Interaction of C-Type Lectin-Like Receptors Nkp65 and KACL Facilitates Dedicated Immune Recognition of Human Keratinocytes

Interaction of C-type lectin-like receptors NKp65 and KACL facilitates dedicated immune recognition of human keratinocytes

Jessica Spreua, Sabrina Kuttruffa, Veronika Stejfovaa, Kevin M. Dennehya, Birgit Schittekb, and Alexander Steinlea,c,1

aDepartment of Immunology, Institute for Cell Biology, and bDepartment of Dermatology, Eberhard-Karls-University Tübingen, 72076 Tübingen, Germany; and cInstitute for Molecular Medicine, Goethe-University Frankfurt, 60590 Frankfurt, Germany

Edited* by Wayne M. Yokoyama, Washington University School of Medicine, St. Louis, MO, and approved February 3, 2010 (received for review November 15, 2009) Many well-known immune-related C-type lectin-like receptors remain unknown, the immunobiology of these NKC-based recep- (CTLRs) such as NKG2D, CD69, and the Ly49 receptors are encoded tor-ligand systems is far from being understood. Also in humans, in the natural killer gene complex (NKC). Recently, we character- corresponding NKRP1–CLEC2 receptor–ligand pairs have ized the orphan NKC gene CLEC2A encoding for KACL, a further recently been characterized: LLT1 (encoded by CLEC2D)was member of the human CLEC2 family of CTLRs. In contrast to the identified as a ligand of the inhibitory NK receptor NKR-P1A/ other CLEC2 family members AICL, CD69, and LLT1, KACL expres- CD161 (11, 12) and another member of the human CLEC2 family, sion is mostly restricted to skin. Here we show that KACL is a non– AICL (CLEC2B), as a ligand of the activating NK receptor NKp80 disulfide-linked homodimeric surface receptor and stimulates cyto- (13). LLT1 is expressed by activated DC and B cells and was shown toxicity by human NK92MI cells. We identified the corresponding to inhibit NK cytotoxicity, whereas AICL on myeloid cells stim- activating receptor on NK92MI cells that is encoded adjacently to ulates NK cytotoxicity as well as a cross-talk between NK cells and the CLEC2A locus and binds KACL with high affinity. This CTLR, monocytes (13, 14). termed NKp65, stimulates NK cytotoxicity and release of proin- Recently, we reported that the orphan gene CLEC2A encodes flammatory cytokines upon engagement of cell-bound KACL.

