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CIS is a potent checkpoint in NK cell–mediated tumor immunity

An Author Correction to this article was published on 12 December 2023

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Abstract

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish−/− mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell–mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

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Figure 1: CIS-deficient NK cells display superior proliferation, survival and killing in response to IL-15.
Figure 2: CIS-deficient NK cells displayed extensive transcriptional changes in response to IL-15.
Figure 3: CIS negatively regulates IL-15 signaling by targeting JAK-STAT signaling.
Figure 4: Cish−/− NK cells have increased active kinase levels following IL-15 stimulation.
Figure 5: CIS-SH2 domain binds to the JAK activation loop targeting JAK1 for proteasomal degradation.
Figure 6: CIS-SH2 binding specifically inhibits JAK1 kinase activity.
Figure 7: Loss of CIS checkpoint induction in NK cells reduces experimental lung metastasis.
Figure 8: CIS deficiency protects against experimental and spontaneous breast cancer metastasis.

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Acknowledgements

We wish to thank N. Nicola for helpful comments and discussion, P. Rueda and C. Quillici for technical assistance, Tracy Wilson for generating the wild-type and SH2-mutant CIS expression plasmids, R. Anderson for the E0771.LMB cells, and L. Town, K. Elder, T. Camilleri and J. Sutton for excellent animal husbandry. We are grateful to the staff of the WEHI Bioservices, Monoclonal antibody facility, Flow cytometry facility and Clinical Translational Centre. This work is supported by program and project grants from the National Health and Medical Research Council (NHMRC) of Australia (1027472 to S.C., N.D.H. and G.T.B., 1013667 and 1098960 to M.J.S., 1047903 to G.T.B., 1016647 to J.G.Z., S.E.N. and W.S.A., 1049407, 1066770 and 1057852 to N.D.H., and 1078763 to D.H.D.G.), as well as an NHMRC Independent Research Institute Infrastructure Support scheme grant and a Victorian State Government Operational Infrastructure Scheme grant. N.D.H. is a recipient of a Melanoma Research Grant from the Harry J Lloyd Charitable Trust and R.B.D. is supported by a Leukemia Foundation scholarship. This work was also supported by fellowships from the NHMRC (GNT0461276 to N.D.H., 1058344 to W.S.A., 545952 to D.S.H., 1090236 to D.H.D.G., 1089072 to C.T., Australia Fellowship 1078671 to M.J.S.), the Australian Research Council (G.T.B.) and the Menzies Foundation (N.D.H.). E.M.P. is supported by a Schroedinger Fellowship from the Austrian Science Fund (FWF): J-3635. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer PHARMA AG, Boehringer Ingelheim, the Canada Foundation for Innovation, the Canadian Institutes for Health Research, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and the Wellcome Trust [092809/Z/10/Z].

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Authors

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R.B.D., T.B.K., L.F.D., J.R., W.S., E.M.P., D.S.H., K.S., J.-G.Z., M.F., T.U., C.T., C.E.A., P.P.S., C.E.S., G.I., N.P.D.L., E.M.L., C.S., A.I.W., J.J.B., S.S.L., A.N.B., S.E.N. and N.D.H. all performed experiments. M.A.D.-E., C.J.B., W.S.A., G.T.B., S.C., D.H.D.G. and M.J.S. provided key reagents and scientific input into experimental design and interpretation of the results. M.J.S., J.J.B., S.E.N. and N.D.H. supervised experimental design and provided input into interpretation of results and writing of the paper.

Corresponding authors

Correspondence to Sandra E Nicholson or Nicholas D Huntington.

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Competing interests

M.J.S. has been supported by a scientific research agreement with Bristol Myers Squibb and Medimmune and is a consultant for Kymab, F-star and AMGEN.

Integrated supplementary information

Supplementary Figure 1 Analysis of NK cells, ILC1s and ILC2s in Cish-deficient mice

(a) Cish expression in NK cell subsets from C57BL/6 mice. Shown as reads per kilobase of exon per million reads (RPKM). Subset definition and RNAseq data have been described previously.1,2,3 (b) Cish+/+ or Cish−/− NK cells were cultured in IL-15, lysed and Cish mRNA analyzed by Q-PCR. Data were normalized to expression of GAPDH mRNA (upper panel). N.D.: not detected. Cish+/+ or Cish−/− NK cells were cultured in IL-15 and the proteasomal inhibitor MG132 for 4 h prior to cell lysis and CIS protein detected in whole cell lysates by Western blotting (lower panel). (c) NK cells (NK1.1+NKp46+TCR-β) were analyzed in the indicated organs from Cish+/+ and Cish−/− mice by flow cytometry. (d) ILC1 (NK1.1+NKp46+TCR-βCD49a+CD49b) in the liver of Cish+/+ and Cish−/− were analyzed by flow cytometry and (e) quantified (vertical axis: % of ILC1). (f) ILC2 Cish+/+ and Cish−/− mice were treated with PBS or IL-2 complexed with anti-IL-2 antibodies (IL-2-JES6.1; expansion of CD25+ cells) every 2 days and were sacrificed after 5 or 7 days (D5, D7). Representative flow cytometry plots of ILC2 in the bone marrow gated on CD3/19/NK1.1/B220/Gr1 negative cells. (g) Frequency of ILC2 in the bone marrow following IL-2-JES6.1 treatment. (b, e, g) Mean ± s.e.m. n=3 biological replicates.

