented the anti-hapten antibody formation when the number of carrier-specific helper T cells was limited. This augmentation was observed only when B and B' cells shared the same set of IgVH genes. The identity of the immunoglobulin gene between T cells and B or B' cells was not necessary for optimum antibody production. The results indicate that the presence of B' cells is necessary for the induction of an optimum antibody response when helper T cells are limited. It is suggested that B' cells deliver an additional signal to the B cell network to magnify the antibody response.