Antigen-induced T suppressor cells regulate the autoreactive T helper-B cell interaction

The T suppressor (Ts) cell population that functions to regulate antigen-specific MHC-restricted T helper (Th)-B cell interactions also regulates the activation of B cells by cloned autoreactive Th cells. Activated Ts cells were generated by in vivo priming and restimulation in vitro with high concentrations of the specific priming antigen. Once generated, this Ts population inhibits the Th-dependent activation of primed B cells by both antigen-specific and autoreactive T cells in an antigen-nonspecific manner. This suppression requires the participation of both Lyt-1+2- and Lyt-1-2+ T cells. It was also demonstrated that accessory cells were required for the induction of Ts cells. Moreover, the generation of suppression was MHC-restricted and required the recognition by T cells of Ia antigens on accessory cells. These studies demonstrate that the same or a very similar Ts cell population can function to inhibit the activation of B cells by antigen-specific as well as autoreactive T cells.     

Analysis of two distinct B cell activation pathways mediated by a monoclonal T helper cell. II. T helper cell secretion of interleukin 4 selectively inhibits antigen-specific B cell activation by cognate, but not noncognate, interactions with T cells

A single monoclonal T helper (Th) clone can activate B cells in two distinct pathways; a cognate pathway requiring a major histocompatibility complex (MHC)-restricted T-B cell interaction, and a noncognate pathway not requiring an MHC-restricted T-B cell interaction. The present study was undertaken to investigate whether Th cells mediating a given immune response provide further regulatory function to B cells other than helper function. It was demonstrated that conditions of high antigen concentration which activate a noncognate B cell activation pathway simultaneously inhibit IgG responses. The inhibition is shown to be mediated by the T cell factor interleukin 4, produced by activated cloned Th cells. The inhibitory effect of this factor is directed to B cells and is MHC-unrestricted, antigen-nonspecific, and IgG class-specific. In addition to being susceptible to the effects of augmenting cells and suppressor cells, cloned Th cell populations can therefore themselves function as regulatory cells to inhibit IgG responses when stimulated with high dose of specific antigen. These results indicate that Th cells function to regulate B cells both positively and negatively, depending upon the activation conditions.     

Lyb-5+ B cells can be activated by major histocompatibility complex-restricted as well as unrestricted activation pathways

It has previously been demonstrated that B cells can be activated through two distinct T helper (Th) cell-dependent pathways, one requiring both carrier-hapten linkage and MHC-restricted T-B interaction and the other requiring neither. In addition, it has been shown that different B cell subpopulations exist and that these subpopulations differ in their activation requirements. Previous studies demonstrated that resting B cells containing an Lyb-5+ subpopulation were activated by MHC-unrestricted T cell signals, whereas resting Lyb-5- B cells were activated only through MHC-restricted T-B interaction. It was suggested that this difference resulted from the ability of Lyb-5+ but not Lyb-5-B cells to respond to soluble MHC-unrestricted Th signals. Because Lyb-5+ B cells were responsive in these previous experiments to MHC-unrestricted Th signals, it could not be determined whether Lyb-5+ B cells were also responsive to MHC-restricted Th signals. Consequently, the present study was undertaken to directly address the question of whether Lyb-5+ B cells can be activated under appropriate conditions by MHC-restricted as well as unrestricted T cell-B cell interactions. It was found that unprimed normal B cells (containing Lyb-5+ and Lyb-5-B cells) but not unprimed xid-defective populations (Lyb-5- only) can be activated by cloned KLH-specific and MHC-restricted Th cells in response to either high or low concentrations of TNP-KLH. The IgM response of Lyb-5+-containing B cells to a high concentration of antigen (10 micrograms/ml) was MHC unrestricted, whereas the IgM response of unprimed Lyb-5+ B cells to a low concentration of antigen (0.001 micrograms/ml) was MHC restricted. Thus, unprimed Lyb-5+ B cells can be activated through both MHC-restricted and unrestricted pathways. It was further demonstrated that the activation requirements of Lyb-5+ and Lyb-5- B cells differed even for MHC-restricted B cell activation.     

