Antigen-specific and nonspecific mediators of T cell/B cell cooperation. III. Characterization of the nonspecific mediator(s) from different sources.

T cell-containing lymphoid populations produce a nonantigen-specific mediator(s) (NSM) which can replace T cell helper function in vitro in the response of B cells to sheep red blood cells (SRBC), but not to the hapten-protein conjugate, trinitrophenyl-keyhole limpet hemocyanin, (TNP-KLH). NSM produced under three conditions: 1) stimulation of KLH-primed cells with KLH; 2) allogeneic stimulation of normal spleen cells; and 3) stimulation of normal spleen cells with Con A (but not PHA) are indistinguishable on the basis of their biologic activity and m.w., estimated as 30 to 40,000 daltons by G-200 chromatography. Production of NSM is dependent on the presence of T cells. The action of NSM on B cells responding to SRBC in the presence of 2-mercaptoethanol is unaffected by severe macrophage depletion. Extensive absorption of NSM with SRBC failed to remove its activity, confirming its nonantigen-specific nature.     

Antigen-specific and nonspecific mediators of T-cell/B-cell cooperation. VI. Distinct patterns of cross-reactivity by two human gamma-globulin-primed helper T-cell subpopulations.

We have previously characterized the activities, in vitro, of two different helper T-cell subpopulations, primed with human γ-globulin (HGG). One T-cell subpopulation helps the response of B cells to determinants (e.g., haptens) bound to the same antigen to which the T cells are primed (specific help); the other helper T-cell subpopulation responds to the same priming antigen by secreting a nonspecific molecule which helps B-cell responses to erythrocyte antigens co-cultured with the priming antigen (nonspecific help). These subpopulations also differ in their frequency and dose response to antigen, both in vivo and in vitro. They are similarly susceptible to the induction of unresponsiveness to HGG. In order to determine whether these T-cell subpopulations share or differ in their ranges of antigen recognition, we have compared the reaction of these two HGG-primed helper T-cell subpopulations to a number of γ-globulins (γG's) from other species. Plaque-forming cells generated in response to HGG shared little or no cross-reactivity with any of the heterologous (γG's) tested. In contrast, HGG-primed nonspecific helper T cells responded with significant cross-reactivity when challenged in vitro with dog γG, but HGG-primed specific helper T cells did not respond with any such cross-reactivity. No other heterologous γG tested stimulated any significant cross-reactivity from either HGG-primed T-cell subpopulation. Thus, these two T-cell subpopulations differ in their antigenic recognition. Possible explanations of these data include: (i) a difference in receptor specificity; (ii) a difference in the receptor affinity; (iii) a difference in Ia determinants of the two subpopulations.     

Cognate interactions between helper T cells and B cells. III. Contact-dependent, lymphokine-independent induction of B cell cycle entry by activated helper T cells

An Ag-specific, IL-2-dependent Th clone induced the growth of B cells in a class II-restricted, Ag-specific, IL-2-dependent manner. The formation of stable Th-3.1-B cell conjugates was restricted by Ag and class II MHC. After activation of Th-3.1 by insolubilized anti-T3 (Th-3.1T3), Th-3.1T3 induced the growth of B cells in a class II unrestricted, Ag nonspecific manner. The formation of stable conjugates between Th-3.1T3 and B cells was also class II unrestricted and Ag nonspecific. Although the interaction of Th-3.1T3 and B cells was class II unrestricted, the interaction was inhibited by the combination of anti-IA and anti-IE mAb. This suggested that monomorphic domains of class II MHC molecules were involved in Th-3.1T3-B cell interaction. Fixed Th-3.1T3 but not fixed resting Th-3.1 induced B cell cycle entry, as measured by an increase in B cell RNA synthesis. Trypsin-treatment of Th-3.1T3 before fixation reduced their ability to activate B cells, indicating that cell surface proteins on Th-3.1T3 were required for enhanced B cell RNA synthesis. Anti-IL-4, anti-IL-2R, or anti-IFN-gamma did not affect the ability of Th-3.1T3 to induce heightened B cell RNA synthesis. Progression into S phase by B cells activated with fixed Th-3.1T3 was supported by the addition of soluble factors. When stimulated with fixed Th-3.1T3, EL4 supernatant (SN) enhanced B cell DNA synthesis. Depletion of IL-4, but not IL-2, from EL4 SN ablated its supportive capabilities. IL-4 alone was completely ineffective in supporting entry into S phase. Therefore, IL-4 and another activity(ies) in EL4 SN were necessary for B cell cycle progression into S phase. Taken together, these data suggest that after Th activation, Th cell surface proteins are expressed that mediate the binding of Th to B cells via recognition of nonpolymorphic domains of class II MHC molecules. Contact of Th-3.1T3 with B cells, not lymphokines, results in the entry of B cells into the cell cycle and heightened B cell lymphokine responsiveness. The addition of exogenous lymphokines supports the progression of Th-3.1T3-activated B cells into S phase.     

