CLEC12A-Mediated Antigen Uptake and Cross-Presentation by Human

CLEC12A-Mediated Antigen Uptake and Cross-Presentation by Human Dendritic Cell Subsets Efficiently Boost Tumor-Reactive T Cell Responses This information is current as of September 29, 2021. Tim J. A. Hutten, Soley Thordardottir, Hanny Fredrix, Lisanne Janssen, Rob Woestenenk, Jurjen Tel, Ben Joosten, Alessandra Cambi, Mirjam H. M. Heemskerk, Gerben M. Franssen, Otto C. Boerman, Lex B. H. Bakker, Joop H. Jansen, Nicolaas Schaap, Harry Dolstra and Willemijn Hobo Downloaded from J Immunol published online 26 August 2016 http://www.jimmunol.org/content/early/2016/08/26/jimmun ol.1600011 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 26, 2016, doi:10.4049/jimmunol.1600011 The Journal of Immunology

CLEC12A-Mediated Antigen Uptake and Cross-Presentation by Human Dendritic Cell Subsets Efﬁciently Boost Tumor-Reactive T Cell Responses

Tim J. A. Hutten,*,1 Soley Thordardottir,*,1 Hanny Fredrix,* Lisanne Janssen,* Rob Woestenenk,* Jurjen Tel,† Ben Joosten,† Alessandra Cambi,† Mirjam H. M. Heemskerk,‡ Gerben M. Franssen,x Otto C. Boerman,x Lex B. H. Bakker,{ Joop H. Jansen,* Nicolaas Schaap,‖ Harry Dolstra,*,1 and Willemijn Hobo*,1

Potent immunotherapies are urgently needed to boost antitumor immunity and control disease in cancer patients. As dendritic cells (DCs) are the most powerful APCs, they are an attractive means to reinvigorate T cell responses. An appealing strategy to use the Downloaded from effective Ag processing and presentation machinery, T cell stimulation and cross-talk capacity of natural DC subsets is in vivo tumor Ag delivery. In this context, endocytic C-type lectin receptors are attractive targeting molecules. In this study, we investigated whether CLEC12A efficiently delivers tumor Ags into human DC subsets, facilitating effective induction of CD4+ and CD8+ T cell responses. We confirmed that CLEC12A is selectively expressed by myeloid cells, including the myeloid DC subset (mDCs) and the plasmacytoid DC subset (pDCs). Moreover, we demonstrated that these DC subsets efficiently internalize CLEC12A, whereupon it quickly translocates to the early endosomes and subsequently routes to the lysosomes. Notably, CLEC12A Ab http://www.jimmunol.org/ targeting did not negatively affect DC maturation or function. Furthermore, CLEC12A-mediated delivery of keyhole limpet hemocyanin resulted in enhanced proliferation and cytokine secretion by keyhole limpet hemocyanin–experienced CD4+ T cells. Most importantly, CLEC12A-targeted delivery of HA-1 long peptide resulted in efficient Ag cross-presentation by mDCs and pDCs, leading to strong ex vivo activation of HA-1–specific CD8+ T cells of patients after allogeneic stem cell transplantation. Collectively, these data indicate that CLEC12A is an effective new candidate with great potential for in vivo Ag delivery into mDCs and pDCs, thereby using the specialized functions and cross-talk capacity of these DC subsets to boost tumor-reactive T cell immunity in cancer patients. The Journal of Immunology, 2016, 197: 000–000.

he prominent role of dendritic cells (DCs) in orchestrating vaccination of patients treated with allogeneic stem cell trans- by guest on September 29, 2021 immune responses has provided the rationale for the de- plantation (alloSCT) (8–11). In this context, T cells could be di- T velopment of DC-based strategies to boost antitumor rected against tumor Ags or recipient-speciﬁc alloantigens immune responses in cancer patients (1). DCs are the most pow- restricted to hematological tumor cells, known as minor histo- erful APCs and efﬁciently initiate and reactivate CD4+ and CD8+ compatibility Ags (MiHAs) (12, 13). T cell responses (2). Importantly, they have the unique capacity to Nevertheless, we and others have observed that often the in- cross-present extracellular Ags, including tumor Ags, in MHC duction and/or reactivation of antitumor immune responses is class I molecules to CD8+ T cells (3, 4). Upon tumor Ag pre- inadequate, contributing to disease progression or relapse (14). sentation and costimulation by the DCs, tumor-reactive T cells This observation illustrates the urgency to develop alternative become activated, expand, and attack tumor cells. Moreover, DC-based strategies to more effectively boost antitumor immu- long-lasting memory against recurrent disease is formed. Impor- nity. One such strategy involves the exploitation of the powerful tantly, tumor regression has been observed following DC-based Ag presentation capacity of natural DC subsets by in vivo tumor vaccination in patients with hematological cancers (5–7). In ad- Ag delivery. Two key populations of human DCs are myeloid DCs dition, productive T cell responses could be boosted by DC (mDCs) and plasmacytoid DCs (pDCs) (15). mDCs are further

