Mixed Lymphocyte Reaction Response Research Articles

Monoclonal antibody analysis of human T lymphocyte subpopulations exhibiting autologous mixed lymphocyte reaction.

In autologous mixed lymphocyte reaction (MLR), T cells proliferate in response to the stimulation by autologous non-T cells. In the present study, human T cell subpopulations, defined by murine monoclonal antibodies OKT4, OKT8, or 9.3, were examined for their capacity to proliferate in autologous and allogeneic MLR. It was observed that the treatment of responder T cells with OKT4 or 9.3 antibody (both defining helper/inducer T cells) and complement (C') ablated their proliferative response in autologous MLR and markedly reduced their allogeneic MLR proliferative response. In contrast, treatment of T cells with OKT8 antibody (defining suppressor/cytotoxic T cells) and C' had no or minimal effect on their proliferative response in autologous MLR. Furthermore, OKT4, 9.3, 9.6 (PAN), or 7.2 anti-human Ia (framework specific) but OKT8 antibody, when present during the entire culture period, in the absence of C' inhibited in a dose-dependent manner the proliferative response in both autologous and allogeneic MLR. Inhibition of proliferation was also observed when the responder T cells were pretreated with OKT4 or 9.3 antibody, washed, and then stimulated with irradiated autologous or allogeneic non-T cells. Pretreatment of T cells with OKT8 or 7.2 anti-Ia antibody in the absence of C' did not influence their proliferative response in autologous MLR. Thus, T cells containing cells with helper/inducer activity defined by OKT4 or 9.3 antibody appear to be the major responder T-cell subpopulation in autologous MLR.

Evidence that strong Mls determinants are nonpolymorphic.

Gene products of the murine Mls locus elicit high primary mixed lymphocyte reactions (MLRs). Although this locus is considered to have four alleles, only two of these alleles, i.e., Mlsa and Mlsd, give strong responses in primary MLRs. This paper presents a series of experiments to show that Mlsa and Mlsd are in fact antigenically indistinguishable. Strong Mls determinants thus appear to be nonpolymorphic.

Reconstitution of Ir genes, Ia antigens, and mixed lymphocyte reaction determinants by gene complementation.

By using clones of murine T cells reactive with alloantigens as well as soluble antigens, it has been possible to demonstrate that Ia antigens, Ir gene phenomena (defined as the ability of immune T cells to recognize antigen), and mixed lymphocyte reaction (MLR)-stimulating determinants encoded within the I-A subregion are different manifestations associated with same product. These studies utilized the I-Ab mutant mouse B6.C-H-2bm (bm12), whose defect in the normal expression of the cell surface products of the I-Ab subregion can be partially circumvented through trans-complementation by deriving hybrids between bm12 and mice expressing normal I-A subregion products. Such mice [e.g., (bm12 X B10.A)F1] express several serologically normal I-Ab products but have defects in certain I-A subregion gene products normally expressed on cells of H-2a X H-2b heterozygote mice that are used as restricting elements for antigen recognition by antigen-reactive T cell clones or recognized as alloantigens by alloreactive T cell clones. This defect in cell surface expression of certain normal I-Ab products on the mutant bm12 cells has allowed us to suggest that there exist trans-complementing products on H-2a X H-2b heterozygote mice consisting of Ab alpha Ak beta and Ak alpha Ab beta that restrict antigen recognition by cloned T cells, stimulate MLR responses by cloned T cells, and react with certain anti-Ia antisera.

Immune Profile of Infectious Bursal Disease. III. Effect of Infectious Bursal Disease Virus on the Lymphocyte Responses to Phytomitogens and on Mixed Lymphocyte Reaction of Chickens

A whole-blood-culture technique was used to sequentially evaluated peripheral blood lymphocyte responses to phytohemagglutinin (PHA) and concanavalin A (Con A) of normal chickens and chickens infected at 1 day or 3 weeks of age with infectious bursal disease virus (IBDV). This method had numerous advantages over the more conventional techniques. A comparative study was made on the percentage of inhibition of responses of peripheral blood lymphocytes to PHA and Con A of 1-day- and 3-week-old IBDV-infected chickens. In both groups, there was a minimum inhibition between 3 and 4 weeks postinfection (PI) and a maximum inhibition at 6 weeks PI. A one-way mixed lymphocyte reaction (MLR) was performed using mitomycin-C-treated cells as stimulator cells obtained from chickens of genetically different strains. Lymphocytes from the experimental birds (control, 1-day-infected, and 3-week-infected groups) were used as the responder cells. The results showed that MLR response of the IBDV-infected chickens was significantly reduced compared with those of the uninfected controls. The degree of lowered response was much more severe in chickens infected at 1 day of age than in those infected at 3 weeks of age.

