Ontogeny of mouse lymphocyte function. II. Development of the ability to produce antibody is modulated by T lymphocytes

The relative functional maturity of neonatal mouse spleen T- and B-cell populations was assessed by comparing the ability to respond to the thymic-independent antigen, DNP-Ficoll, or thymic-dependent SRBC by producing antibody in vitro. Although mouse spleen cells responded to DNP-Ficoll at an earlier age than they responded to SRBC or TNP-SRBC, the reason for the lag in the T-dependent response was confounded by the finding of high numbers of suppressor T lymphocytes in the neonatal spleen. Thus, small numbers of neonatal spleen T cells or thymocytes significantly decreased the in vitro antibody response of adult spleen cells. Although B lymphocytes appear to be functionally mature soon after birth, their acitivity may be modulated by an excess of suppressor T cells; e.g., the reconstitution of helper cell function in the neonatal spleen required anti-theta treatment before addition of adult helper cells. Suppressive activity attributable to T cells seems to play a dominant role in determining the ability of the neonatal animal to react positively or negatively to antigenic stimulation.     

Virus-replicating T cells in the immune response of mice. I. Virus plaque assay of the lymphocytes reactive to sheep erythrocytes

Virus plaque-forming cell assay with vesicular stomatitis virus (VSV), which had been originally introduced by Bloom and his colleagues as a tool for the enumeration of activated lymphocytes, was first applied to the immune response of mice to a widely used antigen, i.e. sheep red blood cells (SRBC). When spleen cells taken from mice previously primed with SRBC were cultured in the presence of the antigen, lymphocytes capable of replicating VSV (antigen-induced virus plaque-forming cells, Ag-V-PFC) were generated in the culture. They seemed to appear as early as 1 day of culture, and the peak was attained by the 2nd day. Most of Ag-V-PFC belonged to T-cell population, since 80-90% of Ag-V-PFC was killed by the treatment of cultured cells with anti-thymocyte serum plus complement. In vitro generation of Ag-V-PFC seemed to be highly cross-reactive (about 40%) with a related antigen (horse red blood cells). Ag-V-PFC detected in the present experiment may not represent helper T cells, effector T cells, or their precursors because of the following: (a) The generation of Ag-V-PFC was completely suppressed by the addition of anti-SRBC mouse serum in the culture, though the helper activity was apparently augmented by the same treatment. (b) Development of Ag-V-PFC was almost completely suppressed by the pretreatment of mice with cyclophosphamide 2 days before immunization, by which delayed-type hypersensitivity (DTH) was markedly augmented. (c) After the immunization of mice, Ag-V-PFC began to develop just when the level of DTH declined, at which point helper activity of the spleen cells also diminished. A possible role of Ag-V-PFC in the immune response was discussed.     

Antigen-binding T cells as helper cells. Separation of helper cells by immune rosette formation

The spleen T cells from mice immunized 6 days earlier with either chicken gamma globulin (CGG) or with donkey erythrocytes (DRC) were rosetted with CGG-coated sheep erythrocytes or with DRC. The immune rosettes (RFC) (antigen-binding cells) were separated from the bulk of nonrosette-forming cells (non-RFC) by 1-g velocity sedimentation and the RFC and non-RFC tested for helper activity in cooperative antihapten responses in vitro. RFC or non-RFC were mixed with normal or hapten-primed spleen cells, challenged with the appropriate hapten-carrier conjugate and cultured for 4 days in Marbrook tissue cultures. The helping activity was quantitated from the numbers of antihapten antibody-producing cells generated per culture. The results show that specific helper cell activity could be selectively recovered in the immune rosette-forming cell population whereas the non-RFC population was depleted of help. These findings indicate that the helper T cells express specific antigen binding receptors.     