*Department of Laboratory Medicine, Laboratory of Hematology, Radboud University T.J.A.H. and S.T. designed and performed experiments and wrote the paper; H.F., L.J., Medical Center, 6500 HB Nijmegen, the Netherlands; †Department of Tumor Immu- and R.W. performed experiments; J.T., B.J., A.C., M.H.M.H., G.M.F., O.C.B., and nology, Radboud University Medical Center, Radboud Institute for Molecular Life L.B.H.B. provided essential materials and advice; J.H.J. and N.S. provided advice Sciences, 6500 HB Nijmegen, the Netherlands; ‡Department of Hematology, Leiden and revised the paper; H.D. and W.H. designed research and wrote the paper. University Medical Center, 2300 RC Leiden, the Netherlands; xDepartment of Nuclear Address correspondence and reprint requests to Dr. Willemijn Hobo, Department of Medicine, Radboud University Medical Center, 6500 HB Nijmegen, the Netherlands; { ‖ Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Cen- Merus N.V., 3584 CH Utrecht, the Netherlands; and Department of Hematology, ter, Geert Grooteplein 8, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands. E-mail Radboud University Medical Center, 6500 HB Nijmegen, the Netherlands address: [email protected] 1T.J.A.H., S.T., H.D., and W.H. contributed equally to this work. The online version of this article contains supplemental material. ORCIDs: 0000-0002-5353-1740 (T.J.A.H.); 0000-0001-5097-7272 (L.J.); 0000-0002- Abbreviations used in this article: alloSCT, allogeneic stem cell transplantation; 7213-3422 (J.T.); 0000-0002-7192-4062 (B.J.); 0000-0001-6320-9133 (M.H.M.H.); CLEC12A, C-type lectin domain family 12 member A; CLR, C-type lectin receptor; 0000-0001-6832-101X (O.C.B.); 0000-0003-4343-3466 (L.B.H.B.); 0000-0001-7696- DC, dendritic cell; hIgG, human IgG; HS, human serum; KLH, keyhole limpet 4752 (N.S.); 0000-0002-8206-8185 (W.H.). hemocyanin; LUMC, Leiden University Medical Center; mDC, myeloid DC; mIgG2, Received for publication January 4, 2016. Accepted for publication July 31, 2016. mouse IgG2; MiHA, minor histocompatibility Ag; MoDC, monocyte-derived DC; pDC, plasmacytoid DC; Poly(I:C), polyinosinic-polycytidylic acid. This work was supported by a grant from the Dutch Cancer Society (Grant KUN 2012-5410). Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600011 2 CLEC12A-MEDIATED Ag CROSS-PRESENTATION BY DCs subdivided based on surface expression of BDCA1+ (CD1c) and after alloSCT (9, 10). Prevaccination leukapheresis material was used to + + BDCA3+ (CD141). Each DC subset has its specialized function in isolate BDCA1 mDCs, BDCA3 mDCs, pDCs, and culture MoDCs. All the induction and maintenance of immune responses (16). Nota- material of patients and healthy donors was obtained after written in- formed consent, according to institutional guidelines. bly, preclinical studies indicate that simultaneous engagement and cross-talk of multiple DC subsets is important for generating more DC isolation and culture potent and broader antitumor responses (17–19). To generate MoDCs, monocytes were isolated from PBMCs via plastic Attractive cell surface molecules for Ab-mediated in vivo tumor adherence in tissue culture flasks (Greiner Bio-One, Alphen a/d Rijn, the Ag delivery into DC subsets are the endocytic C-type lectin receptors Netherlands) and cultured in X-VIVO 15 medium (Lonza, Verviers, Belgium) (CLRs) (20–25). CLRs are pattern-recognition receptors that me- supplemented with 2% human serum (HS) (PAA laboratories, Pasching, Austria), 500 U/ml IL-4 and 800 U/ml GM-CSF (both from Immunotools, diate recognition and uptake of pathogens, as well as Ags exposed Friesoythe, Germany). At day 3, immature MoDCs were harvested and di- or released upon cell death. Furthermore, their ligation can induce rectly used in experiments or routinely matured for 48 h with 500 U/ml IL-4, intracellular signaling pathways regulating DC function. In this 800 U/ml GM-CSF, 5 ng/ml IL-1b, 15 ng/ml IL-6, 20 ng/ml TNF-a (all Immunotools), and 2.5 mg/ml PGE2 (conventional cytokines; Pfizer). study, we investigated the potential of a relatively unexplored CLR, + + C-type lectin domain family 12 member A (CLEC12A), as Ag PBMCs containing naturally occurring BDCA1 mDCs, BDCA3 mDCs, and pDCs were labeled with fluorophore-conjugated aCD11c, aBDCA1, delivery receptor. CLEC12A is also known as MICL (myeloid in- aBDCA2, aBDCA3, and aCD123 (all from BioLegend, San Diego, CA). hibitory C-type lectin-like receptor), CLL-1 (C-type lectin-like Subsequently, CD11c+BDCA1+ mDCs, CD11c+BDCA3+ mDCs, and molecule-1), DCAL-2 (dendritic cell–associated C-type lectin 2), CD123+BDCA2+ pDCs (Supplemental Fig. 1A) were sorted using the ARIA . + and CD371. Notably, CLEC12A is an attractive candidate, as it SORP (Becton Dickinson, Franklin Lakes, NJ) with 95% purity. BDCA1 and BDCA3+ mDCs were matured with 800 U/ml GM-CSF, 10 mg/ml is broadly expressed by all human DC subsets (26). The function polyinosinic-polycytidylic acid [Poly(I:C)] (Sigma-Aldrich), and 5 mg/ml Downloaded from of CLEC12A has not been fully elucidated, although recently R848 (Enzo Life Sciences, Raamsdonkveer, the Netherlands), and pDCs Neumann et al. (27) found CLEC12A to be involved in the control with 10 ng/ml IL-3 (Immunotools) and 5 mg/ml R848. of sterile inflammation by neutrophils. They identified that Expression and internalization CLEC12A binding to uric acid crystals, released by dying cells, inhibits neutrophil-derived reactive oxidant species production, CLEC12A and DEC205 expression and their internalization capacity were analyzed by flow cytometry. Healthy donor PBMCs or day 3 MoDCs were thereby reducing infiltration of immune cells to the site of damage. http://www.jimmunol.org/ incubated for 30 min with 10 mg/ml aCLEC12A (clone 687317; R&D sys- More recently, CLEC12A was reported to be involved in microbial tems, Minneapolis, MN) or mouse IgG2b (mIgG2b) isotype control (Bio- defense in myeloid cells, in particular in bacterial autophagy (28). Legend) at 4˚C. Subsequently, cells were incubated for 0–90 min at 37˚C to It was postulated that CLEC12A is involved early during bacte- allow endocytosis. Then, goat anti-mouse Alexa Fluor 647 (Life Technologies, rial autophagy at the level of pathogen recognition initiated Carlsbad, CA) was added. To inhibit clathrin-mediated endocytosis, cells were incubated in hypertonic 450 mM sucrose solution (Boom, Meppel, the Neth- upon membrane damage. Notably, Chen et al. (29) demonstrated erlands) (32). Blockade of clathrin-mediated uptake was reversed by sucrose that targeting of human monocyte-derived DCs (MoDCs) with washout and resuspension in PBS. Subsequently, cells were labeled with aCLEC12A Ab modulated TLR-mediated maturation and cyto- fluorophore-conjugated aBDCA1, aBDCA2, aBDCA3, aCD123, and kine secretion. In addition, Lahoud et al. (30, 31) have proposed aCD11c (Miltenyi Biotec, Bergisch Gladbach, Germany; BD or BioLegend).

