Lectin-induced regulation of human thymus-independent anti-TNP responses in vitro

Recently we showed that human hapten-specific T cell-independent (TI) antibody responses could be elicited in vitro with the antigen trinitrophenylated Brucella abortus (TNP-Ba). Although by definition T cells are not required for TI responses, they have also shown capable of regulating such responses in the mouse. In this study we examine the ability of the T cell lectins Con A and PHA to modulate TI responses of human tonsil cells. Addition of the lectins to cultures on day 0 or day 3 resulted in inhibition (greater than 90%) or enhancement (greater than 150%) of the anti-TNP PFC response, respectively. The inhibition was only apparent if the lectins were added at the onset of culture but not 24 hr later, and was T cell-dependent, because T cell-depleted cultures (less than 0.5% E-rosetting cells) could not be inhibited. The enhancement observed was even greater in T cell-depleted cultures and was antigen-specific because TNP-SRBC but not PC-SRBC targets were lysed and the PFC were inhibited by soluble TNP (greater than 70%). This finding suggested that anti-TNP PFC precursors were proliferating more vigorously or that additional, previously antigen-insensitive cells were being recruited in the presence of lectin.     

The role of nonactivated and interferon-gamma activated monocytes in regulating normal and SLE patient B cell responses to TNP-Brucella abortus

We have previously characterized the human B cell response to trinitrophenol (TNP)-Brucella abortus (Ba) response as being T cell independent. In this report we examine the role of monocytes in the TNP-Ba antibody response of human peripheral blood mononuclear cells (PBMC). Depletion of monocytes by sequential adherence to plastic and Sephadex G-10 passage did not result in decreased plaque-forming cell responses to TNP-Ba, suggesting that monocytes were not required. On the contrary monocytes were probably inhibitory because their removal resulted in enhanced responses. This was confirmed by showing that adding monocytes back reconstituted the inhibition. When interferon-gamma (IFN-gamma), a potent activator of monocytes, was added to TNP-Ba-driven PBMC cultures, marked inhibition (greater than 90%) of the responses ensued. This IFN-gamma-mediated suppression was monocyte dependent because it was completely abrogated by monocyte, but not T cell depletion. Previously, we described a concanavalin A (Con A), T cell inhibition pathway of the TNP-Ba response. Both the Con A and IFN-gamma pathways were tested for their ability to inhibit systemic lupus erythematosus (SLE) patient responses to TNP-Ba. The B cell response of SLE patients was inhibitable by both pathways. In all of the patients, the inhibition was complete (greater than 95%) when IFN-gamma was added to the cultures. In the presence of Con A, greater than 95% inhibition was observed in six of 10 patients, the remainder being inhibited to a lesser extent. Thus the hyperactive B cells from SLE patients can be down-regulated, particularly in the presence of IFN-gamma.     

Newborn and Wiskott-Aldrich patient B cells can be activated by TNP-Brucella abortus: evidence that TNP-Brucella abortus behaves as a T-independent type 1 antigen in humans

TNP-Brucella abortus (TNP-Ba) has been classified as a T-independent type 1 (TI-1) antigen in the mouse on the basis that it activates neonatal and CBA/N (X-linked immunodeficient) murine B cells in contrast to T-independent type 2 (TI-2) antigens. Therefore, it was of interest to determine whether human newborn and X-linked Wiskott-Aldrich syndrome B cells could be triggered by TNP-Ba. Previous studies had shown that human B cells from both these latter sources were relatively insensitive to stimulation with T-dependent and polysaccharide antigens (TI-2 in mouse). In this study, we show that TNP-Ba can trigger human cord blood B cells to differentiate into anti-TNP plaque-forming cells (PFC) in a hapten-specific and T-independent manner. The dose response and kinetics were similar to those previously seen with adult cells. The newborn responses, however, were lower than adult PFC responses. Precursor frequency and clone size analyses revealed that this lower response was not due to newborn cells containing fewer precursors but was the result of a reduced ability of these anti-TNP clones to expand. The ability of TNP-Ba to activate immature newborn B cells implies that this antigen can be used to assess B cell function in very young children. It also implies that TNP-Ba behaves as a TI-1 antigen in humans as well as in mice. This was supported by the finding that B cells from Wiskott-Aldrich patients, which were unreactive to polysaccharide antigens, were generally responsive to TNP-Ba. Therefore, it would appear that human newborn and Wiskott-Aldrich patients do possess a functionally competent B cell subset possibly equivalent to Lyb-5- immature murine B cells.     

