An assay for colony-stimulating factor (CSF) production by single T lymphocytes: estimation of the frequency of cells producing granulocyte-macrophage CSF and multi-lineage CSF within a T lymphocyte clone.
The frequency of cells producing hemopoietic colony-stimulating factors (CSF) in a murine T lymphocyte clone has been determined by using a simple microassay that does not require clonal expansion or the addition of accessory cells. When stimulated with concanavalin A (Con A), the clone LB3 produced both granulocyte-macrophage CSF (GM-CSF) and multi-lineage CSF (Multi-CSF), which could be detected by using the cell line FDC-P1, whose proliferation is dependent on the presence of either of these factors. Limiting dilution analysis of Con A-stimulated LB3 cells indicated a requirement for cell-cell contact for optimal production of CSF, which could be bypassed by preincubation of the cells at high density with Con A for 4 hr before dilution in the assay. Limiting dilution estimates of the frequency of CSF-producing cells among Con A-pretreated LB3 cells ranged from 20 to 50%. Direct measurement of CSF production by single Con A-pretreated cells isolated by micromanipulation revealed that 10 to 20% could secrete detectable CSF. However, when isolated Con A-pretreated two-cell and three-cell aggregates were assayed, 50 to 99% were positive, indicating that 30 to 80% of the cells in the aggregates secreted CSF. Assay of the supernatants from single cells and two-cell aggregates on both FDC-P1 cells and another cell line, 32D c13, which responds only to Multi-CSF, demonstrated that many cells produced GM-CSF only, and others varied in the relative quantities of GM-CSF and Multi-CSF produced.
D000818:Animals; D002449:Cell Aggregation; D002450:Cell Communication; D002478:Cells, Cultured; D002999:Clone Cells / Q000276:immunology / Q000378:metabolism; D003115:Colony-Stimulating Factors / Q000096:biosynthesis* / Q000145:classification; D003208:Concanavalin A / Q000494:pharmacology; D004306:Dose-Response Relationship, Immunologic; D006098:Granulocytes; D007958:Leukocyte Count / Q000379:methods; D008213:Lymphocyte Activation; D008264:Macrophages; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*
D003115:Colony-Stimulating Factors; D003208:Concanavalin A