Regulation of B lymphocyte activation by the Fc portion of immunoglobulin

Murine splenic B lymphocytes are induced to proliferate and undergo polyclonal activation in the presence of Fc fragments, AHGG, antigen-antibody complexes, and CH₃ fragments derived from plasmin digestion of human Ig. The unifying feature of the polyclonal antibody response induced by these agents is that in all cases a portion of the constant region of the Ig molecule (ie, Fc region) is present. Fragments of Ig lacking the Fc piece, such as Fab and F(ab′)₂ were found not to be stimulatory. In addition, a model is proposed to account for the regulatory effects of antigen-antibody complexes on an ongoing humoral immune response.     

The induction of immunological unresponsiveness in previously immunized mice.

HGG unresponsiveness can be induced in primed A/J mice; however, such induction is difficult and requires multiple injections of large doses of soluble HGG (SHGG). Although single injections (1.5–25 mg) of deaggregated HGG (DHGG) did not result in a significant secondary immune response, an unresponsive state to a subsequent injection of aggregated HGG (AHGG) was not induced. When the dose of DHGG was even smaller (0.5 mg), a normal secondary response was obtained similar to that observed following injection of AHGG. Evidence is presented here which suggests that the difficulty encountered in inducing unresponsiveness may be in part due to partial aggregation of DHGG by persisting antibody in the circulation. The PFC to HGG produced after injection of either AHGG or DHGG or during induction of unresponsiveness to SHGG apparently involved cell division, since all three responses were inhibited by vinblastine. The reduction in PFC in primed mice injected with DHGG or SHGG was not due to selective inhibition of PFC secreting certain classes or subclasses of immunoglobulins.     

Polyclonal activation of murine B lymphocytes by Fc fragments. I. The requirement for two signals in the generation of the polyclonal antibody response induced by Fc fragments

Fc fragments derived from human IgG1 induce murine splenic B lymphocytes to undergo proliferation and differentiation to antibody-secreting cells. The polyclonal antibody response was found to require both the presence of macrophages and T cells. Spleen cell cultures from nude mice or T cell-depleted normal mice proliferate to the level of untreated control mice but do not produce polyclonal antibody unless T cells are added. Regulation of the Fc fragment induced B cell differentiation to antibody synthesis apparently occurs through two distinct signals. One signal is provided by Fc fragments for proliferation and the other by T cells for differentiation. This suggestion is supported by the observation that spleen cell preparations, devoid of T cells, are capable of proliferation to the level of normal spleen cell cultures in response to Fc fragments, but are incapable of making a polyclonal antibody response. The cell population that responds to the differentiation signal also responds to the proliferative signal. "Hot pulse" experiments demonstrated that proliferation precedes polyclonal activation.     

Establishment of unresponsiveness in primed B lymphocytes in vivo

As an approach to examine the influence of the state of cellular activation on the ability to tolerize B cells, the induction of unresponsiveness in human gamma-globulin-(HGG) primed B lymphocytes was studied in an adoptive transfer system. In contrast to transferred normal spleen cells, spleen cells from HGG-primed mice are not readily rendered unresponsive when exposed to the tolerogen, deaggregated HGG (DHGG), in irradiated recipients. A kinetic study showed that unfractionated primed spleen cells do not respond to an antigenic challenge given between 6 and 10 days after cell transfer and injection of DHGG, indicating that they are transiently depressed. In contrast, isolated primed B cells are tolerized when transferred to recipients and treated with DHGG in the absence of T cells. Furthermore, primed B cells exposed to tolerogen in the recipients do not recover the ability to respond to HGG either after a secondary challenge with AHGG given up to 14 days after transfer, or after 2 consecutive challenges given on days 14 and 24 after transfer. The presence of primed T cells at the time of tolerization interferes with the induction of unresponsiveness in these primed B cells. These studies suggest that the presence of primed T cells is responsible for the inability to tolerize unfractionated primed spleen cells populations and that primed B cells themselves are not intrinsically resistant to the induction of unresponsiveness.     

