Hapten-specific responses to the phenyltrimethylamino hapten. V. A single chain antigen-binding I-J+ first-order T suppressor factor requires antigen to induce anti-idiotypic second-order suppressor T cells

We have previously shown that a single i.p. injection of the monovalent antigen, L-tyrosine-p-azophenyltrimethylammonium in complete Freund's adjuvant induces a Ly-1+2-, idiotype-bearing, and antigen-binding first-order T suppressor (Ts1) population. We showed that soluble factors extracted from these cells could suppress delayed-type hypersensitivity responses if administered at the induction phase of the response. In this paper we additionally characterize the suppressor factor, TsF1, with respect to its biologic, serologic, and chemical properties. The studies show that the TsF1 is neither allotype nor H-2 restricted and can induce anti-idiotypic T suppressor cells (Ts2), but it requires the presence of antigen to do so. The factor binds antigen, bears I-J encoded determinants, is resistant to reduction and alkylation, and elutes as a single chain factor after adsorption onto monoclonal anti-I-J antibody-coupled Sepharose beads in the presence of dithiothreitol (DTT). This is in marked contrast to TsF2 (derived from Id-specific Ts2-containing spleen cells), which lost its suppressive activity after reduction and alkylation, and behaves as a two chain factor after adsorption and elution from anti-I-J-coupled beads in the presence of DTT. The TsF1 is discussed with respect to the properties of it and those of TsF1 from other similar idiotype-dominated antigen systems.     

An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM)

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.     

Requirements for suppressor T cell activation

Third-order (Ts3) suppressor cells are generated after conventional immunization. These cells, however, will not mediate suppressor cell function unless specifically triggered by an activating signal, termed TsF2. This report analyzes the mechanism of this TsF2-mediated triggering event. TsF2-mediated suppression is genetically restricted by genes in the I-J and Igh-V regions. The target of the I-J restrictions is a firmly adherent accessory cell, which appears to express I-J-related determinants. These accessory cells are sensitive to cyclophosphamide treatment and 500 R irradiation. In contrast, the target of the Igh-V restriction of TsF2 appears to be the Ts3 cell, which carries antigen-specific, idiotype-related receptors. The mechanism of suppressor cell activation appears to involve two stages. Presentation of I-J-restricted TsF2 by I-J-compatible presenting cells and a second step involving idiotype-anti-idiotype interactions between TsF2 and the Ts3 cell. I-J compatibility is not required with the accessory cell for Ts3 activation. Finally, we hypothesize that the anti-idiotypic determinants expressed on TsF2 can serve as an internal image of antigen, thereby permitting specific targeting of the factor.     

TMA-specific first-order T-suppressor hybridoma. I. Characterization of the hybridoma-derived single-chain inducer suppressor factor, TsF1

Experiments described in this report will characterize a monoclonal phenyltrimethylammonium (TMA) specific, first-order T-suppressor factor (TsF1) produced by a T-cell hybridoma, 8A.3. The hybridoma expressed the Thy-1, Lyt-1, Lyt-2 antigens as well as cross-reactive idiotypic (CRI) determinants but did not express I-J encoded epitopes. It was also found to bear determinants recognized by a monoclonal antibody raised against single-chain GAT-specific TsF1. The hybridoma-derived factor was capable of suppressing primary in vitro trinitrophenol (TNP)-specific responses induced with the Brucella abortus antigen, conjugated with TMA and TNP haptens (TMA-BA-TNP). In addition, in vivo administration of 8A.3 culture supernatant resulted in the specific suppression of TMA-specific delayed-type hypersensitivity (DTH) responses. Analysis of this factor revealed it to be an induction-phase, antigen-binding, CRI+, and I-J+ single chain polypeptide. Our results represent only the second such described single chain, antigen binding, I-J+ suppressor factor derived from a monoclonal T-cell hybridoma.     

Antigen-specific T cell-mediated suppression. II. In vitro induction by I-J-coded L-glutamic acid50-L-tyrosine50 (GT)-specific T cell suppressor factor (GT-T8F) of suppressor T cells (T82) bearing distinct I-J determinants.

