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.     

Cell-Cooperation in Anti-Sheep Red Blood Cell Antibody Response in Mouse Spleen Cell Cultures

Restoration of impaired antibody response to sheep red blood cells (SRBC) in spleen cell cultures from mice treated with heterologous antilymphocyte globulin (ALG) was studied by adding normal cells from various sources, to explore the problems of cell-cooperation in anti-SRBC antibody response and the target of ALG. When spleen cells from ALG-treated mice were separated into macrophage-rich and lymphoid cell-rich subpopulations, only the latter was found to be impaired in the ability for anti-SRBC antibody response. Addition of even a small number of normal allogeneic spleen cells sufficiently restored the impaired anti-SRBC antibody response of the spleen cells from ALG-treated mice. By use of allo-antisera, most hemolysin plaque-forming cells (PFC) generated in such cultures were proved to be derived from the cells of ALG-treated mice. Restoration was also achieved by adding thymus-derived cells, which were obtained from spleens of mice heavily irradiated and repopulated with syngeneic thymus cells, or lymphoid cells directly collected from thymuses. All results indicate that ALG selectively depletes the thymus-derived antigen reactive cells (ARC) in the spleen cell population, and that ARC supplied from normal spleen or thymus can interact with plaque-forming cell precursors (PFCP) that remain intact in the spleen cell population of ALG-treated mice. The results also suggest that a single ARC interacts with more than one PFCP and makes them develop into PFC.     

Acute hepatotoxin exposure effects lymphoid and accessory cell types in inbred mice

The effect of acute hepatotoxin exposure on in vivo and in vitro immune responses were investigated in inbred mice. Splenic anti-SRBC PFC responses were slightly enhanced by carbon tetrachloride or galactosamine administration 5 hr prior to immunization. Whereas splenic anti-SRBC PFC responses were slightly enhanced in euthymic mice exposed to carbon tetrachloride 5 hr prior to immunization, immune responses to the TI antigens, Fl-LPS, Fl-Ficoll, and TNP-LPS, were significantly suppressed. Athymic mice receiving similar hepatotoxin exposure elicited enhanced immune responses to the TI immunogens, thereby suggesting that the activities of B cells and macrophages are enhanced in treated animals and in euthymic mice, T suppressor cells are also activated. By admixture of purified B- and T-cell and macrophage populations from either carbon tetrachloride-treated or control animals, it was demonstrated that hepatotoxin exposure also induces suppressor T cells regulating immune responses to the T-dependent antigen, SRBC, and that macrophages from treated animals are more functional. Further, B-cell responsiveness is enhanced. In addition to these observations, an active factor could be demonstrated in sera from hepatotoxin-treated animals which augments immune responses to SRBC in normal mice and promotes immune responses to this antigen in athymic mice. These findings indicate that the effects of acute hepatotoxin exposure are multifocal, influencing the activity of lymphoid and accessory cells.     

The role of Ia antigens and Fc receptor-bearing T-cells in the inhibition of macrophage receptors for cytophilic antibody induced by soluble immune complexes

Soluble antigen-antibody complexes composed of 3 M KCl-extracted L1210 antigens and alloantibody to L1210 given to C3H mice caused immunosuppression in the mice. This was reflected in part by the inhibition of cytophilic antibody receptors on macrophages which could be used as a measure of the suppression. Thymocytes or splenic T cells from mice treated with immune complexes could adoptively transfer the suppression to normal syngeneic mice. These cells, which we have termed suppressor inducers, were found to be Ia positive: specifically, I-A⁺, I-J^?. Thus, treatment of the inducers with anti-la or anti-I-A antibodies and complement in vitro abrogated their ability to transfer the suppression to normal mice. In contrast treatment with anti-I-J serum and complement had no effect. Through a similar approach, the cooperating (acceptor) T cells were found to be I-A⁺, I-J^?. Pretreatment of mice with anti-Ia or anti-I-A serum before the administration of antigen-antibody complexes prevented the inhibition of macrophages. This was due at least in part to steric hindrance of adjacent Fc receptors on the FcR⁺ T cells with which the complexes interacted. Early interaction of immune complexes with FcR⁺ T cells was in fact demonstrated directly by the inability of the complexes to induce suppression when FcR⁺ T cells were depleted. The thymocytes or splenic T cells from anti-Ia-pretreated mice failed to transfer the suppression to recipient mice. In contrast, treatment with either anti-Ia or anti-I-A after the immune complexes did not abrogate the generation of suppressor inducers. Treatment of normal recipient mice with anti-Ia serum in vivo before they received the suppressor inducer cells did not prevent cooperation between the two types of cells. By the same token, blocking of Ia antigens of the inducers in vitro with anti-Ia serum (without complement) also did not impair the cooperative interaction. These results indicate that antigen-antibody complexes generate I-A-positive, I-J-negative T-suppressor inducer cells from FcR⁺ naive T cells. These in turn interact with Ia-positive (I-A⁺ and I-J^?) normal thymocytes or spleen T cells. This interaction most likely generates the ultimate suppressor T cells that suppress cytophilic antibody receptors on macrophages in vivo. However, the I-region determined antigens did not appear to be directly involved in the T-T interaction of suppressor inducer and acceptor cells.     