Polyclonal activation of murine B lymphocytes by immune complexes

Murine splenic B lymphocytes are stimulated by homologous immune complexes to proliferate and secrete polyclonal antibody. The use of antibody from whole serum or monoclonal antibodies to form complexes resulted in the stimulation of mouse B lymphocytes. The ratio of antibody to antigen appears to be critical for the generation of the polyclonal antibody response. Because antigen and antibody are added independently at culture initiation, the exact nature of the complex is unknown, but optimal polyclonal antibody formation occurs in slight antigen excess. Immune complex-induced polyclonal antibody production requires the presence of both macrophages and T cells, whereas B cell proliferation requires only macrophages. The role of the macrophage appears to be to cleave a low m.w. (17,000) fragment from the complex, which is responsible for lymphocyte activation.     

Induction of tolerance in athymic mice with an antigen which is highly immunogenic in euthymic mice.

Adult congenitally athymic (nu/nu) mice were found to be unable to respond to aggregated human γ-globulin (AHGG), the normally immunogenic form of HGG, unless first reconstituted with specific T cells. However, pretreatment of nude mice with AHGG prior to T-cell reconstitution resulted in the induction of unresponsiveness. This state of tolerance was specific since pretreated animals responded normally to the noncross-reacting antigens turkey γ-globulin or DNP-Ficoll. Transfer of spleen cells from nude mice pretreated with AHGG into normal littermates did not significantly affect a subsequent anti-HGG response of the recipients. Conversely, nude mice pretreated with AHGG and reconstituted with normal littermate spleen cells were hyporesponsive to challenge with AHGG. The results of these experiments are discussed in reference to various models for the induction of B-cell unresponsiveness.     

The induction of peripheral T cell unresponsiveness in adult mice by monomeric human gamma-globulin

Monomeric human gamma-globulin (HGG), when injected into adult mice, induces a state of specific immunologic unresponsiveness to further challenge with immunogenic forms of HGG. In this report we have directly determined the role of the thymus in the induction of HGG tolerance and the proliferative responsiveness of T cells from normal and HGG-tolerant mice. Draining lymph node T cells were isolated from HGG-tolerized and -challenged mice, and tested for their proliferative response to HGG in vitro. T cells from untreated but challenged adult CBA/CaJ and A/J mice proliferate in response to HGG, whereas such mice given monomeric HGG before challenge fail to show an HGG-specific proliferative response. APC from tolerant or nontolerant mice were equally effective in the support of Ag-specific proliferation of primed T cells. The influence of the thymus gland on HGG-induced T cell unresponsiveness was assessed by determining whether thymectomized mice could be tolerized to HGG. The results suggest that the generation of T cell tolerance to HGG is independent of thymic function as assayed by both antibody production in vivo and T cell proliferation in vitro. Unresponsiveness of T cells from tolerant mice was not a result of the presence of CD8+ cells since removal of CD8+ cells from lymph node T cells did not alter unresponsiveness to HGG in vitro. Further, mixing tolerant T cells with normal HGG-primed T lymphocytes did not inhibit proliferation of the HGG-primed cells. The results of this investigation suggest that this mouse model of tolerance to HGG represents a thymus-independent unresponsiveness of mature peripheral T cells to a nonself-Ag. Understanding the regulation of tolerance to HGG may give additional insight into the mechanisms required for the maintenance and possibly the induction of tolerance to certain self-Ag in peripheral lymphoid organs.     

Maturation of the lymphoid system. I. Induction of tolerance in neonates with a T-dependent antigen that is an obligate immunogen in adults.

