A reverse effect of in vitro and in vivo concanavalin A-induced spleen cells on anti-sheep red blood cells response detected with a new method based on flow cytometry

A simple flow cytometric method for detecting humoral immunity against sheep red blood cells (SRBC) is described. The SRBC were incubated with the serum from SRBC-immunized mice, monoclonal anti-SRBC, or the supernatant which was obtained from the in vitro primary culture of spleen cells with SRBC. The antibodies which bound to SRBC were estimated by means of an immunofluorescence and a flow cytometry. When the channel number of the peak in the histogram of flow cytometry was measured as an index of fluorescence intensity of SRBC, the number significantly correlated with the concentration of IgM and IgG classes of anti-SRBC. The flow cytometry method and hemagglutination (HA) test, as a classic method, were compared in SRBC-immune sera and monoclonal anti-SRBC antibody. The sensitivity determined with flow cytometry was much higher than that with HA. The minimum detectable concentration of anti-SRBC antibody was found to be 3.4 ng/ml by the flow cytometry. The dose response of SRBC in in vitro primary culture was detected by the flow cytometry, not by HA, and the response increased with the dose of SRBC. Using this method, the effect of in vitro and in vivo concanavalin A (Con A)-induced spleen cells on humoral response against SRBC was examined in an in vitro culture system. Anti-SRBC response (IgM and IgG) was found to be suppressed by in vitro Con A-induced lymphocytes, but enhanced by in vivo Con A-induced lymphocytes. Thus, this new approach is found to be a good method for detecting the in vitro primary humoral antibody response, which is known to have a low reactivity.     

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.     

Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2

Antibody production to sheep erythrocytes (SRBC) or hapten-conjugated SRBC (TNP-SRBC) was studied in mice with chronic Trypanosoma cruzi infections. Studies in vivo demonstrated that both IgM and IgG anti-SRBC responses were suppressed during chronic infection. Secondary IgG responses were suppressed regardless of whether the primary immunization was given before or after infection. The ability of cells from infected mice to provide help for antibody production was examined in vitro. Anti-SRBC responses were restored to cultures of whole spleen cells from infected mice by the addition of interleukin 2 (IL 2)-rich supernatants, indicating that these cells were capable of antibody production when sufficient help was provided. T cells from SRBC-primed infected mice were unable to provide significant help to normal B cell/M phi cultures for in vitro anti-TNP or anti-SRBC responses. The percentages of Thy-1+, Lyt-1+, and Lyt-2+ spleen cells were not significantly different between normal and infected mice. Anti-TNP and anti-SRBC responses were restored to cultures that contained T cells from infected mice and normal B cell/M phi by the addition of IL 2-rich spleen cell supernatants. The suppression of in vitro antibody responses in mice with chronic T. cruzi infections was associated with a lack of T cell help, which was provided by exogenous spleen cell supernatant.     

An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM)

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.     

Effects of antigen-feeding on intestinal and systemic immune responses. III. Antigen-specific serum-mediated suppression of humoral antibody responses after antigen feeding.

Feeding mice sheep erythrocytes (SRBC) caused a significant decrease in splenic IgM antibody responses to SRBC given ip. Reduced IgM responses were due to a suppressor factor in the serum of fed mice rather than due to a lack of IgM antibody-forming cell precursors or to the presence of suppressor T cells. Although feeding initially primed mice to produce greater IgA and IgG anti-SRBC responses after SRBC challenge, the initial primed state was transitory. Mice fed SRBC for longer than 8 weeks had significantly reduced splenic IgG and IgA responses after SRBC challenge.Suppression of IgM responses by serum from fed mice was antigen-specific and not H-2 restricted. Serum from fed mice inhibited the induction of IgM anti-SRBC responses but did not block the expression of already established responses. The size of the suppressor factor and the ability to remove suppressor activity from serum by anti-mouse immunoglobulin suggested that suppression was mediated by antibody. However, the determinants against which the antibody was directed appeared to differ among batches of suppressor sera. Suppressor activity did not appear to be mediated by immune complexes, or soluble antigen. Oral feeding of antigen can have a marked influence on host systemic immune responses when the antigen used for feeding is subsequently administered parenterally. Thus, oral antigen administration may provide a way for specifically manipulating systemic immune responses in vivo. In addition, antigen-feeding may provide a means for producing transferable factors that suppress humoral antibody responses.     

