Avidities of hapten-specific antibodies when the responses are modulated by anti-carrier antibodies.

Either alone or in combination with antibodies having specificity for the carrier erythrocyte, TNP-ORBC were injected i.p. into CBA/J mice. Five days later, their spleens were removed and evaluated for TNP-specific PFC. The spleens from animals receiving 19S antibody (IgM) with carrier specificity displayed 3-4-fold more direct and indirect hapten-specific PFC than control animals receiving the TNP-erythrocyte conjugate only. Animals receiving 7S antibodies (IgG) with carrier specificity displayed very little change in their direct PFC response to the hapten. However, the indirect response to the hapten was suppressed as much as 16-fold by carrier specific IgG. Evaluation by haptenic inhibition of the relative avidity of the antibodies being secreted by these cells revealed the following: the relative avidity of antibodies secreted by indirect PFC in the spleens of animals receiving TNP-ORBC only was approximately 20-fold higher than antibodies secreted by the direct PFC. The 3-4-fold potentiation of the hapten-specific PFC response by carrier-specific IgM antibody did not result in a change in relative avidity of direct or indirect PFC. IgG with carrier specificity did not change the relative avidity of the antibodies secreted by direct PFC having specificity for the hapten. However, evaluation of the remaining PFC in spleens from animals whose indirect hapten-specific PFC response had been suppressed by carrier-specific IgG revealed that the remaining PFC had a lower avidity than indirect PFC from animals receiving TNP-ORBC only. In other words, carrier-specific IgG selectively induced suppression of high avidity hapten-specific IgG antibody secreting cells.     

Affinity of T and B lymphocyte receptors for hapten determinants

The affinity of anti-hapten antibodies, produced by single cells, was studied by hapten inhibition of plaque-forming cells using the local hemolysis in gel assay. The shift in affinity of anti-hapten antibodies, that occurs with time after immunization, was parallelled by an analogous shift, at the cellular level, with regard to indirect plaque-forming cells (PFC). Thus, there was a gradual decrease of the hapten concentration which was needed to suppress indirect plaque formation with time after immunization, indicating a gradual increase in the number of high affinity cells. No such shift was observed with cells causing direct plaque formation. Analogous studies were performed with hapten-specific antigen-binding cells of T and B origin by using hapten-inhibition of rosette-forming cells. Hapten-specific antigen-binding cells were present in both cell populations. Lymphocyte binding of haptenated red cells could be specifically inhibited by free hapten. It was found that antigen-binding B lymphoid cells became gradually more susceptible to hapten inhibition with time after immunization, whereas no such change was observed in antigen-binding T lymphoid cells. Possible reasons for this discrepancy are discussed, as well as the implications for these findings regarding the specificity and structure of the T cell receptors.     

CD8+ T cells produce IL-2, which is required for CD(4+)CD25+ T cell regulation of effector CD8+ T cell development for contact hypersensitivity responses

Interleukin (IL)-2 functions to promote, as well as down-regulate, expansion of antigen-reactive CD4+ and CD8+ T cells, but the role of IL-2 in hapten-specific CD8+ T cell priming for contact hypersensitivity (CHS) responses remains untested. Using enzyme-linked immunospot to enumerate numbers of hapten-specific CD4+ and CD8+ T cells producing IL-2 in hapten-sensitized mice, the number of IL-2-producing CD8+ T cells was tenfold that of CD4+ T cells. Hapten-primed CD4+ T cells produced low amounts of IL-2 during culture with hapten-presenting Langerhans cells, whereas production by hapten-primed CD8+ T cells was fivefold greater. CD8+ T cells did not express CD25 during hapten priming, but treatment with anti-IL-2 or anti-CD25 monoclonal antibodies during hapten sensitization increased hapten-specific effector CD8+ T cells as well as the magnitude and duration of the CHS response. These results indicate that CD8+ T cells are the primary source of IL-2 and that this IL-2 is required for the function of a population of CD(4+)CD25+ T cells to restrict the development of the hapten-reactive effector CD8+ T cells that mediate CHS responses.     

Carrier and hapten functions in immune deviation. I. Contrary effects of hapten and carrier on cellular and helper functions of T cells.

Carrier and hapten functions have been studied in the immune deviation phenomenon. Delayed hypersensitivity to the carrier and anaphylaxis and Arthus hypersensitivities to the hapten and to the carrier were studied in guinea-pigs injected intravenously with large doses of carrier, homologous and heterologous hapten-carrier conjugates and subsequently immunized with the hapten-carrier conjugate in Freund's complete adjuvant. Pretreatment with DNP-BSA or with HGG were found to modify, in opposite directions, the hypersensitivity reactions induced by DNP-HGG in adjuvant. It is suggested that the hapten and carrier moieties of the antigen molecule might have antagonistic effects on the T cells responsible for cellular immunity as well as on T cells involved in helper functions for B cells.     