T cell regulation of antibody responses: an I-A-specific, autoreactive T cell collaborates with antigen-specific helper T cells to promote IgG responses

In earlier studies we showed that hapten-specific inducer T cell clones specifically induce B cells from immunized donors to secrete IgM antibodies. However, IgG responses were not observed, suggesting that an additional signal(s) was required. In this report, we show that an autoreactive T cell clone produces a factor(s) that collaborates with antigen-specific inducer T cells to promote specific IgG responses. This factor is not restricted by antigen or MHC determinants and promotes IgG production both in vivo and in vitro. These findings suggest that autoreactive cells may play an important role in the regulation of isotype expression.     

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. I. Destruction of B16 melanoma cells mediated by bystander cytolysis in vitro

Two Lyt-1+, L3T4a+ autoreactive T cell clones specific for self-class II major histocompatibility complex (MHC) gene products were established from lymph node cells and spleen cells of C57BL/6J mice, respectively, by different methods. They were stimulated to proliferate in culture in response to I-Ab antigen-bearing syngeneic spleen cells in a class II MHC-restricted manner. This stimulation was inhibited completely by the addition of anti-L3T4a (GK1.5) or anti-I-Ab (3JP) monoclonal antibodies. The autoreactive T cell clones lysed syngeneic I-Ab+ target cells such as lipopolysaccharide (LPS) blasts. They also lysed I-A- bystander cells such as Cloudman and B16 melanoma and lymphoid tumor cells in the presence of I-Ab+ stimulator cells but not I-Ad+ cells. This bystander killing was most likely mediated by soluble factors released from the autoreactive T cells in response to I-Ab antigens, because culture supernatants from activated autoreactive T cells inhibited the proliferation of B16 melanoma cells in vitro and also had significant cytolytic activity. Both lymphotoxin and interferon-gamma were released from activated autoreactive T cells, suggesting that these cytotoxic lymphokines were responsible for autoreactive T cell-mediated cytolysis. The finding that the two clones, established independently and by different methods, show self-class II MHC antigen-restricted cytolysis, and bystander cytolysis suggests that these properties are not restricted to a unique population of autoreactive T cells. These results favor the concept that in vivo, autoreactive T cells may express not only regulatory activity in regard to antibody responses, but also anti-tumor activity via bystander cytolysis.     

IL 2 restores memory B cell activation by antigen-specific T cell clone variants

It has recently been demonstrated that there are at least two separate pathways by which a single keyhole limpet hemocyanin (KLH) reactive T cell clone can induce B cell differentiation. With the use of the high-dose antigen-driven system (10 micrograms/ml trinitrophenyl (TNP)-KLH), a KLH-specific T cell clone was able to induce a primary anti-TNP response in unprimed B cells. In the presence of aliquots of the same T cell clone, a low-dose of antigen (5 X 10(-2) micrograms/ml TNP-KLH) induced an immunoglobulin (Ig)G response in primed B cells. It has also been demonstrated that there are variant subclones of such KLH-specific helper T cell clones that are unable to provide antigen-specific help in the presence of low-dose antigen but maintain the high-dose antigen-driven helper response. This study was undertaken to investigate whether interleukin 2 (IL 2) had some activity in the low-dose, antigen-driven response induced by the T cell clone. With the use of a variant T cell clone (which lost low-dose, antigen-driven helper activity), it was demonstrated that IL 2 was capable of reconstituting the low-dose, antigen-driven helper activity. To investigate whether accessory cells were required in this system, we removed the adherent cell population from the primed spleen cells added to culture. Interestingly, removal of the G10-adherent cells eliminated the low-dose, antigen-driven response induced by IL 2. Additionally by add-back experiments, we were able to demonstrate that the necessary adherent cell population did not require major histocompatibility complex (MHC) restriction for reconstitution of the IL 2-dependent, low-dose, antigen-driven response. Furthermore, 1% concanavalin A (Con A) supernatant (Sn), but not interleukin 1 (IL 1), could replace this adherent cell function. These data suggest that in this system, IL 2 bypasses the MHC-restricted interaction between T cells and antigen-charged adherent cells; B cells can present antigen to cloned helper T cells efficiently for primary responses but need an added factor(s) to induce IgG production; and adherent cells are essential for IgG production in primed B cells, possibly through the release of soluble factor(s) included in Con A Sn.     

