T cell regulation of B cell activation. T cells independently regulate the responses mediated by distinct B cell subpopulations

The present studies have been carried out to characterize the regulatory influences acting upon defined pathways of T cell-dependent B cell activation. In these studies, it was demonstrated that high concentrations of free carrier strongly inhibited the MHC-restricted in vitro T cell-dependent antibody responses of primed Lyb-5- B cells to the corresponding carrier-hapten conjugate. In contrast, these same concentrations of free carrier failed to inhibit the T cell dependent responses of Lyb-5+ B cells to the same antigen. The inhibition of Lyb- 5- B cell responses by free carrier was shown to result from active suppression mediated by carrier-specific primed Lyt-1+2- T cells and to require the additional participation of unprimed Lyt-1-2+ T cells. The activation of this suppression was antigen-specific, but suppression once activated was antigen nonspecific in its effect. These findings thus demonstrate that distinct pathways of B cell activation can be independently regulated by T suppressor network influences, and that these pathways therefore constitute potentially independent components of the immune response to a given antigenic stimulus.

BALB/c mice were provided by the Small Animal Section, National Institutes of Health, Bethesda, MD.

Preparation of ceUs
T CELLS. KLH or FGG-primed T cells were obtained as spleen cells nonadherent to antimouse immunoglobulin (anti-MIg) coated plastic dishes by the method of Mage et al. (8) modified as described previously (5).
(B + ACCESSORY) CELLS. (B + accessory) cells were prepared by depleting TNP-KLHprimed spleen cells of T cells by treatment with a T cell-specific cytotoxic rabbit anti-mouse brain serum (RAMB) + complement (C). The T cell specificity of this reagent has been previously characterized (7). CULTURE CONDITIONS. Cultures were carried out as previously described (5). Cells were harvested and assayed for TNP-specific hemolytic plaque-forming cells (PFC).
PFC Assay. Sheep erythrocytes (SRBC) were conjugated with TNP (TNP-SRBC) and direct PFC (IgM) as well as total PFC (facilitated by rabbit anti-MIgG) were assayed on these conjugated target cells (5). IgG PFC were calculated as total PFC minus direct PFC. All points shown represent the arithmetric mean responses of triplicate or quadriplicate cultures.

High Concentrations of Free Carrier Inhibit the T Cell-Dependent Activation of Lyb-5-B
Cells. T-dependent and predominantly IgG PFC responses are generated in vitro by primed B cells responding to low concentrations of carrier-hapten conjugate. These responses have been shown to require MHC-restricted T-B interaction as well as carrier-hapten linkage, and to be mediated by Lyb-5-B cells (5,6). In contrast, predominantly IgM responses activated at higher concentrations of antigens are equally T dependent but require neither carrier-hapten linkage nor MHC-restricted T-B interaction, and are mediated by the activation of Lyb-5 + B cells (5,6). Consistent with these findings, it was observed in the present studies that KLH-primed T cells cooperated equally well with T-depleted (B + accessory) spleen cell populations from TNP-primed CBA/N (a mutant defective strain expressing only the Lyb-5-B cell subpopulation) or CBA/CaHN (a nondefective strain expressing both Lyb-5 + and Lyb-5-B cell subpopulations) animals to generate predominantly IgG PFC in response to 0.001 pg/ml TNP-KLH (Table I). In contrast, a predominantly IgM response to 10/~g/ml TNP-KLH was generated only in normal CBA/CaHN B cells and not in defective CBA/N populations, demonstrating a requirement for Lyb-5 + B cells in the pathway activated under these conditions (Table I).
To further analyze the regulatory role of antigen concentration in these pathways of B cell activation, graded concentrations of free carrier KLH were added to cultures of TNP-KLH-primed CBA/N or CBA/CaHN spleen cells responding to either high or low concentrations of TNP-KLH. The IgG responses of both CBA/N and CBA/  CaHN spleen cells to 0.001 #g/ml TNP-KLH were consistently inhibited by concentrations of 10-100 #g/ml KLH (Fig. 1A). In contrast, the IgM response of CBA/ CaHN spleen cells to 20 #g/ml TNP-KLH ( Fig. 1 B) or to 0.001 #g/ml TNP-KLH (data not shown) was not significantly affected by the addition of free KLH (Fig.

