Delayed maturation of the antibody response to type 2 thymus-independent antigens in a partially inbred strain of chicken

The ontogeny of antibody responses to trinitrophenylated (TNP) thymus-independent (TI) antigens was compared in two partially inbred strains of chicken: the SC strain (B2/B2 genotype) and the FP strain (B15/B22 genotype). In the SC chicken, maturation of both the splenic anti-TNP plaque-forming cell (PFC) response and the 19S hemagglutinating antibody response to TI type 2 (TI-2) antigens, TNP-Ficoll and TNP-dextran, were delayed to a significantly later time in ontogeny (20 wk of age) than in the FP chickens (9 wk of age). Four- to 6-wk-old SC chickens were virtually immunologically unresponsive to stimulation with TI-2 antigens. The TI-1 antigen TNP-Brucella abortus was equally immunogenic in both FP and SC chickens of different age groups tested. Kinetic studies of the primary PFC response to TNP-Ficoll in immunologically mature chickens of the SC and FP strains demonstrated a peak PFC response 4 days after antigen injection, followed by a rapid decline in numbers of splenic PFC/spleen on day 6. The results of these studies are discussed in relation to earlier observations that suggested there may be a delay or a defect in the ontogeny of the thymus in the SC chicken.     

LPS regulation of the immune response: suppression of immune responses to orally administered T-independent antigen

The regulation of immune responses to gastrically administered TI antigens has been investigated, and the characterization of a regulatory cell population has been performed. Intragastric administration of TNP-haptenated homologous erythrocytes (TNP-MRBC) induced splenic IgM anti-TNP PFC responses in LPS nonresponsive C3H/HeJ mice that were higher than those in LPS-responsive C3H/HeN mice and similar to those noted in athymic (nu/nu) C3H/HeN animals. The simultaneous intragastric administration of LPS with TNP-MRBC augmented immune responses in a manner similar to that previously reported for parenterally administered LPS and antigen. Further, LPS-induced augmentation of TNP-MRBC responses was greater in athymic mice. These findings were substantiated using in vitro spleen cultures. Intragastric challenge with a 2nd TI antigen, TNP-LPS, induced approximately 8-fold higher splenic anti-TNP PFC responses in athymic C3H/HeN mice compared with those in euthymic littermates. By admixture of B and T cell populations, it was demonstrated that the host responsiveness to TNP-LPS was negatively regulated by suppressor cells. Suppressive activity resided in a Thy 1.2-bearing, irradiation-resistant, nylon wool-nonadherent cell population. These cells could be demonstrated in spleen and Peyer's patches from young or old LPS-responsive C3H/HeN mice, but not in tissues from LPS nonresponsive C3H/HeJ mice. The specificity of the regulator cells was not limited to TNP-LPS responses, since immune responsiveness to another TI antigen, TNP-dextran, was also under the control of this cell population. These studies confirm the TI nature of TNP-MRBC and indicate that immune responses to gastrically administered antigens such as TNP-LPS, TNP-dextran, and possibly TNP-MRBC are negatively regulated by a suppressor T cell population. A role for endogenous LPS in the generation of regulator cells and the effect of these cells on host responses to gut-derived antigens is discussed.     

Immunoregulation in the rat: ontogeny of B cell responses to types 1, 2, and T-dependent antigens

The development of rat B cells has been examined in neonatal and adult Fischer rats through the use of type 1 (TNP-Brucella abortus), type 2 (TNP-LPS(Ph), TNP-Ficoll) and T cell-dependent (TD) (SRBC) antigens. In vivo splenic PFC responses to TNP-Brucella abortus could be induced in newborn rats and by 12 days of age had reached adult levels. In contrast, the responses to the type 2 and TD antigens were 30% and 70%, respectively, of the adult levels at 30 days of age. Adoptive transfer of the B cells from neonatal and young rats into irradiated adult hosts demonstrated that the kinetics in the development of responses to these antigens (early for type 1, intermediate for TD, and late for type 2) were not due to limiting accessory cell or T cell help in immature rats. In vitro cultures of purified B cells from neonatal and adult rats were responsive to TNP-BA and TNP-LPS(Ph) but not to TNP-Ficoll and SRBC. However, the addition of spleen cell-derived Con A supernatant to the B cell cultures resulted in responses to all four antigens, which arose as a function of B cell age, with kinetics that were identical to those observed in vivo. Fluorescent staining of B cells from rats of various ages for cell surface IgM and analysis on the fluorescence-activated cell sorter (FACS) revealed that all splenic B cells from rats 4 days of age expressed a relatively high level of sIgM, and that a subpopulation that expressed a relatively low level of sIgM increased with age until it represented approximately 50% of the adult splenic B cells. Challenging Con A supernatant-supplemented cultures of FACS-prepared low sIgM+ and high sIgM+ cells revealed that B cells responsive to TNP-Ficoll were confined to the ontogenically late-arising low sIgM+ subpopulation but that B cells responsive to TNP-BA, TNP-LPS(Ph), and SRBC were present in both subpopulations.     

