The macrophage tetraspan ms4a4a enhances dectin-1-dependent nk cell–mediated resistance to metastasis

ABSTRACT The plasma membrane tetraspan molecule MS4A4A is selectively expressed by macrophage-lineage cells, but its function is unknown. Here we report that MS4A4A was restricted to murine

and human mononuclear phagocytes and was induced during monocyte-to-macrophage differentiation in the presence of interleukin 4 or dexamethasone. Human MS4A4A was co-expressed with

M2/M2-like molecules in subsets of normal tissue-resident macrophages, infiltrating macrophages from inflamed synovium and tumor-associated macrophages. MS4A4A interacted and colocalized

with the β-glucan receptor dectin-1 in lipid rafts. In response to dectin-1 ligands, Ms4a4a-deficient macrophages showed defective signaling and defective production of effector molecules.

In experimental models of tumor progression and metastasis, Ms4a4a deficiency in macrophages had no impact on primary tumor growth, but was essential for dectin-1-mediated activation of

macrophages and natural killer (NK) cell–mediated metastasis control. Thus, MS4A4A is a tetraspan molecule selectively expressed in macrophages during differentiation and polarization,

essential for dectin-1-dependent activation of NK cell–mediated resistance to metastasis. Access through your institution Buy or subscribe This is a preview of subscription content, access

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Article Open access 07 February 2024 TREM2 MACROPHAGES DRIVE NK CELL PAUCITY AND DYSFUNCTION IN LUNG CANCER Article 20 April 2023 SPHINGANINE RECRUITS TLR4 ADAPTORS IN MACROPHAGES AND

PROMOTES INFLAMMATION IN MURINE MODELS OF SEPSIS AND MELANOMA Article Open access 18 July 2024 DATA AVAILABILITY The data that support the findings of this study are available from the

corresponding author upon reasonable request. _Ms4a4a__fl/fl_ animals have been developed by the research team and are available under a material transfer agreement after evaluation of

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Scholar  Download references ACKNOWLEDGEMENTS The authors thank C. Garlanda and M. Kallikourdis (Humanitas Clinical and Research Center, Scientific Institute for Research and Healthcare

(IRCCS)) for providing cDNAs of murine leukocyte subsets, and T. Irimura (Juntendo University School of Medicine, Tokyo, Japan) and R. Giavazzi (Mario Negri Institute, Milan, Italy) for

providing MC38 and SL4 cells. Technical assistance from A. Fontanini, C. Perrucchini, T. Schorn, R. Porte and F. Pasqualini is acknowledged. A. Inforzato (Humanitas Clinical and Research

Center, IRCCS), A. Diefenbach (Charité – Universitätsmedizin Berlin, Germany) and L. Florin (University Medical Centre of the Johannes Gutenberg University, Mainz, Germany) are gratefully

acknowledged for their support and discussion. Financial support came from Fondazione Cariplo (grant no. 2015–0564 to A.M.), Cluster Alisei (grant no. MEDINTECH CTN01_00177_962865 to A.M.),

the European Research Council (grant no. 669415-PHII to A.M.), the Italian Association for Cancer Research (AIRC IG-2016 grant no. 19014 to A.M.; AIRC 5 × 1000 grant no. 21147 to A.M.; AIRC

IG-2016 grant no. 19213 to M.L.), Medical Research Council (Pathobiology of Early Arthritis Cohort grant no. 36661 to C.P.) and Arthritis Research UK Experimental Treatment Centre (grant no.

20022 to C.P.). I.M. was supported by a Mario and Valeria Rindi fellowship and a Fellowship for abroad from the Italian Foundation for Cancer Research, and by a European Federation of

Immunological Societies-IL short-term fellowship. B.S. was supported by Ministero della Salute (progetto Finalizzata GR-2013-02356522). R.S.G. was supported by a PhD studentship

(PD/BD/114138/2016) from Fundação para a Ciência e Tecnologia, Portugal. S.K.B. was supported by core funding from Singapore Immunology Network—Agency for Science, Technology & Research

(A*STAR), Singapore. F.T. was supported by a fellowship from the A*STAR Research Attachment Program, Singapore. S.L. was supported by Fondazione Beretta, Italy. AUTHOR INFORMATION Author

notes * Maria De Pizzol Present address: Dompé Farmaceutici, Milan, Italy * Tamara Gulic Present address: Medical Faculty, Department of Physiology and Immunology, University of Rijeka,

Rijeka, Croatia * Martina Molgora Present address: Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA * Diego Morone Present address: Faculty

of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland * These authors contributed equally: Irene Mattiola, Federica

