Differential engagement of Tim-1 during activation can positively or negatively costimulate T cell expansion and effector function

Abstract

It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed on T cells serves to positively costimulate T cell responses. However, crosslinking of Tim-1 by its ligand Tim-4 resulted in either activation or inhibition of T cell responses, thus raising the issue of whether Tim-1 can have a dual function as a costimulator. To resolve this issue, we tested a series of monoclonal antibodies specific for Tim-1 and identified two antibodies that showed opposite functional effects. One anti-Tim-1 antibody increased the frequency of antigen-specific T cells, the production of the proinflammatory cytokines IFN-gamma and IL-17, and the severity of experimental autoimmune encephalomyelitis. In contrast, another anti-Tim-1 antibody inhibited the generation of antigen-specific T cells, production of IFN-gamma and IL-17, and development of autoimmunity, and it caused a strong Th2 response. Both antibodies bound to closely related epitopes in the IgV domain of the Tim-1 molecule, but the activating antibody had an avidity for Tim-1 that was 17 times higher than the inhibitory antibody. Although both anti-Tim-1 antibodies induced CD3 capping, only the activating antibody caused strong cytoskeletal reorganization and motility. These data indicate that Tim-1 regulates T cell responses and that Tim-1 engagement can alter T cell function depending on the affinity/avidity with which it is engaged.

Figure 1.

Opposite effects of the anti–Tim-1 antibodies 3B3 and RMT1-10 on T cell responses. (A) Anti–Tim-1 antibody 3B3 enhances, whereas RMT1-10 inhibits, antigen-specific T cell proliferation. Spleen cells from PLP_139-151-immunized SJL mice were cultured in vitro with different concentrations of PLP_139-151 plus 10 μg/ml RMT1-10, 3B3, or rIgG2a. Proliferation was measured after 48 h by ³[H]thymidine incorporation. The mean ± SEM of three independent experiments are shown. *, P < 0.0001; **, P < 0.01, relative to isotype control. (B) Treatment with RMT1-10 skews the Th1/Th17 responses toward a Th2 response. Spleen cells from PLP_139-151-immunized SJL mice were cultured in vitro with 1 μg/ml PLP_139-151 plus 10 μg/ml RMT1-10, 3B3, or rIgG2a. Supernatants were taken from the culture at 48 h and assessed by cytokine ELISA for IFN-γ, IL-17, -4, and -10. Data are representative of three independent experiments. Error bars represent the SEM of triplicate measurements in the same experiment.

Figure 2.

Characterization of 3B3 and RMT1-10 anti–Tim-1 antibodies. (A) ELISA plates were coated with goat anti–mouse IgG. Full-length (Fl) or mucinless (Ml) Tim-1-Ig fusion proteins were used to coat ELISA plates, and the binding of 3B3 and RMT1-10 to the fusion proteins was determined. Tim-4-Ig was included as a negative control. Data are representative of three independent experiments. Error bars represent the SEM of triplicate measurements in the same experiment. (B) EL-4–Tim-1 transfectants were incubated with 0.5 μg/10⁶ cells of unlabeled rIgG2a, 3B3, RMT1-10, or anti–Tim-3 antibody (clone 2C12) for 1 h on ice, and 0.5 μg/10⁶ cells of either biotin-labeled RMT1-10 or PE-labeled 3B3 was added into the reaction for another 30 min. The binding of 3B3 or RMT1-10 (followed by PE-conjugated streptavidin) was detected by flow cytometric analysis. The green lines show isotype control staining; the blue lines show Tim-1 staining blocked with rIgG2a; the red lines show the Tim-1 staining blocked with unlabeled 3B3, RMT1-10, or anti–Tim-3; the yellow line shows the Tim-1 staining blocked with 5 μg/10⁶ cells of unlabeled RMT1-10.

Figure 3.

Effects of treatment with 3B3 or RMT1-10 on early T cell responses. CD4⁺ T cells were stained with Alexa Fluor 488–conjugated anti-CD3 on ice and treated with rIgG2a, 3B3, or RMT1-10 in a Live Imaging Microincubator at 37°C and 5% CO₂. The responses of labeled CD4⁺ T cells were recorded for 1 h in 15-min intervals. CD3 capping at the 15-min time point and time courses have been shown in A. The arrows represent cells with CD3 capping. Pictures of the cell mobility were taken from 15 to 22 min, with 104-s intervals, and are shown in B. The arrows represent the cells with active body movement. The time course of the cell mobility has been shown in B. The video of T cell mobility from 15 to 22 min is shown Video 1. Green, CD3; red (A) and purple (B), differential interface contrast. Video 1 is available at http://www.jem.org/cgi/content/full/jem.20062498/DC1.