In vitro alloreactivity of mouse bone marrow lymphocytes

Before characterizing alloreactive cells of the bone marrow, it was necessary to reevaluate the alloantigen response in this tissue. The results of previous studies using the parental-F 1 system in the mixed-lymphocyte reaction (MLR) are open to question because of the recently documented proliferation of F 1 stimulator cells (W. H. Adler, T.Takiguchi, B. Marsh, and R. T. Smith, J. Immunol. 105, 984, 1970; P. F. Piguet, H. K. Dewey, and P. Vassalli, J. Exp. Med. 146, 735, 1977). The culture system was optimized for measuring the MLR of bone marrow lymphocytes enriched on sucrose density gradients. The proliferative response of the enriched fraction (BML) to 2000-R irradiated allogeneic spleen cells was three times as high as the response of unfractionated bone marrow. For maximal responses, antigen concentration had to be twice as high for the BML as for the lymph node, and in a time course study the highest [ 3H]TdR uptake occurred on Day 3 in BML cultures and on Day 5 in the LN. In lymph node semiallogeneic cultures stimulator cell proliferation can be disregarded, while semiallogeneic BML MLR err significantly on the high side. When BML were matched with allogeneic stimulator cells at the H-2 locus, they gave good MLR responses, provided there was a minor Mls histocompatibility locus difference, while in the lymph node the response was greatly diminished in similar mixtures. The differences in the BML and lymph node alloantigen responses with respect to antigen concentration, kinetics and susceptibility to F 1 and Mls stimulation, suggest that the bone marrow alloantigen response is mediated by a cell population that is different than alloresponsive cells in the lymph node.

Modifications of cell-mediated immune responses by moderate dietary protein stress in immunologically immature and mature BALB/c mice

Female BALB/c mice at 5, 6 (immature) and 35 weeks of age (mature) were placed on isocaloric control (20% protein) or experimental (4% protein) diets. Cell-mediated immune responses [theta-bearing cell percentages, phytohemagglutinin (PHA) induced mitogenesis, mixed lymphocyte reaction (MLR) and antibody-dependent lymphocyte-mediated cytotoxicity (ADLMC)] of splenic lymphocytes were measured. A significant decline in percentages of theta-bearing cells, PHA and MLR responses occurred as early as two weeks for the immature mice on the restricted diet and continued for up to 8 weeks. In mature mice, protein restriction did not influence theta-bearing cell percentages, but did suppress PHA and MLR responses between 7 and 13 weeks. A significant suppression of ADLMC responses was also noted at 16 weeks. These results indicate that developing cellular immune systems of immature BALB/c mice are more sensitive to moderate protein restriction ( i.e. immunosuppression occurs after a shorter interval) than those of mature mice. They suggest that moderate protein malnutrition retards but does not prevent immunological maturation.

Ontogeny of Human T Cells

Current knowledge of human T cell ontogeny is reviewed in terms of appearance of T cells in central and peripheral lymphoid organs, maturation of T cell markers, and development of immune functions. Extrapolation of growth curves derived from cell counts from fetal thymus, spleen, and bone marrow indicates the appearance of lymphocytes at 3.5 weeks gestation. E-rosette-forming cells are present in thymus at 11 weeks and in peripheral organs 15 to 16 weeks gestation. beta-2-Microglobulin is associated with all lymphoid cells by 13 weeks gestation. Lymphocyte responses to the T cell mitogen phytohemagglutinin (PHA) are first detected in thymus at 10 weeks and in spleen and blood 3 to 4 weeks later. Allogeneic responses in mixed lymphocyte reactions develop at about 7.5 weeks in fetal liver and later in thymus and peripheral organs. Lymphocytotoxicity for xenogeneic cells is a property of bone marrow cells and not thymocytes. Several aspects of development of a suppressor T cell in human neonates is discussed and related to similar findings in the mouse. These studies indicate a relatively high degree of maturation of human T cells during fetal life.

