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.     

Comparison of T suppressor factors from tumour-bearing mice and mice immunized with a monoclonal anti-idiotypic antibody

Summary Certain dosage schedules of a monoclonal antiidiotypic antibody (related to a murine bladder carcinoma) were found to induce suppressor factor production by syngeneic mice. This suppressor factor resembled the factor from tumour-bearing mice with respect to idiotype specificity, possession of molecular markers (reactive with anti-IJ and B16G antibodies) and production by Lyt2⁺IJ⁺ T cells in spleen cell cultures. The two factors differed with respect to Igh restriction in an in vitro assay (leucocyte adherence inhibition) and ability to suppress the induction of delayed-type hypersensitivity to tumour antigen.     

The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells

Cultures of normal spleen cells with anti-idiotypic antibody (anti-Id) or antigen B (AgB)-specific T suppressor factor (Tsf1) in mini-Marbrook chambers for 4 days at 37 degrees C lead to the in vitro induction of AgB-specific T suppressor (TS) cells. These TS cells significantly suppress a secondary AgB-specific IgE response, but they do not affect a secondary AgB-specific IgG response. Depletion of both B cells and macrophages from normal spleen cells by panning on anti-Ig-coated petri dishes provides an enriched T cell population. These enriched T cells when cultured with anti-Id or Tsf1 in mini-Marbrook chambers do not produce AgB-specific TS cells, and mice treated with cells harvested from the mini-Marbrook chambers have normal secondary AgB-specific IgG and IgE responses. The addition of as few as 1000 bone marrow-derived macrophages (BMDM) to cultures of the enriched T cells with anti-Id, or Tsf1 restores the ability of these cultures to produce significant levels of AgB-specific TS cells. Further studies reveal that the macrophage population must be histocompatible and express a cell surface I-J antigen. Attempts to pulse BMDM with anti-Id or Tsf1 at 4 degrees C and to culture in mini-Marbrook chambers 10(3) pulsed BMDM with enriched T cells were unsuccessful in producing AgB-specific TS cells. However, pulsing BMDM with anti-Id or Tsf1 at 37 degrees C, and adding 10(3) of these pulsed BMDM to enriched T cells in culture led to the formation of significant levels of AgB-specific TS cells.     

Characterization of anti-idiotypic suppressor T cells (Tsid) induced after antigen priming

The induction and fine specificity of idiotype-specific suppressor T cells (Tsid) were studied. Spleen cells from C57BL/6 mice, immunized 4 wk previously with NP-KLH, failed to express NPb3 idiotype-bearing PFC when challenged in vitro with NP-Ficoll or NP-Brucella abortus. After treatment of NP-primed responder cultures with anti-Thy-1.2 anti-serum + C, NPb idiotype-bearing B cells could be detected. This B cell subset was preferentially suppressed by the addition of T cells from NP-primed mice. With this reconstitution protocol, it was determined that suppression of the NPb idiotype-bearing portion of the B cell response was mediated by a specifically induced T cell population (Tsid) that directly suppressed NPb-bearing B cells. As with a previously described suppressor population induced with hapten-modified syngeneic spleen cells (Ts2), the Tsid population bound and was lysed by NPb idiotype-bearing serum antibodies. However, the Tsid could be distinguished from the Ts2 population because it lacked I-J determinants and functioned as an effector T cell, not an intermediary suppressor cell. Furthermore, fine specificity studies with monoclonal NP-specific antibodies expressing various levels of serologically detectable NPb idiotypic determinants indicated that unlike the Ts2, the Tsid population reacts with conventional, serologically detected members of the NPb family. The combined idiotype binding studies for the Tsid and Ts2 populations demonstrate that the fine specificity of suppressor T cell populations reflects their independent mechanisms of regulation.     

Reactivity and regulatory properties of human anti-idiotypic antibodies induced by T cell vaccination

Immunization with irradiated autoreactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that preferentially recognize complementarity-determining region 3 sequences, contributing to clonal depletion of autoreactive T cells. However, it remains unknown whether T cell vaccination elicits anti-idiotypic humoral responses and whether the anti-idiotypic Abs play a similar role in the regulatory mechanism induced by T cell vaccination. In this study we examined the occurrence, the reactivity pattern, and the regulatory role of anti-idiotypic Abs elicited by T cell vaccination in patients with multiple sclerosis. We demonstrated for the first time that B cells producing anti-idiotypic Abs could be isolated from vaccinated patients. These EBV-transformed B cell lines were selected for specific reactivity to a 20-mer TCR peptide incorporating a common complementarity-determining region 3 sequence of the immunizing T cell clones. The resulting anti-idiotypic Abs were found to react with the original immunizing T cell clones and exhibit an inhibitory effect on their proliferation. The findings suggest that anti-idiotypic Ab responses can be induced by T cell vaccination in humans and that their regulatory properties are likely to contribute to the suppression of myelin basic protein-reactive T cells in vaccinated patients. The study has important implications in our understanding of the regulatory role of the anti-idiotypic humoral responses induced by T cell vaccination.     

