Human Mixed Lymphocyte Reaction Research Articles

Effects of bacteria-produced human alpha, beta, and gamma interferons on in vitro immune functions

The effects of bacteria-produced human interferons (HuIFN) α, β, and γ on in vitro immune functions of human peripheral blood mononuclear cells (PBMC) were studied. Proliferative response to phytohemagglutinin was significantly inhibited by the addition of HuIFN-α2 or HuIFN-β at 10, 100, or 1000 U/ml. In contrast, HuIFN-γ showed suppressive activities only when added at 1000 U/ml. HuIFN-α2 or HuIFN-β caused significant inhibition of human mixed-lymphocyte reaction (MLR) as measured by [ 3H]thymidine incorporation. Similar inhibition was caused by HuIFN-γ when it was added only at very low concentrations (1 U/ml); 10, 100, or 1000 U/ml resulted in no or only a modest increase in MLR. All three interferons exhibited dose-related effects on PWM-induced immunoglobulin synthesis in cultures of PBMC. These data demonstrate that purified interferons produced by recombinant DNA technology can significantly alter in vitro immune functions and that HuIFN-γ has properties which are different from those of HuIFN-α2 or HuIFN-β.

Reactivity of inducer cell subsets and T8-cell activation during the human autologous mixed lymphocyte reaction

To characterize the responding T cells in the autologous mixed lymphocyte reaction (AMLR), T cells were fractionated into purified subpopulations employing monoclonal antibodies and a variety of separation techniques including fluorescence-activated cell sorting. It was found that isolated T4 cells, but not T8 cells, proliferated in response to autologous non-T cells. More importantly, within the T4 subset, the autoreactive population was greatly enriched in a fraction reactive with an autoantibody from patients with juvenile chronic arthritis (JRA) or the monoclonal antibody anti-TQ1. Although T8 cells themselves were unable to proliferate in the AMLR, they could be induced to respond in the presence of either T4 cells or exogenous IL-2 containing medium. This was demonstrated by direct measurement of tritiated thymidine uptake by T8 cells during the course of the AMLR as well as by analysis of their relative DNA content. Taken together, these data indicate that the AMLR represents a complex pattern of immune responsiveness distinct from that observed in response to soluble antigen or alloantigen. The precise function of this T-cell circuit remains to be determined.

Inhibition of in vitro immunosuppressive effects of glucocorticosteroids by a competitive antagonist RU-486

The 19-nor-steroid RU-486 is an antagonist of glucocorticosteroids acting competitively at the receptor level. Pharmacological and endocrinological studies have already shown that RU-486 is able to reverse glucocorticosteroid effects. The present study shows that RU-486 can counteract the inhibitory effect of glucocorticosteroids in two highly corticoid-sensitive in vitro immune responses: murine in vitro antibody response and human autologous mixed lymphocyte reaction.

Bacteria-derived human leukocyte interferons alter in vitro humoral and cellular immune responses

Cultures of gradient-purified human peripheral blood mononuclear cells (PBMC) have been employed to examine the effects of three bacteria-derived human leukocyte interferon subtypes on certain aspects of in vitro immune responses. The addition of highly purified IFN-α1, -α2, -α2/α1 to PMBC cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen resulted in a significant suppression of the mitogenic response. This suppression required the presence of interferon in the cultures because pretreatment of cells and removal of interferon had no effect on their response to PHA. The presence of these interferons at 200 U/ml also caused a substantial reduction of human mixed-lymphocyte reactions (MLR) as measured by [ 3H]thymidine incorporation by responder cells. Interestingly, pretreatment of stimulator cells was sufficient for this reduction to occur whereas pretreatment of responder cells had no effect on their ability to respond to allogenic stimulation. In contrast to these suppressive effects, the three interferons enhanced human in vitro primary immune response to sheep red blood cells (SRBC). These data demonstrate that both purified interferon subtypes and genetic hybrids of human interferons produced by recombinant DNA technology have effects on in vitro immune responses.

Human autologous mixed lymphocyte reactions (a review).

