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IL15 stimulation with TIGIT blockade reverses CD155-mediated NK-Cell dysfunction in Melanoma

June 2020
Clinical Cancer Research 26(20):clincanres.0575.2020

June 2020
26(20):clincanres.0575.2020

DOI:10.1158/1078-0432.CCR-20-0575

Authors:

Joe-Marc Chauvin

University of Pittsburgh

Mignane Ka

University of Pittsburgh

Ornella Pagliano

University of Pittsburgh

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Purpose: Natural Killer (NK) cells play a critical role in tumor immunosurveillance. Multiple activating and inhibitory receptors regulate NK cell-mediated tumor control. The inhibitory receptor TIGIT and its counter-receptor CD226 exert opposite effects on NK cell-mediated tumor reactivity. Experimental design: We evaluated the frequency, phenotype and functions of NK cells freshly isolated from healthy donors and melanoma patients with multiparameter flow cytometry. We assessed TIGIT and CD226 cell surface expression and internalization upon binding to CD155. We evaluated the role of Interleukin (IL)-15 and TIGIT blockade in increasing NK cell-mediated cytotoxicity in vitro and in two mouse models. Results: NK cells are present at low frequencies in metastatic melanoma, are dysfunctional and downregulate both TIGIT and CD226 expression. As compared with TIGIT- NK cells, TIGIT+ NK cells exhibit higher cytotoxic capacity and maturation but paradoxically lower cytotoxicity against CD155+ MHC class I-deficient melanoma cells. Membrane-bound CD155 triggers CD226 internalization and degradation, resulting in decreased NK cell-mediated tumor reactivity. IL-15 increases TIGIT and CD226 gene expression by tumor-infiltrating NK cells (TiNKs) and, together with TIGIT blockade, increases NK cell-mediated melanoma cytotoxicity in vitro and decreases tumor metastasis in two mouse melanoma models. Specific deletion of TIGIT on transferred NK cells enhances the anti-metastatic activity of IL-15, while CD226 blockade decreases the effects of IL-15 and TIGIT blockade. Conclusion: Our findings support the development of novel combinatorial immunotherapy with IL-15 and TIGIT blockade to promote NK cell-mediated destruction of MHC class I-deficient melanoma, which are refractory to CD8+ T cell-mediated immunity.

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IL-15 stimulation with TIGIT blockade reverses CD155-mediated NK cell

dysfunction in melanoma

Joe-Marc Chauvin1, Mignane Ka1, Ornella Pagliano1, Carmine Menna1, Quanquan Ding1,

Richelle DeBlasio1, Cindy Sanders1, Jiajie Hou2, Xian-Yang Li3, Soldano Ferrone4,

Diwakar Davar1, John M. Kirkwood1, Robert Johnston5, Alan J. Korman5, Mark J.

Smyth3, and Hassane M. Zarour1,6.

1Department of Medicine and Division of Hematology/Oncology, University of

Pittsburgh, School of Medicine, Pittsburgh, PA 15213, USA. 2Department of Liver

Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai

200127, China. 3Immunology in Cancer and Infection Laboratory, QIMR Berghofer

Medical Research Institute, Queensland, Australia. 4Department of Surgery,

Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

5Biologics Discovery California, Bristol-Myers Squibb, Redwood City, CA 94063, USA.

6Department of Immunology, University of Pittsburgh, School of Medicine, Pittsburgh,

PA 15213, USA.

Running title: IL-15 and TIGIT blockade reverse NK dysfunction in melanoma

Keywords: Melanoma, Immunotherapy, TIGIT, IL-15, NK cells

This work was supported by NIH/NCI grants R01CA228181 and R01CA222203 (to

HMZ), a research grant by Bristol-Myers Squibb BMS (to HMZ), a cancer vaccine

collaborative clinical strategy team grant (to HMZ), and NCI grant P50CA121973 (to

Research.

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Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

JMK). MJS was supported by a National Health and Medical Research Council of

Australia (NH&MRC) Senior Principal Research Fellowship (1078671), a NH&MRC

Program grant (1132519), a NH&MRC Project grant (1124784), a CLIP award from the

Cancer Research Institute (New-York, NY), and a Project Grant from the Cancer Council

of Queensland (1140251). This work benefited from ImageStreamX MARKII grant NIH

1S10OD019942-01.

Address correspondence and reprint requests to Dr. Hassane M. Zarour, Hillman Cancer

Center, Research Pavilion, Suite 1.32a, 5117 Centre Avenue, Pittsburgh, PA 15213-2582,

Phone: 412 623 3272 Fax: 412 623 7704 Email: zarourhm@upmc.edu

MJS has a scientific research agreement with Bristol Myers Squibb and Tizona

Therapeutics and is on the scientific advisory board of Tizona Therapeutics and Compass

Therapeutics.

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Translational relevance

Here, we show that membrane-bound poliovirus receptor (PVR)/CD155 triggers CD226

internalization and degradation, resulting in decreased NK cell-mediated tumor reactivity.

We also show that IL-15 increases TIGIT and CD226 gene expression by tumor-

infiltrating NK cells, and together with TIGIT blockade, increases NK cell-mediated

melanoma cytotoxicity in vitro and decreases tumor metastasis in vivo. Collectively, our

findings support the novel combinatorial therapy with IL-15 together with TIGIT

blockade to promote NK cell-mediated destruction of MHC class I-deficient melanoma,

which are refractory to CD8+ T cell-mediated immunity and PD-1 blockade.

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Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

Abstract

Purpose: Natural Killer (NK) cells play a critical role in tumor immunosurveillance.

Multiple activating and inhibitory receptors regulate NK cell-mediated tumor control.

The inhibitory receptor TIGIT and its counter-receptor CD226 exert opposite effects on

NK cell-mediated tumor reactivity.

Experimental design: We evaluated the frequency, phenotype and functions of NK cells

freshly isolated from healthy donors and melanoma patients with multiparameter flow

cytometry. We assessed TIGIT and CD226 cell surface expression and internalization

upon binding to CD155. We evaluated the role of Interleukin (IL)-15 and TIGIT

blockade in increasing NK cell-mediated cytotoxicity in vitro and in two mouse models.

Results: NK cells are present at low frequencies in metastatic melanoma, are

dysfunctional and downregulate both TIGIT and CD226 expression. As compared with

TIGIT- NK cells, TIGIT+ NK cells exhibit higher cytotoxic capacity and maturation but

paradoxically lower cytotoxicity against CD155+ MHC class I-deficient melanoma cells.

Membrane-bound CD155 triggers CD226 internalization and degradation, resulting in

decreased NK cell-mediated tumor reactivity. IL-15 increases TIGIT and CD226 gene

expression by tumor-infiltrating NK cells (TiNKs) and, together with TIGIT blockade,

increases NK cell-mediated melanoma cytotoxicity in vitro and decreases tumor

metastasis in two mouse melanoma models. Specific deletion of TIGIT on transferred

NK cells enhances the anti-metastatic activity of IL-15, while CD226 blockade decreases

the effects of IL-15 and TIGIT blockade.

Conclusion: Our findings support the development of novel combinatorial

immunotherapy with IL-15 and TIGIT blockade to promote NK cell-mediated destruction

Research.

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of MHC class I-deficient melanoma, which are refractory to CD8+ T cell-mediated

immunity.

Research.

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Introduction

There is ample evidence that NK cells exhibit tumor-killing capacity and play a critical

role in mediating tumor immunosurveillance of primary tumors and controlling

metastases (1). NK cells express multiple activating receptors (ARs) and inhibitory

receptors (IRs) that regulate their function. Hence, therapeutic strategies to engage ARs

and/or counteract NK cell inhibition have the potential to promote NK cell-mediated

tumor reactivity (2). Solid tumors are usually poorly infiltrated by NK cells (3), thus the

phenotypic and functional studies of TiNKs remain very challenging in humans. TiNKs

in ovarian, breast, lung and prostate tumors downregulate multiple ARs, including

DNAM-1/CD226, CD16, NKG2D, NKp30, NKp46, and 2B4, and are dysfunctional (4-

9). NK cells upregulate multiple IRs that are also expressed by activated T cells,

including CD94/NKG2A (10) and the T cell immunoglobulin and ITIM domain (TIGIT)

(11). TIGIT binds with high and low affinity to CD155 (PVR) and CD112 (Nectin-2),

respectively which are expressed on monocytes, dendritic cells, and tumor cells,

including melanoma, and can also bind to the adhesion protein CD113 (Nectin-3) (12).

TIGIT competes with its costimulatory counter-receptor CD226 (DNAM-1), which binds

to CD155 with lower affinity (11,12). CD226 competes with CD112R for binding to

CD112 (Nectin-2) (13,14). In mouse-bearing tumors and in humans, dual PD-1/TIGIT

blockade potently augmented tumor-antigen CD8+ T cell functions and promoted tumor

rejection (15,16). TIGIT acts in regulatory T cells (Tregs) to promote tumor growth (17),

and CD226 opposes TIGIT to disrupt Treg stability in melanoma (18). Several lines of

evidence support the critical role of the TIGIT/CD226 axis in regulating NK cell-

mediated antitumor activity. First, TIGIT is highly expressed by circulating human NK

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cells (cNKs) and impedes NK cell-mediated killing of tumor cells (11). Upon CD155

binding to TIGIT, the ITT-like motif is phosphorylated and binds to Grb2 to recruit the

SH domain-containing inositol-5-phosphatase (SHIP1), impeding PI3 and MAP kinase

pathways, and NFKB signaling (19,20). Second, CD226 associates with LFA-1 and is

recruited to the immunological synapse to promote NK cell-mediated tumor cytotoxicity

(21,22). In vivo, CD226 is involved in NK cell-mediated tumor surveillance and control

of melanoma metastases and NK cell-mediated lysis of melanoma (23-26). Third, one

recent study in mouse tumor models has suggested that TIGIT acted primarily in NK

cells to regulate CD8+ T cell-mediated tumor reactivity (27). Among the various

cytokines that expand and activate NK cells, IL-15 potently enhances NK cell-mediated

tumor killing and is being actively investigated in many clinical trials (1). NK cell-based

therapies represent a powerful approach to kill MHC class I-deficient tumors that may

arise upon CD8+ T cell-mediated immune destruction of MHC class I-presenting tumor

cells, and may therefore counteract some of the mechanisms of resistance to PD-1

blockade (28,29). However, therapeutic strategies to potently reinvigorate NK cells in

human tumors remain to be developed. Here, we show that membrane-bound (mb)

CD155 triggers CD226 internalization and degradation, resulting in decreased NK cell-

mediated tumor reactivity. We also show that IL-15 with TIGIT blockade augments the

functional capacities of TiNKs in vitro and decreases tumor metastasis in mouse

melanoma models. Altogether, our findings provide the rationale for combining IL-15

with TIGIT blockade to counteract melanoma-induced NK cell dysfunction and promote

NK cell mediated lysis of MHC class I-deficient melanoma, which may prove useful in

anti-PD-1 refractory patients (28,29).

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Materials and Methods

Study subjects and cell lines. PBMCs from 30 HDs, and PBMCs and tumor samples

from 30 stage IV MPs were obtained under the Internal Review Board (IRB)–approved

protocols UPCI 05-140 and UPCI 96-099. Biopsies were obtained from 29 stage IV

melanoma patients, including 22 males and 7 females, ranging from age 31 to 82.

Metastatic sites included skin or soft tissue (20%), nodes (45%), lung (20%) and other

visceral locations (20%). Ten out of the 29 patients had received prior interferon-

adjuvant therapy. The samples were collected before therapy for stage IV melanoma and

more than three years after the end of interferon- adjuvant therapy. The melanoma cell

lines were derived from metastatic lesions of melanoma patients at the University of

Pittsburgh. FO-I is a β2m-deficient human melanoma cell line recognized by NK cells

(30). K562 and L cells were purchased (ATCC). Human CD155/PVR transcript variant 1

Gene cDNA Clone (full-length ORF Clone, Sino Biological Inc.) was transfected into L

cells using lipofectamine 3000 (Invitrogen) according to manufacturer’s instructions. L-

CD155 transfectants were selected upon exposure to Hygromycin (400μg/ml),

transfectants and L-CD155+ clonal cell lines were obtained with limiting dilution.

