# CLEC12B Regulates Melanocyte Immunity and Homeostasis in the Skin through the Signal Transducer and Activator of Transcription 1/IRF1 Axis

Lauriane Blot, Hanene Bzioueche, Marjorie Heim, Yann Cheli, Laura

Sormani, Els Verhoeyen, Alexandre Stella, Odile Burlet-Schiltz, Stéphane

Rocchi, Lionel Larue, et al.

► To cite this version:

Lauriane Blot, Hanene Bzioueche, Marjorie Heim, Yann Cheli, Laura Sormani, et al. CLEC12B Regulates Melanocyte Immunity and Homeostasis in the Skin through the Signal Transducer and Activator of Transcription 1/IRF1 Axis. 2024, Online ahead of print. 10.1016/j.jid.2024.11.016. hal-04908237

HAL Id: hal-04908237

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# CLEC12B Regulates Melanocyte Immunity and Homeostasis in the Skin through the Signal Transducer and Activator of Transcription 1/IRF1 Axis

Lauriane Blot<fnref n="1" />, Hanene Bziouche<fnref n="1" />, Marjorie Heim<fnref n="1" />, Yann Cheli<fnref n="1" />, Laura Sormani², Els Verhoeyen<fnref n="1" />, Alexandre Stella³, Odile Burlet-Schiltz³, Stéphane Rocchi<fnref n="1" />, Lionel Larue⁴, Yoann Rombouts³, Bernd Lepenies⁵, Thierry Passeron<fnref n="1" />,⁶ and Meri K. Tulic<fnref n="1" />

**Journal of Investigative Dermatology** (2025) **145**, 2007–2020; doi:10.1016/j.jid.2024.11.016

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JID Open

CLEC12B is a C-type lectin receptor involved in the inhibition of NKs-mediated cytotoxicity. We have previously shown that CLEC12B is predominantly expressed on melanocytes and inhibits melanin production and pigmentation as well as proliferation of melanoma. To date, the role of CLEC12B in skin immunity is unknown. Upon phosphorylation of its immunoreceptor tyrosine-based inhibitory motif, we demonstrate that CLEC12B increases the production of innate chemokines from human melanocytes through the activation of the signal transducer and activator of transcription 1/IFN regulatory factor 1-mediated IFNγ pathway, resulting in che-moattraction of immune cells. We have demonstrated CLEC12B to potentiate the effect of IFNγ in primed melanocytes. Furthermore, CLEC12B recognizes cutaneous (*Staphylococcus aureus* and *E coli*) but not gut (*Listeria monocytogenes*) bacteria and is capable of modulating their chemokine responses. Finally, we have shown that CLEC12B senses motifs present on human melanocytes and fibroblasts but not keratinocytes. Together, these results demonstrate that CLEC12B plays an important function in the human skin, bridging innate and adaptive immunity. This mechanism is of great interest because IFNγ and cellular recruitment are key initial steps involved in inflammation of many skin pathologies, making this receptor an interesting ther-apeutic target.

**Keywords:** CLEC12B, C-type lectin, Immunity, Melanocytes, Skin

## INTRODUCTION

Melanocytes located in the epidermis basal layer of the skin are on the front line to encounter harmful stimuli or pathogens from the outside as well as UVR. In addition to their well-known role in producing melanin and determining skin, hair, and eye color, an antigen-presenting function of melanocytes has been described. Le Poole et al (1993a, 1993b) have shown that melanocytes possess the phagocytic

machinery necessary for antigen processing, that their melanosomes can act as specialized lysosomal organelles for phagolysosome fusion, that melanocytes can process and present antigens to cytotoxic proliferative T cells, and that they can be targeted by T-cell-mediated cytotoxicity. Melanocytes have been demonstrated to trigger autoimmunity in psoriasis by direct presentation of epidermal melanocyte autoantigen ADAMTS5 to the HLA-C*06:02 TCR, the major risk allele (Arakawa et al, 2015; Ishimoto et al, 2024). We know that epidermal melanocytes and melanocytes in hair follicles express different antigens, and this may explain why epidermal melanocytes are targeted for destruction in vitiligo and hair follicles in alopecia aerata (Tobin and Bystryn, 1996). Moreover, melanocytes express major histocompatibility complex class I and II molecules and cell adhesion molecules and are capable of producing T helper 1 (IFNγ) (Tulic et al, 2019) and T helper 2 cytokines and chemokines (Martins et al, 2022), playing a critical role in cutaneous pathophysiology.

C-type lectin receptors (CLRs) are part of the immune pattern recognition receptors and play an important role in immunity by sensing exogenous and endogenous ligands present on pathogens and host cells. Most of CLRs are expressed on antigen-presenting cells and are involved in the production of cytokines and chemokines but also in antigen processing and presentation and subsequent T-cell activation

<footnote n="1">¹INSERM U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), University of Côte d'Azur, Nice, France; ²The University of Queensland, Brisbane, Australia; ³CNRS, UPS, Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, Toulouse, France; ⁴UMR3347 CNRS, INSERM U1021, Institut Curie, Orsay, France; ⁵Institute for Immunology & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany; and ⁶Department of Dermatology, University of Côte d'Azur, Centre Hospitalier Universitaire de Nice (CHU), Nice, France</footnote>

<footnote>Correspondence: Meri K. Tulic, INSERM U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), Bâtiment Archimed, 151 Rue Saint-Antoine, Nice 06200, France. E-mail: Meri.TULIC@univ-cotedazur.fr</footnote>

<footnote>Abbreviations: 3D, 3-dimensional; CLR, C-type lectin receptor; Fc, fragment crystallizable; IRF1, IFN regulatory factor 1; ITIM, immunoreceptor tyrosine-based inhibitory motif; NHM, normal human melanocyte; shRNA, short hairpin RNA; STAT, signal transducer and activator of transcription; TLR, toll-like receptor</footnote>

<footnote>Received 11 July 2024; revised 21 October 2024; accepted 17 November 2024; accepted manuscript published online 28 December 2024; corrected proof published online 20 January 2025</footnote>
(Blot et al, 2022). CLEC12B was initially identified as a CLR expressed on myeloid cells that counteracts NK-meditated cytotoxic activity through the recruitment of phosphatases SHP1 and SHP2 on its immunoreceptor tyrosine-based inhibitory motif (ITIM) (Hoffmann et al, 2007). We recently showed that CLEC12B is predominantly expressed by melanocytes in the skin and negatively regulates melanogenesis as well as melanoma proliferation (Montaudié et al, 2022; Sormani et al, 2022). However, the role of CLEC12B in skin immunity is still unknown.

In this study, we have shown that CLEC12B modulates melanocytes immune function through the phosphorylation of its ITIM domain. In particular, CLEC12B modulates the IFNγ-induced production of chemokines and subsequent chemoattraction of immune cells through the signal transducer and activator of transcription (STAT)1/IRF1 axis. Moreover, we demonstrated that CLEC12B interacts with *Staphylococcus aureus*, *S. epidermidis*, and *E. coli* and modulates an immune response against these opportunistic skin bacteria through the STAT1/IRF1/CXCL9 axis. Finally, we showed CLEC12B to recognize protein structures on melanocytes that may turn out to be its potential ligands, thus opening previously unreported avenues for the identification of novel therapeutic targets.

