FoxP3 in Treg cell biology: a molecular and structural perspective.

Regulatory T cells (Tregs ) are specialized in immune suppression and play a dominant role in peripheral immune tolerance. Treg  cell lineage development and function maintenance is determined by the forkhead box protein 3 (FoxP3) transcriptional factor, whose activity is fine-tuned by its post-translational modifications (PTMs) and interaction partners. In this review, we summarize current studies in the crystal structures, the PTMs and interaction partners of FoxP3 protein, and discuss how these insights may provide a roadmap for new approaches to modulate Treg  suppression, and new therapies to enhance immune tolerance in autoimmune diseases.

In vivo molecular imaging of HER2 expression in a rat model of Barrett's esophagus adenocarcinoma.

Human epidermal growth factor receptor 2 (HER2) is involved in the malignant progression of several human cancers, including esophageal adenocarcinoma (EAC). The purpose of this study was to evaluate HER2 overexpression and to explore the feasibility of confocal laser endomicroscopy for in vivo molecular imaging of HER2 status in an animal model of Barrett's-related EAC. Rats underwent esophagojejunostomy with gastric preservation. At 30 weeks post-surgery, the esophagus of 46 rats was studied; endoscopic and histological findings were correlated with HER2 immunofluorescence on excised biopsies and gross specimens. At this age, 23/46 rats developed Barrett's esophagus (BE), and 6/46 had cancer (four EAC and two squamous cell carcinomas). A significant overexpression of HER2 was observed in esophageal adenocarcinoma compared with normal squamous esophagus (9.4-fold) and BE (6.0-fold). AKT and its phosphorylated form were also overexpressed in cancer areas. Molecular imaging was performed at 80 weeks post-surgery in four rats after tail injection of fluorescent-labeled anti-HER2 antibody. At this age, 3/4 rats developed advance adenocarcinoma and showed in vivo overexpression of HER2 by molecular confocal laser endomicroscopy with heterogeneous distribution within cancer; no HER2 signal was observed in normal or Barrett's tissues. Therefore, HER2 overexpression is a typical feature of the surgical induced model of EAC that can be easily quantified in vivo using an innovative mini-invasive approach including confocal endomicroscopy; this approach may avoid limits of histological evaluation of HER2 status on 'blinded' biopsies.

Prevention of breast tumour development in vivo by downregulation of the p185neu receptor.

Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial cells develop breast tumours at an average of 44 weeks of age. In this study, intraperitoneal injection of a monoclonal anti-receptor antibody specific for the rat neuT oncogene product dramatically affected tumour development in these transgenic mice in a dose-dependent manner. A significant proportion (50%) of mice, when injected with anti-receptor antibodies, did not develop tumours even after 90 weeks of age. The phosphotyrosine levels of the membrane fraction of breast tissues in the anti-receptor antibody-treated mice were almost completely abolished when a higher dose of antibodies was used. This study demonstrates, for the first time, that immunologic manipulation of an oncogene product can effectively prevent the development of tumours in a rodent transgenic model.

Inhibition of cellular DNA synthesis by reovirus occurs through a receptor-linked signaling pathway that is mimicked by antiidiotypic, antireceptor antibody.

Mammalian reovirus type 3 binds to a 67-kD surface glycoprotein on the membrane of neuronal cells. This interaction initiates the infective reovirus cycle. The physiological function of this virus receptor is not known, however, initial studies illustrate a striking structural and antigenic homology to the beta adrenergic receptor family. The earliest known pathologic effect of reovirus type 3 is an inhibition of host cell DNA synthesis within 8-10 h after virus attachment. The studies reported here demonstrate that binding and aggregation of reovirus receptor molecules provides the signal for this inhibitory process. Inhibition of DNA synthesis in the neuroblastoma cell line B104.G4 was unaffected by using replication-defective virus or when lysosomal processing of normal virus was blocked. Inhibition was mimicked by an antiidiotypic, antireceptor mAb. Inhibition was not observed when monovalent mAb fragments were bound to receptors, but was reconstituted when these fragments were aggregated by the addition of anti-Ig. The signal for the inhibitory effect was generated within the first 60 min after mAb binding. These observations demonstrate that reovirus and antiidiotypic pathogenicity can result from the perturbation of membrane proteins that may perform normal physiological functions.

Mechanisms of regulation of cell-mediated immunity. III. The characterization of azobenzenearsonate-specific suppressor T-cell-derived-suppressor factors.

