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.     

Murine bone marrow IgA responses to orally administered sheep erythrocytes

Specific immunization protocols have been established for the induction of murine bone marrow IgA responses to the T cell-dependent (TD) antigen sheep red blood cells (SRBC). Systemic immunization, either i.p. or i.v., followed by a second injection, induced splenic IgM and IgG responses and a bone marrow IgM response. No significant IgA responses were observed in either lymphoid tissue compartment. Oral immunization with SRBC by gastric intubation for 2 days, followed 1 wk later by an i.p. injection of SRBC resulted in a splenic IgA plaque-forming cell (PFC) response, but did not elicit a bone marrow IgA response. Repeated daily gastric intubation of SRBC to C3H/HeN and C3H/HeJ mice led to the previously reported pattern of systemic unresponsiveness in C3H/HeN mice and good anamnestic type IgM, IgG, and IgA splenic anti-SRBC PFC responses in the C3H/HeJ strain upon parenteral challenge. Oral administration of SRBC for 14 days to C3H/HeN mice, followed by systemic SRBC challenge, resulted in diminished splenic PFC responses of all isotypes, whereas gastric intubation of SRBC for 28 days led to complete systemic unresponsiveness to antigen in C3H/HeN mice. Interestingly, the repeated oral administration of SRBC resulted in significant bone marrow IgA PFC responses upon i.p. challenge in both C3H/HeN and C3H/HeJ mouse strains. The bone marrow IgA responses were clearly dependent upon chronic oral exposure to SRBC, because gastric intubation with SRBC for 2 consecutive days/wk for 10 wk also induced bone marrow and splenic IgA anti-SRBC PFC responses in C3H/HeN mice. These results suggest that memory B cells reside in the bone marrow of orally immunized mice and can yield anamnestic-type responses to challenge with the inducing antigen. The memory cells may arise in the Peyer's patches of the gut and migrate to the bone marrow. The possibility that the bone marrow is a component of the common mucosal immune system in mammals is suggested by this study.     

Isotype-specific immunoregulation. Systemic antigen induces splenic T contrasuppressor cells which support IgM and IgG subclass but not IgA responses

In the present study, we have isolated and characterized the Lyt-1+, -2- T contrasuppressor (Tcs) cells from mice systemically primed with SRBC. Adoptive transfer of splenic Tcs cells from these mice abrogates oral tolerance and supports IgM and IgG anti-SRBC plaque-forming cell (PFC) responses; however, unlike the responses seen after transfer of Tcs cells derived from orally primed mice, low IgA responses were seen. Mice systemically primed with lower SRBC doses (0.01 to 1%) exhibited contrasuppression only within the L3T4- T cell subset, whereas mice primed with a high dose of SRBC (10%), harbored Lyt-1+, -2- Tcs cells in both the L3T4+ and L3T4- subsets. Both the L3T4- and L3T4+ Tcs cell subsets supported IgM and IgG responses when adoptively transferred to orally tolerized mice, and when added to tolerized spleen cell cultures. Splenic Tcs cells from systemically primed mice supported mainly IgG1 and IgG2b subclass anti-SRBC PFC responses, a pattern also seen with Tcs cells derived from orally primed mice. Both L3T4+ and L3T4- Tcs cells from systemically primed mice exhibited well established characteristics of contrasuppressor cells including binding to Vicia villosa lectin and expression of I-J. The splenic effector Tcs cells which support IgM, IgG1 and IgG2b anti-SRBC PFC responses are antigen-specific, since both L3T4- and L3T4+ Tcs cells from spleens of mice primed with 10% SRBC reverse tolerance to SRBC, but not to horse erythrocytes (HRBC). Further, both L3T4- and L3T4+ Tcs cells from HRBC-primed mice reverse tolerance to IgM and IgG anti-HRBC, but not to anti-SRBC responses. Isolation of T3-positive Lyt-1+, -2- and L3T4- Tcs cell subsets by flow cytometry followed by adoptive transfer, showed that effector Tcs cells express T3 and presumably contain an Ag-R (TCR-T3 complex). These studies show that systemic priming with heterologous RBC induces splenic Ag specific Tcs cells in a dose-dependent manner, which support IgM and IgG subclass responses, but not IgA responses.     