## RESULTS

### CLEC12B silencing in melanocytes decreases their immune function

IFNγ is a key immune cytokine that increases melanocyte immunocompetency by enhancing the expression of immune response accelerators (Wang et al, 2014) and induces the production of CXCL9, CXCL10, and CXCL11 by melanocytes (Tulic et al, 2019). To determine whether CLEC12B can modulate the production of IFNγ-induced chemokines in melanocytes, we silenced CLEC12B with short hairpin RNA (shRNA) (termed SH). The efficiency of shRNA was analyzed by RT-qPCR (Figure 1a) and western blot (Figure 1b). Our results confirmed that melanocytes are capable of producing IFNγ, CXCL9, CXCL10, and CXCL11 and that CLEC12B silencing significantly inhibited their production in vitro (Figure 1c). Using these melanocytes in coculture with keratinocytes in a 3-dimensional (3D) model of reconstructed human epidermis, we demonstrated reduced IFNg, CXCL-9, and CXCL-10 immunofluorescence in SH compared with that in scrambled shRNA control skin (denoted as CT) (Figure 1d). These results suggest that lack of CLEC12B in human melanocytes reduces their immune function.

### CLEC12B overexpression in melanocytes increases their immune response through its ITIM domain

Transmembrane CLRs possess intracellular signaling motifs that can modulate signaling pathways. Sequence data analysis revealed that CLEC12B contains an ITIM sequence (VTYATL) in its intracellular domain (Hoffmann et al, 2007). To determine whether CLEC12B ITIM domain is involved in the regulation of IFNγ, CXCL9, CXCL10, and/or CXCL11 production, we generated an ITIM domain mutant by exchanging the tyrosine by a phenylalanine (F) using site-directed mutagenesis, so that the ITIM domain can no longer be phosphorylated. We used a lentiviral-mediated overexpression of *CLEC12B* or a lentiviral-mediated

overexpression of ITIM mutant *CLEC12B* (overexpression mutant) in melanocytes whose efficiency was analyzed by qPCR (Figure 2a) and western blot (Figure 2b). *CLEC12B* overexpression induced a significantly higher production of CXCL-9, CXCL-10, and CXCL-11 chemokines as well as an increase in IFNγ but failed to reach statistical significance (*P* = .0621). Overexpression mutant was able to prevent these increases (Figure 2c). Increased IFNγ and chemokine immunodetection was observed in sections of 3D reconstructed human epidermis that contained normal human keratinocytes and melanocytes overexpressing CLEC12B compared with that in reconstructed human epidermis containing keratinocytes and melanocytes containing empty vector controls (CT) (Figure 2d). Overall, these results showed that overexpression of CLEC12B in melanocytes upregulates their immune function and that this regulation occurs through the phosphorylation of CLEC12B's ITIM domain.

### CLEC12B modulates the IFNγ-induced production of chemokines and subsequent chemoattraction of immune cells through its ITIM domain

IFNγ binds to IFNGR to induce activation of the downstream signaling pathway resulting in production of IFN-stimulated genes (Schroder et al, 2004). We set out to determine whether CLEC12B can modulate the IFNγ-induced production of CXCL9, CXCL10, and CXCL11. Results showed an increased chemokines production by melanocytes after IFNγ stimulation (Figure 3a and b). Silencing *CLEC12B* significantly decreased the IFNγ-induced production of CXCL9 and CXCL11; however, the levels remained significantly above those seen in nonstimulated melanocytes (Figure 3a). The reduction in CXCL10 production did not reach significance (*P* = .074). On the contrary, *CLEC12B* overexpression further potentiated the IFNγ-induced production of all chemokines, which was completely abolished in overexpression-mutant melanocytes, back to baseline CT levels (Figure 3b). These results demonstrate that CLEC12B is capable of potentiating IFNγ-induced cutaneous immunity in human melanocytes through the phosphorylation of its ITIM domain.

IFNγ is known to induce the expression of costimulatory and adhesion molecules, namely HLA-DR, CD40, and ICAM-1, at the surface of melanocytes (Le Poole et al, 1993a; Tulic et al, 2019). To examine whether CLEC12B is capable of modulating their expression, we used FACS analysis. Our results have confirmed IFNγ to significantly increase the expression of HLA-DR and ICAM-1; however, *CLEC12B* silencing had no effect on HLA-DR, CD40, or ICAM-1 at baseline or after IFNγ stimulation (Supplementary Figure S1).

IFNγ-induced chemokines regulate immune cell migration and induce chemotaxis to recruit immune cells to the site of inflammation to induce adaptive immune responses (Tokunaga et al, 2018). To determine whether CLEC12B can modulate the migration of PBMCs, we measured their chemoattraction toward secretome of *CLEC12B*-overexpressing melanocytes unstimulated (−IFNg) or IFNγ primed (+IFNg) for 24 hours. We demonstrated overexpression to significantly increase melanocyte capacity to attract PBMCs compared with CT, and chemotaxis was completely abolished in overexpression-mutant melanocytes (Figure 3c). In all groups of melanocytes, IFNγ stimulation results in
<figure><img src="image_3.png" /><figcaption>**Figure 1.** *CLEC12B silencing in melanocytes decreases their immune function.* Lentiviral infection was used to silence *CLEC12B* (denoted as SH) in primary human melanocytes. (a) *CLEC12B* mRNA relative quantity was measured by qPCR. Results were normalized to HKG *SB34* and expressed as fold change relative to control (denoted as CT). (b) *CLEC12B* protein expression was analyzed by western blot. β-Actin was used as the loading control. (c) Production of IFNγ, CXCL9, CXCL10, and CXCL11 in melanocyte supernatant was measured by ELISA, and (d) IFNγ, CXCL9, and CXCL10 were detected by immunofluorescence in 3D model of reconstructed human skin using *CLEC12B*-silenced melanocytes (SH) or control melanocytes (scramble shRNA, CT) cocultured with human keratinocytes. Results are shown as mean ± SEM (n = 10–12). Bar = 5 mm. Differences between groups were tested using nonparametric Wilcoxon matched-pairs signed-rank test (for a) or Mann–Whitney test (for c) and were considered significant when *P* ≤ .05. \* *P* ≤ .05, \*\* *P* ≤ .01, \*\*\* *P* ≤ .001, and \*\*\*\* *P* ≤ .0001. 3D, 3-dimensional; HKG, housekeeping gene; shRNA, short hairpin RNA.</figcaption></figure>

increased chemoattraction compared with that in their non-
stimulated controls. Moreover, *CLEC12B* overexpression
further potentiated the chemoattraction, and overexpression
mutant abrogated the increase (Figure 3c). Altogether, these
results demonstrate that CLEC12B modulates cellular
recruitment through its ITIM domain, and this recruitment is
potentiated by IFNγ priming of melanocytes.

