Two-way Mixed Lymphocyte Culture Research Articles

In Vitro Evaluation of Graft-versus-Graft Alloreactivity as a Tool to Identify the Predominant Cord Blood Unit before Double Cord Blood Transplantation

The transplantation of two cord blood (CB) units obtained from unrelated donors (double CBT) is an effective strategy for adult patients with hematologic malignancies. Sustained hematopoiesis after double CBT is usually derived from a single donor, and only a few transplantation recipients displaying a stable mixed donor-donor chimerism have been reported. We investigated the mechanisms underlying single-donor predominance in double CBT by studying invitro the role of the graft-versus-graft cell-mediated immune effect in two-way mixed-lymphocyte culture, along with the contribution of differential hematopoietic progenitor (HP) potency in HP mixed cultures. Results for the two-way mixed-lymphocyte culture showed that despite the weak and variable alloantigen-specific cytotoxic potential displayed by CB mononuclear cells, an immune-mediated dominance for one of the two CB units was detected in the majority of experiments. Alloantigen-induced cytotoxic activity was directed toward both CB-HP and phytohemagglutinin (PHA)-activated T lymphoblastoid cells. The CB unit with the higher fold expansion of CD34(+) cells in single-expansion culture was prevalent in the HP mixed-expansion culture, as shown by DNA chimerism evaluation. Based on these data, we hypothesize that the dominant CB unit is able to develop prevalent cytotoxic activity toward activated lymphocytes of the other CB unit, thereby preventing them from exerting alloantigen-specific cytotoxic potential against both activated lymphocytes and HPs of the dominant unit. In accordance with this hypothesis, we propose the evaluation ofalloantigen-induced cytotoxic activity generated in two-way mixed-lymphocyte culture and directed toward PHA-activated T lymphoblastoid cells as a tool to identify the potentially predominant CB unit before double CBT.

The Imbalanced Mixed Lymphocyte Reaction Between Maternal and Fetal Lymphocytes As Well As Between the Lymphocytes of Adult Children and Their Parents is Mediated by Activated CD8+ T Cells

The mixed lymphocyte reaction (MLR) between maternal and fetal lymphocytes as well as between adult children and their parents is imbalanced in bidirectional mixed lymphocyte cultures. The present investigation was aimed at examining which type of T cells has primary responsibility for this phenomenon. Two-way mixed lymphocyte cultures between lymphocytes of five female and five male adults and five male and five female newborns against lymphocytes of their parents were performed. Unrelated adults served as the controls. Before and after 72 hr incubation the mixed leukocytes cultures (MLCs) were sorted by flow cytometry according to the surface markers CD4 and CD69 or CD8 and CD69. The single cell populations within the four resulting fractions were discriminated by fluoresence in situ hybridization (FiSH) using xy-DNA probes. Before incubation the share of CD8(+) CD69(+) cells in the MLCs between mothers and their newborn infants was significantly shifted toward the newborn cells (P < 0.01). After 72 hr incubation the CD8(+) CD69(+) ratio in the MLCs between mother and newborn cells and between adult children (male as well female) and their parents showed a significant shift of the CD8(+) CD69(+) cells toward the children's direction. All other MLCs showed a balanced cell population for all investigated cell types, both before and after incubation. The imbalanced MLR between maternal and fetal lymphocytes and lymphocytes of adult children against their parents is mediated by a specific imbalance of the activation of CD8(+) but not of CD4(+) T cells.

The cellular immune response of children is specifically decreased against their parents but not vice versa, independent of pregnancy, age, or HLA or HY antigens.

The fetal immune response against the mother and the father is specifically altered. Up to now it is unclear whether the immune response between adult children and their parents is also specifically altered. Seven female and seven male adults and five male and five female newborns and all their parents were enrolled in the study. Cyotoxicity, apoptosis and proliferation properties of the parents' lymphocytes were measured against those of their children and vice versa using two-way mixed lymphocyte cultures (MLCs). The immune response of adult children against both their parents is reproducibly decreased compared with that of both parents against their children in cytotoxicity, apoptosis and proliferation, independent of sex or age. The cellular immune response of children is decreased against both their parents but not vice versa independent of pregnancy, age, or HLA or HY antigens. This might be acquired during pregnancy or genetically determined.