Preferential recognition of TCR hypervariable regions by human anti-idiotypic T cells induced by T cell vaccination

T cell responses to myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). Immunization with irradiated MBP-reactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that suppress circulating MBP-reactive T cells. This T cell-T cell interaction is thought to involve the recognition of TCR expressed on target T cells. The study was undertaken to define the idiotypic determinants responsible for triggering CD8+ cytotoxic anti-idiotypic T cell responses by T cell vaccination in patients with MS. A panel of 9-mer synthetic TCR peptides corresponding to complementarity-determining region 2 (CDR2) and CDR3 of the immunizing MBP-reactive T cell clones were used to isolate anti-idiotypic T cell lines from immunized MS patients. The resulting TCR-specific T cell lines expressed exclusively the CD8 phenotype and recognized preferentially the CDR3 peptides. CDR3-specific T cell lines were found to lyze specifically autologous immunizing MBP-reactive T cell clones. The findings suggest that CDR3-specific T cells represented anti-idiotypic T cell population induced by T cell vaccination. In contrast, the CDR2 peptides were less immunogenic and contained cryptic determinants as the CDR2-specific T cell lines did not recognize autologous immunizing T cell clones from which the peptide sequence was derived. The study has important implications in our understanding of in vivo idiotypic regulation of autoimmune T cells and the regulatory mechanism underlying T cell vaccination.     

Isolation and characterization of a tumor-specific T suppressor factor from a T cell hybridoma

In our laboratory we have described a monoclonal antibody, B16G, which has been shown to bind to suppressive T cell factors (TsF) in DBA/2 mice. Therefore, B16G was used as a probe to identify T cell hybridomas secreting putative TsF. Hybridomas were obtained by the fusion of DBA/2 thymocytes stimulated in vivo by P815 tumor membrane extracts with the thymoma BW5147. One such hybridoma, A10, was selected and used for additional studies. From both the supernatants and ascites fluid of this hybrid a factor could be obtained that could specifically bind to both B16G and P815 antigen immunoadsorbent columns, and that scored positively with B16G in an ELISA after elution. Such reactivity could not be obtained from A10 supernatants or ascites absorbed over irrelevant columns, nor was it obtained from supernatants or ascites from other T cell hybrids that had scored B16G nonreactive in the original screening. In vivo studies indicated that affinity-purified A10 material injected into DBA/2J mice enhanced significantly the growth of P815 tumor cells, but not the growth of other DBA/2 syngeneic tumor lines such as L1210 or M-I. Additionally, this material did not inhibit the in vitro mixed leukocyte reaction (MLR) between DBA/2 splenocytes and allogeneic B10.BR target cells (unlike B16G purified material from whole DBA/2 spleens, which has been demonstrated to be suppressive in this type of MLR). Biochemical analysis of this tumor-specific TsF from A10 was undertaken; the native m.w. was found to be in the region of 80,000 and 90,000. Under reducing conditions, affinity-purified A10 TsF was found to resolve in SDS-PAGE as what appeared to be a heterodimer of 45,000 and 43,000. In most preparations, an associated molecule resolving at about 25,000 was observed. The implications of these observations are discussed.     

Recognition of a B cell lymphoma by anti-idiotypic T cells

Idiotypic IgM derived from a B cell lymphoma can act as a tumor-associated Ag, in that immunization with this purified protein generates an anti-idiotypic immune response that specifically suppresses tumor development. Spleens of immune mice contain T cells that proliferate in response to idiotypic IgM. However, idiotypic Ag is presented to the T cells most efficiently in its natural form at the surface of the lymphoma cells, than as soluble IgM plus presenting cells. Variant tumors that display either little or no idiotypic IgM at the cell surface, but which are otherwise indistinguishable from parental tumor, induce a weak response or fail to stimulate the T cells, respectively. Anti-idiotypic lines and clones have been derived from the splenic T cells by growth in the presence of irradiated tumor cells. Phenotypic analysis revealed that cells from both lines and clones express CD3 and CD4 Ag, but not CD8. Recognition of tumor Id, which required no added presenting cells, was inhibited by antibody against MHC class II Ag, and variably by anti-CD4. Proliferative responses were inhibited by anti-idiotypic antibodies, but also by antibodies against the constant region of the mu H chain, indicating that perturbation of the surface IgM abrogates availability of idiotypic determinants to the T cells.     

