Cytosolic free calcium concentration in the mitogenic stimulation of T lymphocytes by anti-CD3 monoclonal antibodies.
The effects of anti-CD3 monoclonal antibodies on cytosolic free Ca2+ concentration, [Ca2+]i, were investigated in freshly isolated lymphocytes, T cell lines, T clones and the leukemic T cell line Jurkat with three different methodologies, i.e. classical cuvette experiments, cytofluorimetry and videoimaging. With any technique, concentrations of anti-CD3 antibodies optimal for stimulation of DNA synthesis were completely ineffective at inducing early increases of [Ca2+]i in freshly isolated lymphocytes. At supraoptimal mitogenic concentrations: (i) anti-CD3 mAb induced negligible increases of [Ca2+]i when tested in suspensions of freshly isolated lymphocytes, but the response increased progressively during in vitro culturing with IL2; (ii) most, but not all, T clones, when tested in suspension, were responsive to these concentrations of anti-CD3 antibodies in terms of [Ca2+]i; (iii) using the videoimaging technique at the single cell level, it was demonstrated that the anti-CD3 antibodies induced large increases of [Ca2+]i in lymphocytes only under conditions which allowed adherence of the antibodies (and of the cells) to the glass surface. In all T cell types investigated, the [Ca2+]i increases were most often composed by multiple, asynchronous oscillations. The buffering of [Ca2+]i increases, obtained by loading the cells with membrane permeant esters of Quin-2 and Fura-2, inhibited anti-CD3 mAb induced DNA synthesis, but this appeared entirely attributable to a toxic side effect of the ester hydrolysis. The relevance of these data is discussed in terms of their methodological and functional implications for the understanding of the role of Ca2+ in mitogenic stimulation of T cells.
Murgia
M
M
Department Biomedical Sciences, CNR Unit for the Study of the Physiology of Mitochondria, University of Padova, Italy.
Mion
M
M
Veronese
L
L
Panozzo
M
M
Coppola
V
V
Rizzuto
R
R
Brini
M
M
Malavasi
F
F
Amadori
A
A
Chieco Bianchi
L
L
D000634:Aminoquinolines; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017252:CD3 Complex / Q000032:analysis* / Q000276:immunology; D002118:Calcium / Q000032:analysis*; D002460:Cell Line; D005470:Fluorometry; D016257:Fura-2; D006801:Humans; D008938:Mitosis; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000634:Aminoquinolines; D000911:Antibodies, Monoclonal; D017252:CD3 Complex; C035739:Quin2; D002118:Calcium; D016257:Fura-2