The literature concerning the autologous mixed lymphocyte reactions has been reviewed. This analysis supports the following conclusions: human subjects have self-responsive cells which are capable of proliferating when co-cultured with irradiated autologous non-T or Ia+ T cells. Since monoclonal antibodies recognizing distinct determinants of Ia antigen have different effects on AMLR with non-T cells and on Ia+ type AMLR, there is the possibility that different Ia molecule determinants have different functional role in the process of cell-to-cell interaction. The presence of AMLR abnormalities in disease strongly suggests that reactivity among different cell subsets plays a role in immunological homeostasis.

Neoplastic cells obtained from Hodgkin's disease are potent stimulators of human primary mixed lymphocyte cultures.

Neoplastic cells obtained from the pleural effusion of a patient with Hodgkin's disease have been maintained in culture since 1978. These tumor cells have been shown to have the cytologic features, cytochemical staining, and cell surface markers of Reed-Sternberg cells. In this study we demonstrate that the cell line termed L428 is a potent stimulator of the primary human mixed lymphocyte reaction. Significant proliferation occurred when mononuclear leukocytes obtained from normal donors were stimulated with radiated L428 cells at responder:stimulator ratios varying from 200:1 to 20:1. Proliferative responses occurred between days 3 and 6 of the cultures with maximal proliferation on day 5. Under optimal culture conditions, mean net proliferative response of 14 normal donors was 51,000 +/- 10,600 dpm. The mixed lymphocyte response was totally blocked by concentrations of monoclonal anti-Ia antibody that had no effect on concanavalin A-induced proliferation. However, the mixed lymphocyte response was not blocked by an anti-K562 cell monoclonal antibody of the same immunoglobulin subclass that binds to the L428 cells. Antigen processing by responder monocytes or Ia-positive cells was not required for the MLC. When responder T cells from two normals were depleted of Ia-bearing cells and monocytes, the mixed lymphocyte reaction between the two normals was eliminated, yet the stimulation of each normal by the L428 cells was not reduced. The cells that proliferated in response to stimulation by the L428 cells were T cells, primarily of the helper subset. No IL 1 activity could be detected in concentrated supernatants of L428 cultures after stimulation of L428 cells by mitogens, phorbol esters, or muramyl dipeptide, or in the MLC. All of these cultures contain fetal calf serum. However, the L428 cells are capable of producing IL 1, because IL 1 was detected when the L428 cells were stimulated with LPS in the absence of fetal calf serum. These neoplastic cells, obtained from Hodgkin's disease, have many similarities to the murine as well as human dendritic cells.

Low-density mononuclear cells. Potent stimulators of the human MLR.

We have applied purification strategies similar to those used to purify murine dendritic cells to human peripheral blood in an attempt to enrich for stimulators of the human mixed lymphocyte reaction (MLR). Equilibrium density centrifugation of peripheral blood mononuclear cells yields a population of low density cells that are potent stimulators of a human MLR. The stimulator cells are Dr+, nonlymphocytic, and weakly adherent. Strongly adherent monocytes, also present in the low-density cell population, do not stimulate a human MLR. This contrasts with other human MLR studies that ascribe stimulatory activity to adherent monocytes, and it indicates functional and morphological heterogeneity among monocytes.

Effects of azathioprine on the human mixed lymphocyte reaction (MLR).

