T cell Ig and mucin 1 (TIM-1) is expressed on in vivo-activated T cells and provides a costimulatory signal for T cell activation

Abstract

Polymorphisms in TIM-1, a member of the T cell Ig and mucin (TIM) domain family, are associated with relative susceptibility to the development of T helper 2-dominated immune responses such as in allergic asthma. Recent data have also suggested that ligation of TIM-1 can augment T cell activation. We have found that the TIM-1 protein is expressed on CD4(+) T cells in vivo after intranasal immunization. Ectopic expression of TIM-1 during T cell differentiation results in a significant increase in the number of cells producing IL-4 but not IFN-gamma. Furthermore, TIM-1 expression provides a costimulatory signal that increases transcription from the IL-4 promoter and from isolated nuclear factor of activated T cells/activating protein-1 (NFAT/AP-1) elements. Finally, we provide evidence that TIM-1 can be phosphorylated on tyrosine and that TIM-1 costimulation requires its cytoplasmic tail and the conserved tyrosine within that domain. These results constitute evidence that TIM-1 directly couples to phosphotyrosine-dependent intracellular signaling pathways.

Fig. 1.

TIM-1 is expressed on lung-draining lymph node CD4 T cells under conditions of airway tolerance and inflammation. Mice were immunized as described in Materials and Methods, with OVA alone to induce tolerance (Left) or with OVA plus cholera toxin to induce inflammation (Right). Five days after the last intranasal treatment, lung-draining lymph nodes were harvested, and cells were stained with antibodies to CD4 and TIM-1. Results are gated on CD4⁺ cells.

Fig. 2.

Costimulation of Th2 differentiation and cytokine transcription by TIM-1. (A) Purified T cells were stimulated in vitro under neutral conditions, then infected with control MSCV-GFP retrovirus (“vector”) or virus encoding Flag-tagged murine TIM-1. Cells were rested and restimulated with anti-CD3 antibody, then stained for intracellular cytokines. Results are the percentage of cytokine-positive cells of the GFP⁺ population in each case and are the average ± SD of six samples from three experiments. P values were derived from Student's t test analyses. (B) D10 T cells were transfected with an IL-4 promoter luciferase reporter and either an empty vector or Flag-TIM-1. The next day, cells were stimulated as indicated (PMA, phorbol 12-myristate 13-acetate) and analyzed for luciferase activity. Results are presented as relative light units (mean ± SD) of four samples, from two separate experiments. Student's t tests were performed: *, P < 0.05; **, P < 0.01. (C) Jurkat T cells were transfected with a murine IFN-γ promoter luciferase construct, along with either an empty vector or Flag-TIM-1 plasmid. Cells were stimulated as indicated (TCR, T cell receptor) and luciferase activity was determined. Results are the mean response, normalized to PMA/ionomycin (2,522 relative light units for vector; 2,111 for TIM-1) for duplicate points of a single experiment, representative of the three performed.

Fig. 3.

TIM-1 costimulates NFAT/AP-1-dependent transcription. D10 (A) or Jurkat (B) T cells were transfected with an NFAT/AP-1 luciferase reporter and the indicated plasmids. Cells were treated the next day with the indicated stimuli and analyzed for luciferase activity, which is expressed as the percentage (mean ± SD) of the response with PMA/ionomycin from triplicate points of a single experiment. (A) NFAT/AP-1 activity in D10 T cells transfected with empty vector or Flag-TIM-1. Data displayed are representative of three experiments that were performed. PMA/ionomycin values were 2,137 relative light units for vector and 2,603 for TIM-1-transfected cells. (B) NFAT/AP-1 activity in Jurkat cells transfected with empty vector, Flag-DAP10, or Flag-TIM-1. Data displayed are the means of triplicate points from a single experiment, representative of >10 experiments. PMA/ionomycin values were 32,186 relative light units for vector, 59,935 for DAP10, and 59,373 for TIM-1-transfected cells.

Fig. 4.

The TIM-1 cytoplasmic tail is required for costimulation of transcription. D10 or Jurkat T cells were transfected with the NFAT/AP-1 luciferase reporter and either the empty vector, Flag-TIM-1, or the truncated Flag-TIM-1, lacking the cytoplasmic tail (“delta cyto”). (A) Expression of the Flag-TIM-1 constructs on transfected D10 cells. (B) Luciferase activity of unstimulated or anti-CD3/CD4-stimulated D10 cells transfected with the indicated constructs. Results shown are the mean of duplicate points from a single experiment, representative of four that were performed. (C) Luciferase activity in Jurkat T cells transfected with the same constructs as in B. Luciferase activity is expressed as the percentage (mean ± SD) of the response with PMA/ionomycin from triplicate points of a single experiment, representative of eight that were performed. PMA/ionomycin values were 139,864 relative light units for vector, 49,773 for TIM-1, and 81,540 for Δ-cyto TIM-1-transfected cells.

Fig. 5.

TIM-1 tyrosine phosphorylation and requirement for tyrosine-276 in costimulation of NFAT. (A) Jurkat or D10 T cells were transfected with Flag-TIM-1. The next day, transfected cells were divided and left unstimulated or stimulated with pervanadate as described in ref. . Immunoprecipitations were performed with anti-Flag mAb, then the precipitates were separated by SDS/PAGE and Western-blotted. Blots were probed with anti-Flag (Lower), then stripped and reprobed with anti-phosphotyrosine antibody 4G10 (Upper). Jurkat (B) or D10 (C) cells were transfected with NFAT/AP-1-luciferase plus empty vector, wild type Flag-TIM-1, or Flag-TIM-1 (Y-F). Luciferase activity is expressed as the percentage of the PMA/ionomycin response from triplicate samples (mean ± SD) of a single experiment, representative of five that were performed.