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.     

Monoclonal antibodies specific for single chain or two chain GAT-specific suppressor factors: production and analysis of in vitro modulating properties

Fusion of spleen cells from rats hyperimmunized with T cell hybridoma derived GAT-specific TsF1 or TsF2 suppressor T cell factors has resulted in the generation of hybridomas secreting monoclonal antibodies reactive with the appropriate GAT-TsF used for immunization, and in several cases, reactive with other GAT-TsF1 and TsF2. The monoclonal anti-TsF1 antibodies are capable of modulating in vitro GAT-specific PFC response in a GAT-specific manner; some suppress responses to GAT directly, whereas others reverse GAT-TsF1-mediated suppression of responses. The monoclonal anti-TsF2 antibodies all reverse suppression but are reactive with combinatorial determinants, I-J+ chains or antigen-binding chains of the GAT-TsF2. The data are discussed in terms of the nature of the determinants recognized by these antibodies as well as the potential uses of these reagents for studying the suppressor T cell pathway and potential relationships between Ts1, Ts2, and T helper cells.     

Activation and immunoregulation of antigen-specific human b lymphocyte responses: multifaceted role of the monocyte

The multifaceted role of the monocyte in the induction and modulation of antigen-specific antibody responses by human B cells was delineated. Monocytes were absolutely required for the induction of specific antibody responses to both TT and KLH in an antigen-induced in vitro assay. Monocytes were also required for the PWM induction of specific antibody in immunized subjects. Pulsing monocytes with specific antigen or with PWM consistently stimulated proliferation of T cells in absence of added antigen and could also stimulate specific antibody synthesis although less consistently. Stimulation of specific antibody responses with antigen required fewer numbers of monocytes than did stimulation of specific antibody responses with PWM. Polyclonal antibody synthesis induced by PWM was also dependent on monocytes. However, polyclonal antibody synthesis induced by supraoptimal concentrations of antigen was usually optimal in the absence of monocytes and was actually suppressed when increased numbers of monocytes were added to monocyte-depleted cultures. Monocyte supernatants could not replace the absolute requirements for monocytes in the induction of specific antibody synthesis. However, monocyte supernatants could profoundly modulate the antigen-specific as well as the polyclonal Ig response of lymphocytes to either antigen or PWM stimulation in a manner closely resembling monocytes themselves. Thus, we demonstrated that monocytes and their products play a critical role in the activation and immunoregulation of antigen-specific antibody responses of human B cells.     

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.     

Purification and analysis of an antigen-specific suppressor factor from a T cell hybridoma specific for phenyltrimethylamino hapten

The synthetic monovalent antigen L-tyrosine-p-azophenyltri-methylammonium (tyr (TMA)) induces in A/J mice, a cascade of regulatory T cells in the absence of any detectable effector function (e.g., CTL, delayed-type hypersensitivity, etc.). An important component of the activated T cells is a first order suppressor T cell or Ts1 that is Ly-1+2-, functions only at the afferent limb of the anti-TMA response, binds the TMA ligand and bears cross-reactive idiotypes associated with anti-TMA antibodies. This Ts1 produces a suppressor factor (TsF1) that binds the TMA ligand, bears the cross-reactive idiotypes and I-J determinants and functions to induce an idiotype-specific Ts2 population. To study the biochemistry of this TsF, use was made of T cell hybridomas that constitutively produce TMA-TsF1 (8A.1 and 8A.3). The TsF1 was purified from culture supernatant or cell extracts by (NH4)2SO4 precipitation, reverse phase HPLC and either affinity chromatography or by preparative IEF. The TsF1 has an isoelectric point of 6.5 and a m.w. of 26,000 or 62,000 as analyzed by SDS-PAGE or high performance molecular sieve chromatography. Its precipitation in 30 to 40% (NH4)2SO4; elution pattern from reverse phase high performance columns; its capacity to bind to a mAb specific for L-glutamic acid 60L-alanine30-L-tyrosine10 (GAT)-TsF1 strongly suggest that this protein belongs to the same family of proteins as do the GAT-TsF1 described previously. Most noteworthy is that although these TsF1 proteins show remarkable similarities, they are absolutely specific in their biologic activity; TMA-TsF1 will not suppress the response to GAT-BA-TNP and GAT-TsF1 will not suppress the response to TMA-BA-TNP. Thus the TMA-TsF1 represents a second example of a unique group of Ag-specific proteins whose function is to induce or activate other suppressor T cells in the primary immune response to Ag.     