Regulation of Carrier-Specific Response via a Haptenic Epitope

Influences of haptenic epitopes on the regulation ofcarrier-specific responses are of importance in vaccination and heterogenization protocols. This situation was modelled by analysing the primary and secondary anti-horse red blood cell (HRBC) response under the influence of a haptenic epilope (trinitrophenol. TNP). Primary and secondary anti-HRBC responses were diminished in the presence of TNP. The primary response against the carrier was delayed in the presence of TNP and primary and secondary anti-carrier responses vanished more rapidly in response to TNP-HRBC than in response to HRBC. When frequencies of HRBC-specific B cells were determined under limiting dilution (LD) conditions, a lower frequency of HRBC-speeific B cells was discovered in the presence of TNP-HRBC as compared lo HRBC. Accordingly, after priming with HRBC, the frequency of B cells was increased by a factor of 49, while after priming with TNP-HRBC the frequency was only increased by a factor of 11. Furthermore, haptenic epitopes influenced the anti-carrier response via regulatory elements. Under physiological conditions, hapten-specific help was insufficient and hapten-specific B cells competed for earrier-specific helper T cells (T_H). Suppression was more efficient in hapten-carrier-primed mice than in carrier-primed mice and in line with this observation, an increased frequency of suppressor T cells (T_s) was observed in LD cultures when TNP-HRBC instead of HRBC were used as antigen. It is concluded that haptenic epitopes are relevant for the anti-carrier response, since hapten-specific B cells competc for carrier-specific help, hapten-specific suppression down-regulates carrier-specific help, and the hapten may hide immunogenie epitopes of the carrier, resulting in incomplete activation of the B-cell repertoire.     

The specificity of antibody formation in mice following immunization with hapten-carrier complexes mixed with the surfactant, dimethyl dioctadecyl ammonium bromide.

Mice were immunized i.c. with various enlarged haptens conjugated to bovine serum albumin and mixed with the cationic, surface active lipid, dimethyl dioctadecyl ammonium bromide (DDA). This immunization generated delayed-type hypersensitivity (DH) in these mice without detectable concomitant antibody formation. The DH was measured as footpad swelling. However, both direct and indirect hapten-specific PFC could be detected in peripheral lymph nodes and in spleens 4 days after a challenge injection. Although the adjuvant, DDA, promotes a strong cross-reactivity in DH between heterologous hapten-carrier complexes, the antibody-forming cells produced 4 days after challenge showed relatively high specificity for the immunizing hapten. This indicates only weak cross-reactivity at the antibody-forming cell level co-existent with high cross-reactivity in DH expression to the same hapten-carrier complexes. These results are consistent with the possibility that B-cell receptors may be capable of expressing a greater degree of hapten specificity than T-cell receptors. It is tentatively concluded that Thelper cells participating in antibody formation may represent a subset of the T cells involved in DH.     

Carrier and hapten functions in immune deviation. II. Role of hapten and carrier on the helper functions of the T cells.

The kinetics of haemagglutinating and haemolytic antibody synthesis to the hapten and to the carrier determinants were studied in guinea-pigs injected intravenously with large doses of the carrier, or of the hapten conjugated to an homogous or heterologous protein carrier and subsquently immunized with the hapten-carrier conjugate in Freund's complete adjuvant. The animals treated with the heterologous conjugates exhibited enhanced reactions to the hapten and supressed reactions to the carrier, whereas the animals injected with the homologous conjugate showed depressed reactions to the hapten but unaffected reactions to the carrier. Pretreatment with the carrier alone-seemed to have no effect. These experiments confirm that the hapten determinant must act at the T-cell level but do not exclude the possibility that it could also act at the B-cell level. On the other hand, they allow the dissociation of the different components of the immune response directed against the hapten and carrier determinants of the antigen molecule in immune deviation.     

Cell co-operation and hapten--carrier complexes.

