One-way Mixed Lymphocyte Culture Research Articles

NK cells suppress the generation of Lyt-2+ cytolytic T cells by suppressing or eliminating dendritic cells.

Cells highly enriched for natural killer activity suppress the generation of Lyt-2+ cytolytic T cells in one-way mixed lymphocyte cultures. Suppression occurs because natural killer cells suppress or eliminate dendritic cells, which are required for proliferation of both Ly-1+ and Lyt-2+ lymphocytes.

Effects of Cyclosporin A on Functions of Specific Murine T Cell Clones: Inhibition of Proliferation, Lymphokine Secretion and Cytotoxicity

Allospecific T lymphocyte clones with different functions were generated from spleen cells of C 57/B16 mice following sensitization in vitro by a one-way mixed lymphocyte culture (MLC) with irradiated DBA/2 spleen cells. The clones were propagated in vitro in the presence of interleukin 2 (IL 2) and restimulation with stimulator cells. In these clones, Cyclosporin A (CSA) was tested for its suppressive effect on different T lymphocyte functions. The antigendependent proliferation of a helper clone (HTL) was totally inhibited by 50 ng/ml CSA. Proliferation induced by simultaneous administration of antigen and IL 2 was partially suppressed in all helper and cytotoxic clones (CTL). The IL 2-driven proliferation in the absence of antigen was also suppressed between 25–70 % by the immunosuppressive drug. Secretion of macrophage activating factor (MAF) and interferon (IFN) by HTL and CTL in response to antigen or mitogen was reduced dose dependently by CSA. Concentrations of 50 ng/ml CSA diminished lymphokine secretion to approximately 10 % of controls, also when excess IL 2 was present. Cytotoxicity, previously described to be insensitive to the drug, could be suppressed by 50 ng/ml CSA to a various extent, from 40–70 %, in different cytotoxic clones when the effector cells were preincubated with CSA for 1 h or more. Conclusively, the data suggest that CSA interferes generally with the activation of T lymphocyte clones.

Transfusions of leukocyte-rich erythrocyte concentrates: A successful treatment in selected cases of habitual abortion

Forty-nine women who suffered recurrent spontaneous abortion of unknown etiology were studied for cellular reactivity and blocking antibody in one-way mixed lymphocyte culture before and after their receipt of three transfusion of leukocyte-rich erythrocyte concentrates from third-party donors. Those 38 of the 49 women who had no blocking antibody all developed significant blocking activity after the transfusion series. Twenty-five of them have become pregnant since, and only one aborted again. The blocking activity demonstrable in 11 of 49 women was increased after the transfusions. Subsequently five of them became pregnant, and all aborted again. We later found that these five women, who considered themselves to be in perfect health, all had serologic signs of autoimmune disease. We advise against transfusion treatment of women who habitually abort without preceding immunologic investigation, because a population of habitual aborters may contain women with yet undiagnosed autoimmune disease, who would be worse off after blood transfusion. We conclude from our results that a selected population of habitual aborters, that is, those without blocking antibody, benefits from transfusion treatment.

Induction of IgE synthesis in normal human B cells. Sequential requirements for activation by an alloreactive T cell clone and IgE-potentiating factors.

Two human alloreactive T cell clones were established from a one-way mixed lymphocyte culture involving two nonatopic donors, and were assessed for their capacity to induce IgE synthesis by B cells obtained from the original stimulator. The two alloreactive T cell clones studied induced IgG but not IgE synthesis in normal B cells. However, one of the two clones, clone 2H6, induced IgE synthesis in the presence of supernatants from T cell lines derived from patients with the hyper-IgE syndrome (HIE), and enriched for T cells bearing receptors for IgE. These supernatants by themselves caused no IgE synthesis in nonatopic B cells. The potentiating factors in these supernatants were shown to bind to IgE. Time sequence experiments indicated that interaction of the B cells with the alloreactive clone 2H6 renders them responsive to the action of the IgE-potentiating factors. These results indicate that induction of IgE synthesis in normal B cells involves at least two sequential T cell derived signals. Furthermore, T cell clones are heterogenous in their capacity to provide these signals.