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.     

Ability of chicken B cells from different compartments to respond to TNP-Ficoll

The responsiveness of chicken B cells from various compartments to T-independent antigens was studied by immune transfers of spleen and bursa cells into immunosuppressed recipients. Bursa cells from 8- to 10-wk-old donors failed to respond to trinitrophenylated Ficoll (TNP-F) even when thymus cells or splenic T cells were added. Spleen cells from the same donors transferred responses, as judged both by anti-TNP plaque-forming cells (PFC) per spleen and serum anti-TNP titers. In contrast, responses to TNP-Brucella abortus (TNP-BA) were transferred at least as well as by bursa as by spleen cells. Rabbit anti-chicken T cell serum plus complement treatment of the spleen cells reduced their ability to transfer responses to sheep erythrocytes, but either did not affect or enhanced serum antibody responses to TNP-BA and TNP-F. In intact animals, responsiveness to i.v. injected TNP-F was found to develop slowly after hatching in the chicken. At the age of 2 and 3 mo, PFC/spleen on day 4 after TNP-F injection were only 20% and 40%, respectively, of the adult response. Thymectomy at hatching further delayed this development, resulting in 12% and 45% of the adult control response at ages of 3 and 4 mo. It is concluded that responsiveness to the TI-2 antigen, TNP-F, develops slower than that to the TI-1 antigen, TNP-BA, and is restricted to the splenic B cell compartment. In addition, this development appears to be faster in the presence rather than in the absence of the thymus. In view of the previously shown effect of thymus on bursa development, these data suggest that the maturation of TI-1 antigen (TNP-F)-respondent chicken B cells requires residence in both the bursa and spleen before the development of responsiveness to such antigens.     

Functional relationships between B-cell subsets: evidence for an antigen-induced B1 leads to B2 switch

Priming of mice with the TI-2 antigen TNP-Ficoll results in an increased secondary anti-TNP response to the TD antigen TNP-KLH. Spleen cells obtained from mice primed with TNP-Ficoll or TNP-LPS, but not TNP-KLH, gave augmented responses to DNP-Dextran, TNP-LPS, or LPS. In vitro treatment of spleen cells with DNP-Dextran results in an increased anti-TNP response to TNP-LPS. This effect is also obtained if mitogenic doses of Dextran and LPS are used to induce cell proliferation. We conclude that the B_i2 subset (responding to TNP-Ficoll and DNP-Dextran) switches to the B2 subset (responding to TNP-KLH) after antigen activation. The B_i1 subset (responding to TNP-LPS) may represent an intermediate stage of B-cell differentiation.     

Streptococcal serotype carbohydrate represents a novel class of type 2 antigen which is T-independent

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyclonal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb-3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.     

Immune responses to T-dependent and T-independent antigens during visceral leishmaniasis in mice: evidence for altered T-cell regulation of immune responses to non-parasite antigens

Antibody responses to T-dependent and T-"independent" antigens were studied in disease-susceptible (BALB/c and C57BL/10) and disease-resistant (A/J) mice infected with Leishmania donovani chagasi. Disease-susceptible mice but not disease-resistant mice showed a transient decrease in PFC responses to TNP on a T-dependent carrier (BGG) during the period of 4-8 weeks after infection. Infected disease-susceptible animals also showed increased responses to TNP on a type II T-independent carrier (Ficoll), which persisted until at least 14 weeks after infection. The increased responses were associated with a significant increase in anti-TNP antibody of the IgG2b subclass. When T-enriched spleen cells from infected mice and B-enriched spleen cells from uninfected mice were transferred to irradiated recipients immunized with TNP-Ficoll, increased anti-TNP PFC were observed over numbers seen in irradiated recipients which received both B and T cells from uninfected mice. Increased responses to TNP-Ficoll were also induced by prior administration of soluble leishmania extract in CFA. Infected mice immunized with TNP-LPS, a T-independent type I antigen, also had increased anti-TNP antibody responses, but had normal anti-LPS antibody responses. The elevated antibody production which occurred in response to the T-"independent" antigens could not be attributed to the relatively low polyclonal response which occurred in both disease-resistant and disease-susceptible mice infected with L. donovani chagasi. The observations are consistent with leishmania induced, transient alterations in some T-cell functions including response to haptens on T-dependent carriers, and a lack of down regulation of T-"independent" responses. Subtle lesions in immunoregulation may be important correlates of successful protozoal infection and may be responsible for some of the immunologic manifestations of the disease.     