Delayed type hypersensitivity to the hapten azobenzenearsonate (ABA) can be induced and suppressed by the administration of hapten-coupled syngeneic spleen cells by the appropriate route. Suppressor T cells stimulated by the intravenous administration of ABA-coupled spleen cells have been shown to produce a discrete subcellular factor(s) which is capable of suppressing delayed type hypersensitivity to azobenzenearsonate in the mouse. Such suppressor factors may be produced by the mechanical disruption of suppressor cells or by placing such suppressor cells in culture for 24 h. The suppressor factor(s) (SF) derived from ABA-specific suppressor cells exhibit biological specificity for the suppression of ABA delayed type hypersensitivity (DTH), but not trinitro-phenyl DTH, as well as the capacity to bind to ABA immunoadsorbents. Passage of suppressor factor(s) over reverse immunoadsorbents utilizing a rabbit anti-mouse F(ab')2 antiserum demonstrated that the antigen-specific T-cell derived SF does not bear conventional immunoglobulin markers. The suppressor factor(s) are not immunoglobulin molecules was further demonstrated by the inability of anti-ABA antibodies to suppress ABA DTH. Gel filtration of ABA suppressor factor(s) showed that the majority of the suppressive activity was present in a fraction with molecular weight ranging between 6.8 x 10(4) and 3.3 x 10(4) daltons. We also analyzed for the presence of determinants encoded by the H-2 major histocompatibility complex (MHC) and found that immunoadsorbents prepared utilizing antisera capable of interacting with gene products of the whole or selected gene regions of H-2 MHC, i.e., B10.D2 anti-B10.A and B10 anti-B10.A immunoadsorbents, retained the suppressive activity of ABA-SF. Elution of such columns with glycine HCl buffers (pH 2.8) permitted recovery of specific suppressive activity. Taken collectively such data supports the notion that suppressor T-cell-derived ABA suppressor factors have antigen-binding specificity as well as determinants controlled by the K end of the H-2 MHC. The distribution of strains capable of making SF has also been analyzed. The relationship of the antigen-binding specificity to VH gene products is discussed in this and the companion paper.

T lymphocyte-mediated suppression of myeloma function in vitro. IV. Generation of effector suppressor cells specific for myeloma idiotypes.

BALB/c mice immunized intravenously with syngeneic splenocytes, to which affinity-purified IgA produced by the MOPC 315 myeloma is covalently coupled, develop suppressor T cells (Ts1) that inhibit IgA secretion by MOPC 315 cells after 3-4 d of co-culture. Immunization with M315-coupled splenocytes subcutaneously, followed by administration of a soluble extract of Ts1 cells, leads to the generation of effector Ts that are also idiotype specific and inhibit myeloma function within 1 d. Moreover, effector Ts are Lyt-1-2+, whereas Ts1 are either Lyt-1+2+ or require Lyt-1+ and Lyt-2+ cells to mature into effector Ts in vitro. Such a protocol should be useful for analyzing the interactions that result in the maturation of Ts and in defining the mechanisms of action of Ts, whose effect can be measured on a homogeneous target population and that are specific for a well-characterized myeloma idiotype.

Conversion of immunity to suppression by in vivo administration of I-A subregion-specific antibodies.

The in vivo administration of antibodies specific for gene products of the I-A subregion represents an immunologically specific approach to the manipulation of Ly-1+ T cell responses to antigen. This has been demonstrated previously by the capacity of anti-I-A antibody treatment to abrogate T cell-mediated delayed-type hypersensitivity (DTH) responses to syngeneic tumor antigen, hapten, and non-H-2 histocompatibility antigens. Evidence obtained in these studies suggested that the primary action of antibody was related to its ability to interfere with macrophage-T cell interactions during antigen presentation, consistent with the demonstration that similar antibodies inhibit T cell binding to antigen-pulsed macrophages in vitro. Results presented in this report provide evidence for an additional consequence of in vivo antibody administration that may be secondary to any direct effects on I-A-restricted antigen presentation. Thus, animals treated with I-A subregion-specific antibodies also develop a population of antigen-specific suppressor T cells (Ts) capable of inhibiting recipient Ly-1+ T cell responses to tumor antigen. The induction of suppression appeared to be an essential component of the total biological activity of these antibodies, because elimination of Ts precursors by cyclophosphamide also abrogated the antibody-mediated inhibition of DTH responsiveness. These results are discussed with respect to the possible mechanisms of Ts activation by anti-I-A antibody administration, and the general applicability of this approach as a means of clinical immunotherapy to limit inappropriate T cell responses in human disease.

