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. 2012 Apr 15;188(8):3869-75.
doi: 10.4049/jimmunol.1103627. Epub 2012 Mar 16.

The TIGIT/CD226 axis regulates human T cell function

Ester Lozano  1 , Margarita Dominguez-Villar, Vijay Kuchroo, David A Hafler
Affiliations

The TIGIT/CD226 axis regulates human T cell function

Ester Lozano et al. J Immunol. .

Abstract

T cell Ig and ITIM domain (TIGIT) is a newly identified receptor expressed on T cells that binds to CD155 on the dendritic cell surface, driving them to a more tolerogenic phenotype. Given that TIGIT contains an ITIM motif in its intracellular domain and considering the potential importance of the TIGIT/CD226 pathway in human autoimmune disease, we investigated the specific role of TIGIT in human CD4(+) T cells. Using an agonistic anti-TIGIT mAb, we demonstrate a direct inhibitory effect on T cell proliferation with a decrease in expression of T-bet, GATA3, IFN regulatory factor 4, and retinoic acid-related orphan receptor c with inhibition of cytokine production, predominantly IFN-γ. Knockdown of TIGIT expression by short hairpin RNA resulted in an increase of both T-bet and IFN-γ mRNA and protein expression with concomitant decrease in IL-10 expression. Increases in IFN-γ with TIGIT knockdown could be overcome by blocking CD226 signaling, indicating that TIGIT exerts immunosuppressive effects by competing with CD226 for the same CD155 ligand. These data demonstrate that TIGIT can inhibit T cell functions by competing with CD226 and can also directly inhibit T cells in a T cell-intrinsic manner. Our results provide evidence for a novel role of this alternative costimulatory pathway in regulating human T cell responses associated with autoimmune disease.

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Conflict of interest statement

Disclosures

The authors have no conflicting financial of interests.

Figures

Figure 1
Figure 1
Upregulation of TIGIT, CD226 and CD155 in human CD4+ T cells after activation. (A) Dot plots displaying the cell surface expression of TIGIT, CD226, CD155 and CD112 on naïve CD4+ T cells (CD45RO−) and memory T cells (CD45RO+) ex vivo and after activation with anti-CD3 and anti-CD28 for 2 days. (B) Kinetics of gene expression of TIGIT, CD226 and their ligands CD155 and CD112 on sorted memory T cells (CD4+ CD62L+ CD25lowCD45RO+). Data are normalized relative to beta 2 microglobulin. Data are representative of four donors.
Figure 2
Figure 2
TIGIT cell-intrinsic signaling inhibits T cell proliferation. (A) Proliferation of human CD4+ T cells labeled with CFSE and activated with anti-CD3 and anti-CD28 with plate-bound agonistic anti-TIGIT or isotype-matched control antibody. On day 5, CFSE dilution was analyzed by flow cytometry after gating on viable cells. (B) Percentages of dividing cells after activation represent the CFSElow cells (n=8). Mean values are shown ± SD. (C) Viability was assessed in all the experiments. (D) No significant differences were observed in the frequency of viable cells. (E) Flow cytometry of the surface expression of CD25 and CD69 as markers of activation of human CD4+ T cells activated in presence of agonistic anti-TIGIT or isotype control, assessed on day 5 after activation. (F) Sorting strategy for memory T cells (CD4+ CD62L+ CD25low CD45RO+) purity. (G) CFSE proliferation assay of memory T cells in the presence of agonistic anti-TIGIT. Proliferation was assessed on day 5 after gating on viable cells. Percentages of proliferating cells are depicted above the histograms. (H) Quantitative real-time RT-PCR analysis of relative IL-2 gene expression in FACS-sorted memory T cells. Data shown are representative of four different donors.
Figure 3
Figure 3
TIGIT cell-intrinsic signaling inhibits T cell cytokine production. (A) Time course of transcription factors expression in memory T cells stimulated with IL-12 (Th1) IL-4 (Th2), TGFβ+IL-4 (Th9) and TGFβ+IL-1β/IL-6/IL-21 (Th17) and cultured in presence agonistic anti-TIGIT or isotype control for 3 days. (B) Intracellular cytokine staining for IFNγ, IL-13, IL-4, IL-9 and IL-17 in memory T cells cultured in the same conditions than A for 4 days. Data shown are representative of at least four independent experiments.
Figure 4
Figure 4
Knockdown of CD226 expression in human CD4+ T cells. (A) CD226 gene expression in human CD4+ T cells transfected with specific siCD226 or negative siRNA, assessed by real-time RT-PCR on day 1 and 2 after electroporation. (B) Flow cytometry of the surface expression of CD226 and IFNγ intracellular staining on human CD4+ T cells transfected with siRNA or control, and stimulated with PMA and ionomycin for 4 hr in the presence of GolgiStop. Cells were stained with LIVE/DEAD Fixable Dead Cell Stain Kit before fixation to allow gating on viable cells. (C) Measurement of IFNγ in the supernatants of cells transfected with siCD226 or negative siRNA. (D) Flow cytometry of the surface expression of CD226 and IFNγ intracellular staining on CD4+ T cells transfected as in B, and then stimulated with IL-12 for 3 days. (E) Flow cytometry of the surface expression of CD226 and IFNγ intracellular staining on human CD4+ T cells transduced with shCD226 or control by lentiviral transduction. Data are representative of three independent experiments. Mean values are shown ± SEM.
Figure 5
Figure 5
Knockdown of TIGIT expression in human CD4+ T cells by lentiviral transduction with two specific shRNAs. (A) Quantitative real-time RT-PCR analysis of relative TIGIT gene expression in human CD4+ T cells infected with lentivirus containing two TIGIT-specific shRNA sequences or an empty vector, assessed on day 3 after puromycin selection. (B) Gene expression of other negative regulators, CTLA-4 and PD-1 was measured to exclude non-target effects. (C) Relative quantification of T-bet (Tbx21), IFNγ and IL-10 mRNA in T cells transduced with shTIGIT-1, shTIGIT-2 or an empty vector. (D) Flow cytometry of the surface expression of TIGIT and IFNγ intracellular staining on human CD4+ T cells infected with TIGIT-specific shRNA or control, selected with puromycin and stimulated with PMA and ionomycin for 4 hr in the presence of GolgiStop. Cells were stained with LIVE/DEAD Fixable Dead Cell Stain Kit before fixation to allow gating on viable cells. Data are representative of four independent experiments with three individual donors. (E) Levels of IL-10 in the supernatants derived from the same cultures as in C quantified by ELISA. Mean values are shown ± SEM.
Figure 6
Figure 6
Blockade of CD226 signaling with anti-CD226 or anti-CD155 decreased the effects of TIGIT knockdown. (A) Quantitative real-time PCR analysis of relative T-bet (Tbx21) expression in TIGIT-depleted CD4+ T cells incubated for 4 days in the presence of increasing concentrations of plate-bound blocking anti-CD226, blocking anti-CD155 or isotype control IgG. (B) IFNγ gene expression. (C) IL-10 gene expression in the same conditions than in A. Data shown are representative of four different donors.
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