CD200 ectodomain shedding into the tumor microenvironment leads to NK cell dysfunction and apoptosis
Huw J. Morgan, Elise Rees, Simone Lanfredini, Kate A. Powell, Jasmine Gore, Alex Gibbs, Charlotte Lovatt, Gemma E. Davies, Carlotta Olivero, Boris Y. Shorning, Giusy Tornillo, Alex Tonks, Richard Darley, Eddie C.Y. Wang, Girish K. Patel
Huw J. Morgan, Elise Rees, Simone Lanfredini, Kate A. Powell, Jasmine Gore, Alex Gibbs, Charlotte Lovatt, Gemma E. Davies, Carlotta Olivero, Boris Y. Shorning, Giusy Tornillo, Alex Tonks, Richard Darley, Eddie C.Y. Wang, Girish K. Patel
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Research Article Oncology

CD200 ectodomain shedding into the tumor microenvironment leads to NK cell dysfunction and apoptosis

Abstract

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor–initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells to suppress MAPK pathway signaling that in turn blocked indirect (IFN-γ release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ-regulated gene transcription and led to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL, which resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell–specific immune checkpoint, which is responsible for NK cell–associated poor outcomes in many cancers.

Authors

Huw J. Morgan, Elise Rees, Simone Lanfredini, Kate A. Powell, Jasmine Gore, Alex Gibbs, Charlotte Lovatt, Gemma E. Davies, Carlotta Olivero, Boris Y. Shorning, Giusy Tornillo, Alex Tonks, Richard Darley, Eddie C.Y. Wang, Girish K. Patel

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Figure 4

CD200 induced NK cell apoptosis.

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CD200 induced NK cell apoptosis. (A) Untreated (UNT) and CD200 peptide–t...
(A) Untreated (UNT) and CD200 peptide–treated NKPOS cells observed for apoptotic events by IncuCyte caspase 3 assay. (B) Apoptosis frequency between untreated and CD200 peptide–treated NKPOS cells was determined as a ratio of viability at 8, 12, 16, and 24 hours. (C) Immunoblots from untreated and CD200 peptide–treated NKPOS cells for various time points probed for PARP, caspase 8, caspase 9, and GAPDH. (D) Immunoblots for PARP, caspase 8, caspase 9, and GAPDH from untreated and CD200 peptide–treated (8 hours) NKPOS cells exposed to caspase inhibitors Z-VAD-FMK (pan), Z-IETD-FMK (caspase 8), and Z-LEDH-FMK (caspase 9). (E) Gene set enrichment plots obtained from differentially expressed genes from NKPOS cells incubated with CD200 peptide for 2 and 4 hours compared to untreated cells. (F) qPCR of NKPOS cells for Fas, FasL, and FADD genes after 2-hour or 4-hour CD200 peptide incubation relative to untreated. Expression was normalized to β-actin. Fold change was calculated relative to untreated NK cells according to the 2–ΔΔCt method. (G) Immunoblots from untreated and CD200 peptide–treated NKPOS cells at various time points probed for Fas, FasL, FADD, and GAPDH. (H) Immunoblots for PARP, caspase 8, caspase 9, and GAPDH from untreated and CD200 peptide–treated (8 hours) NKPOS cells also exposed to anti-Fas monoclonal antibody (clone ZB4) at increasing concentrations. (I) Immunoblots for PARP and GAPDH from untreated and CD200 peptide–treated (8 hours) NKPOS cells also exposed to GW9662 at increasing concentrations. (J) Schematic summary of CD200-induced apoptosis. Western blot quantification is shown as a mean of 3 independent experiments. Data are presented as mean ± SD of 3 independent experiments. **P < 0.01; ****P < 0.0001 by 2-way ANOVA with Bonferroni’s post hoc test.