British Journal of Clinical PharmacologyVolume 15, Issue 2 p. 203-209 Free Access Effects of azathioprine on the human mixed lymphocyte reaction (MLR). SA Al-Safi, SA Al-SafiSearch for more papers by this authorJL Maddocks, JL MaddocksSearch for more papers by this author SA Al-Safi, SA Al-SafiSearch for more papers by this authorJL Maddocks, JL MaddocksSearch for more papers by this author First published: February 1983 https://doi.org/10.1111/j.1365-2125.1983.tb01487.xCitations: 15AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References AL-SAFI, S.A. & MADDOCKS, J.L. (1982). The mechanism of action of azathioprine on the mixed lymphocyte reaction (MLR). Br. J. Pharmac., 76, 191P. BACH, M.A. & BACH, J.F. (1972). Activities of immunosuppressive agents in vitro. II. Different timing of azathioprine and methotrexate in inhibition and stimulation of mixed lymphocyte reaction. Clin. exp. Immun., 11, 89– 98. BACH, J.F. & DARDENNE, M. (1971). Activities of immunosuppressive agents in vitro. I. Rosette inhibition by azathioprine. Eur. J. clin. biol. Res., 16, 770– 777. BACH, J.F., DARDENNE, M., FOURNIER, C. (1969). In vitro evaluation of immunosuppressive drugs. Nature, 111, 998– 999. BACH, F.H. & VOYNOW, N.K. (1966). One-way stimulation in mixed leukocyte cultures. Science, 153, 545– 547. BOYUM, A. (1968). Separation of leukocytes from human blood. Scand. J. clin. lab. Invest, (suppl. 97), 21, 9– 89. BOYUM, A. (1976). Isolation of lymphocytes, granulocytes and macrophages. Scand. J. Immun., 5 (Suppl. 5), 9– 15. CALNE, R.Y. (1961). Inhibition of the rejection of renal homografts in dogs by purine analogues. Transplant. Bull., 28, 445– 460. CHALMERS, A.H. (1974). Studies on the mechanism of formation of 5-mercapto-l-methyl-4-nitroimidazole. A metabolite of the immunosuppressive drug azathioprine. Biochem. Pharmac., 23, 1891– 1901. CHALMERS, A.H., KNIGHT, P.R. & ATKINSON, M.R. (1967). Conversion of azathioprine into mercaptopurine and mercaptoimidazole derivatives in vitro and during immunosuppressive therapy. Aust. J. exp. Biol. med. Sci., 45, 681– 691. CHALMERS, A.H. & MURRAY, A.W. (1972). Immunosuppression by 9-alkyl-6-thiopurines. Biochem. Pharmac., 21, 2662– 2664. ELION, G.B. (1967). Biochemistry and pharmacology of purine analogues. Fed. Proc, 26, 898– 904. ELION, G.B., CALLAHAN, S., BIEBER, S., HITCHINGS, G.H. & RUNDLES, W. (1961). A summary of investigations with 6-[(l-methyl-4-nitro-5-imidazolyl)thiol] purine (B.W. 57–322). Cancer Chemother. Rep., 14, 93– 98. KENDALL, P. A. & HUTCHINS, D. (1978). The effect of thiol compounds on lymphocytes stimulated in culture. Immunology, 35, 189– 201. MADDOCKS, J.L. (1979). Progress in immunosuppression In Topics in Therapeutics, eds D.M. Davies & Rawlins, M.D., pp. 33– 44. London: Pitman. MEDZIHRADSKY, J.L., HOLLOWELL, R.P. & ELION, G.B. (1981). Differential inhibition by azathioprine and 6-mercaptopurine of specific suppressor T cell generation in mice. J. Immunopharmac, 3, 1– 16. MERRYMAN, P.F., NOWAKOWSKI, J. & JAFFE, L.A. (1979). Alteration of lymphocyte response by sulfhydryl and disulfide compounds. Biochem. Pharmac., 28, 2297– 2302. MIRANDA, P., BEACHAM III, L.M., CREAGH, T.A. & ELION, G.B. (1973). The metabolic fate of the methyl-nitroimidazole moiety of azathioprine in the rat. J. Pharmac. exp. Ther., 187, 588– 601. RYZEWSKI, J., ROSZKOWSKI-SUZ, W. & KRZYSTYNIATE, K. (1977). The action of cytseine on PHA-stimulation of lymphocytes. I. Influence on DNA synthesis. Arch. Immun. Ther. Exp. (Warsz), 25, 377– 383. SCHUMACHER, K., MAERKER-ALZER, G. & PREUSS, R. (1975). Effect of D-penicillamine on lymphocyte function. Arzneimittel-Forsch. (Drug Res.), 25, 603– 606. SCHWARTZ, G.H., STENZEL, K.H. & RUBIN, A.L. (1969). Depression of lymphocyte transformation by drugs in vitro. Effects of a new immunosuppressant, cinanserin. Transplantation, 8, 704– 711. SKOPINSKA, E., RYCHLIKOWA, M. & NOUZA, K. (1968). Immunosuppressive drugs in lymphocyte mixed cultures. Bull. Acad. pol. Sci. CI. VI Ser. Sci. biol., 16, 385– 389. SMITH, J.L. & FORBES, I.J. (1970). Inhibition of protein synthesis in human lymphocytes by thiopurines. Aust. J. exp. Biol. med. Sci., 48, 267– 276. VAS, M. & LOWENSTEIN, L. (1965). The effect of prednisolone and azathioprine in mixed leukocyte cultures In Histocompatability testing, eds D.B. Amos & VAN Rood, V., pp. 213– 215. Copenhagen: Williams & Wilkins. ZOLLER, K.M., CHO, S.I., COHEN, J.J. & HARRINGTON, J.T. (1980). Cessation of immunosuppressive therapy after successful transplantation: A national survey. Kidney Int., 18, 110– 114. Citing Literature Volume15, Issue2February 1983Pages 203-209 ReferencesRelatedInformation