The co-operation of spleen cells of carrier- and hapten--carrier-primed mice in antibody formation against the hapten part of complexes was studied in 550 rad whole body irradiated mice. Hapten--carrier complexes were prepared with the 2,4-dinitrophenyl group (DNP) as a hapten and heterologous bovine serum albumin (BSA) and isologous mouse immunoglobulin (MIg) as carriers. Priming of donor mice with carrier alone did not prepare for a secondary (IgG) response in the recipients of hapten--carrier. Priming of donors and challenge of recipients with the same hapten--carrier complex resulted in high IgG responses. Whereas donor and recipient immunization with complexes differing in the carrier did not give a secondary response, addition of cells of donors immunized with the carrier of the complex used for challenge, resulted in a secondary response. This was only possible when at least one of the complexes had an intermediate hapten:carrier ratio. Only an IgM or a low IgG response was obtained if both complexes had a high hapten:carrier ratio. Three determinants, namely hapten and carrier groups and new antigenic determinant (NAD), are suggested for antibody formation against hapten--protein complexes. In vivo treatment of donor cells with anti-thymocyte serum (ATS) or anti-plasma cell serum (APCS) and complement (C) suggested that: (1) T-cell epitopes are present on the carrier; (2) DNP groups are B-cell epitopes; (3) NAD and possibly DNP are T-cell epitopes; (4) synergism exists in the collaborative antibody response of B cells recongnizing DNP, T cell recognizing carrier and T cells recognizing NAD. Mitomycin treatment of donor cells was used to test whether cell division was mandatory. While the B cells were sensitive to mitomycin treatment, no effect of this drug was found on the helper activity of T cells.     

Functional Characterization of Hapten-Specific Suppressor Cells

T cells from animals suppressed against a given hapten, trinitrophenyl (TNP), were analysed for their capacity to inhibit the humoral B-cell response against the hapten and/or a complex antigen, horse erythrocytes (HRBC), coupled or not to the hapten. As expected, suppression of the response to HRBC in all experiments required coupling to TNP. However, the suppressing capacity of the T cells varied with the stage of the B cells, with no detectable suppression occurring if already primed carrier-specific B cells were used. Hapten-specific T helper cells could, however, be induced in hapten-suppressed mice, when hapten-carrier conjugates were used as immunogen. Under these conditions normal efficiency of induction of carrier-specific T helper cells was observed, and this probably also applied to hapten-specific T helper cells to the same extent. This assumption was strengthened by using the hapten-binding capacity of the suppressor T cells and Lyt-1/2-specific sera to subdivide physically helper and suppressor function.     

Anti-DNP antibody response after the topical application of DNFB in mice.

A single painting or daily paintings for 5 days with dinitrofluorobenzene (DNFB) on the abdominal skin of mice induced both contact sensitivity, detectable by ear swelling, and, hapten-reactive helper T cells, detectable by the augmented anti-bovine serum albumin (BSA) antibody response on challenge with dinitrophenyl-BSA. Contact sensitivity was induced within 7 days and helper activity within 14 days after the sensitization. Anti-hapten antibody response in the spleen or regional lymph nodes of such mice, however, was negligibly small during the 15 days after a single painting. Failure to respond with anti-hapten antibody production of mice given only a single painting was shown to be due to the shortage of B cells reactive to the hapten. Daily paintings for 5 days did not necessarily result in the augmented antibody response. By contrast, a strong anti-hapten antibody response was observed in mice receiving two paintings at an interval of 10 days. In these mice, hapten-specific B memory cells as well as hapten-reactive T cells were detected. Thus, the anti-hapten antibody response after the topical applciation of simple chemicals may depend upon the priming of B cells, and the response must be mediated by the cooperation of T and B cells both reactive to the antigen.     

Characterization of the homo- and heterotypic immune responses after natural norovirus infection

Noroviruses (NoV) are a genetically and antigenically diverse group of viruses that are common causes of outbreaks of gastroenteritis in humans of all ages. Limited information has been obtained on type specificity of the NoV immune response. In this study, we characterized the homologous and heterologous antibody responses in adults from 13 outbreaks, representing 4 different NoV genotypes. NoV specific IgG and IgA antibodies were determined as well as the increase of antibody avidity. In addition, antibody-mediated blocking of NoV binding to its putative receptor was evaluated. Both homologous and heterologous serological responses were detected after NoV infection. The avidity of antibodies could not be used to distinguish between homologous and heterologous antibody responses. However, a homologous blocking response but not a heterologous response was detected after infection with NoV belonging to genogroup II.4 by a NoV ligand binding inhibition assay. Infection with NoV induces antibodies that can block virus ligand interactions. In contrast with all currently known antibody detection assays for NoV, this can be used as a type specific assay and may be an alternative for studying neutralizing antibodies.     