a fourth member of the human CLEC2 family of NKC-encoded IMMUNOLOGY NKp65, a distant relative of the human activating NK receptor CTLR (15) that we now term KACL (keratinocyte-associated NKp80, possesses an amino-terminal hemITAM that is required C-type lectin). KACL transcripts were almost exclusively detected for NKp65-mediated cytotoxicity. Finally, we show that KACL in human skin, clearly contrasting the broad presence of tran- expression is mainly restricted to keratinocytes. Freshly isolated scripts of other CLEC2 family member in hematopoietic cells keratinocytes express KACL and are capable of stimulating (15–17). In the present study, we addressed expression and a NKp65-expressing cells in a KACL-dependent manner. Thus, we potential immune-related function of KACL. We find KACL report a unique NKC-encoded receptor-ligand system that may specifically expressed on keratinocytes stimulating NK cytotoxicity fulfill a dedicated function in the immunobiology of human skin. by engaging a hitherto unknown activating CTLR, thereby indicating that this receptor–ligand pair may specifically contribute to CLEC2 family | natural killer cells | NKRP1 receptors | skin the immunosurveillance of human skin. atural killer (NK) cells express a diverse array of activating Results Nand inhibitory receptors regulating NK activity depending on Ectopic KACL Stimulates NK Cytotoxicity. In a previous study, we “ ” their respective environmental niche (1, 2). Major inhibitory NK characterized the NKC-resident orphan gene CLEC2A encoding for – receptors are the human killer Ig like receptors, the mouse Ly49 a CTLR that we have now termed KACL. Skin-associated mRNA receptors, and CD94/NKG2A which all bind MHC class I mole- expression clearly distinguished KACL from the other human – fi cules and avert cytolysis of MHC class I pro cient cells (3). CLEC2 family members AICL, LLT, and CD69 (15). To analyze NKG2D, in contrast, is an activating NK receptor stimulating NK protein expression and function of KACL, we generated the mAb – cytotoxicity upon ligation of diverse stress-induced MHC class I OMA1 that specifically binds to soluble KACL ectodomains related molecules thereby promoting NK-mediated elimination of (rKACL) and to KACL ectopically expressed on mouse and primate – infected or malignant cells (4 6). NKG2D, CD94/NKG2A, and cell lines, but not to the KACL relatives AICL and LLT1 (Fig. 1A Ly49 molecules all are dimeric type II transmembrane receptors and Fig. S1). In a screen of a panel of human cell lines, only a few cell with a single C-type lectin-like domain (CTLD) (7) and are lines of myeloid origin specifically bound OMA1, with U937 encoded in the NKC on human chromosome 12 (7, 8). The human expressing highest levels of KACL (Fig. 1B). This is in line with our NKC contains approximately 30 genes encoding for C-type lectin- previously reported mRNA expression profile and paralleled like receptor (CTLR) expressed on NK cells, T cells, myeloid cells, fi expression of AICL on cell lines (13, 15). Next, we assessed KACL and other immune-related cells as well as some poorly de ned expression by human peripheral blood mononuclear cells (PBMCs) orphan genes. from healthy donors. As previously reported (15), no significant Similar to human killer Ig–like receptor and Ly49 receptor families, the NKC-encoded NKRP1 family comprises both inhibitory and activating NK receptors (9). However, in contrast to the Author contributions: A.S. designed research; J.S., S.K., V.S., K.M.D., B.S., and A.S. per- former, NKRP1 receptors do not engage MHC class I molecules. formed research; J.S., B.S., and A.S. analyzed data; and J.S. and A.S. wrote the paper. Rather, at least some mouse NKRP1 receptors ligate members of Conflict of interest statement: A.S. and J.S. have filed a patent application on NKp65. the CLEC2 family with NKRP1 and CLEC2 genes interspersed in a None of the other authors declare a conflict of interest. distinct subregion of the NKC establishing a unique system of *This Direct Submission article had a prearranged editor. – genetically linked C-type lectin-like receptor ligand pairs. For Data deposition: The sequence reported in this paper has been deposited in the GenBank example, the inhibitory Nkrp1d and the activating Nkrp1f receptors database (accession no. GQ398770). bind the CLEC2 family members Clr-b and Clr-g, respectively (9, 1To whom correspondence should be addressed. E-mail: [email protected]. 10). However, as expression of most mouse Clr proteins is poorly This article contains supporting information online at www.pnas.org/cgi/content/full/ characterized and ligands of other Nkrp1 receptors such as NK1.1 0913108107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0913108107 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 A B P815 COS-7 293T U937 AML01 monocytes Fig. 1. KACL is a non–disulﬁde-linked homodimeric CTLR and stimulates NK cytotoxicity. (A)MAbOMA1speciﬁcally detects ectopic KACL on mouse and primate cells. P815 stably transfected

OMA1 OMA1 with KACL (solid), but not mock-transfected P815 (dotted), were stained with OMA1. COS-7 transiently and 293T stably transfected with KACL (solid), but not the respective AICL transfectants (dot- C D E rAICL [80.7 ml] Fe rNKG2D s ted) were stained by OMA1. Shaded histograms are isotype con- aGN rKACL [83.2 ml] P [80.6 ml]

mock AICL mn mock KACL mock KACL AICL KACL trols. (B) KACL is expressed on some myeloid cell lines, but not on kD KACL + kD Chymo 36 082ecn trypsinogen 55 BSA monocytes. U937, AML01, and monocytes isolated from PBMCs [76.1 ml] [93.8 ml] 29 36 were stained with OMA1 (solid). Pretreatment of OMA1 with

abros 29 17 rKACL (dotted) reduced stainings to isotype levels (shaded). (C) ba Monomeric KACL is a glycoprotein of approximately 32 kD. Lysates reducing non-reducing of KACL-transfected COS-7 (with or without PNGase F treatment) elution volume analyzed by immunoblotting with anti–histidine-tag antibody. (D) F Lysates of COS-7 transfected with myc-tagged KACL and FLAG- 25 25 25 25 tagged AICL cDNA separated by SDS/PAGE were analyzed by P815 YT NK92MI NKL NK cells 20 P815-KACL 20 20 20 immunoblotting with anti-c-myc/anti-FLAG mAbs. KACL migrates

sisyL% 15 15 15 15 similarly to a cross-reactive protein also apparent in mock trans- 10 10 10 10 fectants. (E) Sizing of rKACL and rAICL by gel ﬁltration compared 5 5 5 5 0 0 0 0 with BSA (66 kDa), rNKG2D (41 kDa), and chymotrypsinogen (25 kDa). (F) NK92MI, but not YT, NKL, or freshly isolated NK cells, 40:1 20:1 10:1 5:1 2.5:1 1.25:1 40:1 20:1 10:1 5:1 2.5:1 1.25:1 40:1 20:1 10:1 5:1 2.5:1 1.25:1 40:1 20:1 10:1 5:1 2.5:1 1.25:1 E/T E/T E/T E/T exhibit an enhanced cytolysis of P815-KACL.

