Research ArticleImmunology

The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+ T cells

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Science Signaling  10 Jul 2018:
Vol. 11, Issue 538, eaar3083
DOI: 10.1126/scisignal.aar3083
  • Fig. 1 Normal T cell development and functions in Vav1OST mice.

    (A) Representative flow cytometry analysis of thymocytes from wild-type (WT) and Vav1OST mice. Graphs represent total numbers of thymocyte subsets. (B) Representative flow cytometry analysis of cells from spleens of WT and Vav1OST mice. Graphs represent total numbers of lymphocyte subsets. (C) Cellularity of thymus, lymph nodes (LN), and spleen from WT and Vav1OST mice. In (A) to (C), data are presented as means ± SD (n = 5 mice per group). (D) Immunoblot showing the abundance of VAV1 and phosphorylated VAV1 (p-VAV1) before and after pervanadate activation of CD4+ T cells from WT and Vav1OST mice. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a loading control. Data are representative of three independent experiments. (E) Changes in intracellular Ca2+ in WT and Vav1OST CD4+ T cells stimulated with a biotinylated monoclonal antibody recognizing CD3 and treated with streptavidin to cross-link and activate CD3. The arrow indicates the addition of streptavidin. Results are representative of two independent experiments. (F) Immunoblot showing phosphorylated tyrosine (p-Tyr) in equal amounts of total lysates of CD4+ T cells harvested from WT and Vav1OST mice and stimulated with pervanadate for the indicated durations. GAPDH is a loading control. Results are representative of five independent experiments. (G) Immunoblots showing p-Try in equal amounts of OST affinity–purified lysates from WT and Vav1OST CD4+ T cells stimulated with pervanadate for the indicated durations. VAV1 is a loading control. Both short and long exposures of the blot are shown. Results are representative of three independent experiments. (H) Immunoblot showing PLC-γ, VAV1, LAT, and GRB2 in equal amounts of whole-cell lysates and lysates that were subjected to OST affinity purification from WT and Vav1OST CD4+ T cells stimulated with pervanadate for the indicated durations. Results are representative of three independent experiments. Numbers to the right of the blot correspond to the molecular size in kilodaltons.

  • Fig. 2 Analysis of the VAV1 interactome in primary Vav1OST CD4+ T cells.

    (A) Volcano plot of proteins identified as interacting with VAV1 by AP-MS. The mean P value is plotted against the corresponding mean fold change for Vav1OST versus WT samples (enrichment). Proteins were classified as VAV1 interactors (blue) if they displayed enrichment greater than fivefold and P < 0.05. Subsequently, P values were corrected using the Benjamini-Hochberg (BH) method to determine the final VAV1 interactome. The VAV1 bait is shown in red, and dashed lines represent thresholds on P value and enrichment to identify specific VAV1 interactors. CD226 is highlighted in orange. (B) Each protein interacting with the Vav1OST bait is represented as a node linked by an edge to the Vav1OST bait. Proteins are identified by their UniProtKB designation and color-coded according to their function or protein family. (C) Label-free quantitative analysis of the kinetics of the binding of various proteins to VAV1 in mouse CD4+ T cells that were either unstimulated or stimulated for 30, 120, or 300 s with pervanadate. The intensity of the association is shown from minimum (0) to maximum (1). Interacting proteins were clustered using Euclidean distance correlation on the basis of the similarities in their VAV1-binding kinetics.

  • Fig. 3 VAV1 is part of the CD226 signaling pathway in T cells.

    (A) Representative flow cytometry histogram of CD226 surface abundance in control Jurkat cells (J.Ctl) and Jurkat cells overexpressing CD226 (J.CD226) and a graph showing the mean fluorescence intensities (MFI) of CD226 in J.Ctl and J.CD226 cells. Data are representative of five independent experiments. (B) Immunoblot showing p-Tyr and VAV1 phosphorylated on Tyr174 (p-VAV1) in equal amounts of total lysates from J.Ctl and J.CD226 cells that were either unstimulated or stimulated with the antibody that cross-links CD226 molecules for the indicated durations. GAPDH is a loading control. Results are representative of three independent experiments. (C) Immunoblot showing p-VAV1, p-ERK1/2, and total ERK1/2 (ERK1/2) in Jurkat cells treated as in (B). Results are representative of three independent experiments (D) Immunoblot analysis of J.CD226 cells left unstimulated or stimulated with the antibody that cross-links CD226 molecules for the indicated times followed by immunoprecipitation of VAV1 and analysis of immunoprecipitates (IP) and crude lysates (CL) with antibody directed against VAV1 or CD226. Results are representative of three independent experiments (E) Immunoblot analysis of primary human CD4+ T cells left unstimulated or stimulated with the CD226 cross-linking antibody for the indicated times followed by immunoprecipitation of p-VAV1 and analysis of IP and CL with antibody directed against VAV1 or CD226. Results are representative of two independent experiments. (F) Immunoblot analysis of equal amounts of total lysates from J.CD226 cells expressing scrambled small interfering RNA (siRNA; siCTL) or siRNA directed against VAV1 mRNA (siVAV1). Cells were unstimulated or stimulated with the CD226 cross-linking antibody for 2 min. Blots were probed antibodies to VAV1, p-ERK1/2, and the loading control GAPDH. Results are representative of three independent experiments.

  • Fig. 4 VAV1 knockdown reduces the synergistic activity of CD3 and CD226 costimulation in T cells.