Phenotypic analysis and cell sorting. Peripheral blood mononuclear cells (PBMCs)

were used for ex-vivo flow cytometry analysis. We performed CD3 positive beads

magnetic separation (Miltenyi biotec) from the single cell suspensions obtained from

metastatic melanoma and used the CD3 negative fraction for ex-vivo flow cytometry.

CD45+CD3-CD56+ TIGIT+ and TIGIT- NK cells were sorted under sterile conditions

(FACSAria cytometer, BD). The following conjugated mAbs were used in flow

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cytometry experiments: TIGIT-PerCPeFluor710, TIGIT-PE, CD14-PacificBlue and

CD19-PacificBlue (ThermoFisher Scientific), NKG2D-PE-Cy7, PD-1-PE-Cy7 and

CD226-PE-Dazzle594 (Biolegend), CD96-APC and Tim-3-Alexa700 (R&D Systems),

CD45-BV510, CD56-BUV395, CD8-BUV737, CD3-APC-Cy7, and CD3-FITC (BD),

HLA-DR-ECD and CD16-PE-Cy7 (Beckman Coulter), CD226-PE or CD226-biotin

(DX11, Abcam) coupled with streptavidin-PE-TexasRed (ThermoFisher Scientific)

and/or IgG control mAbs. Viability was assessed using LIVE/DEAD violet, aqua kits

(ThermoFisher Scientific) or Zombie-NIR (Biolegend). Intracellular staining was

performed as previously reported (18). NK cells were labeled for Granzyme-A-

PacificBlue (Biolegend), Granzyme-B-APC (ThermoFisher Scientific), Perforin-FITC

(BD). Samples were acquired on a FACS LSR-II machine (BD) and analyzed using

FlowJo software v9 (Tree star).

NK cell stimulation and functional assays. NK cells were obtained from PBMCs and

MM single cell suspensions by a two-step cell separation. CD3 negative cells obtained

after CD3 positive selection with magnetic beads (Miltenyi Biotec), were used for CD56

positive selection (magnetic beads, Miltenyi Biotec) to isolate CD56+ NK cells. NK cells

were stimulated for 16 h with 100 U/mL IL-2 and/or 10 ng/mL IL-15 (Peprotech) prior to

incubation for 4 h with with FO-I (1:1 ratio) +/- blocking aTIGIT 10D7.G8 (IgG4, BMS)

(15), aCD226 (DX11, Abcam) or aCD155 (D171, ThermoFisher Scientific) mAbs. In

some experiments, NK cells were stimulated (16 h, 48 h, 4 d or 6 d) with IL-2

(100U/mL), IL-15 (10 ng/mL), IL-7 (10ng/mL) or IL-21 (10ng/mL) (Peprotech) prior to

4 h-co-culture with FO-I, or in presence or absence of STAT5 inhibitor (CAS 285986-31-

4, Sigma-Aldrich) or STAT3 inhibitor VI, S3I-201 (CAS 501919-59-1, Sigma-Aldrich)

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prior to receptor expression analysis. In other experiments, NK cells were incubated 48 h

with IgG- or CD155-Fc beads +/- blocking antibodies. Cells were washed in PBS 4 μM

EDTA and beads were removed with magnetic separation prior to the functional assays.

NK cell degranulation capacity and intracellular IFN- expression were evaluated with

flow cytometry using CD107a-PerCPcy5.5 and IFN--PE/Cy7 (Biolegend), as previously

reported (15). NK cell-mediated cytotoxicity (specific lysis) was evaluated using standard

51Chromium-release assay as previously reported (31). Briefly, 51Cr-(GE HealthCare)

labeled FO-I cells were incubated 4 h at 37oC either alone (spontaneous release), with

2.5% Triton-X-100 (maximum release), or with NK (E:T=30:1) +/- aTIGIT and/or

aCD226 blocking mAbs in triplicate wells.

Downregulation of CD226 and TIGIT expression by NK cells. 1 x 105 purified NK

cells were cocultured 48 h with L cells, L-CD155, FO-I, K562, HLA-I+ melanoma cell

lines, immature dendritic cells (ratio 1:1) or CD155-Fc-coated beads (ratio 1:4). In some

additional wells, L-CD155 and CD155-Fc beads were incubated 30 min with either

blocking anti-CD155 mAbs (D171, ThermoFisher Scientific), 20 μM of the inhibitor of

metalloproteases and ADAM17, TAPI-2 (Sigma-Aldrich), or 9 μM of the specific

inhibitor of ADAM10, GI254023X (Tocris) prior to coculture with NK cells and flow

cytometry using the following antibodies: CD56-PeCy7, CD226-PE-Dazzle594

(Biolegend) and TIGIT-PerCPefluor710 (ThermoFisher Scientific). Immature dendritic

cells were obtained from CD14+ monocytes isolated from PBMCs of normal donors with

magnetic separation (Miltenyi Biotec) and cultured 6 d with 1000 U/mL IL-4 and GM-

CSF (Peprotech). CD155-Fc beads were obtained by coating a chimeric CD155-Fc

(BMS) on M-450 Tosylactivated beads (Dynal) following the manufacturer’s protocol.

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Cell viability was assessed with LIVE/DEAD violet.

RT-PCR. 5 x 105 NK cells were incubated 48 h either with L cells or L-CD155 +/- 10

ng/mL IL-15. RNA was extracted with RNeasy mini Kits (Qiagen) and cDNA was

prepared by reverse transcription using M-MLV Reverse Transcriptase (ThermoFisher

Scientific). RT-PCR was performed with StepOne System (ThermoFisher Scientific). All

samples were analyzed and normalized to the expression of β-glucuronidase (β-Gus).

CD226 and TIGIT expression were detected using previously described primers (18).

ELISA. sCD155 levels in sera were evaluated by sandwich ELISA. High-binding 96-

well plates were coated with 2 μg/mL aCD155 mAb (D171, Abcam) for capture antibody

for 30 min at 37oC in sodium phosphate buffer (pH 7.6) and blocked for 1 h at room

temperature with blocking buffer (PBS 3% BSA and 0.05% Tween). Plates were washed

three times (PBS 0.05% Tween). Human CD155-Fc and 1:10 diluted samples were plated

at 100 μL for 2 h at room temperature and washed three times. Plates were incubated with

100 μL of secondary rabbit polyclonal aCD155 antibody (2 μg/mL in blocking buffer,

Lifespan) for detection, washed, then incubated for 30 min at room temperature with 100

μL HRP-conjugated goat anti-rabbit (1:1000 in blocking buffer, ThermoFisher

Scientific), washed, and reacted with 100 μL of substrate solution (1:1

tetramethylbenzidine and hydrogen peroxide, BD). The reaction was stopped after 5 min

with 50 μL of 2 N sulfuric acid and absorbance was read at 450 nm. All values were

determined in triplicates.

Imagestream flow cytometry. 1 x 105 NK cells isolated from PBMCs were incubated 1

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h at 37oC either with medium alone, L cells, L-CD155, or FO-I (1:1 ratio). Cells were

washed in cold PBS and in refrigerated centrifuge to prevent further receptor

internalization. Viability was assessed with the Zombie-NIR kit (Biolegend). NK cells

were stained with CD45-BV510 (BD), CD56-PeCy7 (Biolegend), CD226-PE (DX11,

Abcam) or TIGIT PerCPefluor710 (MBSA43, ThermoFisher Scientific), then

permebealized and stained with CD226-biotin (DX11, Abcam) and streptavidin PE-

TexasRed, or with TIGIT-PE (MBSA43, ThermoFisher Scientific) prior to analysis on

ImagestreamX MARKII Imaging flow cytometer with INSPIRE software (Amnis, EMD

Millipore). The flow rate was set at minimum and the objective magnification was set at

60x for all samples. A multifluorophore-labeled sample was used to determine accurate

laser settings and avoid oversaturation. Gradient RMS and aspect ratio versus area on the

brightfield channel were used during acquisition to ensure collection of focused single

cells. At least 5 x 103 live CD45+CD56+ NK cells were acquired per sample. Data

analysis was performed using IDEAS software (Amnis, EMD Millipore). CD226 and

TIGIT internalization ratios were calculated using CD45 membrane expression as a mask

(a region of interest) to determine the membrane and intracellular sections of the cells.

Intracellular receptor degradation. 1 x 105 NK cells were incubated 30 min either with

or without 0.5 μM bafilomycin A1 (Sigma-Aldrich), 25 ng/mL concanamycin-A (Sigma-

Aldrich), or 1% DMSO as control, prior to 16 h or 48 h coculture with L cells, L-CD155

or FO-I (1:1 ratio) +/- IL-2 (100 U/mL), IL-15, IL-7 or IL-21 (10 ng/mL). Cells were

washed with PBS and fixed with 1.3% paraformaldehyde. All cells were stained for

surface CD45-BV510 (BD) and CD56-PeCy7 (Biolegend). Cells were either stained for

surface CD226 and TIGIT expression, or permeabilized and stained for total CD226 and

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TIGIT expression prior to flow cytometry. Experiments were repeated and performed in

triplicates.

Mice and experimental metastasis models. C57BL/6 wild-type (WT) and C57BL/6

Rag2−/−



c−/− mice were bred in-house. C57BL/6 Tigit−/− mice were kindly provided by

Bristol Myers Squibb. All mice were bred and maintained at the QIMR Berghofer

Medical Research Institute and used when more than 6 weeks of age. No mice were

excluded based on pre-established criteria in all studies and no active randomization was

applied to any experimental group. The investigators were not blinded to the group

allocation during the experiment and/or when assessing the outcome. All experiments

were approved by the QIMR Berghofer Medical Research Institute Animal Ethics

Committee.

Mouse B16F10 melanoma cells were grown in Dulbecco’s Modified Eagle Medium

(DMEM) supplemented with 10% Fetal Calf Serum (Bovogen), 1% Glutamine (Gibco),

1% HEPES (Gibco) and 1% Penicillin/Streptomycin (Gibco). LWT1 melanoma cells

were cultured in RPMI 1640, supplemented with 10% Fetal Calf Serum (Bovogen), 1%

Glutamine (Gibco), and 1% Penicillin-Streptomycin (Gibco). All cell lines were

maintained at 37°C, 5% CO2. Cell injection and monitoring procedures were described in

previous studies (32-34). All cell lines were routinely tested negative for Mycoplasma,

but cell line authentication was not routinely performed. B16F10 melanoma (5 x 105) or

LWT1 melanoma (7.5 x 105) cells were injected intravenously into the tail vein of WT or

Tigit−/− mice. On days 0 and 3 after tumor inoculation, some mice were treated

intraperitoneally (i.p.) with PBS or IL-15/IL-15Ra complexes (R&D Sytems) or cIg or

anti-mouse TIGIT (4B1) at the indicated doses. Some groups of mice were additionally

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treated with an aCD226 mAb (480.1) to block CD226. In some experiments, CD3-

NK1.1+ NK cells were sorted by flow cytometry from spleens of WT or Tigit−/− mice to

95% purity and WT or Tigit−/− NK cells (2 x 105) were injected i.v. into Rag2−/−



c−/−

mice. After 6 d, blood was collected to check the equivalent reconstitution of NK cells by

flow cytometry and B16F10 (5 x 105 or 1 x 104) melanoma cells were injected i.v. into

Rag2−/−



c−/− mice. Lungs were harvested on day 14 and metastatic colonies on the

surface of the lungs were counted using a dissecting microscope.

Statistical analysis. Statistical analyses were performed in Prism software (Graphpad).

The normality of each variable was evaluated using the Shapiro-Wilk test. In case of

normally distributed data, the comparison was performed using unpaired or paired two-

tailed t-tests, one-way ordinary or repeated-measures ANOVA tests followed by Tukey’s

multiple comparisons test to compare all data together or Dunnett’s multiple comparisons

test to compare all data with control. Data that were not normally distributed were

compared with Wilcoxon matched-pairs signed rank tests (two paired groups) or Kruskal-

Wallis test followed by Dunn’s multiple tests (more than two groups, unpaired). Linear

regressions were evaluated with Pearson correlation tests. Significant differences were

indicated for each figure and defined as ns (non-significant), P>0.05; *, P<0.05; **,

P<0.01; ***, P<0.001 and ****, P<0.0001.

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Results

NK cells downregulate TIGIT and CD226 in metastatic melanoma. We first

evaluated the expression of TIGIT and CD226 by NK cells (CD45+CD3-CD56+ cells,

Supplemental Figure 1A) in the periphery and at tumor sites in melanoma patients (MPs).

