Mixed Leukocyte Culture Reaction Research Articles

Ontogeny of Cell-Mediated Immunity of Murine Thymocytes and Spleen Cells: In vitro Mixed Leukocyte Culture and Cell-Mediated Lympholysis Reactions

The ontogeny of cell-mediated immunity of spleen cells and thymocytes from B10 mice was studied in both in vitro mixed leukocyte culture and cell-mediated lympholysis reactions. Results show that newborn spleens contain cells competent to respond to X-irradiated allogeneic spleen cells in mixed leukocyte culture. The mixed leukocyte culture response of spleen cells, in terms of both index of stimulation and increment of tritiated thymidine incorporation, seems to be higher for mice four weeks or older than for mice less than four weeks old. The cell-mediated lympholysis response of spleen cells is not detectable until two days postpartum. It reaches adult levels in terms of % cytolysis by day four after birth. Thus, the transition period of the ontogeny of cell-mediated lympholysis response of spleen cells is apparently 0–4 days of age. Newborn and early postnatal thymocytes (0–7 days of age) respond in mixed leukocyte culture at least as strongly as adult thymocytes (2–3 months of age). The cell-mediated lympholysis response of thymocytes is already detectable at birth, but weaker in terms of % cytolysis when compared with the cell-mediated lympholysis response of thymocytes from two days to six weeks of age. The cell-mediated lympholysis response of thymocytes starts to decline at 6–8 weeks of age. Thus, around the time of birth, there is a transition period in the cell-mediated lympholysis response of thymocytes during which thymocytes start to show high cell-mediated lympholysis reactivity. There is a second transition period between six and eight weeks of age during which the cell-mediated lympholysis response of thymocytes diminishes. The early, as well as late, postnatal cell-mediated lympholysis response of both spleen cells and thymocytes seems to be specific in nature.

Differential effects of polyadenylic: polyuridylic acid and lipopolysaccharide on the generation of cytotoxic T lymphocytes.

In a mixed leukocyte culture (MLC) reaction of allogenic mouse spleen cells differing for H-2K or H-2D, only a weak cytotoxic response is generated. This cytotoxic response is augmented significantly if bacterial lipopolysaccharide (LPS), 5 microgram/ml, or polyadenylic acid (poly A):polyuridylic acid (poly U), 20 microgram/ml, is present in the culture. The cytotoxic cells generated in the presence of these two agents are specific for sensitizing H-2K or H-2D antigen. Two lines of evidence suggest that these two agents exert their effect at different steps in the development of cytotoxic lymphocytes: (a) the effect of poly A:U depends on the presence of adherent cells, whereas the effect of LPS is independent of the presence of adherent cells and (b) LPS promotes the development of cytotoxic cells when ultraviolet light-treated stimulating cells are used in the MLC whereas poly A:U does not.

Genetic control of allogeneic interactions in the guinea pig. I. Evidence from mixed leukocyte culture and normal lymphocyte transfer reactions.

AbstractThe role of gene products of the B region of the guinea pig major hist ocompatibility complex (MHC) in the production of mixed leukocyte culture mitogenic factor (MLC‐MF) has been investigated. Lymphoid cells of partially inbred and outbred guinea pigs which bear the serologically defined B.2 alloantigen, respond poorly or not at all in the guinea pig MLC. Since MLC culture supernatants from B.2+ cells do not stimulate B.2− third party lymphoid cells, it is possible that either B.2+ lymphoid cells do not elaborate MLC‐MF or they produce a suppressor factor. Genetic studies have demonstrated that genes closely linked to or identical with the gene(s) coding for the B.2 alloantigen, regulate “MLC unresponsiveness”. By contrast to their unresponsiveness in the MLC, B.2+ lymphoid cells produce migration inhibition factor at least as well as cells bearing the B.1 and B.3 specificities, and they respond in vitro to antigens which are under the control of specific immune response genes.The genetic restrictions for responsiveness in the MLC are supported by in vivo studies which strongly suggest that the degree of interaction between allogeneic lymphoid cells (normal lymphocyte transfer, immune lymphocyte transfer and direct reactions) are also under the control of genes which map in or near the B region of the guinea pig MHC. Preliminary evidence suggests that the B.2+ donor cells rather than B.2+ host cells are responsible for initiating the unresponsiveness associated with the normal lymphocyte transfer reaction. These studies should provide a basis for the reproducible production of MLC‐MF and a system for the study of some molecular aspects of allogeneic interactions in vitro and in vivo.