Persistent suppression of humoral and cell-mediated immunity in mice following exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene

Carcinogen-induced immunosuppression has been implicated as an epigenetic mechanism in promoting the outgrowth and metastasis of neoplastic cells. It has previously reported that the complete carcinogen 7,12-dimethylbenz[a]antracene (DMBA) suppresses both humoral immunity (HI) and cell-mediated immunity (CMI) 3–5 days following exposure. Since persistent systemic immunosuppression may be more relevant in tumor outgrowth, assays quantitating HI and CMI, including those functions involved in tumor resistance, were performed 4 and 8 weeks following exposure to tumorigenic doses of DMBA. Adult B6C3F1 female mice were administered DMBA dissolved in corn oil subcutaneously at 5, 50 and 100 μg/g body weight in ten equal doses over 2 weeks (corn oil = vehicle control). The number of splenocytes producing IgM antibody to the T-dependent antigen, sheep erythrocytes, was suppressed up to 95% and 98% at 4 and 8 weeks, respectively. The IgG response was similarly depressed 75% and 98% at 4 and 8 weeks, respectively. Lymphoproliferation of splenocytes in response to the mitogens LPS, PHA and Con A were depressed up to 88%, 78% and 83% at 4 weeks and 63%, 63% and 67% respectively, at 8 weeks. In addition, alloantigen-induced proliferation of splenocytes in a one-way mixed lymphocyte culture was suppressed up to 90% at 8 weeks. The ability to generate cytotoxic T-lymphocytes (CTL) in vitro against P815 tumor cells was depressed at both time periods (88% and 60%, respectively) as was natural killer (NK) cell cytolysis of YAC-1 tumor targets (84% and 55%, respectively). The immunosuppression noted in these parameters was similar to that observed within 3–5 days following DMBA dosing. The persistent immunosuppression induced by the PAH carcinogen DMBA, including CTL and NK cell tumoricidal functions, may represent an important epigenetic mechanism contributing to tumor outgrowth or metastasis by this class of agents.

Blood transfusions generate/increase previously absent/weak blocking antibody in women with habitual abortion.

Women who suffered recurrent spontaneous abortions of unknown cause were studied for cellular reactivity and blocking antibody in a one-way mixed lymphocyte culture. A defined group of 20 women whose serum displayed no blocking capacity was given three transfusions of leukocyte-rich erythrocyte concentrates. Serum from all women displayed significant blocking capacity 2 months after the third transfusion. Because blocking antibody seems to be one of the necessary prerequisites for successful pregnancy, leukocyte transfusions from third-party donors ought to be an effective cure for habitual abortion in selected cases.

Effect of human syncytiotrophoblast extract on in vitro proliferative responses.

The authors studied the effects of soluble syncytiotrophoblast extract (STE) on the proliferative responses of lymphocytes to different lectins (PHA and Con A) and to allogeneic cells in one-way mixed lymphocyte cultures. STE suppressed lymphocyte reactivity to lectins and to allogeneic cell cultures. The inhibitory effect was dose dependent. Increased concentrations of lectins failed to overcome the inhibitory effect of STE. Lymphocytes preincubated with STE for 18 hr, then washed and exposed to lectins still exhibited an inhibition of cellular proliferation. STE added to lymphocyte cultures at various times in the presence of both mitogens or of allogeneic cells continued to inhibit lymphoproliferative responses even when it was added after 43 hr of cell culture. Furthermore, STE was able to reduce the spontaneous proliferation of tumor cell lines K562 and LHN13 maintained in vitro culture prior to testing. In all cases, the inhibition observed was not due to lymphocytotoxicity or to tumor cell mortality. The inhibitory effect of STE on the proliferative responses of lymphocytes to stimulants may be due to a cytostatic effect that may represent a contributing factor in the nonrejection of the fetus by a competent immune system.