B).
To test the antigen specificity of this observcd inhibition, the effects of gradcd doses of KLH or FOG were examined on the IgG responses of primed spleen cells to either TNP-KLH or TNP-FGG. Responses to 0.001 #g/ml TNP-KLH were generated by TNP-KH-primed B10.A spleen cells, but not by TNP-FGG-primed cells, confirming the specificity of priming in thcse populations ( Fig. 2A). These responses were inhibited by high concentrations of KLH but not by equivalent concentrations of FGG ( Fig. 2 A). In contrast, responses of specifically primed spleen cells to TNP-FGG were inhibited by FGG but not by KLH (Fig. 2B). These results thus dcmonstrate that ffce carrier induces in an antigen-specific fashion thc inhibition of rcsponses by Lyb-5-B cells to carrier-hapten conjugates.

Response Inhibition by Free Carrier is Mediated by Antigen-specific Suppressor Cells. Further
studies were next carried out to examine the mechanism by which high concentrations of free protein carrier inhibit the IgG responses of Lyb-5-B cells. Experiments were first designed to distinguish between an active mechanism of response suppression and inhibition resulting from competition between free carrier and carrier-hapten conjugate. KLH-primed B10.A T cells cooperated with syngeneic TNP-primed (B + accessory) cells for responses to 0.001/~g/ml TNP-KLH; and, in the absence of FGGprimed T cells, this response was not inhibited by the irrelevant carrier FGG (Fig.  3 A). However, when graded numbers of FGG-primed B10.A T cells were added to these cultures, high doses of FGG efficiently suppressed the response to TNP-KLH (Fig. 3A). KLH-primed T cells were similarly able to actively suppress the response to TNP-FGG in the presence of high doses of KLH (Fig. 3 B). These results indicate that the inhibition of PFC responses by free carrier is in fact mediated by active suppression, and that this suppression is generated by carrier-primed T cells only in the presence of the specific carrier protein. Moreover, whereas the induction of this suppression in primed T cells is antigen specific, the effect of suppression, once generated, is antigen nonspecific.