DCs were analyzed on a CyAn ADP or a Gallios flow cytometer (both by guest on September 29, 2021 CLEC12A as a potential target for specific Ag delivery. They + + + + + + Beckman Coulter) and gated as CD11c BDCA1 mDCs, CD11c BDCA3 showed in vivo induction of OVA-specific CD4 and CD8 T cell mDCs, and BDCA2+CD123+ pDCs (Supplemental Fig. 1B). Data were ana- responses after administration of OVA-conjugated aCLEC12A Ab lyzed with Kaluza V1.3 (Beckman Coulter). in mouse models. Yet, little is still known about CLEC12A’s internalization mechanism, intracellular routing and fate, and, most Intracellular routing importantly, whether CLEC12A can efficiently facilitate Ag cross- To study CLEC12A internalization and investigate its intracellular routing, presentation by human DC subsets. Our aim was to acquire insight day 3 MoDCs were adhered to RetroNectin-coated (Takara, Otsu, Japan) into these mechanisms and to explore CLEC12A’s potential for coverslips. After blocking Fc receptors with total human IgG (hIgG) + + (Sanquin) and goat serum (CDL, Nijmegen, the Netherlands), MoDCs were targeted Ag delivery into human DCs to promote CD4 and CD8 incubated for 30 min with 10 mg/ml aCLEC12A (mIgG2b, clone 687317; T cell responses. In this study, we validated the discriminative ex- R&D systems) or aDEC205 (mIgG2b, clone MG38; eBioscience) at 4˚C. pression pattern of CLEC12A, and showed that it is efficiently in- Subsequently, cells were labeled with Alexa Fluor 488–conjugated goat anti- ternalized by human BDCA1+ mDCs, as well as BDCA3+ mDCs mouse IgG2b (Life Technologies) and DCs were incubated for 0–60 min at 37˚C. Next, cells were fixated with 4% paraformaldehyde and incubated with and pDCs, upon targeting with specific Abs. We observed that aHLA-DR/DP (mIgG2a, clone Q5/13). Thereafter, HLA-DR/DP staining CLEC12A initially translocates to the endosomes, whereupon it was visualized using Alexa Fluor 564–conjugated goat anti-mouse IgG2a quickly routes to the lysosomes. Notably, CLEC12A Ab binding did (Life Technologies). Then, after permeabilization, aEEA-1 (rabbit IgG; not negatively affect DC phenotype or allogeneic T cell stimulation Abcam, Cambridge, U.K.) or aLAMP-1 (rabbit IgG; Sigma-Aldrich, St. capacity. Importantly, we showed that Ags delivered via CLEC12A Louis, MO) was added for 30 min, followed by staining with Alexa Fluor 647–conjugated goat anti-rabbit IgG (polyclonal; Life Technologies). Cov- are efficiently processed by DCs and (cross-)presented to Ag- erslips were mounted on slides with Mowiol (Sigma-Aldrich). Images were experienced T cells. These findings demonstrate that CLEC12A is acquired with an Olympus FV1000 Confocal laser scanning microscope an appealing targeting receptor for selective in vivo delivery of (Olympus America, Melville, NY) or an LSM 510 META confocal micro- tumor Ags into mDC and pDC subsets to boost tumor-reactive scope (Zeiss, Jena, Germany). Isotype controls mIgG2b, mIgG2a, and rabbit IgG (BioLegend, Miltenyi Biotec, and Jackson ImmunoResearch Labora- immune responses in cancer patients. tories, respectively) did not show fluorescent staining. Pictures were analyzed with Image J V1.46R and quantified with JACoP plugin V2.1.1. Materials and Methods DC phenotype and function upon CLEC12A Ab binding Patient and donor material Day 3 MoDCs were preincubated with 10 mg/ml aCLEC12A (hIgG1) or PBMCs were isolated from healthy donor buffy coats (Sanquin, Nijmegen, hIgG1 isotype control (Merus N.V., Utrecht, the Netherlands) and matured the Netherlands) or from leukapheresis products using Ficoll-Hypaque with conventional cytokines; 1 mg/ml LPS (Sigma Aldrich); and 10 mg/ml density centrifugation. PBMCs containing keyhole limpet hemocyanin Poly(I:C) or 5 mg/ml R848. After 48 h, IL-6, IL-10, and TNF-a concen- (KLH)–specific T cells were obtained from multiple myeloma patients trations were quantified by ELISA according to the manufacturer’s following vaccination with KLH-pulsed tumor Ag-presenting DCs. instructions [Human IL-6 and IL-10 ELISA Ready-SET-Go! Kit PBMCs containing HA-1–specific T cells were obtained from patients (eBioscience, San Diego, CA); PeliPair human TNF-a ELISA reagent set The Journal of Immunology 3