864 SUPPRESSOR T CELL FUNCTION IN DIABETES MELLITUS

Autoaggression against pancreatic islet cells has been described in patients with diabetes mellitus. Based on the hypothesis that a loss of immune regulatory suppressor T cells could be important in the development of these autoaggressive reactions, we examined suppressor T cell function in 14 children with insulin-dependent diabetes mellitus. Suppressor cell activity was determined by assessing the effect of concanavalin A-treated lymphocytes on the one-way mixed leukocyte culture reaction. Suppressor cell activity was demonstrated in 15 of 15 normal controls. However, 11 of 14 patients with insulin-dependent diabetes mellitus lacked suppressor T cell function. In our sample, age, sex, duration of diabetes mellitus and control of the disease did not correlate with the presence or absence of suppressor cell activity. Our data suggest that decreased suppressor T cell function may be important in the pathogenesis of some forms of diabetes mellitus. The relationship of HLA (HLA-A,B,C,D) and suppressor T cell function in diabetes mellitus is being investigated.

Regulation of the mixed leukocyte culture reaction by suppressor cells.

Regulation of the mixed leukocyte culture (MLC) reaction by suppressor cells raised and tested in MLC has been evaluated. The experiments described suggest that suppressors are distinct from cytotoxic T lymphocytes (CTL), both in terms of conditions optimal for their activation, as well as through demonstration of efficient suppression in the total absence of cytotoxic activity. Suppressor cells inhibit the activation of the precursors of CTL, and may also prevent the production of the lymphokine blastogenic factor; they do not appear to interfere with the reaction of primed cells. It is suggested that suppressor cells may regulate the MLC reaction by preventing recruitment of fresh competent cells, and by limiting the duration of the reaction of cells already activated.

Blastogenic factor: its role in the mixed leukocyte culture reaction.

The MLC-CML reaction depends on cooperation between different subsets of T lymphocytes in which soluble mediators may play a critical role. Evidence is presented that cell-free supernatants from mixed cultures, containing blastogenic factor (BF) activity can, together with solubilized membrane preparations, induce a primary cell-mediated lympholysis (CML) reaction of the same order of magnitude as intact allogeneic stimulating cells. Furthermore, BF alone can induce secondary mixed leukocyte culture (MLC) and CML reactions very efficiently. The results described suggest that BF is produced in response to activation by LD determinants, probably by helper T cells, and that the target of BF is the cytotoxic lymphocyte. It would appear that regulation of CML activity may be mediated by BF, and exhaustion of the mediator may be responsible for the decline of cell proliferation and effector function in the MLC reaction.

Macrophage proteases and rheumatic diseases: Regulation of plasminogen activator by thymus-derived lymphocytes

Macrophages in culture secrete a variety of products including neutral protease activities such as plasminogen activator(s) (P.A.), collagenase and elastase. These products are not made by unstimulated macrophages, but only after induction by inflammatory stimuli, phagocytosis and lymphokines. Phagocytosis induces the prompt release of high levels of P.A. by endotoxin-primed macrophages and prolonged secretion follows uptake of non-degradable particles. Stimulation of lymphocytes results in the release of a supernatant product which enhances P.A. secretion by unstimulated mouse macrophages up to 5-fold. The production of the P.A. inducer (P.A.I.) is immunologically specific and is found in allogeneic mixed leukocyte culture (MLC) reactions, but not in syngeneic controls. The P.A. is also induced in activated macrophages from animals infected with BCG of T. cruzi and challenged with specific antigen. Production of the P.A.I. in MLC reactions depends on the presence of thymus-derived (T) lymphocytes and is closely correlated with the appearance of macrophage migration inhibition factor (MIF). The induction of macrophage P.A. and other proteases provides an important pathway for activating macrophages in delayed hypersensitivity reactions and could contribute significantly to tissue destruction in chronic inflammatory diseases in joints.