REGULATION OF THE IMMUNE RESPONSE : II. QUALITATIVE AND QUANTITATIVE DIFFERENCES BETWEEN THYMUS- AND BONE MARROW-DERIVED LYMPHOCYTES IN THE RECOGNITION OF ANTIGEN

The specificity of antigen recognition by thymus-derived helper cells (T cells) and antibody was examined in mice, heterologous erythrocyte antigens from sheep (SRBC), goat (GRBC), burro (BRBC), chicken (CRBC), and toad (TRBC) being used. Antibody specificity was tested by a number of functional assays: hemagglutination, hemolysis, and immune suppression. The specificity of T cells was determined by titrating their ability to help the in vitro antitrinitrophenol (TNP) responses of mouse spleen cultures immunized with the hapten coupled to the various test erythrocytes as carrier. Anti-SRBC antibody cross-reacted with GRBC, but not with BRBC, CRBC, or TRBC. In contrast, SRBC-primed helper T cells cross-reacted with both GRBC and BRBC, but not with CRBC or TRBC, indicating a difference in the specificity of antigen recognition between the cellular and the humoral immune responses.     

REGULATION OF CELLULAR ANTIBODY SYNTHESIS : CELLULAR 7S PRODUCTION AND LONGEVITY OF 7S ANTIGEN-SENSITIVE CELLS IN THE ABSENCE OF ANTIBODY FEEDBACK

Transfer of spleen cells from mice immunized against sheep red blood cells (SRBC) into irradiated (600 R) nonimmune, syngeneic mice in the presence of antigen resulted in excessive cellular 7S production 7 days later. The number of 7S plaque-forming cells usually exceeded 10⁶ per spleen and the mean proportion varied between 1 and 70%. In occasional animals all spleen cells were producing antibodies to SRBC. Serum antibody synthesis was also excessively increased, the titers in agglutination after 2-ME treatment and in hemolysis varying between 2¹⁵ and 2²⁵. The generation time of the 7S PFC was found to be 9.6 hr in the secondary hosts. It seemed possible that the excessive production of 7S PFC and antibodies in the irradiated nonimmune recipients was caused by the absence of feedback inhibition of the immune response by antibody, a mechanism which would normally function to restrict antibody synthesis. This conclusion was strengthened by the demonstration that transfer of antigen-stimulated immune cells into actively or passively immunized irradiated recipients resulted in a marked suppression of cellular 7S synthesis. Serial transfers of antigen-stimulated immune cell populations in irradiated hosts resulted in an equally high number of 7S PFC during the first four transfer generations. However, after the fifth to seventh transfer generation the number of 7S PFC rapidly declined and disappeared within one to three passages. Serum antibodies and 7S PFC declined in parallel during the last transfer generations. Further passages of antigen-stimulated spleen cells lacking 7S PFC did not lead to reappearance of PFC. Thus, antigen-sensitive cells have a limited lifespan and/or multiplication capacity. From the hypothesis that the 7S PFC developed by division from antigen-sensitive precursors it was calculated that 38–40 divisions occurred, Thus, one antigen-sensitive precursor has the potential to give rise to 10¹² 7S PFC.     

Cytotoxic immune cells with specificity for defined soluble antigens. IV. Antibody as mediator of specific cytotoxicity

Spleen cells from mice immunized against ovalbumin (OA) or dinitrophenylated mouse serum albumin (DM) were found to be specifically cytotoxic in vitro towards target cells (chicken red blood cells) coated with these antigens. Inhibition of specific cytotoxicity was observed when free soluble antigen was added to the incubation mixtures. DM-immune cell cytotoxicity could be specifically and completely inhibited by DNP-lysine and was thus shown to be hapten specific. Complete and specific inhibition was also observed for OA-immune cell cytotoxicity using OA as inhibitor, but compared with the inhibition curves obtained with DNP-lysine, the OA cytotoxicity inhibition curves were shifted by a factor of about one hundred towards lower molar inhibitor concentrations. Very similar results were observed when the serum antibodies of DM- and OA-immune animals were analyzed by passive hemagglutination inhibition. With increasing time after immunization, both cytotoxicity inhibition curves and agglutination inhibition curves, shifted to lower antigen or hapten concentrations. Specific cytotoxicity against antigen-coated target cells was induced in nonimmune spleen cells (a) by serum from immune animals, and (b) by supernatants from in vitro immune cell cultures. In both instances, the factor which induced antigen-specific cytotoxic activity could be absorbed on anti-mouse Ig columns, thus demonstrating its immunoglobulin nature. The ability of target cell bound antibodies to induce cytotoxicity in nonimmune spleen cells was restricted to the 7S antibody class.     