Differential alloreactivity at SLA- DR and - DQ matching in two-way mixed lymphocyte culture

The major histocompatibility complex ( MHC) class II molecules are heterodimeric cell surface glycoproteins important for antigen presentation to CD4 + T lymphocytes. Class II molecules of the pig MHC, termed SLA, identified so far include DR and DQ. Thus far, functional differences between products of different loci in SLα class II have not been well characterized. For detailed research on this issue, SLα- DRβ1 and - DQβ typings were newly developed by restriction fragment length polymorphism (RFLP) of the reverse transcriptase-polymerase chain reaction (RT-PCR) products. Using this method, several RFLP types were chosen from 13 CSK miniature pigs, and alloreactivities in two-way mixed lymphocyte culture (MLC) derived from these pigs were examined by cell proliferation assay using flow cytometry. The responses in MLC varied according to the degree of phenotype difference. In MLC from individuals of the same RFLP type in both SLA- DRβ1 and - DQβ, the proliferative responses showed slight reaction indicating that they were not so stimulated by each other. On the other hand, for the RFLP type-mismatching combination, the responses were strong indicating that they recognized each others alloantigens. The reactivity of only the DQβ mismatching combination was as strong as those of only the DRβ1 mismatching combination. These data indicate the important role of the DQ as well as DR molecule on alloreactivity in MLC.

Platelet immunoregulatory factors.

A number of soluble and membrane-associated proteins are known to mediate platelet:leukocyte interactions. Platelet-derived factors that have attracted the most attention to date include transforming growth factor beta, interleukin 1 and platelet factor 4. Recently, we have uncovered another protein within platelets that has leukocyte modulatory activity. It was previously characterized as an endometrial glycoprotein named placental protein 14 (PP14) with suppressive effects upon lymphocyte proliferation, pro-inflammatory cytokine production and natural killer cell function. The "hematopoietic" PP14 derived from cells of the megakaryocytic lineage shares this immunosuppressive property, as evaluated by two-way mixed lymphocyte cultures. Interestingly, two alternatively spliced hematopoietic PP14 mRNAs have been cloned which differ in their encoded proteins. Cell-free translation and transfection analyses have verified the translatability of both PP14 mRNA species and allowed for the analysis of their glycosylation properties. PP14, a member of the lipocalin structural superfamily of proteins, now offers an intriguing new link between the coagulation and immune systems.

Inhibition of lymphocyte proliferation by the anti-arthritic drug sinomenine

The effect on lymphocyte proliferation of sinomenine, a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum was investigated in vitro using mouse spleen cells and human peripheral blood mononuclear cells. It could be demonstrated that sinomenine markedly inhibited [ 3H]thymidine incorporation in mouse spleen cells activated with concanavin A (IC 50 = 400 μM) or by two-way mixed lymphocyte culture (IC 50 = 60 μM) and also in human peripheral blood mononuclear cells activated with phytohemagglutinin, 12- O-tetradecanoylphorbol-13-acetate plus ionomycin, or mixed lymphocyte culture (IC 50 ranging from 34 to 129 μM). Time kinetic experiments revealed that sinomenine was effective only when added within the first 48 h after the onset of mixed lymphocyte culture, which lasted for 5 days. Inhibition of lymphocyte proliferation by sinomenine was reversible. Accordingly, the drug showed no direct cytotoxicity in our cellular systems and had no inhibitory effect on the proliferation of the cytokine-independent growth of the human leukaemic T-cell lymphoblast cell line Jurkat. It can be considered that these anti-proliferative effects are part of the anti-inflammatory and anti-arthritic mechanisms of sinomenine obvious in clinical trials.

Cytokine gene expression in the mixed lymphocyte culture in allogeneic bone marrow transplants as a predictive method for transplantation‐related complications

We have investigated cytokine gene expression in the two-way mixed lymphocyte cultures (MLC) enhanced by concanavalin A to assess whether this is a useful predictive method for severe graft-versus-host disease (GVHD) and graft failure in nine allogeneic bone marrow transplantation (allo-BMT) patients. Our present study revealed that increased IL-2, IL-5 and IFN-gamma mRNA expression and IL-2 and IFN-gamma production in the MLC in two cases with severe lethal transplantation-related complications (graft failure and grade III acute GVHD). These findings suggest that increased cytokine mRNA expression and cytokine products in this assay may be predictive of the development of transplantation-related complications.