Histoincompatibility in couples with unexplained infertility

Class I human leukocyte antigens (HLA-A, -B) and class II (HLA-DR) antigens were determined in 18 carefully selected couples suffering from unexplained infertility and subsequently compared with 30 normal fertile couples with no history of secondary sterility and with a control group consisting of randomly matched women and men from our laboratory cell panel. No significant differences in the frequencies of HLA antigens were detected between infertile and control groups. The frequency of shared HLA-A, -B, and -DR antigens among members of the couples was similar in all the groups. Finally, the one-way mixed lymphocyte culture showed normal reactivity of both infertile parental pairs in all combinations tested.

1042 INTERFERONOPATHY IN THE BARE LYMPHOCYTE SYNDROME

The Bare Lymphocyte Syndrome (BLS) is a rare immunodeficiency in which surface expression of HLA-DR antigens is absent. In the absence of expression of Major Histocompatibility Complex (MHC) there is an abnormal interaction between immunocytes with a resultant combined immunodeficiency. We report here of a 4 year old female with the BLS. This child is agammaglobulinemic and has poor cell mediated immunity with recurrent bacterial and viral infections. The child persistently excretes the polio vaccine virus type II in the stool. In vitro lymphocyte studies revealed normal responses to phytohemagglutinin and in the one-way mixed lymphocyte culture. Mitomycin C treated patient's cells were, however, unable to stimulate allogeneic lymphocytes, and the patient's lymphocytes did not respond to antigenic stimuli. In vitro treatment of patient's lymphocytes with α or γ interferon restored the expression of the MHC and improved antigenic and allogeneic responses. The patient's lymphocytes did not, however, secrete α or γ interferon. It is postulated that in this instance the BLS may be due to a primary interferonopathy.

Abnormal immunoregulation in remission Hodgkin disease.

Peripheral blood mononuclear cells from 11 patients with remission Hodgkin disease and 20 normal controls were incubated with irradiated allogeneic lymphocytes in one-way mixed lymphocyte cultures. Simultaneously, modified assays were performed by adding supplemental irradiated PBM, T lymphocytes, or adherent cells autologous to the responders. Baseline allogeneic responsiveness of patients and controls was not different. However, significant suppression (p less than .01) was demonstrated when the cultures were supplemented with patient mononuclear cells or adherent cells, an effect not found with similar supplemental cells from controls. Conversely, T-cell supplementation of control cultures produced more than twofold increases in proliferation but significantly less augmentation in the patients' cultures (p less than .01). T-cell subset analysis in six patients showed decreased helper: suppressor cell ratios. Hodgkin disease patients have adherent suppressor cells, which persist during remission, as well as a defect in T-cell helper function.

Activation of cytotoxic T lymphocytes in HLA-A, -B and -C-identical responder-stimulator pairs. I. Variations in generation of anti-class-II CTL in primary mixed lymphocyte cultures.

Cytotoxic T lymphocytes were activated in primary one-way mixed lymphocyte cultures of cells matched for serologically defined HLA-A, -B and -C antigens. In 16 out of the 29 combinations mismatched for the HLA-D/DR antigens, cell-mediated lympholysis of the stimulator cells occurred. The specificity of 5 selected cytotoxic T lymphocytes was studied in detail. Three of these cytotoxic T lymphocytes recognize antigenic determinants associated with HLA-Bw35 (Breuning et al. 1984, II). The 2 other cytotoxic T lymphocytes failed to lyse T-target cells enriched by rosetting with sheep red blood cells, whereas target cells from the 'non-T' fraction were strongly lysed, indicating that antigenic determinants associated with Class-II HLA molecules were the targets recognized by these cytotoxic T lymphocytes. This notion was supported by a study of a panel of HLA-typed third-party target cells. One cytotoxic T-lymphocyte population preferentially lysed HLA-DR2-positive target cells. Family studies, including a family with a recombination between HLA-B and -D, showed that the target antigen recognized by the latter cytotoxic T lymphocyte segregated with DR2. The second cytotoxic T-lymphocyte population recognized a determinant associated with DRw8. However, in 13 of the 29 HLA-A-, -B- and -C-identical, D/DR-different combinations, cell-mediated lympholysis of stimulator target cells could not be detected, not even on enriched 'non-T' target cells. Thus, after primary mixed lymphocyte culture of HLA-A-, -B- and C-identical, HLA-D/DR-non-identical cells, cytotoxic T lymphocytes directed against sensitizing Class-II molecules can be detected in some combinations, but not in others.