Concanavalin A supernatant recruits antigen-insensitive IgG memory B lymphocyte precursors into an antigen-sensitive precursor pool

Spleen cells from mice primed to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) generate IgG anti-TNP memory responses when stimulated in vitro with either thymus-dependent (TD) or thymus-independent (TI) forms of the hapten. When supernatants from Con A-stimulated spleen cells (Con A Sup) were added to such secondary cultures the TI responses to DNP-dextran or TNP-T4 were augmented; the TD response to TNP-KLH was suppressed. Passage over Sephadex and addition of alpha-methyl-D-mannoside did not inhibit augmentation by Con A Sup, indicating that augmentation did not result from direct action of the lectin on the responding cells. Augmentation occurred equally well in cultures that had been depleted of T cells by treatment with anti-Thy-1.2 and complement. Limiting dilution analyses revealed that Con A Sup increased the frequency of TI-responding precursors approximately threefold while causing a concomitant decrease in TD-responding precursors. To determine the relationship of the additional TI precursors and those normally detected in the absence of Con A Sup, the TI-responding IgG precursors were first eliminated through selective suicide by using DNP-dextran plus BUdR and light treatment; subsequently no TI-responding IgG PFC could be detected to DNP-dextran unless Con A Sup was also added. The data suggest Con A Sup may augment the TI responses to DNP-dextran and TNP-T4 by recruiting additional precursors from a memory cell pool formerly insensitive to these forms of antigen.     

Characterization of a concanavalin A supernatant-derived idiotype-specific T helper cell factor

We have previously demonstrated the requirement of two T helper (Th) populations for the expression of plaque-forming cells (PFC) that bear the dominant cross-reactive idiotype (CRI) associated with the phenyltrimethylammonium (TMA) response (1). In addition to the classic major histocompatibility complex-restricted Th cell, the response was also dependent upon the so-called second order Th2 population, which binds to idiotypic determinants, is carrier specific, but does not require hapten linked to carrier for function. This cell type can be replaced by supernatant (Sn) media from concanavalin A (Con A)-stimulated naive spleen cells. This report involves the study of the Con A Sn derived factor(s) responsible for the expression of CRI bearing PFC populations. When the Brucella abortus (BA)-trinitrophenol (TNP) conjugated antigen is added to TNP-ovalbumin-primed A/J-derived spleen cells in culture, anti-TNP PFC are generated of which only less than or equal to 5% bear the CRI normally associated with anti-TMA antibodies. Upon addition of Con A Sn, the total number of generated anti-TNP PFC doubles, whereas the percentage and number of CRI+ PFC increases approximately eightfold to 10-fold. The factor(s) responsible for this activity are T cell derived, bear Jk serologic determinants, and can be detected in the Sn as early as 4 hr after Con A stimulation. The material appears to be late acting, because it can augment the CRI+ anti-TNP response when added as late as 24 hr before termination of the cultures. In addition, the factor(s) can be bound to and eluted from CRI+ anti-TMA and anti-TNP monoclonal antibodies coupled to Sepharose 4B beads, but not from their CRI- counterparts (i.e., CRI- anti-TMA and anti-TNP antibodies), nor from A/J normal mouse immunoglobulin-coupled beads. Most interestingly, the factor(s) also bind to and can be eluted from the TMA ligand coupled to Sepharose 4B, but not from TNP-Sepharose conjugates. All of these results are consistent with the support the contention that the factor(s) is derived from a Th2-like subpopulation. As assayed by standard protocols, the isolated material contains no T cell replacing factor, interleukin 2, or B cell growth factor activity.     