The induction of new suppressor T cells (Ts2) by suppressive extracts (TsF) from L-glutamic acid50L-tyrosine50 (GT) nonresponder mice was examined. Incubation of normal spleen cells with allogeneic GT-TsF for 2 days in vitro led to the generation of Ts2 cells able to suppress subsequent responses to the immunogen GT-methylated bovine serum albumin (GT-MBSA) in vivo. This induction occurred efficiently when TsF donor and target cells differed at all of H-2, including the I-J subregion. B10.BR (H-2k) GT-TsF, adsorbed on, then acid eluted from GT-Sepharose and anti-I-Jk [B10.A (3R) anti-B10.A (5R)]-Sepharose in a sequential fashion could induce BALB/c (H-2d) spleen cells to become Ts2 only if nanogram quantities of GT were added to the purified GT-TsF. This indicates a requirement for a molecule or molecular complex possessing both I-J determinants and antigen (GT)-binding specificity, together with GT itself, for Ts2 induction. The induced Ts2 are I-J+, since their function can be eliminated by treatment with anti-I-Jk plus C. These I-J determinants are coded for by the precursor of the Ts2 and do not represent passively adsorbed, I-J coded TsF, since anti-Ijk antiserum [(3R X DBA/2)F1 anti-5R] which cannot recognize the BALB/c (I-Jd) TsF used for induction still eliminates the activity of induced A/J (I-Jk) Ts2. These data provide further evidence for and information about the minimum of two T cells involved in antigen-specific suppressor T cell systems.     

A monoclonal antibody raised to tumor-specific T cell-derived suppressor factors also recognizes T suppressor inducer factors of the 4-hydroxy-3-nitrophenyl acetyl hapten suppressor network

A monoclonal antibody (mAb), B16G, was raised from BALB/c mice immunized with affinity-purified T suppressor factors (TsF) specific for the murine mastocytoma P815. This mAb was found to bind to polyclonal TsF isolated from the spleens of tumor-bearing animals, and to the TsF released from a P815-specific T cell hybridoma. In this study, B16G was tested for its reactivity with TsF produced in the 4-hydroxy-3-nitrophenyl acetyl hapten system. The factors from three types of suppressor T cell hybridomas, each representing the immortalized analogues of the inducer T suppressor cell (Ts1), transducer suppressor cell (Ts2), and effector suppressor cell (Ts3) network populations, were tested. B16G was found to be reactive with two sources of TsF1 as assayed by enzyme-linked immunosorbent assay and delayed-type hypersensitivity bioassay. By contrast, TsF2 and TsF3 were nonreactive with B16G. These results indicate that B16G recognizes class-specific suppressor factor determinants, and that the transducer/effector factors of the network are apparently serologically distinct. Because the B16G mAb fails to recognize 4-hydroxy-3-nitro-phenyl acetyl-specific TsF3 that share idiotype-related determinants with TsF1 yet binds to TsF1 molecules that have interacted with antigen, the binding is apparently independent of the site of antigen recognition. Additionally, the results show that the tumor-specific TsF1 raised in one suppressor system share serologic determinants with anti-hapten TsF1 raised in another.     

T suppressor factor activity is due to two separate molecules. The Lyt-1(-)2+I-J+ cells of mice primed with antigen make an antigen binding molecule which is only active when complemented by cofactor made by Lyt-1+2(-)I-J+ cells

Mice primed with picrylsulfonic acid (PSA) and then painted on the skin with picryl chloride produce antigen-specific T suppressor factor (TsF). In contrast unpainted primed mice fail to produce active TsF. This is not due to the absence of the antigen binding part of TsF but to the absence of a cofactor. This cofactor is (a) antigen nonspecific and occurs in potassium chloride extract of normal spleen cells. It also occurs in the 24 hr supernatant of normal cells modified by haptenisation with picryl or the unrelated NP antigen (4-hydroxy-3-nitrophenylacetyl), and in preparations of conventional TsF (PSA/PCl) from painted PSA-primed mice; (b) bears I-J determinants; and (c) is produced by Lyt-1+2(-)I-J+ cells. The antigen binding molecule occurs alone in the supernatant of PSA-primed mice. It lacks I-J determinants and has a molecular weight around 35,000 and 75,000. It is produced by Lyt-1(-)2+I-J+ cells and is only active when complemented by cofactor. However, the complementation is genetically restricted and the restriction maps to the I-J subregion of the MHC.     