B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells

The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.     

Antigen-specific B cells efficiently present low doses of antigen for induction of T cell proliferation

Previous experiments suggested a role for specific B cells in the induction of antigen (SRBC)-specific T cell proliferation. Two models were proposed: in the first, B cells directly presented antigen to T cells; alternatively, B cells secreted antibody, which opsonized antigen for presentation by macrophages. Experiments to distinguish between these possibilities are presented here. Three lines of evidence support the conclusion that antigen is presented directly by specific B cells. First, nonimmune splenic adherent cells (SAC), which efficiently induced proliferation of appropriately primed T cells to antigens such as OVA and GAT, were unable to induce SRBC-specific proliferation. Secondly, a slope analysis of the logarithmic plot of T cell proliferation vs the number of irradiated B cells suggested that two cells were limiting within the presenting population. The addition of IL 1 or SAC reduced the slope to 1 (although in serum-free conditions, the addition of IL 1, but not SAC, reduced the slope of the line). Specificity of the B cells for the antigen continued to be required in the presence of exogenous IL 1 or SAC. These results suggested that presentation by specific B cells and the amount of IL 1 were the limiting requirements for the induction of SRBC-specific T cell proliferation. The third line of evidence was the demonstration of a restricted interaction between T cells and B cells. The addition of irradiated, allogeneic SRBC-specific B cells to T cell lines and syngeneic SAC failed to support proliferative responses. We further show that a GAT-specific T cell clone was triggered to proliferate by either SAC or B cells, but that antigen-specific B cells were necessary at low doses of antigen. This finding is important in two respects. First, the T cell clone previously has been shown to act as a helper; secondly, when low doses of antigen are used, the requirement for priming of the B cells to the specific antigen is true for a soluble, as well as a particulate, antigen. We propose that at low (physiologic) doses of antigen, presentation to secondary T cells takes place mainly at the surface of antigen-specific B cells. At high doses of antigen,h presentation can also be accomplished by nonspecific cells such as other B cells, macrophages, or dendritic cells.     

Antigen-specific T helper clones in a nonresponder strain require augmentation for expression of helper activity. Evidence for a possible antigen presentation defect in B cells

Hen egg-white lysozyme (HEL)-specific Thy-1+, Lyt-1+2- T cell lines and clones were derived from the nonresponder C57BL/6 strain. Although the antigen-specific proliferative response of these T cells in the presence of syngeneic irradiated spleen cells as a source of antigen-presenting cells (APC) was normal, the same cells were incapable of stimulating B cells to secrete antibody in vitro. This deficiency could, however, be corrected by the addition of an excess of normal T cells or a supernatant from concanavalin A-stimulated rat spleen cells. Alternatively, the use of highly cross-reactive ring-necked pheasant lysozyme in the cultures allowed expression of efficient help, ruling out any inherent deficiency in the T cells. The antibody response was specific and required MHC compatibility between the T lines and responding B cells. By using (H-2b X H-2d)F1 B cells and another H-2d-restricted HEL-specific T line, it was shown that only the H-2b-restricted T-B collaboration required exogenous factors, and the H-2d-restricted collaboration did not. Because both proliferative and helper responses are dependent upon MHC-restricted antigen presentation by macrophage-APC and B cells, respectively, these results suggest that the defect in the nonresponder H-2b-restricted T-B collaborative pathway may relate to the inability of B cells to adequately process and present HEL to clonal T cells.     