Enhancement of suppressor T cell activity by injection of anti-IFN-gamma monoclonal antibody

In the present study, the contribution of IFN-gamma to the generation of helper activity in mice was investigated by use of anti-mouse IFN-gamma rat mAB (AN 18.17.24). This mAb was alum precipitated and injected i.p. before or after carrier priming. Results show that spleen cell helper activity is markedly inhibited by anti-IFN-gamma mAb injection. This inhibition is time and dose dependent, and counteracted by IFN-gamma administration. Thus, the anti-IFN-gamma mAb appears to inhibit helper cell activity by neutralization of the IFN-gamma required for the antibody response. Moreover, AN 18.17.24 mAb injection results in increased activation of Lyt-2+ T cells which markedly suppress Th activity. These findings altogether indicate that besides the activation of macrophages and Th, IFN-gamma seems to exert a negative interference in suppressor T lymphocyte circuits and, as a consequence, to inhibit immunosuppression.     

抗独特型抗体与T细胞记忆的关系

记忆T细胞作为人体免疫系统中的一个组成部分,在免疫应答中发挥着至关重要的作用,因此利用抗独特型抗体制备诱导产生记忆T细胞的疫苗是免疫学领域的一个重要方向。抗独特型抗体Fab段具有与特异性抗原相似的抗原决定簇的结构,其作为抗原替代物制备的疫苗所激发机体产生的记忆T细胞具有特异性强和安全性高的特点,成为一种比较理想的疫苗.就抗独特型抗体与T细胞记忆之间的联系及其应用效果作一简要综述。     

Identification of a T cell subset using a rabbit antibody raised against a T suppressor molecule

Rabbit antiserum to a unique component of an Ag-binding Ts-factor was generated by repeated immunization with purified 30-kDa protein isolated from Fd11 Ts factor (11). This antiserum (anti-p30) was shown to recognize cell surface determinants expressed on the Ts hybridomas Fd11 and A10 but not the fusion partner BW5147. Furthermore, this antiserum was shown to bind to approximately 4% of thymocytes and 10% of nylon wool-purified splenic T cells from all strains of mice tested. Sorting nylon wool-purified T cells from DBA/2 mice for the CD4+ and CD8+ subsets revealed both populations contained cells that bound anti-p30. In addition, when CD4-8- thymocytes were examined for anti-p30 labeling, it was found that about 30% of this enriched population also expressed p30 molecules. In a functional study, anti-p30 was able to neutralize the suppressive effects of Fd11 on a specific assay for in vitro antibody synthesis against ferredoxin.     

Suppressor T cell growth and differentiation: evidence for induced receptors on suppressor T cells that bind a suppressor T cell differentiation factor

T suppressor cell differentiation factor (TsDF) induces the differentiation of alloantigen-primed suppressor T cells (MLR-Ts) to expression of their effector function, i.e., to active TsF production. The initial activation stimulus to Ts is provided by alloantigen binding; after this binding, Ts are functionally responsive only for a period of hours to the additional stimulus provided by TsDF. The present studies addressed the possibility that MLR-Ts responsiveness to TsDF reflects the induced and transient display of TsDF-binding receptors. TsDF receptor expression was investigated by determining the capacity of TsDF-responsive MLR-Ts to adsorb TsDF activity and to respond to that TsDF pulse by TsF production. Primed Ts populations that were alloantigen restimulated for 8 hr adsorbed TsDF in a cell dose-dependent fashion and produced TsF in response to that adsorption, whereas alloantigen-stimulated naive cells or primed but nonrestimulated cells neither responded to nor bound TsDF. Primed and restimulated L3T4-Ly-2+ but not L3T4+-Ly-2--enriched T cells bound TsDF. TsDF adsorption was saturable and time and temperature dependent. Glutaraldehyde fixation did not prevent TsDF adsorption by restimulated MLR-Ts, whereas pronase treatment abolished their TsDF-binding capacity. Kinetic analyses demonstrated that the capacity to bind TsDF developed rapidly after alloantigen reexposure, with maximal binding within 8 hr, followed by rapid decay with loss of TsDF binding by 36 hr. The kinetics of TsDF-induced TsF production correlated precisely with those of TsDF binding. These observations provide strong evidence that TsDF affects primed alloantigen-reactive Ts by interaction with antigen-induced and transiently expressed cell surface receptors. TsDF-receptor binding is then the stimulus for expression of Ts effector function.     

