Antigen-specific T-cell Factors Research Articles

Nitric oxide production by macrophages stimulated by antigen-binding T-cell factors

Contact sensitivity to small molecular weight compounds is accompanied by the production of antigen-specific T-cell factors (TCF) shortly after skin application of the sensitizing agents. In this study, we show that macrophages can be activated by these TCF to generate large amounts of nitric oxide (NO). Incubation of the murine macrophage cell line J774 for 24 h with TCF raised against dinitrofluorobenzene (DNFB) or picryl chloride (PCL) resulted in a nitrite accumulation in the culture medium. Priming of J774 with rIFN- γ synergistically enhanced stimulation of NO synthesis by DNFB-F and PCL-F. A possible contribution of lipopolysaccharide (LPS) as a contaminant of the TCF was excluded. The enhanced production of NO after stimulation with TCF was accompanied with an increased expression of inducible NO synthase. Inclusion of inhibitors of protein tyrosine kinase and protein kinase C inhibited the TCF-induced NO production by macrophages, indicating the involvement of both protein kinases in the signaling pathway activated by TCF. Since NO is an important biological mediator with many immunoregulatory properties, our results suggest a potential role for increased NO production by macrophages in the elicitation of contact sensitivity to small molecular weight compounds.

Specific T-cell factor production and lymphocytes in the direct surroundings of a subcutaneous allogeneic tumor

Antigen-specific T-cell factors (TCF) play a role in the initiation of cellular immune responses. In allogeneic mouse-tumor models lymphocytes from the direct tumor surroundings of both euthymic and nude mice produce TCF. These lymphocytes produce TCF when collected already 1 day after subcutaneous (sc) injection of tumor cells. In contrast to euthymic mice, draining lymph nodes and spleen of nude mice did not contain TCF-producing lymphocytes at any stage after sc tumor cell injection. In sensitized euthymic mice TCF production by lymphocytes is significantly higher in the direct tumor surroundings than in draining lymph nodes or spleen. At 2 and 5 days after tumor cell injection, the mononuclear cell infiltrate of the tissue surrounding the tumor in euthymic mice showed low expression of Thy 1, CD3, TCR αβ, TCR γδ, CD4, CD8, and asialo GM1, whereas several lymphocytes and mast cells were positive for monoclonal antibody (mAb) 14–30 (directed against TCF). In both euthymic and nude mice, sc injected tumor cells showed apoptosis. In conclusion, the direct tumor surroundings are the first (and, for nude mice, the only) site of TCF production, sc injection of tumor cells attracts mAb 14–30-positive lymphocytes and renders mast cells positive for mAb 14–30.

Antigen-specific T-cell factors and drug research.

The central feature of the immune system is the specificity of antigen-binding proteins for the ligand that induced their synthesis. It is this specificity that has intrigued medical scientists for the last 100 years and has led to repeated attempts to use this feature to combat disease. One of the earliest, successful uses of immune specificity involved the production of specific anti-toxins to combat a variety of ills from bacterial infections to snake bites [1]. In addition, advantage was taken of the fact that by providing appropriate antigens in the proper form (eg. viruses, bacteria), specific immune responses could be elicited [2]. Thus, over the last 80 years or so the primary focus of the therapeutic use of antigen-specific proteins has been the antibody molecule [3]. With the development of monoclonal antibody methodology, there has developed a renewed interest in the use of specific single antibodies as therapeutic agents; “magic bullets” as they are often termed [4]. In addition, the capacity to produce monospecific reagents is providing invaluable tools to probe the structure and function of other proteins; to use in the specific detection of cellular or tissue specific antigens and for purifying and identifying a wide variety of normal body components (eg. peptides, lipids, etc.) and/or ingested drugs [5]. In the early 1970s several groups of investigators reported that, in addition to antibodies, there exist other antigen-binding moieties that appear to function primarily as immune regulators.KeywordsDrug ResearchHelper FactorTissue Specific AntigenImmune Response ModelModulate Factor ActivityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Structural analysis of antigen-specific Ia-bearing regulatory T-cell factors: gel electrophoretic analysis of the antigen-specific augmenting T -cell factor.