Induction of IgG and IgE synthesis in normal B cells by autoreactive T cell clones

During the course of generating tetanus toxoid (TT)-specific T cell clones frm an HLA-DR2,7 donor, four clones were obtained which proliferated in the presence of autologous monocytes alone without the addition of TT antigen. This proliferation was specifically inhibited by anti-HLA-DR framework mouse monoclonal antibody, and appeared to be HLA-DR-restricted. Two of the clones proliferated in response to HLA-DR2-bearing monocytes, and the other two clones proliferated in response to HLA-DR7-bearing monocytes. The capacity of these four autoreactive human T cell helper clones to induce IgE synthesis in B cells was studied. All four clones stimulated autologous peripheral blood B cells to synthesize IgE and IgG antibody. Induction of IgE synthesis in B cells by the autoreactive T cell clones followed the same pattern of HLA-DR restriction which governed the proliferative response of these clones. These results suggest that the interaction of autoreactive helper T cells with B cell HLA-DR antigens may be important in the activation of IgE immune responses in humans.     

An autoreactive T cell clone that can be activated to provide both helper and suppressor function

To understand further the biologic significance of the autologous mixed lymphocyte reaction, we determined the functional properties of autoreactive T cell lines and clones. Initially, we found that cells in an uncloned autoreactive Leu-3+ T cell line helped immunoglobulin production when added to cultures containing fresh T and non-T cells in the absence of pokeweed mitogen (PWM) but suppressed immunoglobulin production in the same cultures in the presence of PWM. To explain this phenomenon, we studied the immunoregulatory potential of an autoreactive T cell clone termed MTC-4. This clone bore the phenotype Leu-3+, 2-, 8-, 11-, DR+ and underwent proliferation when co-cultured with autologous, but not allogeneic non-T cells. Of interest, the immunoregulatory potential of the MTC-4 cells varied according to how the cells were activated. When MTC-4 cells were cultured with autologous non-T cells in the absence of antigen or mitogen (unactivated non-T cells), polyclonal immunoglobulin production (detected by reverse PFC assay) was observed. This helper activity was MHC-restricted in that it was elicited only by autologous non-T cells or MHC-matched allogeneic non-T cells; however, once activated by autologous non-T cells, it could also help allogeneic non-T cells. In contrast, when MTC-4 cells were cultured with autologous non-T cells in the presence of PWM (activated non-T cells), immunoglobulin production was greatly suppressed. This suppression was also observed when MTC-4 cells were added to cultures containing exogenous T cell help (such as that provided by autologous fresh T cells) and was not due to a direct effect of PWM on the T cell clone, because preincubation of MTC-4 cells with PWM before culture with non-T cells did not result in suppression. On the basis of these data, we conclude that autoreactive T cells can have dual immunoregulatory function that is manifest, at least in part, at the single cell level. Moreover, these regulatory functions are differentially elicited depending on the state of activation of the stimulating autologous non-T cells: when stimulated by MHC antigens present on unactivated B cells, they provide helper activity; and when stimulated by MHC antigens present on activated B cells, they act as suppressor cells. Autoreactive T cells with dual regulatory potential appear to make up a substantial proportion of all autoreactive T cells and are cells that are uniquely adapted to maintain immunologic homeostasis.     

Cellular interactions of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor factors. I. Inhibition of the activity of GAT-specific helper T cell clones by monoclonal GAT-specific suppressor T cell factors