Mixed lymphocyte reactivity in cats.

A method was developed to perform primary one-way mixed lymphocyte cultures (MLC) with cat cells. A polymorphic system of MLC reactivities was found within a cat population. In family studies, alloreactivity segregated as a single genetic locus with codominantly expressed products, designated feline lymphocyte defined (Fld)--although closely linked multigene control remains possible. The relationship of Fld to a putative feline major histocompatibility complex is discussed.

Comparative study of proliferative activity of human lymphocytes from different pairs of donors in mixed culturein vitro

The proliferative response of human lymphocytes was studied in one-way and two-way mixed lymphocyte cultures (MLC). The maximal proliferation was shown to be attained in a two-way MLC containing unequal quantities of the cells from two paired donors. It was also demonstrated in a one-way MLC that the cells of one of the two paired donors are more effective responders. The most powerful proliferation in the MLC of these donors was observed in the cultures containing the excess of more effective cells. Thymosine increased the response of human lymphocytes in the two-way MLC. In vitro cell preincubation reduced the response in the cultures with a high proliferative response in the control. It has been thus demonstrated that lymphocytes from paired donors possess different functional activity in the MLC.

Immunomodulating activity of p-hydroxyphenyl-lactic and ascorbic acids

p-Hydroxyphenyl lactic acid (PHA) in a concentration of 5 . 10(-5) M produced a significant inhibition of cell proliferation in response to alloantigens in a one-way mixed lymphocyte culture (MLC) in colonic cancer patients and in blast transformation in response to suboptimal doses of Con A. Multiple administration of ascorbic acid in an optimal concentration to the culture increased the proliferative response of lymphocytes to alloantigens and Con A. PHA and ascorbic acid did not exhibit any immunomodulating action during the use of healthy donors' lymphocytes or lymphocytes from colonic cancer patients, transformed with optimal mitogen doses. PHA did not affect the production of cytotoxic T lymphocytes in the MLC of the spleens of allogeneic mice but inhibited lymphocyte proliferation in response to alloantigens in the MLC of the spleens obtained from B6 and vitamin A deficient animals.

Mixed Lymphocyte Reactivity Nonresponsiveness in Couples with Multiple Spontaneous Abortions

We have endeavored to ascertain the possible existence of deviant recognition of antigens controlled by the major histocompatibility complex by lymphocytes originating in couples with a history of multiple spontaneous abortions of unknown cause. Human leukocyte antigen (HLA) typing, as well as one-way mixed lymphocyte culture (MLC) tests, were performed on 33 couples with multiple spontaneous abortions and subsequently compared to 30 fertile couples with no abortion history. No significant differences in the frequency of HLA were detected between the fertile and infertile groups. The frequencies of shared HLA-A and -B antigens among members of the couples were similar in both groups. In 70% of the cases studied, the husbands revealed a depressed cellular response to their respective wives; whereas reactivity to cells from other women proved normal. However, the response of allogeneic controls to these infertile women was normal. This implies that women with multiple abortions are not poor stimulators. The specific one-way decrease in the MLC reactivity appeared to be mediated by female-derived suppressor mechanisms and may be considered as one of the causes of the interrupted development of pregnancy in vivo.