CLEC12B modulates IFNγ response through the STAT1/IRF1
axis

Upon IFNγ binding to the IFNGR, the receptor is phosphor-
ylated, allowing recruitment and phosphorylation of STAT1.
Activated STAT1 dimers can then translocate to the nucleus
and act as transcription factors that bind to the GAS (IFN-
γ-activated site) elements of IFNγ-inducible genes, such as
CLEC12B in melanocyte immunity

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**Figure 2. CLEC12B overexpression in melanocytes increases their immune response through its ITIM domain.** Lentiviral infection was used to overexpress CLEC12B (OV) or to overexpress an ITIM-mutated CLEC12B (OV mutant) in primary human melanocytes. (a) CLEC12B mRNA relative quantity was measured by qPCR. Results were normalized to HKG SB34 and expressed as fold change relative to control (CT). (b) CLEC12B protein expression was analyzed by western blot. β-Actin was used as the loading control. (c) Melanocyte production of IFNγ, CXCL9, CXCL10, and CXCL11 in culture supernatant was measured by ELISA, and (d) IFNγ, CXCL9, and CXCL10 were detected by immunofluorescence in 3D model of reconstructed human skin using melanocytes overexpressing CLEC12B (OV) or control melanocytes (empty vector, CT) cocultured with human keratinocytes. Bar = 5 mm. Results are shown as mean ± SEM (n = 4–12). Differences between groups were tested using nonparametric Wilcoxon matched-pairs signed-rank test (for a) or Mann–Whitney test (for c) and were considered significant when *P* ≤ .05. \* *P* ≤ .05, \*\* *P* ≤ .01, \*\*\* *P* ≤ .001, and \*\*\*\* *P* ≤ .0001. CT denotes control; OV denotes CLEC12B overexpression. 3D, 3-dimensional; HKG, housekeeping gene; ITIM, immunoreceptor tyrosine-based inhibitory motif; pSTAT1, phosphorylated signal transducer and activator of transcription 1; STAT1, signal transducer and activator of transcription 1.
<figure><img src="image_11.png" /><figcaption>**Figure 3.** CLEC12B modulates the IFNγ-induced production of chemokines and subsequent chemoattraction of immune cells through its ITIM domain. (a, b) Production of cytokines and chemokines was measured by ELISA in (a) SH or (b) OV and OV mutant primary human melanocytes after IFNγ stimulation (50 ng/ml for 24 h). (c) Chemotaxis assay was performed to examine the migration of PBMCs toward secretome from OV and OV mutant with or without IFNg stimulation. Results are expressed as fold change relative to IFNγ-unstimulated CT and shown as mean ± SEM (n = 3–12). Differences between groups were tested using nonparametric Mann–Whitney test (a and b) or Wilcoxon matched-pairs signed-rank test (for c) and were considered significant when $P \le .05$. *$P \le .05$, **$P \le .01$, ***$P \le .001$, and ****$P \le 0.0001$. OV denotes CLEC12B overexpression; SH denotes shRNA-silenced CLEC12B. CT, control; h, hour; ITIM, immunoreceptor tyrosine-based inhibitory motif; pSTAT1, phosphorylated signal transducer and activator of transcription 1; RQ, relative quantity; shRNA, short hairpin RNA; STAT1, signal transducer and activator of transcription 1.</figcaption></figure>

CXCL9, CXCL10, and CXCL11. IRF1 is the most important of these IFNγ-inducible genes, which in turn acts as a transcription factor binding IRF1-binding site-containing promoters of target genes, including CXCL9, CXCL10, and CXCL11 (Forero et al, 2019; Kanda et al, 2007; Schroder

et al, 2004). To understand the mechanisms underlying the regulatory properties of CLEC12B in the production of chemokines, the effects of CLEC12B modulation on the expression of STAT1 (phosphorylated and total forms) and IRF1 was analyzed by western blot. In unstimulated melanocytes, the
constitutive expression of phosphorylated STAT1 and IRF1 is negligible as shown on western blot analysis (Figure 4a-c) and in its quantification (Figure 4b-d). In contrast, in CT melanocytes, IFNγ stimulation significantly increased phosphorylated STAT1, STAT1, IRF1, and CLEC12B expression, and this expression was decreased in SH melanocytes (Figure 4a and b), whereas all IFNg-induced responses were potentiated in overexpressed melanocytes (Figure 4c and d). These later overexpression-induced responses were abolished in ITIM-mutant melanocytes (Figure 4c and d). IFNγ stimulation increased CLEC12B expression in CT melanocytes, which was further potentiated in overexpression and overexpression-mutant melanocytes (Figure 4c and d). Together, these results showed that CLEC12B modulates the IFNγ pathway through the STAT1/IRF1 axis through the phosphorylation of its ITIM domain. Moreover, these data

suggest that there is a positive feedback between CLEC12B and IFNγ whereby IFNγ increases CLEC12B expression, and CLEC12B potentiates the effect of IFNγ in the downstream signaling pathway and target genes.

CLEC12B interacts with *S aureus* and *E coli* and modulates
an immune response against these opportunistic skin
bacteria through the STAT1/IRF1/CXCL9 axis

CLRs can recognize pathogen-associated molecular patterns to induce immune responses and fight against pathogens (Mayer et al, 2017). To determine whether CLEC12B can sense exogenous ligands, we incubated different bacteria with the fragment crystallizable (Fc) protein alone (as a negative control) or the fusion protein CLEC12B-Fc and visualized a possible interaction using confocal microscopy. We looked at bacterial strains that are typically found on the

<figure><img src="image_12.png" /><figcaption>**Figure 4.** CLEC12B modulates IFNγ response through the STAT1/IRF1 axis. (a–c) pSTAT1, STAT1, IRF1, and CLEC12B protein expressions were analyzed by western blot in (a) SH or (c) OV and OV-mutant primary human melanocytes after IFNγ stimulation (50 ng/ml for 24 h). β-Actin was used as the loading control. (b–d) Western blot relative quantification of pSTAT1, STAT1, IRF1, and CLEC12B expression relative to β-actin expression was analyzed using ImageJ. CT denotes control, OV denotes CLEC12B overexpression, and SH denotes shRNA-silenced CLEC12B. h, hour; IRF1, IFN regulatory factor 1; pSTAT1, phosphorylated signal transducer and activator of transcription; RQ, relative quantity; shRNA, short hairpin RNA; STAT1, signal transducer and activator of transcription 1.</figcaption></figure>
skin, including *S aureus*, *E coli*, and *Bifidobacterium bifidum*. In addition, we also looked at *L monocytogenes* mainly found in the gut. Our immunofluorescence results showed that CLEC12B-Fc interacts with *S aureus* and *E coli* but not with *B bifidum* or *L monocytogenes* (Figure 5a) nor with *B breve*, *B lactis*, or *B longum* (data not shown). To assess whether CLEC12B is involved in melanocyte response to *S aureus* and/or *E coli*, we stimulated CT or SH melanocytes with bacteria and measured their production of IFNγ, CXCL9, CXCL10, and CXCL11 in supernatants 24 hours later. Our results demonstrated that different bacterial strains induce differential chemokine production. In CT melanocytes, the production of CXCL9 was significantly increased in response to *S aureus*, and the production of CXCL10 was significantly increased in response to *E coli*, whereas the production of IFNγ and CXCL11 remained insensitive to bacterial stimulation (Figure 5b). CLEC12B silencing significantly inhibited the *S aureus*– and *E coli*–induced CXCL9 production; however, no significant effect was observed for CXCL10, IFNγ, or CXCL11 (Figure 5b). Regarding the downstream signaling pathway, phosphorylated STAT1, STAT1, and IRF1 expression was significantly increased in response to both *S aureus* and *E coli* stimulation in CT melanocytes, and CLEC12B silencing prevented this increase (Figure 5c). Together, these data showed that CLEC12B senses exogenous ligands present on *S aureus* and *E coli* and modulates an immune response against these opportunistic skin bacteria through the STAT1/IRF1/CXCL9 axis.

## CLEC12B does not recognize glycan structures on tested arrays

CLRs can recognize glycan structures expressed by pathogens and host cells to induce immune responses (van Vliet et al, 2008). In search of CLEC12B ligand(s), we set out to determine whether CLEC12B recognizes a carbohydrate structure. Although glycans numbers 198, 308, and 313 showed higher binding than other glycans, these targets had very low specific binding (average relative fluorescent units < 1000, hence below cut-off value) with high background noise for both mammalian (Supplementary Figure S2a and b) and microbial (Supplementary Figure S2c and d) glycan arrays, suggesting that CLEC12B does not recognize any specific mammalian nor microbial glycan structure present on those particular tested arrays.

