Functional characteristic of TTK-lymphocytes in vitro, cytotoxicity and mixed lymphocyte culture (MLC)

A simple method of cryopreservation (TTK) of lymphocytes is presented. The functional properties of TTK-lymphocytes are examined by the lymphocyte toxicity micro test and in the mixed lymphocyte culture (MLC). 51Cr-release technique is performed as a measure for reversible and irreversible cell damages in the course of the Cryopreservation process.     

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.     

Analysis of mixed leucocyte culture (MLC) reactive cells after in vitro priming. Changes in avidity of T cell receptors.

Mixed leucocyte cultures were examined for populations of T cells responding to secondary stimulation with the priming antigen. Two such populations are described, one of which is stimulated optimally by low, the other by high doses of antigen. Both cell populations are sensitive to anti brain θ serum and complement, but are physically separable by size and by adherence on macrophage monolayers. The anti-brain θ-sensitive population stimulated by low antigen doses consists of larger cells with immunoglobulin-moieties on their surfaces.     

Serological typing of HLA-D: Predictive value in mixed lymphocyte cultures (MLC)

Locally produced antisera and antisera received through the Seventh International Histocompatibility Workshop exchange were investigated for specific B-cell cytotoxic activity in a panel of 95 unrelated HLA-D-typed donors. A number of sera formed clusters defining eight B-cell specificities which were strongly associated (p<0.001) to the HLA-D determinants Dw1–8. In panel investigations, only four triplets occurred. In five HLA recombinant families, these B-cell specificities followed the HLA-B-D chromosomal region, and in one —B/D recombination, DRw1 traveled with —Dw1. In MLCs between panel donors sharing zero, one, or two HLA-D-related B-cell specificities, significantly weaker MLC stimulation was observed with increasing compatibility, the median relative responses being 100, 52, and 17 percent, respectively. It is concluded that B cell-specificities HLA-DRw1–7 and WIA8 are probably coded for by HLA-D; they are excellent markers for the HLA-D determinants, which can thus be typed for by serological means; and serological typing for HLA-D has great value in predicting the outcome of MLCs.     

Detection of lymphotoxin produced in mixed lymphocyte cultures (MLC): variation in target cell sensitivity

In an attempt to resolve some of the uncertainty as to whether soluble cytotoxin—lymphotoxin (LT)—is produced in mixed leukocyte cultures (MLC), we established one-way MLC between the lymphocytes of normal individuals and two lines of lymphoblastoid cell lines (LCL). Cell-free culture fluid harvested after 5 days was tested for LT employing not only the stimulating LCL cells as targets, but also two lines of cells known to be sensitive to LT. The LCL cells—RPMI 8866 and NHDL-2—were found to be completely resistant to LT under the conditions of our assay. By contrast, when the sensitive cells were used in the cytotoxicity assay, LT was readily detected. These results emphasize the importance of intrinsic target cell sensitivity in the study of lymphocyte-mediated cytotoxic phenomena and raise questions as to the mechanism whereby cells may be resistant to LT.     

E rosette forming lymphoblasts fail to stimulate allogeneic cells in mixed leukocyte culture (MLC)

Summary Lymphoblasts from patients with acute lymphatic leukemia were examined for the presence of surface markers and for their capacity to stimulate allogeneic donors in MLC. Lymphoblasts from eight patients, which made E rosettes, consistently failed to stimulate allogeneic donors on at least three separate occasions despite the vigorous response of these same allogeneic donors to remission cells from the patients.There were eleven patients who had lymphoblasts with no detectable markers or null lymphoblasts. Three of these also failed to stimulate in MLC. The null lymphoblasts from the remaining eight patients produced vigorous allogeneic responses. Since serologic data is now available suggesting that null lymphoblasts from some ALL patients have serologically detectable T cell antigens [16] while others have antigens found predominantly on B cells [20], it is conceivable that the capacity of these cells to stimulate in MLC may distinguish lymphoblasts within the null category with those which fail to stimulate representing early T cell precursors and those which do stimulate being early B cell precursors.     