Characterization of functionally distinct lymphoid and myeloid cells from human blood and bone marrow. II. Separation by velocity sedimentation

Velocity sedimentation in a zonal rotor using gradients of uniform osmolarity was used to separate leukocyte subpopulations from human blood and bone marrow. The separations were performed at high sedimentation rates having the advantage of rapidity over conventional unit gravity separations. Myeloid stem cells (CFU-C) and cells reactive with phytohemagglutinin (PHA), Concanavalin A (Con A), and in mixed leukocyte culture (MLC) were separated and their sedimentation profiles obtained. CFU-C sedimented ahead of lymphoid cells and behind mature myeloid elements. Two distinct marrow subpopulations separated by velocity sedimentation were consistently stimulated by Con A, and variably stimulated by PHA and MLC reactions. Both large cells (predominantly myeloid) and small cells (predominantly lymphoid) from bone marrow were stimulated by Con A in [ 3H]thymidine incorporation assays. When separated subpopulations showing stimulation by Con A were mixed, inhibition of [ 3H]thymidine incorporation resulted.

Significance of mixed leukocyte culture testing in cadaver kidney transplantation.

A significant correlation of kidney transplant outcome with the strength of mixed leukocyte culture stimulation was found in a series of 131 cadaver donor transplants. Graft survival rates in patients whose cultures with donor cells resulted in less than 20,000 cpm were statistically significantly better than those in patients with greater than 20,000 cpm (at 6 months and 1 year P less than 0.001). Cultures of recipient with normal control lymphocytes also correlated with clinical outcome, albeit to a somewhat lesser degree. Bidirectional cultures gave a slightly better correlation than unidirectional cultures. Whether the patient's plasma contained factors that blocked the mixed leukocyte culture reaction or not was not found to have a significant effect on transplant outcome.

Genetic and cellular aspects of xenogeneic mixed leukocyte culture reaction.

The nature of the antigens stimulating xenogeneic lymphocytes was studied using "primed LD typing". Human lymphocytes were sensitized in vitro against mouse spleen cells and restimulated with spleen cells of mouse strains sharing non-H-2 antigens or various regions of H-2 with the initial stimulating strain. The largest thymidine uptake was caused by restimulation with cells from the specific primary stimulator or an H-2-identical strain. Species-specific antigens or strain-specific antigens carried in the C57BL/10 background account for less than 15% of the total stimulation; a non-H-2 antigen associated with the Mlsalpha genotype caused moderate restimulation, amounting to 25% of the average H-2 response. Within H-2, the strongest restimulation was caused by antigens controlled by the I-A subregion; the K and D regions caused moderate, the I-C and S regions very weak, and the I-B subregion no restimulation. Thus, the genetic control of antigens stimulating xenogeneic and allogeneic MLC responses requires T cells and adherent cells, but in the human-mouse MLC, both cell types must come from the human responder; the majority of the proliferating cells are T cells. It is suggested that allograft and xenograft reactions are fundamentally identical processes, and that the relative vigor of alloaggression may be explained by secondary potentiating mechanisms depending on species-specific interactions between aggressor and target cells.

Circadian rhythm of stimulated lymphocyte blastogenesis: A 24 hour cycle in the mixed leukocyte culture reaction and with SKSD stimulation

The in vitro measurement of stimulated T lymphocyte blastogenesis by the incorporation of 14C-thymidine can be shown to be an acceptable and precise measurement with highly significant variations in blastogenesis over a 24 hr period. This circadian rhythm for human T lymphocyte blastogenesis has been demonstrated with specific antigen (SKSD) and in mixed leukocyte culture (MLC). The hours of peak reactivity were different for the SKSD stimulation and the MLC reaction. This biologic cycle in T cell reactivity has significant implications for the immunologic response.

Suppression of B-cell differentiation by leukocytes from hypogammaglobulinemic patients.

Leukocytes from patients with several forms of immunodeficiency characterized by apparently differing defects in B-lymphocyte maturation produced few or no plasmacytoid cells in vitro, and were capable of suppressing the generation of plasma cells in co-culture with cells of normal persons, in the presence of pokeweed mitogen. Such inhibition was commonly observed in cultures which included cells from patients with primary immunoglobulin deficiency, but was not seen to a significant degree in identical co-cultures of cells from normals. The suppression observed was not dependent upon mixed leukocyte culture reactivity. Both sheep erythrocyte-rosetting lymphocytes and adherent cells appeared to participate in these effects in some patients. In one patient with common variable immunodeficiency, but not in several others, removal of suppressing cells permitted the patient's remaining cells to differentiate into plasma cells in vitro. Because of the diverse syndromes in which suppression was observed, it is likely that, in at least some hypogammaglobulinemic patients, the suppression is secondary to the disease process rather than being the primary pathogenic mechanism.