We measured lower NK cell frequencies in metastatic melanoma (MM) than in PBMCs

of MPs (Figure 1A). In sharp contrast with CD8+ T cells (15), TiNKs exhibited lower

TIGIT expression (frequency and MFI) than cNKs isolated from MPs and HDs (Figure 1,

B and C). TiNKs exhibited lower CD226 expression (frequency and MFI) than cNKs,

resulting in an increased TIGIT to CD226 expression ratio (Figure 1D). We observed a

positive correlation between the percentages and MFI of TIGIT expression in cNKs and

TiNKs for individual donors. We also observed a positive correlation between the

percentages and MFI of CD226 expression in TiNKs, which downregulate CD226

expression, but not in cNKs. (Supplemental Figure 1B). CD56dim NK cells were enriched

in MM and downregulated both CD226 and TIGIT expression as compared with the

periphery (Supplemental Figure 1C). CD56bright NK cells, which represent a minority of

NK cells in the periphery and at tumor sites, expressed low TIGIT level (Supplemental

Figure 1C). TiNKs and cNKs exhibited similar levels of CD96 and CD57 expression and

both expressed low-level PD-1 (Figure 1E). In addition, TiNKs displayed lower

frequencies of activation/maturation markers such as Tim-3, NKG2D, CD16 (Figure 1E).

Notably, TiNKs exhibited lower 2B4, NKp46, NKp30 expression (frequencies) as

compared with HD cNKs but not MPs cNKs (Figure 1E).

Collectively, our findings show that TiNKs are present at low frequencies in MM and

downregulate both TIGIT and CD226 but not CD96 as compared with cNKs.

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TIGIT+ NK cells exhibit higher lytic potential but lower degranulation capacity

than TIGIT- NK cells in melanoma. We next investigated the functional capacities of

TIGIT+ and TIGIT- NK cells isolated from the periphery and tumor sites of MPs (Figure

2A). The frequencies of granzyme A+TIGIT- NK cells were significantly lower than

granzyme A+TIGIT+ NK cells, both for cNKs and TiNKs (mean frequency 70% ± 9% vs.

90% ± SD 6%, respectively, for cNKs, P<0.01, and 41 ±14% vs. 62 ± 12%, respectively,

for TiNKs, P <0.0001). The frequencies of granzyme B+ cells were also significantly

lower in TIGIT+ TiNKs as compared with TIGIT+ cNKs (mean frequency 62 ± 10% vs.

86 ± 6.3%, P<0.01, respectively). Finally, the frequencies of perforin+ cells were also

significantly lower in TIGIT+ TiNKs as compared with TIGIT+ cNKs (mean frequency

64 ± 11 vs. 81 ± 10%, P<0.001). Perforin+ NK cell frequencies positively correlated with

TIGIT+ NK cell frequencies both in the periphery and at tumor site (Figure 2B). Also,

TIGIT+ TiNKs displayed lower expression of granzymes and perforin than TIGIT+ cNKs.

To evaluate the implication of these findings, cNKs and TiNKs were isolated from

PBMCs and tumors of MPs, respectively (Supplemental Figure 1A), before degranulation

assay in the presence of the MHC class I-deficient melanoma cell line FO-I or K562,

which express high-level CD155 and CD112 but low-level MICA/B and ULBPs

(Supplemental Figure 1D). TiNKs exhibited lower degranulation capacity than cNKs and

CD107a+ TiNK frequencies inversely correlated with the TIGIT/CD226 expression ratio,

supporting the role of the TIGIT/CD226 axis in modulating NK cell functions (Figure 2C

and Supplemental Figure 1E). We then evaluated the lysis of FO-I by TIGIT- and TIGIT+

NK cells sorted from PBMCs of MPs. TIGIT+ NK cells exhibited lower lytic activity and

CD107a expression than TIGIT- NK cells (Figure 2D). Altogether, our findings show that

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TiNKs are more dysfunctional than cNKs and that TIGIT+ NK cells display higher lytic

potential but paradoxically lower lytic activity than TIGIT- NK cells, against CD155+

MHC-class I deficient melanoma cells.

TIGIT blockade alone failed to reverse NK cell dysfunction in melanoma. We next

evaluated the role of TIGIT and CD226 in regulating NK cell-mediated reactivity against

MHC class I-deficient melanoma (Figure 2). NK cells isolated from PBMCs and tumor of

MPs were stimulated with IL-2 and IL-15 prior to coculture with FO-I in the presence of

blocking anti-(a)TIGIT and/or aCD226 as compared with IgG control mAbs. In line with

previous published findings (11,27,35), TIGIT blockade increased the frequencies of lytic

cNKs, while CD226 blockade impeded NK cell-mediated cytotoxicity and degranulation

in the presence of FO-I (Figure 2E and Supplemental Figure 1F). The effects of TIGIT

blockade on NK cell-mediated cytotoxicity and degranulation capacity in the presence of

FO-I were abrogated by CD226 blockade (Figure 2E and Supplemental Figure 1F).

CD155 blockade had no significant impact on the degranulation capacity of cNKs against

FO-I (Supplemental Figure 1F). However, and in sharp contrast with the periphery,

neither TIGIT blockade nor CD226 blockade significantly changed the low TiNK lytic

activity against FO-I (Figure 2F). TIGIT blockade modestly increased IFN- production

by cNK cells but had no significant effect on TiNKs (Supplemental Figure 1G).

Altogether, our findings show that, in sharp contrast with cNKs, TiNKs exhibit poor lytic

function against MHC-deficient melanoma cells, which cannot be rescued upon TIGIT

blockade.

Membrane-bound CD155 induces CD226 internalization/degradation and NK cell

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dysfunction. Because NK cells downregulate CD226 cell surface expression upon

binding to membrane-bound (mb) CD155 (7), we next investigated the role of mb and

soluble (s) CD155 in mediating the downregulation of CD226 and TIGIT by NK cells.

To this end, cNKs were incubated in the presence of either CD155-Fc-coated beads,

CD155-expressing cells including L-cells transfected or not with human CD155 (L-

CD155), K562, FOI and dendritic cells, or sCD155 (Figure 3). cNKs strongly

downregulated CD226 expression and to a lesser extent TIGIT expression in presence of

L-CD155, FO-I, K562, immature DCs and CD155-Fc-coated beads, but not L-cells,

resulting in an increased TIGIT/CD226 expression ratio (Figure 3, A and B). CD226 and

TIGIT downregulation were abrogated in the presence of blocking aCD155 mAbs

(Figure 3, A and B and Supplemental Figure 2A), supporting that CD226 and TIGIT

downregulation occurred in a CD155-mediated fashion. In contrast, NK cells did not

significantly downregulate CD96 nor Tim-3 expression as control (Supplemental Figure

2B). CD226 and TIGIT downregulation correlated with the level of CD155 expression

(Supplemental Figure 2, C and D). Similarly, TiNKs showed decreased CD226 and

increased TIGIT/CD226 expression ratio in correlation with the level of CD155

expression by the tumor cells in the TME (Supplemental Figure 2E). Notably, sCD155 or

supernatants of melanoma cell lines containing high-level sCD155 (Supplemental Figure

2C), did not induce CD226 downregulation by NK cells (Figure 3C). CD155-induced

CD226 downregulation by NK cells occurred at a post-transcriptional level since CD226

mRNA relative expression did not significantly change after 48-h co-culture with L-

CD155 (Figure 3D). CD226 downregulaton by NK cells was not abrogated in the

presence of protease inhibitors (TAPI-2 and GI254023X), suggesting that it did not occur

upon ectodomain shedding (Supplemental Figure 2F). Notably, NK cells expressed

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similar Annexin-V expression in the presence of L-CD155 cells or CD155-Fc-coated

beads, respectively, and compared with L cells or IgG beads, respectively (Supplemental

Figure 2G).

To investigate the fate of TIGIT and CD226 upon binding to mbCD155, including

internalization, degradation and recycling, cNKs were co-incubated (1 h) with FO-I, L-

CD155, L-cells or no cells prior to intracellular and extracellular staining of TIGIT,

CD226, and CD45 followed by Imagestream flow cytometry. In presence of L-CD155

and FO-I but not L cells, the cell-surface expression of CD226 and TIGIT strongly

decreased together with an increased intracellular expression of both molecules (Figure 3,

E and F), resulting in increased internalization ratios (Figure 3G). To investigate whether

CD226 and TIGIT undergo ligand-induced endocytosis followed by recycling or

degradation (36), we next evaluated their surface and total cellular expression in cNKs

incubated with L-cells or L-CD155 with or without V-ATPases inhibitors (16 h, Figure

4). In the presence of L-CD155 but not L cells, total CD226 but not TIGIT expression in

NK cells (MFI) sharply decreased as compared with L cells (Figure 4, A, B and C). V-

ATPase inhibitors, which inhibit protein degradation by acidified lysosomes (37),

abrogated the decrease in total cellular CD226 expression (Figure 4B and Supplemental

Figure 3, A and B).

We next assessed the functional implication of CD155-mediated CD226 degradation on

NK cell functions. To this end, cNKs isolated from MPs were incubated in the presence

of CD155-Fc- or IgG-coated beads and/or aTIGIT and/or aCD226 mAbs prior to

stimulation with IL-2 and IL-15, and incubation with FO-I. CD155-Fc-treated cNKs

exhibited lower CD107a expression as compared with IgG-treated cNKs (Figure 4D).

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TIGIT blockade partially reversed the effect of CD155-Fc on cNKs while CD226

blockade had no significant effect (Figure 4E and supplemental Figure 3C). As controls,

IgG-coated beads and sCD155 had no significant impact on cNK cytolytic activity.

Altogether, our data show that mbCD155 but not sCD155 induced both TIGIT and

CD226 internalization, and CD226 but not TIGIT degradation. They also show that

mbCD155-mediated CD226 degradation promoted NK cell dysfunction that was only

partially reversed upon TIGIT blockade.

IL-15 together with TIGIT blockade reverses CD155-mediated NK cell exhaustion

and impedes experimental melanoma metastasis in vivo. IL-15 upregulates CD226

expression by NK cells (38,39). In addition, IL-15 together with IL-12 also upregulates

TIGIT expression by cNKs isolated from HIV-infected patients (40). We next

investigated whether IL-15, which increases NK cell-mediated antitumor activity,

regulates CD226 and TIGIT expression by cNKs and TiNKs as compared with other -

chain cytokines including IL-2, IL-15, IL-7 and IL-21. IL-15 induced higher CD226 and

TIGIT expression after 6 d stimulation than IL-2, IL-7, or IL-21 (Supplemental Figure

4A). We then incubated cNKs in the presence of IL-15 and L cells or L-CD155 prior to

flow cytometry and RT-PCR for CD226 and TIGIT expression. IL-15 increased CD226

and TIGIT cell surface expression that was abrogated in the presence of STAT3 or

STAT5 inhibitors (Figure 5A). IL-15 also increased Tim-3 and NKG2D expression by

NK cells (Supplemental Figure 4A) as previously reported (41-43). IL-15 increased

CD226 and TIGIT gene expression with or without L-CD155 (Figure 5B). To evaluate

whether IL-15 reversed the downregulation of CD226 cell surface expression upon

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mbCD155, cNKs were incubated in the presence of IL-15 and L cells, L-CD155 or FOI

prior to flow cytometry and Imagestream. In the presence of L-CD155 or FO-I, IL-15

significantly increased both cell surface and total CD226 expression by cNKs (Figure 5C)

in contrast with the other -chain cytokines (Supplemental Figure 4B) but did not

significantly impede CD226 internalization (Figure 5D). Notably, IL-15 also increased

the expression of both CD226 and TIGIT by TiNKs (Figure 5E).

We next assessed whether IL-15 alone or together with TIGIT blockade increased TiNK-

mediated antitumor activity and counteracted CD155-mediated NK cell dysfunction. To

this end, cNKs pre-incubated with CD155-Fc beads or IgG beads, or TiNKs were treated

with IL-15 (16 h or 6 d) prior to evaluating their level of granzyme and perforin

expression as well as CD107a expression in the presence of FO-I with or without aTIGIT

mAbs. Prolonged IL-15 (6 d) increased cNK and TiNK degranulation in the presence of

FO-I (Figure 5F) as well as perforin, granzyme A and granzyme B expression

(Supplemental Figure 4, B and C) as compared with overnight stimulation with IL-15.

