Enrichment of effector T lymphocytes specific to H‐2 antigens by elution from allogeneic target cells and characterization of the eluted lymphocyte population

Abstract

The technique o f elution b y pronase of murine immune lymph node cells adherent to relevant allogeneic target cells has been developed for enrichment of effector T cell population. A fraction of killer cells has been obtained possessing cytotoxic activity which exceeds that of the initial immune lymphocyte population b y a factor of 6-8 as judged by a number of lymphocytes required for 50 % cytotoxic effect (CE). T h e gain in CE is fairly reproducible in different H-2 systems and under various lymphocyte-target incubation conditions. It appears to be due t o the quantitative enrichment of the population in cells bearing receptors to the particular H-2 antigens and giving rise to specific CE rather than t o an increase in cytotoxic activity per cell resulting from treatment with the target monolayer and pronase. The eluted killer cell fraction is characterized by a twofold increase in percentage of cells killed by anti-@ antibody, a 4-5-fold increase in the number of DNA-synthesizing cells and b y an increase in medium and largc lymphocyte content. A further enrichment in effector lymphocytes is reached by depletion of B cells a t the expense of their receptors t o Fc fragment of Ig. Macrophage monolayer fixed by glutaraldehyde lost its capacity t o absorb effector lymphocytes. T h e implications of the data obtained for further studies o n cell-mediated immunity are discussed. 1 I ] and the rosetteHowever, one should not disregard the possibility that in all the above-mentioned cases passive absorption of Ig on the T cell surface is revealed rather than its true (endogenic) receptors. In fact, receptors to both F c fragment of IgM Label release upon incubation with immune and normal lymphocytes EA: Erythrocytes-antibodies 281 the possibility is not excluded that such antibodies are actually produced by small B cell contaminants present in suspension. Experimental analysis [29, 301 of these and other ambiguous points in the interpretation of such results have raised doubts whether the above cited reports really concern the endogenic T receptors. .Introduction'Meanwhile, T cell receptors responsible for interaction with target cells (TC) in the H-2 system have been shown to differ from Ig of the known classes [31-331. Such receptors are manifested in the ability of cytotoxic lymphocytes t o get selectively attached for several hours at 25-37 OC t o the naturally immunosorbent-monolayer of the relevant TC ' [34-361. To study the properties o f the effector T cells, including the nature of their receptors specific to the H-2 antigens, it seems t o be essential t o isolate these cells from the whole population where they occur in a ratio of 1-4 ' %, [ 3 7 -~3 9 ] , and to concentrate them.The present work describes a technique developed for the enrichment of the effector T lymphocyte population in cells reactive to the particular H-2 antigens. T h e ability of pronase to completely and reversibly prevent immune lymphocytes from absorption on T C monolayer with no damage of the latter described earlier [40] is t...

Comparative Study of Cytotoxic T‐Lymphocytes and Producers of the Macrophage Migration Inhibition Factor (MIF) in the H‐2 System

“…B cells adhered to the monolayer of sheep erythrocytes fixed by poly-L-lysine onto the surface of plastic Petri dishes (M3OO3, Falcon Plastics) and pretreated with rabbit antibodies. This technique is applicable for the H-2 system as shown earlier [7].…”

“…Absorption and elution of immune lymphocytes were performed as described earlier [7] using the macrophage monolayer cultivated in 250 ml plastic bottles N 3024 (Falcon Plastics, USA). After elution with pronase the latter was neutralized by excess of bovine serum and removed by repeated washing of the cells.…”

“…The specific adherence of T-killers to the relevant target cell monolayers [4] and their selective enrichment by pronase elution of the absorbed lymphocytes [7] revealed the existence of antigen-binding receptors on the surface of T-killers and allowed concentration of specific T-killers obtained by in vivo immunization. By these techniques the peculiarity of T-killer receptor specificity has been established: Tkillers are shown to be absorbed selectively onto third-party target cells bearing a private serologically defined (SD) specificity of the H-2K or H-2D immunizing complex, but they are absorbed only very faintly onto the targets bearing SD public H-2 cross-reactive specificities [8].…”

Transplantational and specific antitumor immunity in retrospective view: new models based on transgenesis of individual chains of T-cell receptor

“…Следующий шаг в исследо-вании природы взаимодействия антигенспецифиче-ских рецепторов Т-лимфоцитов с молекулами гисто-совместимости стал возможным, когда Б.Д. Бронд разработал метод выделения лимфоцитов, специфи-чески прикрепившихся к монослою аллогенных ма-крофагов [17]. Удаление неприкрепившихся клеток и последующая обработка лимфоцитов, прикрепив-шихся к монослою, проназой позволяла смыть их с монослоя макрофагов и добиться существенного обогащения антигенспецифических клеток с полным сохранением ими функциональных свойств.…”

Specific Suppressor T‐Cells Immune to Antigens of the H‐2 Complex: Receptors, Clonal Structure, Genetic Restriction and Antigenic Markers

“…To prepare the enriched SSTC population, monolayer-adherent d anti-b lymphocytes were eluted with pronase as described for CTL elution (Brondz et al 1975b), washed and mixed with normal syngeneic responder lymphocytes at ratios from 1:2.5 to 1:160. It appeared that, after elution from BIOTC monolayer, the SSTC were still active when mixed with the responders at a ratio of about 0.6% for DNA synthesis inhibition ( Fig.…”