Specificity,avidity, and size of B-lymphocyte populations during ontogeny

The size and specificity of plaque-forming cell precursors (PFC) in murine fetal liver, neonatal, and adult spleen were studied in an adoptive transfer system. In this system, anti-4-hydroxy-3-iodo-5-nitrophenylacetic acid (anti-NIP) and anti-2,4,6-trinitrobenzene sulfonic acid (anti-TNP) direct PFC are generated from bone marrow-derived (B) cell precursors in fetal liver between 17 and 20 days of gestation and in 6- or 14-day neonatal spleen. PFC generated from fetal liver and neonatal and adult spleen cells are specific in that they lyse either NIP-coupled SRBC or TNP-coupled SRBC but not both. The generation of specific anti-NIP and anti-TNP PFC from precursors in fetal liver is primarily independent of antigenic stimulation. In contrast, the anti-NIP and anti-TNP responses generated from neonatal and adult spleen are antigen dependent. Both high-avidity PFC (detected with SRBC indicators coupled at low hapten density) and low-avidity PFC (detected with SRBC coupled at high hapten density) are generated from fetal liver and neonatal spleen cells; however, the proportion of high-avidity PFC precursors in adult spleen is at least threefold greater than in fetal liver or neonatal spleen. Analysis by velocity sedimentation indicates that most high-avidity PFC precursors are small lymphocytes in fetal liver, medium lymphocytes in 6-day neonatal spleen, and small lymphocytes in 14-day-old and adult spleen. Low-avidity PFC precursors are primarily medium-sized lymphocytes in fetal liver and 6-day neonatal spleen. In 14-day-old and adult spleen almost all high- and low-avidity PFC precursors are small lymphocytes. The results are discussed in terms of relative changes in the pool sizes of these lymphocyte populations.     

Production of auto-anti-idiotype antibody during the normal immune response. X. Response to TNP-Ficoll in the chicken

The spontaneous production of auto-anti-idiotype (Id) was demonstrated after injection of chickens with trinitrophenylated Ficoll (TNP-F) by: (a) the presence of hapten-augmentable plaque-forming cells (PFC), (b) the ability of serum and of hapten eluates from immune spleen cells to cause hapten-reversible inhibition of anti-TNP plaque formation, and (c) an enzyme-linked immunosorbent assay (ELISA). Tests for anti-Id using the ELISA and hapten-reversible inhibition of PFC correlated very well. As in the mouse, the incidence of hapten-augmentable PFC was reduced by thymectomy and increased by the transfer of TNP-F-immune spleen cells. Hapten-augmentable PFC were also observed during the immune response of chickens to p-azobenzene arsonate-conjugated Brucella abortus.     

Modulation of mouse anti-trinitrophenyl plaque-forming cell affinity by adjuvants or lectins.

The efffects of several kinds of adjuvants or lectins, such as Corynebacterium parvum, dextran, poly AU, poly IC, dibutyryl cAMP, concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. The numbers of anti-TNP PFC in the spleens of mice which had been injected with C. parvum 7 days in advance were greater than those in controls after immunization with TNP-coupled heterologous erythrocytes, while the affinity of antibodies released by these PFC. Copolymers of nucleotides, poly AU and poly IC, were capable of enhancing splenic anti-TNP PFC responses, but showed almost no altering of PFC affinity. Dibutyryl cAMP did not have any effect on this system. Con A had potencies to both augment the number of anti-TNP PFC and heighten the PFC affinity, while PHA seemed to lack these potencies. Injection of PWM in the presence of antigen increased the number of anti-TNP PFC and heightened slightly the PFC affinity. These results indicate that the heightening of the affinity at the cellular level is regulated in ways different from the augmenting effects on the number of anti-TNP PFC by adjuvants or lectins. These results are discussed in the light of the mode of action of the substances used.     

Selective suppression by auto-anti-idiotypic antibody of B-cell idiotype repertoires generated after stimulation with the same hapten on T-dependent and T-independent carriers

