Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

Abstract

It has been shown that macrophage and granulocyte colonies can be induced m semisolid agar (1-3) in the presence of substances termed colony-stimulating factors (CSF), which are released predominantly by monocytes (4). However, attempts to induce formation of lymphoid colonies with CSF have so far proved unsuccessful. In the mouse, B lymphoid colonies are formed in the presence of 2-mercaptoethanol (5), and T lymphoid colonies can be induced with the plant lectins phytohemagglutinin (PHA) and concanavalin A (6). T lymphoid colonies can also be established from human peripheral blood lymphocytes in the presence of PHA (7-9), whereas with pokeweed mitogen mixed T and B lymphoid colonies are formed (9). Established human lymphoid cell lines multiply spontaneously in the absence of plant lectins or mercaptoethanol, and it seemed possible that such cells might release growth-stimulating substances into the culture medium. We have therefore investigated the ability of conditioned medium (CM) obtained from lymphoid cell lines to induce normal human peripheral blood lymphocytes (PBL) to form lymphoid colonies in agar. Materials and MethodsThree B-cell lines-4061 (provided by Dr. J. Sinkovics, Houston, Tex.), PGLC 33H, and RPMI 1788-were used as sources of CM. After 3-4 days of culture in serum-free medium at 37°C, cellfree supernates were obtained by centrifugation at 800 g for 10 rain, filtration through 0.22-~m filters (Millipore Corp., Bedford, Mass.), and ultracentrifugation at 47,500 g for 1 h.Peripheral blood was collected from adult healthy volunteers in pyrogen-and preservativefree heparin. Lymphocytes were obtained by centrifugation over Ficoll Isopaque gradients and washed three times in RPMI 1640 medium (Grand Island Biological Co., Grand Island, N.Y.). Contamination with monocytes varied between 3 and 20% as assessed by latex ingestion and staining for peroxidase (10). Cells were seeded at various concentrations in 2 ml 0.3% agar made by the addition of I vol of double-strength RPMI 1640 (containing 200 gg/ml streptomycin, 200 U/ ml benzyl penicillin, and 20% fetal calf serum [Gibco]) to 1 vol of 0.6% agar (Difco Laboratories, Detroit, Mich.) made up in pyrogen-free distilled water. Various amounts of CM were added directly to the 0.3% agar mixture. After culture in 35-ram plastic Petri dishes at 37°C in 5% CO2 in air for various times, the plates were counted under an inverted microscope. Colonies were defined as clumps containing more than 50 cells. Colony cells were tested for their ability to form E rosettes, by using the technique of Wybran et al. (11), and for the presence of surface * Publication no. 145 from the

Essential and Nonessential Cooperative Signals in Lymphocyte Triggering in Solid Cultures

“…This would suggest that the essential cooperative effects of autologous RBC were specific only for those lymphocyte subpopu lations capable of being stimulated in solid cultures. Kurland et al [HI and Metcalf [ 14] reported RBC to potentiate colony for mation in semisolid culture systems, while others have not found it necessary [7,11,19], Our results differ from those of Claesson et al [4] who found that SRBC in creased human T lymphocyte colony forma tion in solid cultures, but could not replace accessory leukocytes as source of essential cooperative signals. This could be due to their use of a low (l°/o) SRBC concentra tion.…”

“…Studies using colony formation after 5 or more days of culture cannot dis tinguish between a requirement for macro phage products in the triggering of lympho cytes from G" into the cell cycle and the maintainance of their progeny in a prolifer ative state, or both. Furthermore, with the exception of Claesson et al [4,5], all other work on colony formation has been done using semisolid culture systems [7,11,14,20,21], where some degree of early cell to cell contact was likely to occur and could play a key role in lymphocyte triggering [8, 10. 16], Such colonies might originate from small cell aggregates, rather than from sin gle cells.…”

Shine a light: Imaging the immune system

“…This problem can be ameliorated by culturing them in ''conditioned medium'', i.e. the supernatant of cell populations [135,136]. This suggests that the cells require endocrine factors to survive, which in the context of a single cell in a vast volume of medium becomes too dilute.…”

Stage-dependent reduction in T colony formation in Hodgkin's disease. Coincidence with monocyte synthesis of prostaglandins.

“…The positive or promoting agent for the latter system is species specific and can overcome inhibition by PGE (24). A parallel mechanism may exist for T cell precursors; in fact, a species-specific soluble agent(s) promoting T lymphocyte proliferation has been described (25)(26)(27).…”