1 Delconte, R. B. et al. The Helix-Loop-Helix Protein ID2 Governs NK Cell Fate by Tuning Their Sensitivity to Interleukin-15. Immunity 44, 103-115, doi:10.1016/j.immuni.2015.12.007 (2016).

2 Revilla, I. D. R. et al. The B-cell identity factor Pax5 regulates distinct transcriptional programmes in early and late B lymphopoiesis. EMBO J 31, 3130-3146, doi:10.1038/emboj.2012.155 (2012).

3 Holmes, M. L. et al. Peripheral natural killer cell maturation depends on the transcription factor Aiolos. EMBO J 33, 2721-2734, doi:10.15252/embj.201487900 (2014).

Supplementary Figure 2 Analysis of T cells, regulatory T cells and MCMV responses in Cish-deficient mice.

(a) T cells (NK1.1NKp46TCR-β+) were analyzed in the indicated organs from Cish+/+ and Cish−/− mice by flow cytometry. (b) Regulatory T cells (Tregs) Expression of FoxP3 and CD25 on CD4+ cells from spleen and lymph nodes of Cish+/+ and Cish−/− mice before and 5 days after IL-2-JES6-1 treatment. Representative flow cytometry plots are shown. (c) Expansion and contraction of Tregs in the spleen and lymph nodes following IL-2-JES6-1 complex treatment (Mean ± S.E.M., n=1-2 mice per group). (d) 50:50 bone marrow chimeras (5x106 Ly5.1+ Cish+/+ and Ly5.2+ Cish−/− cells) were injected intraperitoneally with 5x103 plaque forming units (PFU) of salivary gland-propagated virus stock of MCMV-K181-Perth and Ly49H+ NK cells frequency monitored by flow cytometry. (Mean ± S.E.M., n=6 mice). (e) Intact Cish+/+ and Cish−/− mice were injected intraperitoneally with 5x103 plaque forming units (PFU) of salivary gland-propagated virus stock of MCMV-K181-Perth. On day 7 spleens were analyzed for MCMV+ CD8+ T cell responses by flow cytometry. Numbers of indicated tetramer+ CD8+ T cells are shown (mean ± s.e.m., n=5 mice).

Supplementary Figure 3 Loss of Socs1 and/or Socs3 does not alter IL-15 responses in NK cells.

(a) Socs3+/+ERT2Cre/+ (Cre+), Socs1−/−Ifnγ−/− (Socs1Δ), Socs3fl/flERT2Cre/+ (Socs3Δ), and Socs1−/−Ifnγ−/−Socs3fl/flERT2Cre/+ (Socs1ΔSocs3Δ) mice were treated with 4-hydroxytamoxifen (4-OHT; to induce Socs3 deletion) by oral gavage and splenic NK cells (TCR-βNK1.1+NKp46+) analyzed 14 days later by flow cytometry. Plots and values (%) are representative of 3 mice analyzed for each genotype. (b) Splenic NK cells from mice in (a) were FACS sorted and cultured in IL-15 (50 ng ml-1) for 7 days before being CFSE labelled and either i.v. transferred into alymphoid Rag2−/−γC−/− recipients or cultured in IL-15 (50 ng ml-1) in vitro. Five and ten days post-transfer, recipient livers were analyzed for donor NK cells by flow cytometry. In vitro cultures were analyzed on day 5. Cish+/+ and Cish−/− NK cell cultures serve as a reference for differential proliferation (lower right panel). (c) Enhanced effector function in Cish−/− NK cells. Cish+/+ and Cish−/− NK cells were cultured for 7 days prior to co-culture with CHO or B16F10 target cells at a ratio of 1:1. Target cell killing at 5 h was determined by relative changes in electrical impedance using the xCELLigence system. Cish+/+ and Cish−/− NK cells achieved maximal killing at 9:1 effector:target ratios (defined as 100% killing). (d) Cish+/+ and Cish−/− mice where injected with RMA-m157 cells i.p and peritoneal NK cells analyzed 18 h later for intracellular granzyme-B production by flow cytometry. Mean and s.d. of two experiments. n = 2 mice. MFI: Mean Fluorescence Index.

Supplementary Figure 4 Transcriptome profiling of ex vivo and in vitro cultured Cish−/− NK cells

100bp single-ended RNAseq was performed on freshly sorted ex vivo Cish+/+ and Cish−/− NK cells, and on Cish+/+ and Cish−/− NK cells that had been cultured for 7 days in IL-15 (50 ng/ml). (a) Relative expression levels (Z-scores) of the top ~100 most differentially expressed genes in Cish−/− cells are shown in the heatmap, color-coded according to the legend. Rows are scaled to have a mean of 0 and s.d. of 1. n=2 biological replicates. (b) Mean-difference plot of the cultured NK cell data generated in Figure 2, showing Log2-fold change versus mean expression. (c) Functional analysis of the 1230 differentially expressed genes observed in IL-15 cultured Cish−/− NK cells. Gene ontology was performed using the PANTHER classification system. Major gene networks are shown as a percentage of total differentially expressed genes in Cish−/− cells.