Nonspecific inhibitor of DNA synthesis elaborated by T acceptor cells. I. Specific hapten- and I-J-driven liberation of an inhibitor of cell proliferation by Lyt-1-2+ cyclophosphamide-sensitive T acceptor cells armed with a product of Lyt-1+2+-specific suppressor cells

Lyt-1+2+ hapten-specific T suppressor cells (Ts) from mice injected and then painted with picryl or oxazolone derivatives produce hapten-specific T suppressor factors (TsF) in vitro. Stimulation by painting with contact sensitizer (which need not be specific) gives rise to Lyt-1-2+, I-J+, cyclophosphamide-sensitive T acceptor cells (Tacc). When the Tacc population is armed with TsF and then is exposed to specific antigen in the context of I-J-controlled determinants (antigen-presenting, haptenized spleen cells and Ts sharing the same I-J subregion), a nonspecific inhibitor of DNA synthesis (nsINH) appears in the supernatant. This inhibitor suppresses the primary DNA synthetic response to concanavalin A, lipopolysaccharide, and alloantigens in both syngeneic and allogeneic lymphocytes. The nsINH is only effective when added to lymphocyte cultures less than 8 hr after the stimulation with concanavalin A. The nsINH, however, affects neither primary nor secondary cytotoxicity in vitro. These data suggest the mouse immune system is capable of selective regulation of the response to specific antigen by the production of nonspecific soluble suppressor factor(s).     

In vitro generation of suppressor T cells. Induction of CD3+, IgH-restricted suppressor cells

Previous studies of the immune response of C57BL/6 mice to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten determined that challenge with antigenic forms of hapten induces both immunity and suppression. The anti-NP plaque-forming cell response can be down regulated by an Ag-induced cascade consisting of three suppressor T cell subsets. These three populations, termed Ts1, Ts2, and Ts3 have been characterized to have inducer, transducer and effector functions, respectively. Although the functions of each of these subsets have been examined in vivo, the cellular requirements for in vitro Ts induction have only been investigated for the Ts3 population. The present study characterizes the cellular events that lead to the induction of the Ts2, suppressor transducer population. Culture of naive C57BL/6 spleen cells with Ts1-derived suppressor factor in the absence of exogenous Ag leads to the generation of Ts2 cells that mediate Ag-specific suppression of NP plaque-forming cell responses. Phenotypic analyses demonstrate that a CD3+, CD4-, CD5+, CD8+, and I-J+ precursor population is stimulated by TsF1 to become mature Ts2 cells that express CD3, CD8, and I-J but not CD5. Although previous studies have reported an essential role for B cells in the induction of other Ts populations, depletion of B cells from Ts2 induction cultures had no effect on Ts2 generation. Despite the absence of B cells in these cultures, the mature Ts2 cells were functionally IgH restricted. Studies with IgH congenic B.C-8 mice suggest that this restriction specificity was imposed by the idiotype-related determinants expressed on the TsF1, not the T cell genotype.     

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.     

A primary in vitro antibody assay for antigen-specific T-suppressor factor: cross-suppression of TNP-specific antibody responses by TMA-specific TsF1