Prolonged IL-15 stimulation alone (mean fold change: 1.49 ± SD 0.32), but not TIGIT

blockade (1.17 ± 0.15), significantly increased TiNK degranulation in the presence of

FO-I as compared with overnight stimulation with IL-15. Prolonged IL-15 together with

TIGIT blockade further increased the degranulation of TiNK in the presence of FO-I

(1.83 ± 0.48) (Figure 5G and Supplemental Figure 4E).

To support the relevance of our findings in vivo, we investigated whether IL-15 and

TIGIT blockade promoted NK cell-mediated control of metastatic tumors in two mouse

melanoma models (Figure 6A). Therapy with aTIGIT mAbs induced anti-metastatic

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activity against established B16F10 and LWT1 lung metastases only in mice treated with

IL-15/IL-15Ra complexes (mean B16F10 metastasis numbers: 72 ± SD: 23, 144 ± 40,

324 ± 43 and 344 ± 36, respectively, for IL-15/IL-15R + aTIGIT mAbs, IL-15/IL-15R,

aTIGITmAbs, and IgG + PBS, respectively) (Figure 6B and Supplemental Figure 5A and

B). IL-15 was more effective in Tigit−/− mice compared with WT mice, despite Tigit−/−

mice having no significant reduction in metastasis compared with WT mice as previously

published (Figure 6B and Supplemental Figure 5A) (32,33). When NK cells from Tigit−/−

and WT mice were transferred into immunodeficient Rag2−/−



c−/− mice (Figure 6A, C,

D), they reduced B16F10 lung metastases by half as compared with no NK cell transfer

(393 ± 38 vs. 186 ± 35 and 192 ± 42, respectively), but IL-15/IL-15R complexes were

more effective in reducing metastases in mice transferred with Tigit−/− as compared with

WT NK cells (31 ± 17 and 100 ± 36, respectively) (Figure 6D). Similar findings were

observed in mice with the LWT1 lung metastases (Supplemental Figure 5C). In line with

previous findings, CD226 blockade significantly increased the number of lung metastases

in mice. Interestingly, CD226 blockade abrogated the effects of IL-15 alone or in

combination with TIGIT blockade (Figure 6E and Supplemental Figure 5B).

Additionally, NK cell but not CD8+ T cell depletion abrogated the antitumor effects of

combined IL-15/IL-15R and TIGIT blockade in both B16F10 and LWT1 models. These

findings support the role IL-15/IL-15R and TIGIT blockade in promoting direct NK cell-

mediated tumor reactivity against lung metastases in vivo (Supplemental Figure 5D).

Altogether, our findings show that IL-15 increased CD226 and TIGIT expression by NK

cells in a STAT3/5-dependent fashion. Prolonged IL-15 stimulation together with TIGIT

blockade increased the TiNK degranulation capacity and lysis of MHC class I-deficient

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melanoma. TIGIT blockade and TIGIT loss in NK cells were effective against tumor

metastasis only in the presence of IL-15. The antitumor activity of IL-15 and TIGIT

blockade were abrogated by CD226 blockade, supporting that TIGIT and CD226 act

antagonistically to regulate the antitumor effector function of NK cells in vivo.

Interestingly the antitumor effects of Tigit−/− NK cells in Rag2−/−



c−/− mice with

melanoma suggest that TIGIT depletion in NK cells enhanced NK cell-mediated

antitumor reactivity, independently of CD8+ T cells.

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Discussion

In the present study, our findings support the development of combinatorial

immunotherapy with IL-15 and TIGIT blockade to reinvigorate TiNKs against MHC

class I-deficient melanoma. We observed that NK cells were present at low frequencies in

human MM, were more dysfunctional and downregulated both TIGIT and CD226 as

compared with cNKs. CD226 expression is downregulated in TiNKs as compared with

cNKs and correlates with the levels of CD155 expression by melanoma cells in the TME.

Noteworthy, CD226 appears to regulate T cell responses to PD-1 blockade and

combinatorial therapy in two mouse tumor models (44). Upon PD-1 blockade, T cells

upregulate CD226, which is the substrate for dephosphorylation by SHP2 upon PD-1

engagement (44). These findings in mouse T cells may not be relevant to human NK cells

for several reasons. First, these data were obtained in mouse tumor models that are

responsive to PD-1 blockade with no significant CD226 downregulation by T cells in the

TME, unlike solid human tumors (15). Second, and most importantly, the reported

CD226 effects are mediated by PD-1 signaling in T cells (SHP2), which upregulate PD-1

in the TME. In sharp contrast with these findings, we show that human NK cells, unlike

mouse NK cells, do not upregulate PD-1 expression in the periphery nor at tumor sites.

TiNKs displayed decreased expression of multiple activation/maturation markers

including CD16 and NKG2D. Our findings are reminiscent of previous studies

supporting that the downregulation of multiple ARs by TiNKs is associated with NK cell

dysfunction and decreased tumor lysis capacity in many human tumors, including breast,

ovarian, lung and prostate cancers (4-7,9). Furthermore, TiNKs exhibit decreased

degranulation capacity that inversely correlated with the percentages of invading

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melanoma tumor cells (26), supporting the role of tumor cells in driving NK cell

dysfunction. The phenotypic features of TiNKs strongly contrasts with those of human

CD8+ tumor-infiltrating lymphocytes, which upregulate TIGIT as well as others IRs like

PD-1 and Tim-3 through Prdm1 and c-Maf activation downstream of TCR activation (45)

while they downregulate CD226 expression in the TME, resulting in an imbalance of

TIGIT/CD226 expression. Strikingly, our findings in melanoma sharply contrast with

those recently published in colon cancers, suggesting increased TIGIT expression by

intratumor NK cells as compared with peritumor NKs (27). In this study including 19

colon tumors, it is unclear how the investigators precisely isolated low-frequency

intratumor and peritumor NKs for flow cytometry. One may also wonder whether the

pathogens in the gut microbiome, which act on innate and adative immunity (46), may

critically influence the activation and phenotype of NK cells in colon tumors. In addition,

and in contrast to humans, mouse cNKs express very low level TIGIT (19,47), which

makes comparative studies of TIGIT expression between mice and humans very difficult.

Several lines of evidence support the role of the TIGIT/CD226 axis in regulating NK

cell-mediated tumor killing capacity. As compared with TIGIT- NK cells, TIGIT+ NK

cells, both in the periphery and at tumor site, upregulated multiple activation/effector

markers including granzymes and perforin, supporting that TIGIT is a marker of NK cell

activation (48). Paradoxically, TIGIT+ NK cells exhibited lower killing activity against

CD155+ MHC class-I deficient melanoma FO-I as compared with TIGIT- NK cells. Such

findings are in line with previous published findings (48,49). They are also reminiscent of

exhausted CD8+ T cells, which upregulate multiple IRs, are dysfunctional, and display

increased levels of perforin and granzymes despite lower degranulation capacities in the

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presence of target cells as compared to non-exhausted CD8+ T cells (50,51). The

degranulation capacity of cNKs and TiNKs against CD155+ MHC class-I deficient

melanoma FO-I inversely correlated with cell surface TIGIT/CD226 ratio, supporting the

role of the TIGIT/CD226 axis in regulating NK cell effector functions. Our findings

showed that mbCD155, but not sCD155, induced both CD226 and TIGIT internalization

with CD226 but not TIGIT degradation. Multiple ARs and IRs, which regulate NK cell

activation, are downregulated upon binding to their respective ligands, including NKG2D

and 2B4 (52,53). Receptor downregulation upon ligand binding contributes to the

regulation of receptor signaling in NK cells (54). In sharp contrast with NKG2D and

soluble MIC-A/MIC-B (52), sCD155, which is present in the serum of patients with

advanced cancers (55), did not induce CD226 endocytosis. In addition, CD226

downregulation correlated with the levels of mbCD155 expression, supporting that

CD226 downregulation electively occurs in the TME. Although CD226 shedding by

metalloproteases has been previously reported in vitro (56), metalloprotease inhibitors

failed to prevent CD155-mediated CD226 downregulation by cNKs. Notably, CD96,

which also binds to CD155, was not significantly downregulated by NKs in presence of

mbCD155 in vitro. Collectively, our findings support that mbCD155 acts as a master

regulator of the TIGIT/CD226 axis to limit NK cell tumor killing capacities in the TME.

This observation adds to the previous findings supporting that the downregulation of

ARs, such as NKG2D, NKp46 and NKp30, impedes the tumor-killing capacity of TiNKs

(4-7). The nature of the intracellular motifs that differentially drive the internalization,

recycling or degradation of TIGIT, CD226 and CD96 remain to be identified.

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TIGIT blockade alone increased cNK cell killing of CD155+ MHC class I-deficient

melanoma, which was abrogated upon CD226 blockade, but had no significant effect on

dysfunctional TiNKs, which downregulate both TIGIT and CD226 and exhibit lower

killing capacities. While IL-15 increased NK cell-mediated killing capacity, it also

increased CD226 and TIGIT expression by NKs in a STAT3/5-dependent fashion.

Prolonged IL-15 stimulation together with TIGIT blockade increased the human TiNK-

mediated lysis of MHC class I-deficient melanoma. TIGIT blockade or TIGIT loss in NK

cells only decreased tumor metastasis in two lung metastasis mouse models in the

presence of IL-15. Interestingly the effects of IL-15 and TIGIT blockade on NK cells in

vivo were abrogated upon CD226 blockade. These findings support that TIGIT and

CD226 exert antagonistic effects to regulate the antitumor effector function of NK cells.

They also suggest that combinatorial therapy with IL-15 and TIGIT blockade promotes

CD226 engagement of CD155 on NK cells to augment their effector functions that may

occur through phosphorylation-mediated inactivation of transcription factor FOXO1 (57).

We cannot exclude that the upregulation of other activating receptors like NKG2D by

NK cells upon IL-15 plays a role in NK cell-mediated tumor reactivity. This will need to

be further investigated. Interestingly, sustained IL-15 stimulation of NK cells in vitro and

in vivo appears to promote initial proliferation and maturation, followed with NK cell

exhaustion with impaired activation, cytotoxicity and proliferative capacity (58,59).

Therefore, IL-15 dosage and administration schedule will need to be carefully designed

to avoid NK cell exhaustion.

In a recent study in mouse-tumor bearing models with lung metastases (27), the

therapeutic effects of PD-1, TIGIT or dual PD-1/TIGIT blockade were reported to act

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primarily on NK cells to enhance antitumor activity mediated by CD8+ T cells. The

mechanisms used by NK cells to regulate adaptive immunity upon TIGIT blockade have

not yet been elucidated. Our findings in mice and in humans do not support these

conclusions for several reasons. First, TIGIT blockade and TIGIT loss in NK cells were

not effective against lung melanoma metastasis in the absence of IL-15. Second, the

antitumor effects of Tigit−/− NK cells in Rag2−/−



c−/− mice with melanoma showed that

TIGIT depletion in NK cells enhanced NK cell-mediated antitumor reactivity, but only in

the presence of exogenous IL-15, and independently of CD8+ T cells. Whether NK cells

participate in the environmental signals guiding CD8+ T cell priming, development of

CD8+ effector T cells into CD8+ memory T cells or CD8+ memory T-cell maintenance

remains to be evaluated. Additional mechanistic studies are needed to thoroughly

investigate these questions.

In summary, the present study shows that mbCD155 triggers CD226 internalization and

degradation by NK cells, resulting in increased cell surface TIGIT/CD226 expression

ratio and decreased NK cell-mediated tumor reactivity. IL-15 together with TIGIT

blockade reinvigorates TiNK-mediated killing of melanoma cells in vitro and in vivo in a

CD226-dependent fashion. Altogether, our findings may support the development of

novel combinational immunotherapy with IL-15 and TIGIT blockade to promote NK

cell-mediated killing of MHC deficient tumors that are refractory to CD8+ T cell-

mediated immunity.

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References

1. Guillerey C, Huntington ND, Smyth MJ. Targeting natural killer cells in cancer

immunotherapy. Nat Immunol 2016;17(9):1025-36 doi 10.1038/ni.3518.

2. Muntasell A, Ochoa MC, Cordeiro L, Berraondo P, Lopez-Diaz de Cerio A, Cabo

M, et al. Targeting NK-cell checkpoints for cancer immunotherapy. Curr Opin

Immunol 2017;45:73-81 doi 10.1016/j.coi.2017.01.003.