Mechanisms of regulation of cell-mediated immunity. VII. Suppressor T cells induced by suboptimal doses of antigen plus an I-J-specific allogeneic effect.

Intravenous injection of 0.01 mM 2,4,6-trinitrobenzene sulfonic acid-derivatized syngeneic lymphoid cells generates a Thy-1-positive, antigen-specific suppressor cell for contact sensitivity which requires an I-J allogeneic effect to become fully activated. It is necessary and sufficient for all allogeneic effect to be directed solely against the suppressor cell, and once activated, the cell can suppress in an H-2-unrestricted fashion. The results are discussed in the framework of entry into the suppressor pathway, the allogeneic effect as a reflection of normal physiologic processes, and the importance of I-J as a receptor and signal among cells in the suppressor pathway.

Characterization of Lyt-2-, L3T4- class I-specific cytolytic clones in C3H-gld/gld mice. Implications for functions of accessory molecules and programmed development.

We report the first demonstration of Thy-1+, Lyt-2-, L3T4- MHC-specific CTL clones derived from the Lyt-2-, L3T4- subset of lymph node cells of C3H-gld/gld mice. These clones express alpha/beta heterodimeric TCRs on the cell surface and specifically recognize class I molecules on target cells. Lyt-2 and L3T4 molecules are therefore not essential for the induction, recognition, and killing of antigen-specific CTL. In addition, these studies suggest that antigen specificity development for class I structures may occur before Lyt-2 gene activation in the differentiation of T cells.

Hapten-specific T-cell responses to 4-hydroxy-3-nitrophenyl acetyl. I. Genetic control of delayed-type hypersensitivity by VH and I-A-region genes.

Hapten-specific delayed-type hypersensitivity (DTH) was induced in several strains of mice. (4-hydroxy-3-nitrophenyl)acetyl-bovine gamma globulin (NP-BGG)-primed mice which did not bear the Ig1b heavy-chain linkage group made a NP-specific DTH response when challenged with NP bovine serum albumin (BSA) and failed to respond to challenge with (4-hydroxy-5-iodo-3-nitrophenyl)acetyl-bovine serum albumin (NIP-BSA). Strains of NP-BGG-primed mice bearing the Ig1b allotype, including SJL, responded to challenges of either NP-BSA or NIP-BSA. F1 hybrids between a cross-reactive strain, C57BL/6, and two other noncross-reactive strains were cross-reactive. Genetic mapping of the NIP-cross-reactive DTH response localized the trait to the VH-region of the Ig1b heavy-chain linkage group. The fine-specificity pattern of the T-cell anti-NP response, and the genetic mapping of this trait, were analogous to the reported fine specificity and mapping data of the humoral heteroclitic anti-NP response. Adoptive transfer studies on the ability to transfer NP-specific DTH between various strain combinations showed that the T-cell donors and the recipient must have homology for at least the I-A subregion. Whenever NP-specific reactivity was transferred from a strain which cross-reactively responded to NIP, the recipient also responded to both NP and NIP. The implications of the control of NP-primed DTH-reactive populations of T cells by two distinct genetic regions, VH and H-2, were discussed.

Mechanisms of regulation of cell-mediated immunity. IV. Azobenzenearsonate-specific suppressor factor(s) bear cross-reactive idiotypic determinants the expression of which is linked to the heavy-chain allotype linkage group of genes.

T-cell derived suppressor factor(s) (SF) specific for azobenzenearsonate (ABA) were prepared by the mechanical disruption of suppressor cells. Such suppressor factors were adsorbed to and recovered from immunoadsorbents prepared from the F(ab')2 fragments of rabbit immunoglobulin directed against the cross-reactive idiotype of A/J anti-ABA antibodies. These ABA-suppressor factors were not retained on Sepharose 4B immunoadsorbent columns which had been coupled with F(ab')2 fragments or normal rabbit immunoglobulins prepared from prebleeds of rabbits used to make anti-idiotypic antiserum. The specificity of the F(ab')2 rabbit anti-idiotypic serum was established by direct idiotypic-binding assays and by affinity purification over an immunoadsorbent consisting of CRI+ anti-ABA immunoglobulin from A/J mice. ABA-suppressor factors were shown to be specifically absorbed and eluted from F(ab')2 anti-idiotypic columns. Futhermore, the eluted suppressor factor can be specifically reabsorbed and recovered from a second anti-idiotypic immunoadsorbent. The concordance between antigen-binding specificity and the presence of idiotypic determinants was demonstrated by adsorbing ABA SF to antigen columns and then fractionating the ABA-specific factor on anti-idiotypic immunoadsorbents. ABA-suppressor factors were shown to be specifically retained on immunoadsorbents directed against major histocompatibility complex (MHC) determinants. Factor eluted from anti-MHC columns could then be specifically adsorbed to anti-idiotypic immunoadsorbents. This suggests that the same molecular complex that is recognized by the H-2 alloantiserum is specifically adsorbed to an anti-idiotypic immunoadsorbent. Genetic analysis of the expression of CRI+ suppressor factor was performed using the C.AL-20 mouse strain which has the AL/N allotype and produces CRI+ anti-ABA immunoglobulins. The implication of these findings to the nature of T-cell-derived regulatory molecules is discussed.