Selective improvement of thymus and some T cell dysfunctions in NZB mice by in utero thymulin treatment

NZB mice were treated during gestation with thymulin, a thymus-secreted, zinc-associated nonapeptide. Control pregnant NZB mice received either zinc alone or saline alone. Offspring from all three groups of NZB mothers, and age-matched DBA/2 mice, were tested for the following immunologic parameters: thymulin serum levels at 2 and 5 wk of age; splenic anti-sheep red blood cell (anti-SRBC) plaque-forming cell (PFC) numbers after immunization at birth or at 2 wk of age; anti-human gamma-globulin (anti-HGG) antibody titers after immunization at 2 wk of age, with or without prior tolerance induction at birth with deaggregated HGG; spontaneous IgM serum levels at 2 and 5 wk of age; spontaneous splenic anti-trinitrophenyl (anti-TNP) PFC numbers at 2 wk of age. As compared with DBA/2 mice, young NZB mice exhibited low circulating thymulin titers, high antibody responses to SRBC and to HGG, resistance to tolerance induction by deaggregated HGG, increased spontaneous IgM serum levels, and increased spontaneous anti-TNP PFC numbers. However, marked reductions in anti-SRBC and anti-HGG antibody production, both thymus-dependent responses, were observed in the young NZB offspring of thymulin-treated mothers as compared with NZB controls born from zinc- or saline-treated mothers. A delay in the postnatal decrease of serum thymulin levels was also noted in the offspring of thymulin-treated mothers. Interestingly, these effects of in utero thymulin treatment tended to become more pronounced with advancing age during the postnatal period. Conversely, IgM serum levels, spontaneous anti-TNP PFC and sensitivity to tolerance induction were not affected by thymulin treatment during fetal life. Taken together, the data suggest that in utero exposure to pharmacologic concentrations of thymulin induces a persistent and selective improvement of some thymus and T cell dysfunctions but has no effect on intrinsic B cell abnormalities of NZB mice.     

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.     

IgM-mediated enhancement of in vivo anti-sheep erythrocyte antibody responses: isotype analysis of the enhanced responses

The direct splenic anti-sheep erythrocyte (anti-SRBC) responses as well as the serum IgG1, IgG2a, IgG2b, and IgG3 anti-SRBC responses of CBA/CaJ mice were monitored 4-35 days after immunization with: (1) a suboptimal dose of SRBC, (2) a suboptimal dose of SRBC plus monoclonal IgM anti-SRBC, or (3) a high dose of SRBC. The direct plaque-forming cell (PFC) responses of mice in treatment group 2 were significantly higher than those in group 1 but similar to the responses in group 3. The serum anti-SRBC antibody responses of all IgG subclasses were significantly enhanced by IgM anti-SRBC and were generally even higher than the responses obtained with high doses of SRBC. The relative proportions of each serum IgG subclass were similar in all three groups. These data suggest that the enhancement of suboptimal anti-SRBC antibody responses by IgM anti-SRBC extends through IgM and all of the IgG subclasses and, further, that the isotype profile in antibody-enhanced responses is similar to that obtained with high doses of SRBC.     

IgA responses in xid mice: oral antigen primes Peyer's patch cells for in vitro immune responses and secretory antibody production