3. Sconocchia G, Arriga R, Tornillo L, Terracciano L, Ferrone S, Spagnoli GC.

Melanoma cells inhibit NK cell functions. Cancer Res 2012;72(20):5428-9;

author reply 30 doi 10.1158/0008-5472.CAN-12-1181.

4. Mamessier E, Sylvain A, Bertucci F, Castellano R, Finetti P, Houvenaeghel G, et

al. Human Breast Tumor Cells Induce Self-Tolerance Mechanisms to Avoid

NKG2D-Mediated and DNAM-Mediated NK Cell Recognition. Cancer Research

2011;71(21):6621-32 doi 10.1158/0008-5472.Can-11-0792.

5. Mamessier E, Sylvain A, Thibult ML, Houvenaeghel G, Jacquemier J, Castellano

R, et al. Human breast cancer cells enhance self tolerance by promoting evasion

from NK cell antitumor immunity. J Clin Invest 2011;121(9):3609-22 doi

10.1172/Jci45816.

6. Platonova S, Cherfils-Vicini J, Damotte D, Crozet L, Vieillard V, Validire P, et

al. Profound coordinated alterations of intratumoral NK cell phenotype and

function in lung carcinoma. Cancer Res 2011;71(16):5412-22 doi 10.1158/0008-

5472.CAN-10-4179.

7. Carlsten M, Norell H, Bryceson YT, Poschke I, Schedvins K, Ljunggren HG, et

al. Primary human tumor cells expressing CD155 impair tumor targeting by

Research.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

down-regulating DNAM-1 on NK cells. J Immunol 2009;183(8):4921-30 doi

10.4049/jimmunol.0901226.

8. Fregni G, Messaoudene M, Fourmentraux-Neves E, Mazouz-Dorval S, Chanal J,

Maubec E, et al. Phenotypic and functional characteristics of blood natural killer

cells from melanoma patients at different clinical stages. Plos One

2013;8(10):e76928 doi 10.1371/journal.pone.0076928.

9. Pasero C, Gravis G, Guerin M, Granjeaud S, Thomassin-Piana J, Rocchi P, et al.

Inherent and Tumor-Driven Immune Tolerance in the Prostate Microenvironment

Impairs Natural Killer Cell Antitumor Activity. Cancer Res 2016;76(8):2153-65

doi 10.1158/0008-5472.CAN-15-1965.

10. Vivier E, Ugolini S, Blaise D, Chabannon C, Brossay L. Targeting natural killer

cells and natural killer T cells in cancer. Nat Rev Immunol 2012;12(4):239-52 doi

10.1038/nri3174.

11. Stanietsky N, Simic H, Arapovic J, Toporik A, Levy O, Novik A, et al. The

interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity.

P Natl Acad Sci USA 2009;106(42):17858-63 doi 10.1073/pnas.0903474106.

12. Yu X, Harden K, Gonzalez LC, Francesco M, Chiang E, Irving B, et al. The

surface protein TIGIT suppresses T cell activation by promoting the generation of

mature immunoregulatory dendritic cells. Nat Immunol 2009;10(1):48-57 doi

10.1038/ni.1674.

13. Zhu Y, Paniccia A, Schulick AC, Chen W, Koenig MR, Byers JT, et al.

Identification of CD112R as a novel checkpoint for human T cells. J Exp Med

2016;213(2):167-76 doi 10.1084/jem.20150785.

Research.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

14. Murter B, Pan X, Ophir E, Alteber Z, Azulay M, Sen R, et al. Mouse PVRIG Has

CD8(+) T Cell-Specific Coinhibitory Functions and Dampens Antitumor

Immunity. Cancer Immunol Res 2019;7(2):244-56 doi 10.1158/2326-6066.CIR-

18-0460.

15. Chauvin JM, Pagliano O, Fourcade J, Sun ZJ, Wang H, Sander C, et al. TIGIT

and PD-1 impair tumor antigen-specific CD8(+) T cells in melanoma patients. J

Clin Invest 2015;125(5):2046-58 doi 10.1172/Jci80445.

16. Johnston RJ, Comps-Agrar L, Hackney J, Yu X, Huseni M, Yang Y, et al. The

immunoreceptor TIGIT regulates antitumor and antiviral CD8(+) T cell effector

function. Cancer Cell 2014;26(6):923-37 doi 10.1016/j.ccell.2014.10.018.

17. Kurtulus S, Sakuishi K, Ngiow SF, Joller N, Tan DJ, Teng MW, et al. TIGIT

predominantly regulates the immune response via regulatory T cells. J Clin Invest

2015;125(11):4053-62 doi 10.1172/JCI81187.

18. Fourcade J, Sun Z, Chauvin JM, Ka M, Davar D, Pagliano O, et al. CD226

opposes TIGIT to disrupt Tregs in melanoma. JCI Insight 2018;3(14) doi

10.1172/jci.insight.121157.

19. Li M, Xia P, Du Y, Liu S, Huang G, Chen J, et al. T-cell immunoglobulin and

ITIM domain (TIGIT) receptor/poliovirus receptor (PVR) ligand engagement

suppresses interferon-gamma production of natural killer cells via beta-arrestin 2-

mediated negative signaling. J Biol Chem 2014;289(25):17647-57 doi

10.1074/jbc.M114.572420.

20. Liu S, Zhang H, Li M, Hu D, Li C, Ge B, et al. Recruitment of Grb2 and SHIP1

by the ITT-like motif of TIGIT suppresses granule polarization and cytotoxicity

of NK cells. Cell Death Differ 2013;20(3):456-64 doi 10.1038/cdd.2012.141.

Research.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

21. Ramsbottom KM, Hawkins ED, Shimoni R, McGrath M, Chan CJ, Russell SM, et

al. Cutting edge: DNAX accessory molecule 1-deficient CD8+ T cells display

immunological synapse defects that impair antitumor immunity. J Immunol

2014;192(2):553-7 doi 10.4049/jimmunol.1302197.

22. Shibuya K, Lanier LL, Phillips JH, Ochs HD, Shimizu K, Nakayama E, et al.

Physical and functional association of LFA-1 with DNAM-1 adhesion molecule.

Immunity 1999;11(5):615-23 doi 10.1016/s1074-7613(00)80136-3.

23. Gilfillan S, Chan CJ, Cella M, Haynes NM, Rapaport AS, Boles KS, et al.

DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional

antigen-presenting cells and tumors. J Exp Med 2008;205(13):2965-73 doi

10.1084/jem.20081752.

24. Iguchi-Manaka A, Kai H, Yamashita Y, Shibata K, Tahara-Hanaoka S, Honda S,

et al. Accelerated tumor growth in mice deficient in DNAM-1 receptor. J Exp

Med 2008;205(13):2959-64 doi 10.1084/jem.20081611.

25. Lakshmikanth T, Burke S, Ali TH, Kimpfler S, Ursini F, Ruggeri L, et al. NCRs

and DNAM-1 mediate NK cell recognition and lysis of human and mouse

melanoma cell lines in vitro and in vivo. J Clin Invest 2009;119(5):1251-63 doi

10.1172/JCI36022.

26. Messaoudene M, Fregni G, Fourmentraux-Neves E, Chanal J, Maubec E,

Mazouz-Dorval S, et al. Mature cytotoxic CD56(bright)/CD16(+) natural killer

cells can infiltrate lymph nodes adjacent to metastatic melanoma. Cancer Res

2014;74(1):81-92 doi 10.1158/0008-5472.CAN-13-1303.

27. Zhang Q, Bi J, Zheng X, Chen Y, Wang H, Wu W, et al. Blockade of the

checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-

Research.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

tumor immunity. Nat Immunol 2018;19(7):723-32 doi 10.1038/s41590-018-0132-

28. Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, Hu-

Lieskovan S, et al. Mutations Associated with Acquired Resistance to PD-1

Blockade in Melanoma. N Engl J Med 2016;375(9):819-29 doi

10.1056/NEJMoa1604958.

29. Gettinger S, Choi J, Hastings K, Truini A, Datar I, Sowell R, et al. Impaired HLA

Class I Antigen Processing and Presentation as a Mechanism of Acquired

Resistance to Immune Checkpoint Inhibitors in Lung Cancer. Cancer Discov

2017;7(12):1420-35 doi 10.1158/2159-8290.CD-17-0593.

30. D'Urso CM, Wang ZG, Cao Y, Tatake R, Zeff RA, Ferrone S. Lack of HLA class

I antigen expression by cultured melanoma cells FO-1 due to a defect in B2m

gene expression. J Clin Invest 1991;87(1):284-92 doi 10.1172/JCI114984.

31. Zarour HM, Storkus WJ, Brusic V, Williams E, Kirkwood JM. NY-ESO-1

encodes DRB1*0401-restricted epitopes recognized by melanoma-reactive CD4+

T cells. Cancer Res 2000;60(17):4946-52.

32. Blake SJ, Stannard K, Liu J, Allen S, Yong MC, Mittal D, et al. Suppression of

Metastases Using a New Lymphocyte Checkpoint Target for Cancer

Immunotherapy. Cancer Discov 2016;6(4):446-59 doi 10.1158/2159-8290.CD-

15-0944.

33. Chan CJ, Martinet L, Gilfillan S, Souza-Fonseca-Guimaraes F, Chow MT, Town

L, et al. The receptors CD96 and CD226 oppose each other in the regulation of

natural killer cell functions. Nat Immunol 2014;15(5):431-8 doi 10.1038/ni.2850.

Research.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

34. Gao Y, Souza-Fonseca-Guimaraes F, Bald T, Ng SS, Young A, Ngiow SF, et al.

Tumor immunoevasion by the conversion of effector NK cells into type 1 innate

lymphoid cells. Nat Immunol 2017;18(9):1004-15 doi 10.1038/ni.3800.

35. Xu F, Sunderland A, Zhou Y, Schulick RD, Edil BH, Zhu Y. Blockade of

CD112R and TIGIT signaling sensitizes human natural killer cell functions.

Cancer Immunol Immunother 2017;66(10):1367-75 doi 10.1007/s00262-017-

2031-x.

36. Cullen PJ, Steinberg F. To degrade or not to degrade: mechanisms and

significance of endocytic recycling. Nat Rev Mol Cell Biol 2018;19(11):679-96

doi 10.1038/s41580-018-0053-7.

37. Huss M, Wieczorek H. Inhibitors of V-ATPases: old and new players. J Exp Biol

2009;212(Pt 3):341-6 doi 10.1242/jeb.024067.

38. Wilson EB, El-Jawhari JJ, Neilson AL, Hall GD, Melcher AA, Meade JL, et al.

Human tumour immune evasion via TGF-beta blocks NK cell activation but not

survival allowing therapeutic restoration of anti-tumour activity. Plos One

2011;6(9):e22842 doi 10.1371/journal.pone.0022842.

39. Levanon D, Negreanu V, Lotem J, Bone KR, Brenner O, Leshkowitz D, et al.

Transcription factor Runx3 regulates interleukin-15-dependent natural killer cell

activation. Mol Cell Biol 2014;34(6):1158-69 doi 10.1128/MCB.01202-13.

40. Yin X, Liu T, Wang Z, Ma M, Lei J, Zhang Z, et al. Expression of the Inhibitory

Receptor TIGIT Is Up-Regulated Specifically on NK Cells With CD226

Activating Receptor From HIV-Infected Individuals. Front Immunol 2018;9:2341

doi 10.3389/fimmu.2018.02341.

Research.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

41. Easom NJW, Stegmann KA, Swadling L, Pallett LJ, Burton AR, Odera D, et al.

IL-15 Overcomes Hepatocellular Carcinoma-Induced NK Cell Dysfunction. Front

Immunol 2018;9:1009 doi 10.3389/fimmu.2018.01009.

42. Vuletic A, Jovanic I, Jurisic V, Milovanovic Z, Nikolic S, Spurnic I, et al. IL-2

And IL-15 Induced NKG2D, CD158a and CD158b Expression on T, NKT- like

and NK Cell Lymphocyte Subsets from Regional Lymph Nodes of Melanoma

Patients. Pathol Oncol Res 2020;26(1):223-31 doi 10.1007/s12253-018-0444-2.

43. Ndhlovu LC, Lopez-Verges S, Barbour JD, Jones RB, Jha AR, Long BR, et al.

Tim-3 marks human natural killer cell maturation and suppresses cell-mediated

cytotoxicity. Blood 2012;119(16):3734-43 doi 10.1182/blood-2011-11-392951.