Suppression by Free Carrier Requires Interaction Between Lyt-l+2 -and Lyt-l-2 + T Cells.
To determine the cellular interactions involved in this antibody response suppression, the Lyt phenotypes of the T cell subpopulations required for suppression were analyzed. To further evaluate the requirement demonstrated above for the participation of Lyt-2 + cells in carrier-induced suppression, TNP-KLH primed B 10.A spleen cells were treated with monoclonal anti-Lyt-2.2 + C and were then tested for their susceptibility to suppression by free carrier. Anti-Lyt-2.2 + C-treated spleen cells were competent to respond to TNP-KLH, demonstrating that T helper cells functioning in this response are Lyt-2- (Fig. 4). In contrast, the ability of free KLH to suppress this response was completely eliminated by this pretreatment, indicating that Lyt-2 + cells are required for suppression (Fig. 4). Because the data presented above demonstrated that a population of specific carrier-primed T cells was required for suppression of antibody responses, the priming requirements of this Lyt-2 + population were examined. The susceptibility of TNP-KLH-primed B 10 spleen cells to inhibition by high concentrations of KLH was first abrogated by treatment of KLH-primed T cells with anti-Lyt-2.2 + C, and the ability of unprimed B10 T cells to reconstitute this suppression was tested. It was found that the addition of unprimed T cells reconstituted the susceptibility to suppression, and that Lyt-l-2 + cells were responsible for this reconstitution (Table II). These results suggested that both an unprimed Lyt-1-2 + T cell population and an additional carrier-primed T cell population were necessary for suppression.
To further characterize the populations involved in carrier-induced suppression,  KLH-primed B10 T cells and FGG-primed B10 T cells were cocultured with TNPprimed (B + accessory) cells stimulated with 0.001 #g/ml TNP-KLH with or without FGG. The helper T cells functioning under these conditions were Thy-1 +, Lyt-l+2 -, because treatment of KLH-primed T cells with either anti-Thy-l.2 + C or anti-Lyt-1.2 + C eliminated their helper activity, whereas anti-Lyt-2.2 + C did not (Table III, groups H, I, N). In the presence of untreated KLH-primed helper T cells, FGGprimed T cells induced suppression in the presence of FGG (Table III, group C), and pretreatment of FGG-primed T cells with anti-Lyt-2.2 + C did not affect this suppression (Table III, group D). Under these conditions, however, treatment of the FGG-primed T cells with either anti-Lyt-l.2 + C or anti-Thy-l.2 + C completely abrogated suppression, and a mixture of these treatment populations was also unable to induce suppression (Table III, groups E, F, G). These results demonstrated that carrier-primed Thy-1 +,Lyt-1 +2-T cells were required for the induction of suppression.
After treatment of the KLH-specific helper T cells with anti-Lyt-2.2 + C, untreated FGG-primed T cells still suppressed responses to TNP-KLH in the presence of FGG (Table III, group J). Treatment of the FGG-primed T cells with either anti-Lyt-l.2 + C or anti-Lyt-2.2 + C eliminated suppression under these conditions (Table III, groups K, L), but a mixture of the residual cells after treatment with anti-Lyt-l.2 + C or anti-Lyt-2.2 + C reconstituted the suppression (Table III, group M). These results indicate that interaction between Lyt-l+2 -cells and Lyt-l-2 + cells is required for the suppression of IgG responses in the presence of free carrier. This interaction further requires that the Lyt-1 +2-cell population be specifically primed to the carrier inducing suppression, whereas the Lyt-1-2 + cells express no such specificity.