The human mixed-lymphocyte culture (MLC) interaction after in vivo allo-immunization. II. HL-A specificity of early proliferative response

Human volunteers were immunized with a single intradermal dose of allogeneic buffycoat cells. When donor and recipient differed for four HL-A antigens belonging to the first and second series, an increased MLC proliferation early in the cultures was observed when responding lymphocytes from the immunized individual were confronted with stimulating cells from his donor. These findings were not observed when the incompatibility between donor and recipient involved only one second series HL-A antigen. The specificity of the altered response was studied by confronting responding lymphocytes from the immunized individual with different third-party stimulating cells. Most of these combinations revealed an early increased proliferation compared to control combinations with responding lymphocytes from nonsensitized individuals. However, the early increased responsiveness was significantly more pronounced in combinations where the stimulating cells shared two or more “private” HL-A determinants with the immunizing donor. It is concluded that a major part of the early increased responsiveness is due to proliferation of lymphocytes with receptors for HL-A (SD) determinants.     

Generation of cytotoxic lymphocytes in the autologous mixed lymphocyte culture.

We have studied the ability of purified B lymphocytes to generate cytotoxic T lymphocytes in autologous mixed leukocyte cultures (MLC). Cytotoxic lymphocytes were produced but only autologous mononuclear cells stimulated by lipopolysaccharide (LPS) were susceptible target cells. Unstimulated mononuclear cells and purified B cells were not susceptible to killing by cytotoxic cells generated in the autologous MLC. This suggests that the target antigen may be expressed on stimulated or dividing B lymphocytes in a way that renders the cells more susceptible to cytolysis. Autologously stimulated cytotoxic effector cells were found to exhibit specificity. Cy totoxicity for autologous LPS-stimulated target cells occurred but not for an allogeneic, B cell, histiocytic lymphoma cell line. It is postulated that cytotoxic T cells generated in the autologous MLC may play a role in immune surveillance or in regulation of the immune system.     

Inhibition of the mixed lymphocyte culture by peritoneal exudate cells.

Inhibition of mixed lymphocyte cultures (MLC) by macrophages and by supernatants of short term cultured macrophages was assessed by incorporation of ³H-thymidine (TdRH3) and also by blast cell counts and by determination of cellmediated lympholysis. Peritoneal exudate cells (PEC) induced by thioglycollate, at concentrations >10%, inhibited all three parameters of MLC. Lower concentrations of PEC, and supernatants from cultured PEC, inhibited TdRH3 incorporation, but had no significant effect on blast cell counts or on generation of cytotoxic effector cells. Inhibition by the supernatants could be reversed by dialysis or by use of low specific activity TdRH3. These data indicate that macrophages can inhibit proliferative responses in MLC, but that this must be carefully distinguished from selective inhibition of TdRH3 incorporation.     

Induction of suppressor cells in autologous mixed lymphocyte culture (AMLC) in humans

T cells stimulated for 6-7 days in autologous mixed lymphocyte culture (AMLC) showed suppressive effects when added to fresh mixed cultures where autologous lymphocytes (A) were stimulated by Mitomycin C-treated allogeneic lymphocytes (Xm), in a ratio of A:Xm:AMLC-activated cells of 1:1:0.5. Both cytotoxic and proliferative activities in second cultures, as assayed after 6 days of incubation, were significantly inhibited (percentage suppression of cytotoxic activity observed in 17 experiments was 75.3 +/- 22.4; percentage suppression of proliferation was 60.6 +/- 18.2). Suppressor cells (SC) generated in AMLC were Mitomycin C sensitive and nonspecific in their action; not only A/Xm but also X/Am and X/Ym cultures were suppressed to the same extent. AMLC-Activated cells showed a considerable degree of proliferation in response to alloantigens but failed to express any cytotoxic activity against autologous or allogeneic phytohemagglutinin blasts. Thus, the inhibitory effect observed in this system is not due to cytotoxic elimination of responding or stimulating cells in the second culture but rather reflects a true regulatory (suppressive) mechanism.     