Tomay, Maria De Pizzol. AUTHORS AND AFFILIATIONS * Humanitas Clinical and Research Center, Scientific Institute for Research and Healthcare, Rozzano, Italy Irene Mattiola, Federica Tomay, 

Maria De Pizzol, Rita Silva-Gomes, Benedetta Savino, Tamara Gulic, Andrea Doni, Roberta Carriero, Diego Morone, Marina Sironi, Barbara Bottazzi, Alberto Mantovani & Massimo Locati *

Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité–Universitätsmedizin Berlin, Berlin, Germany Irene Mattiola * Berlin Institute of

Health, Berlin, Germany Irene Mattiola * Mucosal and Developmental Immunology, Deutsches Rheuma-Forschungszentrum, Berlin, Germany Irene Mattiola * Department of Medical Biotechnologies and

Translational Medicine, University of Milan, Milan, Italy Irene Mattiola, Federica Tomay, Maria De Pizzol, Benedetta Savino, Tamara Gulic & Massimo Locati * Graduate Program in Areas of

Basic and Applied Biology, Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Porto, Portugal Rita Silva-Gomes * Department of Molecular and Translational Medicine, School

of Medicine, University of Brescia, Brescia, Italy Silvia Lonardi & William Vermi * Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and the

London School of Medicine and Dentistry, Queen Mary University of London, London, UK Marie Astrid Boutet, Alessandra Nerviani, Costantino Pitzalis & Alberto Mantovani * Humanitas

University, Pieve Emanuele, Italy Martina Molgora, Matteo Stravalaci & Alberto Mantovani * Singapore Immunology Network, Agency for Science, Technology & Research, Singapore,

Singapore Irina N. Shalova, Yunquin Lee & Subhra K. Biswas * Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Endocrinology Unit, Department of Clinical Sciences and Community

Health, University of Milan, Milan, Italy Giovanna Mantovani * Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA William Vermi Authors *

Irene Mattiola View author publications You can also search for this author inPubMed Google Scholar * Federica Tomay View author publications You can also search for this author inPubMed 

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inPubMed Google Scholar CONTRIBUTIONS I.M., F.T. and M.D.P. performed in vitro experiments. I.M., B.S. and F.T. performed in vivo experiments. R.S.-G., M.M. and M. Sironi provided support

for in vivo and in vitro experiments. A.D. conducted the imaging analysis. D.M. and M. Stravalaci provided support for the analysis of molecular interactions. R.C. performed bioinformatics

analysis. I.N.S. and Y.L. performed gene expression experiments. S.L., W.V., M.A.B., A.N. and T.G. performed histology. G.M. and C.P. provided access to patients’ samples. S.K.B., B.B.,

C.P., A.M. and M.L. contributed to the experimental design and the supervision of the study. CORRESPONDING AUTHORS Correspondence to Alberto Mantovani or Massimo Locati. ETHICS DECLARATIONS

COMPETING INTERESTS A.M. is a recipient of commercial research grants from Novartis; a consultant/advisory board member for Novartis, Roche, Ventana, Pierre Fabre, Verily, AbbVie, Compugen,

Macrophage Therapeutics, AstraZeneca, Biovelocita, BG Fund, Third Rock and Verseau; an inventor of patents related to PTX3 and other innate immunity molecules; and also receives royalties

for reagents related to innate immunity. ADDITIONAL INFORMATION PEER REVIEW INFORMATION: Laurie Dempsey was the primary editor on this article and managed its editorial process and peer

review in collaboration with the rest of the editorial team. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 EXPRESSION LEVELS OF MS4A4A, MS4A6A AND MS4A7 IN HUMAN MONOCYTE-DERIVED MACROPHAGES AND DCS. Representative dot

plots and Mean Fluorescence Intensity (MFI) of the expression of MS4A4A (A, B), MS4A6A (C, D), and MS4A7 (E, F) in monocyte-derived macrophages (Mɸ) and dendritic cells (DC) treated (Dex,

open symbol) or not (NT, closed symbol) with 10−6 M Dex for 24 h. Ctrl represents isotype control staining. Results are shown as mean ± SEM. Three independent experiments were performed for

panels A to D (_n_ = 5 donors) and four independent experiments were performed for panels E to H (_n_ = 6 donors). Statistical analysis by one-way ANOVA. SUPPLEMENTARY FIGURE 2 TRANSCRIPT

LEVELS OF _MS4A4A_ IN HUMAN TISSUES. _MS4A4A_ transcript levels in human tissues (A) and cell types (B) based on the Laboratory for Systems Biology and Medicine database RefExA. EnC,

endothelial cells, EpC, epithelial cells, SMC, smooth muscle cells. SUPPLEMENTARY FIGURE 3 _MS4A4A_ TRANSCRIPT LEVELS IN HUMAN TUMORS AND ITS CORRELATION WITH TAM MARKERS. A) Boxplot of

_MS4A4A_ mRNA expression in human tumors (closed boxes) and corresponding normal tissues (open boxes) among different TCGA cancer datasets. B) Heatmap correlation between mRNAs encoding

_MS4A4A_ and TAM markers in tumor tissues among different TCGA cancer datasets. Genes are represented according to their average Pearson correlation with _MS4A4A_ among all cancer datasets.