An antigen-specific T-cell factor (TaF) that specifically augments the antibody response was purified and biochemically analyzed by NaDodSO4/polyacrylamide gel electrophoresis and isoelectric focusing. Biosynthetically labeled TaF was separated from the Nonidet P-40 extract of T-cell hybridoma FL10, which produces a keyhole limpet hemocyanin-specific TaF, by affinity chromatography either with antigen or with monoclonal anti-I-A antibodies. The material thus obtained was composed of two different types of molecules of molecular weights of 67,000 and 33,000 under nonreducing conditions. After reduction with dithiothreitol, all the molecules migrated to the position of molecular weight 33,000. The absorption studies with immunoadsorbents of antigen and antibodies revealed that the intact TaF is a heterodimer of two discrete polypeptide chains, one carrying a determinant detectable by a monoclonal anti-Tindd directed to an Igh-I -linked allotypic structure of T cells and being associated with the antigen-binding site and the other expressing a unique determinant controlled by the I-A subregion of murine H-2 major histocompatibility complex but being different from known class II polypeptide chains. The antigen-binding polypeptide has an isoelectric point of pH 5.6, and the I-A polypeptide has an isoelectric point of pH 6.3.

Constant region determinants on the antigen-binding chain of the suppressor T-cell factor.

Antigen recognition units on T cells, including isolated antigen receptors and antigen-specific T-cell factors, have been found to have antigen-binding moieties like conventional immunoglobulins and to carry the determinants analogous to the variable (V) region of the immunoglobulin1–5. However, the immunoglobulin heavy chain constant region (Igh-C) determinants have not been demonstrated on T-cell receptor molecules6–9. If they possess unique constant region determinants, they must therefore be entirely distinct from the Igh-C. By production of an antiserum between Igh allotype congeneic mice Owen et al. suggest that a T-cell constant region determinant (Tsud) is encoded by a gene located near the Igh-C genes on the 12th chromosome10,11. In addition, we have also demonstrated that a BALB/c anti-CB-20 antiserum detects at least two distinct allotypic determinants, either of which is expressed on the antigen-specific suppressor or augmenting T-cell factor12. We now demonstrate that the new allotypic determinants detected by the BALB/c anti-CB-20 antiserum are expressed on the constant region of the antigen-binding molecule of an antigen-specific suppressor T-cell factor (TsF), and that this molecule is encoded by genes located on the right side of the Igh- V gene locus on the 12th chromosome.

Monoclonal antibodies against unique I-region gene products expressed only on mature functional T cells

The immune response (I) region of the murine major histo-compatibility complex (MHC) regulates specific immune responses against antigens1 and encodes cell-surface molecules designated as Ia antigens2. Ia antigens are generally detected on B cells and macrophages, and have important roles in immunocyte interactions. On the other hand, it has been reported that some T cells and their products (antigen-specific T-cell factors) carry unique I region-encoded determinants which are different from B-cell Ia antigens3–10. As T-cell factors are known to mediate T–T and T–B cell interactions11, elucidation of such unique I-region determinants on T cells is obviously important for understanding the regulatory cell interactions of the immune response. However, this has long been hampered by the lack of reagents specific for the determinants and due to the small number of T cells expressing them. To overcome this difficulty, cell hybridization techniques have been used to develop T-cell hybrids which continuously express I-region products, and monoclonal antibodies specific for these determinants have been produced. Here we report the production of monoclonal antibodies which detect a unique set of I-region determinants expressed only on functional subsets of T cells and T-cell hybridomas.

Detection of T cells that secrete molecules which share determinants with antigen-specific T-cell factors.