Antigen- and receptor-driven regulatory mechanisms. III. Induction of delayed type hypersensitivity to azobenzenearsonate with anti-cross-reactive idiotypic antibodies.

Delayed-type hypersensitivity (DTH) to p-azobenzenearsonate (ABA) can be induced in A/J mice with intravenous injection of minute amounts of anti-cross-reactive idiotypic (CRI) antibodies, providing that the animals have been pretreated 2 d earlier with low doses of cyclophosphamide (50 mg/kg). However intravenous injection of the F(ab')2 fragments of the anti-CRI antibodies or subcutaneous administration with anti-CRI antibodies induces comparable immunity in both cyclophosphamide-pretreated and normal nontreated animals. Furthermore adoptive transfer experiments indicate that lymph node cells taken from animals sensitized with anti-CRI 4 d earlier can adoptively transfer immunity to naive recipients. Transfer of immunity is mediated by a population of thymus-dependent (T) cells, which express idiotypic structures on their surface. Treatment of effector cells with either anti-theta serum or anti-idiotypic antibodies plus complement completely abrogated their ability to transfer immunity. In addition idiotype-bearing suppressor T cells induced with ABA-coupled spleen cells inhibit the development of ABA-specific DTH induced with anti-CRI antibodies. Genetic analysis revealed that the ability of anti-CRI antibodies to induce ABA-specific DTH was linked to Igh-1 heavy-chain allotype. Anti-idiotypic antibodies to the major CRI associated with anti-ABA antibodies in A/J mice failed to induce significant immunity in BALB/c mice (H-2d, Igh-1a). Nevertheless, they were able to induce significant immunity in C.AL20 mice (H-2d, Igh-1d) which possess a heavy-chain allotype similar to that of A/J mice.

The I-J subregion codes for determinats on suppressor factor(s) which limit the contact sensitivity response to picryl chloride.

The cell-mediated immune reactivity (CMI) of mice to contact chemicals such as picryl chloride (PCI) is influenced by thymus-derived suppressor T lymphocytes (1,2). The development of these suppressor T lymphocytes is stimulated by the intravenous administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Zembala and Asherson have further demonstrated that a specific suppressor factor(s) can be detected in the supernates of cultured suppressor T cells. This factor suppresses the transfer of contact sensitivity (CS) to PCl (1,2). In experiments reported elsewhere (3), we have shown that the PCl suppressor supernates of Zembala and Asherson can also suppress the development of contact sensitivity to PCl. The immunochemical analysis of suppressor factor (SF) operative in the CS response to PCl has revealed many similar properties (3) to other suppressive moieties functioning to limit the plaque-forming cell (PFC) response to dinitrophenylated-keyhole limpet hemocyanin (DNP-KLH) as well as the strict antigen specificity of each respective suppressive factor, suggested that there might be a common origin of these substances. Indeed, in each case these respective factors were found to bear determinants controlled by the H-2 gene complex (4,5). Recently, in selected systems, the I-J subregion has been found to code for the Ia determinants present on suppressor cells (6) and suppressor factors (4,5). In accord with these findings, we report that antigen-specific SF which limit the CS response to PCl bear I-J determinants, implying that analogous suppressive regulatory mechanisms in CMI as well as antibody responses may be determined by genes of one subregion of the H-2 complex.

Identification of an I-J+ antigen-presenting cell required for third order suppressor cell activation.

We have found that an I-J+ I-A- antigen-presenting cell (APC) is required for Ts3 activation in vivo. Together with the I-J restriction previously reported for Ts3 induction (Takaoki, M., M.-S. Sy, A. Tominaga, A. Lowy, M. Tsurifiji, B. Benacerraf, R. Finberg, and M. I. Greene, 1982, J. Exp. Med., 156:1325), it appears that this I-J+ APC is responsible for I-J restriction in the triggering of Ts3. This restriction may be exerted via a pre-Ts3 associative recognition of antigen and I-J encoded determinants, analogous to the T helper recognition of antigen in the context of I-A and I-E determinants.