Figure 4.

Biacore analysis of 3B3 and RMT1-10 interactions with Tim-1 molecules. (A) 3B3 and RMT1-10 were captured on the surface of a biosensor chip, and a concentration series of Tim-1-Ig was used to generate high-resolution kinetic data. Tim-1 association and dissociation data were acquired and rate constants (K _a and K _d) were generated from a global fit of the experimental binding data using a bivalent analyte model (mean χ² = 0.329 for 3B3 and 0.471 for RMT1-10). Sensogram data are representative of five independent experiments. (B) Association/dissociation rate plot showing resolution of binding affinities for each antibody. Isoaffinity diagonals indicate constant affinity values that increase from bottom left to top right. Mean affinities (K _D = K _d/K _a) for 3B3 and RMT1-10 are indicated within the plot. Data are the mean ± the SEM of five independent experiments. (C) Antibody blocking was used to examine the 3B3 and RMT1-10 epitopes on Tim-1. Tim-1-Ig was preincubated with anti–Tim-1 antibodies and observed for binding to 3B3 captured on the surface of a biosensor chip. RMT1-10 significantly reduced, but did not eliminate, Tim-1 binding to 3B3. Data are representative of three independent experiments. The RUs of immobilized anti–Tim-1 for the experiment in A were 137–141 for 3B3 and 108–113 for RMT1-10. In the experiment shown in C, the RUs were 360–365. In all cases, the “DeltaRUs” in the Tim-1 antibody immobilized on the chips was <5%.

Figure 5.

Treatment with 3B3 enhances PLP_139-151-specific Th1 and Th17 responses. (A) Spleen cells from immunized SJL mice treated in vivo with 3B3, or with rIgG or PBS as control, were cultured in vitro for 48 h with PLP_139-151 restimulation. Proliferation was measured in triplicate wells after 48 h by ³[H]thymidine incorporation. Supernatants were taken at 48 h from the culture and assessed by cytokine ELISA for IL-2, IFN-γ, IL-17, -6, -4, and -10. Splenocytes from individual mice (n = 4) were analyzed separately, and mean data for all mice are shown. (B) Lymphocytes from spleen and lymph nodes of immunized SJL mice treated with 3B3 or rIgG were cultured in vitro for 5 d with PLP_139-151 restimulation. Live cells were obtained by Ficoll-Hypaque density gradient centrifugation and used for triple color staining with PE-conjugated IA^s tetramer, APC–anti-CD4, and 7-AAD. The PLP_139-151/IA^s tetramer-positive cells were determined in the live CD4⁺ cell population (CD4⁺7-AAD⁻). Cells were also reactivated with PMA and ionomycin for 4 h and used for four-color staining with APC-conjugated IA^s tetramer, FITC–anti-CD4, 7-AAD, and PE-cytokine. (C) The number of cytokine-producing cells was determined in the PLP_139-151/IA^s tetramer-positive CD4⁺ cell population. Data are representative of three independent experiments.

Figure 6.

Administration of 3B3 anti–Tim-1 antibody enhances EAE severity. 8–12-wk-old female SJL mice were actively immunized with PLP_139-151 emulsified in CFA, and treated with 3B3, rIgG, or PBS every other day from day 0 to 8. Mice were evaluated daily for the signs of EAE.

Figure 7.

Treatment with RMT1-10 anti–Tim-1 antibody inhibits PLP_139-151-specific T cell responses. (A) Spleen cells from immunized SJL mice treated in vivo with RMT1-10 or rIgG were cultured in vitro for 48 h with PLP_139-151 restimulation. Proliferation was measured in triplicate wells after 48 h by ³[H]thymidine incorporation. (B and C) Lymphocytes from spleen and lymph nodes of immunized SJL mice were cultured for 5 d with PLP_139-151 restimulation. Live cells were then obtained by Ficoll-Hypaque density gradient centrifugation and used for determining the frequency of PLP_139-151/IA^s tetramer-positive CD4⁺ cells and cytokine production from these cells as described in Fig. 5 (B and C). Data are representative of three independent experiments.

Figure 8.

Administration of RMT1-10 anti–Tim-1 antibody inhibits EAE. Female SJL mice were actively immunized with PLP_139-151 emulsified in CFA and treated with RMT1-10 or rIgG every other day from day 0 to 10. Mice were evaluated daily for the signs of EAE.