Separation of helper T cells from suppressor T cells expressing different Ly components. II. Activation by antigen: after immunization, antigen-specific suppressor and helper activities are mediated by distinct T-cell subclasses.

Cells of the Lyl subclass generate helper activity in both primary and secondary responses to sheep erythrocytes (SRBC). In contrast, after priming with SRBC, cells of the Ly-2+ subclasses, in particular Ly23 cells, have suppressive activity. The degree of Ly23-mediated suppression is directly proportional to the amount of antigen (SRBC) used for priming. Suppression by Ly23 cells is specific, because Ly23 cells from SRBC-primed animals do not suppress the response to horse erythrocytes, and vice versa. Thus, both cytotoxic and specific suppressor functions are mediated by T cells of a subclass, provisionally designated TCS, which can be distinguished from helper T cells (TH), by their Ly phenotypes. It remains to be determined whether killing and suppression are functionally interrelated properties of a single Ly23 subclass, or whether the Ly23 population comprises two subclasses whose surface phenotypes are not yet distinguishable by immunogenetic criteria.     

Rosette-forming ability of thymus-derived lymphocytes in cell-mediated immunity. I. Delayed hypersensitivity and in vitro cytotoxicity

Effector cells in delayed hypersensitivity and in vitro cytotoxicity were studied in lymph node cells from animals immunized with sheep erythrocytes (SRBC) in complete Freund's adjuvant. Delayed hypersensitivity response (DHR) was assayed by the increase in foot pad swelling after the intrafoot pad injection of immune cells plus antigen. Cell-mediated cytotoxicity against SRBC was assayed by a microcytotoxicity test with sheep fibroblasts as target cells. Effector cells were antigen specific, sensitive to anti-theta serum plus complement (C), and insensitive to anti-Ig serum plus C. A nonrosette- forming (non-RFC) small lymphocyte effector T cell and a rosette- forming medium lymphocyte effector T cell were isolated by velocity sedimentation. The small lymphocyte non-RFC required a longer time than the medium lymphocyte RFC effector cell to produce maximum activity. Buoyant density failed to distinguish medium lymphocyte effector cells in DHR and in vitro cytotoxicity.     

Emergence of insulin receptors upon alloimmune T cells in the rat.

Insulin, as well as other ligands which increase intracellular guanosine 3',5'-cyclic monophosphate (cGMP), augments thymic-derived (T)- lymphocyte effector activity as revealed by alloimmune lymphocyte-mediated cytotoxicity. The observation that insulin binds only to monocytes among circulating nonimmune human mononuclear cells fosterd reexamination of the mechanism by which insulin augments T-lymphocyte function. This report concerns a test of the hypothesis that the T cell is directly affected by insulin and that an insulin receptor emerges upon T lymphocytes consequent to immune activation. Spleens were removed from rats skin grafted across a major histocompatibility barrier. Lymphocytes were harvested from Ficoll-Hypaque density gradients and subsequently enriched for T cells by passage over one or two nylon wool columns. This population was composed of more than 98% T cells as assessed by surface marker techniques (Ig staining, erythrocyte antibody, and erythrocyte antibody complement rosetting, anti-T staining). There was no loss of augmentation of lymphocyte-mediated cytotoxicity induced by insulin, carbamycholine, and 8-bromo-cGMP in the purified cells when compared to unfractionated cells 7 days after transplantation. 125I-insulin bound saturably to the allostimulated T-enriched lymphocytes with maximum binding at 12.8 +/- 0.2 pg and a dissociation constant at equilibrium of 1.3 nM. In contrast, insulin receptors were not present on nonimmune T-enriched cells or on T cells from animals that received syngeneic grafts. The affinity of the lymphocyte insulin receptor was similar to that of more conventional insulin-sensitive tissues e.g., liver, adipocyte. After 89% of T cells from spleens on day 7 were lysed with anti-thy 1.1 antibody and complement, the ability to measure specific insulin binding was lost. These data confirm a physiologic role for insulin in T-lymphocyte effector function and describe the emergence of insulin receptors concomitant with cell sensitivity to ligand. Such receptors may play a role in hormonal modulation of the immune response.     

