Abstract
Leukocyte functional antigen 1 (LFA-1), with intercellular adhesion molecule ligands, mediates T cell adhesion, but the signaling pathways and functional effects imparted by LFA-1 are unclear. Here, intracellular phosphoprotein staining with 13-dimensional flow cytometry showed that LFA-1 activation induced phosphorylation of the β2 integrin chain and release of Jun-activating binding protein 1 (JAB-1), and mediated signaling of kinase Erk1/2 through cytohesin-1. Dominant negatives of both JAB-1 and cytohesin-1 inhibited interleukin 2 production and impaired T helper type 1 differentiation. LFA-1 stimulation lowered the threshold of T cell activation. Thus, LFA-1 signaling contributes to T cell activation and effects T cell differentiation.
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Acknowledgements
We acknowledge support from BD Biosciences-Pharmingen and the Baxter Foundation; and D. Parks and the Herzenberg Laboratory for the resources of the Stanford FACS facility. We thank N. Hogg for mAb 24; J.F. Fortin for the NFAT-luciferase–transfected cells; and C.C. Gahmberg for the gift of phosphorylated β2 integrin antibodies. We thank I. Weissman, M. Davis, H. Blau and R. Smith for discussions; G. Fathman for review of this manuscript; and K. Vang for administrative help. O.D.P. was supported by the Bristol-Meyer Squibb Irvington Institute and the National Heart, Lung and Blood Institute (N01-HV-28183I). G.P.N. was supported by the National Institutes of Health (P01-AI39646, AR44565, AI35304, N01-AR-6-2227, A1/GF41520-01), the National Heart, Lung and Blood Institute (N01-HV-28183I) and the Juvenile Diabetes Foundation.
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Supplementary Fig. 1.
(a) We tested if LFA-1 stimulation differentially induced activation of PKC isozymes. We monitored serine/threonine phosphorylation of PKCs (as a measure of their activation status) that were immunopurified from unstimulated and LFA-1 stimulated cells. We noted increased detection of serine/threonine phosphorylation of PKCα, β, δ, and ιafter LFA-1 stimulation. We noted no increase in phosphorylation of PKCε, η, Θnor λafter LFA-1 stimulation. (b) Loading controls for immunopurified PKC isozymes in Fig 1c. Immunopurified PKCs were quantified by BCA protein assay, and 1 μg was resolved by SDS-PAGE. Gel was stained by coomassie blue, and imaged using a Biorad Versadoc imaging system. (c) To verify the kinase activity PKCs by LFA-1 in Jurkat cells, we used PKC kinase assays with PKCs immunopurified from unstimulated and LFA-1-stimulated cells. We detected phosphothreonine incorporation on the myelin basic protein substrate by PKCβ, δ, and ε. We detected minimal activity by PKC Θ and α. This PKC isozyme kinase assay was done using immunopurified PKC isozymes from ICAM-2 stimulated Jurkat cells and the myelin basic protein peptide as a substrate. Phosphorylation was assessed by anti-phosphorylated (T98)-MBP-HRP based ELISA. Values are shown as relative fluorescent units (RFU). (d) These results were confirmed by intracellular flow cytometry for phospho-specific residues of PKCδ (T505), PKCΘ (T538), and PKC α/β (T638/641) using the only available antibodies for these sites. Intracellular detection of phosphorylated PKCδ(T505), phosphorylated PKCΘ(T538) and phosphorylated PKCα/β(T638/641) in unstimulated and ICAM-2 stimulated Jurkat cells. Detection was made using an anti-rabbit Alexa647 secondary. These results do not exclude the possibility of different PKC isozymes phosphorylating residues other than β2-integrin ser745 as PKCβ, εand αwere also activated by LFA-1. (e) Intracellular flow cytometric staining of PKC α, β, δ, and Θto assess the relative amounts of these PKC isozymes in CD4+ human T cells, as normalized in vitro biochemical assays may not reflect intracellular biology. Using highly specific monoclonal antibodies, we detected the presence of all PKC isozymes inside of human T cells in the order of PKCΘ >> PKCα > PKCβ > PKCΘ. Cells had been stained for intracellular PKCs isozymes followed by secondary anti-mouse Ig-Alexa488 at pre-titred concentrations of 0.1 μg per 1 x 106 cells. Cells were extensively washed and blocked with 5% FCS containing 1 μg/ml of mouse IgG for 30 min, before being stained with CD4-cychrome, and CD3-PE in the same buffer. Cells were gated on CD3+CD4+ populations and displayed? for fluorescence in the AX488 channel. These results do not predict, irrespective of stoichiometry, which isozyme may represent the more active PKC isozyme fraction for any given reaction. (PDF 370 kb)
Supplementary Fig. 2.
Multidimensional gating flow diagram for Figure 5a and 5b. (PDF 507 kb)
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Perez, O., Mitchell, D., Jager, G. et al. Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1. Nat Immunol 4, 1083–1092 (2003). https://doi.org/10.1038/ni984
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DOI: https://doi.org/10.1038/ni984
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