T cell Ig and mucin domain-1-mediated T cell activation requires recruitment and activation of phosphoinositide 3-kinase

Abstract

Ligation of the transmembrane protein T cell Ig and mucin domain (Tim)-1 can costimulate T cell activation. Agonistic Abs to Tim-1 are also capable of inducing T cell activation without additional stimuli. However, little is known about the biochemical mechanisms underlying T cell stimulation or costimulation through Tim-1. We show that a tyrosine in Tim-1 becomes phosphorylated in a lck-dependent manner, whereupon it can directly recruit p85 adaptor subunits of PI3K. This results in PI3K activation, which is required for Tim-1 function. We also provide genetic evidence that p85 expression is required for optimal Tim-1 function. Thus, we describe a pathway from Tim-1 tyrosine phosphorylation to the PI3K signaling pathway, which appears to be a major effector of Tim-1-mediated T cell activation.

Figure 1. Role of Lck in Tim-1 tyrosine phosphorylation and function

(A) Jurkat T cells expressing Flag-Tim-1 were stimulated as indicated, with or without the Src kinase inhibitor PP2. Anti-Flag IP's were separated by SDS-PAGE and western blotted with anti-Flag (lower panel), then stripped and re-probed with anti-phosphotyrosine antibody 4G10 (P-Tyr - upper panel). (B) Lck-deficient JCaM1 or Lck-reconstituted JCaM1 cells expressing Flag-Tim-1 were stimulated and analyzed as in part A. (C) JCaM1 or JCaM1-Lck cells were transfected with an NFAT/AP-1 luciferase reporter and either empty vector or Flag-Tim-1. Cells were stimulated the next day and luciferase activity was determined. Each experiment is representative of two (A-B) or three (C) that were performed.

Figure 2. Specificity of SH2 domain binding to phosphorylated Y276 in the Tim-1 cytoplasmic tail

(A) Sequence of the peptide, and modifications, used to determine the SH2 specificity of phopsho-Y276. (B) Layout of the SH2 domain array. SH2 domains are present as duplicate spots. For proteins with more than one SH2 domain, “D1” indicates the more N-terminal SH2. (C) Results of the SH2 domain array screen, using the peptide illustrated in part A. The dashed box indicates the location of negative control spots; the bottom and right edge contain positive control spots. These results are representative of those obtained in four separate experiments, using two different lots of membrane. (D) Competitive inhibition of Tim-1 peptide binding by the phosphotyrosine analogue phenylphosphate. The blot shown is representative of two experiments.

Figure 3. Recruitment and activation of PI3 kinase by Tim-1

(A) Jurkat T cells expressing Flag-Tim-1 were stimulated as indicated, lysed and IP'd with anti-Flag. Whole cell lysates (WCLs) and IP's were separated by SDS-PAGE and western blotted for p85α/β. (B) Jurkat T cells expressing the indicated wild-type or mutant Flag-Tim-1 constructs were stimulated and analyzed for p85 binding as described for part A. (C) Jurkat T cells expressing wild-type Flag-Tim-1 were stimulated in the presence of 10 μM PP2 (Src kinase inhibitor) or 10 μM Ly294002 (PI3k inhibitor) then analyzed for p85 binding as above. (D) T cell blasts (left panels) or D10 Th2 T cells (right panels) were stimulated as indicated. Lysates were separated by SDS-PAGE and western blotted with a rabbit mAb to phospho-Akt (S473; upper panels). Blots were stripped and re-probed with a mouse mAb to β-actin (lower panels), to control for protein loading. The location of phospho-Akt is indicated by the arrows, while the star indicates a non-specific band that is only observed in primary T cells. Results in each part are representative of three experiments.

Figure 4. Role of the PI3K pathway in Tim-1 activation of NFAT/AP-1

T cells were transfected with an NFAT/AP-1 luciferase reporter and the indicated constructs, then stimulated the next day, as indicated, followed by determination of luciferase activity. (A) Cells were stimulated in the presence or absence of the PI3K inhibitor LY294002. (B) Cells were stimulated in the presence or absence of the Akt inhibitor Akti 1/2 (20 μM). (C-D) T cells were transfected with an NFAT/AP-1 reporter, plus either empty vector or Flag-Tim1, and the indicated amounts of siRNA SmartPool oligos (Dharmacon) specific for p85α and p85β. Twenty-four hours after transfection, cells were stimulated for six hours, followed by determination of luciferase activity. Error bars indicate standard deviation for triplicate points in a single experiment. Results shown in each part are representative of three experiments.

Figure 5. PI3K activity is required for upregulation of early activation markers by endogenous Tim-1 on primary T cells

Purified CD4+ T cells from C57 Bl/6 spleen and lymph node were stimulated for 24 hours with the indicated concentrations of anti-CD3 antibody (and a fixed concentration of anti-CD28 antibody - 0.5 μg/ml), with or without anti-Tim-1 antibody (6 μg/ml), in the presence or absence of PI3K inhibitor LY294002 (10 μM). Cells were stained with fluorescently labeled antibodies to CD69 (A) or CD25 (B) and analyzed by flow cytometry. Results in each panel are representative of three experiments that were performed.

Figure 6. p85 is required for Tim-1-mediated co-stimulation of CD69 expression

(A) Expression of Tim-1 on resting or activated CD4+ T cells from spleens of p85β or p85α/β knockout mice. Splenocytes from p85β (B) or p85α/β (C) deficient mice were stimulated overnight with various concentrations anti-CD3 antibody (0.05, 0.2 and 1 μg/ml), along with a fixed concentration of anti-CD28 (1 μg/ml). Anti-Tim-1 antibody was also added to some cultures, at a concentration of 8 μg/ml. Cells were stained the next day for CD4 and CD69 and analyzed by flow cytometry. Results shown were gated on CD4+ cells.

Figure 7. p85 is required for Tim-1-mediated CD69 and CD25 upregulation

Purified CD4+ T cells from p85β or p85α/β deficient mice were stimulated in vitro with anti-CD3/CD28 antibodies (20 ng/ml and 0.5 μg/ml, respectively) and the indicated concentrations of Tim-1 antibody (A-D) or with anti-Tim-1 antibody alone (E-F). Cells were harvested and stained for CD69 (A,C,E) or CD25 (B,D,F) expression, and analyzed by flow cytometry. Results in A-B and E-F are single experiments, representative of three, or two, separate experiments, respectively. Panels C-D show effects of Tim-1 co-stimulation (+/- S.D.) averaged over those experiments, with p values indicated.

Figure 8. p85 is required for Tim-1-mediated IL-2 production

Purified CD4+ T cells from p85β or p85α/β deficient mice were stimulated in vitro with anti-CD3/CD28 antibodies (20 ng/ml and 0.5 μg/ml, respectively) and the indicated concentrations of Tim-1 antibody (A) or with anti-Tim-1 antibody alone (B). Supernatants were harvested after one day and analyzed by ELISA for IL-2. Error bars (obscured by the marker is most cases) indicate standard deviation for triplicate points within a single experiment, which is representative of three that were performed.