SURFACE MARKERS ON HUMAN T AND B LYMPHOCYTES : I. A LARGE POPULATION OF LYMPHOCYTES FORMING NONIMMUNEROSETTES WITH SHEEP RED BLOOD CELLS

By using the two criteria (a) high density of immunoglobulin determinants on the cell surface and (b) presence of receptors for C'3 on the cell surface for defining bone marrow-derived lymphocytes, it is indirectly shown that all or at least a major population of human thymus-derived lymphocytes under certain conditions will form nonimmune rosettes with sheep red blood cells (SRBC). Almost all thymocytes tested from two different donors formed rosettes. The SRBC rosettes are not formed by virtue of immunoglobulin receptors and form only around living cells. Positive bivalent ions are required for rosette formation since EDTA will block rosette formation. Sodium iodoacetate will also block rosette formation demonstrating the dependence on an intact glycolytic pathway. Rosette formation is temperature dependent and will not appear at 37°C. Trypsin treatment of lymphocytes will abolish their SRBC-binding ability which cannot be restored by treating them with fresh donor serum or fetal calf serum, but which will reappear after culturing the lymphocytes. It is suggested that these rosettes are formed by a rapidly released or metabolized receptor substance on the living cell surface which behaves as a trypsin-sensitive structure produced by the cells themselves.     

Heterogeneity of antibody-forming cells. An electron microscopic analysis

Cell suspensions from draining lymph nodes of immune and nonimmune rats were reacted in vitro with ¹²⁵I-labeled antigens. In light microscopic radioautographs of smears, 17% of the immunized cells were tagged by specific antigen; 2.0% of control cells were positive. In electron microscopic radioautographs, 90% of the labeled elements from immune donors were lymphocytes, blast and plasma cells; 10% were monocytes-macrophages or other elements, including naked nuclei. 15% of the labeled cells from control materials were lymphocytes and plasma cells, while 85% were monocytes-macrophages and naked nuclei. Within cell suspensions derived from immunized animals there were almost twice as many lymphocytes marked by isotope as plasma cells, and the lymphocytes ranged in morphology from mature monoribosomal elements to immature polyribosomal cells. Antibody-forming cells fixed labeled antigen at their surfaces. The monocyte-macrophage class was distinguished by a high mean grain count and by distribution of grains within cytoplasmic vacuoles and lysosomes.     

The role of H-2-linked genes in helper T cell function. VII. Expression of I region and immune response genes by B cells in bystander help assays

The mode of action by bystander helper T cells was investigated by priming (responder X nonresponder) (B6A)F1 T cells with poly-L-(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys [(TG)-A--L] and titrating the ability of these cells to stimulate an anti-sheep red blood cell (SRBC) response of parental B cells and macrophages in the presence of (TG)-A--L. Under limiting T cell conditions, and in the presence of (TG)-A--L, (TG)-A--L-responsive T cells were able to drive anti-SRBC responses of high-responder C57BL/10.SgSn (B10) B cells and macrophages (M0), but not of low-responder (B10.A) B cells and M0. Surprisingly, the (TG)-A--L-driven anti-SRBC response of B10.A B cells was not restored by addition of high-responder acessory cells, in the form of (B6A)F1 peritoneal or irradiated T cell-depleted spleen cells, or in the form of B10 nonirradiated T cell-depleted spleen cells. These results suggested that (TG)-A--L-specific Ir genes expressed by B cells controlled the ability of these cells to be induced to respond to SRBC by (TG)-A--L-responding T cells, implying that direct contact between the SRBC-binding B cell precursor and the (TG)-A--L-responsive helper T cells was required. Analogous results were obtained for keyhold limpet hemocyanin (KLH)-driven bystander help using KLH-primed F1 T cells restricted to interact with cells on only one of the parental haplotypes by maturing them in parental bone marrow chimeras. It was hypothesized that bystander help was mediated by nonspecific uptake of antigen [(TG)-A--L or KLH] by SRBC-specific b cells and subsequent display of the antigen on the B cell surface in association with Ir of I-region gene products, in a fashion similar to the M0, where it was then recognized by helper T cells. Such an explanation was supported by the observation that high concentrations of antigen were required to elicit bystander help. This hypothesis raises the possibility of B cell processing of antigen bound to its immunoglobulin receptor and subsequent presentation of antigen to helper T cells.     