Human suppressor T cells induced by concanavalin A: suppressor T cells belong to distinctive T cell subclasses.

Human peripheral blood lymphocytes were stimulated by concanavalin A (Con A) and then evaluated by their suppressive activity for thymus-derived (T) cell- and bone marrow-derived (B) cell-proliferative responses to mitogen and allogeneic cells. Con A-activated T cells markedly suppressed these responses, but Con A-activated B cells failed to demonstrate suppressor activity. Discontinuous bovine serum albumin (BSA) density gradient separation of T cells which had been activated by Con A demonstrated that a fraction containing blast cells as well as fractions containing unproliferated cells manifest the same degree of suppressor capabilities. However, when density gradient separation of T cells followed by subsequent incubation with Con A was performed, fractions of proliferating cells of low density exhibited no suppression; a fraction containing high density T cells produced marked suppression, but this fraction incorporated only little thymidine in response to Con A. Thus, these studies indicate that Con A-induced suppressor T cells belong to a distinctive subpopulation which has already been programmed to express this function before exposure to Con A and that cell proliferation may not be a prerequisite for the development of such suppressor T cells.     

Preliminary characterization of human T cell suppressor factor (HTsF) isolated from tonsil cells by monoclonal antibody immunoadsorbence

We have shown that a murine monoclonal antibody, B16G, recognizes a constant region determinant of a T cell suppressor factor (TsF) in DBA/2 mice. The molecule recognized by B16G was shown to be a heterodimer with a native m.w. in the region of 80,000. We now show that B16G also reacts with a similar molecule derived from human lymphoid tissue. Yields of about 100 micrograms could be obtained from the solubilized membranes and cytosol from about 10(10) tonsillar cells by elution of adsorbed materials from B16G immunoadsorbent columns. As with the murine system, the human TsF (HTsF) thus derived was capable of suppressing the mixed leukocyte reaction (MLR) of homologous effector T lymphocytes. However, this same material was not suppressive across the HL-A barrier, that is, when allogeneic effector cells were used in the MLR. Preliminary characterization of the HTsF showed it to have a native m.w. of 80,000 to 90,000 to be composed of a heterodimer with subunit m.w. in the region of 45,000 to 50,000, and to have an associated peptide of approximately 25,000. These observations provide evidence for the conserved nature of genes encoding TsF and correlate with observations of other investigators on the considerable homology between genes encoding the murine and human T cell receptor.     

Partial purification and biochemical characterization of a T cell suppressor factor produced by human glioblastoma cells

The human glioblastoma cell line 308 constitutively secretes a soluble factor with biologic and biochemical characteristics of human monocyte-derived interleukin 1 (IL 1). The 308 cells also produce a 97,000 m.w. factor that inhibits the effects of IL 1 and interleukin 2 (IL 2) on T lymphocytes. By using sequential chromatography on Blue Affigel, hydroxyapatite, and Ultrogel AcA54, the inhibitory factor, termed glioblastoma-derived T cell suppressor factor (G-TsF), was separated from IL 1 and purified 2000-fold with respect to the protein present in the crude 308 cell supernatant. This G-TsF preparation was sensitive to tryptic proteolysis, showed a peak of pI 4.6 on isoelectric focusing, and when labeled with 125I, revealed six protein bands in the range of 30 to 100 kdaltons on SDS gel.     

T cell antigen receptor phosphorylation induced by an anti-receptor antibody

In previous studies we demonstrated that the antigen receptor complex on murine T cells is phosphorylated after antigen or mitogen activation. After the clonotypic structures bind antigen, the invariant subunits or CD3 molecules are the target of dual kinase activation. The antigen receptor CD3-gamma-chain subunit is phosphorylated on serine residues by activated protein kinase C and the p21 subunit is phosphorylated by a tyrosine kinase. Herein we demonstrate that another mechanism of receptor activation by the stimulatory monoclonal antibody 145-2C11, which binds the CD3-epsilon chain, results in a similar pattern of kinase activation and receptor phosphorylation.     