(Sanquin)]. MoDC phenotype was analyzed by flow cytometry using the 3-h incubation at 37˚C, maturation stimuli and 10% HS were added and DCs following: aCD14, aCCR7, aCD80, aCD86, aHLA-ABC (all Bio- were cultured overnight. Thereafter, DCs were cocultured for 7 d with CD14- Legend), aHLA-DR (Beckman Coulter), aCD83 (BD), and appropriate depleted PBMCs containing HA-1–specific memory T cells obtained from isotype controls (all BioLegend). Cells were analyzed using an FC500 flow HA-1–mismatched patients after alloSCT, at a ratio of PBL:DC of 1:0.2 in a cytometer (Beckman Coulter). For allogeneic MLR, T cells were prepared total volume of 1 ml. After 4 d, 1 ml IMDM supplemented with 10% HS, by depleting healthy donor PBMCs of CD14+ and CD19+ cells with 100 U/ml IL-2, and 10 ng/ml IL-15 (Immunotools) was added. At day 7, cells magnetic beads (BD), and were subsequently labeled with 1.25 mM CFSE were harvested and counted, and the percentage of HA-1–specific CD8+ (Life Technologies) (33). Next, 1 3 105 T cell–enriched leukocytes and T cells was determined by staining with aCD3 (BioLegend), aCD8 (Beck- 1 3 104 mature MoDCs were seeded in 200 ml IMDM (Life Technologies) man Coulter), HA-1 PE, and allophycocyanin tetramers (both from LUMC). supplemented with 10% HS in round-bottom 96-well plates (Corning Cells were analyzed on a Gallios flow cytometer. Costar) in triplicate or sixplicate. After 5 d of coculture, supernatant was collected for IFN-g ELISA (Pierce Endogen, Rockford, IL). Furthermore, Statistical analysis a a cells were harvested; stained with CD4, CD3 (both BioLegend), and Data were analyzed using one-way ANOVA followed by a Bonferroni post a CD8 (Life Technologies); and subsequently analyzed for CFSE dilution hoc test. The p values ,0.05 were considered significant. within CD3+CD4+ and CD3+CD8+ T cells using a Gallios flow cytometer. KLH-specific T cell activation Results CLEC12A is broadly expressed and efficiently internalized by Day 3 MoDCs were generated, and BDCA1+ mDCs and pDCs were isolated from leukapheresis material. DCs were incubated for 30 min at 4˚C with human DC subsets total hIgG (Sanquin) in the presence of 10 mg/ml aCLEC12A (mIgG2b or To study the suitability of CLEC12A as a targeting receptor for Ag biotinylated hIgG1) or isotype control Ab. Then, in the case of mouse Abs, secondary labeling with 10 mg/ml biotinylated goat anti-mouse IgG (Life delivery, we first validated its expression on human DC subsets Downloaded from Technologies) was performed for 30 min at 4˚C. Subsequently, DCs were using flow cytometry. We confirmed that MoDCs and naturally washed and 2 mg/ml KLH-conjugated abiotin Ab (21) was added for 30 min occurring BDCA1+ mDCs, BDCA3+ mDCs, and pDCs highly at 4˚C. Finally, cells were washed and MoDCs were matured for 48 h, mDCs expressed CLEC12A (Fig. 1A, Supplemental Fig. 2A). Because and pDCs for 24 h. Next, patients’ PBMCs containing KLH-specific T cells expression of CLEC12A was progressively lost during differenti- were thawed, depleted from monocytes using CD14 IMag beads (BD), labeled with 1.25 mM CFSE, and resuspended in IMDM/10% HS to a con- ation of MoDCs (Supplemental Fig. 2B), we selected day 3 MoDCs centration of 0.5 3 106 cells/ml. These were added to the DCs at a PBL:DC for further experiments. ratio of 1:0.1 in 96-well round-bottom plates (in triplicate). Cells were Next, we investigated the CLEC12A internalization potential of http://www.jimmunol.org/ cocultured in a volume of 200 ml at 37˚C. After 4 d, supernatant was har- day 3 MoDCs, as well as BDCA1+ and BDCA3+ mDCs and pDCs, vested and pooled for analysis of IFN-g production by cytokine bead array (BD), and CD4+ T cell proliferation was evaluated, as described before. using flow cytometry. Following incubation at 37˚C, CLEC12A was quickly and efficiently internalized by all DC types (Fig. 1B). Al- HA-1 cross-presentation assay though CLEC12A expression was lower on pDCs than on mDCs, To evaluate receptor-mediated Ag cross-presentation, the synthetic long their CLEC12A internalization efficacy was similar. These findings peptide for the MiHA HA-1 [azido propionic acid-KLKECVLHDDL- were confirmed by confocal microscopy. At the start, CLEC12A LEARRPRAHE-biotin; Leiden University Medical Center (LUMC), the was located on the plasma membrane and costained with MHC-II Netherlands] was conjugated to aCLEC12A (hIgG1), aDEC205 (mIgG2b, molecules (Fig. 1C). Notably, after 1-h incubation at 37˚C, all DC clone MG38; BD), or respective isotype controls (hIgG1 and mIgG2b), using by guest on September 29, 2021 the copper-free click reaction (34). In short, a highly reactive succinimidyl- types showed clear clusters of CLEC12A in intracellular compart- cyclooctyne variant, BCN—2,5-dioxopyrrolidin-1-yl4-((((1R,8S,9s)-bicyclo ments, with hardly any CLEC12A expression on the cell surface. [6.1.0]non-4-yn-9-ylmethoxy)carbonyl)amino) butanoate—(Synaffix, Oss, These data demonstrate that human BDCA1+ mDCs, BDCA3+ the Netherlands) was incubated with the corresponding Ab at a 15 M excess, mDCs, pDCs, and day 3 MoDCs highly express CLEC12A and following the manufacturer’s instructions. Thereafter, the BCN-conjugated efficiently internalize this CLR following Ab targeting. Ab was incubated overnight with a 3 M excess of HA-1 long peptide and purified using a 10-kDa dialysis cassette (Thermo Scientific). Conjugation Clathrin-independent CLEC12A translocation to the efficiency was determined with ELISA. Furthermore, binding capacity and endosomal/lysosomal compartments specificity of the HA-1–conjugated aCLEC12a and aDEC205 Abs, as compared with the isotype controls, were validated by flow cytometry by Intracellular fate and Ag release are essential parameters determining staining the biotin tag on the HA-1 long peptide with abiotin-FITC. DC theefficacyofAgprocessingandpresentationbyDCs.Therefore,we subsets, isolated from leukapheresis material of HA-1–negative healthy donors, were preincubated for 30 min at 4˚C with 1 mg/ml total hIgG (Sanquin) investigated the internalization mechanism and intracellular routing of to block Fc receptors, followed by 30-min incubation at 4˚C with 10 mg/ml CLEC12A. The classical pathway for receptor-mediated endocytosis aCLEC12A/HA-1, aDEC205/HA-1, or corresponding isotype/HA-1 conju- is dependent on clathrin-coated pits (36). The CLR DEC205 is known gates. Furthermore, as an additional control, DCs were labeled with to internalize via this classical route. To evaluate whether the same aCLEC12A/HA-1 or aDEC205/HA-1 conjugate in the presence of endocytosis mechanism applies to CLEC12A, we disrupted clathrin- 100 mg/ml excess unconjugated aCLEC12A or aDEC205 Ab to demonstrate receptor-specific uptake. As the peptide conjugation efficiency slightly dif- coated pits using hypertonic solution (32). Interference with clathrin- fered with the corresponding isotype control Abs, the aCLEC12A or mediated uptake almost completely blocked DEC205 internalization aDEC205 block with excess unconjugated Ab was used in all cross- by day 3 MoDCs, as well as BDCA1+ mDCs and pDCs (Fig. 2A), presentation assays. Subsequently, DCs were washed and incubated at a whereas CLEC12A endocytosis was not affected. By means of su- concentration of 0.1 3 106 cells/ml in IMDM in 96-well round-bottom plates at 37˚C (in triplicate). As positive control, DCs were pulsed with 1 mMHA-1 crose washout, we could restore DEC205 internalization, indicating short peptide (VLHDDLLEA). After 3 h-incubation, maturation stimuli and that the observed effects are the result of the hypertonic milieu. 10% HS were added, and DCs were incubated overnight. Thereafter, DCs Next, we studied the intracellular routing of CLEC12A, in were cocultured overnight with HA-1 TCR-transduced T cells (35) at a T:DC comparison with DEC205, in time on day 3 MoDCs by analyzing ratio of 1:1 in a volume of 200 ml in the presence of aCD107a (BD). Then, their colocalization with EEA-1, an early endosome marker, or supernatant was harvested for IFN-g ELISA, and T cells were stained with aCD8 (Beckman Coulter) and aCD137 (BioLegend) and analyzed for ac- LAMP-1, a marker of the late endosomal/lysosomal compartment. tivation using a Gallios flow cytometer. At the start, both DEC205 and CLEC12A were localized on the cell In the Ag recall assay using primary patient T cells, BDCA1+ mDCs and membrane, yet already after 5 min of endocytosis, colocalization was pDCs were labeled with the aCLEC12A/HA-1 conjugate, as described be- observed with the early endosomes (Fig. 2B, Supplemental Fig. 3). fore, and plated at a concentration of 0.8 3 106 cells/ml in IMDM in a 24-well plate. Owing to limited availability of material from patients, these Notably, whereas DEC205 colocalization with the early endosomes experiments were performed only with the aCLEC12A/HA-1 conjugate in was lost after 15 min, CLEC12A still colocalized with EEA-1. the presence or absence of excess unconjugated Ab as negative control. After Gradually, both receptors routed to the lysosomes with clear 4 CLEC12A-MEDIATED Ag CROSS-PRESENTATION BY DCs