Considerable information concerning the serology and biochemistry of antigen-specific, T cell-derived suppressor factors has been obtained with the use of T cell hybridomas as a source of homogeneous material. Similarly, knowledge of helper T cell products and receptors is accumulating from studies of helper T cell clones and hybridomas. Our strategy for studying the mechanisms by which suppressor factors inhibit responses was to determine whether monoclonal suppressor factors could inhibit antibody responses specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in cultures containing unprimed splenic B cells, macrophages, and GAT-specific T cell clones as a source of helper activity. The MHC-restricted, two chain suppressor factors, GAT-TsF2, inhibited these responses if the helper T cell clones and suppressor factor were derived from H-2-compatible mice. Furthermore, responses were inhibited by briefly pulsing T cell clones with GAT-TsF2 in the presence of GAT, indicating that suppressor factors need not be present continuously. In addition, helper T cell clones adsorbed syngeneic, but not allogeneic, GAT-TsF2 in the presence of GAT. Adsorption also requires a shared antigenic specificity between the H-2b-derived helper T cells and TsF2 factor. Thus, helper T cells can serve as the cellular target of antigen-specific, MHC-restricted GAT-TsF2, and cloned helper T cells can be used as a homogeneous target population for analysis of the molecular mechanisms of T cell suppression.     

Cloned allospecific human helper T cell lines induce an MHC-restricted proliferative response by resting B cells

To analyze helper T (Th) cell-induced B cell proliferation in man, we have cloned allospecific Th cells, grown them as long-term IL 2-dependent T cell lines (TCL), and analyzed their phenotypic and functional properties. The two TCL described in this report, A-7 and A-57, are both composed exclusively of T3+, T4+, T8- T cells blasts. In proliferative assays, with a panel of x-irradiated allogeneic stimulator cells, A-7 was found to proliferate in response to DR3-bearing cells, whereas A-57 responds to DR2-positive stimulators. Both TCL are capable of providing MHC-restricted polyclonal help for allogeneic B cells, as measured in the reverse hemolytic plaque assay. Of greater interest, x-irradiated A-7 and A-57 cells are capable of inducing a proliferative response by allogeneic B cells that is absolutely MHC restricted at the inductive (Th-APC) level. Thus, x-irradiated A-7 cells only trigger proliferation by DR3+ B cells, whereas A-57 cells selectively activate DR2+ B cells. In contrast, after antigen-specific activation, x-irradiated A-7 and A-57 cells can recruit a significant proliferative response by allogeneic B cells bearing "irrelevant" DR antigens. The possibility that Th-induced B cell proliferation may be restricted at the effector (Th-B cell) level was addressed by fractionating B cell populations into "activated" and "resting" subsets by discontinuous Percoll density gradient centrifugation and further purification by employing a monoclonal antibody directed against an antigen expressed on activated B cells (4F2). These studies demonstrate that activated B cells are readily and nonspecifically recruited to proliferate by activated Th cells, whereas optimal proliferative responses by resting B cells require MHC restricted Th-B cell interaction.     

Analysis of two distinct B cell activation pathways mediated by a monoclonal T helper cell. I. MHC-restricted activation of B cells by an IL 2-dependent pathway

The present study was carried out to determine whether the MHC-restricted and MHC-unrestricted B cell activation pathways mediated by a single cloned Th cell are separable, and whether these two pathways are mediated by distinct mechanisms. It was demonstrated that the two B cell activating functions of a single cloned Th cell could be separated by their sensitivity to irradiation. It was shown that MHC-restricted B cell activation is mediated by a radiosensitive Th cell function, whereas MHC-unrestricted B cell activation is mediated by a radioresistant function of the same Th cell. In addition, it was shown that recombinant IL 2 can restore or replace the radiosensitive component of MHC-restricted cognate helper function.     

Antigen processing requirements for T cell activation: differential requirements for presentation of soluble conventional antigen vs cell surface MHC determinants

The present studies were undertaken to characterize the antigen-processing requirements involved in the responses to T cells to soluble antigen (antigen specific), to allogeneic cell surface MHC determinants (alloreactive), and to syngeneic MHC determinants (autoreactive). T cell clones were used that have dual cross-reactive specificities either 1) for self MHC plus soluble antigen and for allogeneic MHC products or 2) for syngeneic MHC and for allogeneic MHC, in order to permit comparison of the processing requirements for responses of the same T cell to distinct antigenic stimuli. The proliferative responses of antigen-specific, Ia-restricted T cell clones to soluble antigens were sensitive to treatment of antigen-presenting cells (APC) with 125 to 250 microM chloroquine, a lysosomotropic agent previously shown to inhibit the processing of soluble antigens. In contrast, the same T cell clones were only minimally affected in their ability to respond to similarly chloroquine-treated APC expressing allogeneic MHC products. The responses of autoreactive T cell clones to syngeneic stimulating cells and their cross-reactive responses to allogeneic cells were both resistant to chloroquine treatment of stimulating cells. The failure of chloroquine to inhibit antigen presentation to autoreactive T cell clones suggests that these clones are specific for self Ia not associated with in vitro processed foreign antigen. Thus, chloroquine sensitivity distinguishes the in vitro antigen-processing requirements for presentation of the soluble antigens tested from the requirements for presentation of syngeneic or allogeneic cell surface MHC determinants to the same T cells.     