Beta2-microglobulin on the cell surface. Specificity of inhibition of the mixed leukocyte reaction and mitogenic properties of antibodies against the two HLA antigen polypeptide chains.

Intact antibodies, and F(ab')2, and Fab' fragments thereof directed against beta2-microglobulin (beta2m) and the HLA alloantigenic polypeptide chain impede the mixed leukocyte culture reaction (MLR). The Fab' fragments are effective only when bound to the responder cell population. The kinetics of their inhibiting action suggest that they interfere with the recognition phase of the MLR. Thus, it seems possible that beta2m may be part of or closely associated with those structures on the responding cells which recognize the stimulatory alloantigens. Antibodies against beta2m or the other HLA antigen polypeptide chain are mitogenic for peripheral blood lymphocytes. The antibodies against beta2m caused greater stimulation than those against the HLA alloantigenic polypeptide chain. Only bivalent antibodies against beta2m were mitogenic and promoted blast transformation of 1 to 2% of the lymphocytes. Although only B lymphocytes were triggered, the mitogenic response of cultures containing various proportions of B and T cells suggests that cooperation of the two cell types is necessary to obtain optimal mitogenic stimulation.

Hodgkin's disease. An immunodepleting and immunosuppressive disorder.

Irradiated leukocytes or mononuclear leukocytes, from 16 out of 30 patients with Hodgkin's disease and from one patient with the Sézary syndrome, stimulated in culture subnormal (3H)thymidine incorporation by allogeneic lymphocytes from normal individuals. This abnormality was not demonstrated in any of 30 other patients with non-Hodgkin's lymphomas. Subnormal mixed leukocyte culture reaction activation was caused by suppression of the mixed leukocyte reaction by patients' cells. Inhibition of the reaction by patient mononuclear leukocytes was corrected when adherent cells were removed or when protein synthesis was inhibited with cycloheximide. The inhibitory cells were probably lymphocytes since selective removal of phagocytic cells did not remove the inhibition by other patient mononuclear leukocytes. The presence in culture of as few as 2,500 granulocytes per mm3 also reduced responses when target cells were from patients with Hodgkin's disease. Patient cells no longer suppressed the mixed leukocyte reaction after patients entered clinical remission which suggests that suppression is a reversible, disease-related abnormality. Thus, the immune deficiency with advance Hodgkin's disease caused by ly lymphocyte depletion may be compounded by a relative excess of suppressor lymphocytes. The overall immunodeficiency may be further compounded by suppression of immune response by granulocytes at even physiologic concentrations.

Specific lymphocyte-activating determinants expressed on mouse macrophages

WHEN two populations of allogeneic lymphoid cells are cultured together, cellular interactions lead to blast transformation and proliferation of a portion of the cultured cells. In mice, this mixed leukocyte culture reaction (MLR) is under the independent control of two genetic systems: the multigenic major (H-2) histocompatibility system (MHS)1,2 and the unigenic (non-H-2) M locus3. The nature and cellular distribution of the gene products which mediate recognition of one cell (the stimulator cell) by another cell (the responder cell) are incompletely defined. The ‘recognising’ structure is likely to be a specific receptor present on thymus-derived (T) lymphocytes, since T cells are generally required as responder cells in MLR3–6, Recently it has been possible to distinguish, from these recognition sites, gene products which are expressed on the stimulator cells7 and which have been termed lymphocyte-activating determinants (LADs)3. Cell fractionation studies revealed that LADs of the MHS are expressed on both B and T lymphocytes8,9, whereas LADs of the M locus are preferentially if not exclusively expressed on B lymphocytes10. In this study we present evidence that LADs of the M locus as well as LADs of the MHS are also expressed on macrophages. These genetic determinants are shown to be stable in long term cultures and to induce specific one-way reactions in mixed lymphocyte–macrophage cultures.