44. Wang B, Zhang W, Jankovic V, Golubov J, Poon P, Oswald EM, et al.

Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8(+)

T cell dysfunction and maintain memory phenotype. Sci Immunol 2018;3(29) doi

10.1126/sciimmunol.aat7061.

45. Chihara N, Madi A, Kondo T, Zhang H, Acharya N, Singer M, et al. Induction

and transcriptional regulation of the co-inhibitory gene module in T cells. Nature

2018;558(7710):454-9 doi 10.1038/s41586-018-0206-z.

46. Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell

2014;157(1):121-41 doi 10.1016/j.cell.2014.03.011.

47. Stanietsky N, Rovis TL, Glasner A, Seidel E, Tsukerman P, Yamin R, et al.

Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR. Eur J

Immunol 2013;43(8):2138-50 doi 10.1002/eji.201243072.

48. Wang F, Hou HY, Wu SJ, Tang Q, Liu WY, Huang M, et al. TIGIT expression

levels on human NK cells correlate with functional heterogeneity among healthy

Research.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

individuals. European Journal of Immunology 2015;45(10):2886-97 doi

10.1002/eji.201545480.

49. Vendrame E, Seiler C, Ranganath T, Zhao NQ, Vergara R, Alary M, et al. TIGIT

is upregulated by HIV-1 infection and marks a highly functional adaptive and

mature subset of natural killer cells. AIDS 2020;34(6):801-13 doi

10.1097/QAD.0000000000002488.

50. Paley MA, Kroy DC, Odorizzi PM, Johnnidis JB, Dolfi DV, Barnett BE, et al.

Progenitor and terminal subsets of CD8+ T cells cooperate to contain chronic

viral infection. Science 2012;338(6111):1220-5 doi 10.1126/science.1229620.

51. Sakhdari A, Mujib S, Vali B, Yue FY, MacParland S, Clayton K, et al. Tim-3

negatively regulates cytotoxicity in exhausted CD8+ T cells in HIV infection.

Plos One 2012;7(7):e40146 doi 10.1371/journal.pone.0040146.

52. Groh V, Wu J, Yee C, Spies T. Tumour-derived soluble MIC ligands impair

expression of NKG2D and T-cell activation. Nature 2002;419(6908):734-8 doi

10.1038/nature01112.

53. Sandusky MM, Messmer B, Watzl C. Regulation of 2B4 (CD244)-mediated NK

cell activation by ligand-induced receptor modulation. Eur J Immunol

2006;36(12):3268-76 doi 10.1002/eji.200636146.

54. Masilamani M, Peruzzi G, Borrego F, Coligan JE. Endocytosis and intracellular

trafficking of human natural killer cell receptors. Traffic 2009;10(12):1735-44 doi

10.1111/j.1600-0854.2009.00973.x.

55. Iguchi-Manaka A, Okumura G, Kojima H, Cho Y, Hirochika R, Bando H, et al.

Increased Soluble CD155 in the Serum of Cancer Patients. Plos One

2016;11(4):e0152982 doi 10.1371/journal.pone.0152982.

Research.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on June 26, 2020; DOI: 10.1158/1078-0432.CCR-20-0575

56. Xu Z, Zhang T, Zhuang R, Zhang Y, Jia W, Song C, et al. Increased levels of

soluble CD226 in sera accompanied by decreased membrane CD226 expression

on peripheral blood mononuclear cells from cancer patients. BMC Immunol

2009;10:34 doi 10.1186/1471-2172-10-34.

57. Du X, de Almeida P, Manieri N, de Almeida Nagata D, Wu TD, Harden Bowles

K, et al. CD226 regulates natural killer cell antitumor responses via

phosphorylation-mediated inactivation of transcription factor FOXO1. Proc Natl

Acad Sci U S A 2018;115(50):E11731-E40 doi 10.1073/pnas.1814052115.

58. Elpek KG, Rubinstein MP, Bellemare-Pelletier A, Goldrath AW, Turley SJ.

Mature natural killer cells with phenotypic and functional alterations accumulate

upon sustained stimulation with IL-15/IL-15Ralpha complexes. Proc Natl Acad

Sci U S A 2010;107(50):21647-52 doi 10.1073/pnas.1012128107.

59. Felices M, Lenvik AJ, McElmurry R, Chu S, Hinderlie P, Bendzick L, et al.

Continuous treatment with IL-15 exhausts human NK cells via a metabolic defect.

JCI Insight 2018;3(3) doi 10.1172/jci.insight.96219.

Research.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Figure Legends

Figure 1. Tumor-infiltrating NK cells downregulate TIGIT and CD226. CD3-

CD45+CD56+ NK cells were evaluated side by side in PBMCs of healthy donors (HD)

and melanoma patients (MP) and in metastatic melanoma (MM) using flow cytometry.

(A) Pooled data showing the frequencies of NK cells within CD3-CD45+ cells (n=16 per

group). (B) Representative dot plots showing the percentages of TIGIT and CD226

expression by NK cells. (C) Pooled data of TIGIT and CD226 expression (% and MFI)

by NK cells in PBMCs (HD: n=30 and n=25, respectively; MP: n=39 and n=32,

respectively) and MM (n=17 and n=14, respectively). (D) TIGIT/CD226 expression ratio

(MFI) by NK cells (from panel C). (E) Percentages of CD96, PD-1, Tim-3, NKG2D,

CD16, 2B4, NKp46, NK30 and CD57 expression by cNKs isolated from HDs (n=9-12)

and MPs (n=7-20), and TiNKs (n=10-11). Horizontal bars depict means and P values

were obtained by one-way ANOVA tests followed by Tukey’s multiple comparisons test

with *, P<0.05; **, P<0.01; ***, P<0.001 and ****, P<0.0001. Data are representative

of ten independent experiments.

Figure 2. TIGIT blockade reinvigorates NK cell-mediated tumor reactivity in the

periphery but not at tumor sites. NK cells were isolated from PBMCs (cNKs) and

tumors (TiNKs) of MPs. (A) Frequency of granzyme A-, granzyme B- and perforin-

expressing NKs according to TIGIT expression (n=14). (B) Pearson correlation between

the frequencies of perforin+ and TIGIT+ NKs. (C) NK degranulation capacity against FO-

I (CD107a %) and Pearson correlation between degranulation capacity and TIGIT/CD226

ratio by NK (n=13). (D) Degranulation against FO-I (CD107a, n=12) and specific lysis of

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FO-I lysis (Chromium 51 release assay, n=8) by TIGIT+ and TIGIT- cNKs. (E) Specific

lysis of FO-I by cNKs +/- aTIGIT, aCD226 blocking mAbs, or IgG control mAbs

(frequency and fold change, n=10) (F). CD107a expression by TiNKs (n=8) in response

to FO-I +/- aTIGIT +/- aCD226, or IgG control mAbs. Horizontal bars depict means and

P values were obtained by unpaired t-tests (A and C) or paired t-tests (A, C-F), with ns

(non-significant), P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001 and ****, P<0.0001.

Data are representative of at least three independent experiments.

Figure 3. Membrane-bound CD155 induces TIGIT and CD226 internalization in

NK cells. (A and B) CD226 (A) and TIGIT (B) expression by cNKs (fold change of

MFI) after 48 h incubation with indicated targets +/- aCD155 blocking mAb, as

compared with control (no target, n=15, or IgG-coated beads, n=8). (C) Fold change of

CD226 expression in MFI by cNKs incubated 48 h with sCD155 or the supernatants of

melanoma cell lines (n=7). (D) CD226 mRNA relative expression by cNKs either alone,

co-cultured for 48 h with L-cells, or with L-CD155 cells. Dots are means of triplicates

(n=7). (E and F) Representative pictures from ImageStream analysis of cNKs alone or

with indicated cell lines (1 h) depicting CD226 (E) and TIGIT (F) membrane (yellow)

and intracellular (violet) expression, as well as surface CD45 expression (turquoise). (G)

Representative histograms gated on total live cNKs (left panels) and statistical analysis

from a pool of 30 randomly selected cNKs (right panels) showing the ratio of CD226 and

TIGIT internalization by cNKs in the presence of L-CD155 and FO-I as compared with L

cells or cNKs alone. Cell surface CD45 expression was used to calculate the

internalization ratio of CD226 and TIGIT intracellular staining using IDEAS software. P

values were obtained from one-way ANOVA tests (A and B) or repeated-measure

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ANOVA tests (D and G) followed by Dunnett’s multiple comparisons test with *,

P<0.05; **, P<0.01 and ***, P<0.001. Horizontal bars depict means (A-C and G) or

means ± SEM (D). Data are representative of at least three independent experiments.

Figure 4. Membrane-bound CD155 induces CD226 degradation and NK cell

dysfunction. (A-C). cNKs isolated from MP were incubated with L cells or L-CD155 for

16 h +/- concanamycin-A (Conca). (A) Representative histograms of flow cytometry

showing the effects of Conca on cell surface and total CD226 expression (MFI) by NK

cells in the presence of L-CD155 cells or L cells as compared with DMSO control. (B

and C) Cell surface and total CD226 (B), TIGIT (C), and surface CD45 (as control)

expressions by cNKs (normalized MFI expression as compared to cNKs + L cells). (D

and E) cNKs were incubated 48 h with either sCD155, IgG- or CD155-Fc-beads +/-

blocking antibodies and washed before functional assay in presence of FO-I. (D) CD107a

expression (%) by cNKs pre-incubated or not with CD155-Fc beads (n=11) (E) CD107a

expression (fold change) by cNKs pre-incubated with either CD155-Fc-beads (n=6), IgG-

beads (n=3), or sCD155 (n=3) +/- aCD226 or aTIGIT blocking mAbs as compared with

IgG-beads (dotted line). Horizontal bars depict means ± SEM (B, C, and E) and P values

were obtained by one-way ANOVA tests followed by Tukey’s (B and C) or Dunnett’s

(E) multiple comparisons test or by paired t-tests (D) with ns (non-significant), P>0.05;

*, P<0.05; **, P<0.01 and ****, P<0.0001. Data are representative of at least three

independent experiments done in duplicates.

Figure 5. IL-15 together with TIGIT blockade reverses CD155-induced NK cell

dysfunction. (A) Pooled data showing the effect of STAT3 and STAT5 inhibitors (Stat3i

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and Stat5i, respectively) on CD226 and TIGIT expression (MFI) by cNKs after 6 d

incubation +/- IL-15 (n=6). (B) Pooled data of from three independent experiments

showing CD226 and TIGIT mRNA relative expression by cNKs from MPs after 48 h

incubation with indicated cell lines +/- IL-15 (n=4 and n=5, respectively). (C) Surface

and total CD226 expression by cNKs after 48 h incubation with L-CD155 or FO-I +/- IL-

15 (normalized MFI ratio as compared with cNKs in medium alone). (D) Pool of 30

randomly selected cNKs from one representative ImageStream analysis (n=3), showing

CD226 internalization ratio after 1 h incubation with indicated cell lines. (E) CD226 and

TIGIT expression (MFI and fold change) by TiNKs, ex-vivo and after 6 d incubation with

IL-15 (n=13). (F) Pooled data comparing CD107a expression by cNKs treated 48 h with

indicated beads (frequency and fold change), and by TiNKs (frequency) after 16 h or 6 d

IL-15 stimulation (n=7 and n=10, respectively), in response to FO-I. (G) Fold change of

CD107a+ TiNKs after 16 h or 6 d IL-15 stimulation prior to incubation with FO-I +/-

aTIGIT mAbs (n=16) as compared with 16 h IL-15 + IgG control mAb (dotted line).

Horizontal bars depict means ± SD (A and B) or ± SEM (C). P values were obtained by

paired t-tests (E and F), ordinary (D) or repeated-measures one-way ANOVA tests

followed by Tukey’s (B, and G) or Dunnett’s (C) multiple comparisons test, with *,

P<0.05; ***, P<0.01 and ***, P<0.001. Data are representative of at least three

independent experiments.

