Analysis of the molecular requirements for T cell recognition and activation by using Ia-containing lipid vesicles and stopped-flow fluorometry.
Using Ia antigen-containing lipid vesicles, we investigated by stopped-flow fluorometry the requirements for helper T cell recognition and activation. When azobenzenearsonate-L-tyrosine (ABA-L-Tyr)-specific, I-Ak-restricted helper T cell hybridomas 2-45-12 were mixed with ABA-L-Tyr and purified I-Ak molecules on lipid vesicles, an increase of intercellular calcium ion concentration ([Ca2+]i) in the T cells were detected within 1-2s. The average increases of [Ca2+]i were not much different when the lipid vesicles were composed of dimyristoylphosphatidylcholine or of egg phosphatidylcholine, but they were dependent on the density of I-Ak molecules on the liposomes. The increase of [Ca2+]i was inhibited in the presence of anti-I-Ak monoclonal antibody 10.2.16, but not by the addition of anti-L3T4 monoclonal antibody GK1.5. However, the addition of anti-L3T4 antibody during the first 3 h of cultivation completely inhibited the T cell activation [interleukin (IL-2) production]. In our experimental system, IL-2 production was observed either when L3T4-positive T cell hybridomas 2-45-12 were stimulated with ABA-L-Tyr and Ia molecules on the vesicles in the presence of phorbol 12-myristate 13-acetate, or when L3T4-negative T cell hybridomas 3H60.12 were incubated with ABA-L-Tyr and Ia molecules on the planar membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Odaka
C
C
Department of Microbiology, Keio University School of Medicine, Tokyo, Japan.
Utsunomiya
N
N
Nakanishi
M
M
Arata
Y
Y
Manabe
S
S
Yasuda
T
T
Tadakuma
T
T
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte; D000949:Histocompatibility Antigens Class II; D006825:Hybridomas / Q000276:immunology; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000096:biosynthesis; D008081:Liposomes; D008213:Lymphocyte Activation; D051379:Mice; D008809:Mice, Inbred C3H; D015398:Signal Transduction / Q000276:immunology; D013050:Spectrometry, Fluorescence; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology; D010132:p-Azobenzenearsonate / Q000031:analogs & derivatives / Q000276:immunology
D000945:Antigens, Differentiation, T-Lymphocyte; D000949:Histocompatibility Antigens Class II; D007376:Interleukin-2; D008081:Liposomes; C000234:azobenzenearsonate-N-acetyl-L-tyrosine; D014443:Tyrosine; D010132:p-Azobenzenearsonate