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The receptors CD96 and CD226 oppose each other in the regulation of natural killer cell functions

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Abstract

CD96, CD226 (DNAM-1) and TIGIT belong to an emerging family of receptors that interact with nectin and nectin-like proteins. CD226 activates natural killer (NK) cell–mediated cytotoxicity, whereas TIGIT reportedly counterbalances CD226. In contrast, the role of CD96, which shares the ligand CD155 with CD226 and TIGIT, has remained unclear. In this study we found that CD96 competed with CD226 for CD155 binding and limited NK cell function by direct inhibition. As a result, Cd96−/− mice displayed hyperinflammatory responses to the bacterial product lipopolysaccharide (LPS) and resistance to carcinogenesis and experimental lung metastases. Our data provide the first description, to our knowledge, of the ability of CD96 to negatively control cytokine responses by NK cells. Blocking CD96 may have applications in pathologies in which NK cells are important.

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Figure 1: Cd96−/− mice develop hyperinflammatory responses to challenge with LPS.
Figure 2: CD96 limits IFN-γ production by NK cells in response to LPS.
Figure 3: CD226 regulates IFN-γ production by NK cells in response to LPS.
Figure 4: CD96 competes with CD226 for binding by CD155.
Figure 5: CD96 regulates NK cell responses to LPS in the absence of CD226.
Figure 6: Binding of CD96 to CD155 regulates the production of IFN-γ by NK cells.
Figure 7: CD96 and CD226 have opposite roles in regulating the antitumor functions of NK cells.

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Acknowledgements

We thank D. Godfrey (University of Melbourne) for CD1d tetramer loaded with α-galactosylceramide; J. Sutton and K. Elder for the care and maintenance of the mouse colonies. Supported by the Leukaemia Foundation of Australia (C.J.C.), the Monash University Faculty of Medicine (C.J.C.), the National Health and Medical Research Council (D.M.A. and M.J.S.; 1013667, 1044392 and 628623) and the US National Institutes of Health (P30AR048335 for the Speed Congenics Facility of the Rheumatic Diseases Core Center at the Washington University School of Medicine).

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Authors

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C.J.C., L.M., S.G., F.S.-F.-G., M.T.C., L.T. and M.J.S. did experiments; C.J.C., L.M., S.G., D.S.R., M.C., D.M.A. and M.J.S. designed experiments and analyzed data; and L.M., C.J.C. and M.J.S. wrote the paper.

Corresponding author

Correspondence to Mark J Smyth.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cd96−/− construct.

A targeting construct designed to replace exons 1 and 2 of Cd96, including the start site, with a MC1-neor gene flanked by loxP sites was electroporated into E14.1 (129P2/OlaHsd) embryonic stem cells. Chimeras transmitting the targeted allele were obtained from two clones following injection into C57BL/6 blastocysts. Mice carrying the targeted allele were bred to C57BL/6 mice expressing a Cre transgene under the CMV promoter to delete the MC1-neor gene. The Cd96 deletion was backcrossed onto a C57BL/6 background, facilitated by a genome-wide screening of polymorphic microsatellite markers at 10-centiMorgan intervals at each generation. Cd96+/- >99% C57BL/6 mice were intercrossed to generate the Cd96-/- mice.

Supplementary Figure 2 Normal homeostasis of the immune system in the absence of CD96.

a. Spleens were harvested from WT and Cd96-/- mice and The expression of CD96 was analyzed by flow cytometry on the indicated spleen lymphocyte populations from C57BL/6 WT (blue) and Cd96-/- mice (red). b. The frequency of lymphocyte subsets that include NK cells, NKT cells, CD8 and CD4 T cells, B cells, macrophages, neutrophils and conventional dendritic cells were analyzed by flow cytometry. All data is representative of at least three independent experiments.

Supplementary Figure 3 Cd96−/− mice have greater susceptibility to LPS-induced endotoxicosis than do wild-type or Cd96+/− mice.

a. WT, Cd96+/- and Cd96-/- mice were challenged with LPS (0.5 mg/30 g) and their survival was monitored over time. b. Cd96-/- mice increased susceptibility to LPS-induced endotoxicosis is abrogated in the absence of NK cells. WT (circle) and Cd96-/- mice (square), injected with either control Ig (solid shape) or NK cell anti-NK1.1 depleting mAbs (PK136, 250 μg -1 and 0, open shape) were challenged with LPS (0.5 mg/30 g) and their survival was monitored over time. The survival curves of experiments involving 5-10 mice per group are shown (* p<0.05, ** p<0.01,*** p<0.001 Mantel-Cox test).

