Cutting edge: CD4 is the receptor for the tick saliva immunosuppressor, Salp15

Abstract

Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.

FIGURE 1

Salp15 binds to CD4. A, Left panels, Analysis of His-tagged Salp15-488 binding to CD4⁺ and CD8⁺ T cells by confocal microscopy compared with unstained CD4⁺ T cells (control). The panels on the right show the same field under brightfield microscopy. B, Murine primary CD4⁺T cells lysates containing His-tagged Salp15 were immunoprecipitated (IP) using anti-His, anti-CD3ε, anti-CD28, anti-CD4, anti-TCRβ, or IgG (control). The immunoprecipitate was immunoblotted (IB) using anti-His, anti-CD4, anti-CD3ε, anti-TCRβ, and anti-CD28 Abs. C, Colocalization of anti-CD4 staining and His-tagged Salp15-488 binding on naive and stimulated CD4⁺ T cells is indicated by the yellow color in the merged confocal micrograph. D, Flow cytometric analysis of CD4 expression using PE-Cy5-labeled anti-CD4 (CD4_PE-Cy5) (upper panel) and His-tagged Salp15-488 binding (lower panel) in 3T3 (red) and 3T3-CD4 (blue) cells. E, HeLa and HeLa-CD4 cell lysates containing His-tagged Salp15 were immunoprecipitated using anti-His Ab. The immunoprecipitates were subjected to immunoblotting using anti-CD4 and anti-His Abs. The reciprocal immunoprecipitation from a HeLa-CD4 cell lysate was performed using anti-CD4 or IgG followed by immunoblotting with anti-CD4 or anti-His Abs. F, Immunoprecipitation from HeLa-CD4 cell lysate containing either His-tagged TR-Salp13 (control) or His-tagged Salp15 using an anti-His Ab followed by immunoblotting with anti-CD4 or anti-His Abs.

FIGURE 2

Salp15 binds to the extracellular domains of CD4. A, Unlabeled Salp15 but not lysozyme pretreatment blocks His-tagged Salp15-488 binding to HeLa-CD4 cells. Control represents HeLa-CD4 cells in the absence of His-tagged Salp15-488. Right panel, The brightfield of the image on the left. B, Preincubation of HeLa-CD4 cells with a polyclonal anti-CD4 Ab abolishes His-tagged Salp15-488 binding compared with control IgG pretreatment. C, His-tagged Salp15 (0.4 µM) was incubated with increasing amounts of immobilized sCD4 (D1D2, ■) or lysozyme (▲) in a microtiter assay showing saturable binding. The results are expressed as mean ± SE of three independent experiments. D, Elution profiles of sCD4 (D1–D4) (blue), His-tagged Salp15 (pink), and His-tagged Salp15-sCD4 (red) from Superdex-200 gel filtration columns (left panel). The Gaussian deconvolution of His-tagged Salp15-sCD4 and sCD4 peaks is shown on the right panel. The results shown are representative of three to five individual experiments performed.

FIGURE 3

The C-terminal peptide of Salp15 binds CD4. A, Binding of His-tagged Salp15 (5 µg) and overlapping synthetic peptides of Salp15 (0.5 µg) to sCD4 (D1–D2). B, Increasing concentrations of P11 (■) but not P8 (▲) show saturable binding to sCD4 (D1–D2). C, Competition of increasing concentrations of free P11 with immobilized P11 (50 nmol) for binding to sCD4 (D1–D2). D, Competition of P11 (50 nmol) binding to sCD4 (D1–D2) by increasing concentrations of His-tagged Salp15. Control represents P11 binding in the absence of Salp15. The results are expressed as mean ± SE of at least three independent experiments.

FIGURE 4

Salp15 inhibits early steps during T cell activation. A, Western blots showing the increase in tyrosine phosphorylation of Lck at the residue 505, and decreased phosphorylation of Lck at Tyr³⁹⁴, Zap70 at Tyr³¹⁹, and PLCγ1 at Tyr⁷⁸³ in anti-CD3/CD28-stimulated mouse CD4⁺ T cells in the presence or absence of His-tagged Salp15. B, Representative immunofluorescence micrographs showing CTB₅₉₄ staining in CD4⁺ and CD8⁺ T cells stimulated with anti-CD3/CD28 in the presence or absence of His-tagged Salp15.