Studies of immune functions of patients with systemic lupus erythematosus

In normal individuals T cells are stimulated to proliferate by autologous non-T cells; this is called the autologous mixed lymphocyte reaction (MLR). Previous studies demonstrated that such an autologous MLR was markedly impaired in patients with active systemic lupus erythematosus (SLE). To determine whether the defect resided in the responding cell or the stimulating cell, mixing experiments were performed using cells from identical twins. We identified two sets of identical twins discordant for SLE activity and correspondingly discordant in their degree of responsiveness in the autologous MLR. Reciprocal mixing experiments were performed in which T cells from one twin of each pair were mixed with non-T cells from the other twin of that pair. These studies indicated that patients with active SLE have a defect in the ability of non-T cells to stimulate as well as a defect in the ability of both Tgamma and Tnongamma cells to respond in the autologous MLR. Patients with inactive SLE have a defect only in responsiveness of Tgamma cells.

Lymphocyte Transformation Induced by Chemical Modification of Membrane Components

Abstract Neuraminidase treatment of responder cells in indirect periodate or indirect neuraminidase-galactose oxidase (NGO) stimulation of rat lymph node cells (LNC) led to proliferative responses that were substantially greater and faster than those of controls. The indirect responses followed bicellular kinetics, i.e., the number of cells that initially responded was a function of the product of the number of stimulator cells and the number of responder cells in the cultures. Indirect NGO stimulation between allogeneic stimulators and responders was very similar to that between syngeneic cells, and neuraminidase treatment of responders had similar effects in both cases. The indirect mitogenic response between allogeneic cells completely abrogated the expected mixed lymphocyte reaction (MLR). Neuraminidase treatment of stimulators had little effect on the kinetics or magnitude of the rat one-way MLR. Treatment of responders, however, led to a faster and stronger response as compared with the normal MLR response. Treatment of both stimulators and responders was essentially the same as treatment of responders alone. These results, and those for indirect stimulation, suggest that the ability of responder cells to receive a mitogenic signal from stimulator cells is modulated by the amount of sialic acid on the responder cell surface. Although more blast cells were produced in indirect NGO cultures with neuraminidase-treated responders, the cytotoxic activity of these cultures was not enhanced over that produced in normal indirect stimulation. In fact, when referred to the extent of cellular proliferation in each culture, the cytotoxic activity produced by indirect stimulation was higher than that produced either by direct NGO stimulation or by indirect stimulation with neuraminidase-treated responders. Thus indirect NGO stimulation selectively activates cytotoxic cells. The additional cells that are activated by direct NGO stimulation, or after neuraminidase treatment of indirect responders, do not appear to be cytotoxic.

Incorporation of murine MHC antigens into liposomes and their effect in the secondary mixed lymphocyte reaction.

THE major histocompatibility complex (MHC) controls the mixed lymphocyte reaction (MLR) by a dual mechanism involving both the I-region and the K/D-loci products (for a review, see ref. 1). Most information about the MLR has so far been obtained by genetic studies using congenic resistant mouse strains that differ only at defined MHC loci. To dissect the events of the MLR further, it would be advantageous to use purified MHC antigens to gain control not only over the genetical but also the physico-chemical nature of the stimulating antigens. We describe here how in a first attempt to use highly purified MHC antigens as stimulatory agents in the MLR, we have succeeded in anchoring the antigens to liposomes in such a way that they give rise to a good response in the secondary MLR.

Immunosuppressive characteristics of human AFP: effect on tests of cell mediated immunity and induction of human suppressor cells.

Murine AFP has been reported to be immunosuppressive in a variety of systems. However, the extent and degree of inhibition has varied in different species and laboratories. Therefore, we have examined the potential suppressive effect of purified human AFP on several in vitro tests of cellular immunity and the potential mechanism of its action. AFP purified from fetal and liver cancer sera significantly inhibited mitogen and antigen-induced proliferative responses but had no effect on lymphocyte E rosetting, MIF production or mitogen induced T cell cytotoxicity to Chang target cells. Purified human AFP induced human suppressor cell activity, capable of suppressing a one-way mixed lymphocyte reaction (MLC). In contrast to Con A induced suppressor cells, AFP induced suppressor cell activity was overcome by mitogen augmentation of the proliferative response in MLC. These data suggest that the inhibition of lymphocyte proliferation by human AFP may be mediated by the induction of a subpopulation of human suppressor cells. Furthermore, mitogen induced cell mediated cytotoxicity was partially inhibited by primary liver cancer serum and completely inhibited by newborn cord serum, in contrast to purified fetal or tumor AFP which had no effect. These data suggest that there are other immunosuppressive factors in fetal and tumor serum which require further characterization. These other serum factors may be responsible for some of the immunosuppressive effects attributed to AFP. Although AFP is unlikely to play a major immunosuppressive role physiologically in vivo, its selective effect on proliferative responses, apparently mediated by suppressor cells, may prove to be a useful pharmacologic probe of the mechanism of these in vitro lymphocyte responses and biological interactions.

