Antigen-specific Factor Research Articles

Role of T-lymphocytes in production of a cancer-associated protein factor in serum from lung cancer patients

Oligoclonal T-cells have been generated by sensitization of peripheral blood mononuclear cells from lung cancer patients to a lung cancer tumor-associated antigen (TAA). A factor similar to the antigen-specific glycoprotein factor in the serum of these cancer patients was found in the supernatant of the oligoclonal T-cells. The factor from the T-cell supernatant had specificity for lung cancer TAA and induced stimulation of normal lymphocytes of the CD8 phenotype when mixed with lung cancer TAA. Furthermore, the factor blocked the ability of lymphocytes from lung cancer patients to recognize lung cancer TAA. Both the factor from lung cancer serum and from the oligoclonal T-cells were absorbed on a lung cancer-associated antigen-coupled immunosorbent column. On FPLC-gel filtration the desorbed fractions from the immunosorbent column from both sources showed activity in the same molecular weight range, 70–90 kD. Heteroantisera raised against the factor from serum and against the factor from the oligoclonal T-cell supernatant bound about the same portion of lymphocytes from lung cancer patients as measured by immunofluorescence, while only a minor fraction of cells from patients with unrelated cancers and from healthy persons were labelled on incubation with the antisera. These results support the hypothesis that an antigen-specific factor found in serum of cancer patients is produced by antigen-stimulated T-cells, possibly of the CD8 phenotype. This putative antigen-specific suppressor factor and the tumor antigen-reactive lymphocytes of the patient seem to share similar idiotopes.

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction

We previously found an antigen-specific factor capable of augmenting delayed-type hypersensitivity (DTH) in the culture supernatant of the mixture of immune spleen cells and erythrocyte antigen, or in the serum of mice immunized with heterologous erythrocytes and exhibiting delayed-type footpad reaction. To elucidate whether this kind of factor (DTH-augmentation factor; DAF) participates in the establishment of DTH to various kinds of antigen besides erythrocyte antigen, we chose a bacterial antigen, Listeria monocytogenes, which is a facultative intracellular bacterium. In the present study, we demonstrated that the immune serum from mice immunized with viable Listeria augmented the delayed-type footpad reaction to Listeria. Furthermore, acquired resistance against Listeria was also augmented by the transfer of such immune serum. Such augmentation of acquired resistance was observed in sites infected locally and in the spleen of mice infected systemically. This effect was also seen in sera from mice immunized with heat-killed Listeria emulsified with complete Freund's adjuvant.

Production of tumor-specific suppressor T cell hybridomas

Spleen cells from late tumor-bearing host mice (TBH) with progressive P815 tumors have been shown to suppress the generation of cytotoxic T lymphocytes (CTL) from spleens of syngeneic immune or early TBH mice in vitro. This suppression is antigen-specific and is mediated by T lymphocytes. By positive selection, a highly enriched population of T cells was obtained from late TBH spleens and fused with the BW5147 HAT-sensitive thymoma. Cloned hybrid cell lines were obtained which released soluble antigen-specific factors capable of suppressing anti-P815 CTL generation. Similar T cell hybrids with suppressive activity were also derived from the fusion of spleen cells of TBHs bearing a HAT-sensitive subline of the P815 tumor.

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction: IV. Effect of delayed-type hypersensitivity augmentation factor on in vitro induction of DTH

We found an antigen-specific factor capable of augmenting delayed-type hypersensitivity (DTH) in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR), or in the culture supernatant of the mixture of sensitized T cells and specific antigens. This factor (DTH augmentation factor; DAF) was confirmed to augment DTH in transferred recipients. In this paper, such an activity of DAF was further investigated using the system with in vitro induction and local transfer of DTH. DAF also augmented the primary in vitro induction of DTH, when spleen cells from mice transferred with the DAF-containing serum 12 hr previously or spleen cells incubated with the DAF-containing serum on ice for 2 hr were cultured with heterologous erythrocytes. DAF acted on the induction phase of DTH and augmented a typical DTH which was dependent on Thy-1-positive T cells. DAF showed antigen specificity, but was not assigned to conventional immunoglobulin. The activity of DAF was detected when nylon-wool nonadherent cells were incubated with DAF prior to the culture of those cells and antigens, but not detected when only nylon-wool adherent cells were incubated with DAF. Thus, DAF exerted its effect through binding to acceptor cells which were included in nylon-wool nonadherent spleen cells from normal mice.