THE EFFECTS OF TISSUE-ASSOCIATED AND MHC CLASS II ANTIGEN PRESENTATION ON IN VITRO LYMPHOPROLIFERATIVE RESPONSES AGAINST CANINE LIVER AND KIDNEY CELL SUBPOPULATIONS

Purified hepatocytes (LH), Kupffer cells (LKu), and intrahepatic biliary duct cells (LD) were isolated from canine livers, as well as tubular cells from canine kidneys, by enzymatic digestion, gradient centrifugation, and tissue culture techniques. Incubation of LH, LKu, and LD for 48 hr in a two-compartment diffusion chamber opposite two-way mixed lymphocyte cultures, or with canine gamma interferon purified and standardized in our laboratory, resulted in a significant increase in class II expression. This was detected in the cell analyzer with directly fluoresceinated B1F6, a monoclonal antibody (mab) generated in our laboratory vs. a canine class II monomorphic epitope. An amplification of the allogeneic mixed lymphocyte liver cell cultures (MLLC) of at least 2-fold was observed by preinduction of canine class II expression with IFN-gamma on LKu and LD cells, but an autologous reaction could not be elicited. However, an autologous as well as allogeneic lymphoproliferation against kidney tubular cells (MLKC) could be easily observed without IFN-gamma and amplified with IFN-gamma to stimulation indices of at least 3 times that of noninduced cultures. Dependence of the allogeneic MLLC and allogeneic and autologous MLKC on class II gene expression was also evidenced by blocking of 3H-thymidine uptake seen by incubation with 5 micrograms of B1F6. Another mab, I1F6, generated against tubular cells and inhibiting the autologous and allogeneic MLKC, had no blocking effect on lymphoproliferation with any of the liver cell preparations. No such tissue-specific mab (analogous to I1F6) has thus far been found in response to mouse immunization with LH, LKu, or LD. In the absence of accepted defined molecular probes in the dog as yet, we conclude that, in contrast to kidney tubular cells, cells of the normal canine liver do not readily stimulate a primary lymphoproliferative autoimmune reaction in vitro despite class II amplification. Thus autoreactivity (as opposed to alloreactivity) is much less prominent in immune recognition of purified cellular components of nondiseased liver tissue than of kidney tissue in which tissue-associated epitopes are more operative.

Human cord blood suppressor T lymphocytes. II. Characterization of inducer of suppressor cells.

Previously, we reported an antigen nonspecific inducer of T suppressor cell factor (TisF) produced by cord blood mononuclear cells (MNC) in 48-hr, two-way mixed lymphocyte cultures (MLC). The target of this factor was a radiosensitive, T4+ (T8-) adult suppressor T cell subset. The cellular origin of this TisF was examined in the present study. IgG production by pokeweed mitogen (PWM)-stimulated adult MNC was used as an assay for TisF activity. It was found that TisF-producing cells formed rosettes with sheep erythrocytes (E+) and were independent of adherent cells (AC) in the production of TisF. They were resistant to irradiation (2500 rads) and phenotypic characterization with T cell reactive monoclonal antibodies indicated that they resided in the T8- (T4+) population. Furthermore, both TQ1- and TQ1+ cells were required for the production of TisF activity and such activity could not be reconstituted by supernatants from TQ1- MLC and TQ1+ MLC. These results indicate that the production of TisF is dependent upon interactions between radioresistant E+, T8-, TQ1- and radioresistant E+, T8-, TQ1+ cells.

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.

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.

Easily suppressed lymphocytes and absence of cytotoxic antibody in three women with habitual abortion.

This investigation involves 3 women with habitual abortion and 2 women with multiple normal pregnancies. Extensive clinical investigations failed to reveal any cause for the abortions. All husbands were ABO-compatible with their respective wives. The women shared three, two, and zero of five identified HLA-A antigens with their respective husbands. None of the women had any cytotoxic antibody. Lymphocytes from all three women reacted normally in one-way mixed lymphocyte cultures to their husbands' alloantigens. Cell-mediated lympholysis was similar in couples with habitual abortion and in those with repeated normal pregnancies. The women's lymphocytes' proliferation was significantly suppressed by lymphocytes from their husbands and by those from most male controls in two-way mixed lymphocyte culture and double chamber experiments. Two women with 6 and 5 live-born children, respectively, served as controls. Their lymphocytes were not suppressed by lymphocytes from their respective husbands or by those from most male controls in two-way mixed lymphocyte cultures. Their reactions in one-way mixed lymphocyte cultures and in cell-mediated lympholysis, however, did not differ from those of the women with habitual abortion.

Suppressor cell activity after major injury: indirect and direct functional assays.