FIGURE 1. CLEC12A is highly expressed and efficiently internalized by human DC subsets. (A) CLEC12A expression on day 3 MoDCs and BDCA1+ mDCs, BDCA3+ mDCs, and pDCs was analyzed by flow cytometry. Open black histograms correspond to cells stained with CLEC12A, whereas filled gray histograms represent the isotype control. Numbers in the plots represent the mean fluorescence intensity. Data of one representative donor of three are shown. (B)Day3MoDCs,mDCs,andpDCswerelabeled Downloaded from with aCLEC12A, followed by 0- to 90-min incubation at 37˚C. Surface expression of bound aCLEC12A was analyzed by flow cytometry following labeling with goat anti- mouse IgG. CLEC12A internalization is calculated relative to the surface expression at the start based on the mean fluorescence intensity. Data are shown as mean 6 SEM of http://www.jimmunol.org/ three independent experiments. (C) Confocal analysis of CLEC12A internalization in day 3 MoDCs and FACS- sorted BDCA1+ mDCs and pDCs. DCs were stained with mouse aCLEC12A, followed by labeling with Alexa Fluor 488–conjugated goat anti-mouse IgG2b (green). Then, internalization was allowed for 1 h at 37˚C, with subsequent staining for extracellular MHC class II with mouse anti-human HLA-DR/DP, followed by Alexa Fluor 564– conjugated goat anti-mouse IgG2a (red). Scale bar length is shown in the figure. by guest on September 29, 2021

colocalization at 60 min after internalization. These colocalization 48-h incubation of aCLEC12A-labeled MoDCs in the presence of patterns were confirmed with the Manders coefficient (Fig. 2C–F). conventional cytokines, LPS, Poly(I:C), or R848 did not influence Together, these data demonstrate that, in contrast to DEC205, expression levels of DC maturation markers (Fig. 3B), as compared CLEC12A is not internalized via the classical clathrin-coated pits with isotype control–treated DCs. In addition, CLEC12A Ab binding but follows a distinct endocytosis pathway. Yet, the intracellular did not affect production of the immunoregulatory cytokine IL-10 routing of both receptors appears highly similar, as both colocalize or the proinflammatory cytokine TNF-a (Fig. 3C). In contrast, we with the early endosome after internalization and subsequently observed an almost 2-fold increase in IL-6 secretion by LPS- and route to the lysosomes. cytokine-matured MoDCs following CLEC12A targeting, as compared with isotype control treatment. Importantly, the capacity of CLEC12A targeting does not affect DC maturation and T cell mature MoDCs to promote allogeneic T cell proliferation and IFN-g stimulatory capacity secretion was not affected by CLEC12A targeting (Fig. 3–F). These As CLEC12A contains an ITIM motif in its cytoplasmic tail, it might data indicate that CLEC12A Ab targeting does not negatively affect function as an inhibitory receptor upon triggering. Therefore, we DC maturation, phenotype, or T cell stimulatory potential. investigated whether CLEC12A Ab binding, alone or in combination CLEC12A efficiently delivers KLH into DCs, resulting in with different maturation stimuli, modulated DC phenotype or func- + tion. CLEC12A Ab binding by itself did not affect expression levels of potent CD4 T cell recall responses maturation marker CD83, costimulatory molecules CD80/CD86, As CLEC12A is efficiently internalized and Ab binding does not coinhibitory ligand PD-L1, or HLA-DR (Fig. 3A). Furthermore, affect DC function, we next investigated whether CLEC12A could The Journal of Immunology 5

FIGURE 2. Clathrin-independent CLEC12A translocation to the endosomal/lysosomal compartments. (A) Day 3 MoDCs, BDCA1+ mDCs, and pDCs, were labeled with aDEC205 or aCLEC12A and incubated for 30 min in isotonic or hypertonic (450 mM sucrose) medium at 37˚C. DEC205 and CLEC12A surface expression was assessed by flow cytometry following Alexa Fluor 647–conjugated goat anti-mouse IgG staining. CLEC12A internalization is calculated relative to the surface expression at the start based on the mean fluorescence intensity. Data are shown as mean 6 SEM of three independent experi- Downloaded from ments. (B) Confocal laser scanning microscopy analysis of DEC205 and CLEC12A internalization in day 3 MoDCs and colocalization with early endosomes and lysosomes in time. MoDCs were incubated with aDEC205 or aCLEC12A, followed by staining with Alexa http://www.jimmunol.org/ Fluor 488–conjugated goat anti-mouse IgG2b (green). Receptors were allowed to internalize for 0–60 min at 37˚C. Then, MoDCs were stained for membrane MHC-II molecules using aHLA-DR/DP, which was visualized with Alexa Fluor 564–conjugated goat anti-mouse IgG2a (blue). Finally, MoDCs were per- meabilized, then stained for early endosomes (EEA-1) or late endosomes (LAMP-1), whereupon visualization with Alexa Fluor 647–con- by guest on September 29, 2021 jugated goat anti-rabbit IgG was performed (red). Images were acquired on an Olympus FV1000 Confocal laser scanning microscope, original magnification 360 oil objective (nu- merical aperture, 1.47). Data of one representative donor of three independent experiments are shown. (C–F) The Manders coefficient was calculated for CLEC12A or DEC205 colocalization with EEA-1 or LAMP-1, using Image J V1.46R and quantified with JACoP plugin V2.1.1. Data are shown as mean 6 SEM of three independent donors where $10 cells were analyzed per condition/donor. Data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test; *p , 0.05, ***p , 0.001.