levels of KACL transcripts were detected in freshly isolated human (Fig. S3). As we failed to identify a receptor for KACL among PBMCs or in PBMCs after treatment with potent stimulatory known activating NK receptors, we considered the possibility that compounds such as phythemagglutinine or phorbol myristate ace- KACL may interact with a yet undescribed receptor encoded in the tate. Accordingly, we failed to detect surface KACL on PBMC NKC in proximity to the CLEC2A locus based on the genetic including T cells using OMA1 (Fig. S2). This was unexpected, linkage of the KACL relatives AICL and LLT1 with their respective because a previous study reported KACL (also known as PILAR) receptors. Computational analysis of the genomic region flanked by expression by activated T cells (18). Immunoblot analyses of KACL- the genes CLEC2B and CLEC2A identified putative exons transfected COS-7 revealed that KACL is a glycoprotein of encoding for a CTLD. Employing exon-spanning oligonucleotides, approximately 32 kDa (Fig. 1C). Removal of N-linked glycosylation we amplified a corresponding partial transcript from activated resulted in a protein of approximately 20 kDa well in accord with the human NK cells. Subsequently, we obtained the sequence of the predicted molecular mass of KACL monomers and the presence of complete ORF by RACE-PCR. This ORF is encoded by a gene, three potential N-glycosylation sites. In contrast to AICL, which termed KLRF2, consisting of six exons extending over 15 kb, and fi forms disul de-linked homodimers, KACL runs as a monomer positioned just approximately 3 kb telomeric from the CLEC2A D under both reducing and nonreducing conditions (Fig. 1 ). How- gene in a tail-to-tail orientation (Fig. 2A). The ORF predicted a fi ever, in gel ltration, rKACL eluted at a similar volume as did typical C-type lectin-like type II transmembrane glycoprotein of 207 ectodomains of NKG2D (rNKG2D) or AICL (rAICL) which are of amino acids sharing the key features of a CTLD, such as six con- similar molecular mass and known to occur as homodimers (Fig. served cysteines and the WIGL motif (Fig. 2C)(7).Comparative E – fi 1 ). This strongly suggested that KACL occurs as a non disul de- sequence analyses of the CTLD with CTLD of other NKC-encoded linked homodimer in contrast to AICL, LLT1, and CD69, which all human CTLR identified NKp80 as closest relative and placed this form disulfide-bonded homodimers (19). Having established that fi CTLR into a subgroup of related CTLRs comprising human KACL is a bona de cell surface CTLR, we addressed its potential NKp80 and NKR-P1A as well as the mouse Nkrp1 receptors, which immune-related function. As the KACL relatives AICL and LLT1 we suggest to term NKRP1 subfamily (Fig. 2B). We named this modulate NK cytotoxicity by engaging NK receptors NKp80 and CTLR NKp65 in view of its molecular mass and homodimeric NKR-P1A, respectively, we tested various human NK cell lines for occurrence (as detailed later), and to indicate its similarity to cytotoxicity against P815 ectopically expressing KACL. No sig- NKp80. The stalk region connecting the CTLD to the cell mem- nificantly increased cytotoxicity of YT or NKL cells toward P815- brane is fairly short in NKp65 (24 aa vs. 52 aa in NKp80), whereas KACL transfectants was observed, and NK cells isolated from the amino-terminal cytoplasmic sequence is again homologous to healthy donors did not specifically react against P815-KACL (Fig. NKp80 (Fig. 2C). Interestingly, a tyrosine at position 7 of the 1F). In contrast, NK92MI cells exhibited a pronounced cytotoxic response against P815-KACL cells, but did not lyse P815-neo con- cytoplasmic domain is present in all three receptors, including NKR-P1A. In NKp65, tyrosine 7 is embedded in a sequence iden- trol transfectants, suggesting that NK92MI cells bear an activating fi receptor stimulating NK cytotoxicity upon KACL engagement tical to the hemITAM motif described for some myeloid-speci c (Fig. 1F). CTLR such as Dectin-1 and Clec1b (21, 22). We next tested whether NKp65 is a bona fide cell surface CTLR. Surface expres- NKp65 Is a CTLR Encoded Adjacent to KACL in the NKC. To charac- sion of NKp65 was demonstrated on COS-7 cells after transfection terize the receptor on NK92MI cells specifically interacting with of a carboxy-terminally tagged NKp65 cDNA (Fig. 2D). Immuno- KACL, we assessed binding of ectodomains of known activating NK blot analysis of NKp65-transfectants revealed that NKp65 is a receptors to 293T or COS-7 cells ectopically expressing KACL (Fig. glycoprotein of approximately 32 kDa under reducing and non- 1A). Neither the ectodomains of NKC-encoded C-type lectin-like reducing conditions that was reduced to approximately 24 kDa after NK receptors such as NKp80 or NKG2D nor the ectodomains of removal of N-linked carbohydrates (Fig. 2E) in line with the pre- the Ig-like receptors NKp30, NK44, NKp46, 2B4, and DNAM-1 dicted molecular mass and two N-linked glycosylation sites flanking bound to KACL-expressing cells (Fig. S3). Further, experiments the CTLD. Purified ectodomains of NKp65 (rNKp65) produced in forcing ligand-induced down-regulation did not provide any evi- 293T cells were subjected to gel filtration and eluted at a similar dence for KACL serving as a ligand for NKp80 or NKR-P1A volume as rNKp80 (Fig. 2F), suggesting that NKp65 forms non–