Antigen-specific suppressor T cell interactions. I. Induction of an MHC-restricted suppressor factor specific for L-glutamic acid50-L-tyrosine50

The synthetic polymers L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) and L-glutamic acid50-L-tyrosine50 (GT) stimulate specific suppressor T cells in certain strains of mice. Extracts from these T cells contain factors (TsF) that inhibit GAT- or GT-specific antibody responses by normal spleen cells or proliferative responses by primed T cells. We constructed T cell hybridomas that constitutively produce GAT-TsF or GT-TsF, which functionally and serologically are identical to factors extracted from suppressor T cells. In this report we demonstrate that monoclonal GT-TsF can induce specific unresponsiveness in vivo or in vitro and that this unresponsiveness is due to development of second-order antigen-specific suppressor T cells. T cell hybridomas were constructed by fusion of BW5147 with GT-TsF1 induced second-order suppressor T cells and clones that produced suppressor factor (GT-TsF2) were isolated and characterized. GT-TsF2 differs from the GT-TsF1 used to induce it in that GT-TsF1 acts across allogeneic barriers whereas GT-TsF2 does not. This restriction is controlled by genes in the H-2 gene complex and maps to the I-J subregion. GT-TsF2 is antigen-specific in suppressive activity and also in its antigen-binding site(s). Thus, GT-TsF2 closely resembles the carrier-specific, I-J+, genetically restricted factor described by Tada and his colleagues. Because GT-TsF2 was induced by GT-TsF1, we suggest cells producing GT-TsF1 are an early cell in the pathway of suppression, and that this cell is required for the activation of antigen-specific, MHC-restricted TsF.     

Induction of antigen-specific antibody responses in primed and unprimed B cells. Functional heterogeneity among Th1 and Th2 T cell clones

CD4+ T cells have been recently divided into two subsets. The functions of these subsets are thought to be distinct: one subset (Th1) is responsible for delayed type hypersensitivity responses and another (Th2) is primarily responsible for induction of antibody synthesis. To more precisely define the roles of both subsets in humoral immune responses, we examined the ability of a panel of nominal antigen specific Th1 and Th2 clones to induce anti-TNP specific antibody synthesis in TNP-primed or unprimed B cells. Four of nine Th1 clones induced little or no antibody synthesis with TNP-primed B cells. However, five other Th1 clones were very effective at inducing IgG anti-TNP plaque-forming cell (PFC) responses in primed B cells. One of these Th1 clones was analysed in detail and found to also provide helper function for unprimed B cells. Cognate B-T cell interaction was required for induction of both primary and secondary responses with this clone, indicating that a Th1 clone could function as a "classical" Th cell. The seven IL-4 producing Th2 clones examined were also heterogeneous in their ability to induce antibody secretion by TNP-primed B cells. Although four of the Th2 clones induced IgG and IgM anti-TNP PFC responses, two Th2 clones induced only IgM and no IgG antibody, and another clone failed to induce any anti-TNP PFC. All Th2 clones failed to induce any anti-TNP PFC. All Th2 clones produced high levels of IL-4, but "helper" Th2 clones produced significantly greater amounts of IL-5 than "non-helper" Th2 clones. These studies indicate that some IL-2- and some IL-4-producing T cell clones can induce TNP-specific antibody in cell clones can induce TNP-specific antibody in primed and unprimed B cells, and that Th1 and Th2 clones are heterogeneous in their ability to induce Ig synthesis. Therefore, although T cell clones can be classified as Th1 or Th2 types according to patterns of IL-2, IFN-gamma, or IL-4 synthesis, the functional capacity to induce antibody synthesis cannot be predicted solely by their ability to secrete these lymphokines.     

Transforming growth factor-beta directs IgA switching in human B cells.