FUNCTIONAL ACTIVITY OF SOLUBLE ANTIGEN-SPECIFIC HELPER T CELL MOLECULES Requirement For Seprable entities for The Induction of cytotoxic T Cell and B Cell Responses

These studies constitute the first report of a comparison of the ability of a population of antigen-specific helper factors (ASHF) to induce cell-mediated and humoral immune responses. Supernatants of helper T lymphocyte (TH) cell lines, were purified by antigen-affinity chromatography and tested for their ability to induce cytotoxic T lymphocyte (CTL) responses and IgM responses in vitro. The supernatant of a noncloned TH line was found to contain two functionally distinct types of ASHF that are separable by anion-exchange chromatography: one factor triggers CTL responses (ASHFCTL), the other triggers IgM B cell responses (ASHFB). Clone 4C6, derived from the parent line CHI, produces ASHFCTL but not ASHFB. These observations could result from two entirely distinct ASHF or from the association of common antigen-specific subunits with different antigen-nonspecific subunits to make ASHFCTL or ASHFB.

Isotype-like suppression of T cell-mediated immunity in vivo. II. Suppression of the early component of contact sensitivity by a Ly-2+ T cell-derived suppressor factor that binds to contact sensitivity-initiating, antigen-specific, Ly-1+ T cell-derived factors that are of different antigen specificities.

Recognition that delayed-type hypersensitivity (DTH) reactions, such as contact sensitivity (CS) in mice, are initiated by Ly-1+ T cell-derived, antigen-specific factors has led to identification of a new kind of suppressor T cell that regulates this initiation phase of CS. Regulation by these suppressor T cells is T cell isotype-like in that initiation of DTH of various antigenic specificities is suppressed, whereas, Ly-1+ T cells mediating the antigen/major histocompatibility complex-restricted, classic delayed phase of CS responses are not affected, nor are other T cell activities. This study shows that these isotype-specific suppressor T cells probably act by release of soluble, isotype-specific, suppressor factors. These isotype-specific T cell factors bind to and can be eluted from columns linked with antigen-specific Ly-1+ T cell factors that initiate CS, and are of different antigen specificities. These T cell regulating, anti-isotypic suppressor factors are derived from Lyt-2+ I-J- T cells and suppress CS-initiating T cells, but do not affect the delayed-acting T cells of CS. This is in contrast with antigen-specific T cell suppressor factors that affect the late-acting and not the early-acting T cells of CS. It is suggested that the antigen-binding, CS-initiating, T cell factors, and their regulatory, anti-isotypic T cell factors are, respectively, T cell analogues of immunoglobulin(Ig)E antibody, and IgE-binding factors, that regulate IgE antibody production by IgE+ B cells.

Isotype-like suppression of T cell-mediated immunity in vivo. I. Delayed-type hypersensitivity specificity of T cell suppression induced by antigen-binding T cell factors that initiate contact sensitivity.