Identification,isolation, and characterization of a human T cell population by a monoclonal antibody

The hybridization of spleen cells from mice immunized with mononuclear leukocytes with the HAT-sensitive nonsecreting myeloma, NS1, resulted in the production of hybrid cell lines secreting monoclonal antibodies to lymphocyte surface antigens. One of these, anti-Ta, was shown by fluorescence-activated cell sorter analysis to be specific for a subpopulation of peripheral human T cells. Anti-Ta did not react with peripheral human B cells. Immunoprecipitation followed by two-dimensional gel analysis demonstrated that the T cell subpopulation-specific antigen recognized by this monoclonal antibody is part of, or firmly associated with, a protein of the plasma membrane.     

Characterization of the antibody response against the type II collagen induced by anti-idiotypic antibody.

We reported that rabbit anti-idiotypic antibody (Ab2) against mAb, termed 1-5 (Ab1) and reactive with human type II collagen (CII) induced antibody response to CII in DBA/1J mice susceptible to collagen-induced arthritis. In the present study, we further characterized the anti-CII antibody response elicited by Ab2 with respect to epitope specificity, putative genetic background, and IgG subclass. Most of anti-CII antibodies (polyclonal Ab3) derived from Ab2-immunized mice were of the IgG1 subclass. We purified polyclonal Ab3, using a CII-coupled immunoadsorbent column and we developed monoclonal Ab3 from Ab2-immunized mice. Both purified polyclonal Ab3 and two monoclonal Ab3s specifically reacted with a selected epitope on CII, recognized by Ab1. The anti-CII antibody response stimulated by Ab2 was observed in DBA/1J (H-2q, Igh-1c) and DBA/2 (H-2q, Igh-1c) mice, but not in the BALB/c (H-2d, Igh-1a) and C57BL/6 (H-2b, Igh-1b) strains, thereby suggesting that the anti-CII antibody response elicited by Ab2 is controlled by the Igh gene.     

Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma

By fusing Con A-activated BALB/c mice spleen cells with AKR thymoma BW5147 cells, we prepared a hybridoma producing a monoclonal nonspecific suppressor factor (MNSF). This factor inhibits a generation of LPS-induced immunoglobulin-secreting cells. We used ELISA for the bioassay of MNSF activity. With this method, a stable E17 hybridoma clone was selected, and its product in culture medium was isolated and characterized. MNSF fractionated on Sephadex G-100 in saline buffer shows a form with multiple m.w., but fractionated in 0.4 M pyridine-acetic buffer, it is limited to two species of approximately 24Kd and 16Kd. The MNSF was purified by hydroxyapatite chromatography, with marked effectiveness. MNSF activity was found exclusively in the 0.35 M sodium phosphate elution, and the content was further fractionated on subsequent gel filtration in the high ionic strength buffer described above. The purified factor exhibited two forms, of 24Kd and 16Kd, and showed peaks of pI 5.3 and 5.7, respectively, on isoelectric focusing. The MNSF preparation described here is stable at 56 degrees C and unaffected by 2-mercaptoethanol, but is unstable at pH 2.0 and is sensitive to tryptic proteolysis. We injected the hybridoma cells into the peritoneal cavity of pristane-primed F1 (AKR/J X BALB/c) mice, and a large amount of pure MNSF was obtained from the ascites, the characteristics of which were similar to those in the culture supernatant. Thus, the MNSF obtained from the E17 hybridoma consists of functionally identical but physicochemically different discrete proteins. This simple method of purification can serve as a probe for further characterization of MNSF and its application in in vivo experiments.     

Idiotype and antigen-specific T cell responses in mice on immunization with antigen, antibody, and anti-idiotypic antibody.

Idiotypic determinants of immunoglobulin molecules can evoke both CD4(+) and CD8(+) T responses and exist not only as the integral components of a bona fide antigen binding receptor but also as distinct molecular entities in the processed forms on the cell surface of B lymphocytes. The present work provides experimental evidence for the concept that regulation of memory B cell populations can be achieved through the presentation of idiotypic and anti-idiotypic determinants to helper and cytotoxic cell. The potential of B cells to present antigens to helper and cytotoxic T cells through class II and class I MHC suggests a mechanism by which both B and T cell homeostasis can be maintained. We provide evidence for the generation of idiotype- and antigen-specific Th and Tc cells upon immunization of syngenic mice with antigen or idiotypic antibody (Ab1) or anti-idiotypic antibody (Ab2). The selective activation and proliferation of the antigen-specific Th and Tc cells mediated by idiotypic stimulation observed in these experiments suggests a B-cell-driven mechanism for the maintenance of antigen-specific T cell memory in the absence of antigenic stimulation, under certain conditions.