Figure 6. IL-15 and TIGIT blockade/deletion combine to suppress experimental

lung metastasis of melanoma. (A) Schematic of the mice tumor immunotherapy

experiments. (B and E) Groups of C57BL/6 WT and Tigit−/− mice (n=5 mice/group) were

injected i.v. with (B) 5 x 105 B16F10 or (E) 2 x 105 B16F10 on day 0. Some groups of

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mice then received PBS or IL-15 (0.5 µg)/IL-15Ra (3.0 µg) i.p., cIg or anti-TIGIT (200

µg) on days 0 and 3 and/or anti-CD226 (250 µg) on days -1, 0 and 7 (E). (C) Freshly

sorted NK cells (TCRβ-NK1.1+) from spleens of wild-type (WT) or TIGIT−/− male mice

were injected intravenously into Rag2−/−γc−/− recipient male mice (2 x 105 cells per

mouse). At day 6, peripheral blood was collected from Rag2−/−γc−/− recipient mice to

check NK cell reconstitution. Data are shown as representative flow and quantitative

results of NK cell reconstitution. (D) Groups of C57BL/6 Rag2−/−



c−/− (n=6-10

mice/group) were i.v. reconstituted with 2 x 105 purified WT or Tigit−/− NK cells. Six

days later, reconstituted mice were injected i.v. with 5 x 105 or 1 x 104 B16F10 (day 0).

Some groups of mice then received PBS or IL-15 (0.5 µg)/IL-15R (3.0 µg) i.p. on d 0

and 3. In B-E on day 14, lungs were harvested, and the metastatic burden was quantified

by counting colonies on the lung surface. Data are presented as mean ± SEM. Each

experiment was performed once. Legends indicates the group of mice and the treatment

per condition. Statistical significance was determined by one-way ANOVA with Tukey’s

multiple comparisons test with *, P<0.05; **, P<0.01; ***P<0.001 and ****, P<0.0001.

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Published OnlineFirst June 26, 2020.Clin Cancer Res

Joe-Marc Chauvin, Mignane Ka, Ornella Pagliano, et al.

NK cell dysfunction in melanoma

IL-15 stimulation with TIGIT blockade reverses CD155-mediated

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Sulfarotene Inhibits Colorectal Cancer via Mitigating Natural-Killer-Cell-Induced Stemness

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Mar 2024

Tumor cell stemness stands out as a pivotal factor driving tumor recurrence or metastasis and significantly contributes to the mortality of patients with colorectal cancer (CRC). Recent research has unveiled a link between immune-active cells and the induction of tumor cell stemness, ultimately leading to heightened resistance to treatment. In this study, stemness in CRC cell lines was assessed after co-culture with natural killer (NK) cells, both with and without sulfarotene administration. Furthermore, a CRC xenograft model was utilized to scrutinize the in vivo efficacy of sulfarotene in overcoming stemness induced by NK cell activation. As a result, CRC cells exhibited significant stemness after NK cell co-culture, as evidenced by the upregulation of several stemness markers associated with cancer stem cells. Moreover, these cells demonstrated remarkable resistance to commonly used chemotherapy agents for CRC, such as oxaliplatin and irinotecan. Importantly, sulfarotene effectively reversed the altered stemness of CRC cells in both in vitro and in vivo assays. In conclusion, sulfarotene emerges as a promising therapeutic strategy for overcoming colorectal cancer resistance to NK cells by effectively inhibiting stemness remodeling. This study underscores the potential of sulfarotene in augmenting NK-cell-mediated immune surveillance, proposing a novel immunotherapeutic approach against colorectal cancer.

synNotch-programmed iPSC-derived NK cells usurp TIGIT and CD73 activities for glioblastoma therapy

Article

Full-text available

Mar 2024

Severe heterogeneity within glioblastoma has spurred the notion that disrupting the interplay between multiple elements on immunosuppression is at the core of meaningful anti-tumor responses. T cell immunoreceptor with Ig and ITIM domains (TIGIT) and its glioblastoma-associated antigen, CD155, form a highly immunosuppressive axis in glioblastoma and other solid tumors, yet targeting of TIGIT, a functionally heterogeneous receptor on tumor-infiltrating immune cells, has largely been ineffective as monotherapy, suggesting that disruption of its inhibitory network might be necessary for measurable responses. It is within this context that we show that the usurpation of the TIGIT − CD155 axis via engineered synNotch-mediated activation of induced pluripotent stem cell-derived natural killer (NK) cells promotes transcription factor-mediated activation of a downstream signaling cascade that results in the controlled, localized blockade of CD73 to disrupt purinergic activity otherwise resulting in the production and accumulation of immunosuppressive extracellular adenosine. Such “decoy” receptor engages CD155 binding to TIGIT, but tilts inhibitory TIGIT/CD155 interactions toward activation via downstream synNotch signaling. Usurping activities of TIGIT and CD73 promotes the function of adoptively transferred NK cells into intracranial patient-derived models of glioblastoma and enhances their natural cytolytic functions against this tumor to result in complete tumor eradication. In addition, targeting both receptors, in turn, reprograms the glioblastoma microenvironment via the recruitment of T cells and the downregulation of M2 macrophages. This study demonstrates that TIGIT/CD155 and CD73 are targetable receptor partners in glioblastoma. Our data show that synNotch-engineered pluripotent stem cell-derived NK cells are not only effective mediators of anti-glioblastoma responses within the setting of CD73 and TIGIT/CD155 co-targeting, but represent a powerful allogeneic treatment option for this tumor.

Challenges and Recent Advances in NK-Mediated Adoptive Cell Therapies against Cancer

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May 2024

Tianxiang Zhang

Natural killer (NK) cells play a crucial role in the innate immune system. Unlike adaptive immune cells that rely on specific antigen receptors, NK cell activity is governed by germline-encoded activating or inhibitory receptors expressed on the cell surface. The integration of signals from these receptors determines the NK cell response. Activated NK cells demonstrate potent cytotoxicity against target cells. The distinctive attributes of NK cells, marked by quick response, robust cytotoxicity, and the absence of antigen receptors, position them as promising tools in cancer immunotherapy. Ongoing clinical trials are actively assessing NK cells and related reagents, showing promising outcomes. However, significant challenges arise from the immunosuppressive mechanisms within the tumor microenvironment, impeding the broader application of NK cells. In-depth studies on these mechanisms are imperative to identify solutions, ultimately paving the way for the widespread clinical utilization of NK cells in cancer immunotherapy.

Combining TIGIT blockade with IL‐15 stimulation is a promising immunotherapy strategy for lung adenocarcinoma

Article

Full-text available

Jan 2024

Background T‐cell immunoglobulin and immunoreceptor tyrosine‐based inhibitory motif domain (TIGIT) is an immune checkpoint molecule that suppresses CD8 ⁺ T‐cell function in cancer. However, the expression profile and functional significance of TIGIT in the immune microenvironment of lung adenocarcinoma (LUAD) remain elusive. Interleukin (IL)‐15 has emerged as a promising candidate for enhancing CD8 ⁺ T‐cell mediated tumour eradication. Exploring therapeutic strategies that combine IL‐15 with TIGIT blockade in LUAD is warranted. Methods We investigated the regulatory network involving coinhibitory TIGIT and CD96, as well as costimulatory CD226 in LUAD using clinical samples. The potential role of TIGIT in regulating the pathogenesis of LUAD was addressed through a murine model with transplanted tumours constructed in Tigit −/− mice. The therapeutic strategy that combines TIGIT blockade with IL‐15 stimulation was verified using a transplanted tumour murine model and a patient‐derived organoid (PDO) model. Results The frequency of TIGIT ⁺ CD8 ⁺ T cells was significantly increased in LUAD. Increased TIGIT expression indicated poorer prognosis in LUAD patients. Furthermore, the effector function of TIGIT ⁺ CD8 ⁺ tumour‐infiltrating lymphocytes (TILs) was impaired in LUAD patients and TIGIT inhibited antitumour immune response of CD8 ⁺ TILs in tumour‐bearing mice. Mechanistically, IL‐15 enhanced the effector function of CD8 ⁺ TILs but stimulated the expression of TIGIT on CD8 ⁺ TILs concomitantly. The application of IL‐15 combined with TIGIT blockade showed additive effects in enhancing the cytotoxicity of CD8 ⁺ TILs and thus further increased the antitumour immune response in LUAD. Conclusions Our findings identified TIGIT as a promising therapeutic target for LUAD. LUAD could benefit more from the combined therapy of IL‐15 stimulation and TIGIT blockade.

Immune Regulation and Immune Therapy in Melanoma: Review with Emphasis on CD155 Signalling

Article

Full-text available

May 2024

Simple Summary For melanoma patients, the most promising curative treatment is immunotherapy. Here, we summarise current immunotherapy strategies and their indications and challenges, and provide future directions of immunotherapy development. Several therapy resistance mechanisms have been described, resulting in primary refractory disease or resistance development in patients. Our increasing knowledge of immune regulation within the tumour microenvironment identifies potential alternate immunotherapy targets, including CD155. CD155 and its receptor, TIGIT, have been shown to be highly expressed in therapy-resistant melanoma cells and interference with both is therefore considered a possible immunotherapeutic strategy. This review describes the immune regulation within the melanoma tumour microenvironment, how and why immunotherapies work, and why CD155 might be an ideal target for the next generation of anti-melanoma immunotherapies. Abstract Melanoma is commonly diagnosed in a younger population than most other solid malignancies and, in Australia and most of the world, is the leading cause of skin-cancer-related death. Melanoma is a cancer type with high immunogenicity; thus, immunotherapies are used as first-line treatment for advanced melanoma patients. Although immunotherapies are working well, not all the patients are benefitting from them. A lack of a comprehensive understanding of immune regulation in the melanoma tumour microenvironment is a major challenge of patient stratification. Overexpression of CD155 has been reported as a key factor in melanoma immune regulation for the development of therapy resistance. A more thorough understanding of the actions of current immunotherapy strategies, their effects on immune cell subsets, and the roles that CD155 plays are essential for a rational design of novel targets of anti-cancer immunotherapies. In this review, we comprehensively discuss current anti-melanoma immunotherapy strategies and the immune response contribution of different cell lineages, including tumour endothelial cells, myeloid-derived suppressor cells, cytotoxic T cells, cancer-associated fibroblast, and nature killer cells. Finally, we explore the impact of CD155 and its receptors DNAM-1, TIGIT, and CD96 on immune cells, especially in the context of the melanoma tumour microenvironment and anti-cancer immunotherapies.

Development of pharmacological immunoregulatory anti-cancer therapeutics: current mechanistic studies and clinical opportunities

Article

May 2024

Immunotherapy represented by anti-PD-(L)1 and anti-CTLA-4 inhibitors has revolutionized cancer treatment, but challenges related to resistance and toxicity still remain. Due to the advancement of immuno-oncology, an increasing number of novel immunoregulatory targets and mechanisms are being revealed, with relevant therapies promising to improve clinical immunotherapy in the foreseeable future. Therefore, comprehending the larger picture is important. In this review, we analyze and summarize the current landscape of preclinical and translational mechanistic research, drug development, and clinical trials that brought about next-generation pharmacological immunoregulatory anti-cancer agents and drug candidates beyond classical immune checkpoint inhibitors. Along with further clarification of cancer immunobiology and advances in antibody engineering, agents targeting additional inhibitory immune checkpoints, including LAG-3, TIM-3, TIGIT, CD47, and B7 family members are becoming an important part of cancer immunotherapy research and discovery, as are structurally and functionally optimized novel anti-PD-(L)1 and anti-CTLA-4 agents and agonists of co-stimulatory molecules of T cells. Exemplified by bispecific T cell engagers, newly emerging bi-specific and multi-specific antibodies targeting immunoregulatory molecules can provide considerable clinical benefits. Next-generation agents also include immune epigenetic drugs and cytokine-based therapeutics. Cell therapies, cancer vaccines, and oncolytic viruses are not covered in this review. This comprehensive review might aid in further development and the fastest possible clinical adoption of effective immuno-oncology modalities for the benefit of patients.

IL-15 as a key regulator in NK cell-mediated immunotherapy for cancer: From bench to bedside

Article

Apr 2024

Overview of tumor immunotherapy based on approved drugs

Article

Jan 2024

Targeting TIGIT for cancer immunotherapy: recent advances and future directions

Article

Full-text available

Jan 2024

As a newly identified checkpoint, T cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is highly expressed on CD4⁺ T cells, CD8⁺ T cells, natural killer (NK) cells, regulatory T cells (Tregs), and tumor-infiltrating lymphocytes (TILs). TIGIT has been associated with NK cell exhaustion in vivo and in individuals with various cancers. It not only modulates NK cell survival but also mediates T cell exhaustion. As the primary ligand of TIGIT in humans, CD155 may be the main target for immunotherapy due to its interaction with TIGIT. It has been found that the anti-programmed cell death protein 1 (PD-1) treatment response in cancer immunotherapy is correlated with CD155 but not TIGIT. Anti-TIGIT alone and in combination with anti-PD-1 agents have been tested for cancer immunotherapy. Although two clinical studies on advanced lung cancer had positive results, the TIGIT-targeted antibody, tiragolumab, recently failed in two new trials. In this review, we highlight the current developments on TIGIT for cancer immunotherapy and discuss the characteristics and functions of TIGIT.