A new form of immunoregulation is described that is based on the recent suggestion that the effector phase of delayed-type hypersensitivity (DTH) responses consists of a cascade of steps that are dependent on the sequential action of two types of antigen-specific Ly-1+ effector cells. According to this formulation, which is based on analysis of contact sensitivity (CS) in mice, DTH consists of at least two T cell-dependent steps that must occur in sequence. The first of these steps occurs within 2 hr of challenge and depends on DTH-initiating, antigen-binding, antigen-specific T cell factors that sensitize the tissues for an obligatory initial vasoactive step, which allows the antigen/major histocompatibility complex (MHC)-restricted, Ly-1+ effector T cells of classic 24 to 48 hr DTH responses to enter the tissues and produce chemoattractant lymphokines. We have now found that nonspecific suppression of CS responses can be induced by i.v. injection of these antigen-binding, CS-initiating T cell factors. Injection of the antigen-binding T cell factor induces Ly-2+, I-J-, cyclophosphamide sensitive, seemingly nonspecific suppressor T cells to inhibit initiation of CS responses. These suppressor cells do not affect the late-acting lymphokine-producing T cells, but probably act by preventing production of antigen-specific factors of the type that are required to initiate DTH responses. Furthermore, injection of CS-initiating antigen-binding T cell factors also induces suppression of sheep red blood cell (SRBC)-specific DTH, but does not affect classic anti-SRBC B cell responses, which are dependent on antigen/MHC-restricted Ly-1+ helper T cells; skin allograft rejection responses are also not affected. Thus, the suppression is DTH-specific. In addition, suppression induced by antigen-binding T cell factors is Igh and not MHC/H-2 restricted. These findings and data in the companion manuscript showing that these suppressor T cells act by production of soluble suppressor factors that bind to antigen-specific T cell factors of different antigenic specificities, cause us to suggest that the antigen-binding T cell factors are T cell isotype-like. Therefore, an isotype-like suppression is induced by these factors. This isotype-like suppression affects factor-producing cells of various antigenic specificities, may be mediated by T cell isotype-binding factors that are Igh restricted and block initiation of DTH responses, but does not affect conventional, antigen/MHC-restricted T cells, which may therefore have antigen receptors of a different isotype.

Differential effect of experimental diabetes on the early and late phase of contact sensitivity reaction in mice.

Contact sensitization induces two different kinds of T cells (both Ly 1) that act in sequence to produce upon challenge with antigen a classical 24-hour local skin swelling reaction. One of these cells produces an antigen-specific factor. It has been suggested that it sensitizes mast cells, similar to IgE antibody, and causes them to release vasoactive amines in the presence of antigen. This results in an early (2-hour) swelling reaction. Increased vascular permeability facilitates the entry of the second, lymphokine-producing Ly 1 cell into the site of reaction to elicit the classical 24-hour delayed-type hypersensitivity reaction. In alloxan diabetic mice, contact sensitivity reactions are reduced significantly, and our experiments show that insulin deficiency affects only the activity of the late acting, lymphokine-producing cell and leaves the factor-producing cell responsible for the early swelling reaction unaffected. Our experiments demonstrate that insulin deficiency has different effects on distinct subpopulations of T lymphocytes.

I-A region genetic restriction in the production and action of an antigen-specific T-helper factor which bears I-A region determinant(s)

Antigen-specific T-helper factor (ThF) augments the contact sensitivity reaction induced by the injection of small numbers of picrylated cells into mice. ThF was produced by injected picrylated spleen cells into the footpads and taking the 24-hr culture supernatant of the regional lymph node cells. Analysis showed that there was a genetic restriction in the induction of ThF, between these picrylated cells and the mouse making the ThF, which maps to the I-A region. This finding suggests that the receptor on the T cell which makes ThF, and by implication ThF itself, bears recognition site(s) for both antigen and I-A. ThF was then prepared from mice painted on the skin with picryl chloride, and the genetic restriction in its action was investigated. There was a requirement for genetic matching between the mouse producing the ThF and the final recipient which mapped to the I-A region. The genotype of the picrylated cell used as a source of antigen was unimportant. This I-A genetic restriction, together with the finding that ThF bears at least some I-A determinants, suggests that ThF may act by binding to the picrylated cells used as a source of antigen through its antigen-binding site and hence provide the I-A determinants needed for the recognition of antigen in the context of self-MHC. The present findings add to the list of antigen-specific factors which have a two-chain structure and show genetic restriction in their induction, action, and in the interaction between their chains which maps to the same region as the MHC-related determinant(s) which they bear.