Mechanisms of T and B cell collaboration in the in vitro production of anti-DNA antibodies in the NZB/NZW F1 murine SLE model

B/W mice spontaneously develop IgG antibodies to DNA that cause lethal immune nephritis. T and B cell interactions in the in vitro anti-DNA antibody response of B/W mice were investigated, and two distinct families of helper T cells that drive these responses were defined. First, the anti-DNA antibody-forming cell (AFC) response was found to be increased in B/W mice with nephritis and was inhibited with the monoclonal antibody anti-L3T4, suggesting a major role for helper T cells. Purified splenic T cells from mice with nephritis were able to augment both the IgG and the IgM anti-DNA AFC response of young B/W B cells. T helper cells were cloned from spleens of NZB/W F female mice with high titer anti-DNA antibodies and nephritis. The cloned T cells augmented both IgG and IgM anti-DNA AFC responses of young B/W B cells. Four clones--27.9, 30.7, 30.8, and 30.10--were selected for further study. These cells proliferated, in the context of syngeneic (H2d/z) antigen-presenting cells (APC) but not to allogeneic APC. Analysis of the mechanism of T helper cell clone-mediated augmentation of anti-DNA AFC revealed two populations: "cognate" T helper cells, which specifically augment anti-DNA AFC (30.7 and 30.10), and non-antigen-specific T helper cells (27.9 and 30.8), which augment the response of B cells of differing specificity by a bystander mechanism, probably through increased release of B cell growth and differentiation factors.     

Staphylococcal enterotoxin B stimulates expansion of autoreactive T cells that induce apoptosis in intestinal epithelial cells: regulation of autoreactive responses by IL-10

T cell responses to self Ags and normal microbial flora are carefully regulated to prevent autoreactivity. Because IL-10-deficient mice develop colitis, and this response is triggered by luminal flora, we investigated whether IL-10 regulates the ability of microbial Ags to induce autoreactive T cells that could contribute to intestinal inflammation. T cells from wild-type mice were primed with staphylococcal enterotoxin B (SEB) in vitro, which induced an autoreactive proliferative response to syngeneic feeder cells. The cells were predominately CD3+ and CD4+. T cells from IL-10-deficient mice were constitutively autoreactive, and SEB priming enhanced this further. The autoreactive, proliferative response of T cells from wild-type mice was suppressed by IL-10 in the primary or secondary culture, and this effect was inhibited by neutralizing Abs to the IL-10R. To confirm that an autoreactive repertoire was expanded after SEB priming, we used CBA/J mice (Mls-1a) in which autoreactive T cells recognizing the endogenous viral superantigen are depleted (Vbeta6, 7, 8.1 TCR-bearing cells). However, SEB rescued these autoreactive T cell repertoires. Adding anti-MHC class II Ab blocked the autoreactive response. SEB-primed splenic or colonic T cells also induced apoptosis in syngeneic intestinal epithelial cells that was blocked significantly by IL-10. Thus, microbial Ags have the potential to abrogate self tolerance by stimulating autoreactive T cells that become cytolytic to target cells. IL-10 plays a protective role in maintaining self tolerance after microbial stimulation by preventing the activation of T cells that contribute to epithelial cell damage.     