Antigen- and receptor-driven regulatory mechanisms. I. Induction of suppressor T cells with anti-idiotypic antibodies.

Delayed-type hypersensitivity (DTH) to the azobenzenearsonate (ABA) hapten can be readily induced in A/J mice injecting ABA-coupled syngeneic spleen cells subcutaneously. To further characterize this T-cell-dependent immunological phenomenon, the effect of passively administered anti-cross-reactive idiotype common to anti-ABA antibodies of A/J mice (CRI) antibodies on the development of ABA-specific DTH was investigated. Animals given daily injections (of minute amounts) of anti-CRI antibodies subsequent to immunization with ABA-coupled cells show significant reduction of ABA specific responses. This inhibition is antigen specific and requires the intact immunoglobulin molecule, as F(ab')2 treatments were ineffective in suppressing the reaction. Investigations of the mechanism of the anti-CRI-induced suppression of ABA DTH revealed that the observed suppression is a result of the activation of suppressor cells. Spleen cells taken from animals which received anti-CRI antibodies were able to adoptively transfer suppression to naive recipients. This suppression was shown to be mediated by T cells, as anti-Thy1.2 plus complement completely abrogated the transfer of suppression. In addition, animals pretreated with low doses of cyclophosphamide were not suppressed by the administration of anti-CRI antibodies. The genetic restriction of anti-CRI-induced suppression was demonstrated. Antibodies to the major cross-reactive idiotype, (CRI) associated with anti-ABA antibodies in A/J mice were unable to suppress the development of DTH to ABA in BALB/c mice (H-2d, Igh-1a). Such antibodies were, however, fully active in suppressing ABA DTH in the allotype-congenic C.AL-20 strain which has an allotype (Igh-1d) similar to that of A/J (Igh-1e) on a BALB/c background, and which produces humoral antibodies with the CRI.

Antigen- and receptor-driven regulatory mechanisms. II. Induction of suppressor T cells with idiotype-coupled syngeneic spleen cells.

Anti-p-azobenzenearsonate (ABA) antibodies, coupled covalently to normal syngeneic spleen cells and then given intravenously to normal animals, were found to be potent tolerogens for delayed-type hypersensitivity (DTH) to ABA. The ability of the antibody-coupled cells to induce tolerance was determined to be a result of the cross-reactive idiotype (CRI+) fraction of the antibodies, because anti-ABA antibodies lacking the CRI+ components when coupled to spleen cells were unable to cause any significant inhibition. Furthermore, genetic analysis revealed that the ability of CRI-coupled cells to inhibit ABA-specific DTH is linked to Igh-1 heavy chain allotype, in as much animals which possess heavy chain allotypes similar to that of A/J were sensitive to this inhibition. Adoptive transfer experiments provided evidence that CRI-coupled cells induce suppressor cells, and spleen cells or thymocytes from animals received CRI-coupled cells were able to transfer suppression to naive recipients. In addition, treatment with anti-Thy1.2 serum plus complement completely abrogated their ability to transfer suppression. Thus, this active suppression is a T-cell-dependent phenomenon. In investigating the specificity of these suppressor T cells, it was found that they functioned in an antigen-specific manner and were unable to suppress the development of DTH to an unrelated hapten 2,4-dinitro-1-fluorobenzene.

Antigen- and receptor-driven regulatory mechanisms. IV. Idiotype-bearing I-J+ suppressor T cell factors induce second-order suppressor T cells which express anti-idiotypic receptors.

Administration of azobenzenearsonate (ABA)-coupled syngeneic spleen cells intravenously to A/J mice leads to the generation of suppressor T cells (Ts1) which exhibit specific binding to ABA-bovine serum albumin (BSA)-coated dishes. These Ts1 share idiotypic determinants with the major cross-reactive idiotype (CRI) of the anti-ABA antibodies of A/J mice, and also produce a soluble suppressor factor (TsF) bearing CRI and I-J subregion-coded determinants. Injection of this TsF into naive A/J mice elicits a second set of specific suppressor cells (Ts2) which are not lysed by anti-CRI antibody plus C, and which do not bind to ABA-BSA-coated dishes. However, in contrast with Ts1, these Ts2 do bind to plates bearing CRI+ anti-ABA immunoglobulin. Thus, Ts2 exhibit anti-idiotypic specificity. These data indicate that antigen elicits the production of a soluble T cell product bearing both variable portion of the Ig heavy chain (VH) and I-J subregion-coded determinants which serves to communicate between T cell subsets to establish an idiotype-anti-idiotype regulatory pathway.