Because the gut-associated lymphoreticular tissue (GALT), e.g., Peyer's patches (PP), of X-linked immunodeficient (xid) mice possesses a subpopulation of mature B cells, we have characterized the ability of xid mice to respond to the thymic-dependent antigen sheep erythrocytes (SRBC) given by the oral route. Gastric intubation of SRBC to xid (CBA/N X DBA/2) F1 male or CBA/N mice, followed by the in vitro culture of dissociated PP cells with SRBC, resulted in IgM, IgG1, IgG2, and high IgA anti-SRBC plaque-forming cell (PFC) responses. The addition of unprimed PP but not splenic T cells to splenic xid B cell cultures resulted in IgM anti-SRBC PFC responses, suggesting the importance of GALT T cells for support of the immune responses to SRBC by splenic B cells from xid mice. Furthermore, purified PP T cells from SRBC orally primed xid mice supported in vitro IgA anti-SRBC PFC responses in B cell cultures from either the PP or the spleens of nonprimed xid mice. Higher IgA responses, however, occurred in PP, when compared with splenic B cell cultures. Additional evidence that the GALT of xid mice contains functional IgA precursor cells was provided by the finding that cloned H-2k PP T helper cells (PP Th A) supported IgA responses in PP B cell cultures derived from (CBA/N X C3H/HeN) F1 male (xid) mice. On the other hand, splenic B cells from these xid mice, in the presence of PP Th A cells, did not support in vitro responses. These results suggest that unique subpopulations of T cells occur in the GALT of xid and normal mice; one T cell subpopulation may induce immature B cells to become precursor IgA cells in the PP. A separate GALT T cell subpopulation, e.g., isotype-specific helper T cells, effectively collaborates with mature IgA B cells for the induction of IgA responses to orally administered antigen. When xid mice were gastric intubated with SRBC, followed by i.p. injection of SRBC, good splenic IgA anti-SRBC PFC responses were seen. Salivary and serum IgA antibodies were also detected in these xid mice. Nevertheless, the magnitude of the anti-SRBC response in xid mice was lower than that seen in similarly treated normal mice. These studies indicate that the GALT of both xid and normal mice possess unique populations of T cells that support in vitro responses in xid B cell cultures from either the spleen or the PP, which direct the mature B cell populations present toward IgA isotype-specific responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Idiotypic properties of the murine anti-arsonate antibody response: B- and T-cell influences

In a previous report characterizing the arsonate (ABA)-specific plaque-forming cell (PFC) responses of A/J mice induced by ABA-KLH, two interesting characteristics of the idiotypic (Id) profile were noted: (1) an apparent Id selectivity in the isotype switch since the earliest appearing IgG PFC in the primary response were significantly more "cross-reactive Id" (CRI)-dominant than the IgM PFC population, and, (2) a temporal waning of CRI dominance with time among IgG PFC, from 75-100% CRI+ PFC to about 25-45% CRI+ PFC in secondary responses. Experiments were performed to determine whether these effects are largely attributable to T or to B cells. Mice were immunized with a T-independent (TI) form of ABA (ABA-Brucella abortus) and apparent Id selectivity was observed; the earliest IgG PFC averaged 75% CRI+ while IgM PFC were only 39% CRI+. Due to the TI nature of the Ag, this provides suggestive, but not conclusive, evidence that the Id asymmetry in the isotype switch may be attributable to the direct interaction of Ag with B cells. Other studies addressed the temporal shift in CRI dominance. First, it was found that preexposure of mice to either KLH or to ABA (on an irrelevant carrier) resulted in diminished CRI dominance in subsequent "primary" responses to ABA-KLH. Secondly, adoptive transfer experiments with B and T cells from virgin mice (Bv, Tv) or ABA-KLH-primed mice (Bp, Tp) showed that recipients of Bv + Tp or Bp + Tv generated anti-ABA PFC responses with intermediate CRI levels. The Tv cells had some preferential tendency to activate CRI+ clones in the Bp population. The results demonstrate that CRI levels are jointly determined by the immune status of both B and T cells. A simple model is offered which accounts for early Id dominance and its gradual decline and has as its central postulate the assumption that CRI+ B cells in the virgin ABA-specific repertoire have an affinity advantage over CRI- clones.     

Differential immunosuppressive effects of ascites fluid from mastocytoma-bearing mice on primary vs secondary immunocyte responses.

The effects of immunosuppressive ascites fluids from mastocytoma-bearing mice on the primary vs secondary immune response to sheep red blood cells (SRBC) was examined. Injection of mice with ascites fluid from tumor-bearing mice markedly depressed the primary immune response of normal syngeneic mice challenged with SRBC. However, there was a preferential depression of the 19S IgM antibody response as compared with the 7S IgG response. Injection of ascites fluid shortly before secondary immunization of mice with SRBC also resulted in depressed IgM PFC responses but only a slight to moderate depression of IgG PFC. Treatment of mice with the ascites fluid before primary immunization had little if any effect on the secondary IgG PFC response, although the IgM response was moderately depressed. These results indicate that the immunosuppressive factor(s) present in the ascites fluid of mastocytoma-bearing mice has a differential effect on distinct classes of immunocytes. Those immunocytes or their precursors involved in formation of low efficiency 7S IgG antibody are more resistant to immunodepression. Such differences appear due to different sensitivities of cells involved in the immune response system.     