Characterization and function of autoreactive T-lymphocyte clones isolated from normal, unprimed mice

Self-Ia-reactive cloned T-cell lines, designated PK, were established by long-term culture of T cells from normal DBA/2 mice with irradiated syngeneic splenic adherent cells (SAC), rich in macrophages and dendritic cells. The cell lines were Thy 1+, Lyt 1+, Lyt 2-, produced IL-2 following stimulation with syngeneic spleen cells, and did not exhibit alloreactivity when screened against six different H-2 haplotypes. Of the five cloned PK cell lines tested, four were I-Ed restricted while one was I-Ad restricted as determined by genetic mapping and blocking studies carried out with monoclonal anti-Ia sera. Extensive specificity studies suggested that the PK cells reacted to syngeneic Ia molecules alone and not to foreign antigens such as fetal calf serum (FCS) used in the culture medium, in association with self-Ia. SAC pulsed with FCS or other protein antigens such as turkey gamma-globulin (TGG) were tested for their ability to induce proliferation of autoreactive T cells and other antigen-specific T cells using culture conditions consisting of serumless medium and interleukin 2 (IL-2). The data showed that the autoreactive T cells proliferated better in response to antigen-unpulsed SAC, while FCS-specific and TGG-specific cell lines, developed independently, proliferated only in response to FCS- or TGG-pulsed SAC, respectively, but not to antigen-unpulsed SAC. These results clearly distinguished the autoreactive T-cell clones from the antigen-specific T-cell clones. Preliminary studies carried out to investigate the functions of autoreactive T cells suggested that these cells helped in the in vitro differentiation of alloantigen-specific cytotoxic T lymphocytes (CTL) from CTL precursors obtained from the thymus and augmented syngeneic, allogeneic, and antigen-specific immune responses in vitro. The autoreactive T cells were also capable of inducing both proliferation and differentiation of antigen-specific populations of B cells in the absence of antigen. The present investigation suggests that autoreactive, non-antigen-reactive T cells can be cloned from normal, unimmunized mice and that such cell lines may provide a powerful tool for analyzing the role of the syngeneic mixed lymphocyte reaction in induction and maintenance of both T-and B-cell immune responses.     

T cell regulation of B cell activation. An antigen-mediated tripartite interaction of Ts cells, Th cells, and B cells is required for suppression

To determine the requirements underlying the antigen specificity observed in T cell-mediated immune response suppression, cloned major histocompatibility complex (MHC)-restricted T suppressor (Ts) cells specific for keyhole limpet hemocyanin (KLH) and cloned MHC-restricted T helper (Th) cells specific for fowl gamma-globulin (FGG) were employed to study the regulation of trinitrophenyl (TNP)-specific B cell responses. Neither antigen bridging between Ts cells and Th cells (FGG=KLH) nor bridging between Ts cells and B cells (TNP-KLH) was sufficient to allow suppression; a mixture of FGG=KLH and TNP-KLH was also insufficient for suppression. In contrast, suppression was induced by KLH-specific Ts cells only when suppressor determinants (KLH), helper determinants (FGG), and B cell determinants (TNP) were covalently linked on the same molecule (TMP-FGG)=(TNP-KLH) or TNP-(FGG=KLH)). These findings imply that a tripartite antigen-mediated interaction of Ts cells, Th cells, and responding B cells is necessary for the mediation of this antigen-specific suppression.     

T lymphocyte heterogeneity in the rat. III. Autoreactive T cells are activated by B cells

Evidence has been presented to show that CD4+ autoreactive T cell lines (ATs)2 in the rat require periodic stimulation with syngeneic spleen cells for in vitro proliferation. This proliferation can be blocked by treatment of the stimulator (spleen) cells with mAb to Ia antigens. Although ATs are Ia+ and can activate the allogeneic MLR, they fail to be autostimulatory. Fractionation of the spleen cells revealed that ATs can be stimulated with B cells and not by macrophages, although the latter were efficient in several accessory cell functions, including antigen presentation, lectin-dependent T cell activation and allogenic MLR response. Moreover, B cells proliferated and differentiated in response to AT cells. These data are compatible with a model in which ATs respond to hitherto undetermined B cell membrane antigen(s) in association with MHC class II antigens. These results may have important implications in understanding autoimmune responses.     