## CLEC12B senses motifs present on fibroblasts and recognizes protein structures on melanocytes

CLRs can sense self-associated molecular patterns on healthy cells to maintain the baseline nonactivated state of innate immune cells and dampen their reactivity after an immune response (Varki, 2011). To assess whether CLEC12B senses endogenous ligands, we incubated different skin cells with the Fc or the fusion protein CLEC12B-Fc and visualized the possible interaction using confocal microscopy. Our results showed that CLEC12B strongly interacts with melanocytes and fibroblasts but not with keratinocytes (Figure 6a).

CLRs can bind a wide variety of ligands other than glycans, such as proteins, lipids, or inorganic compounds, on host cells (Zelensky and Gready, 2005). To determine whether CLEC12B recognizes a protein structure on melanocytes, we treated the cells with 0.05% trypsin (serine protease) or 5 µg/

ml papain (cysteine protease) for 5 minutes prior to cell incubation with the Fc protein alone (as a negative control) or the fusion protein CLEC12B-Fc and visualized possible interactions using confocal microscopy. This short, low-dose trypsin or papain pretreatments prevented the interaction between CLEC12B-Fc and melanocytes (Figure 6b), indicating that CLEC12B recognizes protein structures on melanocytes. To identify these protein structures, we performed a coimmunoprecipitation assay using lysates of Hermes melanocytic cell lines. Hermes have CLEC12B expression (at both mRNA and protein levels) and immune responses similar to those of primary human melanocytes (Supplementary Figure S3); hence, they are good substitutes to use in immunoprecipitation experiments where large cell numbers are required. After lysis of Hermes cells, CLEC12B ligands were immunoprecipitated using the CLEC12B-Fc fusion protein (or Fc protein as negative control) followed by analysis by nanoscale liquid chromatography coupled to tandem mass spectrometry. Results identified several potential CLEC12B ligands, including 5 proteins that were recognized in both Hermes 2A and Hermes 3A cell lines (classified in descending order of their peptide enrichment) (Figure 6c and d): IGF2R, lipoprotein lipase, CRLF1, out at first homolog, HscB mitochondrial iron-sulphur cluster cochaperone.

## DISCUSSION

We have previously demonstrated that CLEC12B is a melanocytic gene that modulates pigmentation and decreases melanoma proliferation (Montaudié et al, 2022; Sormani et al, 2022); however, its role in skin immunity was unknown. In this study, we demonstrate that CLEC12B is an important player in cutaneous immunity, directly regulating cytokine and chemokine production in primary melanocytes. Our in vitro data were confirmed in 3D reconstructed human epidermis where both keratinocytes and melanocytes were present (and in correct proportions), suggesting that they are likely to have important biological relevance. We have demonstrated that upon phosphorylation of its ITIM domain, CLEC12B increases the production of innate chemokines from human melanocytes through the activation of the STAT1/IRF1-mediated IFNγ pathway, resulting in chemoattraction of immune cells. Moreover, our study has shown that CLEC12B potentiates the effect of IFNγ in primed melanocytes, thus further amplifying the production of chemokines and therefore their subsequent capacity to attract immune cells. Together, these results suggest that CLEC12B in melanocytes is likely to be an important contributor to local IFNγ content in the skin and may be an attractive target in diseases where its excessive production leads to pathogenesis such as those seen in vitiligo or cutaneous lupus. Along with previous work published by Arakawa et al (2015) demonstrating that melanocytic autoantigen ADAMTSL5 can directly activate T cells and trigger antimelanocytic immunity in psoriasis, our data in normal skin suggest that CLEC12B on melanocytes is implicated in recruitment of immune cells through its ITIM domain.

Even though ITIM-containing CLRs are usually thought to act as negative regulators of immune cell signaling, they are also able to activate some pathways. For instance, CLEC4A has been shown to sustain the type I IFN signaling pathways
CLEC12B in melanocyte immunity

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<figure><img src="image_15.png" /><figcaption>Figure 5. CLEC12B interacts with *S. aureus* and *E. coli* and modulates an immune response against these opportunistic skin bacteria through the STAT1/IRF1/CXCL9 axis. (a) Bacteria were incubated with Fc protein or CLEC12B-Fc. Bacterial DNA was stained with Hoechst solution. The binding of the CLEC12B-Fc fusion protein to bacteria was detected using a FITC-conjugated antihuman IgG Fc secondary antibody. Bacteria were fixed onto cover slips, mounted onto microscopic slides, and visualized using confocal microscopy. Bar = 10 µm. (b, c) SH primary human melanocytes were stimulated with *S. aureus* or *E. coli* (MOI = 10) for 24 h. (b) Production of IFNγ, CXCL9, CXCL10, and CXCL11 was measured by ELISA. Results are shown as mean ± SEM (n = 6). Differences</figcaption></figure>
CLEC12B in melanocyte immunity

<figure><img src="image_16.png" /><figcaption>**Figure 6. CLEC12B senses motifs present on fibroblasts and recognizes protein structures on melanocytes. (a, b) Primary human melanocytes, fibroblasts, or keratinocytes were incubated with Fc protein or CLEC12B-Fc. DNA was stained with Hoechst solution. The binding of the CLEC12B-Fc fusion protein to cells was detected using a FITC-conjugated antihuman IgG Fc secondary antibody. Cells were grown onto cover slips, mounted onto microscopic slides, and visualized using confocal microscopy. Bar = 10 µm. (b) Prior to cell incubation and staining, melanocytes were treated with trypsin (0.05 %) or papain (5 µg/ml) for 5 min. (c, d) Lysates from melanocytic cell line (c) Hermes 2A and (d) Hermes 3A were incubated with Fc protein or CLEC12B-Fc and immunoprecipitated using protein G magnetic beads. Samples were analyzed by NanoLC-MS/MS, and a label-free quantification was performed with PROLINE 2.0 software. Fc, fragment crystallizable; min, minute; NanoLC-MS/MS, nanoscale liquid chromatography coupled to tandem mass spectrometry.**</figcaption></figure>

<footnote>between groups were tested using nonparametric Mann-Whitney test and were considered significant when $P \le .05$. *$P \le .05$, **$P \le .01$, ***$P \le .001$, and ****$P \le .0001$. (c) STAT1, IRF1, and CLEC12B protein expression levels were analyzed by western blot. β-Actin was used as the loading control. CT denotes control, and SH denotes shRNA-silenced CLEC12B. Fc, fragment crystallizable; h, hour; IRF1, IFN regulatory factor 1; MOI, multiplicity of infection; shRNA, short hairpin RNA; STAT1, signal transducer and activator of transcription 1.</footnote>
in dendritic cells as demonstrated in the context of tubercu-
losis infection (Troegeler et al, 2017). Likewise, CLEC12A
activation by monosodium urate released by host dead cells
after viral infection positively activates type I IFN signaling,
thereby amplifying an antiviral immune response (Li et al,
2019). CLEC12B was named after CLEC12A owing to their
high similarity in structure, function, and chromosomal
location; however, CLEC12A is not expressed on melano-
cytes. CLEC12A was also the first identified myeloid inhibi-
tory CLR that can recruit both SHP1 and SHP2 through its
ITIM domain (Marshall et al, 2004). Considering this infor-
mation, it is not surprising that CLEC12B can in turn act as a
positive regulator of immune signaling. Overall, we have
demonstrated that CLEC12B positively regulates IFNγ
pathway through the activation of the STAT1/IRF1 axis;
however, the exact molecular mechanism remains unclear.
Whether CLEC12B delivers a signal on its own through the
ITIM domain or whether participation of other coreceptors
such as CLEC12A is required for optimal antimelanocyticy
immunity remains to be investigated.