We have previously shown that phenyltrimethylammonium (TMA)-specific, first-order suppressor T cells (Ts1) and soluble factors extracted from these cells (TsF1) can suppress delayed-type hypersensitivity (DTH) responses. The TsF1, as monitored in the DTH system, was characterized and found to be a single-chain, antigen-binding, I-J+, and Id+ molecule. To monitor TsF1 in an efficient manner, an in vitro antibody system was developed. The studies show that in vitro stimulation of naive A/J spleen cells with the thymic-independent antigen, Brucella abortus, to which TMA and trinitrophenol (TNP) or fluorescein (FL) are coupled (TMA-BA-TNP or TMA-BA-FL), induces significant numbers of anti-TNP or anti-FL plaque-forming cell (PFC) responses. The addition of TMA-specific TsF1 results in the cross-suppression of 30-50% of the total anti-TNP and FL PFC responses. This activity is antigen (TMA) dependent since suppression occurs only when the TMA ligand is present in the culture media. Analysis of the TNP-specific PFC responses in nonsuppressed cultures revealed that 20-35% of the PFC bear the cross-reactive idiotype(s) (CRI) normally associated with anti-TMA antibodies. In cultures containing TMA-TsF1, CRI+PFC are suppressed by 90-100% while the CRI-PFC are suppressed only by 10-30%. Our studies further show that an induction-phase, antigen-binding, CRI+, and I-J+ single-chain factor is responsible for the observed in vitro suppression. The possibility of utilizing this assay to monitor a variety of antigen-specific suppressor factors is discussed.     

Insulin-specific suppressor T cell factors

Murine antibody responses to heterologous insulins are controlled by MHC-linked immune response genes. Although nonresponder mice fail to make antibody when injected with nonimmunogenic variants of insulin, we have recently shown that nonimmunogenic variants stimulate radioresistant, Lyt- 1+2- helper T cells that support secondary antibody responses. However, the helper activity can not be detected unless dominant, radiosensitive Lyt-1-2+, I-J+ suppressor T cells are removed. In this paper we report that extracts of primed Lyt-2+ suppressor T cells contain insulin-specific suppressor factors (TsF) that are capable of replacing the activity of suppressor T cells in vitro. The activity of these factors is restricted by MHC-linked genes that map to the I-J region, and immunoadsorption studies indicated that they bind antigen and bear I-J-encoded determinants. Insulin-specific TsF consists of at least two chains, one-bearing I-J and the other the antigen-binding site. Furthermore, mixing of isolated chains from different strains of mice indicates that the antigenic specificity is determined by the antigen-binding chain and the MHC restriction by the H-2 haplotype of the source of the non-antigen-binding, I-J+ chain. Moreover, mixtures containing antigen-binding chain from allogeneic cell donors and I-J+ chain from responder cell donors have activity in cultures containing responder lymphocytes. This suggests that preferential activation of suppressor T cells, rather than differential sensitivity to suppression, results in the nonresponder phenotype to insulin.     

Isolation and characterization of a T suppressor factor by using a monoclonal antibody

We have developed a monoclonal antibody to a T cell-derived suppressor factor (TsF) found in the serum of C57BL/6 mice hyperimmune to sheep red blood cells (SRBC). The antibody binds to the SRBC-specific TsF as well as to a TsF (TNP-TsF) from another system differing in both antigen specificity and MHC. It does not bind to unrelated proteins. The antibody inhibits the activity of the SRBC-specific TsF in vitro. By using the monoclonal anti-TsF, we can isolate sufficient quantities of TsF to demonstrate that it fulfills several properties that have been attributed to TsF, namely, MHC restriction, antigen specificity, and the requirement for a second chain. Also, the purified TsF gives a single 68,000 dalton band upon SDS-PAGE gel analysis under reducing conditions. We conclude, therefore, that we have a method of the isolation of pure TsF, as well as a probe for the genetic, biochemical, and biologic analysis of TsF.     

Anti-idiotypic treatment of BALB/c mice induces CRIa-bearing suppressor cells with altered Igh-restricted function

The ABA-specific antibody response of A/J mice (Igh Ie) is dominated by the CRIa idiotype. In contrast, BALB/c mice (Igh Ia) do not produce CRIa-bearing anti-ABA antibodies after antigenic challenge. We have shown previously that treatment with rabbit anti-CRIa (R-anti-CRIa) induces the expression of "CRIa-like" anti-arsonate antibodies in BALB/c mice. In the present report, we demonstrate that R-anti-CRIa treatment enables BALB/c mice to respond to A/J ABA-specific first-order suppressor molecules (TsF1). Manipulated BALB/c also produced CRIa bearing ABA-specific immune response. Thus, R-anti-CRIa treatment induces a change in the characteristic Igh restriction pattern typically seen in this system. These data suggest that Igh restriction in the ABA-specific T suppressor cell pathway is the result of CRIa+ dominance in the T suppressor cell response of A/J mice. The effectiveness of idiotypic manipulation in inducing the expression of a given idiotype at both the B cell and T suppressor cell levels is discussed.     