Immunoregulatory activity of T-cell subsets activated in human mixed lymphocyte reaction

Unseparated human T cells or isolated OKT4 + and OKT8 + subsets were stimulated in allogeneic MLR and subsequently tested for their capacity to suppress fresh autologous responding cells in a second MLR. Contrary to the bulk of evidence regarding the immunoregulation of B-cell function, both OKT4 + and OKT8 + cells could be activated to form suppressors of the MLR and were equally radioresistant. An early peak of blastogenesis was observed when OKT4 + suppressors were used, and this event was completely radiosensitive. When the fresh responding cells were also fractionated into OKT4 + and OKT8 + subsets, no preference for suppressibility of either subset could be demonstrated, indicating that OKT4 + suppressors were acting directly and not through an inductive mechanism requiring OKT8 + effectors. It is suggested that when alloantigenic responses are involved, the human T-cell subsets defined by these monoclonal antibodies may be more accurately defined by the class of MHC antigen to which they respond than by their effector function.

Bacteria-immune system interactions. IX. Modulation of human and murine cytotoxic reactions by crude lipoteichoic acid extracted from Staphylococcus aureus.

Crude lipoteichoic acid extracts of Staphylococcus aureus (SA-LTA) or Bacillus glogigii (Bg-LTA) inhibited cytotoxic reactions when added at day 0 to human or murine mixed lymphocyte reactions (MLR). The inhibition was dose dependent, and at low Sa-LTA concentrations it did not require reduction of thymidine incorporation into effector lymphocytes. In the murine system, Sa-LTA was a much more potent inhibitor than Bg-LTA. LTAs of both origin also could inhibit cytotoxic reactions when bound to stimulator cells. However, the LTA derivatized stimulator cells were much more inhibitory in the human than in the murine MLR. The inhibition of human cytotoxic reaction by Sa-LTA derivatized stimulator was not associated with a significant reduction of thymidine incorporation in effector cells.

Failure of the human autologous mixed lymphocyte reaction in the absence of foreign antigens.

The autologous mixed lymphocyte reaction (AMLR) is thought to represent the in vitro proliferation of T cells in response to stimulation by autologous histocompatibility antigens present on non-T cells. We have investigated the possibility that xenogeneic determinants act as the stimulating antigens in the human AMLR. Exposure of lymphocytes to fetal calf serum and/or sheep red blood cells during fractionation triggers substantial proliferation of T cells, plaque-forming cell responses, and interleukin 2 production. In contrast, lymphocytes isolated and cultured in the absence of any known foreign substances failed to respond in the AMLR, yet responded significantly when cultured in the presence of fetal calf serum. These results suggest that without exposure to foreign antigens, the human AMLR is difficult to demonstrate in vitro.

The human autologous mixed lymphocyte reaction. III. Immune circuits.

The human autologous mixed lymphocyte reaction represents the proliferative response of T cells to determinants presented on autologous non-T cells. Purified T4+ cells vigorously proliferate in response to stimulation by either (B + null) cells or M phi, whereas purified T8+ cells proliferate very little without a source of help. Such help can be provided by mitomycin C-treated T4+ cells, which indicates that proliferation of T4+ cells is not necessary for the help. M phi suppress the human AMLR, as measured by the response of T cells to stimulation by (B + null) cells. The target of this M phi-induced suppression was found to be the T4+ inducer cell. In contrast to the suppressive effects of M phi on T4+ cells, M phi did not suppress T8+ cells. The available data suggest that the human AMLR is a two-part inducer circuit. One part can be stimulated preferentially by M phi and the other by (B + null) cells; however, M phi prevent activation of the (B + null) cell part of the circuit. These results may provide an explanation for the differential activities of different regulatory circuits without and also with antigenic stimulation.