Antigen-specific augmentation of murine immediate hypersensitivity-like footpad reaction by a T-cell factor in the culture supernatant of immune spleen cells

An antigen-specific factor capable of augmenting delayed-type hypersensitivity (DH) in the culture supernatants from immune spleen cells and erythrocyte antigen has been found. These culture supernatants also augmented an immediate hypersensitivity-like reaction which appeared in advance of the classical DH reaction. In this paper, the basic characteristics and cells producing the augmentation factor (IAF) involvd in immediate hypersensitivity-like reaction were investigated, (i) Maximum activity of IAF was detected in a supernatant from 24-hr culture of immune spleen cells and antigen, (ii) In vitro antigen stimulation was essentially required for the production or release of IAF. (iii) IAF showed antigen-specificity, (iv) IAF was produced or released by T cells. In addition to these facts, the DH-augmentation factor proved to be a T-cell product.

21] Antigen-specific factors: An overview

Publisher Summary This chapter provides an overview of antigen-specific factors, which are produced by T-lymphocytes. Antigen-specific factors may be divided into two broad categories: antigen-specific helper factors (THF), produced by T-helper (T-H) cells; and antigen-specific suppressor factors (TSF) produced by T-suppressor (T-S) cells. It has proven to be easier to obtain T lymphocyte clones or T lymphocyte hybridomas which release TSF than it has been to establish T cell clones or T cell hybridomas which make antigen-specific THF. Most of the T-H clones or T-H hybridomas produced so far have shown a requirement for specific antigen for activation but release the non-antigen-specific interleukin 2 (IL-2) (or T cell growth factor) rather than antigen-specific THF. It should also be noted that THF and TSF which display antigen specificity may be idiotype-binding rather than antigen binding in some experimental systems. Thus, it is possible to have either idiotype- or antigen specific THF or TSF acting to modify the response to a given antigen.

T-Lymphocyte regulation of humoral immunity in [formula omitted], the South African clawed toad

Helper function was the first of the regulatory activities to be investigated in the lower vertebrates (I). Initially, it was found to be carrier dependent and specific when tested in responses to haptenatedheterologous erythrocytes (2). Subsequent studies using adult (3) and early (4) thymectomy suggested T-cell involvement in vivo. Experiments in vitro strengthened the view that T-cells were responsible for amplification in Amphibia (5). The most commonly studied amphibian in this regard was Xenopus laevis, the South African clawed toad. By taking advantage of the newly developed isogeneic strains, it was possible to demonstrate that helper function in Xenopus was genetically (Major Histocompatabillty Complex~C) restricted (6). Recently, it has been shown that the cells which engage in helper function in Xenopus may reside in and derive from the cortical region of the thymus (7). Both the function of carrier priming and the presence of the thymus can be substituted for by a lectin, Concanavalin A (Con) A. Other lectins, wheatgerm agglutinin (WGA) and peanut agglutinin (PNA), which bind Xenopus T-cells were not effective in this regard (8). Thus, there are Con A sensitive T-cells in the periphery capable of affecting helper function. Both the thymic and peripheral subsets are sensitive to N-methyl-N-nitrosourea (NMU) and hydrocortisone. Thymic helper cells may be distinguished from those with suppressor inducer function resident within the medulla as they are less sensitive to irradiation than are the suppressor inducer cells (9). While it is possible to characterize helper function in this way, we know of no evidence to suggest that T-cells secrete antigen specific helper factors such as those observed in mammalian model systems(10).

Suppressor T cell circuits in contact sensitivity. II. Induction and characterization of an efferent-acting, antigen-specific, H-2-restricted, monoclonal T cell hybrid-derived suppressor factor specific for DNFB contact hypersensitivity.