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)     

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.     

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.     

Immunosuppressive effect of Entamoeba histolytica extract on hamsters

The immune response to sheep red blood cells (SRBC) in mice and hamsters injected with an extract of entamoeba histolytica was studied. Both the primary and secondary immune response, measured by anti-SRBC antibody titers, were unaltered in the mouse, while a significant depression of the primary, but not the secondary, response was observed in the hamster. The effect was greatest when the amebic extract (AE) and SRBC were injected on the same day. The number of anti-SRBC rosettes formed in the spleen cells of hamsters treated with both AE and SRBC on day 0 was measured from days 1-16. The response peaked on day 13, while cells from animals injected with SRBC alone gave a maximal response on day 5. The formation of anti-SRBC rosettes in T-lymphocyte-enriched spleen cells treated with anti-gamma globulin serum and complement was almost abolished for the duration of the experiment. It is suggested that the mechanism responsible for this immunosuppressive phenomenon could involve early interference in the afferent limb of the immune response.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

In vivo suppression of the primary immune response by a species of low density serum lipoprotein.

An apparent subspecies of normal human serum low density lipoprotein (LDL-In) has been identified with suppressive activity for early or facilitating events of human lymphocyte mitogen and allogenic cells stimulation in vitro. This report describes the effects of in vivo administration of LDL-In on the mouse anti-SRBC immune response. Human LDL-In is not species specific and was capable of suppressing the in vivo mouse anti-sheep erythrocyte (SRBC) hemagglutination response by 88% after the administration of 500 to 600 mug LDL-In IV, whereas human serum high density lipoproteins and fibrinogen had no effect. Maximal suppression occurred only when LDL-In was injected 24 to 48 hr before antigen administration. Simultaneous or subsequent injection of LDL-In had no effect. The activity of LDL-In was influenced by antigen dose and maximal at low antigen doses. The number of splenic plaque-forming cells was also reduced indicating a suppression of the clonal expansion of primary B cells to antibody-secreting cells rather than only suppression of antibody synthesis by differentiated B cells and their progeny. These observations suggest the hypothesis that endogenous LDL-In could play an important immunoregulatory role in the maintenance of immune homeostasis and the "natural" suppression of non-productive lymphocyte proliferation.     

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.     

Antibody formation in mouse bone marrow. VII. Evidence against the migration of plaque-forming cells as the underlying cause for bone marrow plaque-forming cell activity: a study with parabiotic mice

After intravenous immunization of mice with Escherichia coli lipopolysaccharide (LPS) or sheep red blood cells (SRBC), the bone marrow can contain large numbers of plaque-forming cells (PFC). By means of parabiosis, it was studied whether or not this appearance of PFC in the bone marrow might be due to a migration of such cells from peripheral lymphoid organs into the marrow, as has been suggested in the literature. Using parabionts consisting of nonimmunized mice and mice immunized with LPS, only background numbers of PFC could be demonstrated in the bone marrow of the nonimmunized mice. In similar experiments, with SRBC as antigen, mice showing high anti-SRBC PFC activity in the bone marrow could only provide for minor numbers of anti-SRBC PFC in the bone marrow of affixed normal mice. These results suggest that migration of PFC can not be the main cause for bone marrow PFC activity in the mouse. This provides additional evidence for our view presented in previous papers of this series that the appearance of PFC activity in the bone marrow is dependent on local maturation of B cells into PFC rather than on immigration of PFC.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

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.     

Immunoregulation by monoclonal sheep erythrocyte-specific IgG antibodies: suppression is correlated to level of antigen binding and not to isotype

Six different monoclonal IgG antibodies with specificities for sheep erythrocytes (SRBC) were tested for immunosuppressive ability. Four of them, one IgG3, two IgG2a, and one IgG1, could yield suppression of more than 90% of the anti-SRBC response. The remaining two antibodies, which were both IgG2a, were found to have no significant effect. The degree of suppression correlated well with the amount of antibodies used that could bind to SRBC, as measured by an ELISA assay. High avidity for SRBC was also a factor making the monoclonal antibody more efficient as an immunosuppressor. The response against antigenic determinants on the SRBC other than those for which the monoclonals were specific, was suppressed to an equal degree. This was established by immunizing mice with SRBC using monoclonal anti-SRBC antibodies that did or did not bind to goat RBC (GRBC). The PFC responses against both SRBC and GRBC were then measured. The anti-SRBC and GRBC responses were suppressed in parallel regardless of whether or not the monoclonal reacted with GRBC. None of the tested antibodies displayed any significant ability to enhance the anti-SRBC response. Thus, IgG1 is not the only murine isotype that can efficiently suppress the immune response against SRBC, but IgG2a and IgG3 can also exert this capacity. The mechanism of IgG-mediated suppression is not one of merely blocking single epitopes but involves the immunogenicity of the entire SRBC.     