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KLRB1 CLEC2D CLECL1 CD69/CLEC2C KLRF1 CLEC2B KLRF2 CLEC2A Ly49L (NKR-P1A/CD161) (LLT1) (DCAL1) (CD69) (NKp80) (AICL) (NKp65) (KACL) 650 kb 100 kb

CLEC7A B hCD69 mCd69 C

mNkg2d Clr b Clr g Fig. 2. NKp65 is an NKC-encoded CTLR distantly related to hNKG2D NKp80. (A) NKp65 is encoded in the NKC by KLRF2 ﬂanked by Clr f CLEC2A and CLEC2B. Schematic representation of a subregion of

NKR P1A the human NKC. Boxes and arrows represent CLEC2 (dark gray) LLT1 and NKRP1 genes (light gray) and transcriptional orientation,

KACL respectively. (B) Phylogenetic analysis of CTLD of NKRP1 and Nkrp1f AICL CLEC2 receptors of man and mouse. NKG2D, CLEC7A, and Ly49D

Nkrp1a are included for comparison. The phylogenetic tree was gen- Nkrp1d Nkrp1c erated using the PHYLIP program. (C) Sequences of human NKRP1 receptors NKp80, NKR-P1A, and NKp65. Tyrosine-based NKp80 Ly49d NKp65 motifs in the cytoplasmic domain are boxed and predicted

NKp80 NKp65 D E F [80ml] transmembrane domains are shaded. Conserved cysteines of the [81ml] NKp65 Chymo NKp65 PNGaseF NKp65 + BSA trypsinogen kD kD m CTLD are underlined. (D) NKp65 is a cell surface CTLR. Myc- or NKp65-myc NKp65-FLAG [93ml]

n [76ml] 50 50 08 FLAG-tagged NKp65 was transfected into COS-7 and surface

36 c expression detected by tag-speciﬁc mAb. (E) NKp65 is a glyco- 36 nab

r protein. Immunoblot analysis of lysates from transfected COS-7

osba with an anti–histidine-tag mAb. (F) Sizing of rNKp65 by gel ﬁl- anti-myc anti-FLAG 22 22 reducing non-reducing tration compared with BSA (66 kDa), rNKp80 (approximately 60 elution volume kDa), and chymotrypsinogen (25 kDa).

disulﬁde-linked homodimers, whereas NKp80 and NKR-P1A form indicative of an activating receptor. A stimulatory function was also