In the present study, we investigated whether auto-anti-idiotypic antibody in the immune sera from old mice could recognize antitrinitrophenyl (TNP) plaque-forming cells (PFC) generated after stimulation with the T-dependent and T-independent forms of the hapten, TNP. Young and old C57BL/6J male mice were immunized with a variety of T-dependent (TNP-bovine gamma-globulin, TNP-BGG; TNP-keyhole Limpet hemocyanin, TNP-KLH; ovalbumin, OVA; bovine serum albumin, BSA; BGG) and T-independent (TNP-Brucella abortus, TNP-BA; TBP-Ficoll; TNP-polyacrylamide beads, TNP-PAA) antigens either in complete Freund's adjuvant (CFA) or in soluble form. Splenic anti-TNP or antiprotein PFC responses were assayed for anti-idiotype-blocked, hapten- or protein-augmentable IgM, IgG and IgA PFC, 1-2 weeks after immunization. It was found that 8-month-old mice produced significantly a higher percentage of hapten augmentable (26-42%) IgM PFC response to T-independent antigens as compared with the 2-month-old mice (3-6% augmentation). Similarly, old mice produced a significantly higher percentage of hapten or protein augmentable (25-129%) IgG PFC response to T-dependent antigens as compared with the 2-month-old group (2-6% augmentation). The data support the view that age-related regulation of auto-anti-idiotypic antibody is a general phenomenon for immune responses to T-dependent and T-independent antigens. Hapten-reversible inhibition of plaque formation was used to determine whether anti-idiotypic antibody containing antisera from old mice could inhibit B-cell idiotype repertoires generated after stimulation with the same hapten, TNP, on T-dependent and T-independent carriers. Pools of immune sera from 8-month-old mice primed with T-dependent TNP-BGG or TNP-KLH antigens but not with T-independent TNP-PAA or TNP-BA antigens, or with the proteins OVA, BSA, or BGG selectively inhibited IgM, IgG, and IgA anti-TNP PFC from 2-month-old mice that were previously primed with either TNP-BGG or TNP-KLH. In contrast, immune sera from old mice primed with TNP on either T-dependent or T-independent carriers inhibited anti-TNP PFC from mice primed with T-independent TNP-PAA or TNP-BA antigens. Immune sera from old mice primed with OVA or BSA only inhibited the respective antiprotein PFC. The immune sera from young mice did not show any appreciable inhibition of PFC generated after stimulation by any of the antigens studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of Mouse Anti-Trinitrophenyl Plaque-Forming Cell Affinity by Adjuvants or Lectins

The effects of several kinds of adjuvants or lectins, such as Corynebacterium parvum, dextran, poly AU, poly IC, dibutyryl cAMP, concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. The numbers of anti-TNP PFC in the spleens of mice which had been injected with C. parvum 7 days in advance were greater than those in controls after immunization with TNP-coupled heterologous erythrocytes, while the affinity of antibodies released by these PFC was not affected. On the contrary, simultaneous injection of dextran with TNP-erythrocytes did not increase the numbers of splenic anti-TNP PFC, but heightened the affinity of antibodies released by these PFC. Copolymers of nucleotides, poly AU and poly IC, were capable of enhancing splenic anti-TNP PFC responses, but showed almost no altering of PFC affinity. Dibutyryl cAMP did not have any effect on this system. Con A had potencies to both augment the number of anti-TNP PFC and heighten the PFC affinity, while PHA seemed to lack these potencies. Injection of PWM in the presence of antigen increased the number of anti-TNP PFC and heightened slightly the PFC affinity. These results indicate that the heightening of the affinity at the cellular level is regulated in ways different from the augmenting effects on the number of anti-TNP PFC by adjuvants or lectins. These results are discussed in the light of the mode of action of the substances used.     

Production of auto-anti-idiotypic antibody during the normal immune response. XII. Enhanced auto-anti-idiotypic antibody production as a mechanism for apparent B-cell tolerance in rabbits after feeding antigen

Rabbits fed trinitrophenylated bovine serum albumin (TNP-BSA) generated fewer anti-TNP plaque-forming cells but greater numbers of hapten (TNP)-augmentable IgM and IgG PFC following immunization with TNP-Ficoll or TNP-Brucella abortus than did animals not previously fed antigen. Spleen and mesenteric and bronchial lymph nodes were similarly affected. In addition more auto-anti-idiotype (Id) antibody (anti-anti-TNP) was eluted by hapten from spleen cells of antigen-fed rabbits than from spleen cells of control rabbits not prefed antigen. Gel filtration studies ruled out the possibility that the Id binding activity in the eluates was due to immune complexes. The isotype of the anti-Id was IgG except in one rabbit where it was IgM. The results are consistent with the interpretation that the production of auto-anti-Id antibody is one of the factors responsible for the specific depression of the IgM and IgG immune responses which follows antigen feeding. In contrast the antigen feeding resulted in priming for an IgA anti-TNP response without detectable hapten-augmentable IgA PFC.     

Priming of peripheral lymph node B cells with TNP-Ficoll: role of lymphokines in B cell differentiation.