Number of samples included in the analysis of both panels (normal tissue, corresponding tumor tissue) are: bladder urothelial cancer: 19, 408; breast cancer: 112, 1100; cervical

adenocarcinoma: 3, 306; cholangiocarcinoma: 9, 36; colon adenocarcinoma: 41, 287; colorectal adenocarcinoma: 51, 382; esophageal cancer: 11, 185; esophageal carcinoma: 46, 600; glioblastoma:

5, 166; glioma: 5, 696; head and neck cancer: 44, 522; kidney cancer: 25, 66; kidney clear cell cancer: 72, 534; kidney renal papillary cell cancer: 32, 291; liver cancer: 50, 373; lung

cancer: 59, 517; lung squamous cell cancer: 51, 501; pan-kidney cohort: 129, 891; pancreatic cancer: 4, 179; paraganglioma: 3, 184; prostate cancer: 52, 498; rectum cancer: 10, 95; sarcoma:

2, 263; stomach adenocarcinoma: 35, 415; thymoma: 2, 120; thyroid cancer: 59, 509; uterine cancer: 24, 177. SUPPLEMENTARY FIGURE 4 _MS4A4A_ EXPRESSION IN MURINE LEUKOCYTE SUBSETS AND

POLARIZED BMDMS. A) _Ms4a4a_ mRNA levels in murine leukocyte subsets sorted form the spleen of wild-type animals (NK cells: CD45+/CD3-/NK1.1+; CD4+ T cells: CD45+/CD3+/CD4+; CD8+ T cells:

CD45+/CD3+/CD8+; B cells: CD45+/B220+; PMN: CD45+/CD11bhi/Gr1hi; mDC: CD45+/CD11b+/CD11c+; pDC: CD45+/CD11b-/CD11c+; macrophages: CD45+/CD11b+/F4/80hi). Results are shown as mean ± SEM of

_Ms4a4a_ relative expression normalized to _Gapdh_ (_n_ = 3 mice). Statistical analysis by one-way ANOVA. B) _MS4A4A_ mRNA levels in wild-type BMDMs exposed or not (M0) to 20 ng/ml IFN-γ

plus 100 ng/ml LPS, 20 ng/ml IL-4, 10−6 M Dex, or 20 ng/ml IL-4 plus 10−6 M Dex for 18 h. Results are shown as fold change over resting macrophages (M0; _n_ = 5). Statistical analysis by

one-way ANOVA. C, _Ms4a4a_ mRNA levels in BMDMs from wild-type (closed symbols) or _Ms4a4a_-/-(open symbols) mice treated (Dex) or not (NT) with 10−6 M Dex for 24 h. Results are shown as

mean ± SEM of _Ms4a4a_ relative expression normalized to _Gapdh_ (_n_ = 3 mice). Statistical analysis by two-tailed unpaired (Mann-Whitney) Student’s t test and one-way ANOVA. D) Percentage

of NK cells (CD45+/CD3-/NK1.1+), T cells (CD45+/CD3+), B cells (CD45+/B220+), PMN (CD45+/CD11bhi/Gr1hi), mDC (CD45+/CD11b+/CD11c+), pDC (CD45+/CD11b-/CD11c+), and macrophages

(CD45+/CD11b+/F4/80hi) in the spleen of wild-type (closed symbols) or _Ms4a4a__-/-_ (open symbols) mice. Results are shown as mean ± SEM (_n_ = 4). No statistical difference by two-tailed

unpaired Student’s t test. SUPPLEMENTARY FIGURE 5 EXPRESSION LEVELS OF MS4A4A AND DECTIN-1 IN HUMAN MONOCYTE-DERIVED MACROPHAGES AND DCS. A, B) Flow cytometry analysis of Dectin-1 expression

in monocyte-derived macrophages (Mɸ) and dendritic cells (DC) treated (Dex, open symbol in B) or not (NT, closed symbol in B) with 10−6 M Dex for 24 h. Ctrl represents isotype control

staining. Results are shown as mean ± SEM. Three independent experiments were performed (_n_ = 5 donors). Statistical analysis by one-way ANOVA. C, D) Flow cytometry analysis of Dectin-1 and