The immune response and the eye. I. The effects of monoclonal antibodies to T suppressor factors in anterior chamber-associated immune deviation (ACAID)

We report the effects of two monoclonal antibodies (mab) specific for murine T suppressor (Ts) factors (TsF) in anterior chamber (AC)-associated immune deviation (ACAID), as induced by AC inoculation of TNP-coupled syngeneic spleen cells (TNP-Spl). One mab (14-12) is specific for Ts effector factor and can block the induction of Ts cells in ACAID if given before or after AC injection of TNP-Spl. The other mab (14-30) is specific for Ts inducer factors and blocks suppression only after given after TNP-Spl. We also studied the surface phenotype of the Ts cells induced by AC injection of TNP-Spl. We show that at least two cells are required for the adoptive transfer of suppression in TNP-ACAID. One is Lyt-2+ and 14-12+, the other is I-J+. These Ts cells have the surface phenotype of Ts effector cells as seen in other systems. These results indicate that mab which bind TsF in other systems affect Ts cells in TNP-ACAID, and that the Ts cells induced in TNP-ACAID are only of the Ts effector type.     

Mechanism of action of a T suppressor factor (TsF) in contact sensitivity: the T cell target for TsF activity in adoptive transfer of immunity is not effector cell

The passive transfer of contact sensitivity (CS) by immune cells into normal animals requires the interaction of two distinct Ly-1+ T cells, one which is Vicia villosa lectin (VV)-nonadherent, the other which adheres to VV. Functional deletion of either cell type abrogates the adoptive transfer of CS into normal animals, whereas VV-nonadherent cells alone can transfer CS into animals pretreated with cyclophosphamide (Cy). An antigen-specific T suppressor factor, designated TNP-TsF, inhibits the transfer of CS into normal adoptive recipients. TNP-TsF mediates its suppressive activity by inducing an I-J+ subfactor (designated I-J2) from the assay population by the interaction of PC1-F (a TNP-binding subfactor of TNP-TsF) with antigen-primed Ly-2+ T cells. This I-J+ subfactor then complements TNBS-F (an antigen-nonbinding subfactor of TNP-TsF) to form an antigen-nonspecific effector molecule which suppresses DTH responses in an antigen-nonspecific fashion. We report here that TNP-TsF suppresses the adoptive transfer of CS into normal animals but not into animals pretreated with Cy. TNBS-F + I-J2, the effector complex of TNP-TsF, also suppresses the transfer of CS into normal but not Cy-treated animals. When the Ly-1 immune cells were separated into VV-adherent and -nonadherent populations, the TNBS-F + I-J2 suppressor complex suppressed the functional activity of the VV-adherent cell population, but not the VV-nonadherent cells. This suppressive activity correlates with the need for VV-adherent cells in the transfer of CS into normal but not Cy-treated recipients. When an I-J+ molecule (I-J1) from an SRBC-specific TsF was used in place of I-J2 to form a suppressor complex with TNBS-F, this TNBS-F + I-J1 TsF suppressed the transfer of CS into both normal and Cy-treated recipients. This difference in functional suppressive activity correlated with a difference in target cell specificity: TNBS-F + I-J1 suppressed the VV-nonadherent TDTH cell, whereas TNBS-F + I-J2 suppressed the VV-adherent T cell of CS. Immune cells which are transferred under conditions which do not require the VV-adherent cell for transfer are not suppressed by TNBS-F + I-J2 or TNP-TsF, but are suppressed by the TNBS-F + I-J1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Information transfer between T cell subsets is directed by I-J+ antigen nonspecific molecules