Comparative study of conditions for demonstrating receptors of memory T cells and other T lymphocyte subpopulations immune to antigens of the H-2 complex

Mice of lines BI0.D2 (H-2d), abbreviated to D2, and C57BL/6 (H-2b), abbreviated to B6, were obtained from the nursery of the All-Union Oncologic Scientific Center, Academy of Medical Sciences of the USSR. During induction of CTL and MC in vitro, D2 mice were immunized by a single intraperitoneal injection of 2.5.107 EL-4 ascites leukemia cells from B6 mice. To induce STS, 9.107 B6 spleen cells irradiated with y-rays in a dose of 1500 rads (laTCs, 740 rads/min), were injected [4]. CTL also were induced in vitro in a one-way mixed lymphocyte culture (MLC), as described previously [6], using B6 lymphocytes irradiated with x-rays as the stimulating cells. Immune spleen cells obtained 10-12 days after immunization in vivo for 5 days after immunization in vitro were used as primary CTL. To obtain secondary CTL, MC induced in vivo 1-3 months before the experiments were stimulated in MLC by B6 lymphocytes, killed by heating for 1 h at 45~ for 4 days [6]. The resulting CTL were washed, counted, and their cytotoxic index (CI) was determined for action on TC (peritoneal macrophages), labeled with =iCr and cultured for 2 days, using a microversion [7] of the test described previously [8]. To determine activity of STS, D2 mouse spleen cells obtained 3-4 days after immunization (normal D2 spleen cells in the control) were treated with mitomycin C, washed, and added to a one-way D2 anti-B6 MLC, in the ratio of 1:1.5 to the reacting cells [4]. Suppressor activity was calculated by means of the index of inhibition (II) of DNA synthesis.

Production of α-lymphotoxin by human T-cell subsets

Human T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed-lymphocyte culture. These sensitized T cells were fractionated on the basis of their possession of Fc receptors for IgG (T G+) or IgM (T M+), or the absence of both IgG and IgM receptors (T G−M−). When restimulated with alloantigen of the same derivation, T G+, T M+, and T G−M− cells yielded almost equal amounts of cytotoxin. Anti-α-lymphotoxin serum neutralized most of this cytotoxic activity indicating that α-lymphotoxin (α-LT) constituted most of this activity. Although T G−M− cells function as effectors in allogeneic cytotoxicity, T G+ cells lyse IgG-coated targets in an antibody-dependent cell-mediated cytotoxic (ADCC) reaction, which has been shown to be mediated in part by α-LT. Whether T M+ cells can be cytotoxic is not clear. In addition, freshly isolated human T-cell subsets were stimulated with phytohemagglutinin-P (PHA-P). After PHA stimulation, T G+, T M+, and T G−M− cells produced similar amounts of soluble cytotoxin, which was largely neutralized by anti-α-LT. The T G+ cells incorporated less thymidine than the T M+ or T G−M− cells. Likewise, OKT4+ and OKT8+ subsets, isolated with the aid of monoclonal OKT8 or OKT4 antibody and complement, yielded lymphotoxin after stimulation with PHA. It is shown that all T-cell subsets, as defined here, can produce lymphotoxin. Furthermore, depending on the assay system, cytotoxicity can be clearly demonstrated in all of these subsets, except in T M+ cells, where positive and negative results have been reported.

Soluble Mlsa antigens: stimulatory effect in vitro versus suppressive effect in vivo.