MS4A4A expression in monocyte-derived macrophages (Mɸ) and dendritic cells (DC) treated (Dex, open symbol in D) or not (NT, closed symbol in D) with 10−6 M Dex for 24 h. Ctrl represents

isotype control staining. Percentage of double-positive cells is shown in d. Results are shown as mean ± SEM. Three independent experiments were performed (_n_ = 5 donors). Statistical

analysis by one-way ANOVA. SUPPLEMENTARY FIGURE 6 MS4A4A IMPACT ON DECTIN-1 SIGNALLING IN BMDMS. A–C) Syk phosphorylation in BMDMs from _Ms4a4a__-/-_ (open symbol) and wild-type (closed

symbol) mice primed for 18 h with 10 ng/ml GM-CSF and stimulated or not with 100 μg/ml depleted zymosan (A), 100 μg/ml curdlan (B), or 100 ng/ml PMA (D). Results are shown as mean ± SEM of

relative MFI (fold on untreated). Three independent experiments were performed. A) time 5: 4 wild-type and 4 _Ms4a4a__-/-_ mice; time 15: 5 wild-type and 8 _Ms4a4a__-/-_ mice. B) time 5: 6

wild-type and 5 _Ms4a4a__-/-_ mice; time 15: 5 wild-type and 6 _Ms4a4a__-/-_ mice. C) time 5: 4 wild-type and 5 _Ms4a4a__-/-_ mice; time 15: 4 wild-type and 4 _Ms4a4a__-/-_ mice. Statistical

analysis by two-tailed unpaired (Mann-Whitney) Student’s t test. D, E) Phosphorylation of ERK (D) and p38 (E) in BMDMs from _Ms4a4a__-/-_ (open symbols) and wild-type (closed symbols) mice

primed for 18 h with 10 ng/ml GM-CSF and stimulated or not (NT) with 100 μg/ml depleted zymosan, 100 μg/ml curdlan, 100 ng/ml LPS, and 100 ng/ml PMA. Results are shown as mean ± SEM of

relative MFI (fold on untreated). Two independent experiments were performed. One dot represents one mouse (2–4 for E, 2–6 for F). F–K) Secretion of IL-6 (F–H) and TNF (I–K) by BMDMs from

_Ms4a4a__-/-_ (open symbol) and wild-type (closed symbol) mice primed for 18 h with 10 ng/ml GM-CSF and stimulated for 24 h (F, G and I, J) or 6 h (H–K) with 100 μg/ml depleted zymosan

(F–I), 100 μg/ml curdlan (G–J) or 100 ng/ml Pam3Cys (H–K). Cytokine levels in untreated cells were below detection limit. Results are shown as mean ± SEM. Two independent experiments for F

and I (9 wild-type and 11 _Ms4a4a__-/-_ mice for F; 8 wild-type and 12 _Ms4a4a__-/-_ mice for I); five independent experiments for G and J (14 wild-type and 16 _Ms4a4a__-/-_ mice for G; 13

wild-type and 16 _Ms4a4a__-/-_ mice for J), three independent experiments for H and K (7 wild-type and 10 _Ms4a4a__-/-_ mice). Statistical analysis by two-tailed unpaired (Mann-Whitney)

Student’s t test. SUPPLEMENTARY FIGURE 7 ROLE OF MS4A4A IN B16F1 METASTATIC SPREADING AND MACROPHAGE INFILTRATION. A) Representative images of lungs from _Ms4a4a__-/-_ and wild-type mice

upon i.v. injection of B16F1 or B16F10 cells. B) Number of metastatic foci in lungs from _Ms4a4a__-/-__Clec7a__-/-_ (open symbol) and wild-type (closed symbol) mice upon B16F1 i.v.

injection. Results are shown as mean ± SEM. Four independent experiments were performed (11 wild-type and 19 _Ms4a4a__-/-__Clec7a__-/-_mice). Statistical analysis by two-tailed unpaired

(Mann-Whitney) Student’s t test. C) Representative immunofluorescence images of the distribution of NK cells (NKp46+), macrophages (F4/80+), and proliferating tumor cells (Ki-67+) in the

lung of wild-type mice injected i.v. with B16F1 cells. Nuclei were counterstained with DAPI and visualized in blue. Images refer to one experiment out of two analyzed. Scale bars: 150 μm in

the confocal images (left panels), 15 μm in the 3D bland images of representative areas (right panels). D–G) Frequency (D, F) and numbers (E, G) of interstitial (D, E) and alveolar (F, G)

macrophages in the lungs of _Ms4a4a__-/-_ (open symbol) and wild-type (closed symbol) mice injected i.v. with B16F1 cells. Resuls are shown as percentage or absolute numbers (n) of

F4/80+/CD11c- (interstitial) or F4/80+/CD11c+ (alveolar) macrophages pregated on Live/CD45+/SSC-Ahi/CD11blo/hi cells that were Ly6C and Ly6G negative. Results are expressed as mean ± SEM.