T cell antigen-specific suppressor factors (TsF) consist of two distinct polypeptide chains: one that binds antigen (ABM) and one that bears I-J region markers (I-J+ chain). We studied the functional role of these two molecules in delivering the biologic message of suppression to its appropriate target cell. Two different biologically active TsF were used in these studies: TsiF, a T suppressor-inducer factor consisting of an ABM secreted by Ly-1 T cells (Ti-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Ji), which initiates the suppressor circuit by inducing an Ly-1,2 T cell; and TseF, a T suppressor-effector factor consisting of an ABM secreted by Ly-2 T cells (Te-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Je), which delivers the biologic message of suppression to the T helper (TH) cell. In both TsF, the ABM and I-J+ chain are noncovalently associated and can be easily separated. Both molecules must be present, however, for biologic activity of the TsF to be manifest. We studied the role of each chain in delivering these biologically active messages by constructing "hybrid" factors made from mixing the ABM from TsiF with I-J+ chains from either TsiF or TseF and determined which of these chains could reconstitute functional TsiF activity. Likewise, we mixed the AMB from TseF with I-J+ chains of TsiF or TseF to determine which I-J+ chain could reconstitute TseF activity. We found that I-J+ chain from TsiF (I-Ji) can reconstitute ABM from TsiF to form a functional TsiF capable of inducing suppression but cannot reconstitute ABM from TseF to form a functional TsiF capable of suppressing the activity of TH cells. Likewise, the addition of I-J+ chain from TseF to ABM from TseF can reconstitute its ability to suppress TH responses, but I-J+ chain from TsiF plus ABM from TseF has no effect on these TH cell responses. We did find, however, that this hybrid TsF composed of the ABM from TseF and the I-J+ chain from TsiF is capable of suppressing the Ly-1,2 Ttrans cell, the cell normally induced by the ABM + I-J+ suppressor inducer complex from T suppressor-inducer cells (TsiF).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Activation of acceptor-suppressor hybridoma with antigen-specific suppressor T cell factor of two-chain type: requirement of the antigen- and the I-J-restricting specificity

The molecular mechanisms of activation of immunoregulatory T cells were characterized by using two complementary suppressor T cell hybridoma systems: the KLH-specific monoclonal suppressor factor (KLH-TsF), and the inducible acceptor-suppressor hybridoma line with anti-idiotypic receptor for KLH-TsF. It was demonstrated that the identity of the KLH specificity and genetic specificity was required for the TsF-acceptor interaction. These specificities were found to be mediated by the two polypeptide chains of TsF: KLH-binding, Ct-bearing heavy chain and I-J+ light chain. These two chains were essential for stimulation of the acceptor hybridoma. The results were also confirmed by the findings that the mixture of the 11S and 13S mRNA translation products reconstituted the active TsF to stimulate the acceptor hybridoma. Furthermore, the genetic restriction observed was found to be mediated by the I-J+ light chain and to be governed by the gene linked to the H-2 complex but not to the Igh genes. The gene controlling the restriction specificity was strongly suggested to be in the intra-H-2 complex, but not outside of the H-2 complex.     

Characterization of an anti-idiotypic T cell hybridoma involved in the regulation of the immune response to the P815 mastocytoma

We report the isolation and characterization of a T cell hybridoma (A29) which secretes a factor that exhibits anti-idiotypic and immune-modulating characteristics. The A29 cell line is thought to represent the hybrid analog of the Ts2 suppressor cell population in the cascade regulating the immune response to the P815 tumor in DBA/2 mice. The putative TsF2 molecule is reactive with the monoclonal antibody B16G, shown previously by us to bind a public specificity of T suppressor factors (TsF). A29 TsF also exhibits specific binding to a TsF1 secreted by another T cell hybridoma, A10, which shows specificity for antigen from the P815 tumor (this has been described previously). A29 itself does not exhibit binding to P815 antigens. Affinity-purified material from A29 appears to share characteristics with A10 molecules in that the predominant material has an apparent m.w. of 70,000. Studies with calcium flux of A29 cells showed that they respond significantly and specifically on exposure to A10 TsF stimulus. We showed further that affinity-purified A29 TsF molecules can specifically suppress the in vitro generation of syngeneic CTL to the P815 tumor, and that panning of DBA/2 splenocytes over A29-TsF-coated plates renders cell populations capable of generating a higher in vitro CTL response to P815 than appropriately treated controls.