We have previously shown that peripheral lymph node (PLN) B lymphocytes of adult DBA/2J mice failed to make an antibody response to type 2 antigen TNP-Ficoll, but exhibited a good antibody response to type 1 antigen TNP-Brucella abortus. In the present study we wanted to find out whether the unresponsiveness of PLN B cells to TNP-Ficoll is due to defects in the early activation and proliferation stage or in the final differentiation stage of B cells. Therefore, we have used a two-step protocol of in vivo immunization of mice with TNP-Ficoll and the subsequent in vitro challenge with TNP-Brucella abortus and studied the anti-TNP plaque-forming cell (PFC) responses. The results indicate a three- to sixfold increase of PFC responses in PLN cell cultures derived from TNP-Ficoll-primed animals compared to saline control mice. This increased antibody response was TNP-specific as 93% of the PFC's were inhibited by TNP-lysine. Limiting dilution experiments confirm that the increase in anti-TNP PFC response from the TNP-Ficoll-primed animals was indeed due to an increase in TNP-specific precursor B cells. Further, the addition of rIL-5 or rIL-6 induced anti-TNP PFC in the TNP-Ficoll-primed and in control PLN cell cultures in the presence of antigen. However, in primed PLN cells lymphokines alone were sufficient to restore anti-TNP PFC response. In conclusion, our results show that in PLN, the TNP-Ficoll can induce proliferation of hapten-specific B cells but not final differentiation. These primed PLN B cells mature into antibody-secreting cells upon stimulation with TNP-BA or lymphokines.     

T cells and the anti-trinitrophenyl antibody response to fetal calf serum and 2-mercaptoethanol

Unprimed murine lymphocytes maintained in culture medium containing fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) developed very high levels of anti-trinitrophenyl (TNP) plaque forming cells (PFC). Both FCS and 2-ME contributed to the response. The development of anti-TNP PFC during culture was accompanied by a 10-fold expansion in the number of immunoglobulin-secreting cells, indicating polyclonal stimulation. However, the number of anti-TNP PFC was disproportionately high and not accompanied by a similar increase in plaques specific for sheep red blood cells. The TNP-specific plaques were not artifacts of the plaque assay since they were 98% inhibited by specific antigen. The in vitro induction of anti-TNP PFC by FCS and 2-ME was common to a number of mouse strains, although some genetic variation occurred. Nylon-wool-separated B cells, nude mouse spleen cells, and bone marrow cells all produced high levels of anti-TNP after culture with medium containing FCS and 2-ME. The addition of T cells to B-cell cultures increased the numbers of anti-TNP PFC by 1.5- to 2.5-fold. The presence of a TNP-cross-reacting antigen in FCS probably contributed to the unexpectedly high anti-TNP response. The response to the antigen in FCS was potentiated by the enhancing activity of 2-ME.     

Restricted maturation of antibody-binding characteristics for hapten-specific IgM-plaque-forming cells in mice

Inhibition of plaque formation was used as a method for studying changes in binding characteristics of IgM antibody specific for the hapten, 2,4,6-trinitrophenyl (TNP). By incorporating successive concentrations of TNP ligand into specific hemolytic plaque assays, we were able to obtain inhibition curves and to determine relative ligand binding of anti-TNP antibodies secreted by plaque-forming cell (PFC) populations. The concentration of free hapten required to obtain 50% reduction in IgM PFC decreased with time after immunization, indicating that an increase in average binding affinity of their secreted antibodes occurred. The slopes of the inhibition curves also increased with time after immunization. Maturation with regard to the above two properties was virtually complete for IgM PFC 5–7 days after immunization. The manner by which the slopes of the inhibition curves changed suggested that maturation occurred as the result of a simple selective process, and it appeared to involve primarily precursor cells existing prior to administration of antigen.     

Ontogeny of murine B-cell responses to thymus-independent trinitrophenyl antigens.

The ontogeny of B-cell responsiveness to three thymus-independent trinitrophenyl (TNP) antigens has been examined in BALB/c mice in vivo and in vitro. When in vivo splenic plaque-forming cell (PFC) responses to TNP-conjugated lipopolysaccharide (TNP-LPS), Ficoll (TNP-Ficoll), and Brucella abortus (TNP-Brucella) were measured in neonatal and adult mice, a defined sequence of responsiveness was observed. Newborn mice responded well to TNP-LPS, but not to TNP-Ficoll or TNP-Brucella. Neonates injected at 1 day of age responded to TNP-LPS and TNP-Ficoll and mice 5 to 14 days of age responded to TNP-LPS, TNP-Ficoll, and TNP-Brucella. Furthermore, the antigen-reactive populations increased at different rates for the three antigens in the first 2 weeks of life. In vitro experiments confirmed the results obtained in vivo although slightly earlier responsiveness to TNP-Brucella was observed in vitro. PFC inhibition assays with free TNP hapten were performed so that avidity profiles could be examined in neonatal and adult anti-TNP PFC responses. The results clearly demonstrate that once a response becomes detectable in neonatal mice immunized with any of the three TI TNP antigens, fully heterogeneous or “adult-like” responses are found. In addition, experiments comparing avidity profiles in athymic (nu/nu) BALB/c mice and their normal (nu/+) littermates demonstrate that T cells are not required for the generation of fully heterogeneous anti-TNP PFC responses. These results indicate that B cells responsive to different TI TNP antigens mature at different times and at different rates during ontogeny. Late maturation events of such B cells do not include the acquisition of additional V-region specificities as detected in a PFC inhibition assay.     

