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CD226 (DNAM-1) is involved in lymphocyte function-associated antigen 1 costimulatory signal for naive T cell differentiation and proliferation.

Shibuya K, Shirakawa J, Kameyama T, Honda S, Tahara-Hanaoka S, Miyamoto A, Onodera M, Sumida T, Nakauchi H, Miyoshi H, Shibuya A - J. Exp. Med. (2003)

Bottom Line: These results suggest that CD226 is involved in LFA-1-mediated costimulatory signals for triggering naive T cell differentiation and proliferation.We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells.Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Immune Receptor, RIKEN Research Center for Allergy and Immunology, 3-1-1 Koyadai, Ibaraki 305-0074, Japan. ashibuya@rtc.riken.go.jp

ABSTRACT
Upon antigen recognition by the T cell receptor, lymphocyte function-associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12-independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1-mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

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Lentiviral vector-mediated gene transfer into resting blood cell subsets. (A) Each resting blood cell subset indicated was separated from PB by MACS. CD4+ T cells were stimulated or not with plastic-coated anti-CD3 and soluble anti-CD28 mAbs for 3 d. Each subset indicated was infected with the retroviral or lentiviral vectors containing EGFP at an MOI of 10 without any exogenous stimuli or cytokines. 3 d after infection, EGFP expressions were analyzed by flow cytometry. (B–D) CD4+ CD45RA+ naive T cells were purified from CB using MACS followed by FACS® sorting. Purified cells were infected with the retroviral or lentiviral vector containing EGFP at MOIs indicated (B) or of 10 (C and D) without any exogenous stimuli or cytokines. The naive T cells were stained with anti-CD45RA or anti-CD62L 3 d after infection, or were stained with Hoechst and Pyronin Y or anti-Ki67 before and 3 d after infection (D) and analyzed by flow cytometry. Staining of CD4+ CD45RA+ naive T cells activated with anti-CD3 and anti-CD28 were used as a positive control (D). The naive T cells 3 d after infection were also stimulated with plate-coated anti-CD3 and anti-CD28 mAbs and proliferation and cytokine production in culture supernatants were analyzed (C).
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fig1: Lentiviral vector-mediated gene transfer into resting blood cell subsets. (A) Each resting blood cell subset indicated was separated from PB by MACS. CD4+ T cells were stimulated or not with plastic-coated anti-CD3 and soluble anti-CD28 mAbs for 3 d. Each subset indicated was infected with the retroviral or lentiviral vectors containing EGFP at an MOI of 10 without any exogenous stimuli or cytokines. 3 d after infection, EGFP expressions were analyzed by flow cytometry. (B–D) CD4+ CD45RA+ naive T cells were purified from CB using MACS followed by FACS® sorting. Purified cells were infected with the retroviral or lentiviral vector containing EGFP at MOIs indicated (B) or of 10 (C and D) without any exogenous stimuli or cytokines. The naive T cells were stained with anti-CD45RA or anti-CD62L 3 d after infection, or were stained with Hoechst and Pyronin Y or anti-Ki67 before and 3 d after infection (D) and analyzed by flow cytometry. Staining of CD4+ CD45RA+ naive T cells activated with anti-CD3 and anti-CD28 were used as a positive control (D). The naive T cells 3 d after infection were also stimulated with plate-coated anti-CD3 and anti-CD28 mAbs and proliferation and cytokine production in culture supernatants were analyzed (C).

Mentions: To examine whether lentiviral vectors can efficiently mediate gene transduction into resting blood cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ NK cells, and CD11b+ monocytes sorted from PB were infected with a retroviral or lentiviral vector containing the EGFP gene and cultured for 72 h in RPMI medium containing 10% FBS without any exogenous stimuli and cytokines. Consistent with previous reports (32), each subset was refractory for transduction with the retroviral vector at an MOI of 10, although PB CD4+ T cells stimulated with anti-CD3 and anti-CD28 mAbs were efficiently transduced at the same MOI (Fig. 1 A). In contrast, the lentiviral vector efficiently transduced the EGFP gene into all blood cell subsets tested at an MOI of 10 (Fig. 1 A).