This report defines a methodology for the production and characterization of an antigen-specific, monoclonal T cell hybrid-derived suppressor T cell factor (TsF) that suppresses the passive transfer of 2,4-dinitrofluorobenzene (DNFB) contact hypersensitivity. Fusion of T cells from BALB/c (H-2d) mice tolerized with syngeneic DNP-spleen cells to BW 5147 thymoma cells resulted in several hybrids that constitutively produce a soluble regulatory molecule. One of these hybrids, 26.10.2, was subsequently cloned, and its soluble factor was characterized with respect to its antigen specificity, biochemical nature, MHC restriction pattern, and identity of its target cell. 26.10.2 TsF suppresses the passive transfer of delayed-type hypersensitivity (DTH) mediated by DNP- but not trinitrochlorobenzene- or oxazalone-primed DTH T cells (TDH) after a 1 hr incubation at 37 degrees C. In contrast, 26.10.2 TsF had no suppressive effect on secondary in vitro DNP-specific T cell proliferative responses. 26.10.2 TsF therefore represents an antigen-specific factor with effector (efferent-acting) function. The monoclonal TsF was shown to consist of a two-chain, disulfide-bonded molecule, and to bear a receptor(s) specific for DNP and determinants encoded by the I region of the H-2 complex. Effector suppressive activity of 26.10.2 TsF was restricted by Class I H-2Dd determinants. One cellular target of this monoclonal factor was shown to be the DNP-specific TDH cell, because DNFB-primed lymph node cells from cyclophosphamide-pretreated donors (lacking Ts-auxiliary (Ts-aux) cells) were efficiently suppressed. The TsF appears to focus on passively bound, TDH receptor-associated, DNP-Class I determinants, as suggested by the observation that freshly prepared, but not overnight cultured, DNP-specific TDH cells were susceptible to suppression.

Delayed-type hypersensitivity is mediated by a sequence of two different T cell activities.

Classical 24- to -48 hr delayed-type hypersensitivity (DTH) skin reactions are preceded by an early skin swelling reaction that peaks 2 hr after challenge. The ability to elicit this early component of DTH is T cell dependent and is also dependent on tissue mast cells and release of serotonin, mainly from these mast cells. The current study presents pharmacologic and kinetic evidence that the late response depends on the occurrence of the early response. A variety of pharmacological agents known to deplete, prevent release of, or block the activity of serotonin, when given just before skin challenge, blocked both the early and late components of DTH, but had no effect when given (even repeatedly) after the occurrence of the early component. Thus, the serotonin-dependence of the 24-hr component of DTH represents a dependence on the early component in which serotonin release is required. A temporal dependence of the late component of DTH on the early component was also demonstrated. The early and late phases occur at different times in recipients of sensitized T cells, depending on the interval between transfer and challenge, but there is a fixed 10- to 12-hr gap. Delayed onset of the late component occurs in recipients challenged immediately after transfer and appears to be due to a delay in the onset of the early component. This delay can be abolished by adoptive cell transfer into mice that are able to elicit a normal early component because of prior transfer of T cells that are able to mediate just an early component. On the basis of these findings, we conclude that DTH consists of a cascade of events. T cells mediating the early aspect of DTH release antigen-specific factors that, upon encountering antigen activate local serotonin-containing cells, such as mast cells, to release serotonin, which opens gaps between adjacent endothelial cells. Through these interendothelial gaps a second T cell population enters the extravascular space and interacts with local antigen to induce the late response by releasing the chemoattractant lymphokines that are classically associated with DTH and that cause recruitment of bone marrow-derived circulating leukocytes to infiltrate the reaction site. The ability of the second T cell population to mediate the late component of DTH is independent of further release of serotonin.

Reconstitution of an inactive antigen-specific T cell suppressor factor by incubation of the factor with prostaglandins.