In this study, we have shown that CLEC12B is capable of
modulating cutaneous bacterial responses to *S aureus* and *E
coli*. A recent study supports our data showing that CLEC12B
from *Oreochromis niloticus* (OnCLEC12B) also recognizes *S
aureus* and *E coli*. Interestingly, OnCLEC12B contains a
conserved extracellular C-type lectin-like domain and shares
high identities with the human CLEC12B (Zhang et al, 2022).
We did not show CLEC12B to interact with *B bifidum*,
commensal bacteria that help in maintaining skin homeo-
stasis, nor with *L monocytogenes*, a pathogenic bacterium
mainly found in the gut. These data suggest that CLEC12B
may preferentially interact with strains such as *S aureus* to
ultimately control the ensuing cutaneous responses associ-
ated with this strain; however, this remains to be formally
tested. It is important to note that ligand binding may also be
pH sensitive, so it might be worth trying different pHs in these
CLR–Fc binding experiments. Another limit to these types of
experiments is that we only used whole heat-killed bacteria,
allowing CLEC12B to bind only external ligands, whereas the
use of bacterial lysates could have rendered accessible in-
ternal components such as glycolipids.

IFNγ production remained relatively unchanged after
stimulation of normal CT melanocytes with *S aureus* or *E coli*.
Although *CLEC12B* silencing significantly reduced IFNγ
production in nonstimulated melanocytes, it had no effect in
bacterial-stimulated melanocytes. These results suggest that
during infection, the role of CLEC12B may be limited. One of
the reasons may be that although *E coli* stimulates both toll-
like receptor 2 and toll-like receptor 4, *Staphylococcal* li-
gands are predominantly recognized by toll-like receptor 2,
and pathogenicity of *S aureus* strains can also induce type I
IFN signaling (Askarian et al, 2018). Considering that IFNγ
production remains insensitive to *S aureus* and *E coli* stimu-
lation in our experiments; that CXCL9 production can be
induced independently of IFNγ (Ding et al, 2016); and that
STAT1 and IRF1 can be part of signaling pathways other than
IFNγ, including toll-like receptors and type I IFN (Feng et al,
2021), we can conclude that CLEC12B-mediated anti–*S
aureus* and –*E coli* innate immune responses are IFNγ in-
dependent in melanocytes. To test this conclusively, the

exogenous ligands that are sensed by CLEC12B on *S aureus* and *E coli* must first be identified.

CLRs have been shown to recognize glycan structures expressed by pathogens and host cells to induce immune responses (van Vliet et al, 2008), and the identification of CLEC12B ligands is a critically important step to having a deeper understanding of how it influences ongoing immune responses. We have made a number of attempts to unravel the CLEC12B ligand(s). Our initial results showed that CLEC12B does not recognize any mammalian nor microbial glycan structure present on the 2 tested arrays. Although we did detect 3 possible candidates just above the background in the microbial screen, including glycans numbers 198 (Shigella boydii type 9), 308 (laminarin), and 313 (1–2 Mannan *Acetobacter methanolieus* MB135), this binding remained below the cut-off value (average relative fluorescent units < 1000) (Supplementary Figure S2). It is interesting to note that a recent study has reported CLEC12B to recognize laminarin, a polysaccharide containing β-(1,3) glucans, in *Pneumocystis murina* cell wall homogenates and in a purified *P carinii* cell wall fraction (Kottom et al, 2023), which are both fungi not included on our microbial screen. In light of this data, we can hypothesize that laminarin could be a potential ligand candidate for CLEC12B; however, a secondary screening must be performed to confirm these results by measuring the binding affinity through biophysical methods such as surface plasmon resonance (Bochner et al, 2005). Furthermore, even though these 2 glycan arrays represent many known important epitopes (585 glycans on the mammalian screen and 313 glycans on the microbial screen), and they contain only a fraction of existing glycans (glycolipids and proteoglycans not included) and only Gram-negative bacteria (hence, no *S aureus* strains), their coverage was the best available, to our knowledge, at the time of this study.

Demonstrating that CLEC12B interacts with protein struc-
tures on melanocytes, using proteomics analysis, we identi-
fied 5 potential candidates, including IGF2R, lipoprotein
lipase, CRLF1, out at first homolog, and HscB mitochondrial
iron-sulphur cluster cochaperone. Of these, IGF2R and
CRLF1 seem most relevant biological targets. IGF2R is a cell
surface glycoprotein that binds many different M6P-
containing ligands, is functionally related to CD-MPR, and
is involved in trafficking of lysosomal (melanosome) enzymes
(Brown et al, 2009), whereas CRLF1 is a soluble cytokine
receptor that is upregulated by proinflammatory cytokines,
including IFNg, and can activate STAT1/SHP2 signaling
pathway (Crisponi et al, 2022). These possible targets as
CLEC12B ligands in melanocytes (and fibroblasts) remain to
be validated in future studies. The finding of cis-interaction
(on the membrane of the same cell) between CLEC12B and its
ligand is not a novel concept because we have previously
demonstrated this to be the case for another ITIM receptors:
dendritic cell immunoreceptor and CLEC4A (Raymond et al,
2004¹). In these cases, ligands can be released from cells
such as melanocytes and fibroblasts to act as damage-

<footnote n="1">¹ Raymond BBA, Sneperger T, Rousset S, Gilles N, Bally A, Stella A, et al. The human and mouse dendritic cell receptor DCIR binds to LRPI N-glycans containing terminal galactoses, including the a-Gal antigen. BioRxiv 2024.</footnote>
associated molecular patterns for CLEC12B, playing an
important role in early immune defense of melanocytes.

In conclusion, our results demonstrate CLEC12B to play an
important function in the human skin immunity, bridging
innate and adaptive responses, and support the key func-
tion of melanocytes in cutaneous immunity. This mechanism
is of great interest because IFNγ and cellular recruitment are
key initial steps involved in inflammation of numerous skin
pathologies and autoimmune diseases, including vitiligo,
alopecia areata, cutaneous lupus, and psoriasis, making this
receptor an interesting therapeutic target.

MATERIALS AND METHODS

Cell culture

Melanocytes, keratinocytes, and fibroblasts were extracted from the
foreskin of healthy children undergoing circumcision as previously
described (Sormani et al, 2022). French law considers these samples
as discarded material, hence no formal written, informed consent
was required. Samples were washed and incubated overnight at 4 °C
with Dispase (4 U/ml, Gibco). The next day, epidermis was sepa-
rated from the dermis, and dermis was transferred to DMEM/F-12
10% fetal bovine serum (Gibco) to obtain normal human fibro-
blasts. Epidermis was incubated with 1:1 trypsin/PBS solution for 20
minutes at 37 °C, and suspension was filtered before the cell pellet
was split for normal human melanocytes (NHMs) or normal human
keratinocytes. For NHMs, cells were resuspended in MCDB (Sigma-
Aldrich, St. Louis, MO) supplemented with 1.18 g/l sodium bicar-
bonate (Invitrogen, Waltham, MA), 0.1 mM calcium chloride, 1%
antibiotic/antimicotic, 2% fetal calf serum, 5 µg/ml insulin (Sigma-
Aldrich), 0.5 µg/ml hydrocortisone (Sigma-Aldrich), 16 nM phorbol
12-myristate 13-acetate (Sigma-Aldrich), 1 ng/ml fibroblast GF
(Promega, Madison, WI), 15 µg/ml bovine pituitary extract (Gibco),
and 10 µM forskolin (Sigma-Aldrich). NHMs were selected with 20
µg/ml geneticin (Invitrogen) for approximately 2 weeks. Once
selected, NHMs were cultured in Cascade 254 (Thermo Fisher Sci-
entific, Waltham, MA) supplemented with Human Melanocyte
Growth Supplement (Gibco). For normal human keratinocytes, cells
were resuspended in keratinocyte serum-free growth medium sup-
plemented with 30 µg/ml bovine pituitary extract, 10 µg/ml trans-
ferrin, 5 µg/ml insulin, 0.3 µg/ml hydrocortisone, 0.39 µg/ml
epinephrine, 0.5 mM calcium chloride, and 0.125 ng/ml epidermal
GF (PromoCell, Heidelberg, Germany). Primary, nondifferentiated
keratinocytes were used for the experiments. All cells were main-
tained at 37 °C in a 5% carbon dioxide atmosphere. Bacterial strains
*S aureus*, *E coli*, and *L monocytogenes* were kindly provided by
Laurent Boyer (C3M, Nice, France). and *B bifidum* was provided by
Hanene Bzioueche (C3M).

