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. 2009 Aug 14;284(33):22149-22154.
doi: 10.1074/jbc.M109.000752. Epub 2009 Jun 19.

LFA-1-dependent Ca2+ entry following suboptimal T cell receptor triggering proceeds without mobilization of intracellular Ca2+

Kwangmi Kim  1 , Lin Wang  1 , Inkyu Hwang  1
Affiliations

LFA-1-dependent Ca2+ entry following suboptimal T cell receptor triggering proceeds without mobilization of intracellular Ca2+

Kwangmi Kim et al. J Biol Chem. .

Abstract

A surge in cytosolic calcium ion concentration by entry of extracellular Ca2+ is a hallmark of T cell activation. According to store-operated Ca2+ entry mechanism, the Ca2+ entry is preceded by activation of phospholipase C-gamma1 (PLC-gamma1) and the consequent mobilization of intracellular Ca2+. Using membrane vesicles expressing the mouse class I major histocompatibility complex, i.e. Ld plus costimulatory ligands, i.e. B7-1 and intercellular adhesion molecule-1 along with 2C T cell receptor transgenic T cells, we investigated the roles of CD28 and LFA-1 (lymphocyte function-associated antigen-1) in the activation of PLC-gamma1 and Ca2+ signaling. Both CD28 and LFA-1 made significant and comparable contributions to the activation of PLC-gamma1 as gauged by the level of its phosphorylation at tyrosine 783. In contrast, their roles in Ca2+ signaling were quite distinct so that LFA-1/intercellular adhesion molecule-1 interaction exerted a determining role, whereas CD28/B7-1 interaction played only a minimal role. In particular, when the T cells were activated by suboptimal T cell receptor stimulation, LFA-1 played an indispensable role in the Ca2+ signaling. Further experiments using Ca2+-free medium demonstrated that the entry of extracellular Ca2+ was not always accompanied by mobilization of intracellular Ca2+. Thus, intracellular Ca2+ mobilization was hardly detected under the condition that LFA-1 played the indispensable role in the entry of extracellular Ca2+, while a distinct level of intracellular Ca2+ mobilization was readily detected under the condition that LFA-1 played only the supporting role. These results ensure the unique role of LFA-1 in T cell Ca2+ signaling and reveal that LFA-1-dependent Ca2+ entry proceeds via a mechanism separate from store-operated Ca2+ entry.

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Figures

FIGURE 1.
FIGURE 1.
Contributing roles of CD28 and LFA-1 in phosphorylation of Akt and PLC-γ1. Cell extracts, prepared from 2C T cells cultured for different periods of time with LdB7-1ICAM-1 pMVs loaded with QL9 (1 μm) or P1A (1 μm) peptide, were subjected to Western blotting analysis for Akt phosphorylated at Thr308 (A, left) and PLC-γ1 phosphorylated at Tyr783 (B, left) along with pan-Akt and total PLC-γ1 as protein loading controls, respectively. Cell extracts, prepared from wild type (WT), LFA-1−/−, and CD28−/− 2C T cells pretreated with isotype control (Cont.), anti-LFA-1 (CD11a), or anti-CD28 mAb before culture with QL9- or P1A-loaded LdB7-1ICAM-1 pMVs for 15 min, were subjected to Western blot analysis for Akt (A, right) and PLC-γ1 (B, right). Note that P1A peptide forms a complex with Ld, but the Ld-P1A complex is not recognized by 2C TCR.
FIGURE 2.
FIGURE 2.
Contributing roles of CD28 and LFA-1 in increase of cytosolic [Ca2+]. Kinetic flow cytometric analyses for changes in cytosolic [Ca2+] were performed with wild type (WT), LFA-1−/−, and CD28−/− 2C T cells being cultured with P1A (1 μm) or QL9 (1 μm) peptide-loaded LdB7-1ICAM-1 pMVs as indicated. 2C T cells were treated with isotype control (black and gray), anti-CD28 (red), or anti-LFA-1 (blue) mAb prior to culture with the pMVs. The flow analyses were initiated 60 s before addition of the pMVs and continued for 720 s afterward.
FIGURE 3.
FIGURE 3.
Extracellular Ca2+ entry accompanied with or without mobilization of intracellular Ca2+. Changes in cytosolic [Ca2+] of wild type 2C T cells and in Ca2+-containing or Ca2+-free medium were monitored after addition of thapsigargin (100 nm) (black) or DMSO alone (gray) (A, left) or QL9-loaded (1 μm) (black) or P1A-loaded (1 μm) (gray) LdB7-1ICAM-1 (A, right), Ld (B, left), or Ld(high)B7-1ICAM-1 (B, right) pMVs as indicated. Flow cytometric analyses were performed as in Fig. 2.
FIGURE 4.
FIGURE 4.
Relationship among levels of pMHC expression, mobilization of intracellular Ca2+, and LFA-1 dependence of extracellular Ca2+ entry. Wild type and LFA-1−/− 2C T cells in Ca2+-containing or Ca2+-free medium were mixed with Ld(high)B7-1ICAM-1 pMVs loaded with P1A (1 μm) (gray) or titrated concentrations of QL9 peptide (black, 200 nm; red, 40 nm; blue, 8 nm; green, 1.6 nm), and changes in cytosolic [Ca2+] were analyzed as in Fig. 2.
FIGURE 5.
FIGURE 5.
Role of PLC-γ1 in LFA-1-dependent Ca2+ entry. Wild type 2C T cells in Ca2+-containing medium were pretreated with a signaling inhibitor (bold gray line) or DMSO (bold and thin black line) as indicated for 30 min before culture with P1A (1 μm) (thin black line) or QL9 (1 μm) (bold black and gray line) peptide-loaded LdB7-1ICAM-1 pMVs. Changes in cytosolic [Ca2+] were analyzed as in Fig. 2.
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