Identification of CD112R as a novel checkpoint for human T cells

Abstract

T cell immunoglobulin and ITIM domain (TIGIT) and CD226 emerge as a novel T cell cosignaling pathway in which CD226 and TIGIT serve as costimulatory and coinhibitory receptors, respectively, for the ligands CD155 and CD112. In this study, we describe CD112R, a member of poliovirus receptor-like proteins, as a new coinhibitory receptor for human T cells. CD112R is preferentially expressed on T cells and inhibits T cell receptor-mediated signals. We further identify that CD112, widely expressed on antigen-presenting cells and tumor cells, is the ligand for CD112R with high affinity. CD112R competes with CD226 to bind to CD112. Disrupting the CD112R-CD112 interaction enhances human T cell response. Our experiments identify CD112R as a novel checkpoint for human T cells via interaction with CD112.

Figure 1.

Characterization of human CD112R protein. (A) Protein sequence encoded by the human CD112R gene. Predicted extracellular IgV-like and transmembrane domains are highlighted in blue and red, respectively. Two tyrosines (Y233 and Y293) in the cytoplasmic domain are underlined with one within an ITIM-like motif underlined. (B) Alignment of the extracellular domains of human and mouse CD112R protein sequences using the MacVector 6.5 program. The shaded boxes refer to the shared amino acids among CD112R orthologues. (C) Guide tree analysis of human CD112R and the known PVR-like proteins via the Clustal W program in MacVector 6.5. (D) Multiple sequence alignment of the IgV domains of PVR-like proteins. Similar and identical residues among this group are shaded in red. The PVR signature motifs are outlined in green frames. Blue boxes mark conserved amino acids. (E) A predicted protein structure model of human CD112R IgV domain (55–150 aa) using human PVRL4 (Protein Data Bank accession no. 4JJH) as the template.

Figure 2.

CD112R expression in immune cells and its effect on TCR signal. (A) Human CD112R transcript in human immune cells. RNAs were isolated from DCs, NK cells, and T cells stimulated by OKT3 plus CD28 mAb. The expression of CD112R was detected by PCR. G3PDH was used as a housekeeping gene. (B) HEK293T cells transduced with control or CD112R gene were stained with control (red) or CD112R mAb (clone 2H6; blue). (C) Cell lysate of HEK293T/CD112R transfectant was run in reducing (+DTT) and nonreducing (−) conditions and detected by CD112R mAb (clone 2H6). (D) Flow cytometry analysis of CD112R expression in human peripheral blood from healthy donors (n = 4 donors) stained with indicated cell surface markers. (E) CD112R expression on different NK cell subsets: CD16⁺ (CD56⁺CD16⁺) and CD16⁻ (CD56⁺CD16⁻). The expression of CD112R (blue) in these two NK subsets is shown. (F) The CD112R expression on CD4⁺CD3⁺ and CD8⁺CD3⁺ T cell subsets. Graph (right) shows mean ± SD frequencies of CD112R-expressing cells in each subset. (G) CD8⁺ T cells were divided into two groups based on the expression of CD112R, and their expression of CD45RA and CCR7 was revealed. (H) Purified CD4⁺ T cells were left unstimulated (day 0) or activated by anti-CD3/CD28 Dynabeads for different times, and the CD112R expression on T cells was detected by biotinylated CD112R mAb. (I) HEK293T cells were transiently transfected with WT or tyrosine mutants of CD112R. Cells were treated with or without pervanadate before analysis for tyrosine phosphorylation on CD112R. (J) Molt4 cell lysates were immunoprecipitated with CD112R mAb or mouse IgG1 (control) and blotted with different phosphatase mAbs as indicated. The presence of CD112R and tyrosine phosphorylation was demonstrated by immunoblotting with CD112R and phosphorylated tyrosine (P-Tyr) mAbs, respectively. Whole cell lysate serves as a detective control. (K) Jurkat-NFAT-Luc cells transfected with different chimeras as indicated were stimulated with OKT3 in the presence or absence of a mouse CD28 agonistic mAb. Data shows mean ± SD of relative luciferase activity upon 4 h of stimulation. All data shown are representative of at least two independent experiments. IP, immunoprecipitation. F and K were analyzed by Student’s t test; *, P < 0.05; **, P < 0.01.

Figure 3.

