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

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.

Figure 1.

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).

Figure 1.

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).

Figure 1.

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).

Figure 2.

Stimulation of CD3 and LFA-1 induces Th1 development from naive CD4⁺ Ths. (A–C) CD4 naive T cells were stimulated with plate-coated anti-CD3 alone or anti-CD3 plus mAbs indicated on days 1 and 8 and cultured for 14 d in IL-2–containing medium. Intracellular IFN-γ, IL-4, and IL-13 syntheses were analyzed in these cells by flow cytometry (A and B). Cytokine concentrations in culture supernatants were also analyzed by ELISA (C). (D) CD4⁺ naive T cells were stimulated with plate-coated anti-CD3 plus either plate-coated anti-CD18 or 10 ng/ml soluble IL-12 on days 1 and 8 and cultured for 14 d in the presence or absence of 40 μg/ml anti–IL-12.

Figure 2.

Stimulation of CD3 and LFA-1 induces Th1 development from naive CD4⁺ Ths. (A–C) CD4 naive T cells were stimulated with plate-coated anti-CD3 alone or anti-CD3 plus mAbs indicated on days 1 and 8 and cultured for 14 d in IL-2–containing medium. Intracellular IFN-γ, IL-4, and IL-13 syntheses were analyzed in these cells by flow cytometry (A and B). Cytokine concentrations in culture supernatants were also analyzed by ELISA (C). (D) CD4⁺ naive T cells were stimulated with plate-coated anti-CD3 plus either plate-coated anti-CD18 or 10 ng/ml soluble IL-12 on days 1 and 8 and cultured for 14 d in the presence or absence of 40 μg/ml anti–IL-12.

Figure 3.

CD226 is involved in LFA-1 signal for Th1 development from naive CD4⁺ T cells. (A) Naive CD4⁺ T cells were stimulated with plate-coated mAbs as indicated for 2 min and were lysed in 1% NP-40 lysis buffer. The lysates were immunoprecipitated with anti-CD226 and analyzed by immunoblotting with anti-phosphotyrosine mAb or anti-CD226. (B–F) Naive CD4⁺ T cells were infected for 72 h with the lentiviral vector containing Flag-tagged WT, Y-F ³²² CD226-IRES-hrGFP, or a mock control vector containing hrGFP alone. (B) The naive T cells were then stimulated with anti-CD3 plus anti-CD18 mAbs or control Ig for 2 min and were then lysed in 1% NP-40 lysis buffer. CD226 transduced by lentiviral vector was immunoprecipitated with anti-Flag mAb and immunoblotted with anti-phosphotyrosine mAb or anti-Flag. (C–F) The naive T cells expressing hrGFP after infection were sorted by flow cytometry and stimulated with anti-CD3 and anti-CD18 for 7 d in the presence of IL-2. These stimulated T cells were then lysed, immunoprecipitated with control Ig or anti-CD226, and analyzed by immunoblotting with anti-phosphotyrosine or control Ig (C), or were stained with anti-Flag mAb (D). These T cells were also further stimulated with anti-CD3 and anti-CD28 for 48 h and analyzed for intracellular IFN-γ (E) by flow cytometry. Cytokine concentrations in culture supernatants after 48 h of restimulation were analyzed by ELISA (F).

Figure 3.

CD226 is involved in LFA-1 signal for Th1 development from naive CD4⁺ T cells. (A) Naive CD4⁺ T cells were stimulated with plate-coated mAbs as indicated for 2 min and were lysed in 1% NP-40 lysis buffer. The lysates were immunoprecipitated with anti-CD226 and analyzed by immunoblotting with anti-phosphotyrosine mAb or anti-CD226. (B–F) Naive CD4⁺ T cells were infected for 72 h with the lentiviral vector containing Flag-tagged WT, Y-F ³²² CD226-IRES-hrGFP, or a mock control vector containing hrGFP alone. (B) The naive T cells were then stimulated with anti-CD3 plus anti-CD18 mAbs or control Ig for 2 min and were then lysed in 1% NP-40 lysis buffer. CD226 transduced by lentiviral vector was immunoprecipitated with anti-Flag mAb and immunoblotted with anti-phosphotyrosine mAb or anti-Flag. (C–F) The naive T cells expressing hrGFP after infection were sorted by flow cytometry and stimulated with anti-CD3 and anti-CD18 for 7 d in the presence of IL-2. These stimulated T cells were then lysed, immunoprecipitated with control Ig or anti-CD226, and analyzed by immunoblotting with anti-phosphotyrosine or control Ig (C), or were stained with anti-Flag mAb (D). These T cells were also further stimulated with anti-CD3 and anti-CD28 for 48 h and analyzed for intracellular IFN-γ (E) by flow cytometry. Cytokine concentrations in culture supernatants after 48 h of restimulation were analyzed by ELISA (F).