Anti-immunoglobulin autoantibodies are not preferentially induced in polyclonal activation of human and mouse lymphocytes, and more anti-DNA and anti-erythrocyte autoantibodies are induced in polyclonal activation of mouse than human lymphocytes

Using a plaque assay with immunoglobulin (Ig)-coated SRBC, we and others have previously reported that the majority of polyclonally activated mouse lymphocytes secreted antibodies that appeared to be IgM anti-IgG autoantibodies. Careful reexamination of this assay, with application of several highly purified mouse serum and myeloma IgG and IgM preparations, revealed that IgM, which was a minor contaminant of Ig preparations, rather than IgG, was responsible for the formation of these plaques. High numbers of plaques could also be detected in assays with polyclonally activated human lymphocytes, Ig-coated SRBC, and anti-Ig developing sera. Of all IgG-, IgM- or IgA-secreting cells, 40 to 100% were detected with SRBC coated with gamma-globulin or Ig of the same isotype as the isotype to which the developing serum was specific; in general, low proportions of all PFC were detected with SRBC coated with Ig of a different isotype. Studies on the sequence of events leading to the formation of plaques with Ig-sensitized SRBC (both in humans and mice) revealed that antibodies detected in these assays were not able to bind to the Ig-coated SRBC (without the presence of developing serum), and therefore were not anti-Ig autoantibodies. It is our conclusion that the plaque assays with Ig-coated SRBC represent another type of a reverse hemolytic PFC assay that detects cells secreting antibodies regardless of their specificity, and these plaques are formed due to the cross-linking by the anti-Ig developing serum of the Ig coated on SRBC and the Ig secreted by lymphocytes. Our results confirmed preferential induction of anti-DNA antibody secreting cells in mice by showing that these antibodies indeed bind to DNA coated on SRBC. In cultures of polyclonally activated human lymphocytes, anti-DNA and anti-erythrocyte autoantibody-secreting cells were over 10 to 100 times less frequent than in mice. These results, therefore, disprove the concept of preferential induction of anti-Ig autoantibodies in the polyclonal activation of mouse and human lymphocytes, and show that anti-DNA and anti-erythrocyte autoantibodies are easily induced in the polyclonal activation of mouse, but not human, lymphocytes.     

Distinct subpopulations of IgG memory B cells respond to different molecular forms of the same hapten.

Spleen cells from mice primed with the thymus-dependent antigen trinitrophenyl keyhold limpet hemocyanin several months earlier can be cultured in vitro to give vigorous IgG antihapten PFC responses to thymus-dependent (TD) and thymus-independent (TI) forms of the same hapten. Here we show that the IgG memory precursors that respond to these two forms of the hapten constitute functionally distinct subpopulations. We have designated these subpopulations as B1gamma and B2gamma to represent secondary precursor cells responding to TI and TD antigens, respectively. Three types of evidence for these subpopulations are presented: 1) In vitro secondary IgG responses to TD and TI forms of the TNP hapten are additive when both forms are added to the same culture. 2) The precursor frequencies for the TD and TI antigens are additive, but addition is not observed between two TD or two TI antigens. 3) Each population can be selectively eliminated by BUdR and light treatment without affecting the other population. The ontogenetic relationships between these subpopulations are discussed in relation to all presently proposed subpopulations B1mu, B2mu, B1gamma, and B2gamma.     

T-Cell-Independent Immunoglobulin G Responses In Vivo Are Elicited by Live-Virus Infection but Not by Immunization with Viral Proteins or Virus-Like Particles

Immunoglobulin G (IgG) responses to viruses are generally assumed to be T-cell dependent (TD). Recently, however, polyomavirus (PyV) infection of T-cell-deficient (T-cell receptor β chain [TCR-β] −/− or TCR-β×δ −/−) mice was shown to elicit a protective, T-cell-independent (TI) antiviral IgM and IgG response. A repetitive, highly organized antigenic structure common to many TI antigens is postulated to be important in the induction of antibody responses in the absence of helper T cells. To test whether the repetitive structure of viral antigens is essential and/or sufficient for the induction of TI antibodies, we compared the abilities of three forms of PyV antigens to induce IgM and IgG responses in T-cell-deficient mice: soluble capsid antigens (VP1), repetitive virus-like particles (VLPs), and live PyV. Immunization with each of the viral antigens resulted in IgM production. VLPs and PyV elicited 10-fold-higher IgM titers than VP1, indicating that the highly organized, repetitive antigens are more efficient in IgM induction. Antigen-specific TI IgG responses, however, were detected only in mice infected with live PyV, not in VP1- or VLP-immunized mice. These results suggest that the highly organized, repetitive nature of the viral antigens is insufficient to account for their ability to elicit TI IgG response and that signals generated by live-virus infection may be essential for the switch to IgG production in the absence of T cells. Germinal centers were not observed in T-cell-deficient PyV-infected mice, indicating that the germinal center pathway of B-cell differentiation is TD even in the context of a virus infection.     