A/J mice displayed a striking ontogenetic difference in the capacity to respond to DNP-Ficoll, a T-independent antigen, and to aggregated human gamma-globulin (AHGG), a T-dependent antigen. Thus, whereas responses to DNP-Ficoll of 4-day-old mice were similar in magnitude to those of adult animals, responses to AHGG did not become pronounced until mice were some 30 to 40 days of age. The inability of young animals to respond to AHGG was reflective of a negative consequence of lymphocyte/antigen interaction, since such mice became specifically unresponsive to subsequent challenges with AHGG. Unresponsiveness induced by neonatal injection of AHGG lasted 50 to 60 days, in contrast to that induced by deaggregated HGG, which persisted some 100 days longer. The unresponsive state induced by injection of neonates with AHGG maintained itself upon adoptive transfer and did not appear to be linked to suppressive factors associated with either serum or lymphoid cells for its maintenance. Finally, AHGG was also shown to be capable of inducing unresponsiveness in neonatal, athymic mice. These results demonstrate that AHGG, the normally immunogenic form of HGG in adult mice, can serve as an effective tolerogen when administered into a neonatal environment.     

The effect of aging on the induction of humoral and cellular immunity and tolerance in two long-lived mouse strains.

Aging is a complex process that adversely affects most if not all components of the immune system. In this report, two long-lived mouse strains have been compared in ability to generate both antigen-specific immunity and tolerance. Although CBA/CaJ mice produced high levels of antibody following injection of aqueous preparations of aggregated human gamma-globulin (AHGG), C57BL/6 mice made only meager antibody responses to such preparations. Age dramatically affects the humoral anti-HGG response to aqueous AHGG in both strains, but the meager response of young C57BL/6 mice was at insignificant levels in aged C57BL/6 mice. Conversely, both mouse strains generated good responses following injection of HGG in complete Freund's adjuvant at both the T and B cell level as evidenced by in vitro antigen-specific T cell proliferation and anti-HGG antibody production. Aged mice of both strains showed a marked decrease in the production of serum anti-HGG antibody in comparison to young mice. Although the antigen-specific T cell proliferative response was significantly decreased in aged CBA/CaJ mice, such proliferation was not affected in aged mice of the C57BL/6 strain. Removal of CD8+ cells from lymph node T cells of either young or aged C57BL/6 mice did not increase the antigen-specific proliferative response, suggesting that loss of CD8+ suppressors during the aging process is not responsible for the high level of antigen-specific T cell proliferation in aged C57BL/6 mice. Tolerance to HGG was readily induced in both young and aged C57BL/6 and CBA/CaJ mice although aged mice demonstrate a modest resistance to tolerance induction when compared to their young counterparts. This resistance was observed in both antibody production and antigen-specific T cell proliferation.     

Blockade of physiologically secreted IFN-gamma inhibits human T lymphocyte and natural killer cell activation

The role of physiologically secreted human IFN-gamma in T lymphocyte and NK cell activation has been probed with a panel of mouse mAb directed against various epitopes of the human IFN-gamma molecule, or human IFN-gamma R. Addition to the culture medium of those mAb that neutralize the antiviral activity of IFN-gamma or interact with its receptor inhibited proliferative and cytotoxic responses elicited in PBL by HLA alloantigens, anti-CD3 mAb, and IL-2, but not the proliferative response to PHA. The IFN-gamma blockade also inhibited IFN-gamma, IL-2, and TNF-alpha release during MLC. Kinetic experiments showed that reduction of proliferative and cytotoxic responses to HLA alloantigens is maximal when IFN-gamma is blocked within the first 48 h. Exogenous rIFN-gamma restored the proliferative response only when added at the beginning. Moreover, when IFN-gamma was blocked, T lymphocytes recovered from 6-day MLC displayed a profound decrease in their expression of p55 and p75 chains of the IL-2R, as well as in the number of high-affinity IL-2 binding sites. These findings strongly suggest that IFN-gamma is required in the early phases of induction of the oligo- and polyclonal proliferative and cytotoxic responses of lymphocytes.     

