Cutting edge: TIGIT has T cell-intrinsic inhibitory functions

Abstract

Costimulatory molecules regulate the functional outcome of T cell activation, and disturbance of the balance between activating and inhibitory signals results in increased susceptibility to infection or the induction of autoimmunity. Similar to the well-characterized CD28/CTLA-4 costimulatory pathway, a newly emerging pathway consisting of CD226 and T cell Ig and ITIM domain (TIGIT) has been associated with susceptibility to multiple autoimmune diseases. In this study, we examined the role of the putative coinhibitory molecule TIGIT and show that loss of TIGIT in mice results in hyperproliferative T cell responses and increased susceptibility to autoimmunity. TIGIT is thought to indirectly inhibit T cell responses by the induction of tolerogenic dendritic cells. By generating an agonistic anti-TIGIT Ab, we demonstrate that TIGIT can inhibit T cell responses directly independent of APCs. Microarray analysis of T cells stimulated with agonistic anti-TIGIT Ab revealed that TIGIT can act directly on T cells by attenuating TCR-driven activation signals.

Figure 1. Expression of CD226 and TIGIT on T cells

CD4⁺ or CD8⁺ T cells were isolated from B6 mice and stimulated with plate-bound anti-CD3 and anti-CD28. Samples were taken at the indicated time points and (A) expression of CD226 and TIGIT mRNA was determined by quantitative RT-PCR (mean ± SD). (B) Surface expression of CD226 and TIGIT protein was assessed by flow cytometry.

Figure 2. TIGIT^−/− mice show increased T cell responses upon immunization

TIGIT^−/− and B6 mice were immunized s.c. with 100μg MOG_35–55 peptide in CFA. On day 8 spleens and lymph nodes (LN) were harvested and cells were re-stimulated with MOG_35–55 peptide (0 – 100μg, 10x dilution steps; s: spleen, d: draining LN, nd: non-draining LN). (A) Proliferation was measured after 48 hours based on ³H-thymidine incorporation (mean ± SD, n=4). (B) Tetramer staining was used to determine the frequencies of MOG_35–55–specific CD4⁺ T cells in spleens and LNs of B6 (WT) and TIGIT^−/− (KO) mice. Dot plots from the draining LNs as well as the frequencies of MOG_35–55–specific cells of one representative experiment are shown (percentage of tetramer⁺ cells within the CD4⁺7AAD⁻ population, n=4). (C) Cytokines were measured in the supernatants derived from the same cultures as in (A) at 48 hours (n=4; *: p-value ≤ 0.05, **: p-value ≤ 0.001).

Figure 3. TIGIT^−/− mice are more susceptible to EAE

(A) B6 and TIGIT^−/− mice were immunized s.c. with 10–15μg MOG_35–55 peptide in CFA, followed by injection of 100ng pertussis toxin i.v. on day 0 and day 2. Mice were monitored daily for EAE. Mean clinical score ± SEM is shown and linear regression curves of the disease for each group is depicted in the inset (the 95% confidence intervals are represented with dashed lines, p< 0.0001; n=12 per group). (B) 2D2 TCR transgenic and 2D2 × TIGIT^−/− mice were monitored for the spontaneous onset of EAE. Mean clinical score ± SEM is shown and linear regression curves of the disease for each group is depicted in the inset (the 95% confidence intervals are represented with dashed lines, p< 0.0001).

Figure 4. TIGIT has T cell intrinsic effects

TIGIT-specific antibodies were generated in Armenian hamsters (clone 4D4). (A) 4D4 was titrated in an ELISA against recombinant mouse TIGIT or a control protein. (B) P815 cells transfected with mouse TIGIT (solid line) or the parental cell line (shaded histogram) were stained with anti-TIGIT antibodies and analyzed by flow cytometry. (C) Primary T cells from B6 or TIGIT^−/− mice were activated for 48h and stained with anti-TIGIT antibody 4D4 (solid line) or isotype control (shaded histogram) and analyzed by flow cytometry (gated on CD4⁺ cells). (D) CD4⁺ T cells were sorted from B6 and TIGIT^−/− mice and stimulated with plate-bound anti-CD3 and anti-CD28 plus 4D4 or isotype control Ab. Proliferation was assessed by ³H-thymidine incorporation (mean ± SD).