Immunoregulatory functions for murine intraepithelial lymphocytes: gamma/delta T cell receptor-positive (TCR+) T cells abrogate oral tolerance, while alpha/beta TCR+ T cells provide B cell help.

Past work has shown that a subset of effector T cells with unique characteristics could abrogate hapten- or antigen-induced tolerance, and the reconstitution of this immune response has been termed contrasuppression. We have studied contrasuppression in a model of oral tolerance (OT) in which adoptively transferred antigen-specific T contrasuppressor (Tcs) cells reverse OT and result in antibody responses to the eliciting antigen. In the present study, we show that murine intraepithelial lymphocytes (IELs) from mice orally immunized with sheep red blood cells (SRBC) contain T cells that exhibit Tcs cell activity. This effect was mediated by CD3+ gamma/delta T cell receptor-positive (TCR+), but not alpha/beta TCR+ T cells, and gamma/delta TCR+ Tcs cells were associated with both the CD4-,CD8+ and CD4-,CD8- (double-negative) IEL fractions. The CD4-,CD8+ gamma/delta TCR+ IELs were further separated into Vicia villosa-adherent and -nonadherent fractions. Adoptive transfer of V. villosa-adherent gamma/delta TCR+ T cells to mice with OT to SRBC resulted in splenic IgA, IgM, and IgG subclass anti-SRBC responses, while V. villosa-nonadherent gamma/delta TCR+ T cells were without activity. The gamma/delta TCR+ IELs did not support in vitro antibody responses in B cell cultures, while alpha/beta TCR+ IELs were effective T helper cells. Further, cytokine production by the gamma/delta TCR+ IELs was examined, and the gamma/delta TCR+ V. villosa-adherent fraction, which possessed contrasuppressor function, contained low levels of IL-5 mRNA and small numbers of IL-5-producing cells when compared with alpha/beta TCR+ IELs and V. villosa-nonadherent gamma/delta TCR+ IELs. Our results now show that mouse IELs contain two distinct types of T cells that function in the immune response, e.g., alpha/beta TCR+ T cells that produce IL-5 and function as helper cells, and gamma/delta TCR+ T cells that restore antibody responses in mice that had been orally tolerized with antigen.     

THYMUS-MARROW IMMUNOCOMPETENCE : V. HYDROCORTISONE-RESISTANT CELLS AND PROCESSES IN THE HEMOLYTIC ANTIBODY RESPONSE OF MICE

Corticosteroids suppress the humoral antibody response of mice to sheep erythrocytes. This response depends on interactions between thymus-derived helper cells and bone marrow-derived antibody-forming cell precursors (AFC precursors). Previous experiments had shown that spleen cells (a mixture of thymus-derived and marrow-derived cells) were sensitive to corticosteroids while AFC precursors in the bone marrow were resistant. The present experiments showed that the thymus of a mouse given 2.5 mg of hydrocortisone acetate, although containing only about 5% of the number of cells of a normal thymus, was as effective as a normal thymus in cooperating with bone marrow when transferred to irradiated syngeneic mice and stimulated with SRBC. The proliferative response of thymus helper cells to SRBC was also resistant to hydrocortisone. In this context, the majority of thymic cells are in the cortex, are rapidly dividing, are sensitive to corticosteroids and are not iminunocompetent. A small number of thymic cells, probably located in the medulla, are resistant to corticosteroids, but are immunocompetent since they can serve as helper cells. The hydrocortisone-sensitive phase of the splenic response to SRBC was found to be the bone marrow-derived AFC precursor since spleens from hydrocortisone-treated donors had immunocompetence restored by normal bone marrow but not by normal thymus cells.     