Suppression of IgE antibody production in SJL mice. II. Expression of Ly-1 antigen on helper and nonspecific suppressor T cells.

Under appropriate conditions of immunization combined with irradiation, SJL/J mice show a high and persistent anti-DNP IgE antibody response. Spleen cells transferred from normal untreated SJL mice suppress this response. Elimination of Ly-1+ cells, but not of Ly-2+ cells, abolished the capacity of spleen cells to suppress the IgE response. Thus of the three T cell Ly subclasses presently identified, Ly-1, Ly-2,3, and Ly-1,2,3, the normal SJL spleen cell which suppresses the IgE response of irradiated-immunized SJL mice belongs to the Ly-1 set. It is not known whether this Ly-1 cell suppresses the IgE response directly or by helping another cell in the recipient. The carrier-specific helper cell activity for IgE and probably IgG1 antibody response belongs to Ly-1 subclass in the SJL strain also.     

Induction of peripheral T cell tolerance by antigen-presenting B cells. II. Chronic antigen presentation overrules antigen-presenting B cell activation

Ag presentation in the absence of danger signals and Ag persistence are the inductive processes of peripheral T cell tolerization proposed so far. Nevertheless, it has never been definitively shown that chronic Ag presentation per se can induce T cell tolerance independent of the state of activation of APCs. In the present work, we investigated whether chronic Ag presentation by either resting or activated B cells can induce tolerance of peripheral Ag-specific T cells. We show that CD4(+) T cells that re-encounter the Ag for a prolonged period, presented either by resting or activated Ag-presenting B cells, become nonfunctional and lose any autoimmune reactivity. Thus, when the main APCs are B cells, the major mechanism responsible for peripheral T cell tolerization is persistent Ag exposure, independent of the B cell activation state.     

Cognate interactions between helper T cells and B cells. II. Dissection of cognate help by using a class II-restricted, antigen-specific, IL-2-dependent helper T cell clone

In order to determine the involvement of T-B cell contact vs lymphokine production in mediating B cell cycle entry and progression, Th cell clones "defective" in lymphokine production were cloned. Th-3.1 is one such clone that required IL-2 to produce significant levels of IL-4 and IFN-gamma. Unlike conventional Th clones, Th-3.1 induced B cell proliferation only in the presence of Ag and IL-2. In contrast to the absolute requirement of IL-2 for Th-3.1-induced B cell proliferation, IL-2 was not required for the formation of stable Th-3.1-B cell conjugates or Th-3.1-induced B cell entry into the G1 phase of the cell cycle. In the absence of IL-2 and under conditions that promoted Th-B cell interactions, Th-3.1 induced 10 to 20% of resting B cells to enter G1. B cell entry into the cell cycle was not inhibited by anti-lymphokine mAb or promoted by exogenous lymphokines, suggesting that endogenous lymphokine activity was not required for Th-3.1-induced G0 to G1 transition. The data suggested that the IL-2-independent induction of B cells into G1 by Th-3.1 was a cell contact-dependent event. Direct proof that Th-3.1-B cell contact was necessary for B cell cycle entry was provided by comparative in situ analysis of the RNA synthetic activity and the RNA content of B cells that were in physical contact with Th-3.1 or not in contact with Th-3.1. In situ autoradiography of RNA synthesis illustrated that a high frequency of B cells in contact with Th-3.1 expressed heightened RNA synthetic activity, whereas "bystander" B cells were less frequently induced into cycle. In situ laser cytometry of B cell size and total RNA content showed that B cells in physical contact with Th-3.1 had a higher RNA content and were larger than "bystander" B cells present in the same microcultures. This model system has allowed the dissection of T cell help into IL-2-dependent and IL-2-independent phases. Early cell contact-dependent events and B cell cycle progression into G1 were IL-2 independent, whereas the production of lymphokines (IL-4, IFN-gamma) by Th-3.1 and Th-3.1-induced B cell proliferation was IL-2 dependent.     