Three independent experiments were performed (9 wild-type and 6 _Ms4a4a__-/-_ mice). Statistical analysis by two-tailed unpaired (Mann-Whitney) Student’s t test. H) NK cell degranulation

towards B16F10 cells. Results are shown as mean ± SEM of percentage of CD107a+ cells gated on Live/CD45+/CD3-/NK1.1+ splenocytes. Two independent experiments were performed (5 wild-type and

4 _Ms4a4a__-/-_ mice). Statistical analysis by two tailed unpaired (Mann-Whitney), Student’s t test. I–K) _Calhm6_ mRNA levels in _Ms4a4a__-/-_ (open symbol) and wild-type (closed symbol)

BMDMs primed for 18 h with 10 ng/ml GM-CSF and stimulated for 24 h with zymosan (I), depleted zymosan (J), or LPS (K). Results are shown as mean ± SEM of fold of induction. Two independent

experiments were performed for i (3 wild-type and 5 _Ms4a4a__-/-_ mice), j (7 wild-type and 9 _Ms4a4a__-/-_ mice), and k (10 wild-type and 5 _Ms4a4a__-/-_ mice). Statistical analysis by

two-tailed unpaired (Mann-Whitney) Student’s t test. L) IL-18 secretion by _Ms4a4a__-/-_ (open symbol) and wild-type (closed symbol) BMDMs primed for 18 h with 10 ng/ml GM-CSF and stimulated

for 24 h with zymosan. Resuts are shown as mean ± SEM. Two independent experiments were performed (6 wild-type and 5 _Ms4a4a__-/-_ mice). Statistical analysis by two-tailed unpaired

(Mann-Whitney), Student’s t test. IL-18 levels in untreated cells and IL-12p70 levels in untreated and stimulated cells were below detection limit and are not shown. SUPPLEMENTARY FIGURE 8

MS4A4A CONTRIBUTION TO DECTIN-1-MEDIATED CONTROL OF METASTATIC SPREAD. A) Representative dot plot (left) and Mean Fluorescence Intensity (right) of WGA binding on the surface of MC38 and SL4

cells assessed by flow cytometry. Results are shown as mean ± SEM of relative MFI (fold on unstained). Three independent experiments were performed (5 independent samples for SL4, 7

independent samples for MC38). Statistical analysis by two-tailed unpaired (Mann-Whitney) Student’s t test. B, C) Number of metastatic foci in liver (B) and lungs (C) of _Clec7a__-/-_ (open

symbol) and wild-type (closed symbol) mice upon intrasplenic injection of MC38 (B) or i.v. injection of SL4 (C) cells. Results are shown as mean ± SEM. One experiment was performed (5

wild-type and 6 _Ms4a4a__-/-_ mice). Statistical analysis by two-tailed unpaired (Mann-Whitney) Student’s t test. D) Number of metastatic foci in liver of _Ms4a4a__-/-_ (open symbol) and

wild-type (closed symbol) mice injected intrasplenic with MC38 cells upon NK cell depletion. Results are shown as mean ± SEM. Two independent experiments were performed (5 wild-type and 9

_Ms4a4a__-/-_ mice). Statistical analysis by two-tailed unpaired (Mann-Whitney) Student’s t test and one-way ANOVA. E) Number of metastatic foci in lungs from _Ms4a4a__-/-_ (open symbol) and

wild-type (closed symbol) mice upon i.v. injection of SL4 cells. Results are shown as mean ± SEM. One experiment was performed (6 mice). Statistical analysis by two-tailed unpaired

(Mann-Whitney) Student’s t test. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–8 and Supplementary Tables 1–3 REPORTING SUMMARY SUPPLEMENTARY NOTE Gating

strategies applied to Figures and Supplementary Figures are reported. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Mattiola, I., Tomay, F., De Pizzol,

M. _et al._ The macrophage tetraspan MS4A4A enhances dectin-1-dependent NK cell–mediated resistance to metastasis. _Nat Immunol_ 20, 1012–1022 (2019).

https://doi.org/10.1038/s41590-019-0417-y Download citation * Received: 13 November 2017 * Accepted: 02 May 2019 * Published: 01 July 2019 * Issue Date: August 2019 * DOI:

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