Sheep erythrocyte rosetting induces multiple alterations in T lymphocyte function: inhibition of T cell receptor activity and stimulation of T11/CD2

When T lymphocytes from human blood or lymphoid organs are prepared by the sheep red blood cell (SRBC) rosetting procedure, glycoproteins of the SRBC membrane interact intimately with the CD2 (T11) molecule on the T cell surface. We now show that rosette formation has measurable short- and long-term effects upon the T cells. First, for a period of 24-48 hr after rosetting, the signal transducing and activation functions of the T3/Ti T cell antigen receptor complex is paralyzed for anti-T3-induced calcium mobilization, with a concomitant decrease in proliferative response to mitogens or stimulatory anti-T3 antibodies. Calcium mobilization through the alternate pathway of T cell activation, the T11/CD2 SRBC receptor, was also inhibited by rosetting. Second, rosetting appears to confer a partial stimulatory signal through the T11/CD2 pathway. Thus, 72 hr or more after rosetting, there was increased expression of the T11(3) activation epitope, and rosetted T cells were stimulated to proliferate in the presence of anti-T11(3) antibodies alone. These results provide further details on the effects of SRBC-T cell interactions, with important methodological implications. Moreover, they suggest a hitherto unrecognized down-regulatory effect of engaging the CD2 molecule, and provide further evidence that the T cell receptor is functionally interconnected to the CD2 activation pathway.     

Lysozyme-induced inhibition of the lymphocyte response to mitogenic lectins

Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 micrograms/ml for HL and 1 microgram/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 micrograms/ml HL and 1 microgram/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.     

Requirement of macrophage metabolic activity for T-lymphocyte activation

It has been recently demonstrated that macrophages are necessary accessory cells for T-lymphocyte activation by antigens, mitogens and allogenic cells. This paper shows the function of metabolically inactive macrophages in the activation of T-lymphocytes by phytohemagglutinin (PHA). Macrophages inactivated by mitomycin C are unable to collaborate with T-lymphocytes in the development of the proliferative response to PHA indicating that the role of the macrophage in T-lymphocyte activation by mitogens goes beyond insolubilizing it or rendering it more stimulatory by another passive mechanism.     

Activation of a human T cell subpopulation bearing receptors for autologous erythrocytes by concanavalin A.

Human lymphocytes from different lymphoid organs were examined for rosette formation with autologous erythrocytes. The autorosette-forming cells (A-RFC) were shown to belong to a T cell subset including less mature lymphocytes. When normal human peripheral blood lymphocytes were stimulated with low doses of the plant lectin concanavalin A (Con A), in the presence of autologous plasma, the A-RFC levels were strongly enhanced. This response gave rise to two peaks: the first one coincided with the peak of thymidine incorporation but the maximum increase occurred 5 or 6 days later when the proliferative response was impaired. Depletion of A-RFC before stimulation with Con A led to a clear-cut decrease in autorosette levels at both peaks of the response. It is concluded that Con A, generally used for polyclonal activation against heteroantigens, may also result, in terms of A-RFC marker, in expansion of an autoreactive T cell population.     

Anaphylatoxin-mediated regulation of human and murine immune responses

C3a and C5a derived from the human complement components C3 and C5, respectively, were found to possess immunoregulatory activities. C3a was found to be capable of suppressing both antigen-specific and polyclonal antibody responses. In contrast, C3a was unable to suppress antigen- or mitogen-induced B or T cell proliferative responses. Helper T cells were found to be the target of C3a-mediated immunosuppression. Suppression occurred via the generation of suppressor T cells. In contrast to the results obtained with C3a, C5a was found to augment both antigen-specific and non-specific in vitro humoral immune responses. Moreover, C5a potentiated antigen- and alloantigen-induced T cell proliferative responses. As opposed to C3ades Arg-77, C5ades Arg retained all of the immunoregulatory activity associated with the intact molecule. Helper T cells are required for C5a-mediated potentiation of the Fc fragment-mediated polyclonal antibody response. Substitution for T cells by a soluble T cell-replacing factor rendered lymphocytes refractory to the enhancing properties of C5a.     