A single-cell secretion assay was used to detect cells that secrete products which react with an antiserum that binds T cell antigen-binding polypeptides. The antiserum (R11), which was produced by immunization of rabbits with a murine trinitrophenyl-specific suppressor factor, reacts with T cells and their products and with a suppressor T-cell clone but not with B cells or their products. The secretory cells this antiserum detected were found to be unevenly distributed among various organs (spleen, lymph node, and thymus) and, to different degrees, in spleens of various strains of mice. In unimmunized CBA/J mice, approximately 1-3% of spleen cells secreted macromolecules precipitable by R11. The majority of the secretory cells could be removed by panning with a mixture of Ly 1 and Ly 2 antibodies but not with either antibody alone. This is consistent with the cells having low surface antigen densities as a result of being either "pre-T" cells or mature secretory cells analogous to B-lineage plasma cells. In agreement with the latter possibility was our finding that the secretory activity of cells detected with antisuppressor factor was comparable to that of Ig-secretory cells as detected with an anti-Ig antiserum. However, higher numbers of R11+ secretory cells were seen in the immunoglobulin-negative fraction of spleen cells from nude mice, which could be interpreted to favor the first possibility. In either case this study shows that the single-cell assay technique is well suited for the detection and characterization of molecules released by immunoregulatory T cells.

Isolation and partial characterization of an antigen-specific T-cell factor associated with the suppression of delayed type hypersensitivity.

Antigen-specific factors associated with immunosuppressive activity, released by cultured T cells from mice tolerant to the haptens trinitrophenyl, dinitrophenyl and oxazolone, were purified by hapten affinity chromatography. Their binding specificity for antigens paralleled their immunoregulatory activity. Like some immunoglobulin molecules, these factors had blocked NH2 termini and could be bound to Fc-like receptors on macrophages. However, neither immunoglobulin constant region determinants (isotypes) nor antigens encoded by the major histocompatibility complex were detected on the suppressive factors. The purified factors occurred as 68,000-dalton proteins and non-covalently linked dimers. No associated immunoglobulin light chain molecules were detected. The factors showed a marked propensity toward degradation with major breakdown products of 45,000-50,000 and 25,000-30,000 daltons. These results suggest that these molecules are the T-cell products analogous to B-cell immunoglobulin (equivalent to heavy chains) and that they may be the antigen-specific components which act in conjunction with major histocompatibility-controlled gene products to perform antigen-specific suppression.

Antigen-specific T-cell factors.

Antigen-specific T-cell factors are mediator molecules which are produced by helper and suppressor T cells and which can perform the function of those cells in an antigen-specific manner. They probably play an important part in immunoregulation. The major histocompatibility complex has a controlling influence on their structure and activity, while their antigen-recognition properties may be conferred by immunoglobulin V regions. Interest in the factors derives from three related areas of research, namely (i) the problem of T-cell recognition of antigen; (ii) the mechanisms of cellular interactions in antibody production and cell-mediated immunity; and (iii) the genetic control of immune responses. This review discusses the literature up to June 1980 on their production, structure, genetic restriction and mechanism of action.

Cellular consequences in the suppression of antibody response by the antigen-specific T-cell factor.

Cellular events mediated by antigen-specific soluble factor extracted from carrier-primed suppressor T cells (TsF) in the suppressive interaction was studied. Keyhole limpet hemocyanin (KLH)-specific TsF directly acts on KLH-primed, I-J positive, nylon-wool-adherent T cells that have an acceptor site for TsF. The nylon-wool-adherent T cells, after accepting TsF in the presence of specific antigen, generate new suppressor T cells acting as an actual effector cell type. Antigen-specificity and syngeneity at I-J between TsF and acceptor T cells are both required for the induction of new suppressor T cells. Newly induced suppressor T cells, however, suppress both syngeneic and allogeneic responses in an antigen-nonspecific fashion.

Structure of an antigen-specific suppressor factor produced by a hybrid T-cell line.