Expression of a putative ligand for CD112R. (A) Immune cells in human blood and monocyte-derived DCs were stained with control (FLAG-Fc; red) or CD112R-Fc (blue) protein by flow cytometry. Immune cells were gated on individual lineage markers. T cell: CD3; B cell: CD19; NK cell: CD56⁺CD3⁻; monocyte: CD14; DC: CD11c. (B) Cultured human tumor cell lines were stained with control (FLAG-Fc) and CD112R protein, and the median fluorescence intensity (MFI) was determined by flow cytometry. CD112R MFI ratios (CD112R MFI/control MFI) are indicated. (C) SK-MEL28 melanoma line with or without trypsin treatment (10 min) was stained with control (FLAG-Fc; red) or CD112R-Fc (blue) protein by flow cytometry. (D) Blockade of CD112R binding by a CD112R mAb. HEK293T cells were stained with control (FLAG-Fc; red) or CD112R-Fc (blue) protein by flow cytometry with or without the presence of a CD112R mAb (clone 2H6). All data shown are representative of at least three independent experiments.

Figure 4.

Identification of CD112 as a ligand for CD112R. (A) HEK293T cells were transiently transfected with different PVR-like gene plasmids as indicated and stained with control (FLAG-Fc; red) or CD112R-Fc (blue) protein. (B) HEK293T cells transduced with CD112R gene were incubated with anti-CD112R mAb (clone 2H6) or control mIgG1 as indicated before being stained with CD112-Fc (blue) or control FLAG-Fc (red). (C) Beads coated with CD112 (right) or control protein (left) were stained with CD112 mAb (blue) or isotype control (red) to confirm the presence of CD112 on beads. Beads were also incubated with CD112R fusion protein (blue) or control (red) for direct interaction. (D) Biacore 3000 analysis of CD112R binding to CD112. The surface plasmon resonance sensorgrams were recorded with threefold serial dilutions starting at the highest concentration of 333 nM. The fitting curves are in orange. (E) RMA-S/mCD112 (blue) or control RMA-S (red) cells were stained for binding by mCD112 mAb or mCD112R, mCD226, and mTIGIT fusion protein, respectively. (F) Competitive binding assay for CD112 among CD112R, CD226, and TIGIT proteins. Beads coated with CD112 were stained by CD112R-Fc protein in the presence of different concentrations of TIGIT or CD226 protein, whereas beads coated with CD112 were stained by CD226-Fc in the presence of different concentrations of CD112R protein. All data shown are representative of at least two independent experiments.

Figure 5.

CD112 binds to CD112R to inhibit T cell response. (A) Human monocyte–derived DCs stimulated with LPS overnight were preincubated with mIgG1 or CD112 mAb (clone TX31) and then stained for CD112R protein binding. DCs stained with control FLAG protein are shown in red. Data shown are representative of three different experiments (n = 3 donors). (B) Human pancreatic cell line PANC198 was stained with isotype control (red) or CD112 mAb (blue) for CD112 expression (left). Cells were preincubated with control mIgG1 or CD112 mAb (clone TX31) before being stained by control (FLAG-Fc; red) or CD112R fusion protein (blue). Data shown are representative of two independent experiments. (C) Purified human T cells were CFSE labeled and stimulated with OKT3 together with plate-coated CD112-Fc or control protein (FLAG-Fc). Control (mouse IgG1) or CD112R mAb was added during cell culture. Cells were gated on CD8⁺ T cells, and their division was analyzed based on the dilution of CFSE. The CFSE-diluted cells indicated were counted as divided T cells. Data shown are representative of at least three independent experiments (n > 3 donors). (D and E) CFSE-labeled CD4⁺ T cells were cultured with mytomycin-treated CHO stimulators expressing CD112 or control CHO stimulator cells. Antibodies as indicated were added from the beginning of culture. After 5 d of culture, cell division was analyzed based on the dilution of CFSE (D). (E) IL-2 (day 2) and other cytokines (day 5) in the supernatant were measured by a human T helper cytokine panel. Data are representative of three independent experiments. (F) Purified human T cells were labeled with CFSE and were co-cultured with autologous DCs in the presence of TT. Control (mouse IgG1), CD112R mAb, or TIGIT mAb was included at the beginning of the culture. The proliferation of TT-specific CD4⁺ T cells was determined by CFSE dilution of the human CD3 and CD4 double-positive cells. Data are representative of three independent experiments (n = 3 donors). (G) In the same culture condition as in F, CD112R-Fc or control protein (FLAG-Fc) was included at the beginning to examine the effect on TT-specific T cell response. n = 5. *, P < 0.05 using two-way ANOVA. All bar graphs in C–F represent the mean ± SD results; *, P < 0.05; **, P < 0.01 (Student’s t test). The numbers in the histograms in C, D, F, and G refer to the percentages of divided T cells.