Immunoregulation in the rat: cellular and molecular requirements for B cell responses to types 1, 2, and T-dependent antigens

The requirements for primary in vitro plaque-forming cell (PFC) development in cultures of purified rat splenic B cells have been examined. Rat B cells were directly responsive to the type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA), but both T cells and adherent accessory cells were required for B cell responses to the type 2 antigen TNP-Ficoll and the T cell-dependent (TD) antigen sheep erythrocytes (SRBC). However, the cellfree supernatants from concanavalin A-induced spleen cells of rat or mouse origin replaced the requirement for T cells and macrophages, and resulted in PFC development in response to TNP-Ficoll and SRBC and augmented PFC numbers in response to TNP-BA. Culture supernatants from induced murine T cell and macrophage cell lines were used to partially deduce the molecular requirements for the support of PFC development by rat B cells to these three antigens. Supernatants from the EL-4 (EL-4 sup) and B151 K12 (B15 sup) T cell lines augmented TNP-BA responses, suggesting that B cell growth factor II (BCGF-II) mediated this effect. An admixture of purified interleukin 2 (IL 2) and B15 sup supported PFC development to SRBC; indicating that IL 2, BCGF-II, and the T cell-replacing factor in B15 sup (B15-TRF) were sufficient to support this response. In addition, the IL 2 plus B15 sup-supported anti-SRBC PFC response was increased by the addition of an interleukin 1-containing fraction from the supernatant of the macrophage line P388D1. PFC development in response to TNP-Ficoll had the most stringent requirements and only occurred in the presence of EL-4 sup and B15 sup (IL 2, BCGF-I, BCGF-II, EL-TRF, B15-TRF). These data indicate that different cellular and molecular requirements exist for PFC development in response to types 1, 2, and TD antigens by rat B cells.     

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.     

Functional heterogeneity of virgin and memory B murine lymphocytes revealed by the utilization of cyclosporin A: an overview

The effects of cyclosporin A on the generation and revelation of B memory cells by thymus-independent (TI) antigens was investigated. A class 1 (TNP-LPS) and a class 2 (TNP-Ficoll) TI antigens were used for priming an elicitation. Evidence is presented that cyclosporin A does not interfere with the generation of hapten-specific (TNP) B memory cells by TNP-LPS or DNP-Ficoll. Cyclosporin A does not affect the revelation of B memory cells by TNP-LPS, but inhibits their revelation by TNP-Ficoll. These findings are discussed in terms of two distinct B cell lineages leading to antibody-forming cells and memory cells precursors, and in terms of heterogeneity of B memory cells.     

T cell dependence and factor reconstitution of in vitro antibody responses to TNP-B. Abortus and TNP-Ficoll: restoration of depleted responses with chromatographed fractions of a T cell-derived factor

In vitro anti-trinitrophenyl (TNP) antibody responses to TNP conjugates of killed Brucella abortus organisms (TNP-BA), an antigen previously designated as a type 1 thymus-independent (TI-1) antigen, are markedly diminished after vigorous depletion of T cells, as are the responses to the type 2 TI (TI-2) antigen, TNP-Ficoll. We, therefore, propose that these antigens be redesignated as type 1 and type 2, respectively, to reflect their T cell dependence but to differentiate them from classical T cell-dependent (TD) antigens. T cell-depleted responses to type 1 and type 2 antigens can be restored by the addition of a) EL4 supernatant, b) phenyl-sepharose-purified fractions of EL4 supernatant that are rich in interleukin 2(IL2), and c) pl 4.5-5.5 isoelectric focused (IEF) fractions of EL4 supernatant which are also rich in IL2 activity. Removal of IL2 activity from EL4 supernatant by absorption on IL2-dependent T cells substantially reduced its restorative ability. Whether the active principle in EL4 supernatant activity responsible for restoring responses to type 1 and type 2 antigens is IL2, and whether it acts directly on B cells or by acting on contaminating T cells, is unresolved.     