Using a pair of congenic strains of mice differing only at the Mls haplotype (Mls locus and closely linked genes), BALB/c (Mlsb) and BALB.D2-Mlsa, we have compared the in vitro proliferative responses of Mlsb lymphocytes to Mlsa antigens presented on either lymph node cells (LNC) or peritoneal adherent cells (PAC). Results showed that Mlsa-PAC are stronger stimulators than Mlsa-LNC, and furthermore, that the supernatant from Mlsa-PAC may be effective in eliciting a lymphocyte proliferative response. The proliferation in response to PAC supernatant is partially due to activation by nonspecific factor(s); however, the response in the presence of Mlsa incompatible PAC supernatant is about three times greater than the response obtained in the presence of syngeneic Mlsb-PAC supernatant, suggesting an additional stimulation by soluble Mlsa antigens. Contrasting with the ability of PAC-supernatant to stimulate a primary proliferative response in vitro, the in vivo immunization of Mlsb mice with Mlsa-PAC supernatant abrogates the specific proliferative response in subsequent one-way mixed lymphocyte cultures. This abrogation of the specific response is comparable to that observed after immunization with intact Mlsa peritoneal or spleen cells, although in the latter case the anti-H-2 proliferative response is also decreased, regardless of whether the H-2 incompatible stimulating cells express an additional incompatibility for Mlsa. The proliferation of untreated, but not of Mlsa-immunized BALB/c LNC, is stronger in cultures with DBA/2 stimulating cells (incompatible for Mlsa and other non-H-2 antigens) than in cultures with BALB.D2-Mlsa cells (incompatible for Mlsa alone), and is comparable in intensity to that activated by H-2 incompatibility. We conclude that Mlsa antigens are more efficiently recognized by unprimed helper T cells when presented on PAC than when presented on LNC. In the primary proliferative response, the effects of Mlsa and other non-H-2 antigens may be cumulative. In vivo immunization against Mlsa antigens results in suppression of the specific proliferative response and, to a certain extent, of the nonspecific proliferative response (directed against both H-2 and other non-H-2 antigens). Since Mlsa antigens are obtainable in soluble form, their physico-chemical purification can now be envisaged.

Inhibition of in vitro lymphocyte blastogenesis by chicken alpha-fetoprotein

Chicken alpha-fetoprotein (ch-AFP), purified from fetal chicken serum and embryo extracts, respectively, was examined for its immunomodulatory effect in vitro. Significant ( P < 0.05) suppression of the allogeneic mixed lymphocyte reaction (MLR) was observed, when these preparations were added to one-way mixed lymphocyte cultures (MLC) in quantities of 62.5–1000 μg/ml. Suppression of the MLR was depending on the presence of ch-AFP for at least 16 h after initiation of the MLC, suggesting that this fetal protein was acting mainly in the early phase of lymphoblastogenesis. Serum of chicken embryos (12th and 15th day of incubation), day-old chickens, and of 10-week-old chickens of four different inbred lines were also found to exert suppression of the MLR. From these data, it is hypothesized that ch-AFP plays an immunoregulatory role by maintaining a certain stage of self tolerance during differentiation of the avian immune system.

Habitual abortion: parental sharing of HLA antigens, absence of maternal blocking antibody, and suppression of maternal lymphocytes.

The immunologic responsiveness of eight women who habitually abort has been investigated. All shared an HLA-A or B antigen with their husbands. Sharing of an HLA-DR antigen was found in seven couples, one of which also had a second DR antigen in common. The probability for this high frequency of HLA-DR sharing is negligible (p = 0.0004), as calculated from the antigen frequencies among Europeans. Cells from the woman with two shared DR antigens displayed a minor response to her husband's cells but reacted strongly to control cells, whereas the other women's cells reacted normally to cells from both their husbands and controls in one-way mixed lymphocyte culture (MLC). Only minor cytotoxicity was displayed by women's cells in a direct cell-mediated lympholysis (CML) assay, but they mounted normal cytotoxic responses against both husbands' cells and control cells in an amplified CML assay. The sera from six of the habitually aborting women displayed no blocking activity in one-way MLC, and seven of them had no cytotoxic antibodies. Cells from all habitual aborters were suppressed in two-way MLC by cells from husbands and most controls. We hypothesize that increases in HLA compatibility between mother and fetus and in maternal susceptibility to suppressive influences are in some way linked to a deficiency in the development of antifetal antibody during pregnancy. As a consequence, the fetus may be deprived of the protection by maternal blocking antibody, which may allow maternal cytotoxic reactions to cause abortion.