Suppressor cells induced in the human autologous mixed lymphocyte reaction

Theophylline, a phosphodiesterase inhibitor, also reversibly inhibits the SRBC receptor of a subpopulation of human T lymphocytes. We have previously reported that the precursor for the Concanavalin A (Con A) inducible suppressor cell (SC) is found predominantly in the theophylline-sensitive (T s) population; whereas, the precursor of the mitomycin resistant suppressor cell induced in the allogeneic mixed lymphocyte reaction (MLR) is predominantly in the theophylline-resistant (T R) subsei. This study examines the theophylline sensitivity and functional characteristics of suppressor cells induced in autologous MLR (AMLR). Both T S and T R proliferate moderately in AMLR but differ in their ability to form suppressors. AMLR activated T S and T R cells demonstrate early blastogenesis and weak suppression in the Con A assay system, but suppression in an MLR assay system appeared to be confined largely to the T R subset. AMLR suppressors were mitomycin sensitive. These data illustrate that suppressors generated in allogeneic and autologous MLR have significant functional and phenotypic similarities, and strengthen the association between these two immunologic phenomena.

Role of Ia antigens in the human autologous mixed lymphocyte reaction.

The role of Ia antigens in the human autologous mixed lymphocyte reaction (MLR) was analyzed. To do this, different mononuclear cell populations were fractionated according to their Ia antigen expression and used as stimulating cells in autologous MLR. Also examined was the effect of two hybridoma monoclonal antibodies specific for human Ia molecules on the autologous MLR. The results show that the stimulating capacity of peripheral blood mononuclear cells is restricted to Ia-bearing cells. While peripheral blood T cells are unable to function as stimulating cells, highly purified Ia+ mixed lymphocyte culture-activated T cells are very effective. The possibility that Ia molecules are directly involved in the autologous MLR is further substantiated by the strong inhibitory effect of monoclonal anti-Ia antibodies.

MHC specified lymphocyte activating and suppressor activating determinants in human mixed lymphocyte reactions.

We have shown (1) the presence of HLA-linked Sad/s on a homozygous typing cell (HTC), FPA (8W321). When we used it to type the P family, which included an HLA-B/DR recombinant sibling, its genes mapped towards the HLA-B-A end of the HLA haplotype separate from HLA-D/DR and was found to be associated with HLA-Bw35 homozygosity; (2) this same recombinant maps the C4 complement genes on the HLA-D/DR end of the chromosome away from HLA-B; (3) that the suppressor T cell subpopulation which was activated by the Sad of this consanguineous HTC FPA was left behind after separating the OKT4 positive cells with a monomorphic monoclonal anti-T-cell antibody (OKT4). These OKT4 positive cells reacted normally in MLC to the FPA Lad Fes 6; (4) that at least part of the genetic control of suppressor recognition is outside the HLA complex.

Responder cells in the human autologous mixed lymphocyte reaction.

Isolated human T4+ cells proliferate in the autologous mixed lymphocyte reaction (AMLR), whereas isolated T8+ cells do not. However, in the presence of Interleukin 2 or T4+ cells, the T8+ cells demonstrated substantial proliferation. These studies suggest that T8+ cells recognize signals from autologous non-T cells, but require an additional factor for the subsequent proliferative response. Since this stimulus can be provided by T4+ cells, the AMLR appears to constitute an inducer circuit. Different defects in this circuit may be responsible for the common abnormality of the AMLR in different diseases.