Induction of antigen-specific proliferation in affinity-purified small B lymphocytes: requirement for BSF-1 by type 2 but not type 1 thymus-independent antigens

We report here the role of B cell stimulatory factors in the induction of antigen-specific proliferation of affinity-purified small B lymphocytes. TI-1 antigens such as TNP-LPS and TNP-BA induced proliferation of hapten-binding B cells in the absence of exogenous B cell stimulatory factors. TI-2 antigens such as TNP-Ficoll required the co-stimulator BSF-1 to induce antigen-specific proliferation, and this response could be augmented by IL 1. TD antigens such as TNP-OVA were unable to induce antigen-specific proliferation either in the absence or presence of B cell stimulatory factors, and showed an absolute activation requirement for carrier-specific helper T cells. No role for IL 2 or BCGF II could be found in the factor-dependent proliferative response of hapten-binding B cells to TI-2 antigens, either as primary co-stimulators or as modulators of the response obtained with TNP-Ficoll, BSF-1, and IL 1. In contrast, concentrations of IFN-gamma that were nontoxic for normal B cells and B cell hybrids effectively abrogated the proliferative response of affinity-purified cells to TNP-Ficoll, BSF-1, and IL 1. By all of these criteria, the B cell activation requirements of TI-2 antigens appear to be identical to those previously published for soluble anti-IgM antibodies.     

Selective effect of irradiation on responses to thymus-independent antigen

Low doses of ionizing radiation have a selective immunosuppressive effect on in vivo B cell responses to thymus-independent (TI) antigens. The B cell response, assayed as direct anti-trinitrophenyl (TNP)-specific plaque-forming cells (PFC), induced by type 2, TI antigens (TNP-Ficoll or TNP-Dextran), was reduced, on the average, by 10-fold in animals exposed to 200 rad of ionizing radiation 24 hr before antigen challenge. In contrast, PFC responses to type 1, TI antigens (TNP-lipopolysaccharide or TNP-Brucella abortus) are unaffected in mice exposed to the same dose of radiation. Adoptive transfers showed that this selective immunosuppression is a result of the specific inactivation of the B cell subpopulation responding to type 2, TI antigens. These experiments suggest that physiologic differences exist in the B cell subpopulations of normal mice which respond to type 1, or type 2, TI antigens.     

Hormonal modulation of responses to thymus-independent and thymus-dependent antigens in autoimmune NZB/W mice

Previous work suggested that gonadal steroids influence immunity through the thymus, but the mechanisms were unclear. To investigate the effects of these hormones on immune responses to T1 and TD antigens in autoimmune mice, we studied hybrid NZB/W mice and the nonautoimmune DBA/2 strain. Mice castrated at 14 days of age were implanted with Silastic capsules releasing, in adults, physiologic levels of E2 in males or Te in females. Sham-operated controls received empty capsules. Splenic PFC were quantified 4 to 5 days after challenge with the TI2 antigen TNP-Ficoll, the TI1 antigen TNP-LPS, or the TD antigen SRBC. Young castrated NZB/W males implanted with E2 had striking enhancement of IgM responses to TNP-Ficoll when compared to castrated Te-treated females and comparable sham-operated controls of both sexes. E2 also stimulated responses to TNP-LPS. In response to challenge with SRBC, young E2-treated NZB/W males had a consistent trend to increased IgM PFC, and the stimulatory effect of E2 on IgG plaques was variable. Physiologic doses of Te had no consistent effect on responses in young mice. In old female NZB/W mice, Te caused PFC response after immunization with TNP-Ficoll to resemble age-matched NZB/W males. As sham-operated NZB/W females grew older, PFC responses to SRBC fell. This age-related phenomenon was delayed, however, in female castrates implanted with Te. In contrast, Te clearly suppressed responses to TNP-LPS. Implantation of E2 did not alter responses to TNP-Ficoll, TNP-LPS, or SRBC in nonautoimmune DBA/2 males. This finding suggested that exogenous E2 given in physiologic doses did not influence immunologic responsiveness in a normal strain to the degree seen in hormone-sensitive NZB/W mice. It was concluded that E2 enhanced responses to a variety of exogenous antigens in autoimmune NZB/W mice. The most consistent E2-induced increase in PFC response was observed with TI antigens, suggesting that E2 exerted its effects on B cells or Ts.     

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)     

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.     