Antibody-mediated modulation of the immune response

We have studied the ability of monoclonal IgM and IgG antibodies to enhance or suppress immune responses and attempted to dissect the underlying mechanisms. Both IgM and IgG1 antibodies increased the rate of clearance of antigen from the circulation. Monoclonal IgM antibody to SRBC was found to specifically increase antibody responses, enhancement being insensitive to low doses of irradiation (150 R). IgM antibody specifically depressed the delayed hypersensitivity response to SRBC in vivo. Following administration of IgM in vivo, in vitro responses to SRBC were also enhanced. This in vitro enhancement appeared to depend on both T cells and B cells. In contrast, monoclonal IgG1 antibody to SRBC specifically depressed antibody responses in vivo. Such depressed antibody responses were also seen in vitro following IgG1 in vivo and did not appear to be due to the induction of suppressor T cells.     

Antigen-reactive cell opsonization: a mechanism of antibody-mediated immune suppression.

Antisera against sheep red blood cells (SRBC) specifically suppressed the direct anti-SRBC plaque-forming cell (PFC) response in mice when passively administered with the antigen. The suppressive activity of mouse and rabbit anti-SRBC sera was found to correlate with anti-SRBC opsonic activity but not with hemagglutination or hemolysin titers. Macrophage depletion of mice, using carrageenan treatment, inhibited antibody-mediated immune suppression. When mice immunized with SRBC were given ¹²⁵I-labeled Udr, radiolabeled spleen lymphocytes were obtained which specifically formed rosettes with SRBC. These radiolabeled antigen-reactive cells (1ARC) were specifically opsonized in mice treated with antigen-antibody complexes but not in mice treated with antigen or antibody alone. These results suggest that antibody-mediated immune suppression may be due to specific opsonization (and subsequent destruction) of ARC in the presence of antigen-antibody complexes.     

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)     

Patterns of immunoenhancement and suppression induced by Chlamydia trachomatis in vivo and in vitro

We investigated the cycle of immune enhancement and suppression seen in mice infected with Chlamydia trachomatis by using in vivo and in vitro model systems. BALB/c mice injected intravenously with chlamydia reveal a three- to seven-fold increase in numbers of plaque-forming cells producing antibodies against sheep red blood cells (SRBC), when immunized with SRBC 0 to 5 days after chlamydia infection. When mice are injected with SRBC 10 to 15 days after initial chlamydia infection, the specific anti-SRBC plaque-forming cell response is suppressed two- to three-fold. In vitro, low numbers (2 to 5 X 10(6) bacteria/ml) of chlamydia stimulate potent proliferative responses by B lymphocytes while high numbers (25 X 10(6) bacteria/ml) of bacteria generate strong, general T suppressor activity. This model has important implications for regulation of immune responses that arise at different times during chlamydial infections, as well as for the potential effectiveness of chlamydial vaccines.     

FcgammaRIIB in IgG-mediated suppression of antibody responses: different impact in vivo and in vitro.

The suppressive effect of IgG on Ab responses to particulate Ags such as erythrocytes is well documented. IgG-mediated suppression is used clinically in rhesus prophylaxis to prevent RhD-negative mothers from becoming immunized against their Rh D-positive fetuses. We have recently shown that IgG anti-SRBC, passively administered together with SRBC, can induce efficient suppression of primary Ab responses to SRBC in mice lacking the known FcRs for IgG (FcgammaRI, FcgammaIII, and FcgammaRIIB or the neonatal FcR). The lack of a demonstrable effect of the inhibitory FcgammaRIIB was particularly surprising, and, in this study, the involvement of this receptor is further investigated during broader experimental conditions. The data show that SRBC-specific IgG administered up to 5 days after SRBC can induce suppression both in wild-type and FcgammaRIIB-deficient mice. Suppression of secondary Ab responses to SRBC in vivo was similar in the two strains. In contrast, IgG-mediated suppression of Ab responses in vitro was impaired in cultures with primed FcgammaRIIB-deficient spleen cells. In conclusion, inhibition of in vivo Ab responses to SRBC by passively administered IgG can take place via an FcgammaRIIB-independent pathway. This pathway causes >99% suppression and operates during all experimental conditions studied so far. The nature of the mechanism can at present only be hypothesized. Masking of epitopes and/or rapid elimination of IgG-Ag complexes would both be compatible with the observations.     

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.     

Nonspecific Elicitation of Antibody-Forming Cells in the Mouse Spleen by Bacterial Lipopolysaccharide