Transforming growth factor (TGF)-beta added to cultures of highly purified human splenic B cells induced high levels of IgA synthesis in the presence of PWM and activated cloned CD4+ T cells. TGF-beta had no effect on IgM or IgG production. The induction of IgA synthesis by TGF-beta reflected IgA switching, because a strong induction of IgA production was also observed, when sIgA- B cells were cocultured with cloned activated CD4+ T cells in the presence of pokeweed mitogen. Resting CD4+ T cell clones or activated CD8+, TCR-gamma delta + CD4-,CD8- T cell clones failed to provide the co-stimulatory signal that in addition to TGF-beta and pokeweed mitogen was required for induction of IgA switching and IgA synthesis. mAb against CD4 or class II MHC molecules inhibited TGF-beta induced IgA synthesis, indicating that CD4-class II MHC interactions are required for productive T-B cell contacts resulting in IgA production. In contrast, anti-LFA-1, anti-CD2, and anti-class I MHC mAb were ineffective. TGF-beta failed to induce IgA synthesis by sIgA+ B cells under these culture conditions. Interestingly, induction of IgA production by sIgA- B cells required neutralization of TGF-beta activity by addition of the anti-TGF-beta mAb 1D11.1G 24 h after onset of the cultures. IgA production was prevented when the anti-TGF-beta mAb was added at the start of the cultures, indicating the specificity of the reaction. IgA synthesis was completely suppressed when TGF-beta was present during the total culture period of 11 days. These findings indicate that TGF-beta can act as a specific switch factor for IgA, provided it is only present at early stages of the cultures.     

Selective induction of B7/BB-1 on interferon-gamma stimulated monocytes: a potential mechanism for amplification of T cell activation through the CD28 pathway.

The B cell activation antigen B7/BB-1 is the natural ligand for the T cell antigen CD28 and these two molecules are capable of mediating T-B cell adhesion. Engagement of the CD28 pathway provides a costimulatory signal to T cells leading to enhanced lymphokine production. We report that interferon-gamma (INF-gamma) induces the expression of B7/BB-1 on monocytes. This induction was very specific since other cytokines and stimuli which activate monocytes including M-CSF, GM-CSF, IL3, TNF-alpha, and LPS were unable to induce B7/BB-1. Following culture of monocytes with INF-gamma, maximal mRNA and cell surface B7/BB-1 expression was detected at 12 and 24 hr, respectively. In addition to antigen presentation, optimal T cell activation and lymphokine synthesis require an additional cell to cell contact signal provided by the antigen presenting cell. The induction of B7/BB-1 on monocytes and subsequent heterophilic interaction of B7/BB-1 with CD28 may provide a mechanism for the amplification of T cell proliferation and lymphokine production by INF-gamma activated monocytes.     

Suppressor cell induction in vitro. VI. Production of suppressor factors to synthetic polypeptides GAT and (T,G)-A--L from cells of responder and nonresponder mice.

The capacity of responder and nonresponder strains of mice to generate suppressor cells and factors to two antigens under MHC linked Ir gene control was investigated. Eight different H-2 types (H-2b,d,f,k,p,q,r,s) as well as seven independently derived strains (B10, BALB/c, CBA/Ca, A/St, DBA/2, P/J, SJL) were tested, and all yielded suppressor factor (SF) to (T,G)-A--L and GAT. This indicated that the genetic control of SF production was different from that of helper cell induction. Unlike previous reports of GAT suppressor extracts that GAT-specific supressor factors acted equally on both responder and nonresponder strains. As reported earlier with in vitro induced protein- (KLH) specific suppressor factors, GAT and (T,G)-A--L specific suppressor factors failed to show any genetic restriction in their function. The implications of these results for the general mechanism of Ir gene control are discussed.     