Discussion
Two distinct pathways have been described for the TH cell-dependent activation of different B cell subpopulations (4)(5)(6). The activation of Lyb-5-B cells requires the MHC-restricted interaction of TH cells with this B cell subset. In contrast, Lyb-5 + B Anti-Thy-I + C --43 ± 21 ND ND * 2 X 106 T cells from KLH-primed BI0 spleens were treated as indicated and co-cultured with 2 X 106 TNP-primed, RAMB + C-treated B 10 spleen cells. :~ 1 X 106 T cells were added. § 0.001 #g/ml TNP-KLH.
II 100 #g/ml FGG. ¶ Responses to TNP-KLH + FGG were compared to responses to TNP-KLH alone. The activation of suppression was antigen specific, requiring the presence of carrierprimed T cells and a high concentration of the corresponding carrier. Once activated, however, the effect of this suppression was antigen nonspecific. The generation of carrier-induced suppression required the interaction of at least two T cell subpopulations, an antigen-specific (carrier-primed) Lyt 1+2 -T cell and an Lyt-l-2 + T cell population which exhibited no such specificity.
A number of alternative explanations may account for the selective effect of carrierinduced suppression on responses of the Lyb-5-B cell subset. In several previously characterized systems, it has been concluded that Ts cells act upon the Tu cell populations involved in antibody response (10)(11)(12)(13)(14)(15)(16). If such a mechanism were involved in the suppression described here, its selective effect could result from the participation of different TH cell populations in the T dependent activation of Lyb-5-and Lyb-5 ÷ B cells. Alternatively, a single TH cell functioning in responses of both B cell subsets might be selectively inhibited in its MHC-restricted activation of Lyb-5-B cells. A second possibility is that carrier-induced suppression acts directly upon responding B cells and acts to specifically suppress the Lyb-5-subpopulation. It is relevant in this regard that the same exposure to high concentrations of free carrier that induces suppression of Lyb-5-B cell responses can also trigger TH cell activity sufficient to activate responses by Lyb-5 + B cells (5,6). Under these conditions, however, the suppression of Lyb-5-responses requires the participation of Lyt-2 ÷ T cells, whereas the activation of Lyb 5 + B cells exhibits no such requirement (Y. Asano, unpublished data), demonstrating that the signals mediating these two regulatory events are in fact different. The activation of Lyb-5-B cells in response to T-dependent antigens such as TNP-KLH requires the function of carrier-primed Lyt-l÷2 -TH cells. The suppression of these same antibody responses by high doses of carrier requires the participation of an antigen-primed Lyt-1+2 -T cell and an unprimed Lyt-1-2 + T cell. The Lyt phenotype of the first Ts population is thus identical to that of the TH ceils functioning in these responses. It is of interest, however, that the activation requirements demonstrated for TH function and for Ts function in these responses are significantly different. Antigenspecific triggering of Lyt-1 +2-Ts activity occurs in response to free carrier and results in the subsequently antigen-nonspecific suppression of Lyb 5-B cell responses. In contrast, the effector function of TH cells for the activation of the same Lyb-5responses has been shown to be highly antigen specific, as reflected in the absolute requirement for carrier-hapten linkage in these responses (6). Efforts to determine whether these functions are mediated by the same or distinct T cell subpopulations are currently in progress through the use of additional cell surface phenotyping and with monoclonal TH cell populations (6,17).
The regulatory network described in this study resembles in several respects the system of antibody response suppression initially defined by Eardley and co-workers (14), as well as the Ts cell pathways subsequently characterized by a number of investigators (10-13, 15, 16). In each of these systems, a requirement has been demonstrated for interactions among T cell subsets in the mediation of suppression. In several systems, this suppressor network is initiated by the antigen-specific triggering of a primed Lyt-l+2 -"afferent suppressor" or Ts1 population (12)(13)(14)16), consistent with the requirement for such a population demonstrated in the present report. In this work, an additional requirement was found for an Lyt-l-2 ÷ cell that did not require specific priming. This second T cell subpopulation is analogous to the "efferent suppressor" or Ts2 also identified in a number of regulatory networks (10,(12)(13)(14)(15)(16). In contrast to a number of previous reports (10,(12)(13)(14)(15)(16), however, no requirement for an Lyt-l+2 + cell was identified in the present work, a finding that may reflect only quantitative differences in the susceptibility of certain Lyt-1 + cells to lysis by antibody and C (18).
Recent findings have demonstrated the existence of two distinct pathways of Tdependent B cell activation. The present studies have revealed an additional dimension of this complexity in demonstrating that distinct B cell activation pathways can be independently regulated by Ts cell network influences. Thus, the MHC-restricted TH cell activation of Lyb-5-B cells is regulated by a Ts cell network, which is without apparent effect on the responses of Lyb-5 + B cells to the same antigen. Studies are currently in progress to further analyze the genetically restricted cell recognition events and effector mechanisms involved in this highly specific mode of immune response regulation.

Summary
The present studies have been carried out to characterize the regulatory influences acting upon defined pathways of T cell-dependent B cell activation. In these studies, it was demonstrated that high concentrations of free carrier strongly inhibited the MHC-restricted in vitro T cell-dependent antibody responses of primed Lyb-5-B cells to the corresponding carrier-hapten conjugate. In contrast, these same concentrations of free carrier failed to inhibit the T cell dependent responses of Lyb-5 + B cells to the same antigen. The inhibition of Lyb-5-B cell responses by free carrier was shown to result from active suppression mediated by carrier-specific primed Lyt-1 +2-T cells and to require the additional participation of unprimed Lyt-l-2 + T cells. The activation of this suppression was antigen-specific, but suppression once activated was antigen nonspecific in its effect. These findings thus demonstrate that distinct pathways of B cell activation can be independently regulated by T suppressor network influences, and that these pathways therefore constitute potentially independent components of the immune response to a given antigenic stimulus.