Tim-1 signaling substitutes for conventional signal 1 and requires costimulation to induce T cell proliferation

Abstract

Differentiation and clonal expansion of Ag-activated naive T cells play a pivotal role in the adaptive immune response. T cell Ig mucin (Tim) proteins influence the activation and differentiation of T cells. Tim-3 and Tim-2 clearly regulate Th1 and Th2 responses, respectively, but the precise influence of Tim-1 on T cell activation remains to be determined. We now show that Tim-1 stimulation in vivo and in vitro induces polyclonal activation of T cells despite absence of a conventional TCR-dependent signal 1. In this model, Tim-1-induced proliferation is dependent on strong signal 2 costimulation provided by mature dendritic cells. Ligation of Tim-1 upon CD4(+) T cells with an agonist anti-Tim-1 mAb elicits a rise in free cytosolic calcium, calcineurin-dependent nuclear translocation of NF-AT, and transcription of IL-2. Because Tim-4, the Tim-1 ligand, is expressed by mature dendritic cells, we propose that interaction between Tim-1(+) T cells and Tim-4(+) dendritic cells might ensure optimal stimulation of T cells, when TCR-derived signals originating within an inflamed environment are weak or waning.

FIGURE 1

Anti-Tim-1 mAb plus mature DC stimulate naive T cells to proliferate in the absence of MHC class II-derived signals. Naive CD4⁺ CD25⁻ T cells were cultured with mature syngeneic BM-DC from wild-type mice (A) or MHC class II (I-A^b)-deficient mice (B), in the presence of increasing doses of anti-Tim-1 mAb or isotype control mAb. Tritiated thymidine incorporation (cpm) was measured after 48 h of culture. Bars represent the mean ± SEM of one of four consecutive experiments yielding similar results.

FIGURE 2

Anti-Tim-1-induced T cell proliferation requires costimulatory signals. Naive CD4⁺CD25⁻ T cells were cultured in medium containing various doses of anti-Tim-1 mAb or isotype control mAb with immature or mature syngeneic BM-DC purified from MHC class II KO mice (A). Naive CD4⁺CD25⁻ T cells were cultured with mature syngeneic BM-DC from CD40 KO mice (B), CD80 plus CD86 double-KO mice (C), or OX40L KO mice (D). Tritiated thymidine incorporation (cpm) was measured after 48 h of culture. Bars represent the mean ± SEM of one of three consecutive experiments yielding similar results.

FIGURE 3

Fixed mature BM-DC function in the anti-Tim-1 mAb proliferation assay. Naive CD4⁺CD25⁻ T cells purified from C57BL/6 mice were cultured with immature or mature MHC class II-deficient DC, in the presence of increasing dose of anti-Tim-1 mAb. Immature and mature MHC class II-deficient DC were fixed with 2% paraformaldehyde in PBS prior addition to the culture. Tritiated thymidine incorporation (cpm) was measured after 48 h of culture. Bars represent the mean ± SEM of one of three consecutive experiments yielding similar results.

FIGURE 4

Activation of Tim-1 on the surface of T cells directly delivers a positive signal that triggers Ca²⁺/calmodulin/NF-AT pathway. A, Naive CD4⁺CD25⁻ T were labeled with fluo-3-AM and IgG2a or agonist anti-Tim-1 mAb was added at the time indicated by (↓). Data are representative of three consecutive experiments yielding similar results. B, Naive CD4⁺CD25⁻ T were cultured with mature syngeneic BM-DC and anti-Tim-1 mAb in the presence or in the absence of cyclosporine (100 ng/ml). Tritiated thymidine incorporation (cpm) was measured after 48 h of culture. Bars represent the mean ± SEM of one of three consecutive experiments yielding similar results. C, Naive CD4⁺CD25⁻ T were cultured with mature syngeneic BM-DC and anti-Tim-1 mAb or isotype control mAb in the presence or absence of cyclosporine (100 ng/ml) for 21 h. NF-AT activity was measured from the nuclear extract of the various experimental settings. Bars represent the mean ± SEM of one of three consecutive experiments yielding similar results. *, p < 0.05 (Mann-Whitney U test). D, Naive CD4⁺CD25⁻ T were cultured with mature allogeneic BM-DC in the presence of IgG2a isotype control mAb or anti-Tim-1 mAb (5 μg/ml), and in the absence or the presence of cyclosporine (100 ng/ml), for 2 days. Expression of IL-2 transcripts was then quantified by quantitative RT-PCR. Bars represent the mean ± SEM of three consecutive experiments.

FIGURE 5

In vivo stimulation of Tim-1 induces polyclonal T cell proliferation. CFSE-labeled splenocytes from C57BL/6 mice were injected into irradiated allogeneic DBA/2 hosts treated with agonist anti-Tim-1 mAb or IgG2a control mAb (250 μg/ml). Three days later, proliferation was assessed by analysis of the CFSE profile of CFSE-labeled CD4⁺ T cells (A). Proliferative T cells that are programmed for apoptosis were identified by flow cytometry as the annexin V⁺ CFSE-labeled CD4⁺ T cells (B). Data are representative of four consecutive experiments yielding similar results.

FIGURE 6

In vivo Tim-1-induced CD4⁺ T cell proliferation occurs in the absence of MHC class II-derived signals. CFSE-labeled splenocytes from C57BL/6 CD45.1 mice were injected into nonirradiated or irradiated C57BL/6 CD45.2 hosts treated with an agonist anti-Tim-1 mAb or the IgG2a control mAb. Three days later, proliferation was assessed by analysis of the CFSE profile of CFSE-labeled CD4⁺ CD45.1⁺ T cells. Data are representative of four consecutive experiments yielding similar results.