disulfide-linked homodimers (23, 24). noted for the putative KACL receptor on NK92MI cells (Fig. 1F). IMMUNOLOGY Hence, we analyzed NK92MI cells for NKp65 expression. Both NKp65 Is a High-Affinity Receptor for KACL. To address whether NK92MI and parental NK92 cells contained significant amounts of NKp65 is a receptor for KACL, we assessed binding of KACL NKp65 transcripts, whereas no NKp65 transcripts were detected tetramers to cells ectopically expressing NKp65. Only NKp65- for YT and NKL cells (Fig. 4A). NK92MI cells also were markedly expressing cells, but not controls or NKp80- or NKR-P1A- stained by KACL tetramers (Fig. 4B), strongly suggesting that expressing cells, were stained by KACL tetramers (Fig. 3A and NKp65 expression on NK92MI cells is accountable for the KACL- Fig. S3). We further investigated specific binding of NKp65 to specific lysis of P815-KACL cells. However, in the absence of a KACL by using the purified, biotinylated ectodomains of these suitable NKp65-specific mAb, this could not be unequivocally and related CTLR described here or elsewhere (13). In line with demonstrated. Therefore, to directly assess functional activity of results described already, KACL tetramers strongly bound to NKp65, we transduced NK92MI with tagged NKp65 (NK92MI- rNKp65 immobilized on streptavidin-coated microspheres NKp65). NKp65 was brightly expressed on transduced cells (Fig. (imNKp65), but not to imNKp80 or imNKR-P1A (Fig. 3B)at 4B), and in a redirected cytotoxicity assay, NK92MI-NKp65 lysed variance to a previous study (18). Conversely, NKp65 tetramers P815 loaded with tag-specific Ab, but not control Ig-loaded cells also bound imKACL, but not to imAICL or imLLT1 (Fig. 3C). (Fig. 4C), demonstrating that NKp65 triggers NK cytotoxicity. Further, NKp80 and NKR-P1A tetramers bound only to their To evaluate the relevance of the aminoterminal hemITAM, tyro- known ligands imAICL and imLLT1, respectively, demon- sine 7 was mutated to phenylalanine and the resulting mutant strating that there is no significant cross-reactivity between (NKp65Y7F) introduced into NK92MI. Although mutant these cognate and genetically coupled receptor-ligand pairs NKp65Y7F was expressed at similar levels as WT NKp65, and (Fig. 3C). Pretreatment of imKACL with mAb OMA1 largely NK92MI-NKp65Y7F responded to NKp44 engagement by redir- abrogated binding of NKp65 tetramers further corroborating ected lysis, no redirected cytolysis was observed when NKp65Y7F the specificity of the NKp65-KACL interaction and validating was engaged (Fig. 4D). Thus, tyrosine 7 is crucially involved in OMA1 as a suitable blocking reagent for functional studies NKp65-mediated cytotoxicity. Next, we investigated NKp65- (Fig. 3D). Pretreatment with OMA1 also completely blocked mediated cytotoxicity and cytokine secretion toward KACL- binding of NKp65 tetramers to U937 cells endogenously expressing cells. P815-KACL elicited a strong cytolytic response of expressing KACL (Fig. 3E). Subsequently, we determined the NK92MI-NKp65 cells and cytotoxicity was abrogated by pretreat- kinetics of KACL-NKp65 interaction by surface plasmon reso- ment with OMA1 (Fig. 4E). Moreover, P815-KACL stimulated nance (SPR). Soluble rNKp65 bound immobilized KACL with a IFN-γ secretion by NK92MI-NKp65 cells in a KACL-dependent 5 −1 −1 fast association rate (kon,5.8× 10 M s ) and dissociated manner, which became particularly apparent in the presence of IL- −3 −1 relatively slowly (koff,6.4× 10 s ), yielding a fairly high 12 or IL-18 (Fig. 4F). Finally, we also showed that cytolysis of U937 calculated affinity (KD,calc of approximately 11 nM at 25 °C; by NK92MI-NKp65 cells is partially KACL-dependent (Fig. 4G). Fig. 3F)thatwasconfirmed in steady-state analyses (KD of Together these data demonstrate that NKp65 is a functional acti- approximately 13 nM at 25 °C; Fig. 3G). Hence, these results vating receptor stimulating NK cytotoxicity and cytokine secretion demonstrate that NKp65 is a high affinity receptor for KACL, upon engagement of its ligand KACL. and further establish and extend the principle of genetically linked C-type lectin-like receptor–ligand pairs recruited from KACL on Keratinocytes Stimulates NKp65-Mediated Degranulation. the NKRP1 and CLEC2 subfamilies, respectively. KACL transcripts are abundantly present in human skin, but barely detectable in other tissues (Fig. 5A and ref. 15). To further NKp65 Engagement Triggers Cytotoxicity and Cytokine Secretion. pinpoint KACL expression in skin, we performed in situ hybrid- Next, we continued with the functional characterization of NKp65. ization of nondiseased human skin samples and observed a fairly The hemITAM present in the cytoplasmic domain of NKp65 was homogenous distribution of KACL transcripts throughout the

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A neo NKp65-EGFP B

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EGFP (rKACL)4

C KACL-coated beads AICL-coatedAICL- beads LLT1-coated beads D KACL-coated beads E OMA1 IgG U937 (rNKp80)4 (rNKR-P1A) (rNKp80)4 4

(rNKp65)4

(rNKR-P1A) 4

fluorescence (rNKp65)4 fluorescence F 200 G 1400 1400 RU RU R 150 1200 eq 1000 1300 100 800 1200 600 50 400 1100 200 0 0 1000 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 1 51 101 151 201 1 101 201 301 63 250 500 1000 Time (s) Time (s) rNKp65 (nM)

Fig. 3. Identification of NKp65 as a high-affinity receptor for KACL. (A) KACL interacts with NKp65. KACL tetramers bind COS-7 transfected with a bicistronic NKp65-EGFP construct (Right), but not control transfectants (Left). (B) KACL tetramers bind imNKp65 (shaded), but not imNKp80 (solid), imNKR-P1A (dashed), or uncoated microspheres (dotted). (C) NKp65 specifically binds KACL. NKp65 tetramers (shaded) bind to imKACL (Left) but not to imAICL (Center) or imLLT1 (Right). NKp80 (solid) and NKR-P1A tetramers (dotted) bind imAICL and imLLT1, respectively, but not imKACL. (D) Binding of NKp65 tetramers to imKACL was blocked by preincubation of imKACL with OMA1 (solid) but not with an isotype control (shaded). Background staining of uncoated microspheres is dotted. (E) NKp65 binds