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.     

Antigen-specific helper factor production by immortalized clone of OVA-specific T cells. I. In vitro activity

Immortalized clones of virally transformed OVA-specific T cells produce antigen-specific helper factor upon stimulation in vitro. The helper factor activate DNP-primed B cells to multiply and synthesize IgG anti-DNP antibodies. The trigger of the helper clone is antigen specific and the B cell-stimulating hapten must be coupled to the specific T cell carrier in order to transfer the help signal from the activated T clone to the B lymphocytes. Activation of the helper clone is performed by antigen-pulsed macrophages and cannot be achieved by the free soluble antigen. However, cell-free supernatant of the antigen-pulsed macrophages can stimulate the helper cells. Thus the antigenic determinant must be presented to the helper cell in the form of macrophage-processed antigen. These requirements for antigenic stimulation and the activity of the secreted helper factor demonstrate that the immortalized helper clone preserved the cellular components which control the antigen-specific immune function of the normal T lymphocyte.     

Monoclonal helper T cells induce B cell responses to T-independent antigens: antigen-specific T cells are directly stimulated by activated B cells in the absence of antigen

Efficient B cell responses to most polysaccharide antigens such as TNP-PAA or TNP-Ficoll require factors produced by activated T cells. However, the mechanism of T cell activation during such responses has not been established, because these antigens do not activate T cells, either directly or in conjunction with I-A gene products. We used a panel of antigen-specific monoclonal helper T cells to study T cell activation during the course of such responses. We show that activated I-A-identical B cells directly stimulate these monoclonal T cells, and that this stimulation is in the absence of nominal antigen. The high frequency of inducer cells that are stimulated by activated B cells suggests a major biologic role for this novel pathway of T cell activation.     

T cells in B cell chronic lymphocytic leukemia. II. Lack of antigen-specific T suppressor cells and their progenitors

Nylon wool-purified T cells (Tn) of two patients with chronic lymphocytic leukemia of the B cell type were phenotyped and tested in various assays for antigen-specific T helper (Th), T suppressor effector (Tse), T suppressor precursor (Tsp), and T suppressor inducer (Tsi) function. Antigen-specific Th as well as Tsi activity could be effectively generated. Although phenotypically CD8+ T cells, carrying the receptor for the Fc part of IgG, were present in mononuclear blood cells and Tn fractions, no antigen-specific Tse cell activity could be induced. In addition, Tsp cells were found to be functionally absent. These findings are discussed in relation to a tumor-induced limited heterogeneity within the T suppressor (Ts) cell compartment.     

Antigen-specific human T cell factors. II. T cell suppressor factor: biologic properties

The in vitro induction of an ovalbumin-specific human T cell suppressor factor is described (TsF120-OA). The antigen-specific suppressive component can be purified by affinity chromatography from supernatants derived from Marbrook-Diener type cultures of peripheral blood T cells stimulated with a high dose of ovalbumin. TsF120-OA suppresses the antigen-induced PFC formation of human blood B cells in vitro in an antigen-specific way. The target of TsF120-OA activity is shown to be the T helper cell. No genetic restriction in the action of the factor is observed.     