Lentivirus production and transduction

Lentiviral particles were produced using vectors as previously
described (Sormani et al, 2022). For CLEC12B overexpression, we
used CLEC12B Lentiviral Vector pLenti-GIII-CMV-GFP-2A-Puro
(Applied Biological Materials, Richmond, Canada) custom (use of
CDS of CLEC12B accession number NM_001129998) and the
control plasmid Blank Vector pLenti-CMV-GFP-2A-Puro-Blank CT
(Applied Biological Materials). For CLEC12B silencing (SH), we used
the MISSION shRNA plasmid DNA pLKO.1-puro-CMV-Tur-
boGFP+shRNA (target sequence CAAGAACTCCACCCTAGTGAA)
(TRCN0000164269, Sigma-Aldrich) and the MISSION nontarget
shRNA Control Vector pLKO.1-puro-CMV-TurboGFP CT (Sigma-

Aldrich). We used the plasmid psPAX2 (Addgene, Watertown, MA) as a lentiviral packaging plasmid and the plasmid pCMV-VSV-G (Addgene) as an envelope protein to produce lentiviral particles. The 293FT cell line (Invitrogen) was transfected with jetPRIME transfection reagent kit (Ozyme, Saint-Cyr-l'École, France), and ViraPower lentiviral expression system was used for generating high-titer lentivirus. Viral supernatant was harvested 24 hours after transfection and filtered through a 0.45-µm cellulose acetate filter. A total of 1.5 × 10⁵ melanocytes were seeded in 6-well plates and reverse infected with the virus for 72 hours. The infected cells were selected for stable expression using 1 µg/ml puromycin every 2–3 days.

Site-directed mutagenesis of CLEC12B

The *CLEC12B* YàF mutation was generated by site-directed muta-
genesis using the QuikChange II XL Site-Directed Mutagenesis kit
(Agilent Technologies, Les Ulis, France). The following primers were
used: 5'-GTCTGAAGAAGTGACCTTCGCGACACTCACATTTC-3' (CLEC12B YàF forward) and 5'-GAAATGTGAGTGTCGCGAAGGTCACTTCTTCAGAC-3' (CLEC12B YàF reverse). Mutations were verified by DNA sequencing (Eurofins Genomics, Louisville, KY).

RT-qPCR

RNAs were extracted from NHMs using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). RNA quality and quantity were determined using the NanoDrop (Thermo Fisher Scientific). The reverse transcriptase reaction was carried out using the Reverse Transcription System kit (Promega) with 1 µg of RNA. The cDNAs were amplified by PCR with primers specific to *CLEC12B* (forward AGGAATTGCCTGTCCATTTCAT; reverse AGGAATTGCCTGTCCATTTCAT) and house-keeping gene *SB34* (forward TGCTCAGTACCCATTCTATCAT; reverse AAGGTAATCCGTCTCCACAGA) using Fast SYBR Green Master Mix (Thermo Fisher Scientific). Samples were run using StepOne Real-Time PCR System (Thermo Fisher Scientific), and analysis was performed by relative quantification with StepOne Software.

Western blot analysis

NHMs were lysed on ice with RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with Mini cOmplete EDTA-Free Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and phosphatase inhibitors PhosSTOP (Roche). Supernatant was collected, centrifuged (13,000g for 10 minutes at 4 °C), and assayed for protein concentration using the BCA kit (Thermo Fisher Scientific). A total of 20–30 µg of proteins were added to 3% SDS recovery buffer (Euromedex, Souffelweyersheim, France), bromophenol blue, and β-mercaptoethanol (Sigma-Aldrich) and then denatured for 5 minutes at 95 °C. The proteins were separated by electrophoresis on SDS-PAGE polyacrylamide gels and then transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA) before saturation with blocking buffer (10 mM Tris, pH 7.4, 150 mM sodium chloride, 1 mM EDTA, 0.5% Gelatin, 3% BSA, 0.1% Tween-20) for 1 hour at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies (Supplementary Table S1) diluted in blocking buffer. After 3 washes in wash buffer (10 mM Tris pH 7.6, 150 mM sodium chloride, 1% Triton X-100), membranes were incubated with secondary antibodies (Supplementary Figure S2) for 1 hour at room temperature. Detection was carried out using enhanced chemiluminescence detection (Dutscher, Bernolsheim, France) and the chemiluminescent image analyzer LAS-3000
(Fujifilm, Tokyo, Japan). Relative expression of target proteins was reported to β-actin expression and quantified using ImageJ software.

## ELISA

Melanocytes were seeded in 12-well plates at a density of 3.5 × 10⁵ cells/ml of medium, and 48 hours later, they were stimulated with IFNγ (50 ng/ml), S aureus (multiplicity of infection = 10), or . coli (multiplicity of infection = 10) for further 24 hours. IFNγ, CXCL9, CXCL10, and CXCL11 production was measured in supernatants using commercially available ELISA kits (Peprotech, Rocky Hill, NJ).

## Reconstructed 3D skin model

The reconstruction of the 3D skin model (skinEthic RHE) was performed by EPISKIN (Lyon, France) using our in-house *CLEC12B* silencing (SH), *CLEC12B* overexpressing, or control melanocytes (denoted as CT) as described earlier. The reconstructed epidermis was embedded in Tissue-Tek optimal cutting temperature medium in cryomolds, snap frozen in liquid nitrogen, and stored at -80 °C before cutting. A total of 7-mm sections were cut on a microtome and collected on SuperFrost Plus slides (J1800AMNZ, Thermo Fisher Scientific). Slides were fixed with 4% paraformaldehyde (Merck, Lyon, France), permeabilized with Tris-buffered saline in the presence of 0.3% Triton (Euromedex, France), blocked for 1 hour with Tris-buffered saline 5% BSA, and incubated overnight at 4 °C using specific antibodies directed against human IFNg (Abcam, 1/100), CXCL9 (Invitrogen, 1/100), or CXCL10 (Abcam, 1/100). The next day, sections were incubated with fluorescent dye-conjugated secondary antibody (Supplementary Table S1), slides were mounted with Prolong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific), and fluorescence was detected using a confocal microscope (Nikon A1R, Nikon Instruments).