KACL on U937. NKp65 tetramer binding to U937 is blocked by preincubation of U937 with OMA1 F(ab)2 (solid) but not with control F(ab)2 (shaded). Direct staining by OMA1 F(ab)2 (stippled) or control F(ab)2 (dotted) is also shown. (F) Kinetics of the binding of rNKp65 (8, 16, 31, 62, and 125 nM) to imKACL measured by SPR at 5 −1 −1 −3 −1 25 °C. Association rate is 5.8 × 10 M s , dissociation rate is 6.4 × 10 s , and calculated KD is 11 nM. (G)Afﬁnity of NKp65 for KACL was determined by injection of rNKp65 (63, 125, 250, 500, and 1,000 nM) over imKACL at 25 °C; KD is 13 nM. Average interval for the best ﬁt curve (Right) is indicated.

epidermis indicating KACL expression by keratinocytes (Fig. 5B). aided in the identification of AICL as a genetically linked ligand for Subsequently, we addressed surface expression of KACL on ker- the human activating NK receptor NKp80 (13). Based on this trait atinocytes isolated from nondiseased surgical skin samples. Using of genetic linkage and the relatedness of the respective CTLD OMA1, we detected marked KACL expression on freshly isolated sequences in mouse and man (Fig. 2B), we would like to suggest to keratinocytes (Fig. 5C). KACL expression by keratinocytes was group these CTLR together into distinct subfamilies (i.e., NKRP1 further corroborated by the specific and distinct binding of NKp65 and CLEC2 families). tetramers that was completely blockable by pretreatment with As our current study provides further documentation that OMA1 (Fig. 5C). Finally, we assessed whether KACL on kerati- humans, like mice, possess several of these genetically coupled nocytes can be functionally recognized by the NKp65 receptor. receptor/ligand pairs of CTLRs, it evokes several questions. First, Coculture with freshly isolated keratinocytes from various donors why are genes of receptors and ligands tightly coupled, although triggered a marked degranulation of NK92MI-NKp65 cells that there is no apparent polymorphism? In contrast to other NKC- was partially blocked by addition of OMA1 (Fig. 5 D and E). Thus, encoded CTLR such as NKG2D, CD94, or Dectin-1, there are no the specific expression of KACL by keratinocytes allows for a clear homologies between certain NKRP1 or CLEC2 genes of dedicated functional recognition of keratinocytes via the activat- mouse and man, apart from CD69. But do some of these genes ing receptor NKp65. encode for CTLR serving a similar function across species, i.e., are there functional homologues? Are there even more functional Discussion members of these CTLR subfamilies to be discovered that may have We describe here the activating immunoreceptor NKp65, the high- escaped description because of a highly restricted expression pat- affinity interaction of NKp65 with the recently reported orphan tern? Does CD69, which, like KACL, AICL, and LLT1, also is clearly CTLR KACL/CLEC2A, a highly restricted expression of KACL by a member of the CLEC2 subfamily, also engage a yet unrecognized human keratinocytes, and functional consequences of NKp65– receptor encoded in this subregion of the NKC? What is the KACL interaction. NKp65 and KACL both are non–disulfide- immunological function of these multiple receptor/ligands pairs? linked homodimeric CTLRs and are encoded by genes that are Are these receptor–ligand pairs functionally redundant or do they separated by only a few thousand basepairs in the human NKC. This exert some specialized function that has not yet been appreciated? report further establishes and extends the principle of genetically With regard to the last issue, this study reporting a tissue-specific linked C-type lectin-like receptor–ligand pairs in humans that was expression of KACL introduces another aspect into this discussion, originally reported in a seminal study by Yokoyama and colleagues raising the possibility that at least some members of the CLEC2 for certain CTLRs encoded in the mouse NKC (9). This latter study subfamily (together with their putative receptors) may fulfill a prompted the characterization of an obviously corresponding dedicated function in tissue-specific immunity. From our data pre- receptor/ligand pair in humans (i.e., NKR-P1A/LLT1) (11, 14), and sented in this study it is evident that NKp65/KACL does not simply