be used for Ag delivery. DC subsets were labeled with CLEC12A- KLH-speciﬁc T cell proliferation (Fig. 4C). Moreover, these biotin and abiotin/KLH conjugates, matured, and then cocultured T cells secreted high levels of IFN-g (Fig. 4D). Interestingly, with patients’ PBMCs containing KLH-experienced CD4+ T cells. despite lower CLEC12A expression, CLEC12A-mediated KLH CLEC12A-mediated KLH delivery into day 3 MoDCs resulted in delivery in pDCs efﬁciently promoted proliferation and IFN-g a 2-fold increase of CD4+ T cell proliferation (Fig. 4A) and en- production of KLH-experienced T cells (Fig. 4E, 4F). Although hanced IFN-g production (Fig. 4B) as compared with isotype T cell proliferation was lower in pDC cocultures as compared with control treatment. Importantly, CLEC12A-mediated KLH uptake mDC cocultures, IFN-g levels were equal in both cocultures, in- and release into BDCA1+ mDCs showed superior induction of dicating that pDC stimulation promotes higher IFN-g secretion 6 CLEC12A-MEDIATED Ag CROSS-PRESENTATION BY DCs Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. CLEC12A ligation does not negatively affect DC function. Day 3 MoDCs were incubated for 48 h with aCLEC12A or hIgG1 isotype control in the absence or presence of different maturation stimuli. (A) Expression of CD83, CD80, CD86, PD-L1, and HLA-DR on nonmatured CLEC12A Ab-treated (black line) or isotype-treated (dark gray line) MoDCs was determined by ﬂow cytometry. Isotype controls for the respective maturation markers are depicted in light gray. Data of one representative donor of four independent experiments are shown. (B and C) Relative expression of the DC maturation markers CD83, CD80, CD86, and CCR7 and (C) relative secretion of IL-6, IL-10, and TNF-a for Ab-treated MoDCs cultured with various maturation stimuli. Data are calculated for aCLEC12A- treated cells relative to cells incubated with hIgG1 (aCLEC12A/IgG). Graphs show the median with a range of seven independent experiments. (D–F)Mature aCLEC12A (black lines/circles) or isotype-treated (gray lines/white circles) MoDCs were cocultured with allogeneic CFSE-labeled T cells at a ratio of 1:10 for 5 d. Subsequently, T cell proliferation was determined by ﬂow cytometry and IFN-g secretion by ELISA. (D) Representative histograms show CSFE dilution of CD4+ and CD8+ T cells of one T cell donor of four donors. (E)CD3+CD4+ and CD3+CD8+ T cell proliferation and (F)IFN-g secretion of four different T cell donors tested. Data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test; *p , 0.05. The Journal of Immunology 7

FIGURE 4. CLEC12A-dependent Ag uptake leads to Ag presentation by different human DC subsets. (A and B) Day 3 MoDCs were labeled with aCLEC12A or mIgG2b isotype control, followed by labeling with biotinylated goat anti-mouse IgG, and subsequently targeted with KLH-conjugated abiotin. Next, MoDCs were matured for 48 h with the conventional cytokines, harvested, and cocultured at a 1:10 ratio with CFSE-labeled KLH- responsive PBMCs. (A) After 4 d of culture, CD4+ T cell proliferation was analyzed by ﬂow cytometry. The mean 6 SD of triplicate measurements is shown for one representative patient of three patients. (B)IFN-g levels at day 4 of coculture were

determined on pooled supernatant using cytokine Downloaded from bead array. Data of one representative patient are shown. (C–F) FACS-sorted BDCA1+ mDCs or pDCs were incubated with biotinylated aCLEC12A or hIgG1 isotype control and subsequently targeted with KLH-conjugated abiotin. DCs were matured overnight and subsequently cocultured with CFSE- labeled KLH-responsive PBMCs at a 1:10 ratio. http://www.jimmunol.org/ (C and E)CD4+ T cell proliferation was measured by ﬂow cytometry and (D and F)IFN-g production was analyzed by ELISA after 4 d of coculture with (C and D) BDCA1+ mDCs or (E and F)pDCs.The mean 6 SD of triplicate measurements is shown for two of three representative patients. Data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test; *p , 0.05, ***p , 0.001. by guest on September 29, 2021

per T cell. These data demonstrate that CLEC12A efficiently TCR-transduced CD8+ T cells, as demonstrated by significant up- enables KLH uptake, and its subsequent processing and presen- regulation of the degranulation marker CD107a (Fig. 5) and activa- tation in MHC-II molecules, eliciting productive CD4+ T cell tion marker CD137 (Supplemental Fig. 4C). Furthermore, DC subsets activation and proliferation. targeted with aCLEC12A/HA-1 or aDEC205/HA-1 conjugates efficiently promoted IFN-g secretion, as compared with the controls CLEC12A-targeted long peptide is efficiently cross-presented + (Fig. 5B–D). Interestingly, CLEC12A and DEC205 showed a similar by natural DC subsets to CD8 T cells capacity to deliver Ags for cross-presentation and activation of HA-1– For effective boosting of antitumor immunity, cross-presentation of specific CD8+ Tcells. internalized tumor Ags to cytotoxic CD8+ T cells is essential (37). To evaluate the efficacy of CLEC12A as an attractive target for Therefore, we investigated whether CLEC12A-mediated delivery of facilitating Ag cross-presentation to patient-derived T cells, we MiHA long peptide leads to effective cross-presentation by BDCA1+ stimulated PBLs of alloSCT patients containing HA-1–reactive CD8+ mDCs, BDCA3+ mDCs, and pDCs. For this investigation, DC memory T cells with BDCA1+ mDCs (Fig. 6A) or pDCs (Fig. 6B) subsets were labeled with aCLEC12A/HA-1 or aDEC205/HA-1 loaded with the aCLEC12A/HA-1 conjugates or short HA-1 peptide conjugates. Subsequently, DCs were matured and cocultured with (i.e., positive control). After 7 d of coculture, increased percentages HA-1 TCR-transduced CD8+ T cells. Notably, we confirmed spe- of HA-1–specific CD8+ memory T cells were observed as compared cific binding of the aCLEC12A/HA-1 and aDEC205/HA-1 conju- with stimulation with non–peptide-loaded DCs. Moreover, when gates to BDCA1+ mDCs, BDCA3+ mDCs, and pDCs, using flow DCs were labeled with the aCLEC12A/HA-1 conjugate in the cytometry by staining the biotin tag on the HA-1 long peptide with presence of excess unconjugated aCLEC12A, no increase in the abiotin-FITC (Supplemental Fig. 4B). DCs labeled with either percentage of HA-1–specific CD8+ T cell was observed. Further- aCLEC12A/HA-1 or aDEC205/HA-1 conjugate in the presence of more, also augmented absolute numbers of HA-1–specific CD8+ excess unconjugated specific Ab or isotype/HA-1 conjugates were T cells were detected upon stimulation with BDCA1+ mDCs incapable of activating HA-1 TCR-transduced CD8+ T cells (Fig. 5, (Fig. 6C) or pDCs (Fig. 6D) labeled with the aCLEC12A/HA-1 Supplemental Fig. 4C). Importantly, CLEC12A- and DEC205- conjugates or short HA-1 peptide. Collectively, these findings mediated HA-1 delivery and cross-presentation by BDCA1+ mDCs, demonstrate that CLEC12A effectively mediates HA-1 long peptide BDCA3+ mDCs, and pDCs resulted in significant activation of HA-1 delivery and cross-presentation by BDCA-1+ mDCs and pDCs, 8 CLEC12A-MEDIATED Ag CROSS-PRESENTATION BY DCs Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. CLEC12A-delivered HA-1 long peptide is efficiently cross-presented by BDCA1+ mDCs, BDCA3+ mDCs, and pDCs. FACS-sorted BDCA1+ mDCs, BDCA3+ mDCs, and pDCs were incubated with long HA-1 peptide chemically conjugated to aCLEC12A (aCLEC12A/HA-1) or aDEC205 (aDEC205/HA-1). As negative controls, DCs were not labeled (DC2), labeled with isotype/HA-1 conjugates, or labeled with the specific conjugate in the presence of excess unconjugated Ab (blocked aCLEC12A/HA1 or aDEC205/HA1). As positive control, DCs were pulsed with 1 mM short HA-1 peptide. BDCA1+ mDCs, BDCA3+ mDCs, and pDCs were subsequently matured overnight, followed by 24-h coculture with HA-1 TCR-transduced T cells (between 40 and 66% HA-1 tetramer positive; data not shown) at a ratio of 1:1. T cell activation was assessed by determining the percentage of CD107a+CD8+ Tcells using flow cytometry, and by analysis of IFN-g secretion using ELISA. (A) Dot plots show CD107a expression after coculture with aCLEC12A/HA-1 targeted or nontargeted BDCA1+ mDCs of one representative donor. (B–D)PercentageofCD107a+CD8+ T cells and IFN-g production after coculture with (B)BDCA1+ mDCs, (C) pDCs targeted with aCLEC12A/HA-1 or aDEC205/HA-1, or (D) BDCA3+ mDCs targeted with aCLEC12A/HA-1. (Figure legend continues) The Journal of Immunology 9