Mechanisms of nonspecific elicitation of anti-sheep erythrocyte (SRBC) hemolytic antibody plaque-forming cells (PFC) in mouse spleens with an injection of bacterial endotoxin (lipopolysaccharide (LPS)) were studied in comparison with the genesis of naturally occurring ‘background’ PFC in normal mouse spleens and of rapidly arising PFC in mouse spleens after immunization with SRBC. The cytokinetic pattern of anti-SRBC PFC response after an injection of LPS was quite different from that of the response elicited after immunization with SRBC. In addition, even though LPS nonspecifically elicited anti-SRBC PFC response in mice, LPS could not confer any immunological memory on mouse immunocytes for a ‘secondary-type’ anti-SRBC PFC response to restimulation with LPS or SRBC. The administration of rabbit anti-mouse thymocyte immunoglobulin or anti-SRBC antiserum in mice markedly suppressed the PFC response after immunization with SRBC, but did not do so after stimulation with LPS. Neonatally thymectomized mice could still respond to stimulation with LPS, producing anti-SRBC PFC in their spleens. Injections of actinomycin D or cyclophosphamide into mice resulted in obvious reductions of the PFC responses elicited by either LPS or SRBC. However, injections of these immunosuppressive antisera or drugs did not affect the number of anti-SRBC PFC in normal mouse spleens. These results suggest that the geneses of anti-SRBC PFC developed under different conditions, i.e., background PFC, LPS-stimulated PFC, and antigen-stimulated PFC, are quite different from each other, and that the nonspecific elicitation of anti-SRBC PFC by LPS does not require the helper function of T lymphocytes. No obvious difference, however, was observed in the time of ontogenic maturation among these three different anti-SRBC PFC in the mouse spleens judging from when they were first manifested after birth.     

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.     

IgG Suppresses Antibody Responses in Mice Lacking C1q,C3, Complement Receptors 1 and 2, or IgG Fc-Receptors

Antigen-specific IgG antibodies, passively administered to mice or humans together with large particulate antigens like erythrocytes, can completely suppress the antibody response against the antigen. This is used clinically in Rhesus prophylaxis, where administration of IgG anti-RhD prevents RhD-negative women from becoming immunized against RhD-positive fetal erythrocytes aquired transplacentally. The mechanisms by which IgG suppresses antibody responses are poorly understood. We have here addressed whether complement or Fc-receptors for IgG (FcγRs) are required for IgG-mediated suppression. IgG, specific for sheep red blood cells (SRBC), was administered to mice together with SRBC and the antibody responses analyzed. IgG was able to suppress early IgM- as well as longterm IgG-responses in wildtype mice equally well as in mice lacking FcγRIIB (FcγRIIB knockout mice) or FcγRI, III, and IV (FcRγ knockout mice). Moreover, IgG was able to suppress early IgM responses equally well in mice lacking C1q (C1qA knockout mice), C3 (C3 knockout mice), or complement receptors 1 and 2 (Cr2 knockout mice) as in wildtype mice. Owing to the previously described severely impaired IgG responses in the complement deficient mice, it was difficult to assess whether passively administered IgG further decreased their IgG response. In conclusion, Fc-receptor binding or complement-activation by IgG does not seem to be required for its ability to suppress antibody responses to xenogeneic erythrocytes.     

Combinations of two synthetic adjuvants: synergistic effects of a surfactant and a polyanion on the humoral immune response

Synergistic effects of two synthetic adjuvants, dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) on the humoral response to sheep red blood cells (SRBC) were investigated. Mice received intraperitoneal (ip) injections of adjuvant and antigen simultaneously. The number of plaque-forming cells (PFC) in the spleen were determined 5 days later and circulating anti-SRBC antibodies were measured till 16 weeks after immunization. Although combinations of DDA and DXS were very effective in enhancing the PFC response to both moderate (2 X 10(7] and low (2 X 10(6] doses of SRBC, synergy between the adjuvants was only observed at the low dose of SRBC. Optimal augmentation of the primary response to the low antigen dose was evoked by the combination of the highest dose tested of either adjuvant (1 mumol DDA and 1 nmol DXS) resulting in a 560-fold increase of the number of PFC in the spleen as compared to controls. Even combinations of relatively small amounts of both adjuvants were very effective in augmenting the response to SRBC. Mice receiving half the amounts of both adjuvants with 2 X 10(6) SRBC displayed increased numbers of PFC in the spleen at Day 5 as well as increased titers of total anti-SRBC antibodies at Week 1 and Week 2 and 2-mercaptoethanol-resistant antibodies from Week 4 till Week 16 as compared to the calculated sum of responses in mice which received either DDA (0.05 mumol per mouse) or DXS (0.05 nmol per mouse). The mechanism behind the synergy between these adjuvants is discussed and the possibility of discerning adjuvants on their modes of action is suggested.     

IgG1 hypergammaglobulinaemia in chronic parasitic infections in mice: evidence that the response reflects chronicity of antigen exposure.