Dual-crosslinking immunostimulatory hydrogel synchronously suppresses pancreatic fistula and pancreatic cancer relapse post-resection

Article

Dec 2023
BIOMATERIALS

CD226 regulates natural killer cell antitumor responses via phosphorylation-mediated inactivation of transcription factor FOXO1

Article

Full-text available

Nov 2018

Significance CD226 is an important activating receptor involved in mediating natural killer (NK) cell responses against tumors, but how CD226 exerts control over NK cell function is not fully understood. CD226 belongs to the poliovirus receptor (PVR)-nectin family that includes TIGIT and CD96, with TIGIT garnering much attention as a key checkpoint in T cell and NK cell antitumor responses and as an immunotherapy target. Thus, it is imperative to determine how CD226 counteracts the actions of TIGIT and CD96 with which it competes for binding to its ligands such as CD155 (PVR). We demonstrate that CD226 engagement of CD155 is required for phosphorylation of transcription factor FOXO1, resulting in inactivation of its negative regulatory control over NK cell effector function.

Expression of the Inhibitory Receptor TIGIT Is Up-Regulated Specifically on NK Cells With CD226 Activating Receptor From HIV-Infected Individuals

Article

Full-text available

Oct 2018

Natural killer (NK) cells are important for maintenance of innate immune system stability and serve as a first line of defense against tumors and virus infections; they can act either directly or indirectly and are regulated via co-operation between inhibitory and stimulatory surface receptors. The recently reported inhibitory receptor, TIGIT, can be expressed on the NK cell surface; however, the expression level and function of TIGIT on NK cells during HIV infection is unknown. In this study, for the first time, we investigated the expression and function of TIGIT in NK cells from HIV-infected individuals. Our data demonstrate that the level of TIGIT is higher on NK cells from patients infected with human immunodeficiency virus (HIV) compared with HIV-negative healthy controls. TIGIT expression is inversely correlated with CD4⁺ T cell counts and positively correlated with plasma viral loads. Additionally, levels of the TIGIT ligand, CD155, were higher on CD4⁺ T cells from HIV-infected individuals compared with those from healthy controls; however, there was no difference in the level of the activating receptor, CD226, which recognizes the same ligands as TIGIT. Furthermore, TIGIT was found to specifically up-regulated on CD226⁺ NK cells in HIV-infected individuals, and either rIL-10, or rIL-12 + rIL-15, could induce TIGIT expression on these cells. In addition, high TIGIT expression inhibited the production of interferon-gamma (IFN-γ) by NK cells, while TIGIT inhibition restored IFN-γ production. Overall, these results highlight the important role of TIGIT in NK cell function and suggest a potential new avenue for the development of therapeutic strategies toward a functional cure for HIV.

CD226 opposes TIGIT to disrupt Tregs in melanoma

Article

Full-text available

Jul 2018

CD4+ Tregs impede T cell responses to tumors. They express multiple inhibitory receptors that support their suppressive functions, including T cell Ig and ITIM domain (TIGIT). In melanoma patients, we show that Tregs exhibit increased TIGIT expression and decreased expression of its competing costimulatory receptor CD226 as compared with CD4+ effector T cells, resulting in an increased TIGIT/CD226 ratio. Tregs failed to upregulate CD226 upon T cell activation. TIGIT+ Tregs are highly suppressive, stable, and enriched in tumors. TIGIT and CD226 oppose each other to augment or disrupt, respectively, Treg suppression and stability. A high TIGIT/CD226 ratio in Tregs correlates with increased Treg frequencies in tumors and poor clinical outcome upon immune checkpoint blockade. Altogether, our findings show that a high TIGIT/CD226 ratio in Tregs regulates their suppressive function and stability in melanoma. They provide the rationale for novel immunotherapies to activate CD226 in Tregs together with TIGIT blockade to counteract Treg suppression in cancer patients.

Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity

Article

Full-text available

Jul 2018
NAT IMMUNOL

Checkpoint blockade enhances effector T cell function and has elicited long-term remission in a subset of patients with a broad spectrum of cancers. TIGIT is a checkpoint receptor thought to be involved in mediating T cell exhaustion in tumors; however, the relevance of TIGIT to the dysfunction of natural killer (NK) cells remains poorly understood. Here we found that TIGIT, but not the other checkpoint molecules CTLA-4 and PD-1, was associated with NK cell exhaustion in tumor-bearing mice and patients with colon cancer. Blockade of TIGIT prevented NK cell exhaustion and promoted NK cell-dependent tumor immunity in several tumor-bearing mouse models. Furthermore, blockade of TIGIT resulted in potent tumor-specific T cell immunity in an NK cell-dependent manner, enhanced therapy with antibody to the PD-1 ligand PD-L1 and sustained memory immunity in tumor re-challenge models. This work demonstrates that TIGIT constitutes a previously unappreciated checkpoint in NK cells and that targeting TIGIT alone or in combination with other checkpoint receptors is a promising anti-cancer therapeutic strategy.

Induction and transcriptional regulation of the co-inhibitory gene module in T cells

Article

Full-text available

Jun 2018
NATURE

The expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated expression of co-inhibitory receptors on CD4+ T cells promotes autoimmunity, whereas sustained overexpression on CD8+ T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and diseases such as cancer1,2. Here, using RNA and protein expression profiling at single-cell resolution in mouse cells, we identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, TIM-3, LAG-3 and TIGIT) but also many new surface receptors. We functionally validated two new co-inhibitory receptors, activated protein C receptor (PROCR) and podoplanin (PDPN). The module of co-inhibitory receptors is co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in several physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors PRDM1 and c-MAF as cooperative regulators of the co-inhibitory module, and this was validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies regulators of T cell function with the potential to control autoimmunity and tumour immunity.

IL-2 And IL-15 Induced NKG2D, CD158a and CD158b Expression on T, NKT- like and NK Cell Lymphocyte Subsets from Regional Lymph Nodes of Melanoma Patients

Article

Full-text available

Jun 2018

Regional lymph nodes (LN)s represent important immunological barriers in spreading of malignant tumors. However, they are the most frequent early metastatic site in melanoma. Immunomodulatory agents including cytokines have been included in therapy of melanoma and have shown severe side effects and toxicity. In this sense, there is a growing need for bringing these agents to further in vitro testing that may enlighten aspects of their regional application. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, the two cytokines with similar immune-enhancing effects, on the expression of activating NKG2D, inhibitory CD158a and CD158b receptors on CD8⁺ T, NKT-like and NK cell lymphocyte subsets from regional LNs of melanoma patients. In this study, we showed significant effects of IL-2 and IL-15 cytokine treatments on the expression of activating NKG2D and on inhibitory CD158a and CD158b receptors on lymphocytes, CD8⁺ T, NKT-like and NK cell lymphocyte subsets originating from regional LNs of melanoma patients. Furthermore, IL-2 and IL-15 by inducing the expression of NKG2D activating receptor on innate and on adaptive lymphocyte subsets and by augmenting NK cell antitumor cytotoxicity that correlated with the cytokine-induced NKG2D expression, increased antitumor potential of immune cells in regional LNs of melanoma patients irrespective of LN involvement. These findings indicate the importance of immune cell population from regional LNs of melanoma patients in the development of immune intervention strategies that may if applied locally increase antitumor potential to the level that controls tumor progressions.

TIGIT is upregulated by HIV-1 infection and marks a highly functional adaptive and mature subset of natural killer cells

Article

Feb 2020

Objective: Our objective was to investigate the mechanisms that govern natural killer (NK) cell responses to HIV, with a focus on specific receptor-ligand interactions involved in HIV recognition by NK cells. Design and methods: We first performed a mass cytometry-based screen of NK cell receptor expression patterns in healthy controls and HIV individuals. We then focused mechanistic studies on the expression and function of T cell immunoreceptor with Ig and ITIM domains (TIGIT). Results: The mass cytometry screen revealed that TIGIT is upregulated on NK cells of untreated HIV women, but not in antiretroviral-treated women. TIGIT is an inhibitory receptor that is thought to mark exhausted NK cells; however, blocking TIGIT did not improve anti-HIV NK cell responses. In fact, the TIGIT ligands CD112 and CD155 were not upregulated on CD4 T cells in vitro or in vivo, providing an explanation for the lack of benefit from TIGIT blockade. TIGIT expression marked a unique subset of NK cells that express significantly higher levels of NK cell activating receptors (DNAM-1, NTB-A, 2B4, CD2) and exhibit a mature/adaptive phenotype (CD57, NKG2C, LILRB1, FcRγ, Syk). Furthermore, TIGIT NK cells had increased responses to mock-infected and HIV-infected autologous CD4 T cells, and to PMA/ionomycin, cytokine stimulation and the K562 cancer cell line. Conclusions: TIGIT expression is increased on NK cells from untreated HIV individuals. Although TIGIT does not participate directly to the response to HIV-infected cells, it marks a population of mature/adaptive NK cells with increased functional responses.

Mouse PVRIG Has CD8 + T Cell–Specific Coinhibitory Functions and Dampens Antitumor Immunity

Article

Jan 2019

A limitation to antitumor immunity is the dysfunction of T cells in the tumor microenvironment, in part due to upregulation of coinhibitory receptors such as PD-1. Here, we describe that poliovirus receptor–related immunoglobulin domain protein (PVRIG) acts as a coinhibitory receptor in mice. Murine PVRIG interacted weakly with poliovirus receptor (PVR) but bound poliovirus receptor–like 2 (PVRL2) strongly, making the latter its principal ligand. As in humans, murine NK and NKT cells constitutively expressed PVRIG. However, when compared with humans, less PVRIG transcript and surface protein was detected in murine CD8+ T cells ex vivo. However, activated CD8+ T cells upregulated PVRIG expression. In the mouse tumor microenvironment, infiltrating CD8+ T cells expressed PVRIG whereas its ligand, PVRL2, was detected predominantly on myeloid cells and tumor cells, mirroring the expression pattern in human tumors. PVRIG-deficient mouse CD8+ T cells mounted a stronger antigen-specific effector response compared with wild-type CD8+ T cells during acute Listeria monocytogenes infection. Furthermore, enhanced CD8+ T-cell effector function inhibited tumor growth in PVRIG−/− mice compared with wild-type mice and PD-L1 blockade conferred a synergistic antitumor response in PVRIG−/− mice. Therapeutic intervention with antagonistic anti-PVRIG in combination with anti–PD-L1 reduced tumor growth. Taken together, our results suggest PVRIG is an inducible checkpoint receptor and that targeting PVRIG–PVRL2 interactions results in increased CD8+ T-cell function and reduced tumor growth. See related article on p. 257

Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8 + T cell dysfunction and maintain memory phenotype

Article

Nov 2018

Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8 ⁺ T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor–related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8 ⁺ T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8 ⁺ T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1–Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.

To degrade or not to degrade: mechanisms and significance of endocytic recycling

Article

Sep 2018

Newly endocytosed integral cell surface proteins are typically either directed for degradation or subjected to recycling back to the plasma membrane. The sorting of integral cell surface proteins, including signalling receptors, nutrient transporters, ion channels, adhesion molecules and polarity markers, within the endolysosomal network for recycling is increasingly recognized as an essential feature in regulating the complexities of physiology at the cell, tissue and organism levels. Historically, endocytic recycling has been regarded as a relatively passive process, where the majority of internalized integral proteins are recycled via a nonspecific sequence-independent 'bulk membrane flow' pathway. Recent work has increasingly challenged this view. The discovery of sequence-specific sorting motifs and the identification of cargo adaptors and associated coat complexes have begun to uncover the highly orchestrated nature of endosomal cargo recycling, thereby providing new insight into the function and (patho)physiology of this process.

IL15 stimulation with TIGIT blockade reverses CD155-mediated NK-Cell dysfunction in Melanoma

Abstract

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