Received: 23 June 2020 | Revised: 20 April 2021 | Accepted: 28 April 2021 DOI: 10.1111/sji.13050 REGULAR ARTICLE Exploring the role of the multiple sclerosis susceptibility gene CLEC16A in T cells Anna M. Eriksson1,2 | Ingvild Sørum Leikfoss1,2,3 | Greger Abrahamsen4 | Vibeke Sundvold4 | Martine Mesel Isom1 | Pankaj K. Keshari1,2 | Torbjørn Rognes5,6 | Ole J. B. Landsverk7 | Steffan D. Bos1,2 | Hanne F. Harbo1,2 | Anne Spurkland4 | Tone Berge3,8 1Department of Neurology, Oslo University Hospital, Oslo, Norway Abstract 2 C- type lectin- like domain family 16 member A CLEC16A Institute of Clinical Medicine, University ( ) is associated with auto- of Oslo, Oslo, Norway immune disorders, including multiple sclerosis (MS), but its functional relevance is 3 Neuroscience Research Unit, Department not completely understood. CLEC16A is expressed in several immune cells, where of Research, Innovation and Education, it affects autophagic processes and receptor expression. Recently, we reported that Oslo University Hospital, Oslo, Norway CLEC16A 4Department of Molecular Medicine, the risk genotype of an MS-associated single nucleotide polymorphism in + Institute of Basic Medical Sciences, intron 19 is associated with higher expression of CLEC16A in CD4 T cells. Here, University of Oslo, Oslo, Norway we show that CLEC16A expression is induced in CD4+ T cells upon T cell activation. 5 Department of Informatics, University of By the use of imaging flow cytometry and confocal microscopy, we demonstrate that Oslo, Oslo, Norway 6 CLEC16A is located in Rab4a- positive recycling endosomes in Jurkat TAg T cells. Department of Microbiology, Oslo University Hospital, Oslo, Norway CLEC16A knock-down in Jurkat cells resulted in lower cell surface expression of 7Department of Pathology, Oslo University the T cell receptor, however, this did not have a major impact on T cell activation Hospital, Oslo, Norway response in vitro in Jurkat nor in human, primary CD4+ T cells. 8Department of Mechanical, Electronic and Chemical Engineering, Oslo Metropolitan University, Oslo, Norway Correspondence Tone Berge, Department of Mechanical, Electronic and Chemical Engineering, Oslo Metropolitan University, Postboks 4, St. Olavs plass, Oslo 0130, Norway. Email: [email protected] Funding information Norges Forskningsråd, Grant/Award Number: 240102; Odd Fellow Society; Novartis; Legatet til Halvor Homans Minde; Helse Sør- Øst RHF, Grant/Award Number: 201008 and 2012008 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology. Scand J Immunol. 2021;00:e13050. wileyonlinelibrary.com/journal/sji | 1 of 15 https://doi.org/10.1111/sji.13050 2 of 15 | ERIKSSON ET AL. 1 | INTRODUCTION Scientific and Merck), anti-GAPDH and anti p62/SQSTM1 (D- 3) (Santa Cruz Biotechnology), anti- actin (Sigma- A single nucleotide polymorphism (SNP) in the C- type Aldrich), allophycocyanin (APC)- conjugated anti- CD69 lectin- like domain family 16 member A (CLEC16A) gene was (Immunotools), APC- conjugated TCR alpha/beta (IP26, among the first genetic variants outside the human leucocyte Molecular Probes, Thermo Fisher Scientific), APC- antigen (HLA) region that showed evidence of association conjugated anti- human CD25 (MEM- 181. Immunotools), in the first genome- wide association study (GWAS) per- phycoerythrin (PE)- conjugated anti- human HLA- DR (L203, formed in multiple sclerosis (MS).1 CLEC16A has since then R&D Systems) and anti- CD3ε (clone OKT3, American been established as an MS- susceptibility gene.2- 11 SNPs in Type Culture Collection (ATCC)). Secondary antibodies CLEC16A are also associated with several other autoimmune used were as follows: Alexa Fluor (AF) 647-conjugated diseases, including type 1 diabetes,5,12- 16 Crohn's disease,17 anti- rabbit IgG, AF647-conjugated anti- mouse IgG and Addison's disease18 and rheumatoid arthritis.19,20 These stud- AF555- conjugated anti- mouse IgG2a (Life Technologies, ies support a general role for CLEC16A in development of Thermo Fisher Scientific), horseradish peroxidase (HRP)- autoimmunity. How CLEC16A contributes to autoimmunity conjugated goat anti- mouse IgG, goat anti- rabbit IgG and inflammation is however poorly understood. and mouse anti- goat IgG (Jackson Immuno Research Ema, the CLEC16A orthologue in Drosophila melano- Laboratories Europe Ltd.). Isotype controls used were gaster, has been shown to regulate endosomal trafficking and APC- conjugated anti- IgG1 (Immunotools). Dynabeads® maturation as well as autophagy.21,22 Also, in mouse and hu- Human T- Activator CD3/CD28 for T Cell Expansion and mans, CLEC16A plays a role in autophagic processes as ob- Activation and