Hashemi B B, Slattery J P, Holowka D, Baird B
School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
J Immunol. 1996 May 15;156(10):3660-7.
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.
我们通过流式细胞术测量可溶性和珠附单抗聚集TCR后T细胞胞质Ca2+变化，研究了表面附着配体对TCR介导的细胞活化的功能优势。用包被抗TCR单抗的细胞大小的珠子结合HPB-ALL人白血病细胞，比相同单抗的可溶性形式产生更强、更持久的Ca2+反应。在用珠子刺激后加入大量过量的可溶性单抗，会导致珠结合细胞的反应显著降低，但仅对结合物有有限的破坏。在该混合物中同时取样的游离（未结合）细胞，对可溶性单抗的反应是Ca2+短暂增加，其下降动力学与加入可溶性单抗后的珠结合细胞相同。抗TCR单抗的Fab片段导致珠结合细胞的反应类似降低，且它们不刺激游离细胞。在Fab介导结合细胞胞质Ca2+降至接近基线浓度后，加入第二种非竞争性抗TCR单抗，引起的Ca2+反应与游离细胞相比幅度大幅降低。结果表明，可溶性和表面附着配体导致Ca2+反应的TCR特异性脱敏。表面附着配体在维持T细胞信号传导方面比可溶性配体更有效，至少部分原因是它们促进TCR在表面接触区域稳定缔合和/或重新缔合到结合状态。