    (A) Immunoblot showing p-VAV1 and p-ERK1/2 in equal amounts of total lysates of J.CD226 cells that were unstimulated or stimulated with increasing amounts of an antibody that cross-links CD3 molecules associated with either a control antibody or an antibody that cross-links CD226 molecules for 2 min. GAPDH is a loading control. Results are representative of three independent experiments. (B) Immunoblot showing p-VAV1, p-ERK1/2, and total ERK1/2 in equal amounts of total lysates of J.CD226 cells expressing scrambled siRNA (siCTL) or siRNA directed against VAV1 mRNA (siVAV1). Cells were unstimulated, costimulated with antibodies that cross-link the CD226 and CD3 molecules (CD3+CD226), or stimulated with an antibody that cross-links either the CD3 (CD3) or the CD226 (CD226) molecules only for the indicated times, and lysates were probed with antibodies recognizing VAV1, p-ERK1/2, or total ERK1/2. The graph shows the relative abundances of p-ERK1/2, determined as a ratio of intensity of CD3+CD226 costimulation to that of CD3 stimulation alone at 2 min. Data are from three independent experiments. (C) Immunoblot analysis of equal amounts of proteins from total lysates of primary human CD4+ T cells expressing siCTL or siVAV1. Cells were treated as in (B), and lysates were immunoblotted to show VAV1, p-ERK1/2, total ERK1/2, phosphorylated AKT (p-AKT), and total AKT. The graph shows the relative abundances of p-ERK1/2 and p-AKT, determined as a ratio of intensity of CD3+CD226 costimulation to that of CD3 stimulation alone at 2 min. Data are from three independent experiments.

  • Fig. 5 VAV1 controls CD226-induced IL-17 secretion.

    (A) Secretion of IL-17, IL-10, GM-CSF, IFN-γ, IL-5, and IL-13 was quantified in supernatants of primary human CD4+ T cells stimulated with CD3 antibody + immunoglobulin G1 (IgG1) isotype control (CD3) or CD3 and CD226 (CD3+CD226) antibodies for 3 days using cytometric beads array assay and expressed as picograms per milliliter. (B) Secretion of IL-17, IL-10, GM-CSF, IFN-γ, IL-5, and IL-13 was quantified in supernatants of primary human CD4+ T cells expressing scrambled siRNA (siCTL) or siRNA directed against VAV1 mRNA (siVAV1) and stimulated with CD3 antibody + IgG1 isotype control (CD3) or CD3 and CD226 (CD3+CD226) antibodies for 3 days. The data are represented as ratio of cytokine production between CD3+CD226 and CD3 stimulations. All data are from eight unrelated donors. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

  • Fig. 6 The CD226 G307S variant enhances CD226-driven IL-17 production and VAV1 activation.

    (A) Cytokine secretion was quantified in supernatants of primary human CD4+CD45RA T cells expressing the Gly307 or the Ser307 variants of CD226, using enzyme-linked immunosorbent assays for IL-17 and IFN-γ (11 donors) or cytometric beads array assays for IL-5, IL-10, IL-13, and GM-CSF (14 donors). Cells were stimulated with antibodies that cross-link CD3 and CD226 molecules (CD3+CD226) or antibodies that cross-link only CD3 molecules (CD3) for 5 days. (B) Representative flow cytometry analysis of cells stained to show intracellular IL-17 and IFN-γ in primary human CD4+CD45RA T cells expressing the Gly307 or the Ser307 variants of CD226. Cells were stimulated as indicated in (A). The graphs summarize the data obtained from 20 unrelated donors. (C) Phosphoflow analysis of ERK, AKT, and VAV1 phosphorylation (p-ERK, p-AKT, and p-VAV1) in CD4+ T cells that were unstimulated (NS) or stimulated with antibodies that cross-link CD3 and CD226 molecules (CD3+CD226) or antibodies that cross-link only CD3 molecules (CD3). Graphs represent data from four individual donors per genotype and are representative of three independent experiments. (D) Jurkat cells expressing the indicated CD226 variants or an empty vector (Mock) were stimulated by antibodies that cross-link the CD226 molecules for the indicated times, and the amount of p-VAV1 was determined by Western blot analysis. The graph shows the relative abundance of p-VAV1, determined as a ratio of the intensity of p-VAV1 to that of GAPDH during the time course of the experiment. The histogram shows the amount of CD226 Gly307 and CD226 Ser307 on the transfected Jurkat cells and are representative of two independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; n.s., not significant.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/538/eaar3083/DC1

    Fig. S1. Structure of the 3′ end of the wild-type VAV1 allele and the VAV1OST allele.

    Fig. S2. Assessment of biological and technical variability across samples.

    Fig. S3. The VAV1 interactome.

    Fig. S4. The synergistic effect of CD3 and CD226 co-engagement is not observed in naïve CD4+ T cells.

    Table S1. List of proteins associated with VAV1 in resting and pervanadate-activated CD4+ T cells.

  • The PDF file includes:

    • Fig. S1. Structure of the 3′ end of the wild-type VAV1 allele and the VAV1OST allele.
    • Fig. S2. Assessment of biological and technical variability across samples.
    • Fig. S3. The VAV1 interactome.
    • Fig. S4. The synergistic effect of CD3 and CD226 co-engagement is not observed in naïve CD4+ T cells.
    • Legend for table S1

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). List of proteins associated with VAV1 in resting and pervanadate-activated CD4+ T cells.

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