Antigen-specific memory and virgin B cells differ in their requirements for conjugation to T cells

The physical interaction between carrier-specific T hybridoma cells and long-term primed, TNP-specific memory B cells (TNP-MABC) exposed to TNP-OVA was compared to that of unprimed, TNP-specific virgin B cells (TNP-ABC). The direct conjugation of the T and B cells was visualized at the light microscopic level and the number of T/B conjugates was quantified directly. The results demonstrate that the TNP-MABC, as compared to TNP-ABC, formed T/B conjugates after a shorter exposure time to antigen and at a 10-fold lower concentration of antigen. Conjugate formation was inhibited almost completely by treating the TNP-MABC with concentrations of chloroquine that only partially inhibited the ability of the TNP-ABC to form conjugates. Exposure of the T hybridoma cells or the TNP-ABC to monoclonal antibodies directed against cell surface antigens prior to conjugation indicated that L3T4, Thy-1.2, and LFA-1 antigens on the T cells and LFA-1 and I-A antigens on the TNP-ABC are involved in conjugate formation. However, in contrast to the TNP-ABC where treatment of the B cells with anti-LFA-1 blocked T/B conjugate formation, pretreatment of the TNP-MABC with anti-LFA-1 had no effect.     

Cognate interactions between helper T cells and B cells. V. Reconstitution of T helper cell function using purified plasma membranes from activated Th1 and Th2 T helper cells and lymphokines.

Th physically interact with B cells and produce lymphokines that influence B cell growth and differentiation. The respective contribution of cell contact and lymphokines to induction of B cell growth and differentiation was addressed using purified plasma membranes (PM) from resting Th (PMrest) and anti-CD3-activated Th (PMCD3) together with lymphokines. Results show that PMCD3, but not PMrest, induce 10% of resting B cells to enter the G1 phase of the cell cycle, with few B cells entering G1b and S/G2. The inclusion of IL-4, but not IL-2, IL-5, or IFN-gamma, amplifies the B cell response to PMCD3 by increasing the total percentage of activatable B cells to greater than 40% and inducing B cell progression into G1b, S, and G2. Direct comparison between PMrest and PMCD3 purified from Th1 and Th2 indicate that both Th1 and Th2 induce similar levels of B cell proliferation in the presence of IL-4. Further, the lymphokine requirements for B cell proliferation induced by PMCD3 from Th1 and Th2 is indistinguishable. B cell differentiation to IgM, IgG1, and IgG2a synthesis by PMCD3 required IL-4 and IL-5. Using lymphokine conditions that supported B cell differentiation, PMCD3 purified from Th1 and Th2 induced similar levels of IgM, and IgG1. Given the functional data on PMCD3 from Th1 and Th2, the data indicate that there are no substantive differences between Th1- and Th2-derived PMCD3, and that the major differences in the ability of viable Th1 and Th2 to activate B cells is the lymphokines produced by the cells.     

Cognate interactions between helper T cells and B cells. IV. Requirements for the expression of effector phase activity by helper T cells.

After activation with anti-CD3, activated Th (THCD3), but not resting Th, fixed with paraformaldehyde induce B cell RNA synthesis when co-cultured with resting B cells. This activity is expressed by Th of both Th1 and Th2 subtypes, as well as a third Th clone that is not classified into either subtype. It is proposed that anti-CD3 activation of Th results in the expression of Th membrane proteins that trigger B cell cycle entry. Kinetic studies reveal that 4 to 8 h of activation with anti-CD3 is sufficient for ThCD3 to express B cell-activating function. However, activation of Th with anti-CD3 for extended periods of time results in reduced Th effector activity. Inhibition of Th RNA synthesis during the anti-CD3 activation period ablates the ability of ThCD3 to induce B cell cycle entry. This indicates that de novo synthesis of proteins is required for ThCD3 to express effector function. The ability of fixed ThCD3 to induce entry of B cell into cycle is not due to an increase in expression of CD3, CD4, LFA-1, ICAM-1, class I MHC or Thy-1. Other forms of Th activation (PMA and A23187, Con A) also induced Th effector function. Furthermore, purified plasma membranes from anti-CD3 activated, but not resting Th, induced resting B cells to enter cycle. The addition of IL-4, but not IL-2, IL-5, or IFN-gamma amplified the DNA synthetic response of B cells stimulated with PM from activated Th. Taken together these data indicate that de novo expression of Th surface proteins on activated Th is required for Th to induce B cell cycle entry into G1 and the addition of IL-4 is required for the heightened progression into S phase.