## Chemotaxis assay

The chemotaxis of PBMCs was assessed using the CytoSelect 96-Well Cell Migration Assay (5 µm, Fluorometric Format) according to manufacturer's instructions (Cell Biolabs, San Diego, CA). Briefly, melanocytes were seeded in 12-well plates at 3.5 × 10⁵ cells/ml of medium, and 48 hours later, they were stimulated with IFNγ (50 ng/ml) for 24 hours. A total of 120 µl of the supernatants were used with 1.3 × 10⁵ PBMCs from healthy donors. Migratory cells were dissociated from the membrane, lysed, and quantified using CyQuant GR Fluorescent Dye.

## Immunofluorescence

Bacteria (10⁸) were fixed with 2% paraformaldehyde onto slides for 10 minutes at room temperature. Cells were grown onto cover slides in plates and then fixed with 2% paraformaldehyde for 10 minutes at room temperature. After 3 PBS washes, both cells and bacteria were incubated with 500 ng of Fc protein alone (negative control) or CLEC12B-Fc fusion protein diluted in lectin-binding buffer (50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 5 mM magnesium chloride, and 5 mM calcium chloride) overnight at 4 °C. The next day, slides were washed in lectin-binding buffer and incubated with the secondary antibody (Supplementary Table S1) diluted in lectin-binding buffer for 2 hours at 4 °C in the dark. After PBS washing, DNA was stained with Hoechst solution (Life Technologies, Carlsbad, CA) for 5 minutes at room temperature. The cover slides were mounted onto Menzel-Glaser Superfrost Plus microscopic slides (Thermo Fisher Scientific) with ProLong Diamond Antifade Mountant (Invitrogen) and visualized using Nikon confocal A1R microscope (Nikon) or Spinning disk W1 microscope (Nikon).

## Fusion protein

CLEC12B-Fc fusion protein (and the Fc alone as a negative control) was produced as previously described (Mayer et al., 2018). Briefly, the cDNA encoding for the mouse CLEC12B C-type lectin-like domain was cloned into the pFUSE expression vector and fused to the cDNA fragment encoding human IgG1-Fc. CHO-S cells were transiently transfected with the expression vector construct, and soluble CLEC12B-Fc fusion proteins were secreted. The cell supernatant was purified using protein G columns, and the presence of CLEC12B-Fc fusion proteins was detected by western blot.

## Immunoprecipitation

For this experiment only, 2 melanocytic cell lines were used (Hermes 2A and 3A) because large quantity of cellular material was needed, which was not attainable with primary melanocytes. Hermes melanocytes express similar amount of *CLEC12B* mRNA and protein and have immune function similar to that of NHMs (Supplementary Figure S3). Hermes were kindly provided by Lionel Larue (Institut Curie, Orsay, France). Hermes 2A is immortalized from Hmel-p16-2 and are p16 null melanocytes immortalized with hTERT. Hermes 3A is immortalized from Nohm-1 normal diploid human melanocyte strain and has hTERT and CDK4 expression vectors. Hermes cell lines were cultured in RPMI 1640 (Gibco) supplemented with 100 units/ml penicillin (Gibco), 100 µg/ml streptomycin (Gibco), 10% fetal calf serum (HyClone), 200 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich), 200 pM cholera toxin (Sigma-Aldrich), 10 nM endothelin-1 (Bachem, Bubendorf, Switzerland), and 10 ng/ml human stem cell factor (Miltenyi, Paris, France) at 37 °C in a 5% carbon dioxide. After washing with PBS, melanocytes were lysed on ice using RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with Mini cOmplete EDTA-Free Protease Inhibitor Cocktail (Roche) and phosphatase inhibitors PhosSTOP (Roche). The supernatant was collected, centrifuged at 13,000g for 10 minutes at 4 °C, and assayed for protein concentration using the BCA kit (Thermo Fisher Scientific). A total of 1 mg of protein lysate was incubated with Dynabeads Protein G (Invitrogen) for 1 hour 30 minutes at 4 °C on a rotating shaker for a preclearing phase. After removing the beads, protein lysate was incubated with 2 µg of CLEC12B-Fc or Fc alone overnight at 4 °C on a rotating shaker. Dynabeads Protein G was then added to the complex antigen/fusion protein for 1 hour 30 minutes at 4 °C on a rotating shaker. After 3 washes in RIPA buffer, the samples were eluted in 5% SDS and heated for 10 minutes at 70 °C, beads were removed, and the eluted antigen/CLEC12B-Fc or antigen/Fc complexes were sent in quadruplicates for each melanocyte cell line to the ProteoToul facility (Toulouse, France). Samples were analyzed using nanoscale liquid chromatography coupled to tandem mass spectrometry, and a label-free quantification was performed with PROLINE 2.0 software by the ProteoToul facility staff.

## FACS analysis

After 24 hours of IFNγ stimulation (50 ng/ml), melanocytes were detached using StemPro Accutase (Gibco) and incubated with FcR-blocking reagent (Miltenyi, Paris, France) for 10 minutes at 4 °C. After washing with PBS, cells were resuspended in MACSQuant Running Buffer (Miltenyi) and seeded in a conical bottom 96-well plate at a density of 5 × 10⁴ cells per well. Cells were then incubated with primary antibodies or control isotypes (Supplementary Table S1) for 15 minutes at 4 °C in the dark. After washing, cells were centrifuged at 300g and resuspended in 170 µl of MACSQuant
Running Buffer before analysis by flow cytometry (MACSQuant10, Miltenyi).

## Glycan array

The affinity of the CLEC12B-Fc fusion protein for carbohydrate structures, along with the Fc alone and the secondary antibody (Supplementary Table S1) as negative controls, was tested using the mammalian glycan array of 585 glycans (version 5.1) or the microbial glycan array of 313 glycans (version 1) developed by the Consortium for Functional Glycomics (Harvard Medical School, Boston, MA). Data were collected and analyzed by the Consortium for Functional Glycomics Core H staff, and results can be viewed in full on the Harvard Dataverse website under the following link: https://doi.org/10.7910/DVN/TICFQW.

## Statistical analyses

Statistical analyses were performed with Graphpad Prism 9 software. Differences between groups were tested using nonparametric Wilcoxon matched-pairs signed-rank test for paired differences or Mann-Whitney test for unpaired differences and were considered significant when $P \le .05: *P \le .05, **P \le .01, ***P \le .001, ****P \le .0001$.

## DATA AVAILABILITY STATEMENT

All data are available from the corresponding author upon reasonable request. The glycan arrays data can be viewed in full on the Harvard Dataverse website under the following link: https://doi.org/10.7910/DVN/TICFQW. The mass spectrometry proteomics data are available through ProteomeXchange with identifier PXD046123. Reviewer account details: Username: reviewer_pxd046123@ebi.ac.uk. Password: 7u3WxOmV

## ORCIDs

Lauriane Blot: http://orcid.org/0000-0002-7375-8096
Hanene Bzioueche: http://orcid.org/0000-0001-7243-0606
Marjorie Heim: http://orcid.org/0000-0002-7858-4822
Yann Cheli: http://orcid.org/0000-0001-9839-4332
Laura Sormani: http://orcid.org/0000-0002-0838-2088
Els Verhoyen: http://orcid.org/0000-0001-9224-5491
Bernd Lepenies: http://orcid.org/0000-0001-9033-5522
Yoann Rombouts: http://orcid.org/0000-0003-4482-2199
Alexandre Stella: http://orcid.org/0009-0009-1849-7565
Odile Burlet-Schiltz: http://orcid.org/0000-0002-3606-2356
Lionel Larue: http://orcid.org/0000-0002-2010-6107
Stéphane Rocchi: http://orcid.org/0000-0002-0943-1304
Thierry Passeron: http://orcid.org/0000-0002-0797-6570
Meri K. Tulic: http://orcid.org/0000-0002-2661-2369

## CONFLICT OF INTEREST

The authors state no conflict of interest.