CD226 (DNAM-1) is involved in lymphocyte function-associated antigen 1 costimulatory signal for naive T cell differentiation and proliferation.

Shibuya K, Shirakawa J, Kameyama T, Honda S, Tahara-Hanaoka S, Miyamoto A, Onodera M, Sumida T, Nakauchi H, Miyoshi H, Shibuya A - J. Exp. Med. (2003)

Lentiviral vector-mediated gene transfer into resting blood cell subsets. (A) Each resting blood cell subset indicated was separated from PB by MACS. CD4+ T cells were stimulated or not with plastic-coated anti-CD3 and soluble anti-CD28 mAbs for 3 d. Each subset indicated was infected with the retroviral or lentiviral vectors containing EGFP at an MOI of 10 without any exogenous stimuli or cytokines. 3 d after infection, EGFP expressions were analyzed by flow cytometry. (B–D) CD4+ CD45RA+ naive T cells were purified from CB using MACS followed by FACS® sorting. Purified cells were infected with the retroviral or lentiviral vector containing EGFP at MOIs indicated (B) or of 10 (C and D) without any exogenous stimuli or cytokines. The naive T cells were stained with anti-CD45RA or anti-CD62L 3 d after infection, or were stained with Hoechst and Pyronin Y or anti-Ki67 before and 3 d after infection (D) and analyzed by flow cytometry. Staining of CD4+ CD45RA+ naive T cells activated with anti-CD3 and anti-CD28 were used as a positive control (D). The naive T cells 3 d after infection were also stimulated with plate-coated anti-CD3 and anti-CD28 mAbs and proliferation and cytokine production in culture supernatants were analyzed (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194159&req=5

fig1: Lentiviral vector-mediated gene transfer into resting blood cell subsets. (A) Each resting blood cell subset indicated was separated from PB by MACS. CD4+ T cells were stimulated or not with plastic-coated anti-CD3 and soluble anti-CD28 mAbs for 3 d. Each subset indicated was infected with the retroviral or lentiviral vectors containing EGFP at an MOI of 10 without any exogenous stimuli or cytokines. 3 d after infection, EGFP expressions were analyzed by flow cytometry. (B–D) CD4+ CD45RA+ naive T cells were purified from CB using MACS followed by FACS® sorting. Purified cells were infected with the retroviral or lentiviral vector containing EGFP at MOIs indicated (B) or of 10 (C and D) without any exogenous stimuli or cytokines. The naive T cells were stained with anti-CD45RA or anti-CD62L 3 d after infection, or were stained with Hoechst and Pyronin Y or anti-Ki67 before and 3 d after infection (D) and analyzed by flow cytometry. Staining of CD4+ CD45RA+ naive T cells activated with anti-CD3 and anti-CD28 were used as a positive control (D). The naive T cells 3 d after infection were also stimulated with plate-coated anti-CD3 and anti-CD28 mAbs and proliferation and cytokine production in culture supernatants were analyzed (C).
Mentions: To examine whether lentiviral vectors can efficiently mediate gene transduction into resting blood cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ NK cells, and CD11b+ monocytes sorted from PB were infected with a retroviral or lentiviral vector containing the EGFP gene and cultured for 72 h in RPMI medium containing 10% FBS without any exogenous stimuli and cytokines. Consistent with previous reports (32), each subset was refractory for transduction with the retroviral vector at an MOI of 10, although PB CD4+ T cells stimulated with anti-CD3 and anti-CD28 mAbs were efficiently transduced at the same MOI (Fig. 1 A). In contrast, the lentiviral vector efficiently transduced the EGFP gene into all blood cell subsets tested at an MOI of 10 (Fig. 1 A).

Bottom Line: These results suggest that CD226 is involved in LFA-1-mediated costimulatory signals for triggering naive T cell differentiation and proliferation.We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells.Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Immune Receptor, RIKEN Research Center for Allergy and Immunology, 3-1-1 Koyadai, Ibaraki 305-0074, Japan. ashibuya@rtc.riken.go.jp

ABSTRACT
Upon antigen recognition by the T cell receptor, lymphocyte function-associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12-independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1-mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

Show MeSH
Related in: MedlinePlus