Tim-1 stimulation of dendritic cells regulates the balance between effector and regulatory T cells

Abstract

We show that the T-cell immunoglobalin mucin, Tim-1, initially reported to be expressed on CD4(+) T cells, is constitutively expressed on dendritic cells (DCs) and that its expression further increases after DC maturation. Tim-1 signaling into DCs upregulates costimulatory molecule expression and proinflammatory cytokine production, thereby promoting effector T-cell responses, while inhibiting Foxp3(+) Treg responses. By contrast, Tim-1 signaling in T cells only regulates Th2 responses. Using a high-avidity/agonistic anti-Tim-1 antibody as a co-adjuvant enhances the immunogenic function of DCs, decreases the suppressive function of Tregs, and substantially increases proinflammatory Th17 responses in vivo. The treatment with high- but not low-avidity anti-Tim-1 not only worsens experimental autoimmune encephalomyelitis (EAE) in susceptible mice but also breaks tolerance and induces EAE in a genetically resistant strain of mice. These findings indicate that Tim-1 has an important role in regulating DC function and thus shifts the balance between effector and regulatory T cells towards an enhanced immune response. By understanding the mechanisms by which Tim-1 regulates DC and T-cell responses, we may clarify the potential utility of Tim-1 as a target of therapy against autoimmunity, cancer, and infectious diseases.

Figure 1. DC constitutively express Tim-1.

(A) Different cell populations in the spleens of SJL mice were isolated and stained with anti-Tim-1 (thick lines) or rIgG2a (thin lines). (B) Purified CD11c⁺ cells were stained for Tim-1 expression and for the indicated surface molecules. The gating strategy of different DC subsets is shown. Right panel shows the MFI (shown as mean ± SEM of four experiments) of DC subsets stained with rIgG2a and anti-Tim-1. * P < 0.01, Student’s t test. (C) Purified CD11c⁺ cells were cultured with LPS/CpG overnight, and then stained for Tim-1 expression. In B and C, numbers in parentheses represent MFI; numbers without parentheses represent percentage. (D) SJL mice were immunized with PLP_139-15/CFA to induce EAE. At the peak of the disease, CNS-infiltrating cells were isolated and stained with Tim-1 (thick lines) or rIgG2a (grey areas). Stained cells were analyzed by flow cytometry. Data represent Tim-1 expression on gated populations and are representative of five experiments for (A), four experiments for (B) and (C), three experiments for (D).

Figure 2. Tim-1 signaling increases NF-κB activity in DC and promotes the expression of costimulatory molecules and production of proinflammatory cytokines.

(A) DC were incubated with the indicated stimuli for 2 h. Cell nuclear extracts were prepared to determine the NF-κB p65 activity (shown as mean ± SEM of triplicates in one experiment) using the TransAM NF-κB p65 Assay Kit. Data are representative of three experiments. (B) DC were treated with the indicated stimuli overnight, stained for surface molecules, and analyzed by flow cytometry. ΔMFI = MFI of indicated surface marker staining on gated CD11c⁺ populations - MFI of isotype staining. (C) The overnight culture supernatants from (B) were collected and assessed by cytometric bead assay (CBA) for IFN-γ, TNF-α, and IL-6. (D) DC were incubated with the indicated stimuli for 6 h. Cells were collected and total RNA was isolated for determining the expression of the indicated cytokine genes. Data in (B-D) are mean ± SEM of four experiments. * P < 0.01, Student’s t test.

Figure 3. Tim-1 crosslinking induces differential T cell responses in the absence or presence of DC.