Characterization of a serum inhibitor of MLC reactions: III. Specificity: HL-A-related antibodies

A mixed leukocyte culture (MLC)-inhibitory serum from a healthy multipara, JH, has been characterized with regard to the specificity of its inhibitory antibody. When added directly to MLC, JH serum will inhibit most combinations. However, when lymphocytes intended as responder or stimulator cells in MLC were preincubated with this serum, the specificity narrowed considerably. Four groups of lymphocyte donors were recognized, depending upon whether their lymphocytes were inhibited as responders, as stimulators, as both, or as neither. Absorptions of inhibitory activity, followed by assay of the absorbed sera in pretested MLC combinations, yielded reliable data for determining which donors' cells shared pertinent antigens. An association of MLC inhibition by JH serum with the HL-A types of the involved lymphocytes was observed and these relationships are summarized in Table 4. The three HL-A specificities identified, W19, W29, and 12, correspond with the HL-A typing of the husband of the serum donor. Various cell types absorbed relevant inhibitory activity (against responder and stimulator functions) in the following order of efficiency: LCL cells, B lymphocytes, T lymphocytes, fibroblasts, and erythrocytes. When the above three HL-A specificities were removed by absorption, the serum was no longer inhibitory in any combinations. Whether the inhibitory activity of JH serum is directly related to anti-HL-A antibodies or to antibodies against closely related MLR determinants will depend to a large extent upon the degree of linkage disequilibria found between W19, W29, and 12 antigens and the MLR locus.

Mixed Leukocyte Culture Reactivity in Operationally Tolerant Rats: Relationship of Lymphocyte-Mediated Reactivity and Serum-Blocking Activity

Tolerance to Brown Norway (BN) allografts was induced in Wistar Furth rats by neonatal inoculation of BN bone marrow cells or mixtures of (W/Fu x BN)F-1 hybrid spleen and bone marrow cells. Lymphoid cells from rats in which operational tolerance had been induced (maintenance of BN skin graft for more than 100 days) were studied for mixed leukocyte culture (MLC) reactivity. Peripheral blood lymphocytes from 10 of 25 W/Fu rats operationally tolerant to BN skin grafts showed MLC reactivity when exposed to BN antigens in vitro, implying that some degree of MLC reactivity is compatible with prolonged skin graft survival. These same rats also showed cytotoxic activity to BN fibroblasts in vitro regardless of their MLC status. MLC is quantitatively decreased and possibly qualitatively altered as compared to that of control W/Fu lymphocytes. The decrease was specific for the tolerated antigens. Serum from the tolerant rats inhibited cytotoxic but not normal MLC reactivity of W/Fu cells against BN antigens.

Differential recognition by thymocytes of H-2 and non-H-2 alloantigens

IN this report we examine the behaviour of mouse thymocytes in the mixed lymphocyte culture. The thymus is believed to contain large numbers of T cell precursors or immature T cells undergoing differentiation before migrating to the peripheral lymphoid areas as mature, immunocompetent cells. Our results suggest that the thymus contains a class of cell possessing the potential to react against products of H-2 and non-H-2 loci, but that mixed leukocyte culture reactivity against H-2 depends on a factor(s) present in normal mouse serum.

Macrophages in the mixed leukocyte culture reaction (MLC)

Two aspects of the mixed leukocyte culture reaction have been investigated. In the first study the capacity of two highly purified adherent cell populations, one consisting of neutrophils the other of macrophages, to support the reactivtiy of purified lymphocytes was compared. Activity was entirely confined to the latter preparation. In the second study, the ability of platelets, neutrophils, lymphocytes and macrophages to provoke a MLC reaction was studied. Macrophages were found to be 10 times as efficient in eliciting a reaction as an equal number of lymphocytes. Neutrophils and platelets appeared to be devoid of stimulating capacity. This lack of stimulation was shown to be due to a true deficiency in stimulating capacity rather than to an inhibition obtained by high concentrations of neutrophils and platelets. The two functions of the macrophage in MLC, its auxiliary helper role, and the elicitation of the reaction, may be dependent on one and the same mechanism.

Functional characteristics of T cell receptors during sensitization against histocompatibility antigens in vitro

Using the mixed leukocyte culture (MLC) reaction as a model system for the generation of cytotoxic effector lymphocytes, we have examined changes which occur in recognition-binding function during T cell sensitization. Properties of recognition-binding units on normal and immune T lymphocytes were assessed through the ability of T cells to bind to specific cellular immunoadsorbants at various stages of in vitro sensitization. While antigen-specific recognition-binding function was readily detected on fully cytotoxic effector cells, we were unable to detect functionally specific binding of unsensitized lymphocytes to cellular immunoadsorbants. The ability of cells undergoing sensitization in MLC to bind specifically to target cell monolayers appeared congruent in time with cytotoxic function. These results suggest that a fundamental membrane-associated change occurring during T cell sensitization may be the development of a strong and specific target-cell binding function.