The IgG1 molecules in the sera of IgG1 hypergammaglobulinaemic mice chronically infected with the larval cestode, Mesocestoides corti, are a heterogeneous population. Although antibodies to M. corti are present, the question of whether a minority or majority of the serum IgG1 molecules has anti-parasite reactivity remains open. The splenic PFC response to an intravenous injection of SRBC in M. corti-infected mice does not consist of an unusually high proportion of IgG1 anti-SRBC PFC. Moreover, the adoptive anti-DNP PFC response of spleen cells from M. corti-infected mice to DNP-M. corti is not biased towards IgG1 antibody production. Since IgG1 hypergammaglobulinaemia is seen in mice with chronic, "high-dose" infections, an attempt has been made to simulate chronic antigenic exposure with SRBC in uninfected mice. A split, high-dose regime of SRBC injections leads to a high number and high proportion of IgG1 anti-SRBC PFC in the spleen in three strains of mice. The results suggest that the extraordinarily high levels of IgG1 seen in the sera of mice chronically infected with the metazoa, M. corti and Nematospiroides dubius, reflect persistent, high-dose, "strong", T cell-dependent stimulation of the B cell system.     

Complement Receptors 1 and 2 in Murine Antibody Responses to IgM-Complexed and Uncomplexed Sheep Erythrocytes

Early complement components are important for normal antibody responses. In this process, complement receptors 1 and 2 (CR1/2), expressed on B cells and follicular dendritic cells (FDCs) in mice, play a central role. Complement-activating IgM administered with the antigen it is specific for, enhances the antibody response to this antigen. Here, bone marrow chimeras between Cr2(-/-) and wildtype mice were used to analyze whether FDCs or B cells must express CR1/2 for antibody responses to sheep erythrocytes (SRBC), either administered alone or together with specific IgM. For robust IgG anti-SRBC responses, CR1/2 must be expressed on FDCs. Occasionally, weak antibody responses were seen when only B cells expressed CR1/2, probably reflecting extrafollicular antibody production enabled by co-crosslinking of CR2/CD19/CD81 and the BCR. When SRBC alone was administered to mice with CR1/2(+) FDCs, B cells from wildtype and Cr2(-/-) mice produced equal amounts of antibodies. Most likely antigen is then deposited on FDCs in a way that optimizes engagement of the B cell receptor, making CR2-facilitated signaling to the B cell superfluous. SRBC bound to IgM will have more C3 fragments, the ligands for CR1/2, on their surface than SRBC administered alone. Specific IgM, forming a complex with SRBC, enhances antibody responses in two ways when FDCs express CR1/2. One is dependent on CR1/2(+) B cells and probably acts via increased transport of IgM-SRBC-complement complexes bound to CR1/2 on marginal zone B cells. The other is independent on CR1/2(+) B cells and the likely mechanism is that IgM-SRBC-complement complexes bind better to FDCs than SRBC administered alone. These observations suggest that the immune system uses three different CR1/2-mediated effector functions to generate optimal antibody responses: capture by FDCs (playing a dominant role), transport by marginal zone B cells and enhanced B cell signaling.     

Immunogenic and antigenic epitopes of immunoglobulins. II. Antigenic differences between secreted and membrane IgG demonstrated using monoclonal antibodies

Twenty-three monoclonal antibodies with specificity for epitopes in the Fc fragment of IgG have been used to investigate antigenic differences between secreted and membrane forms of IgG produced by 2 human B lymphoblastoid cell lines (LCL). All of the monoclonals reacted with IgG secreted by the cell lines, as demonstrated by their ability to agglutinate SRBC coated with immunoglobulin isolated from culture supernatants. Membrane IgG expression was studied using direct and indirect rosette assays with antibody-coated ORBC. A surprisingly high number of antibodies, 13 on EB2 and 9 on EB4, did not bind to the cell surface immunoglobulin. These included antibodies with specificities for both C gamma 3 and C gamma 2 domain determinants. Similar results were obtained with an indirect radiobinding assay, indicating that negative results with the rosette test were not due to steric hindrance by the red cell carrier. Their performance in indirect hemagglutination indicated that most of the antibodies that did not bind to membrane IgG were of high avidity. It is concluded that the epitopes for which these antibodies are specific are not available on the cell surface. Possible explanations for the apparent antigenic differences between secreted and membrane forms of IgG are discussed against the background of previous work on the structure and mode of insertion of cell surface immunoglobulin.     

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.     

Adjuvant effects of crystal proteins from a Mexican strain of Bacillus thuringiensis on the mouse humoral response

In this study, we determined the adjuvant effects of the crystal (Cry) proteins, p130, p98, and p64-62, on the immune response of mice to both sheep red blood cells (SRBC) and ovalbumin (OVA). The administration of p130, p98, and p64-62 Cry proteins to Balb/c mice induced a significant (p<0.01) increase in the production of anti-SRBC antibody-secreting cells (ASC). The p64-62 Cry proteins demonstrated the best ability to induce the production of IgA and IgG antibodies to SRBC (p<0.05), and IgM, IgA, and IgG antibodies to OVA (p<0.05). Additionally, Cry proteins did not produce any side effects associated with their administration to Balb/c mice. We suggest the potential use of the p64-62 Cry proteins as adjuvants for the administration of heterologous antigens.