Functional analysis of two human T-cell subpopulations: help and suppression of B-cell responses by T cells bearing receptors for IgM or IgG

Subpopulations of thymus-derived T lymphocytes bearing receptors for either IgM or IgG molecules were isolated from human peripheral blood. Those with receptors for IgM (T.M) provided help in a cell dose-dependent fashion for the pokeweed mitogen-induced differentiation of B lymphocytes in vitro, whereas cells with receptors for IgG (T.G) did not. T.G cells, on the hand, efficiently suppressed the differentiation and proliferation of B cells in the pokeweed system in the presence of helper T.M cells. This suppressive activity of T.G cells required prior interaction of the T.G cells with immune complexes. The helper activity of T.M cells was relatively radioresistant while the suppressor activity of T.G cells was radiosensitive. The results indicate that helper and suppressor functions of human T lymphocytes in this model system are mediated by different subpopulations of T cells which can be distinguished by their ability to bind IgM or IgG immune complexes, respectively.     

The postnatal development of the white pulp in the rat spleen and the onset of immunocompetence against a thymus-independent and a thymus-dependent antigen.

The spleen of the rat contains 5 well delineated compartments: a central and peripheral part of the periarteriolar lymphatic sheath (PALS), the marginal zone and the follicle with centre and corona. A study was made on the development of these compartments in correlation with the onset of immunocompetence of the spleen. The spleens of animals in the age range from 1-40 days were studied with the light and electron microscopes. Immunocompetence was assessed by measuring serum antibody levels 5 days after intravenous antigen administration. Comparable doses of paratyphoid vaccin (PTV) and sheep red blood cells (SRBC) were used: PTV as a thymus-independent, SRBC as a thymus-dependent antigen. At the first day of life few lymphocytes are present around small arterioles. The marginal zone is first present at 9 days of age. At 14 days of age the typical thymus-dependent area develops. Interdigitating cells, which seem to develop from monocytes, and lymphocytes are present in close apposition. Primary follicles were first seen on 20 days of age, follicle centres at 25 or 30 days of age. The appearance of "typical" dendritic cells coincided with the appearance of follicle centres. The first titre against PTV was seen after antigen administration at 9 days after birth, against SRBC after injection in 14 days old animals. As PTV is a thymus-independent antigen it needs only B-cells for a primary IgM response. The appearance of functional B-cells in sufficient numbers to give a measurable response therefore coincides with the appearance of the marginal zone. Although T-cells are present from birth, the response against SRBC is delayed until the thymus-dependent area has developed. Thus, in the first 2 weeks of life, T-cells are either immature, or they are not able to react, because their microenvironment is not yet adequate.     

Priming of T helper cells by antigen-activated B cells. B cell-primed Lyt-1+ helper cells are restricted to cooperate with B cells expressing the IgvH phenotype of the priming B cells

Activated B cells isolated shortly after primary immunization of BALB/c donor mice with sheep erythrocytes (SRBC), were transferred to normal syngeneic recipients or to low-dose cyclophosphamide-pretreated syngeneic recipients. In pretreated recipients, the transfer of activated B cells, but not of T cells or macrophages, resulted in an augmented production of indirect plaque-forming cells in the primary immune response to SRBC but not to horse erythrocytes. It was shown in double-transfer experiments that T helper cells (Lyt-1+) had been stimulated by the transfer of antigen-activated B cells. Criss-cross double-transfer experiments using the mouse strains CB20 and BAB14 (congenic to BALB/c at the loci coding for the immunoglobulin heavy chain) indicate that those T helper cells are primed after recognition of B cell products that are encoded for by genes linked to the loci coding for the variable region of the immunoglobulin heavy chain (IgVH). The thus-primed Ig-dependent T helper cells (THIg) are adaptively restricted to cooperate with B cells that display IgVH- linked gene products similar to those that originally stimulated the THIg. These findings suggest that in the course of an immune response to T cell-dependent antigens, help for the production of specific IgG can be provided by THIg that have been primed and/or clonally expanded after recognition of IgVH-linked gene products by (e.g., complementary) T cell receptors.     