Supplementary Figure 4 Normal NK cell development and function in the absence of CD96.

a. Expression of major activating and inhibitory receptors on NK cells from the spleen from WT and Cd96-/- mice were analyzed by flow cytometry. b, c. Primary NK cells were isolated from the spleens of WT (black bars) and Cd96-/- mice (white bars) and stimulated with the indicated concentrations of IL-12 and IL-18. The supernatant (b) and the intracellular content (c) of IFN-γ were measured after 24 h.

Supplementary Figure 5 Increased NK cell activation by TLR-activated macrophages and DCs in the absence of CD96.

Cd96-/-NK cells express more CD69 and produce more IFN-γ than WT NK cells when co-cultured with TLR ligands treated APCs. We analyzed the intracellular production of IFN-γ (a, d) and the expression of CD69 (b, c, e) by NK cells purified from WT and Cd96-/- mice cultivated with BMDCs or with Bone marrow derived macrophages (BMDM) treated with poly IC (50 μg/ml; c, d) or LPS (100 ng/ml; a, b, e) for 24 h. The mean ± SD of triplicates wells from one representative experiment out of three is shown. * p<0.05, ** p<0.01, Student T test.

Supplementary Figure 6 Neutralization of CD96 enhances LPS-induced inflammation.

a. WT mice were injected with either: control Ig (solid circle), anti-CD96 (solid square, clone 3.3, 250 μg day -2 and -1), anti- CD226 (solid triangle, clone 480.1, 250 μg day -1 and 0) or with anti-CD155 (open triangle, clone 4.24.3, 250 μg day -1 and 0). On day 0 mice were challenged with LPS (0.5 mg/30 g) and their survival was monitored over time. The survival curves of 2 pooled experiments involving 20 mice per group are shown (* p<0.05, ** p<0.01,*** p<0.001 Mantel-Cox test).

Supplementary Figure 7 Host CD96 has no effect on cytotoxicity but suppresses lung metastasis.

a. Primary NK cells were harvested from the spleens of WT (solid circles) and Cd96-/- mice (open circles), cultured for 3 days in IL-2 and then exposed to RMA-S and YAC-1 cells in 4 h 51Cr release assays. The mean specific lysis ± SD of triplicate wells from one experiment out of 3 are shown. b. Cd96-/- mice control of B16F10 metastasis is abrogated in the absence of NK cells. WT and Cd96-/- mice injected with either control Ig or anti-NK1.1 depleting mAbs (PK136 250 μg) were challenged with B16F10 (2x105) and metastatic burden was quantified in the lungs after 14 days by counting colonies on the lung surface. C57BL/6 wild type (WT), Cd226-/-, Cd96-/-, and Cd226-/- Cd96-/- mice were injected intravenously with either (c) RM1 prostate carcinoma cells (104 cells) or (d) 3LL lung carcinoma cells (105) and metastatic burden was quantified in the lungs after 14 days by counting colonies on the lung surface. Mean ± SD of 5-19 mice per group are shown. Each symbol represents an individual mouse (**p<0.01; ***p<0.001; Mann-Whitney test).

Supplementary Figure 8 Opposing roles of CD226 and CD96 in immunosurveillance against MCA-induced fibrosarcoma

a-e Groups of 15−32 male, WT, Cd226-/- and Cd96-/- and Cd226-/-Cd96-/- mice were injected with (a) 5, (b) 25, (d) 100 or (c, e) 400 g/mouse of MCA. The survival (a-c) and the tumor growth from individual mice with sarcoma (d, e) are shown. WT mice were treated with either a control antibody or with anti-CD96 mAbs * p<0.05, *** p<0.001 Mantel-Cox test.

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Chan, C., Martinet, L., Gilfillan, S. et al. The receptors CD96 and CD226 oppose each other in the regulation of natural killer cell functions. Nat Immunol 15, 431–438 (2014). https://doi.org/10.1038/ni.2850

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