A capture enzyme-linked immunosorbent assay for species-specific detection of Bothrops venoms

A direct sandwich enzyme-linked immunosorbent assay (ELISA), employing affinity purified antivenom antibodies specifically recognizing the homologous venom, was developed for species-specific detection of bothropic venom. The method is based on a two-step affinity purification of the specific antibodies. A species monovalent antivenom is adsorbed onto a venom adsorbent containing heterologous venoms from the Bothrops, Crotalus and Lachesis genera. The species-specific antibodies obtained, are then adsorbed onto a second venom adsorbent containing only the homologous venom for the removal of non antivenom antibodies. Venom concentrations of 0.1 and 1,000 ng/ml were specifically identified for Bothrops jararacussu and B. alternatus venom respectively.     

Hapten-specific helper T cells. I. Collaboration with B cells to which the hapten has been directly coupled

A new system to obtain large numbers of functionally competent hapten-specific effector helper T cells has been developed. Mice were primed in the tail with syngeneic spleen cells derivatized with one of three different haptens: 2,4,6-trinitrophenyl, fluorescein isothiocyanate and 3-(p-sulfophenyldiazo)-4-hydroxyphenyl. Draining lymph node cells were subsequently restimulated in culture with the homologous antigen for periods up to 6 weeks. More than 99% and 90% of the cells recovered from these preparative cultures, expressed Thy-1 and Lyt-1 antigens, respectively. These cells proliferated specifically in response to the homologous stimulation by haptenated syngeneic spleen cells, and they were found to display very high levels of hapten-specific helper activity, as assayed in a new, universal method for testing T - B cell cooperation, where every B cell can potentially respond to specific T cell help. Coculture of hapten-derivatized, functionally competent B cells with hapten-specific T cells obtained from the preparative cultures, resulted in the appearance, and exponential increase with time, of very large numbers of IgM and IgG plaque-forming cells. This helper activity could be shown to be: specific for the immunizing hapten and to increase with the time of in vitro helper cell enrichment, which also resulted in higher average avidities expressed by the helper cell populations. This methodology appears valuable for structural studies on hapten-specific helper cells, as well as for the functional analysis of effector helper - B cell interactions.     

High-level expression of B7-H1 molecules by dendritic cells suppresses the function of activated T cells and desensitizes allergen-primed animals

A body of evidence indicates that expression of the programmed cell death 1 (PD-1) receptor by activated T cells plays an important role in the down-regulation of immune responses; however, the functions of its known ligands, B7-H1 (PD-L1) and B7-dendritic cell (DC; PD-L2), at the effector phase of immune responses are less clear. In the current study, we investigated the roles of B7-H1 in DC-mediated regulation of hapten-activated T cells and the delayed-type contact hypersensitivity response in primed animals. We found that the expression of B7-H1 and B7-DC was induced on activation of DC by hapten stimulation. Blockade of B7-H1, but not B7-DC, enhanced the activity of hapten-specific T cells. Interaction with a DC line that expresses high cell-surface levels of B7-H1 (B7-H1/DC) suppressed the proliferation of, and cytokine production by, activated T cells. In vivo administration of hapten-carrying B7-H1/DC desensitized the response of sensitized animals to hapten challenge, and this desensitization was hapten-specific. These data indicate that B7-H1 expressed by DC mediates inhibitory signals for activated T cells and suppresses the elicitation of immune responses. The ability of B7-H1/DC to inhibit the function of preactivated T cells in vivo suggests novel strategies for the treatment of immune response-mediated disorders.     

Effect of carrier priming on antibody avidity in the in vivo and in vitro immune response.

The effect of carrier priming on antibody avidity was investigated under several experimental conditions. Basically, mice were carrier primed with HRBC (horse red blood cells) prior to immunization with TNP (2,4,6-trinitrophenyl) conjugated to HRBC. Immunization was performed either in vivo or in spleen cell culture, and avidity of anti-TNP antibodies was estimated from inhibition of direct PFC (plaque-forming cells) by free TNP-BSA (-bovine serum albumin).The data indicate the appropriate conditions under which carrier priming can enhance antibody avidity. The carrier effect is maximized by priming the animals with 10⁴-10⁵ HRBC 3-7 days before immunization with a low dose of TNP-HRBC. Hyper-immunization by repeated injections of a high dose of the conjugate does not modify the carrier effect on avidity but it delays the fall of avidity in both carrier primed and unprimed animals. These results are interpreted in terms of T- and B-cell co-operation within the framework of the maturation theory of antibody affinity.Carrier priming was also found to increase the number of direct PFC of the IgM and, mostly, of the non-IgM classes, a finding in agreement with the notion that T cells can help IgM production and the shift to IgG.     

Increased production of antigen-specific B lymphocytes during in vitro immunization using carrier-specific T helper hybridomas.

