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. 2009 Jun 25;4(6):e6044.
doi: 10.1371/journal.pone.0006044.

A novel flow cytometric high throughput assay for a systematic study on molecular mechanisms underlying T cell receptor-mediated integrin activation

Kwangmi Kim  1 , Lin Wang, Inkyu Hwang
Affiliations

A novel flow cytometric high throughput assay for a systematic study on molecular mechanisms underlying T cell receptor-mediated integrin activation

Kwangmi Kim et al. PLoS One. .

Abstract

Lymphocyte function-associated antigen 1 (LFA-1), a member of beta2-integrin family, exerts multiple roles in host T cell immunity and has been identified as a useful drug-development target for inflammatory and autoimmune diseases. Applying the findings that primary resting T cells absorb nanometric membrane vesicles derived from antigen presenting cells (APC) via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide-major histocompatibility complex (MHC) complex (pMHC) and LFA-1 with its ligand, intercellular adhesion molecule-1 (ICAM-1), and that signaling cascades triggered by TCR/pMHC interaction take a part in the vesicle-absorption, we established a cell-based high throughput assay for systematic investigation, via isolation of small molecules modulating the level of vesicle-absorption, of molecular mechanisms underlying the T cell absorption of APC-derived vesicles, i.e., structural basis of TCR/pMHC and LFA-1/ICAM-1 interactions and TCR-mediated LFA-1 activation. As primary T cells along with physiological ligands expressed in biological membrane are used and also individual cells in assay samples are analyzed by flow cytometry, results obtained using the assay system hold superior physiological and therapeutic relevance as well as statistical precision.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Importance of intracellular signaling cascades in the pMV-absorption.
(A) Wild type (WT), CD28−/−, and LFA-1−/− 2C T cells, pre- treated with the respective mAbs, were cultured with or without the peptide-loaded LdB7-1ICAM-1 pMVs, as indicated, followed by mAb staining for B7-1 and flow cytometric analysis. Mean fluorescence intensities (MFIs) of B7-1 staining relative to that of WT 2C T cells, pre-treated with control mAb and cultured with the QL9-loaded pMVs, were plotted. Note that P1A peptide forms complex with Ld but Ld/P1A complex is not recognized by 2C TCR . (B) WT 2C T cells, pre-treated with the respective mAbs, were cultured with the peptide-loaded pMVs for different periods of time, as indicated, and stained for B7-1. (C) WT 2C T cells were treated with PP2 (PP), Wortmannin (WM), Pervanadate (PV), Cytochalasin D (CT), BAPTA/AM (BA), Forskolin (FS), Cyclosporin A (CC) and Nocodazole (NZ), respectively, as indicated, before culture with the QL9-loaded pMVs. MFIs of the B7-1 staining relative to that of 2C T cells treated with DMSO alone were plotted.
Figure 2
Figure 2. Analysis of F-actin polymerization occurring during culture of 2C T cells with the pMVs.
(A) Wild type (WT) 2C T cells were cultured with the peptide-loaded pMVs for different periods of time and stained for F-actin with FITC-labeled phalloidin followed by flow cytometric analysis. Fold changes in the level of F-actin relative to that of 2C T cells fixed immediately after mixing with the QL9-loaded LdB7-1ICAM-1 pMVs were plotted. Histograms represent the F-actin staining of 2C T cells cultured with P1A- (solid grey), P2Ca- (dotted black) and QL9- (solid black) loaded pMVs for 5 min, respectively. (B) WT, CD28−/− and LFA-1−/− 2C T cells were treated with the respective mAbs, as indicated, before 5 min culture with the QL9-loaded pMVs. Fold changes in the level of F-actin during the culture were plotted. (C) WT 2C T cells were pre-treated with the respective mAbs and cultured with the peptide-loaded pMVs, as indicated, for 20 min on poly-L-lysine-coated coverslips followed by staining for F-actin with FITC-labeled phalloidin.
Figure 3
Figure 3. Schematic representation of the mechanistic model for 2C T cell absorption of the pMVs.
The whole process of the pMV-absorption commences as 2C TCRs interact with cognate pMHCs expressed on the pMVs. The TCR/pMHC interaction activates intracellular signaling cascades (i.e., “inside-out” signaling) resulting in promotion of LFA-1 function and thereby stabilization of T/pMV contact. According to that model, the pMV-absorption can be inhibited by three different routes: (i) interruption of TCR/pMHC interaction, (ii) interruption of LFA-1/ICAM-1 interaction, (iii) inhibition of intracellular signaling cascades for LFA-1 activation.
Figure 4
Figure 4. High throughput screening for systematic study on molecular mechanisms underlying the pMV-absorption.
(A) Flow diagram of the high throughput screening (HTS) for isolation of small molecules modulating the pMV-absorption. (B) Z-factor was calculated with averages and standard deviations of 32 sets of assays; each set included assays with control mAb plus QL9-loaded pMVs used as the positive control, anti-LFA-1 mAb plus QL9-loaded pMVs used as the negative control and with control mAb plus P1A-loaded pMVs. (C) IC50s (or ED50) and pharmacological uses of hits isolated from the pilot scale HTS were summarized.
Figure 5
Figure 5. Structure-activity relationship study on the agonistic activity of Brompheniramine on the pMV-absorption.
(A) Chemical structures of structurally- or functionally-related compounds of Brompheniramine are shown. Refer to Supplementary Figure 4 for their names and pharmacological uses. (B) 2C T cells were treated with compounds shown in (A) at titrated concentrations and cultured with the QL9-loaded pMVs followed by mAb staining for B7-1. MFIs of the B7-1 staining relative to that of 2C T cells treated with DMSO alone were plotted. Experiments were performed in duplicate and standard deviations of duplicate samples were all within 10% of the averages. Shown is one of three representative experiments. (C) ED50s of the compounds were calculated from the experiments described in (B) and plotted against Kis of the compounds for 5-HT transporter. Note: Ki values used in this plot were adopted from PDSP Ki data base (http://pdsp.med.unc.edu/pdsp.php). To ensure data consistency, only the Ki values obtained using rat frontal cortex as the source of 5-HT transporter and [3H]-5-HT as the hot ligand were used .
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References

    1. Pribila JT, Quale AC, Mueller KL, Shimizu Y. Integrins and T cell-mediated immunity. Annu Rev Immunol. 2004;22:157–180. - PubMed
    1. Kuhn JR, Poenie M. Dynamic polarization of the microtubule cytoskeleton during CTL-mediated killing. Immunity. 2002;16:111–121. - PubMed
    1. Mousa SA. Cell adhesion molecules: potential therapeutic and diagnostic implications. Methods Mol Med. 2004;93:157–174. - PubMed
    1. Nishibori M, Takahashi HK, Mori S. The regulation of ICAM-1 and LFA-1 interaction by autacoids and statins: a novel strategy for controlling inflammation and immune responses. J Pharmacol Sci. 2003;92:7–12. - PubMed
    1. Hogg N, Henderson R, Leitinger B, McDowall A, Porter J, et al. Mechanisms contributing to the activity of integrins on leukocytes. Immunol Rev. 2002;186:164–171. - PubMed

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