FIGURE 6. CLEC12A-delivered HA-1 long peptide is efficiently cross-presented by BDCA1+ mDCs and pDCs to stimulate patient CD8+ T cells. FACS-sorted BDCA1+ mDCs and pDCs were incubated with long HA-1 peptide chemically conjugated to aCLEC12A (i.e., aCLEC12A/HA-1 conjugate). As controls, DCs were not labeled (DC2) or labeled with the conjugate in the presence of excess unconjugated aCLEC12A (blocked aCLEC12A/HA1; negative control) or with 1 mM short HA-1 peptide (positive control). BDCA1+ mDCs and pDCs were subsequently matured overnight, and cocultured with patient PBLs at a ratio of 1:5. At day 7, the expansion of HA-1– specific CD8+ memory T cells was assessed by Downloaded from determining the percentage of HA-1 tetramer– positive CD8+ T cells and absolute counts. (A and B) Dot plots show the percentage of HA-1 tetramer– positive CD8+ T cells out of total CD8+ T cells after coculture with aCLEC12A/HA-1 targeted or control (A) mDCs or (B) pDCs. (C and D) Absolute number of HA-1–specific CD8+ T cells http://www.jimmunol.org/ after coculture with (C) mDCs or (D) pDCs; one of two donors is shown. by guest on September 29, 2021 resulting in strong activation of HA-1–reactive CD8+ T cells of not involve clathrin-coated pits. Alternatively, similar to Langerin patients after alloSCT. (CD207) (46), CLEC12A might be endocytosed via the caveolin pathway. However, so far we were not able to demonstrate caveolin Discussion involvement in CLEC12A internalization (data not shown). Next Although the immune system is capable of eliciting tumor-reactive to the capacity to internalize, the intracellular fate and cargo immune responses, the magnitude and functionality of these re- release are key parameters determining the suitability and effi- sponses are often inadequate (38, 39). Therefore, powerful strategies cacy of CLEC12A for targeted Ag delivery and presentation via are needed to boost antitumor immunity to prevent disease pro- MHC-I and MHC-II molecules. In general, receptor/Ag traf- gression. As DCs are the most powerful APCs, they are highly at- ficking to the lysosomes is considered to result in loading onto tractive vehicles for boosting T cell responses (40, 41). An appealing MHC-II molecules and subsequent CD4+ T cell activation (47). strategy to make use of the specialized functions and cross-talk of In contrast, prelysosomal localization has been postulated to be natural DC subsets and exploit their potent Ag processing and more favorable for Ag processing and cross-presentation via presentation machinery is targeted delivery of tumor Ags in vivo via MHC-I molecules to CD8+ T cells (48–52). Nevertheless, DEC205, endocytic receptors (24, 25, 42). In this study, we evaluated the which quickly routes to the lysosomes, can efficiently facilitate potential of CLEC12A, an endocytic CLR, as a new targeting Ag cross-presentation (52–55). To elucidate the intracellular fate candidate for simultaneous Ag delivery into all human DC subsets. of CLEC12A, we performed colocalization studies. CLEC12A We confirmed that the expression pattern of CLEC12A is re- and DEC205 showed similar intracellular routing: initial coloc- stricted to hematopoietic cells of myeloid origin, including alization with the early endosomes, followed by further traf- monocytes, granulocytes, BDCA1+ mDCs, BDCA3+ mDCs, and ficking to the lysosomes, where they were retained. However, pDCs, as has been reported by others (26, 43). Next, we demon- CLEC12A colocalization with the early endosomes lasted longer. strated that CLEC12A is efficiently internalized by all human DC In accordance, Begun et al. (28) recently reported CLEC12A subsets. Most CLRs, such as DCIR (44), DC-Sign (45), and colocalization with Rab5, another early endosome marker. These DEC205 (21), endocytose via the classical clathrin pathway. Of findings suggest that CLEC12A might provide a good window for interest, CLEC12A endocytosis was not affected in hypertonic endosomal escape of targeted Ag, thereby facilitating effective conditions, indicating that its mechanism of internalization does cross-presentation.