Generation of immune memory by haptenated derivatives of thymus-independent antigens in C57BL/6 mice. I. The differentiation of memory B lymphocytes into antibody-secreting cells depends on the nature of the thymus-independent carrier used for memory induction and/or revelation

It has been previously reported that trinitrophenylated lipopolysaccharide (TNP-LPS), a thymus-independent (TI)-1 antigen, elicits an anamnestic response to TNP in C57BL/6 mice. The ability of these mice to mount a secondary response to TI-2 antigens was analyzed. Priming with DNP-Ficoll or DNP-Dextran, both TI-2 antigens, resulted in an increased frequency of TNP-binding B lymphocytes. Evidence is presented that memory cell-induction by DNP-Ficoll does not require functional T cells. The differentiation into antibody-forming cells (AFC) of memory cells generated by DNP-Dextran or DNP-Ficoll cannot be obtained by a challenge with either antigen. There was no indication that the lack of a secondary response to TI-2 antigens was related to suppressive T cells interfering with memory expression. Memory cells induced by DNP-Dextran or DNP-Ficoll can nevertheless be activated by TNP-LPS. In contrast to the restricted sensitivity of TNP-memory cells generated by TI-2 antigens, TNP-LPS-induced memory cells are indifferently susceptible to TI-1 or TI-2 antigenic stimulation. These results are discussed in terms of memory B-cell subpopulations.     

Immunopotentiating effects of the adjuvants SGP and Quil A. I. Antibody responses to T-dependent and T-independent antigens

The present study was undertaken to compare the effects of two adjuvants, SGP (a starch-acrylamide polymer) and Quil A (purified saponin), with that of aluminum hydroxide (Al(OH)3) on murine primary antibody responses to T-independent (TI) and T-dependent (TD) antigens. All three adjuvants augmented the responses to the TD antigens, dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH), and sheep erythrocytes (SRBC). SGP was the most potent adjuvant and increased the primary IgG response to DNP-KLH as much as 90-fold. Quil A and Al(OH)3 had comparable effects on the primary response to DNP-KLH, but Quil A was less effective than Al(OH)3 for augmenting the primary response to SRBC. Quil A and SGP both augmented the primary IgM and IgG responses to trinitrophenyl-lipopolysaccharide (TNP-LPS), TNP-Brucella (TI-1 antigens), and TNP-Ficoll (TI-2 antigens). Al(OH)3, like most commonly used adjuvants, had little or no effect on responses to TI antigens. The kinetics of the response to TNP-Ficoll was altered by SGP, since peak responses were maintained for at least 7 days, while the response to TNP-Ficoll alone peaked on Day 4 and had declined considerably by Day 7. Both SGP and Quil A could augment responses to both optimal and suboptimal doses of antigen. The adjuvant activity of SGP was diminished, but still effective, when smaller amounts of SGP were used with the immunizing antigen, and all three adjuvants were able to augment primary responses when given in separate injections from the antigen. These results demonstrate that SGP is a very effective adjuvant, and show that both Quil A and SGP have a unique ability to increase antibody responses to TI antigens, suggesting that their effects may be mediated at least partially through B cells.     

The early postnatal development of the primary immune response in rat popliteal lymph node,stimulated with thymus-independent type-1 and type-2 antigens

Summary To examine the development of the postnatal immune response to thymus-independent type-1 (TI-type 1) and TI type-2 antigens, respectively, trinitrophenyl-lipopolysaccharide (TNP-LPS) or TNP-Ficoll was injected subcutaneously into the hind footpads of young rats of various ages. After 5 days the popliteal lymph nodes (PLNs) were removed and the localization pattern of specific anti-TNP antibody-containing cells was studied. The first specific antibody-containing cells elicited in rats by TNP-LPS appeared in animals at day 19 after birth. The results suggest that the development of these cells from lymphocyte to plasma cell occurs while they migrate from cortex to medulla. An unexpected finding was the low response to TNP-Ficoll in PLN; from 6 weeks after birth only very few specific antibody-containing cells were found. However, in the spleen numerous anti-TNP antibody-containing cells were found in the periarteriolar lymphocyte sheaths. To test the exclusive role of the spleen in the appearance of anti-TNP antibody-containing cells in lymph node after subcutaneous administration of TNP-Ficoll, the experiment was repeated in rats that had been splenectomized. Evidence from these experiments suggests that the spleen plays a major role in the appearance of the above-mentioned cells in lymph nodes.Abbreviations KLH keyhole-limpet haemocyanin - LPS lipopolysaccharide - PBS phosphate-buffered saline - PLN popliteal lymph node - TD thymus-dependent - TI thymus-independent - TNP trinitrophenyl