Inhibitory effect of tumor promoting phorbol esters and mezerein on human mixed lymphocyte reaction

The effect of phorbol ester-type tumor promoters on the mixed reaction of human lymphocytes was investigated. When a low concentration (10 ng/ml, 1.67 × 10 −8 M) of 12- O-tetradecanoylphorbol-13-acetate (TPA) was added at the beginning of the mixed lymphocyte reaction, this, as measured by [ 3H]thymidine incorporation, was almost completely inhibited. The inhibitory effect is apparently not related to a toxic effect, since [ 3H]uridine incorporation was affected to a lesser extent by the addition of TPA. TPA is also inhibitory when added after the reaction has started. The inhibition of the reaction by TPA is very rapid and complete inhibition can be seen within 2 h after TPA addition. Another promoting phorbol ester, phorbol-12,13-didecanoate (PDD), also inhibited the reaction, whereas inactive derivatives, phorbol and 4α-PDD, did not. Mezerein, a weak promoter on mouse skin, was also a potent inhibitor of the human mixed lymphocyte reaction.

An Fab-related surface component of some normal and neoplastic human and marmoset T cells: Demonstration, functional analysis and partial characterization

Chicken antibodies raised to the (Fab') 2 fragment of pplyclonal human IgG recognize a set of configurational determinants, including idiotypic determinants of individual myeloma proteins, formed by certain heavy chain Fd regions in association with κ light chains. Chicken anti-Fab binds, as shown by indirect immunofluorescence, to a surface component of human and marmoset B cells and to some neoplastic T cell lines, as well as to approximately 50% of T cells isolated from human peripheral blood. It does not bind to normal or neoplastic ‘null’ cells. The generation of human alloreactive cytotoxic T cells in vitro is substantially inhibited by incubation with chicken anti-human (Fab') 2; whereas chicken anti-mouse (Fab') 2 has no effect. Conversely, chicken anti-mouse (Fab') 2 completely inhibits the in vitro induction of alloreactive mouse T cells, but does not affect the human mixed lymphocyte reaction. Components bearing Fab-related antigenic determinants were isolated from culture fluids in which two marmoset T cell lines had been grown. When analysed by polyacrylarnide gel etectrophoresis under reducing conditions in sodium dodecyl sulfate-containing buffers, the major Fab-related component from line 70-N2 had an apparent mass of 70,000 dallons and that from line 22-CM-37 had an apparent mass in the range of 44,000–50,000 daltons.

Evaluation of the Role of C4 in the Cellular Immune Response in Vitro

Abstract The demonstration that antisera to human C4 inhibit the human mixed lymphocyte reaction and the response to the mitogen, phytohemagglutin, suggested a possible role of C4 in the afferent phase of immune recognition. An attempt was made to reproduce and extend these observations in the guinea pig. Monospecific antisera to C4 were raised in C4-deficient guinea pigs, rabbit, or goat. The antisera were tested for inhibition of the unidirectional mixed leukocyte reactions between strain 13 and strain 2 guinea pigs, for inhibition of proliferation induced by the mitogens, phytohemagglutinin and concanavalin A, and for inhibition of the proliferative response of immune T lymphocytes to soluble protein antigens. In a large number of experiments no reproducible inhibition was found when the various cultures were performed in the presence of antiserum to C4. Furthermore, lymphocytes from C4-deficient guinea pigs responded as well as lymphocytes from strain 13 animals to specific antigen or in mitogen-induced proliferation, indicating a normal capacity of T cells from C4-deficient animals for recognition of antigen or mitogen and for proliferation. Antigen- or mitogen-pulsed macrophages from C4-deficient guinea pigs were able to effectively stimulate T cell proliferation, suggesting a normal capability for antigen or mitogen presentation. These data argue against a possible role for C4 in antigen, mitogen, or alloantigen-induced T lymphocyte activation and indicate that the demonstrated functional association of C4 and MHC-mediated reactions in the human does not generally apply to another species.

Human leucocyte antigens and mixed lymphocyte reaction in severe pre-eclampsia.

Human leucocyte antigens (HLA) and mixed lymphocyte reactions (MLR) were studied in 38 women with severe pre-eclampsia and their husbands. Thirty-nine women with normal pregnancies and their husbands served as controls. Thirty-three of the control women were matched for age and parity with members of the study group. Infants were studied when possible. HLA compatibility was increased in the pre-eclamptic group compared with matched controls and with theoretical estimates for possible matings. The one-way MLR at delivery showed diminished response of maternal to paternal and cord cells in pre-eclamptic women. This reduced maternal reactivity in women with pre-eclampsia may have a role in the illness, and paternal/maternal histocompatibility may be a feature of the severe form.