Data are depicted as the mean 6 SD of triplo measurements of one representative donor. For the aCLEC12A/HA-1 conjugate, n = 6 (BDCA1+ mDC), n =5 (pDC), or n = 1 (BDCA3+ mDC) independent experiments were performed. For aDEC205/HA-1, n = 2 independent experiments were performed. Data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test; ***p , 0.001. 10 CLEC12A-MEDIATED Ag CROSS-PRESENTATION BY DCs

CLRs are known to shape immune responses via the signaling CLEC12A. CLEC12A is efficiently internalized via a clathrin- motifs in their cytoplasmic tails or via the association with adaptor independent mechanism and is quickly translocated from the molecules that elicit downstream signaling cascades. CLEC12A early endosomes to the lysosomes. Notably, we did not observe contains an inhibitory ITIM motif in its cytoplasmic tail. Recently, negative effects of CLEC12A Ab binding on DC phenotype or Neumann et al. (27) identified that CLEC12A expressed on neutro- functionality. Most importantly, we demonstrated that CLEC12A- phils is involved in controlling sterile inflammation. They discovered targeted Ag delivery results in potent activation of KLH-reactive that CLEC12A, upon triggering by uric acid crystals, inhibits Syk- CD4+ T cell responses and highly effective cross-presentation to dependent reactive oxygen species production, thereby limiting in- HA-1–reactive CD8+ T cells of alloSCT patients. These data infiltration of immune cells to the site of damage. Furthermore, Chen dicate that CLEC12A is an attractive candidate for in vivo tar- et al. (29) observed positive as well as negative effects on DC phe- geting of human DC subsets with tumor Ags, thereby using the notype and function upon aCLEC12A binding, dependent on the specialized functions and cross-talk of these DCs to boost tumor- type of TLR activation. In this report, we showed that binding of our reactive T cell immunity in cancer patients. aCLEC12A did not promote DC maturation. Moreover, no effect on CD86, CD80, CD83, or CCR7 expression was observed following Acknowledgments aCLEC12A binding in combination with different maturation stim- We thank Anoek van Rijn (Radboud University Medical Center) and Eric uli. In contrast, Chen et al. (29) observed increased expression of Mul (Sanquin, Amsterdam, the Netherlands) for technical assistance and CCR7 following aCLEC12A ligation in combination with TLR4 Michel Kester and Fred Falkenburg (LUMC) for providing HA-1 tetramers. stimulation. These opposite findings might be attributed to differences in epitope recognition of the used Abs or isotype-related ef- Disclosures Downloaded from fects. Surprisingly, we observed augmented IL-6 secretion upon L.B.H.B. is an employee of Merus N.V. The other authors have no conflict- CLEC12A targeting in the presence of conventional cytokine mat- ing financial interests. uration or LPS-mediated TLR-4 stimulation. Despite this effect on IL-6 secretion, no difference in allogeneic T cell stimulatory capacity was observed for aCLEC12A versus isotype control-treated MoDCs. References 1. Palucka, K., and J. Banchereau. 2012. Cancer immunotherapy via dendritic cells. Because of its efficient internalization capacity and the lack of Nat. Rev. Cancer 12: 265–277. http://www.jimmunol.org/ negative effects following Ab binding, we consider CLEC12A to be 2. Steinman, R. M., and J. Banchereau. 2007. Taking dendritic cells into medicine. an interesting candidate for targeted Ag delivery into DCs. Impor- Nature 449: 419–426. 3. Joffre, O. P., E. Segura, A. Savina, and S. Amigorena. 2012. Cross-presentation tantly, we demonstrated that targeted delivery via CLEC12A enabled by dendritic cells. Nat. Rev. Immunol. 12: 557–569. KLH presentation onto MHC-II molecules of human BDCA1+ 4. Rosenberg, S. A. 1997. Cancer vaccines based on the identification of genes mDCs, pDCs, and MoDCs, thereby promoting profound prolifera- encoding cancer regression antigens. Immunol. Today 18: 175–182. + 5. Engell-Noerregaard, L., T. H. Hansen, M. H. Andersen, P. Thor Straten, and tion and IFN-g secretionbyKLH-experiencedCD4 T cells. Fur- I. M. Svane. 2009. Review of clinical studies on dendritic cell-based vaccination thermore, to our knowledge, we provided the first mechanistic of patients with malignant melanoma: assessment of correlation between clinical response and vaccine parameters. Cancer Immunol. Immunother. 58: 1–14. evidence that human mDCs and pDCs are capable of cross- 6. Van de Velde, A. L., Z. N. Berneman, and V. F. Van Tendeloo. 2008. Immu- presenting natural tumor Ags delivered via CLEC12A, facilitating notherapy of hematological malignancies using dendritic cells. Bull. 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Greupink-Draaisma, B. Esendam, effective targets than CLEC12A. In contrast, in our studies CLEC12A T. de Witte, F. Preijers, H. Levenga, B. van Rees, et al. 2013. Immunogenicity of performed equally well as DEC205 in Ag cross-presentation assays dendritic cells pulsed with MAGE3, survivin and B-cell maturation antigen by human DC subsets. These opposing findings could possibly be mRNA for vaccination of multiple myeloma patients. Cancer Immunol. Immunother. 62: 1381–1392. attributed to differences in epitope recognition of the used Abs and 10. Levenga, H., N. Schaap, F. Maas, B. Esendam, H. Fredrix, A. Greupink- functionality after cargo conjugation or to differences in conjuga- Draaisma, T. de Witte, H. Dolstra, and R. Raymakers. 2010. Partial T cell- tion efficiency, or may be related to differences between the murine depleted allogeneic stem cell transplantation following reduced-intensity conditioning creates a platform for immunotherapy with donor lymphocyte in- and human model systems. fusion and recipient dendritic cell vaccination in multiple myeloma. Biol. Blood In addition to other CLRs for which Abs are currently in phase Marrow Transplant. 16: 320–332. I/II clinical investigation, CLEC12A holds great promise for future 11. Broen, K., A. Greupink-Draaisma, H. Fredrix, N. Schaap, and H. Dolstra. 2012. Induction of multiple myeloma-reactive T cells during post-transplantation im- cargo delivery applications. Using nanoparticles, the combined munotherapy with donor lymphocytes and recipient DCs. Bone Marrow Trans- delivery of tumor Ags and immunomodulatory agents, such as TLR plant. 47: 1229–1234. 12. Hobo, W., K. Broen, W. J. van der Velden, A. Greupink-Draaisma, N. Adisty, ligands or small interfering RNA against coinhibitory molecules, Y. Wouters, M. Kester, H. Fredrix, J. H. Jansen, B. van der Reijden, et al. 2013. could be realized to further improve DC immunogenicity in vivo. Association of disparities in known minor histocompatibility antigens with Previously, we showed that silencing the coinhibitory molecules relapse-free survival and graft-versus-host disease after allogeneic stem cell transplantation. Biol. Blood Marrow Transplant. 19: 274–282. PD-L1/L2 on DCs resulted in superior T cell stimulatory potential 13. de Rijke, B., A. van Horssen-Zoetbrood, J. M. Beekman, B. Otterud, F. Maas, (56, 57). Another interesting application for CLEC12A targeted R. Woestenenk, M. Kester, M. Leppert, A. V. Schattenberg, T. de Witte, et al. cargo delivery is in the setting of acute myeloid leukemia and my- 2005. 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