## ACKNOWLEDGMENTS

We would like to thank all the volunteers and patients enrolled in this study as well as the clinical staff and nurses at the Department of Pediatric Surgery, Lenval Hospital (Nice, France). We thank the Centre Méditerranéen de Médecine Moléculaire (C3M, INSERM U1065) microscopy and imaging platforms. The authors thank Laurent Boyer and Orane Visvikis for their provision of bacterial strains. We gratefully acknowledge Silke Schöneberg for the production of C-type lectin receptor-fragment crystallizable fusion proteins and Olivier Neyrolles and Thomas Botton for their intellectual input and fruitful scientific discussions. We acknowledge participation of the Protein-Glycan Interaction Resource of the Consortium for Functional Glycomics and the National Center for Functional Glycomics (Harvard Medical School, Boston, MA) supporting grants P41 GM103694 and R24 GM137763. The authors also acknowledge participation of the ProteoToul services (proteomics platform of Toulouse, France). This study was supported by a Federal Student PhD Scholarship, the Institut National de la Santé et de la Recherche Médicale (INSERM), and La Ligue Nationale contre le Cancer.

## AUTHOR CONTRIBUTIONS

Conceptualization: LB, MKT, TP; Data Curation: LB, HB, MH, YC, EV, AS, OB-S, MKT; Formal Analysis: LB, HB, YC, AS, OB-S, MKT; Funding Acquisition: TP, MKT, SR; Investigation: LB, HB, MH, YC, LS, EV, BL, YR, AS, OB-S,

LL, SR, TP, MKT; Methodology: LB, MH, LS, EV, BL, YR, AS, OB-S, MKT; Project Administration: TP, SR, MKT; Resources: BL, AS, OB-S, YR, SR, TP, MKT; Supervision: MKT, TP; Validation: MKT, TP; Visualization: LB, MKTBL, YR; Writing - Original Draft Preparation: LB, MKT, TP; Writing - Review and Editing: LB, HB, YR, MKT, TP

## SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at www.jidonline.org, and at https://doi.org/10.1016/j.jid.2024.11.016.

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<figure><img src="image_17.png" /></figure>

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
<figure><img src="image_18.png" /><figcaption>**Supplementary Figure S1.** CLEC12B silencing has no effect on immunological and adhesion markers on melanocytes. Proportion of HLA-DR-, CD40-, or ICAM-1-positive cells (%) in SH primary human melanocytes after IFNγ stimulation (50 ng/ml for 24 h) were counted by FACS. Results were shown as mean ± SEM (n = 3). SH denotes shRNA-silenced CLEC12B. CT, control; h, hour; shRNA, short hairpin RNA.</figcaption></figure>
CLEC12B in melanocyte immunity

**Supplementary Figure S2. CLEC12B does not recognize glycan structures on tested arrays.** (a–c) Fc (50 µg) or (b–d) CLEC12B-Fc (50 µg) proteins were incubated with (a, b) the mammalian glycan array of 585 glycans or (c, d) the microbial glycan array of 313 glycans developed by the Consortium for Functional Glycomics. Protein binding was revealed by FITC fluorescence using a FITC-conjugated antihuman IgG Fc secondary antibody and expressed as RFU. The average intensity detected from all glycans was calculated and set as baseline. Glycans with average RFU > 1000 were considered significant. Results are shown as mean ± SD. Fc, fragment crystallizable; RFU, relative fluorescent unit.

<figure><img src="image_19.png" /></figure>
CLEC12B in melanocyte immunity

**Supplementary Figure S3. CLEC12B expression and function in primary melanocytes and in melanocytic cell line.** (a) Expression of CLEC12B mRNA in NHMs and Hermes 2A melanocytic cell line, (b) their chemokine production in culture supernatant (measured by ELISA), and (c) recognition of mCLEC12B-hFc on the surface of Hermes 2A melanocytes. Bar = 5 mm. NHM, normal human melanocyte.

<figure><img src="image_20.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_24.png" /></figure>
<table><caption>Supplementary Table S1. List of Antibodies Used</caption><thead><tr><th>Target Gene</th><th>Conjugated</th><th>Supplier</th><th>Reference</th><th>Dilution</th></tr></thead><tbody><tr><td colspan="5">Primary antibodies</td></tr><tr><td>CLEC12B</td><td>—</td><td>Abcam</td><td>ab211452</td><td>1/200</td></tr><tr><td>CLEC12B</td><td>—</td><td>Proteintech</td><td>26077-1-AP</td><td>1/500</td></tr><tr><td>pSTAT1</td><td>—</td><td>CST</td><td>7649</td><td>1/500</td></tr><tr><td>STAT1</td><td>—</td><td>CST</td><td>9172</td><td>1/1000</td></tr><tr><td>IRF1</td><td>—</td><td>CST</td><td>8478</td><td>1/500</td></tr><tr><td>β-Actin</td><td>—</td><td>Santa Cruz</td><td>sc-47778</td><td>1/3000</td></tr><tr><td>HSP90</td><td>—</td><td>Santa Cruz</td><td>sc-13119</td><td>1/1000</td></tr><tr><td>CD40</td><td>APC</td><td>Miltenyi</td><td>130-102-547</td><td>1/10</td></tr><tr><td>HLA-DR</td><td>APC-Vio770</td><td>Miltenyi</td><td>130-111-792</td><td>1/200</td></tr><tr><td>ICAM-1</td><td>PE-Vio770</td><td>Miltenyi</td><td>130-104-031</td><td>1/1000</td></tr><tr><td>REA control (S) APC</td><td>APC</td><td>Miltenyi</td><td>130-113-434</td><td>1/50</td></tr><tr><td>REA control (S) APC-Vio770</td><td>APC-Vio770</td><td>Miltenyi</td><td>130-113-435</td><td>1/50</td></tr><tr><td>REA control (S) PE-Vio770</td><td>PE-Vio770</td><td>Miltenyi</td><td>130-113-440</td><td>1/50</td></tr><tr><td>CXCL9</td><td>—</td><td>Thermo Fisher Scientific</td><td>PA5-34743</td><td>1/100</td></tr><tr><td>CXCL10</td><td>—</td><td>Abcam</td><td>ab8098</td><td>1/100</td></tr><tr><td>IFNg</td><td>—</td><td>Abcam</td><td>ab9657</td><td>1/100</td></tr><tr><td colspan="5">Secondary antibodies</td></tr><tr><td>Antimouse Ig</td><td>HRP</td><td>Dako</td><td>P0447</td><td>1/2000</td></tr><tr><td>Antirabbit Ig</td><td>HRP</td><td>Dako</td><td>P0448</td><td>1/2000</td></tr><tr><td>Antihuman IgG Fc</td><td>FITC</td><td>Invitrogen</td><td>A18818</td><td>1/500</td></tr><tr><td>Goat antimouse IgG</td><td>AF647</td><td>Invitrogen</td><td>A21235</td><td>1/1000</td></tr><tr><td>Goat antirabbit IgG</td><td>AF647</td><td>Invitrogen</td><td>A21244</td><td>1/1000</td></tr></tbody></table>

Abbreviations: APC, allophycocyanin; CST, Cell Signaling Technologies; Fc, fragment crystallizable; HRP, horseradish peroxidase; PE, phycoerythrin; pSTAT1, phosphorylated signal transducer and activator of transcription 1; STAT1, signal transducer and activator of transcription 1.