Figure 3.

CD226 is involved in LFA-1 signal for Th1 development from naive CD4⁺ T cells. (A) Naive CD4⁺ T cells were stimulated with plate-coated mAbs as indicated for 2 min and were lysed in 1% NP-40 lysis buffer. The lysates were immunoprecipitated with anti-CD226 and analyzed by immunoblotting with anti-phosphotyrosine mAb or anti-CD226. (B–F) Naive CD4⁺ T cells were infected for 72 h with the lentiviral vector containing Flag-tagged WT, Y-F ³²² CD226-IRES-hrGFP, or a mock control vector containing hrGFP alone. (B) The naive T cells were then stimulated with anti-CD3 plus anti-CD18 mAbs or control Ig for 2 min and were then lysed in 1% NP-40 lysis buffer. CD226 transduced by lentiviral vector was immunoprecipitated with anti-Flag mAb and immunoblotted with anti-phosphotyrosine mAb or anti-Flag. (C–F) The naive T cells expressing hrGFP after infection were sorted by flow cytometry and stimulated with anti-CD3 and anti-CD18 for 7 d in the presence of IL-2. These stimulated T cells were then lysed, immunoprecipitated with control Ig or anti-CD226, and analyzed by immunoblotting with anti-phosphotyrosine or control Ig (C), or were stained with anti-Flag mAb (D). These T cells were also further stimulated with anti-CD3 and anti-CD28 for 48 h and analyzed for intracellular IFN-γ (E) by flow cytometry. Cytokine concentrations in culture supernatants after 48 h of restimulation were analyzed by ELISA (F).

Figure 4.

CD226 is involved in LFA-1 signal for CD4⁺ and CD8⁺ naive T cell proliferation. Naive CD4⁺ and CD8⁺ T cells were infected for 72 h with the lentiviral vector containing Flag-tagged WT, Y-F ³²² CD226-IRES-hrGFP, or a mock control vector containing hrGFP alone. The naive T cells were stained with anti-Flag mAb and analyzed by flow cytometry (A). The naive CD4⁺ and CD8⁺ T cells after infection were also stimulated with plate-coated mAbs indicated for 48 h in the presence (B) or absence (B and C) of IL-2. Cell proliferations (B) and IL-2 production in culture supernatants (C) were analyzed by BrdU uptake and ELISA, respectively. IL-2 was not detected in culture of CD8⁺ naive T cells after stimulation with any mAbs (not depicted).

Figure 4.

CD226 is involved in LFA-1 signal for CD4⁺ and CD8⁺ naive T cell proliferation. Naive CD4⁺ and CD8⁺ T cells were infected for 72 h with the lentiviral vector containing Flag-tagged WT, Y-F ³²² CD226-IRES-hrGFP, or a mock control vector containing hrGFP alone. The naive T cells were stained with anti-Flag mAb and analyzed by flow cytometry (A). The naive CD4⁺ and CD8⁺ T cells after infection were also stimulated with plate-coated mAbs indicated for 48 h in the presence (B) or absence (B and C) of IL-2. Cell proliferations (B) and IL-2 production in culture supernatants (C) were analyzed by BrdU uptake and ELISA, respectively. IL-2 was not detected in culture of CD8⁺ naive T cells after stimulation with any mAbs (not depicted).

Figure 5.

Relationship of LFA-1 signaling molecules with the lipid raft. (A) CD4⁺ and CD8⁺ T cells were incubated with beads precoated with anti-CD3 plus either anti-CD28, anti-CD11a, or anti-CD18 for 30 min at 37°C and then stained with FITC-conjugated cholela toxin subunit B (GM1), Alexa 594–conjugated anti-CD11a (LFA-1), and anti-CD226 mAbs. T cells were also stained with rabbit anti–Fyn polyclonal antibody, followed with Alexa 594–coupled anti–rabbit secondary antibodies and analyzed by confocal laser scanning microscopy. (B) CD4⁺ and CD8⁺ PB T cells were treated or not with MβCD at 10 mM for 30 min at 37°C. Cells were then stimulated with control Ig or anti-CD3 for 5 min and lysed in 1% digitonin buffer. The lysates were immunoprecipitated with anti-CD18 or control Ig and analyzed by immunoblotting with anti-CD226. (C and D) CD4⁺ and CD8⁺ PB T cells were labeled (C) or not (D) with CFSE and treated or not with MβCD at 10 mM for 30 min at 37°C. Cells were then stimulated or not with plastic-coated mAbs as indicated for 3 d and analyzed for proliferation by flow cytometry (C) or BrdU uptake (D).