Thymus-derived rosettes are not "helper" cells

Rosettes against SRBC were made from normal spleen cells. Although T rosettes tend to dissociate, they could be stabilized with 0.05% sodium azide. A clear separation of nonrosettes, T rosettes, and B rosettes was obtained by subjecting the suspension of splenic rosettes to velocity sedimentation at unit gravity. Each fraction was injected with either normal bone marrow cells or normal thymus cells with antigen into 650-R-irradiated hosts. Direct plaque-forming cells (PFC) were assayed in the spleens 7 days later. Synergism with thymus cells occurred only in the B-rosette fraction; PFC precursors therefore sedimented as B rosettes. Synergism with bone marrow cells occurred only in the nonrosette small lymphocyte fraction; helper cells therefore did not bind detectable numbers of sheep red blood cells (SRBC). Thus T rosettes are not helper cells in the direct PFC response of bone marrow B cells to SRBC.     

Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. II. Selective inactivation of hapten-reactive suppressor T cells by hapten-nonimmunogenic copolymers of D-amino acids, and its application to the study of suppressor T-cell effect on helper T-cell development

An experimental condition was established in vivo for selectively eliminating hapten-reactive suppressor T-cell activity generated in mice primed with a para-azobenzoate (PAB)-mouse gamma globulin (MGG)-conjugate and treated with PAB-nonimmunogenic copolymer of D-amino acids (D- glutamic acid and D-lysine; D-GL). The elimination of suppressor T-cell activity with PAB-D-GL treatment from the mixed populations of hapten- reactive suppressor and helper T cells substantially increased apparent helper T-cell activity. Moreover, the inhibition of PAB-reactive suppressor T-cell generation by the pretreatment with PAB-D-GL before the PAB-MGG-priming increased the development of PAB-reactive helper T-cell activity. The analysis of hapten-specificity of helper T cells revealed that the reactivity of helper cells developed in the absence of suppressor T cells was more specific for primed PAB-determinants and their cross-reactivities to structurally related determinants such as meta-azobenzoate (MAB) significantly decreased, as compared with the helper T-cell population developed in the presence of suppressor T lymphocytes. In addition, those helper T cells generated in the absence of suppressor T cells were highly susceptible to tolerogenesis by PAB-D- GL. Similarly, the elimination of suppressor T lymphocytes also enhanced helper T-cell activity in a polyclonal fashion in the T-T cell interactions between benzylpenicilloyl (BPO)-reactive T cells and PAB- reactive T cells after immunization of mice with BPO-MGG-PAB. Thus inhibition of BPO-reactive suppressor T-cell development by the BPO-v-GL- pretreatment resulted in augmented generation of PAB-reactive helper T cells with higher susceptibility of tolerogenesis to PAB-D-GL. Thus, these results support the notion that suppressor T cells eventually suppress helper T-cell activity and indicate that the function of suppressor T cells related to helper T-cell development is to inhibit the increase in the specificity and apparent affinity of helper T cells in the primary immune response. The hapten-reactive suppressor and helper T lymphocytes are considered as a model system of T cells that regulate the immune response, and the potential applicability of this system to manipulating various T cell-mediated immune responses is discussed in this context.     

Production of human heterophile antibodies. III. In vitro specific stimulation and immortalization of human lymphocytes

The production of primary human specific antibody against an exogenous antigen by in vitro system was achieved in this study. Human lymphocytes were prepared from the lymphocytes of human spleen (SPL), tonsil (TL), and peripheral blood (PBL). These lymphocytes were stimulated by sheep red blood cells (SRBC) co-cultured with the appropriate number of allogeneic lymphocytes or fractionated T cells. Significant numbers of plaque forming cells (PFC) against SRBC were obtained. A major population of PFC responded to the stimulator RBC and a minor population of PFC responded to both SRBC and bovine red blood cells (BRBC). The culture of allogeneic combination of whole SPL or TL stimulated with SRBC produced PFC, but not that of whole PBL. Reconstitution of equal numbers of allogeneic separated B cells and T cells from PBL was required for a significant response. These specific antibody forming cells (AFC) were immortalized by Epstein Barr virus (EBV) infection and culture supernatant containing antigen-specific antibodies (anti-SRBC: Forssman antibody) were obtained after repeated cloning.