Characterization of Background Anti-Trinitrophenyl Plaque-Forming Cells Observed in Several Strains of Mice

Normal mice have a large number of background anti-trinitrophenyl (TNP) antibody-forming cells (AFC) in their spleens (about 40–50 anti-TNP PFC/10⁶ cells). We investigated this among several mouse strains, i.e., C57BL/6, C3H/He, Balb/c, ddd, and ICR mice, and found that all strains had a similar number of anti-TNP PFC (plaque-forming cells). Developmental aspects of background anti-TNP PFC in the ontogenic process were also investigated. The number of anti-TNP PFC increased logari thmically during the first few days of age, reached a peak on the 13th day and attained a constant value within 30 days. Neonatal thymectomy did not decrease the number of background anti-TNP PFC but such treatment decreased the anti-TNP PFC response to TNP-HRBC (horse red blood cells) immunization. Germ-free ICR mice had a number of background anti-TNP PFC similar to that of conventional ICR mice. Avidity of background anti-TNP PFC was compared among mice of several ages and it was shown that there were no differences among them. These results suggest that the occurrence of these background anti-TNP PFC is not elicited by the immune response but by the natural maturation of precursors of AFC without antigenic stimulation.     

LPS-induced autoantibody response. I. Ontogenic development of PFC response to bromelain-treated syngeneic erythrocytes

Normal adult mice have been shown to contain a large number of cells secreting antibodies against bromelain-treated syngeneic erythrocytes (Br.MRBC) and the numbers remarkably increase by the stimulation with LPS. In this report development of the anti-Br.MRBC response during ontogeny was examined and it was shown that on the injection of LPS suckling mice responded little to generate splenic plaque-forming cells (PFC) against Br.MRBC in vivo and in vitro. The responsiveness of suckling mice to produce anti-Br.MRBC was shown to be less developed than the anti-TNP response or the mitotic response to LPS. The low responsiveness of suckling mice was analyzed in terms of suppressor activity in the spleen cell population, proliferative capacities of the precursors of anti-Br.MRBC PFC, and their frequencies in the spleen. In the coculture experiment of suckling and adult spleen cells or culture of anti-brain-associated Thy 1-treated, macrophage-depleted spleen cell population, no evidence was obtained to show that suckling spleen cells contained suppressor cells. Kinetic profiles studied in vitro showed that anti-Br.MRBC PFC in the suckling spleen did not increase during the culture as those in the adult spleen. Studies on the precursor frequencies revealed that spleen cells of 15-day-old mice contained precursors of anti-Br.MRBC PFC amounting to 20.5% of the adult precursors whereas the PFC response in vitro by the former was only 4% of the latter. From these experimental data, it was concluded that the low responsiveness of suckling mice was partly due to the low frequency of the precursors in the spleen and, in addition, to the defective nature of the precursors in proliferating to differentiate into PFC.     

In vivo immune response to TNP hapten coupled to thymus-independent carrier lipopolysaccharide