Experiments were performed to test the hypothesis that prostaglandins are crucial to the ability of an antigen-specific T cell suppressor factor to deliver a suppressive signal. In the system employed, T suppressor cells release an antigen-specific factor (TsF) that suppresses the ability of effector cells to transfer contact sensitivity (CS) skin swelling responsiveness to adoptive recipients. Culture of TsF-producing cells in the presence of indomethacin caused production of an inactive TsF that could be reconstituted by incubation of this inactive factor with low concentrations of certain prostaglandins such as PGE2 or PGE1. Subsequently, nearly all the prostaglandins were removed by dialysis, and the reconstituted TsF then acted as an antigen-specific suppressor of CS effector cells. Neither the inactive factor nor prostaglandins were suppressive alone. Furthermore, the prostaglandins are crucial to the constitution of TNBSA-F, the non-antigen-binding subunit of the TsF that probably delivers the ultimate suppressive signal. These results provide a new type of antigen-specific role for prostaglandins in immunoregulation and indicate that simple, local, hormonal molecules in physiologic concentrations can have a crucial and long-lasting role in constituting the suppressive activity of antigen-specific regulatory macromolecules released by suppressor T cells.

Receptor-specific mediation by immunoglobulin E of antigen-induced contraction of tracheal and lung parenchymal strips isolated from the guinea pig.

The guinea pig is much like humans in the cells and mediators involved in immediate hypersensitivity reactions. However, the major anaphylactic antibody in this species is IgG1, not IgE. Recently, we have been successful in producing IgE antibody in guinea pigs. The current study examined whether guinea pig IgE antibody could mediate pulmonary smooth muscle contraction. IgE antibody to picryl and oxazolone determinants was induced by immunizing Hartley strain guinea pigs pretreated with cyclophosphamide. Hyperimmune serum from these animals was passed through a heavy chain-specific anti-IgG1 affinity column. The presence of IgE anti-hapten antibody in the filtrate fraction was verified by passive cutaneous anaphylaxis (PCA) testing with a 7-d period of local passive sensitization and by heat lability (56 degrees C X 4 h) of PCA activity. This IgE-rich fraction, and purified IgG1 anti-hapten antibody were transferred to normal guinea pigs. Both fractions sensitized trachea and pulmonary parenchyma for antigen-induced smooth muscle contraction. The IgG1-mediated antigen-induced contractile response was not affected by heat (56 degrees C X 4 h) and was inhibited in a dose-dependent fashion by IgG1 blocking antibody (anti-OA). The IgE-mediated antigen-induced contractile response was significantly decreased by heat and was not affected by the anti-OA blocking antibody even at a concentration of 100 mg/kg. Thus, two antigen-specific factors in guinea pig serum can mediate antigen-induced pulmonary smooth muscle contraction: IgG1 and IgE antibodies. Our data also suggests that these antibodies mediate the contractile response through separate receptors. The finding that guinea pig IgE can mediate pulmonary smooth muscle contraction suggests this species can be a model for IgE-mediated events in the lung.

Quantitation of helper factor activity. II. Inhibition of antibody binding

A sensitive direct assay has been developed to quantitate antigen specific helper factors (ThF). The assay depends on the ability of ThF to block the binding of free antibody to immobilized antigen. It has been used to follow the course of purification of ThF produced in short-term cultures of in vitro primed helper cells and purified by affinity chromatography on antibody and antigen immunoabsorbents. The identity of the ThF was confirmed by assaying its biological activity and by its reactivity with anti-Ia antibody and with a monoclonal anti-ThF.

IgE antibody-mediated cutaneous basophil hypersensitivity reactions in guinea pigs.