T lymphocyte-mediated suppression of myeloma function in vitro. III. Regulation of antibody production in hybrid myeloma cells by T lymphocytes.

To investigate the mechanisms by which T lymphocytes regulate myeloma function in vitro, the effects of regulatory T cells on antibody secretion by a hybrid myeloma cell line were examined. Suppressor T cells (Ts) specific for idiotypic determinants on M315 (IgA, lambda 2 anti-2,4-dinitrophenol and anti-2,4,6-trinitrophenol [TNP]) and MPC 11 (IgG2b, kappa) myeloma proteins inhibit antibody secretion by the appropriate parental myeloma cells. When cocultured with a hybrid cell line derived by fusion of MOPC 315 and MPC 11 myelomas, the idiotype-reactive Ts inhibit secretion of only the immunoglobulin (Ig) bearing the relevant idiotype. In contrast, syngeneic TNP-reactive cytolytic T lymphocytes (CTL) inhibit antibody secretion by TNP-binding MOPC 315 cells but not by MPC 11 cells in the presence of soluble TNP-keyhole limpet hemocyanin (KLH), and this inhibition probably represents a prelytic effect of the CTL. Such TNP-reactive CTL, in the presence of TNP-KLH, inhibit both IgA and IgG secretion by the MOPC 315-MPC 11 hybrid, which is consistent with a prelytic effect. Thus, myeloma hybrids are a useful tool for investigating the effector function of regulatory T cells. These results are discussed with reference to the mechanisms of action of regulatory T cells and their relevance to modulation of physiologic humoral immune responses.

Syngeneic monoclonal antiidiotype can induce cellular immunity to reovirus.

A syngeneic monoclonal antiidiotypic antibody was generated in BALB/c mice after repeated immunization with a BALB/c monoclonal anti-reovirus hemagglutinin (HA) antibody. The resultant syngeneic monoclonal antiidiotypic antibody, in the absence of adjuvant, was found to be capable of priming both BALB/c (H-2d, Igh-1a) and C3H/Hej (H-2k, Igh-1j) mice for Lyt-1+- and Lyt-2+-dependent responses against the mammalian reovirus. By the use of intertypic reassortants and variant virus analysis, the specificity of the response was finely mapped to the neutralization domain of the viral hemagglutinin (HA). Using purified monoclonal antiidiotype, we were able to compare the potency of antiidiotype to virus in terms of induction of immunity. 8 X 10(8) protein molecules were able to prime for cellular responses to reovirus. These studies indicate that in the reovirus system, T cells and B cells share idiotypic configurations, and that antiidiotypic antibodies of the type described herein may be useful in the development of vaccines against certain viral infections.

Igh variable region-restricted T cell interactions. Genetic restriction of an antigen-specific suppressor inducer factor is imparted by an I-J+ antigen-nonspecific molecule.

Immunized Ly-1 T cells secrete an antigen-specific molecule that will induce Ly-2+ T cells to express suppressive activity. In two separate systems, factors that suppress the primary anti-sheep erythrocyte (SE) plaque-forming cell response of spleen cells in vitro (Ly-1 TsiF) or the contact sensitivity of azobenzenearsonate (ABA)-TsF1 consist of two macromolecules, one which binds antigen and is IJ-, the other which is I-J+ and does not bind antigen. Both of these chains are required for the factor's biological activity. These factors show a genetic restriction in their ability to induce suppression that is linked to the variable region of the Ig heavy chain gene complex (Igh-V). The I-J+ chain from the ABA-specific TsF1 could replace the I-J+ chain needed by the SE-specific Ly-1 TsiF for biological activity. Mixtures of ABA-binding chain with I-J+ material obtained from the SE-specific Ly-1 TsiF had no effect on the primary anti-SE response in vitro. In mixtures of SE antigen-binding chain from Ly-1 TsiF and I-J+ material from the ABA-specific TsF1, it is the I-J+ molecule that determined the factor's Igh-V restriction. Thus, the antigen-combining site of the factor determined the antigen specificity of this factor but is irrelevant to its Igh-V-linked genetic restrictions. The implications of these results for the idiotype network hypothesis are discussed.