Macrophages are required for the dextran-sulfate induced activation of B lymphocytes.

The proliferative response induced by the B cell activating ligand dextran-sulfate (DxS) requires the presence of macrophage-produced factors or the macrophage-substituting compound 2-mercaptoethanol. Thus, the mechanism for activation of mouse B splenic lymphocytes is different for DxS than for other polyclonal B cell activators such as lipopolysaccharides, which can trigger B cells directely in the absence of the helper factors. It is suggested that the disappearance of the DxS-induced response could be studied in a simple functional tests for the efficiency of macrophage depletion obtained by different techniques.     

Immunological unresponsiveness of self-recognizing B lymphocytes to the PBA property of a soluble T cell factor.

Spleen cells cultured in the presence of Con A became activated to polyclonal anti-body synthesis. This effect was found to be mediated by a polyclonal factor released by the activated T cells. Such a factor, contrary to any other polyclonal B cell activator, so far tested, failed to induce resting B cells to synthetize antibodies capable of lysing autologous albumin coupled SRBC. The Unresponsiveness was found to be specific since Con A activated spleen cells were capable of lysing heterologous albumin and HGG coupled SRBC. Moreover, neither active suppressor cells, nor soluble suppressor factors seem to be involved in the lack of response to autologous antigens. It is concluded that the clones of B cells carrying the polyclonal receptor for the T factor and Ig receptors for self are eliminated or functionally inactivated. The implication of these findings for the mechanism of self-non-self discrimination are discussed in relation to the mechanism of immunocyte triggering.     

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

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

Maturation of the lymphoid system. II. Characterization of the cellular levels of unresponsiveness induced in neonates by a T-dependent antigen that is an obligate immunogen in adults.

It has previously shown that AHGG, a form of HGG that is highly immunogenic in euthymic adult mice, is capable of inducing specific unresponsiveness when injected into neonatal animals. This report extends this finding and indicates that such a neonatal treatment results in the induction of tolerance in T as well as B cells. Furthermore, a similar conclusion was reached regarding specific T lymphocyte function in animals treated as neonates with OVA. The ability of LPS to modulate responses of neonatal animals to AHGG or DHGG was also examined. It appeared that such mice were not susceptible to the adjuvant effects of LPS until the 4th week of life. Furthermore, LPS was incapable of inhibiting the unresponsiveness induced in mice by either DHGG or AHGG until the 3rd or 4th week of life.     

In vivo cyclophosphamide-mediated augmentation and aqueous HGG-induced suppression of murine delayed hypersensitivity to HGG are reflected by in vitro HGG-stimulated proliferation of lymph node cells from equivalent mice

The question addressed in this report was whether immunological processes which culminate in in vivo expression and augmentation as well as suppression of delayed effector cell activity are mirrored by events that can be quantified in vitro. For this purpose the previously characterized murine model of delayed hypersensitivity (DHS), which employs SJL/J mice immunized with aggregated human γ-globulin (AHGG) in complete Freund's adjuvant (CFA) was employed. The results indicated that lymph node cells (LNC) from cyclophosphamide (CY)-pretreated, AHGG-CFA immunized mice expressed increased proliferation in the presence of HGG and concanavalin A (Con A) but decreased LPS responsiveness compared with LNC from equivalently immunized but non-CY-treated animals. It was also found that LNC from CY-treated, AHGG-CFA immunized mice that were pretreated with aqueous HGG (aqHGG), but not aqueous bovine serum albumin (aqBSA), evidenced a markedly decreased capacity to proliferate in the presence of HGG compared with LNC from equivalent animals that were not pretreated with aqHGG. This suppressive effect was not attributable to antibody production. These findings support the conclusion that in vitro quantitation of antigen-induced proliferation by LNC from HGG-DHS mice appears to correlate with modulatory effects which are observed in in vivo expression of DHS responses.