A method for analysing the clonal precursors of concanavalin A-induced suppressor cells.
Spleen cells from 3 different strains of mice (C57 (H-2b), CBA (H-2k) and BALB/c (H-2d] were stimulated in vitro with different concentrations of concanavalin A (CA) for 48 h. This resulted in the production of cells capable of inhibiting the generation of alloantigen-specific cytotoxic T lymphocytes (CTL) in a mixed lymphocyte culture (MLC). 1 microgram/ml was an effective concentration of CA to induce C57 and BALB/c suppressor cells (SC), but 5 micrograms/ml CA was required to induce CBA SC. SC precursors (SC-P) were shown to be radiosensitive and the results suggest that SC themselves may be radiosensitive. SC were effective in the presence of added interleukin-2 (IL-2). SC were then induced at limit dilution in microwells in a volume of 25 microliter. A MIC (200 microliter) was then (after 48 h) added to each microwell. This resulted in a dilution of the concentration of CA to a level below which it was effective at inducing suppression. Cytotoxicity was then assessed 7 days later. It was thus possible to analyse SC-P at the clonal level and estimate their frequency. The frequency of C57 splenic SC-P (active against a C57 anti-BALB/c MLC) was 14.4 X 10(-6), the frequency of CBA splenic SC-P (active against a CBA anti-BALB/c MLC) was 92.3 X 10(-6), and the frequency of BALB/c splenic SC-P (active against a BALB/c anti-CBA MLC) was 15.8 X 10(-6). It was possible to analyse SC-P at a clonal level whether or not the MLC contained added IL-2. SC and SC-P were shown to be sensitive to anti-Thy-1 and complement.
Good
M F
MF
Halliday
J W
JW
Powell
L W
LW
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D002452:Cell Count; D002999:Clone Cells / Q000145:classification; D003208:Concanavalin A / Q000494:pharmacology*; D008213:Lymphocyte Activation / Q000528:radiation effects; D007959:Lymphocyte Culture Test, Mixed / Q000379:methods; D008222:Lymphokines; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D010641:Phenotype; D051381:Rats; D011919:Rats, Inbred Strains; D013154:Spleen / Q000166:cytology; D013234:Stem Cells / Q000145:classification*; D050378:T-Lymphocytes, Regulatory / Q000145:classification* / Q000276:immunology; D018800:Thy-1 Antigens
D000954:Antigens, Surface; D008222:Lymphokines; D018800:Thy-1 Antigens; C021649:concanavalin A-induced helper factors; D003208:Concanavalin A