Sustained T cell receptor-mediated Ca2+ responses rely on dynamic engagement of receptors.
We have investigated the functional advantage of surface-attached ligands for TCR-mediated cell activation with flow cytometric measurements of cytoplasmic Ca2+ changes in T cells after aggregation of TCR by soluble and bead-attached mAb. Conjugation of HPB-ALL human leukemia cells with cell-sized beads coated with anti-TCR mAb causes a stronger, more sustained Ca2+ response than that produced by the soluble form of the same mAb. Addition of a large excess of the soluble mAb subsequent to stimulation with the beads causes a marked reduction in the response of the bead-conjugated cells, but only limited disruption of the conjugates. Free (nonconjugated) cells, sampled simultaneously in this mixture, respond to the soluble mAb with a transient Ca2+ increase that declines with the same kinetics as the bead-conjugated cells after addition of the soluble mAb. Fab fragments of the anti-TCR mAb cause a similar reduction in the response of the bead-conjugated cells, and they do not stimulate free cells. Following the Fab-mediated decline in cytoplasmic Ca2+ of conjugated cells to near-baseline concentrations, the addition of a second, noncompetitive, anti-TCR mab causes a Ca2+ response that is substantially reduced in magnitude compared with that for the free cells. The results indicate that soluble and surface-attached ligands cause TCR-specific desensitization of the Ca2+ response. Surface-attached ligands are more effective than soluble ligands in sustaining signaling in T cells at least in part because they facilitate steady association and/or reassociation of TCR into the bound state in the surface contact area.
Hashemi
B B
BB
School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
Slattery
J P
JP
Holowka
D
D
Baird
B
B
D017607:Aluminum Compounds / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D015458:Leukemia, T-Cell; D008213:Lymphocyte Activation / Q000187:drug effects; D008863:Microspheres; D011940:Receptor Aggregation / Q000187:drug effects / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D017607:Aluminum Compounds; D000911:Antibodies, Monoclonal; D011948:Receptors, Antigen, T-Cell; D005459:Fluorides; D002118:Calcium; C032311:aluminum fluoride