THE preceding report1 described the production by the hybrid T-cell line A1 of a suppressor factor with specificity for sheep red blood cells (SRBC). The factor, which binds specifically to SRBC and suppresses the antibody response to this antigen in culture, does not react with anti-mouse immunoglobulin (mIg) reagents, but is removed by mouse alloantisera (anti-H–2). In these respects it resembles the antigen-specific T-cell factors which have been obtained from T cells after stimulation by antigen and which play an important part in regulation of the antibody response2,3. We report here our study of the structure of the A1 suppressor molecule by incorporation of 3H-leucine, followed by SDS–polyacrylamide-gel electrophoresis (SDS–PAGE). The molecule seems to be composed of two types of polypeptide chain in noncovalent association, a large chain of molecular weight 85,000 and a small chain of 25,000. The MW of the native molecule is about 200,000. The isolated large chain binds to SRBC. The H–2 determinants are apparently present on the small chain.

Suppressive and Enhancing T-cell Factors as I-region Gene Products: Properties and the Subregion Assignment

This paper describes two distinct antigen-specific T-cell factors having opposite functions in the regulation of antibody response, one suppressive and the other enhancing for a T-cell-dependent antibody response. Both factors are found to have I-region gene expressions. Available evidence indicates that they are coded for by genes present in distinct I subregions, and that the targets for these factors are probably T cells also carrying I-region determinants. Incorporating the new target cell type in the network of immunocompetent cell interactions, possible mechanisms of T-cell-mediated regulation of antibody response will be discussed.

Antigen-Specific T-cell factors in the immune response to poly(Tyr,Glu)-poly(Pro)-poly(Lys)

The production by T cells of an antigen-specific factor capable of replacing the T-cell function in specific antibody formation was used as a tool for studying the cellular aspects of the genetic control of immune responses. The ability of different T-cell populations to produce a cooperative signal and the ability of B-cell populations to react to this signal were studied in different mouse strains. The antigen used was the synthetic polypeptide poly(LTyr,LGlu)-poly-(LPro) —poly(lXys), (T,G)-Pro -L, the response to which was found not to beH-2-linked. It was found that the SWR strain of mice, a low responder to (T,G)-Pro -L, is not capable of producing a T-cell factor specific to this antigen, but its B cells react normally to an active factor produced in a high responder strain. In the DBA/1 strain, also a low responder to (T,G)-Pro -L, the bone marrow cells are not able to cooperate with an active T-cell factor to produce anti-(T,G)-Pro —L-specific antibodies, while their T cells do produce a (T,G)-Pro -L-specific factor. The SWR (low responder) B cells can be triggered by DBA/1 (low responder) T cells factor specific to (T,G)-Pro —L to produce an antibody response to this immunogen. These results suggest that the immune response to (T,G)-Pro -L is controlled by two genes which are expressed in different lymphocyte populations.

Antigen-specific T-cell factor in cell cooperation. Mapping within the I region of the H-2 complex and ability to cooperate across allogeneic barriers.

Further mapping of the mouse T-cell factor specific for poly(Try,Glu)-polyD-LAla--polyLys is reported. It is shown to be a product of the I-A subregion of the H-2 complex by the use of antisera either raised specifically against or made specific, by absorption, for different regions of the H-2 complex. The factor cooperates across allogeneic barriers, e.g., when factor produced by one strian is combined with bone marrow cells of other H-2 incompatible strains.

Antigen-specific T-cell factors in the genetic control of the immune response to poly(Tyr,Glu)-polyDLAla--polyLys. Evidence for T- and B-cell defects in SJL mice.

The cellular basis of the genetic control of the immune response to poly(LTyr, LGlu)-polyDLAla--polyLLys [(T,G)-A--L] in SJL (H-2s, low responder) mice has been investigated using T-cell factors. Thymocytes of SJL origin were educated to (T,G)-A--L and tested for their ability to produce an antigen-specific factor capable of cooperating in vivo with bone marrow cells of either SJL or C3H.SW (high responder) origin. SJL T cells were found to be incapable of producing such a cooperative factor, in contrast with results previously obtained with C3H/HeJ (low responders) and C3H.SW strains. Moreover, SJL bone marrow cells did not produce an antibody response to (T,G)-A--L, even when combined with factor produced by high responder (C3H.SW) mice. Thus, both T and B cells appear to be defective in the SJL strain in the response to (T,G)-A--L.