Accessory cell stimulation of T cell proliferation requires active antigen processing, Ia-restricted antigen presentation, and a separate nonspecific 2nd signal

The roles of Ia+ accessory cells in H-2-restricted stimulation of antigen-specific T cell proliferation were explored in an in vitro model. L-glutamic acid60-L-alanine30-L-tyrosine10-(GAT) primed BALB/c nylon wool-passed T cells were depleted of Ia+ antigen-presenting cells (APC) by treatment with monoclonal anti-Ia antibody plus complement. Such cells failed to respond to soluble GAT, or to soluble GAT in the presence of phorbol myristic acetate (PMA), which is known to stimulate production of, or replace, IL-1 in vitro. Addition of gamma-irradiated syngeneic spleen cells reconstituted the response to soluble GAT, but addition of ultraviolet (UV) light-irradiated spleen cells did not, even in the presence of PMA. Preincubation of cells with GAT for 24 hr, followed by washing, then gamma irradiation, generated a cell population able to stimulate GAT-primed T cells to proliferate. The same pulsed cells exposed to UV irradiation failed to stimulate T cell responses unless PMA was added to the cultures. The relevant cells in this UV-irradiated population are Ia+. It is concluded that a finite period of time for interaction of metabolically intact APC with antigen is required before creation of an appropriate (Ia + antigen) signal recognized by the T cell. In addition to such Ia-restricted antigen presentation, however, a 2nd nonspecific signal, again requiring metabolically active APC for elaboration, is necessary for detectable T cell activation. These studies thus define 3 separable activities of APC during the process of H-2 restricted T cell activation.     

In vitro studies linking induction of spontaneous antibody synthesis to an accessory cell with persisting antigen on its surface

Antibody synthesis was spontaneously induced in lymphoid cell cultures prepared from the draining lymph nodes of immunized rabbits. Induction of this response was attributed to cell-associated persisting antigen. The identity of the cell with which the antigen was associated was sought. Removal of the macrophage-rich adherent cell population did not diminish spontaneous induction. However, removal of a different cell population by the carbonyl iron method markedly inhibited spontaneous antibody synthesis. In addition, treatment of cultured lymph node cells with ethylenediaminetetraacetate, which is known to remove antigen bound to cell surfaces, also inhibited spontaneous induction. Induction of antibody synthesis could be restored in either of these situations by the addition of a suitable quantity of specific antigen. Although removal of the cell type which adhered to carbonyl iron depressed spontaneous antibody synthesis, the treatment had no effect on concanavalin A-mediated lymphocyte transformation. The combined evidence indicates that the persisting antigen responsible for induction of the spontaneous response is bound to the surface of an accessory cell which is probably a sessile macrophage or a dendritic-type cell.     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

Nonproductive Infection and Induction of Cellular Deoxyribonucleic Acid Synthesis by Bovine Adenovirus Type 3 in a Contact-Inhibited Mouse Cell Line

Bovine adenovirus type 3 (BAV-3), which has been reported to produce tumors in newborn hamsters, induced cellular deoxyribonucleic acid (DNA) synthesis in a contact-inhibited mouse kidney cell line (C3H2K). In this system, the virus did not multiply, whereas virus-specific tumor antigen (T antigen) was detected in nearly all cells. Replication of viral DNA could not be detected by DNA-DNA hybridization on membrane filters. The cellular DNA synthesis induced by BAV-3 did occur in the absence of added serum. Extent of induction of cellular DNA synthesis was closely correlated with the multiplicity of infection. Cells activated to synthesize DNA in the serum-free medium by the virus infection progressed to cell division without noticeable cell killing.     

The functional helper T cell repertoire specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

Experiments have been carried out to examine the potential helper T cell repertoire specific for the random terpolymer GAT on responder, nonresponder, and (responder x nonresponder)F1 murine strains. The ability of GAT-MBSA immunized T cells to collaborate with DNP-specific primary and secondary B lymphocytes of each strain in response to the antigen DNP-GAT was tested with the splenic fragment culture system. The results of these experiments show that there are GAT-specific T lymphocytes in the responder, nonresponder, and F1 strains but that these 3 GAT-specific T cell populations differ in their collaborative potential. In sum, these findings present new evidence that the nonresponder status to the terpolymer GAT is due, in part, to a functional deletion of helper T cells capable of recognizing the antigen in the context of the nonresponder haplotype. Further, a new responsive phenotype is evidenced when F1 secondary B cells are stimulated in nonresponder GAT-MBSA-primed recipients. In this case, rather than the IgG1 responses observed in such strain combinations to other antigens such as DNP-Hy or DNP-Gl phi 9, only IgM responses were obtained. This new phenotype may be the result of GAT-specific suppression of isotype switching by B cells bearing the nonresponder cell surface alloantigens.     