Lipopolysaccharide has been utilized as a carrier for the TNP hapten, producing an antigen which induces an in vivo thymus-independent antibody response to TNP as determined using athymic nude mice and their normal littermates. The immune response to TNP-LPS was investigated at both the antibody-forming cell and the serum antibody levels.The primary response to an optimal dose of TNP-LPS (1.0 μg) exhibited unusual kinetics reaching a sharp peak on day 3 of 58,000 anti-TNP PFC/spleen. Serum antibody to TNP was first detected on day 3 and reached a maximum log₂ titer of 17.5 on day 5, an uncommonly high level for hapten-carrier conjugates and most carriers. Both the anti-TNP serum antibody and PFCs were exclusively IgM. No IgG antibody was detected in the primary response through 28 days postimmunization, nor was any detected in any experiment described in this paper. The primary PFC response to 1.0 μg of TNP-LPS was specific for TNP, producing no evidence of polyclonal antibody synthesis. The relative affinities of PFC-secreted antibody were investigated using hapten inhibition. The hapten inhibition curves for TNP-LPS and TNP-SRBC were very similar, indicating that relatively high affinity antibody was elicited by TNP-LPS. The secondary response to this dose following priming with TNP-SRBC or TNP-LPS was similar to the primary response, though the peak was less sharp in both cases. The response to the homologous secondary challenge shifted somewhat, reaching a peak on days 3–4. The effect of various doses in priming or challenging for the secondary response to TNP-LPS was investigated. Using an increased PFC response as a criterion, no dose was optimal for priming or immunological memory to TNP-LPS. While the adoptive primary response to TNP-LPS reached a low level peak on day 7, the adoptive secondary attained a maximum on day 6. This shift in kinetics in intact mice and in adoptive hosts in comparing primary to secondary responses indicated that a state of B cell priming may be induced. However, its full expression may be suppressed by endogenous factors at the time of priming, such as the high level of circulating anti-TNP antibody or residual antigen. Adoptive transfer would remove the cells from these influences, allowing such B cell priming to manifest itself fully.     

In vitro immune response to the 2,4,6-trinitrophenyl determinant in aged C57BL/6J mice:changes in the humoral immune response to, avidity for the TNP determinant and responsiveness to LPS effect with aging.

An in vitro anti-TNP response of the spleen cells from aged C57BL/6J mice showed approximately 4-fold less PFC than did that from young adult mice. Anti-theta serum-treated young spleen cells gave an anti-TNP response that was definitely greater than the response of the anti-theta serum-treated aged spleen cells in the presence of the exogenous activated thymus cells as helper cells. These results suggest that the deficits in B cells may be partly responsible for the imparied anti-TNP response of the aged spleen cells. To examine further the capacity of stem cells in the bone marrow to generate B cells responsible for anti-TNP response in the spleen, we injected i.v. 1.5 to 2.0 times 10(7) bone marrow cells from young or aged mice into lethally irradiated syngeneic recipients that had previously been thymectomized. Four to 6 weeks later, 10(7) spleen cells from the two groups of these recipient mice were immunized with TNP-SRBC in the presence of the exogenous activated thymus cells and assayed for anti-TNP PFC. The response of the aged marrow-derived B cells was approximately one-half of that of the young marrow-derived B cells.The avidity for TNP determinant of the antibodies produced by the PFC was determined by the plaque-inhibition technique. The avidity of the antibodies produced by the aged mice was approximately 33 times lower than that by the young mice. Anti-TNP response of the young spleen cells were markedly enhanced by the addition of LPS to the cultures, whereas no or little enhancement of the response was induced in the aged spleen cells even in the presence of high concentration of LPS. In contrast, DNA synthesis of both the young and aged spleen cells was comparably stimulated by 1 mug/ml and 10 mug/ml of LPS, however, it was rather less in the aged spleen cells at a concentration of 100 mug/ml. Mechanisms responsible for the changes in avidity and responsiveness to LPS with aging are discussed.     

Cellular events in tolerance. VII. Decrease in tolerant spleens of PFC precursors stimulated in vitro by specific antigen or mitogen.

Spleen cells from adult mice rendered tolerant to the fluorescein (FL) hapten (as FL-sheep γ-globulin) were analyzed at limiting dilution for the numbers of precursors stimulatable either by specific antigen (FL-polymerized flagellin; FL-POL) or by a polyclonal B-cell activator (E. coli lipopolysaccharide; LPS). As expected, the number of PFC presursors activated by FL-POL was reduced more than fourfold in the spleens of FL-tolerant mice compared to normal controls. In contrast, LPS was able to trigger equivalent numbers of “FL-specific” PFC precursors in both normal and tolerant spleens. However, the clones stimulated by LPS were predominantly the “low-avidity” precursors in FL-tolerant spleens as shown by plaque inhibition studies. In addition, after FL-gelatin enrichment of normal or tolerant spleen cells, which contain equal numbers of antigen-binding cells, we found that purified cells from tolerant mice were in fact reduced in the numbers of clonable precursors upon LPS stimulation. Two other B-cell mitogens, POL and PPD, also failed to activate PFC precursors from FL-gelatin-purified tolerant spleen cells. Our results suggest that some high-avidity clones may be functionally deleted even in adult B-cell tolerance as previously noted for neonatal tolerance.