Figure 5.

Relationship of LFA-1 signaling molecules with the lipid raft. (A) CD4⁺ and CD8⁺ T cells were incubated with beads precoated with anti-CD3 plus either anti-CD28, anti-CD11a, or anti-CD18 for 30 min at 37°C and then stained with FITC-conjugated cholela toxin subunit B (GM1), Alexa 594–conjugated anti-CD11a (LFA-1), and anti-CD226 mAbs. T cells were also stained with rabbit anti–Fyn polyclonal antibody, followed with Alexa 594–coupled anti–rabbit secondary antibodies and analyzed by confocal laser scanning microscopy. (B) CD4⁺ and CD8⁺ PB T cells were treated or not with MβCD at 10 mM for 30 min at 37°C. Cells were then stimulated with control Ig or anti-CD3 for 5 min and lysed in 1% digitonin buffer. The lysates were immunoprecipitated with anti-CD18 or control Ig and analyzed by immunoblotting with anti-CD226. (C and D) CD4⁺ and CD8⁺ PB T cells were labeled (C) or not (D) with CFSE and treated or not with MβCD at 10 mM for 30 min at 37°C. Cells were then stimulated or not with plastic-coated mAbs as indicated for 3 d and analyzed for proliferation by flow cytometry (C) or BrdU uptake (D).

Figure 5.

Relationship of LFA-1 signaling molecules with the lipid raft. (A) CD4⁺ and CD8⁺ T cells were incubated with beads precoated with anti-CD3 plus either anti-CD28, anti-CD11a, or anti-CD18 for 30 min at 37°C and then stained with FITC-conjugated cholela toxin subunit B (GM1), Alexa 594–conjugated anti-CD11a (LFA-1), and anti-CD226 mAbs. T cells were also stained with rabbit anti–Fyn polyclonal antibody, followed with Alexa 594–coupled anti–rabbit secondary antibodies and analyzed by confocal laser scanning microscopy. (B) CD4⁺ and CD8⁺ PB T cells were treated or not with MβCD at 10 mM for 30 min at 37°C. Cells were then stimulated with control Ig or anti-CD3 for 5 min and lysed in 1% digitonin buffer. The lysates were immunoprecipitated with anti-CD18 or control Ig and analyzed by immunoblotting with anti-CD226. (C and D) CD4⁺ and CD8⁺ PB T cells were labeled (C) or not (D) with CFSE and treated or not with MβCD at 10 mM for 30 min at 37°C. Cells were then stimulated or not with plastic-coated mAbs as indicated for 3 d and analyzed for proliferation by flow cytometry (C) or BrdU uptake (D).

Figure 5.

Relationship of LFA-1 signaling molecules with the lipid raft. (A) CD4⁺ and CD8⁺ T cells were incubated with beads precoated with anti-CD3 plus either anti-CD28, anti-CD11a, or anti-CD18 for 30 min at 37°C and then stained with FITC-conjugated cholela toxin subunit B (GM1), Alexa 594–conjugated anti-CD11a (LFA-1), and anti-CD226 mAbs. T cells were also stained with rabbit anti–Fyn polyclonal antibody, followed with Alexa 594–coupled anti–rabbit secondary antibodies and analyzed by confocal laser scanning microscopy. (B) CD4⁺ and CD8⁺ PB T cells were treated or not with MβCD at 10 mM for 30 min at 37°C. Cells were then stimulated with control Ig or anti-CD3 for 5 min and lysed in 1% digitonin buffer. The lysates were immunoprecipitated with anti-CD18 or control Ig and analyzed by immunoblotting with anti-CD226. (C and D) CD4⁺ and CD8⁺ PB T cells were labeled (C) or not (D) with CFSE and treated or not with MβCD at 10 mM for 30 min at 37°C. Cells were then stimulated or not with plastic-coated mAbs as indicated for 3 d and analyzed for proliferation by flow cytometry (C) or BrdU uptake (D).