Immunogenetic Analysis of H-2 Mutations. VII. Cells Responding to H-2 Kk Gene Products Give Rise to an Allogeneic Supernatant

Abstract The mixed lymphocyte reaction (MLR) in the mouse is a response by thymus-derived (T) cells against genetically determined alloantigens (1). Two genetic regions have been shown to control the elicitation of strong MLR responses: the I region of the H-2 complex and the Mls region (2–4). Genes in these regions presumably control the expression of cell surface antigens that are recognized by a large number of responding T cells (5). In addition to the cell proliferation that results from the coculture of cells differing at these loci, a factor, termed allogeneic supernatant (AS),2 is also generated very early during the MLR in the culture supernatants. The AS enhances the response of normal spleen cells to T-dependent antigens (6–8), and has the ability to substitute for T cells in the splenic B cell humoral response to the same class of antigens.

T Cell-Mediated Cytotoxicity against Trinitrophenyl-Modified Cells: Effect of Glutaraldehyde Treatment on the Immungenicity and Antigenicity of Trinitrophenyl-Modified Cells

Abstract Cells treated with low concentrations of glutaraldehyde for a 10-sec interval were unable to incorporate 3H-leucine into TCA precipitable protein, respond to H-2 allogeneic cells in mixed lymphocyte reactions (MLR) and cell-mediated lympholysis (CML) assays, or display capping of cell surface immunoglobulin (Ig) with a fluoresceinated anti-Ig reagent. Such cells could stimulate and specifically block H-2 allogeneic CML activity but could not stimulate an H-2 allogeneic MLR response. Trinitrobenzenesulfonic acid (TNBS) treated spleen cells were used to sensitize syngeneic splenocytes into displaying a cytotoxic effect against trinitrophenyl (TNP)-modified target cells. Treatment of the stimulator cells with glutaraldehyde immediately after modification with TNBS did not impair their immunogenic activity. Similar treatment of TNP-modified concanavalin A-stimulated lymphoblasts that were used as inhibitors in a CML cold target competition assay allowed such cells to retain their antigenicity. Cells treated with glutaraldehyde before TNP-modification, however, were not antigenic in the cold target competition assay. These data are compatible with TNBS acting on plasma membrane molecules directly to cause cells to be antigenic and immunogenic in the CML assay rather than affecting internal cellular components.

Fractionation of Lymphocyte Subpopulations Which Regulate Mixed Lymphocyte Reactions

Four days after injection of allogeneic lymphocytes BALB/c splenic T cells suppress proliferation of syngeneic cells in mixed lymphocyte reactions (MLR). Conversely, lymph node cells from the same mice amplify MLR responses. To further characterize these functional subpopulations, alloantigen-primed lymphocyte suspensions from both organs were fractionated by velocity sedimentation at unit-gravity. After fractionation MLR suppressor cells from spleens localized exclusively in rapidlly sedimenting fractions of large cells. MLR suppressor activity of cells from these fractions, as well as that of unfractionated spleen cell suspensions, was abolished by treatment with anti-Thy-1.2 serum and complement. Spleen cell fractions of similar sedimentation velocity also secreted a soluble MLR suppressor into culture supernatants. Although inhibitory of MLR, spleen cells of rapid sedimentation velocity did not suppress responses to T cell mitogens. In marked contrast with the effects of spleen cells, large 4-day-alloantigen-primed lymph node cells had no suppressive activity in MLR. MLR amplifier cells of uncertain derivation were found in fractions of medium sedimentation velocity from both spleens and lymph nodes. Fractionation of alloantigen-primed lymph node cell suspensions did reveal, however, a subpopulation of small cells with MLR suppressor acitivty which was unaffected by treatment with anti-Thy-1 serum and complement. The data thus indicate that large alloantigen-activated lymphocytes are not intrinsically suppressive nor are cells which suppress MLR necessarily large. We consequently conclude that regulation of MLR responses by alloantigen-primed lymphocytes involves a complex interaction between distinct functional subpopulations of cells which are separable both by physical and biologic properties.