An in vitro method to increase the production of hapten-specific antibody-forming B cells (AFC) using a carrier-specific T helper hybridoma and murine splenocytes is described. Naive splenocytes (6 x 10(6)/ml) are cultured in vitro in the presence of a hapten-carrier conjugate (DNP.OVA) and OVA-specific T helper hybridomas (0.5 x 10(6)/ml). After 4-5 days in vitro immunization (IVI), the maximum number of DNP-specific AFC were found using a spot-ELISA with twice the number of IgM positive cells as IgG positive AFC. The presence of antigen in the form of a hapten-carrier complex and the use of a carrier-specific Th hybridoma resulted in more hapten-specific AFC than when neither antigen nor Th hybridoma were present or when antigen alone or T help alone were used. Also when the hapten was conjugated to a carrier not recognised by the carrier-specific Th hybridoma there were considerably fewer (less than 50%) hapten specific AFC formed. When in vivo primed splenocytes (DNP) were boosted in vitro (IVB) under the same conditions as for IVI most hapten-specific AFC were found on day 4 and both anti-DNP IgM and IgG AFC were increased relative to IVI. Again most AFC were found when hapten was bound to the relevant carrier. In conclusion, carrier-specific T hybridomas can be used in an in vitro immunization procedure with naive or primed splenocytes to increase the frequency of anti-hapten AFC. This method offers an improvement over the current in vitro immunization procedures for the production of monoclonal antibodies.     

T cell suppression in vitro. II. Nature of specific suppressive factor

The mechanism of specific T cell suppression in vitro was investigated. It was found that suppression of hapten-reactive B cells only occurred in the presence of hapten linked to the carrier to which the T cells were reacting. Thus, there was the same requirement for linked recognition in T cell suppression as was previously described in cooperation. The nature of the suppressive factor was investigated, and like the cooperating factor, it was found to have specificity for the immunizing carrier protein and to be fully absorbed by peritoneal macrophages. The ratio of helper to suppressor activity obtained from activated T cells did not change between 4 and 8 days after injection of antigen and thymocytes. Both activities were absorbed by Sepharose beads conjugated with polyvalent anti-mouse Ig, anti-? or anti-μ-chain antibody. There was thus a resemblance between the T cell suppressive factor and the immunizing factor, although it is not yet known whether both properties are expressed by one and the same molecule. This must await further biochemical characterization of specific T cell factors.     

Age-related changes in antibody repertoire: contribution from T cells

Summary: The immune system of aged mice produces antibodies that are characterized by low affinity, diminished protection against infections and autoreactivity. It has been shown that these antibodies may be encoded by different immunoglobulin V genes and that the mechanism of somatic hypermutation in the V genes is inefficient. Studies on scid mice reconstituted with B and T cells from donors of different ages suggested that both lymphocyte subsets may contribute to the age-related changes in antibody repertoire. With help provided by T cells from young mice, the response to a hapten, nitrophenyl(acetyl), became gradually dominated by B-cell clones that rearranged a particular germline V_H gene (V186.2). However, help from the aged T cells resulted in A heterogeneous response of B cells expressing many different V segments. Analysis of discrete foci of primary antibody-forming cells suggested that the aged T-helper cells are unable to govern the normally-occurring competition between the B-cell clones that have different affinities for the hapten. It is proposed that a signaling disequilibrium from the aged T cells, which provide less efficient help in quantitative terms, supports the growth of low-affinity B cells. This process may be exacerbated due to the apparent hyperactivity of aged B cells to CD40-mediated mitogenic signal.     

Affinity-based selection and the germinal center response

Interactions between B-cell antigen receptors (BCRs) and their ligands have a complexity and variability that is unparalleled within known biology. Each developing B cell undergoes gene rearrangements to generate a BCR encoded by a unique pair of immunoglobulin (Ig) variable region genes, which serves to make the antigen-binding capabilities of primary BCRs incredibly diverse. Further diversification of the BCR repertoire takes place when antigen-activated B cells enter the germinal center (GC) response and undergo somatic hypermutation (SHM) of their Ig variable region genes. To develop optimal antibody responses against foreign antigens, the key B-cell survival and differentiation decisions made in the GC are based primarily on the affinity of the BCR (and therefore subsequent antibodies) for foreign antigen. However, the secondary diversification of BCRs by SHM also carries the risk of generating new self-reactive specificities and thus autoantibody production. Herein, we review the role of antigen affinity/avidity in controlling pivotal events both leading up to and during the GC response. The emergence of self-reactivity during the GC response is also examined, with particular focus on the threat posed by cross-reactive GC B cells that bind both self and foreign antigen.