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Fig. 4. NKp65 triggers cytolytic activity and stimulates cytokine secretion. 5 (A) NK92 and NK92MI, but not YT or NKL, express NKp65. Real-time PCR 0 analysis of NKp65 transcripts (n.d., not detectable). (B) NKp65 expression on NK92MI. NK92MI transduced with tagged NKp65 or untransduced cells were stained for NKp65 with anti-FLAG mAb (solid) or KACL tetramers (shaded) or donor 1 donor 2 donor 3 donor 4 donor 5 isotype control (dotted). (C) NKp65 triggers redirected cytolysis. Redirected Fig. 5. KACL on keratinocytes stimulates NKp65-mediated degranulation. (A) lysis assay using P815 as targets and NK92MI-NKp65 or mock transductants as Predominance of KACL transcripts in human skin. Relative levels of KACL tran- effectors either in the presence of anti-FLAG mAb M2 or isotype control. (D) scripts in human tissues were determined by real-time PCR. (B) KACL is expressed Tyrosine 7 of NKp65 is critically involved in NKp65-mediated cytotoxicity. in the epidermis of human skin. In situ hybridization of human skin with a sense Redirected lysis assay using P815 as targets and NK92MI-NKp65 (WT) or KACL probe (control, Upper) and an antisense KACL probe (Lower). (C) Kerati- NK92MI-NKp65Y7F (Mut) as effectors in the presence of anti-NKp44 mAb, nocytes express surface KACL. Freshly isolated human keratinocytes were stained anti–histidine-tag mAb or isotype control. (E) P815-KACL, but not P815-neo, with OMA1 F(ab)2 or control F(ab)2, or with NKp65 tetramers after preincubation are lysed by NK92MI-NKp65. Lysis of P815-KACL was inhibited to background with control F(ab)2 or OMA1 F(ab)2.(D and E) KACL-mediated activation of levels in the presence of OMA1, but not in presence of an isotype control. (F) NK92MI-NKp65 by keratinocytes. Degranulating (CD107a+) NK92MI-NKp65 KACL stimulates IFN-γ secretion by NK92MI-NKp65. NK92MI-NKp65 were (NKp44+ cells) after coculture with freshly isolated keratinocytes in presence of cocultured with P815-neo or P815-KACL in presence or absence of OMA1, IL- OMA1 or isotype control. (D) One representative dot plot analysis and (E)sum- 12, or IL-18, and subsequently, IFN-γ secretion was measured by ELISA. (G) mary of ﬁve independent experiments using keratinocytes of different donors. KACL-dependent lysis of U937. Cytolysis of U937 in presence of OMA1 or isotype control by NK92MI-NKp65.

receptors (21, 22). Tyrosine 7 of this hemITAM is essential for represent “just another” receptor–ligand pair involved in NK cell NKp65-stimulated cytotoxicity and it will be addressed in upcoming activation. Rather, KACL is clearly distinct by its skin-specific studies whether NKp65 signals through Syk kinase as reported for expression from other CLEC2 family members that are broadly other hemITAM-bearing receptors (22). Interestingly, tyrosine 7 is expressed by hematopoietic cells. Further, both NKp65 and KACL also shared by NKp80 and NKR-P1A, likewise suggesting a crucial are non–disulfide-linked homodimers and thereby differ from involvement of tyrosine 7 in signaling of these latter receptors. other members of the human NKRP1 and CLEC2 subfamilies At present, our knowledge on the physiological expression of including NKp80, NKR-P1A, AICL, LLT1, as well as CD69, all NKp65 is rather limited. We isolated the full-length NKp65 cDNA being disulfide-linked homodimers (19, 20, 23, 24). It remains to be from primary NK cells, but in contrast to NK92MI cells, human addressed whether this trait is related to a particular function or a peripheral blood NK cells contain only low levels of NKp65 tran- skin-associated function of this receptor–ligand pair. Noteworthy is scripts, suggesting that NKp65 is barely expressed on blood NK cells also the strong affinity of the NKp65–KACL interaction (KD of (Fig. S4). Accordingly, we did not obtain evidence for NKp65 sur- approximately 0.01 μM at 25 °C) which is considerably higher face expression on freshly isolated or IL-2/IL-12–stimulated (approximately 400-fold) than the NKp80–AICL interaction (KD of peripheral blood NK cells by staining with KACL tetramers (Fig. approximately 4.1 μM at 25 °C), the only CTLR–CTLR interaction S4). Taking into consideration that KACL expression is restricted to for which affinity data are available (13). Further, to the best of our skin, in contrast to other CLEC2 family members, one may speculate knowledge, NKp65 is the first NK-associated receptor that contains that expression of NKp65 likewise is restricted to a subset of skin- a so-called hemITAM previously reported for myeloid-specific associated lymphocytes. For example, a subset of CD4 memory T