Naïve CD4⁺ T cells from 5B6 TcR Tg SJL mice were activated with either plate-bound anti-CD3/anti-CD28 or with PLP_139-151 plus purified syngeneic DC in the (A, B) absence or (C, D) presence of TGF-β. (A) Cytokine production from T cells was determined on d6 by intracellular cytokine staining. Number in upper right quadrants indicates the percentage of cytokine-producing cells in CD4⁺ populations. (B) DC purified from SJL mice were cultured with PLP_139-151 plus anti-Tim-1, rIgG2a or LPS for 6 h. Cells were then washed with PBS and subcutaneously transferred into SJL mice on d0 and d7. Draining LN cells were isolated on d14 and restimulated with PLP_139-151. After 4 days, cytokine production was measured by intracellular cytokine staining. (C) Naïve 5B6 CD4⁺ T cells were activated with either plate-bound anti-CD3/CD28 or with PLP_139-151 plus purified syngeneic DC or subsets in the presence of TGF-β. Cells were then measured for CD103 and Foxp3 expression on CD4⁺ T cells by flow cytometry. (D) Supernatant from the cell cultures described in (C) was collected after 48 h and measured for IL-17 production by ELISA. Data are mean ± SEM of triplicates in one experiment. * P <0.01, Student’s t test. All above data are representative of three experiments.

Figure 4. High-avidity anti-Tim-1 as a co-adjuvant enhances antigen-specific Th1 and Th17 responses and worsens EAE in SJL mice.

(A-C) SJL mice were immunized with PLP_139-151/CFA/3B3 emulsion to induce EAE (25 μg of PLP_139-151 per mouse). Draining LN cells were isolated from one group of mice on d10 and restimulated with PLP_139-151. (A) Proliferation and (B) cytokine production were measured by ³[H]-thymidine incorporation and intracellular cytokine staining, respectively. Data are mean ± SEM (A) and representative (B) of six mice in each group. (C) Clinical EAE score in another group of six mice was evaluated daily. * P < 0.01, Fisher’s exact test.

Figure 5. High-avidity anti-Tim-1 as a co-adjuvant breaks tolerance and induces EAE in disease-resistant B10.S mice.

(A-C) B10.S mice were immunized with PLP_139-151/CFA/3B3 emulsion to induce EAE (80 μg of PLP_139-151 per mouse). Draining LN cells were isolated on d10 in one group of six mice and restimulated with PLP_139-151. (A) Proliferation (Mean ± SEM) and (B) cytokine production were measured by ³[H]-thymidine incorporation and intracellular cytokine staining, respectively. * P < 0.01, Student’s t test. (C) Clinical EAE score (shown as mean ± SEM) in another group of six mice were evaluated daily. # P < 0.05; * P < 0.01, Fisher’s exact test.

Figure 6. Effect of high-avidity anti-Tim-1 as a co-adjuvant on DC, Teff, and Treg in B10.S mice during EAE.

B10.S Foxp3/GFP “knockin” mice were immunized with PLP_139-151/CFA/3B3 emulsion. At the peak of the disease, Teff (CD4⁺Foxp3/GFP⁻), Treg (CD4⁺Foxp3/GFP⁺) and DC were purified from spleens and lymph nodes of mice treated with 3B3 anti-Tim-1 or control rIgG, and CNS-infiltrating mononuclear cells were isolated from brains and spinal cords of the mice. (A) Different mixtures of Teff and DC as indicated were cultured in the presence of antigen, and cell proliferation and cytokine production in the culture supernatant were measured by ³[H]-thymidine incorporation and ELISA, respectively. (B) Teff and DC from rIgG-treated mice were co-cultured with different ratios of Treg (as indicated) in the presence of antigen, and cell proliferation was measured by ³[H]-thymidine incorporation. Data in (A) and (B) are mean ± SEM of triplicates for one mouse and representative of four mice in each group. * P < 0.05, Student’s t test. (C) DC were stained and analyzed for CD80, CD86, and MHC class II expression. Numbers in Italic font represent MFI of rIgG-treated samples; Numbers in the normal font represent MFI of 3B3-treated samples; (D) CNS-infiltrating cells were stained to determine the percentage of DC, CD4⁺ T cells, and Foxp3⁺ Treg by flow cytometry. Cytokine production of CNS-infiltrating Foxp3⁺ and Foxp3⁻ CD4⁺ T cells were determined by intracellular staining.