Antigen-specific and polyclonal immunoglobulin production induced by a cloned tetanus toxoid-specific T cell line

This report describes the isolation and the phenotypic and functional characterization of a cloned, IL 2 dependent, TT-specific human helper T cell line (TCL), designated 1A1. 1A1 was derived by limiting dilution culture of a bulk IL 2-dependent TCL that was found to contain both TT and trinitrophenyl (TNP) altered self-reactive T cells. Specifically, 1A1 represents the outgrowth of one well of a microwell cloning plate initially seeded at 1 TCL/well, from which less than 4% (3/96) wells grew. Phenotypic analysis, utilizing a battery of monoclonal antibodies, demonstrates that all 1A1 cells are T cells belonging to a stable and discrete T cell subset: T3+, T4+, T17+, T8-. In proliferative assay, 1A1 responds specifically to TT but not to other soluble antigens against which the donor is sensitized, a panel of allogeneic stimulators, nor to TNP-modified-self. Moreover, 1A1 is HLA-DRw-restricted, proliferating only to TT in association with DRw3+ antigen-presenting cells. Of greater interest is the observation that 1A1 is an antigen-specific helper T cell line. Thus, by utilizing ELISA systems to quantitate class-specific immunoglobulin and antigen-specific antibody, it was determined that co-culture of autologous B cells, 1A1 cells, and a low concentration (1 to 10 ng/ml) of TT results in an IgG response that is predominantly, if not exclusively, antigen-specific antibody. In contrast, the presence of high concentrations of TT (4 micrograms/ml) triggers a polyclonal immunoglobulin response comprised of IgM with a small IgG component that is essentially devoid of anti-TT antibody. These results demonstrate that depending on the mechanism of activation, a cloned antigen-specific helper T cell line can mediate antigen-specific or polyclonal help for autologous B cells.     

Antigen-specific, MHC-restricted B cell activation by cell-free Th2 cell products. Synergy between antigen-specific helper factors and IL-4

Ag-specific and MHC-restricted Th clones of different Ag specificities and MHC haplotypes were tested for their ability to produce soluble factors capable of providing the signals required for B cell activation and IgG antibody production. Each of five Th clones tested generated significant helper activity in supernatants derived from coculture of the T cell clone with specific Ag and syngeneic APC. The same helper activity was detected in supernatants of clones stimulated with immobilized anti-CD3 antibody in the absence of Ag or APC. The secreted helper activity resembled the activity of the intact Th cells in that it was Ag-specific, carrier-hapten-linked and MHC-restricted. These T cell products functioned to activate only those B cells expressing MHC products which corresponded to the specificity of each Th clone. Thus, the specificity of the cell-free T cell product mimicked precisely that expressed by the intact Th cell and presumably mediated by the cell surface TcR. In addition to the apparent presence of specific helper factor in Th clone supernatants, a role for nonspecific lymphokines was also identified in these preparations. Although recombinant or purified IL-4 alone was not sufficient to stimulate hapten-primed B cells to secrete hapten-specific IgG antibodies, mAb specific for IL-4 blocked the induction of antibody secretion by Th cell supernatant. These results indicate that stimulation of B cells to produce hapten-specific IgG antibody requires at least two distinct signals: an Ag-specific T cell signal which is restricted by MHC products expressed on the B cells, and a nonspecific signal mediated at least in part by the lymphokine IL-4.     

T cell activation of antigen-specific antibody responses by large B cells is MHC restricted

The activation of small, resting B cells for antibody synthesis by helper T cells has been proposed to require an MHC-restricted interaction between the T and B cells. Large, activated B lymphocytes were, in contrast, thought to be activated by an unrestricted pathway. We re-examined this issue and found that both large and small size fractionated murine B lymphocytes required an MHC-restricted interaction with helper T cells to be activated for specific antibody synthesis. Polyspecific antibody synthesis in the same cultures was not dependent upon an MHC-restricted T-B interaction for any size category of B cell. These results are interpreted as reflecting the ability of antigen-specific B cells to focus and present antigen to T cells, in contrast to B cells of random specificity, which have no effective focusing mechanism for a given experimental antigen. We found that the polyspecific response required much higher antigen concentrations than the antigen-specific response, a result consistent with the antigen-focusing hypothesis.