Suppressor T-cell activity in MRL/Mp-lpr/lpr mice: differential effects on primary and memory antibody responses of MRL/Mp-lpr/lpr and MRL/Mp-+/+ spleen cells to thymus dependent and thymus independent antigens in vitro

We have previously shown that suppressor-T-cell (TS) activity in the spleens of autoimmune MRL/Mp-lpr/lpr (MRL/l) mice is increased after 2 months of age. The TS suppress the in vitro primary IgM response to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC) of B and T cells from young congenic MRL/Mp-+/+ (MRL/n) mice which lack the lymphoproliferation (lpr) gene. The TS are nylon wool nonadherent, Thy 1.2 positive, and radiation sensitive. The studies presented here were done to further characterize the TS and to attempt to determine the mechanism of action of these cells. We found that increased TS activity was also present in the proliferating lymph nodes of old MRL/l mice but not in lymph nodes of young MRL/l or MRL/n mice. The splenic TS equally suppressed the primary IgM SRBC response of both young MRL/l and MRL/n B and T cells, indicating that MRL/l SRBC-specific B and T cells are not resistant to suppression. The IgM response of MRL/n B and T cells to the T-independent (TI) antigen trinitrophenyl conjugated to Brucella abortus (TNP-BA) was not suppressed by the TS, although the IgM response to TNP was suppressed when TNP was coupled to the TD carrier SRBC. The results of kinetics studies of TS expression showed that when the TS were added on Day 0 of culture the SRBC response was suppressed as early as Day 2 of culture; however, when the TS were added on Days 1, 2, or 3 of culture, the suppression was reduced. The TS suppressed the in vitro memory IgG response of spleen cells from MRL/n mice which had been primed with SRBC; the memory IgG responses of spleen cells from MRL/l mice were variably suppressed. Taken together, these results suggest that the TS suppress TH function in early events of antibody production and that some activated B or T cells may be resistant to the effects of the TS. Increased TS activity was not present in the spleens of aged New Zealand Black X NZ White (NZB/W) F1 mice. Possible reasons for the presence of increased TS activity in MRL/l mice and its relation to autoimmune disease is discussed.     

Variations in the responses of C57BL and a mice to sheep red blood cells: II. Analysis of plaque-forming cells

The number of direct and indirect plaque-forming cells (PFC) and the serum hemolytic activity was determined for A/He, C57BL/6J, and B6AF1 mice responding to multiple injections of sheep red blood cells (SRBC). Although the kinetics of the primary response differed, all mice had high numbers of both direct and indirect PFC and low-titered 2-mercaptoethanol (2-ME) sensitive serum antibody. Following multiple SRBC injections, the A/He spleens contained predominantly IgG producing PFC. Their serum antibody activity was resistant to 2-ME signifying the presence of IgG. The serum activity of both the C57BL/6J and B6AF1 mice was sensitive to 2-ME (IgM antibody) over the course of immunization, and although there was a definite IgM PFC memory response, the presence of 7S memory PFC was questionable. The results are discussed in terms of the maturation of the antibody response to SRBC and of the question of the postulated IgM and IgG switch.     

Characterization of the primary IgM response to GAT and GT: conditions required for the detection of IgM antibodies.