Mastocytoma-medicated suppression of mixed lymphocyte culture and mitogen responsiveness.

Murine spleen cells, stimulated in vitro by allogeneic spleen, display a strong proliferative response with the subsequent development of cytotoxic cells. This proliferation and sensitization can be abrogated by the addition of mitomycin-treated or X-irradiated murine DBA/2 mastocytoma cells (P-815). The substance required for this depression of lymphocyte responsiveness is present in the cell-free supernatant fluids of P-815 cultures. The suppression appears to be due to interference with cell proliferation in the mixed lymphocyte culture, because the P-815 also prevents spleen cells from proliferating in response to the mitogens concanavalin A (Con A), lipopolysaccharide (LPS), and phytohemagglutinin (PHA). The significance of these findings is discussed.     

The mixed lymphocyte reaction (MLR) stimulatory capacity of human lymphocyte subpopulations.

Using various cell separation techniques and combinations of these, suspensions were obtained highly enriched or depleted with respect to their content of E-rosette-forming T cells, Ig-bearing B cells, Fc-receptor-bearing cells, or monocytes. These purified populations were tested for their capacity to stimulate allogeneic cells in a mixed lymphocyte reaction (MLR). It could be demonstrated that the Ig-bearing B cells provide the strongest stimulus in the MLR.     

Inhibitory effect of ethylenediaminetetraacetate (EDTA) on the porcine lymphocyte response to mitogens and reversal of the effect with metal ions

The effect of ethylenediaminetetraacetate (EDTA) on the mitogen response of porcine lymphocytes and the role of metal ions in reversal of the inhibitory effect of EDTA were determined. Porcine lymphocyte responses to mitogens were totally suppressed when serum used to supplement Ca²⁺, Mg²⁺-free minimum essential medium (MEM) was dialyzed against saline or saline with 0.2 or 0.60 mM EDTA, but the responses were only partially reduced when the same serum was added to RPMI-1640 medium. The inhibition observed in MEM could be reversed by adding 1×10⁻³ M Ca²⁺ and 1×10⁻³ M Mg²⁺ to the dialyzed serum. Serum treated directly with 0.60 mM EDTA completely suppressed blastogenesis in lymphocyte cultures maintained in RPMI-1640 or Ca²⁺, Mg²⁺ free MEM. The inhibitory effect of EDTA-treated serum could be completely reversed by adding Zn²⁺ or a combination of Zn²⁺ with other cationic ions, or partially reversed by adding Ni²⁺ or Fe³⁺. Zn²⁺ was the most effective ion, in that it was the only ion that, when alone added to the serum, could completely restore lymphocyte responses to phytohemagglutinin (PHA) or pokeweed mitogen (PWM).     

Weak mixed lymphocyte culture response in the Mongolian gerbil (Meriones unguiculatus).

A study was made to determine whether the mixed lymphocyte culture test could detect histocompatibility differences between two strains of the Mongolian gerbil, Meriones unguiculatus. A semi-micro mixed lymphocyte culture was developed using 6 X 10(5) stimulating and responding cells per 0.12 ml culture volume at a ratio of 1:1. A culture period of 120 hours was found to be optimal. Although a weak allogeneic response was demonstrated with the Uclp:(MON) strain responding to stimulating cells from the MON/Tum strain, the reverse was not seen. A mixed lymphocyte reaction to xenogeneic (mouse) cells was demonstrated, and response to the mitogen, phytohemagglutinin, was strong. These data and the low stimulation index obtained in allogeneic culture supported the view that histocompatibility differences among different strains of the Mongolian gerbil are weak.