Mechanisms of idiotype suppression. V. Anti-idiotype antibody inhibits early B cell triggering induced by antigen

To investigate the relationship between antigen-mediated B cell commitment and induction of idiotype (id) suppression, anti-id antibody directed against the major id (TEPC-15 idiotype or T15id) of the anti-phosphorylcholine (PC) antibody was added at various time intervals to BALB/c spleen cell cultures stimulated with a T-independent PC antigen, R36a. The suppressive effect of anti-T15id antibody on the anti-PC response was rapidly decreased as addition of the antibody was delayed; when anti-T15id antibody was added 6 hr after the initiation of the cultures, only partial suppression was induced, whereas the addition of anti-id antibody after 24 hr did not result in significant suppression of the anti-PC response when compared with similar cultures treated with mock anti-id antibody. This acquisition of resistance to id suppression was completely inhibited by treatment with either sodium azide or colchicine, as well as at temperatures below 20 degrees C. The induction of resistance to id suppression during the preincubation period was dependent on the presence of an immunogenic level of specific antigen. This antigen-mediated B cell commitment did not appear to require macrophages because preincubation of macrophages with antigen did not affect the sensitivity of the B cells to anti-id antibody. These results support the possibility that anti-id antibody inhibits early B cell triggering, which involves an energy-dependent, epitope-mediated, lateral mobility of antigen receptors possibly followed by repolymerization of microtubules.     

The nuclear matrix protein, numatrin (B23), is associated with growth factor-induced mitogenesis in Swiss 3T3 fibroblasts and with T lymphocyte proliferation stimulated by lectins and anti-T cell antigen receptor antibody

Numatrin is a tightly bound nuclear matrix protein (40 kD/pI-5) whose synthesis is markedly and promptly increased in association with cellular commitment for mitogenesis in B lymphocytes. (Feuerstein, N., and J.J. Mond. 1987. J. Biol. Chem. 262:11389-11397). To study whether this event is exclusively associated with proliferation of B lymphocytes, we examined the synthesis of numatrin in T lymphocytes (murine and human) activated by lectins or by anti-T cell antigen receptor monoclonal antibody and in Swiss 3T3 fibroblasts stimulated by growth factors. We showed a close correlation between induction of DNA synthesis and induction of numatrin synthesis in T lymphocytes stimulated by concanavalin A, anti-T cell antigen receptor monoclonal antibody, and IL-2 in murine T cells. Similar results were observed in Swiss 3T3 fibroblasts, thus only combinations of growth factors (insulin/EGF or insulin/B subunit of cholera toxin) or serum, which induced a significant increase in DNA synthesis, were also associated with a significant increase in synthesis of numatrin. Similar to B cells, the increase in numatrin synthesis in fibroblasts was found to occur at early G1 phase. The calcium ionophores, A23187 and ionomycin, previously shown to induce an increase in c-myc and c-fos mRNA levels in fibroblasts, induced a marked increase in the synthesis of a nuclear protein at 80 kD/pI-5 but failed to induce an increase in the synthesis of numatrin indicating that an increase in intracellular Ca++ level is not sufficient for induction of the synthesis of numatrin. This further indicates that the increase in synthesis of numatrin may be more closely correlated with cellular commitment for mitogenesis as compared with other biochemical parameters. Using a polyclonal numatrin antibody we demonstrated that mitogen stimulation is also associated with a marked increase in numatrin abundance, which reached a peak at the onset of S phase and declined at the end of S phase. Evidence is presented to show that numatrin synthesis and abundance is elevated in various lymphoma cell lines. Using indirect immunofluorescence assays we showed that numatrin is abundant in other malignant cells: KB, epidermoid carcinoma, and Hep2 human hepatoma. Immunofluorescence studies further showed that mitogen stimulation of B lymphocytes induced a marked accumulation of numatrin in the nucleoli. This observation is in accord with the recent finding of identity of numatrin with the nucleolar protein B23 (Feuerstein et al. 1988. J. Biol. Chem. 263:10608-10612).(ABSTRACT TRUNCATED AT 400 WORDS)     

Different antigen-presenting cells differ in their capacity to induce lymphokine production and proliferation of an apo-cytochrome c-specific T cell clone

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.     