In vitro responses to streptokinase-streptodornase (SKSD), mumps, and mixed lymphocyte culture (MLC) in 19 burned and 13 multitrauma patients were studied sequentially by lymphocyte tritiated thymidine incorporation and compared to responses of 28 normals. Mean responses to SKSD remained significantly depressed from normal for up to 14 to 28 days following injury: MLC responses, significantly depressed at 48 hours, recovered promptly to normal levels. Because of evidence that the proliferative capacity of the T-cell population to soluble antigens is contained within the inducer subpopulation while both inducer and suppressor subpopulations respond in MLC, the observed increase in MLC responses, coupled with a sustained depression of SKSD and mumps responses, suggests activation of a population of suppressor cells. In a direct assay of suppressor cell function, lymphocytes from three or four multitrauma patients incubated in a two-way MLC with normal lymphocytes significantly suppressed PHA responsiveness, confirming the findings of the direct assay.

Formation of IgE binding factors by human T lymphocytes.

Peripheral blood mononuclear cells from ragweed-sensitive individuals formed soluble factors that inhibited rosette formation of Fc epsilon receptor (+) cells with IgE-coated ox erythrocytes when the cells were incubated with ragweed antigen E and human IgE. Either antigen E alone or IgE alone failed to induce the formation of the rosette inhibiting factors. Two-way mixed lymphocyte culture from 2 normal individuals followed by incubation of the activated lymphocytes with IgE induced the formation of the rosette-inhibiting factor. Normal human mononuclear cells cultured in the presence of T cell growth factor (TCGF) formed the soluble factor when they were incubated with IgE. The rosette-inhibiting factor in culture filtrates was specifically absorbed with IgE-coupled Sepharose and was recovered from the beads by elution at acid pH, indicating that the soluble factors have affinity for IgE. Because the lymphocytes cultured with TCGF were mostly T cells and contained neither B cells nor monocytes, it appears that the IgE-binding factors are derived from T cells.

Modification of the mixed lymphocyte reaction for pharmacokinetic assessment of immunosuppresive activity in human plasma

A modification of the two-way mixed lymphocyte culture (MLC) for the assessment of immunosuppressive activity in human plasma is proposed. Adding 50% of plasma instead of 10% to micro MLC increases the sensitivity and decreases the variability of the system used for pharmacodynamic analysis of prednisoloneand azathioprine combined with prednisolone. Harvesting the cultures on Day 3 augments the area under the inhibition time curve when compared with Day 5 and reduces the variability of the response of the MLCs. The method provides a time dependent measure of immunosuppression which may be correlated with pharmacokinetic parameters.

Demonstration of a dog liver immunosuppressive factor active in vitro.

These studies define the presence of an immunosuppressive factor in dog liver that is active in vitro. A crude dog liver extract was prepared by saline extraction and ultracentrifugation. In vitro immunosuppressive activity of the extract was assayed by its influence on standard phytohemagglutinin mitogen stimulation assays (PHA), two-way mixed lymphocyte culture (MLC) reactions, and primed lymphocyte culture (PLC). In six experiments with four extracts, the mean percentage of inhibition (MPI) of dog lymphocytes in PHA mitogen stimulation assays was 102.0 +/- 3.6. In two human MLC experiments, the extract produced an MPI of 102.3 +/- 0.3. In one canine MLC the MPI was 102.5. In three PLC experiments the MPI was 92.7 +/- 8.0, indicating that the extract inhibited sensitization. Cytotoxicity of the extract was shown not to be the mechanism of proliferation inhibition by restimulation of cells washed free of extract, concurrent eosin viability studies, and documentation of normal base proliferation of cells after extract was washed from them. We concluded that there is a naturally occurring immunosuppressive agent in dog liver that is active in vitro as demonstrated by inhibition of PHA MLC, and PLC cellular proliferation assays. The activity is not attributable to cytotoxicity.

Mitogen-Stimulated Lymphoid Cells from Human Newborns Suppress the Proliferation of Maternal Lymphocytes across a Cell-Impermeable Membrane