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.     

Estimation of relative antibody affinity at the level of the antibody-secreting cell during maturation of the immune response

The relative affinity of specific antibody secreted by mouse spleen cells following primary immunization with SRBC was estimated by competitive inhibition assay of antibody secreted by PFC as well as by inhibition of observed PFC number. Inhibition of direct and of indirect anti-SRBC plaque assays by the addition of specific antigen (SRBC stromata) gave sigmoid inhibition profiles from which the concentration of antigen required to inhibit 50% of the plaques (PI₅₀) was determined, Alternatively, the sum of the cube of individual plaque diameters (Σd³) provided a measure of total anti-SRBC antibody secreted by PFCs from which the concentration of antigen required to inhibit 50% of the antibody (Ab₅₀) was determined. Ab₅₀, rather than PI₅₀: (a) was a more sensitive measure of inhibition by antigen; (b) decreased following immunization indicating a progressive increase in mean antibody affinity; and (c) correlated with the results of hemolysin transfer experiments, an independent measure of mean affinity of circulating anti-SRBC antibody. From theoretical considerations, estimation of mean antibody affinity requires quantitative analysis of fractional antibody inhibition by antigen. Determination of Ab₅₀, rather than PI₅₀, provides an estimate of bound and of free antibody and therefore should provide a more valid estimate of the relative antibody affinity at the cellular level. Experimentally, utilizing Ab₅₀ analysis, the IgM and IgG responses of C3H mice to immunization with SRBC demonstrated a progressive increase in affinity during maturation of the immune response.     

Suppression and augmentation of the primary in vitro immune response by different classes of antibodies

The capacity of purified γG₁ and γG₂ anti-sheep red blood cell (SRBC) antibodies to exert antigen-specific feedback regulations on the primary in vitro immune response to SRBC was studied. Antibodies were administered to the culture in the native form, as sheep erythrocyte-antibody complexes or as pepsin-derived F(ab′)₂ antibody fragments. Marked differences in the feedback regulatory effects of γG₁ and γG₂ antibodies were found. Antibodies of the γG₁ class suppressed the immune response to SRBC, whereas γG₂ antibodies isolated from the same serum exerted an augmenting effect on antibody synthesis. These opposing feedback effects on in vitro antibody synthesis were immunologically specific, relatively insensitive to changes in antigen concentrations, and could be elicited by either adding antibodies and antigen separately to the culture or as preformed antigen-antibody complexes. Experiments comparing the activities of the F(ab′)₂ antibody fragments with the parent γG₁ and γG₂ antibodies suggested that the Fc fragments may be involved in these regulatory effects on the immune response. It is concluded that the antigen-specific suppressive and augmenting effects on antibody synthesis shown here are determined by the antibody class. In addition, we suggest that these opposing antibody-mediated feedback effects may represent one of the important elements of the immune response.     

Alterations in the Immunogenic Properties of Sheep Erythrocytes by Sonic Disruption

A solubilized sheep red blood cell (SRBC) antigen (supernatant fraction obtained by centrifuging 10^7-2 × 10⁸ sonicated SRBC at 6 × 10⁴ g for 30 min [Sup-SRBC]), whose ability to inhibit anti-SRBC plaque formation was 70% of that of the original sonicated SRBC, was unable to elicit a detectable antibody response in either unprimed or SRBC-primed mice. However, Sup-SRBC as well as intact SRBC antigens generated memory for the secondary response, which was transferable to irradiated syngeneic recipients by injection of immune spleen cells. The memory generated by Sup-SRBC involved helper memory for anti-trinitrophenyl group (TNP) response to challenge with TNP-conjugated SRBC. Increase in the helper T cell memory in the spleens of Sup-SRBC-primed mice was also demonstrated by an in vitro culture experiment and by an adoptive cell transfer experiment. In contrast, no detectable B cell memory was generated by Sup-SRBC. Repeated stimulation with Sup-SRBC never induced significant antibody response but reduced the level of memory. A single injection of a low dose (10⁶) of SRBC also failed to induce a definite primary antibody response generating memory for the secondary response. However, repeated stimulation with this dose of SRBC induced a high antibody response and generated good memory. From these results it is suggested that the intact structure of SRBC is required for the activation of B cells, but is not necessary for the stimulation of T cells.