Spreu et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 cells has recently been reported which is prone to home to the skin, streptavidin-coated microspheres (Bangs Laboratories) or tetramerized using PE- produces IL-22, and is found in lesions of psoriatic skin (25, 26). It labeled streptavidin (Molecular Probes) (13). KACL-specificmAbOMA1was will be of immediate interest to address expression and function of generated by standard hybridoma technology (28). In brief, BALB/c mice were fi NKp65 on these T cells and on skin-associated NK cells (27) when immunized with P815-KACL and resulting hybridoma screened for speci creac- ’ NKp65-specific mAb become available. At any rate, our findings tivity against COS-7 expressing a KACL-pIRES-DsRed construct. F(ab )2 fragments demonstrate that NKp65 is an activating receptor capable of were generated by pepsin digestion (Pierce). mediating the dedicated immunorecognition of keratinocytes via SPR. Using a BIAcore X apparatus (BIAcore) KACL ectodomains were immo- engaging the genetically linked CTLR KACL. fl μ fi fi bilized to CM5 chips by amine coupling. In kinetic analyses ( ow rate, 50 L/ In summary, we nd KACL speci cally expressed on human min), RU from the control flow cell (imAICL) were subtracted from response keratinocytes and detected by a previously unknown genetically units (RU) of the KACL-derivatized surface (black traces) with overlaid gray linked activating CTLR capable of stimulating cytotoxicity and traces representing fitting of a 1:1 Langmuir model to the association and cytokine secretion via a hemITAM. Considering the particular dissociation phases. In steady-state analyzes (15 μL/min), RU from the KACL- immunobiology of human skin, it will be of considerable interest derivatized surface were corrected by RU from the LLT1-derivatized control to address functional implications of this unique NKC-encoded cell. Raw data were analyzed and illustrated using BIAevaluation software receptor–ligand pair in skin-affecting diseases such as psoriasis (BIAcore). and graft-versus-host disease, as well as in wound healing. Cytotoxicity, Degranulation, and Cytokine Analyses. Direct cytotoxicity of NK Materials and Methods cells as well as redirected lysis of P815I was assessed in a standard 51Cr release μ Cells. Surgical samples of human skin were obtained from the Dermatology assay (28). In redirected lysis assays, P815 were loaded with 5 g/mL antihistidine μ μ Department of the University of Tübingen with approval of the local ethics (Qiagen), 2 g/mL anti-NKp44 (R&D Systems), or 5 g/mL anti-FLAG. In blocking fi committee. After removing the cutaneous fat layer, skin pieces were incu- experiments, antibodies (OMA1, isotype) were used at a nal concentration of bated for 16 h at 37 °C in trypsin solution. Subsequently, the epidermal layer 10 μg/mL Degranulating NK92MI-NKp65 were quantified by analysis of surface was separated from the dermis and resuspended keratinocytes filtered CD107a after overnight coculture with freshly isolated keratinocytes in presence through a cell strainer before used in experiments. Human NK cell lines NKL, of mAb OMA1 or an isotype control (10 μg/mL). Golgi-Stop (BD Biosciences) was YT, and NK92MI (ATCC) and freshly isolated human NK cells were used in NK added for the last 4 h. IFN-γ secreted by NK92MI-NKp65 after 16-h coculture with effector assays. NK92MI cells were transduced with retroviruses generated P815 transfectants was quantified using a sandwich ELISA (BD Biosciences). by transfection of phoenix-ampho cells (Nolan Laboratory, Stanford, CA) Assays were performed in presence or absence of blocking anti-KACL mAb with the vector pMXsIP kindly provided by Toshio Kitamura (Tokyo, Japan) OMA1 (10 μg/mL), IL-12 (1 ng/mL), or IL-18 (10 ng/mL), respectively. All samples and containing cDNA of NKp65 or NKp65Y7F with carboxy-terminal FLAG- were done in triplicates. and six-histidine tags. Transduced cells were selected in puromycin- containing medium. In Situ Hybridization. In situ hybridization was performed on sections of paraffin-embedded skin with digoxigenin-labeled RNA probes generated by Recombinant Proteins and Antibodies. The complete NKp65 cDNA was isolated in vitro transcription of KACL full-length cDNA in pBluescript using DIG RNA from IL-2/IL-12–stimulated human NK cells by a 5′RACE kit (Invitrogen) according labeling kit (Roche) and T7 (sense) and T3 polymerases (antisense), respec- to the manufacturer’s instruction (accession no. GQ398770). Soluble recombinant tively. Tissues were surgical samples of nondiseased human skin. For more fi rKACL (Ile-46 through Leu185) and rNKp65 (Ser63 through Val207) were puri ed details, see SI Materials and Methods. from supernatants of 293T stably transfected with the respective cDNA containing BirA, c-myc, and six-histidine tags as previously reported for rNKp80, rNKR- ACKNOWLEDGMENTS. We thank Birgit Sauer, Wiebke Ruschmeier, and fi P1A, rAICL, and rLLT1 (13). For gel ltration analysis, rCTLR were loaded onto a Beate Pömmerl for excellent technical assistance. This work was supported Superdex 200 gel filtration column at a flow rate of 0.5 mL/min. Ectodomains by Grants SFB 685/A1 and STE 828/5-1 from the Deutsche Forschungsgemein- were biotinylated using BirA ligase and, before use, either immobilized on schaft (to A.S.).

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