Cutaneous basophil hypersensitivity (CBH) reactions are a heterogeneous group of delayed time course basophil-rich immune responses that can be mediated in the guinea pig by T cells, B cells, or IgG1 antibody. This study examined whether guinea pig IgE antibody could also mediate CBH reactions. IgE antibody to picryl or oxazolone determinants was induced by immunizing Hartley strain guinea pigs pretreated with cyclophosphamide. Hyperimmune serum from these animals was passed through a heavy chain-specific anti-IgG1 affinity column. The presence of IgE anti-hapten antibody in the filtrate fraction was verified by passive cutaneous anaphylaxis (PCA) testing with a 7-day period of local passive sensitization and by the heat lability (56 degrees C, 4 hr) of PCA activity. This IgE-rich fraction and the IgG1 fraction eluted from the column with base (0.2 M Na2CO3, pH 11.3) were transferred i.v. to separate groups of normal guinea pigs. Both fractions mediated delayed time course reactions that contained basophils. Macroscopic and microscopic reactions mediated by the IgE-rich fraction were abolished with heat (56 degrees C, 4 hr). Thus, two antigen-specific factors in guinea pig serum can mediate delayed time course basophil-containing reactions: IgG1 and IgE antibodies. IgE-mediated CBH reactions are similar to the late-phase reaction that follows IgE-dependent wheal-and-flare reactions in humans. The finding that guinea pig IgE can mediate a late reaction that contains basophils makes this a possible model for the human late-phase response, and suggests that some forms of CBH may play a role in human allergic disease.

Partial purification and characterization of an antigen-specific helper factor synthesized by a T-cell continuous line.

Antigen-specific factors produced by the T-cell growth factor-dependent T-cell continuous line E-9M(+) were partially purified. Gel analysis of the twice-affinity-purified eluate of a poly(Tyr,Glu)-poly(DLAla)--poly(LLys) [(T,G)-A--L] column revealed the existence of iodinated bands with molecular weights of 17,000 and 15,000, in addition to a diffuse band of high molecular weight. The specific helper activity of the E-9M(+) supernatants was associated with a precipitate from 65-75% ammonium sulfate. Gel electrophoresis of either the eluate of a (T,G)-A--L column or of the 65-75% salt precipitate indicated that in both preparations two fractions contained the biological activity of the factor, one of a high (less than 67,000) molecular weight and the other of a low (15,000-17,000) one. Culture supernatants of the internally [35S]methionine-labeled E-9M(+) line were subjected to a combined purification of sequential ammonium sulfate precipitations, followed by affinity chromatography. Polyacrylamide gel patterns obtained of the eluates of the different salt precipitates demonstrated that the 65-75% ammonium sulfate precipitate contained two 35S-labeled bands with apparent molecular weights in the range of 60,000 and 15,000, similar to the activity patterns obtained by the gel electrophoresis fractionation experiments. Thus, it is suggested that a fraction of low molecular weight preserves the antigen specificity and the helper activity of the factor produced by the T-cell line.

Genetically regulated human (T,G)-AL specific helper T cell replacing factors possess HLA-DR and V h determinants

Human (T,G)-AL specific T cell helper factors secreted by in vitro activated peripheral blood lymphocytes of normal donors were characterized. Factors were passed through columns of Sepharose coupled either to antibodies against human immunoglobulin or antibodies against the variable region of the heavy (V h ) and light (V l ) chains of human immunoglobulin. In addition, the same factors were applied to columns of Sepharose coupled to anti-HLA-DR antibodies or to monoclonal antibodies against human Ia or β 2-microglobulin. The activity of the antigen specific factors was removed by the anti-V h antibodies and not by anti-V l or anti-human immunoglobulin antibodies. The factors passed through Sepharose coupled to anti-DR antibodies could be removed and eluted from columns of anti-DR antibodies relevant to the donors' DR antigens. The same factors were also removed by a monoclonal antibody (anti-Ia) which recognizes a monomorphic determinant on HLA-DR, but not by monoclonal anti- β 2-microglobulin. The results suggest that the genetically regulated (T,G)-AL specific helper factors possess HLA-DR as well as V h determinants in their active moiety.