Isolation and characterization of an I-A-restricted T cell clone with dual specificity for poly(Glu60Ala30Tyr10) (GAT) and Mlsa,dl

We have isolated a BALB/c (H-2d, Mlsb) T cell clone (JTL-G12) specific for the synthetic polypeptide antigen poly(Glu60Ala30Tyr10) (GAT) in the context of self I-A determinants and for Mlsa,d antigens in the absence of GAT. JTL-G12 proliferation in response to GAT was mapped to the Kd, I-Ad subregions by using inbred H-2 congenic and recombinant strains. In addition, monoclonal antibody directed against I-Ad but not Kd or I-As determinants blocked JTL-G12 proliferation in response to GAT presented by syngeneic splenocytes, indicating I-A restriction. The Mls cross-reactivity of this clone was verified by using a panel of inbred strains bearing the Mlsa,b,c,d alleles and by using BXD recombinant inbred strains bearing the Mlsa allele or the Mlsb allele. All of the Mlsa BXD strains of the H-2d or H-2b haplotypes stimulated JTL-G12 in the absence of GAT, whereas all of the Mlsb BXD strains were nonstimulatory. This response pattern is in complete accordance with recognition of the Mlsa determinant encoded by Mls or closely linked loci on chromosome 1. JTL-G12 proliferation in response to GAT/I-Ad and Mlsa,d determinants could be blocked with a monoclonal antibody (GK1.5) directed against L3T4, a structure involved in class II major histocompatibility complex antigen recognition. These results suggest that antigen/class II responsiveness, Mls reactivity, and expression of L3T4 can be properties of a single T cell population.     

IgE production by normal human B cells induced by alloreactive T cell clones is mediated by IL-4 and suppressed by IFN-gamma

Seven T cell clones were established from mixed leukocyte cultures in which PBMC from two healthy donors and from one patient suffering from the hyper-IgE syndrome were stimulated by the irradiated EBV-transformed B cell lines JY or UD53. Five of seven T cell clones, after activation by co-cultivation with JY or UD53 cells, induced a low degree of IgE production by normal blood B cells. In one experiment in which the normal B cells could activate the T cell clones directly, IgE production was also observed in the absence of the specific stimulator cells. IgE production was also obtained with supernatants of the T cell clones collected 4 to 5 days after activation by their specific stimulator cells. In addition, the supernatants induced IgG, IgA, and IgM synthesis. All seven clones produced variable concentrations of IL-4 and IFN-gamma. The clones FA-28 and BG-39, which failed to induce IgE synthesis, produced, compared with the other clones tested, relatively high quantities of IFN-gamma (4700 and 2500 pg/ml, respectively). These high levels of IFN-gamma accounted for the lack of induction of IgE synthesis, because in the presence of a polyclonal anti-IFN-gamma antiserum, supernatants of FA-10 and BG-39 induced significant IgE production. In addition, the low degree of IgE production induced by supernatants of two other T cell clones (FA28 and BG24) was 15- and 3-fold enhanced, respectively, in the presence of the anti-IFN-gamma antiserum. IgE synthesis by normal B cells was also induced by rIL-4, indicating that IL-4 present in T cell clone supernatants was responsible for induction of IgE production. This notion was supported by the finding that IgE production induced by supernatant of BG-24 was strongly inhibited by a polyclonal anti-IL-4 antiserum. In contrast, IgG and IgA production induced by supernatant of BG-24 were not significantly affected by the anti-IL-4 antiserum. Only a slight inhibition of IgM synthesis was observed. Collectively, our results indicate that both recombinant and naturally produced IL-4 induce normal human B cells to synthesize IgE. However, final IgE production induced by T cell clone supernatants is the net result of the inducing and suppressive effects of IL-4 and IFN-gamma respectively, that are secreted simultaneously by the T cell clones upon activation.