Supplementary Figure 5 Cish−/− NK cells display increased JAK-STAT signaling and normal respiration and glycolysis.

(a) Cish−/− NK cell respiration and glycolysis is unperturbed. Cish+/+ and Cish−/− NK cells were cultured in the presence of IL-15 and the extracellular acidification rate (ACR; glycolysis) and oxygen consumption rate (OCR; mitochondrial respiration) measured using the XF Analyzer system. Glucose (1), Oligomycin (2), FCCP and pyruvate (3) and Antimycin A/Rotenone (4) were added at times indicated by the numbered arrows. (b) Overview of the proteomic workflow used in this study. Equal numbers of cultured NK cells derived from Cish+/+ and Cish−/− mice were lysed and subjected to kinase enrichment using NHS-CYT-387 beads. Protein eluates from the CYT-387 resin, in addition to a portion of whole cell lysate (WCL; pre-kinase enrichment) were subjected to trypsin digestion and nanoLC-MS/MS. (c) Label-free quantification of global protein expression. Volcano plot showing the Log2 protein ratios following the quantitative pipeline analysis (Cish+/+ vs Cish−/−) from WCL. The red and yellow lines represent a 2-fold change in protein expression (log2 ratio of 1), while blue and green lines represent a 4-fold change (log2 ratio of 2); dots are colored accordingly and represent individual proteins. Proteins with a -log10 p-value of 1.3 or greater (corresponding to a p-value of ≤ 0.05) were deemed differentially abundant. (d) Heat map displaying Log2-transformed summed peptide intensities (non-imputed) for proteins with significantly differential expression in (d). Data from individual biological replicates are shown (n=3). Green to red indicates increasing expression levels. See also Extended Data Table 2.

Supplementary Figure 6 CIS targets JAK and the IL-2R complex.

(a) Cultured NK cells from wild-type and Cish−/− mice were lysed, mRNA purified and analyzed by RNAseq. Mean RPKM values for duplicate samples (left panel). JAK1 mRNA levels were analyzed by Q-PCR (right panel). Mean and s.d., n=3. (b) 4-12% Coomassie-stained SDS-PAGE gel showing purified hCIS-SH2-SB, elongin B and elongin C complex (CIS-SH2-BC). (c) Isothermal calorimetry (ITC) was used to measure the affinity of hCIS-SH2-BC binding to phosphopeptides corresponding to tyrosines within the JAK1/3 kinase domain activation loops and IL-2Rβ and γ cytoplasmic domains. 300 µM phosphopeptides were titrated into a 30 μM solution of the GST-CIS-SH2-BC ternary complex. ITC titration curves and tabular view of some results (inset) showing average and range from two independent experiments. N.D.=Not detectable, p=phosphorylated. The titration curves all fitted well to a single-site model. (d) Kinase inhibition assays were performed with the kinase domain (JH1) of JAK1 in the presence of CIS-SH2-BC with and without excess JAK1-Y1034 phosphopeptide as a competitor. The pY1034 peptide partially reduced CIS-mediated inhibition. Data were normalized to no-CIS controls. (e) Diagram illustrating the in vitro E3 ligase ubiquitination components and proposed model for CIS-mediated inhibition of JAK activity, whereby CIS recruitment to the receptor complex promotes binding to active JAK1 and results in kinase inhibition and proteasomal degradation. eloB: elongin B; eloC: elongin C.

Supplementary Figure 7 CIS-null mice resist tumor metastasis.

(a) Metastatic burden in lungs of Cish+/+ and Cish−/− mice 14 days following i.v injection of B16F10 melanoma cells (as in Figure 7a). (b) Metastatic burden in the lungs of NK cell-deficient (Ncr1Mcl1Δ/Δ) mice injected i.v. with B16F10 melanoma cells and Cish+/+ or Cish−/− NK cells or PBS (as in Figure 7e). (c) Metastatic burden in the lungs measured by imaging (IVIS; mCherry fluorescence) of Cish+/+, Cish−/− and Ncr1Mcl1Δ/Δ (NK-null) mice 14 days following i.v injection of E0771-mCherry+luciferase+ breast cancer (as in Figure 8a). (d) Orthotopic E0771.LMB tumors generated as in (Figure 8c) were surgically removed at 400–600 mm3 and spontaneous lung metastases measured by IVIS (mCherry fluorescence) 14 days later.

Supplementary Figure 8 Generation of modified CYT-387 compound (S6).

Schematic workflow (S1-S6) showing generation of the modified CYT-387 compound.

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Supplementary Figures 1–8 and Supplementary Tables 1 and 2 (PDF 2283 kb)

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Delconte, R., Kolesnik, T., Dagley, L. et al. CIS is a potent checkpoint in NK cell–mediated tumor immunity. Nat Immunol 17, 816–824 (2016). https://doi.org/10.1038/ni.3470

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