LIVE/DEAD Fixable Near- IR Dead Cell served in thymic epithelial cells,23 Purkinje cells,24 pancreatic Stain Kit were from Thermo Fisher Scientific. β- cells,25,26 adipose tissue27 and in the kidney epithelial cell C- type lectin- like domain family 16 member A - specific line HEK293T.28 Furthermore, in splenic B and T cells from siRNA (ON- TARGET plus SMARTpool siRNA) and con- CLEC16A knockout mice, mitophagy is disrupted.29 In human trol siRNA (ON- TARGET plus Non- targeting Pool) were antigen- presenting cells, CLEC16A regulates HLA-II expres- obtained from Thermo Fisher Scientific and Hoechst from sion via late endosomal biogenesis,30 and in murine natural Life Technologies. Phorbol 12-myristate 13- acetate (PMA), killer (NK) cells, CLEC16A modulates receptor expression.31 paraformaldehyde (PFA), saponin, sodium azide, ionomy- Even though functional and epigenetic annotation stud- cin calcium salt from Streptomyces and dimethyl sulphoxide ies suggest that multiple components of the immune system (DMSO) were obtained from Sigma- Aldrich. Bovine serum are involved in MS; T cells, and in particular CD4+ T cells, albumin (BSA) was obtained from Bio-Rad and Bafilomycin are considered major players in MS pathogenesis.10,32,33 We A1 (BafA1) from Bio Vision. recently showed that the MS-risk allele of an MS-associated SNP in intron 19 of CLEC16A correlates with higher expres- sion levels of CLEC16A in human CD4+ T cells.34 In the cur- 2.2 | Plasmids rent study, we analysed the function of CLEC16A in T cells using the Jurkat TAg T cell line and primary human CD4+ T pCMV6- CLEC16A- myc- DDK was obtained from Origene cells. We show that CLEC16A localizes to Rab4a- positive Technologies (TrueORF™ Precision Shuttle Vector sys- recycling endosomes in Jurkat cells and its expression is in- tem), pEGFP- N3 from Clontech Laboratories (Takara Bio), duced upon activation of CD4+ T cells. Knocking down its pcDNA3- Rab4a (cloneID: RAB04A000) in from UMR expression did not influence starvation-induced autophagy cDNA Resource Center and pmax- GFP from Lonza. pMep4- and had only a marginal impact on T cell activation. Our Rab5- mCherry has been described previously.35 Rab4a was data indicate a T cell-specific localization of CLEC16A to subcloned into EcoRI- XhoI sites of pEGFP- C2 (Clontech Rab4a- positive endosomes; however, its role in T cells is still Laboratories). EGFP was replaced with mCherry using Gibson enigmatic. assembly multiplex PCR. C- terminally and N- terminally GFP- tagged CLEC16A was generated by subcloning a blunted EcoRI/NotI digest of pCMV6- CLEC16A- myc- DDK 2 | MATERIALS AND METHODS into the SmaI site of pEGFP- N3 (Clontech Laboratories; C- term tag) and SmaI and blunted XbaI sites of pEGFP-C1 2.1 | Antibodies and reagents (Clontech Laboratories; N- term tag). The pCMV6- empty vector was generated by removing CLEC16A from pCMV6- Primary antibodies used were as follows: anti- LAMP1 CLEC16A- myc- DDK by digestion with BamHI and PmeI, (Abcam), anti- TGN46 (Bio- Rad), anti- LC3B (Cell blunting and re- ligation. The subcloned constructs were con- Signaling Technologies), anti- CLEC16A (Thermo Fisher firmed by DNA sequencing (GATC Biotech AG). ERIKSSON ET AL. | 3 of 15 2.3 | RNA isolation, synthesis of cDNA and human T cells, Lonza). After transfection, cells were incu- quantitative real- time PCR bated with preheated LGM-3 medium (Lonza) supplemented with 10% FCS at 37°C, 5% CO2. Twenty hours later, the RNA was isolated using the RNeasy Plus Mini Kit and CD4+ T cells were activated with anti- CD3/CD28- coated QiaShredder spin column (both from Qiagen, Hilden) accord- beads (one bead per cell) in X- VIVO 15. ing to the manufacturer's protocol. RNA concentration, qual- ity and integrity were measured using the NanoDrop 2000 c Spectrophotometer (Thermo Fisher Scientific Inc) and Agilent 2.6 | Flow cytometry 2100 Bioanalyzer (Agilent Technologies), respectively. RNA with RNA integrity number values above 6.0 was included. Cells were blocked in PBS with 5% FCS (for staining of Reverse transcription was performed as previously de- Jurkat cells) or human serum (for staining of CD4+ T cells) scribed with minor adjustments34; 100 ng RNA in 10 µL (H4522; Sigma- Aldrich) and 0.05% sodium azide prior to reaction and a standard curve with 1:2 fold dilution se- staining with indicated antibodies diluted in PBS with 1% ries (500- 7.8 ng). The quantitative real-time PCR (RT- FCS and 0.05% sodium azide for 30 minutes on ice. Flow qPCR) was performed as previously described34 using 18S cytometry was performed using Attune Acoustic Focusing rRNA (Hs99999901_s1), TATA- box binding protein (TBP;