Primary IgM antibody responses to synthetic linear copolymers of L-glutamic acid, L-tyrosine, and L-alanine were investigated. The appearance of primary IgM anti-GAT antibodies was detected in BALB/c mice by using a solid phase radioimmunoassay (SPRIA) procedure. The finding was verified for GAT in responder mice and GAT-MBSA and GT-MBSA in nonresponder mice in an indirect plaque forming cell (PFC) assay by using a rabbit antiserum directed against the mulambda myeloma protein, MOPC 104E. Facilitated IgM PFC could be inhibited by a purified muK myeloma protein, TEPC 183. Maximal facilitated IgM plaque response was found to precede the IgG response by several days. A direct plaque assay was developed for the detection of IgM anti-GAT plaques using poly-L-lysine (PLL) to couple GAT to sheep erythrocytes (SRBC). GAT-SRBC coupled by the PLL method optimally couple 4 to 5 times less antigen to the indicator cell surface than does the CrCl3-coupling method routinely employed in our laboratory. These findings were extended to a conventional antigen, chicken gamma globulin (CgammaG). We found that a less dense epitope coat on the indicator cell surface favors detection of direct IgM PFC, whereas a more densely coated indicator cell favors the detection of facilitated IgM and IgG PFC responses.     

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10⁶ SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10⁸ SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10⁶ SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.     

In vivo allogeneic effects: shift in the isotype profile of primary TI-2 responses in mice undergoing graft-vs-host reaction

We showed previously that primary responses to T-dependent (TD) and T-independent type 2 (TI-2) antigens were differentially affected by allogeneic effects induced in vivo during a graft-vs-host reaction (GVH). TD responses were greater than or equal to 80% suppressed, whereas the TI-2 responses were greatly enhanced, particularly the IgG component, which normally is very low. We have analyzed the IgG subclass distribution in primary responses of normal and GVH F1 mice in order to determine whether the strong T cell signals that occur during GVH reactions also induce shifts in the isotype profile. The effect of GVH on responses to TI-2 antigens was of particular interest because they are usually dominated by IgM and IgG3 classes in normal mice. We found a threefold to 10-fold increase in the PFC numbers of all four IgG subclasses in the response to TI-2 antigens, with an apparent shift from the usual IgG3 dominance to IgG1 in GVH mice. This IgG1 dominance was not found in serum antibodies where IgG3, IgG1, and IgG2b were equally expressed, although total IgG was increased greater than 20-fold. No isotype shift was found in either the TNP-KLH response, which was greater than or equal to 75% suppressed (IgG1 dominance was retained), or in the TI-1 response to TNP-Ba. The latter response was reduced (25 to 50%) in GVH mice and continued to be dominated by IgG2b/2a and IgG3. Unlike the unique isotype patterns found in primary responses, TNP-KLH primed mice challenged with TD, TI-1, or TI-2 antigens gave memory responses with identical isotype profiles that were dominated by IgG1 PFC. The role of T cells in B cell differentiation and isotype expression is discussed.     

Dose-dependent induction of immunologic enhancement and suppression after oral administration of antigen

The effects of feeding various quantities of a particulate antigen, sheep red blood cells (SRBC), on plaque-forming cells (PFC) in the spleen were determined. Mice were given various numbers of SRBC orally daily for 14 days, then injected with SRBC intravenously. Splenic IgA PFC responses to SRBC were enhanced in the mice fed 5 X 10(8) SRBC and splenic IgG PFC responses to SRBC were depressed in the mice fed 5 X 10(9) SRBC. Adoptive transfer experiments showed that enhancement of splenic IgA PFC responses and suppression of splenic IgG PFC responses were induced by the T-cell rich fraction from Peyer's patches (PP) and the spleen in 5 X 10(8) SRBC- and 5 X 10(9) SRBC-fed mice, respectively. Kinetic studies revealed that IgA helper cells or IgG suppressor cells appeared in PP 2 days after oral administration and 4 days after it in the spleen.     

Subclass restriction of murine antibodies: V. The IgG plaque-forming cell response to thymus-independent and thymus-dependent antigens in athymic and euthymic mice

Antigens differ in their abilities to stimulate antibodies of various isotypes. Many thymus-independent (TI) polysaccharide antigens stimulate largely IgG3 and IgM antibodies while thymus-dependent (TD) protein antigens stimulate predominantly IgG1 and smaller amounts of other isotypes. Here we determine whether thymus dependence or independence is a property of antigens which is expressed equally by all isotypes. To do this nu/+ and nu/nu mice were immunized with several TI and TD antigens and antibody responses of IgM and the four IgG subclasses measured. We found that, within the conditions of these experiments, all IgG isotypes were influenced equally by the presence or absence of T lymphocytes. Second, in agreement with J. L. Press (J. Immunol.126, 1234, 1981), we propose a division of TD antigens into two types based upon the ability to stimulate responses in the CBA/N mouse.     

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.