Suppression of mixed lymphocyte reactions by alloantigen-activated spleen-localizing thymocytes

Heavily irradiated BALB/c and C57BL/6 mice were injected intravenously with BALB/c thymocytes. At varying times thereafter spleen and lymph node cell suspensions from these animals were treated with mitomycin C and added as regulator populations to mixed lymphocyte reactions (MLR) with syngeneic responder cells. Alloantigen-activated spleen-localizing thymocytes suppressed MLR responses 40 to 95%. Suppressor activitity, manifested as a quantitative reduction in peak proliferative responses, was maximal 4 days after cell transfer. Antigenic specificity for the stimulator cell strain in MLR was not demonstrated. The effect of lymph nodelocalizing thymocytes as regulators in MLR was variable, but in most experiments these cells slightly enhanced responses. We conclude that splenic localization is an intrinsic property of the thymocyte subpopulation capable of suppressing MLR responses, and that the suppressor activity of this subpopulation is substantially enhanced by activation in allogeneic hosts.

Regulatory mechanisms in cell-mediated immune responses. II. A genetically restricted suppressor of mixed lymphocyte reactions released by alloantigen-activated spleen cells.

The mechanism of alloantigen-activated spleen cell suppression of mixed lymphocyte reaction (MLR) is explored in this report. Activated murine suppressor spleen cells elaborated a soluble noncytotoxic factor which suppressed MLR responses by 55-95%. Generation of suppressor factor required both in vivo alloantigen sensitization and specific in vitro restimulation. Suppressor factor was not produced by activated spleen cells which had been treated with anti-Thy-1.2 serum and complement. Antigenic specificity toward alloantigens of the stimulator cells was not demonstrable. In contrast, suppressor factor effectively inhibited MLR response only of responder cells of those strains that shared the D-end and the I-C subregion of the H-2 complex with the cells producing suppressor factor. Therefore, active suppression appears to require an MHC-directed homology relationship between regulating and responder cells in MLR.

Biological Expressions of Lymphocyte Activation

Abstract Thymus-derived lymphocytes (T cells) from mouse spleen, activated in vitro or in vivo with concanavalin A (Con A), suppress proliferative responses of syngenic lymphocytes in mixed lymphocyte reactions (MLR). Replication in vitro was not required for expression of suppressor activity by Con A-activated cells and was blocked in MLR by treating suppressor cells with mitomycin C or irradiation. Kinetics of MLR responses and viability of cultures were not altered by addition of activated suppressor cells. The data are consistent with a direct inhibitory effect of suppressor T cells on antigen-induced DNA replication. These observations extend a model previously described for regulation of antibody synthesis by Con A-activated T cells to control of cell-mediated immune responses. This model should be particularly useful in further definition of regulatory T cell subpopulations, and in investigation of interactions and relationships between such populations.

Dissociation of responses to phytohaemagglutinin and adult allogeneic lymphocytes in human foetal lymphoid tissues.

IN vitro reactivity to phytohaemagglutinin (PHA) and to allogeneic lymphocytes in the one way mixed lymphocyte reaction (MLR) is associated with lymphocytes which are thymus derived1–4 and usually correlates well with cell mediated immune competence in vivo5–8. That these responses may be the property of separate cell populations has been suggested by observations that (a) a fraction of rat thymocytes separated on an albumin density gradient reacted in an MLR but not to PHA, while other fractions were responsive in both tests9; (b) human lymphocytes treated with trypsin and Vibrio cholera extract react in an MLR but have a depressed response to PHA10; and (c) rare reports of PHA-responsive MLR-nonresponsive lymphocytes in immune deficient states11. We wished to determine whether MLR and PHA responses are possessed by the same or different populations of lymphoid cells during human foetal development. Results indicate that the MLR is acquired earlier in development and is independent of the PHA response and that foetal liver is the site of MLR-responsive PHA-nonresponsive cells.