Abstract The immunologic consequences of the interaction between lymphocytes from human newborns and their mothers were investigated in vitro. Maternal and male baby lymphocytes were cocultured in the presence or absence of PHA, and the resulting cellular proliferation was assessed by a) the fluorescent chromosome technique with the Y chromosome as a marker for dividing male baby cells, and by b) measuring the incorporation of 3H-thymidine in one- and two-way mixed lymphocyte cultures (MLC). In confirmation of previous reports, we found that approximately 90 to 100% of the dividing cells in two-way MLC were of baby origin, indicating that lymphocytes from human newborns effectively inhibit the proliferative capacity of maternal lymphocytes in 6-day MLC. The simultaneous measurement of 3H-thymidine uptake in the co-cultures revealed that the maternal cells with an inhibited proliferative capacity did cause a strong allogeneic stimulation in the newborn cell population. The suppressor activity associated with newborn lymphocytes was completely abrogated by irradiation with 6000 rads, showing a requirement for cell division in the in vitro development of the anti-proliferative effect on maternal cells. In contrast to the suppressive effect mediated by actively dividing newborn lymphocytes, irradiated cells from newborns exerted a strong stimulating effect on maternal lymphocyte proliferation in one-way MLC. Experiments using a double chamber culture system showed that mitogen-stimulated cells from newborns could effectively suppress the proliferation of lymphocytes from recently delivered mothers and nonpregnant adult females across a cell-impermeable membrane. PHA-stimulated lymphocytes from nonrelated adults did not, however, inhibit the division of maternal lymphocytes or lymphocytes from another nonpregnant woman when physically separated in double chamber cultures. Furthermore, mitogen-stimulated cells from one newborn failed to inhibit the proliferation of cells from another newborn. These findings suggest that proliferating newborn cells inhibit maternal lymphocyte proliferation by releasing a low m.w.-soluble suppressive factor(s). The characterization of the inhibitory substance released from mitogen- or alloantigen-activated human newborn suppressor cells is in progress.

Role of MLC compatibility in intrafamilial kidney transplantation.

The role of compatibility at the HL-A and mixed lymphocyte culture (MLC) loci for graft survival was analysed in 45 recipients of intrafamilial kidneys. MLC tests performed after transplantation when the recipients were on maintenance immunosuppressive therapy did not show a reduced reactivity of the recipient lymphocytes as compared to tests performed before surgery. The results with the two-way MLC test was paradoxical: patients with functioning grafts had a higher mean stimulation than did those with nonfunctioning grafts. Subsequent clinical correlations were based on one-way MLC carried out before or after transplantation. The 1-year survival of grafts from HL-A compatible donors was 94% and that of graft from HL-A incompatible donors was 75%. When the comparison was between grafts from MLC-negative and MLC-positive donors the figures were 100 and 75%, respectively. If the cases were divided in those displaying a low relative reactivity (RR), indicating identity at the major (LD-1, HL A-D) MLC locus, towards their donors in MLC and those with a high RR, the graft survival was 100% versus 70% (P less than 0.05). The prognosis seemed to be worse the higher the relative reactivity. At 3 years all grafts from donors with negative reaction or low RR in the MLC were still functioning. Analysis of the few exceptional cases in which there was compatibility for either the HL-A or MLC locus but not for the other points to the major MLC locus as being most important for graft survival.

The influence of glutamine, its decomposition products, and glutaminase on the transformation of human and mouse lymphocytes

The extent of blast transformation for human and BALB/c mouse lymphocytes has been examined over a wide range of glutamine concentrations with several agents which initiate blastogenesis. Maximum [ 3H]thymidine incorporation was seen at 0.5 mM glutamine for lymphoid tissues stimulated in the following manner: human and BALB/c splenic peripheral blood lymphocytes with phytohemagglutinin, BALB/c splenic lymphocytes with lipopolysaccharide, and BALB/c vs C3H/HeJ two-way mixed lymphocyte cultures. The inhibition of blastogenesis exerted by glutamine concentrations greater than 0.5 mM could not be reversed by washing and reculturing the cells at 0.5 mM glutamine. To elucidate the reason for inhibition by higher glutamine concentrations, the products of spontaneous glutamine decomposition, L-2-pyrrolidone-5-carboxylic acid and ammonia were tested for their in vitro influence on BALB/c splenocyte blastogenesis. Pyrrolidone-carboxylic acid, in concentrations up to 5 mM, was without effect. In contrast, ammonia concentrations exceeding 1 mM became increasingly more inhibitory. The genesis of inhibitory levels of ammonia in culture medium was confirmed and has been considered as primarily responsible for inhibition by high glutamine. Addition of Escherichia coli glutaminase (pH optimum 4.9) to cultures of BALB/c splenocytes or human peripheral blood lymphocytes had no effect on either the extent of blastogenesis of these tissues or the glutamine levels in their culture medium.

Differential production of interferon and lymphotoxin by human tonsil lymphocytes

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [ 3H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [ 3H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.