Antigen-Specific Transfer of Functional Programmed Death Ligand 1 from Human APCs onto CD8+ T Cells via Trogocytosis

This information is current as Regina Gary, Simon Voelkl, Ralf Palmisano, Evelyn Ullrich, of October 1, 2021. Jacobus J. Bosch and Andreas Mackensen J Immunol 2012; 188:744-752; Prepublished online 14 December 2011; doi: 10.4049/jimmunol.1101412 http://www.jimmunol.org/content/188/2/744 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2011/12/14/jimmunol.110141 Material 2.DC1 http://www.jimmunol.org/ References This article cites 44 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/188/2/744.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antigen-Specific Transfer of Functional Programmed Death Ligand 1 from Human APCs onto CD8+ T Cells via Trogocytosis

Regina Gary,* Simon Voelkl,* Ralf Palmisano,† Evelyn Ullrich,* Jacobus J. Bosch,* and Andreas Mackensen*

Upon specific interaction with APCs, T cells capture membrane fragments and surface molecules in a process termed trogocytosis. In this study, we demonstrate that human Ag-specific CD8+ T cells acquire the coinhibitory molecule programmed death ligand 1 (PD-L1) from mature dendritic cells (mDC) and tumor cells in an Ag-specific manner. Immature dendritic cells were less effective in transferring surface molecules onto CD8+ T cells than mDCs. Interestingly, trogocytosis of PD-L1 requires –cell contact and cannot be induced by uptake of soluble proteins obtained from mDC lysates. The transfer process is impaired by inhibition of vacuolar ATPases in T cells as well as by fixation of dendritic cells. Of importance, CD8+ T cells that acquired PD-L1 complexes Downloaded from were able to induce apoptosis of neighboring programmed death 1–expressing CD8+ T cells. In summary, our data demonstrate that human CD8+ T cells take up functionally active PD-L1 from APCs in an Ag-specific fashion, leading to fratricide of programmed death 1–expressing, neighboring T cells. The transfer of functionally active coinhibitory molecules from APCs onto human CD8+ T cells could have a regulatory role in immune responses. The Journal of Immunology, 2012, 188: 744–752.

he exchange of proteins is an important process in the observed for T cells, DCs, B cells, NK cells, tumor cells, and other http://www.jimmunol.org/ communication between immune cells. Besides exosomes immune cells (13–18). Moreover, trogocytosis of membrane and nanotubes, the phenomenon of trogocytosis has re- patches was not only described in vitro, but also in vivo in mice T + cently become subject of renewed interest in cell–cell interactions (19). Of interest, Ag-specific membrane capture by CD8 T cells (1). In 2002, the term trogocytosis was defined by Hudrisier and is a feature of highly functional CTL, and has also been described Bongrand (2) as intercellular transfer of membrane patches con- as a new option to identify and isolate tumor-specific T cells (20). taining membrane-anchored proteins from presenting cells to Besides this, the functional relevance of trogocytosis by acquired . Of note, the phenomenon itself has already been HLA-G molecules has been demonstrated in NK and T cells described for the first time in 1973 (3). conferring temporary suppressive function (15, 21). by guest on October 1, 2021 Most data on trogocytosis have been generated in murine mod- Trogocytosis may also have clinical implications: the transfer of els showing the acquisition of both peptide-MHC class I and II complexes of the anti-CD20 Ab and CD20 Ag from complexes and costimulatory molecules by T cells after Ag rec- targeted B cells onto monocytes has been demonstrated in CLL ognition in a TCR-dependent manner (4–7). Human T cells have patients and induced a substantial loss of targeted CD20 on cir- also been described to acquire MHC and costimulatory molecules culating B cells (18). In addition, the uptake of multidrug resis- upon –dendritic cell (DCs) interaction as well as adhesion tance proteins by ovarian cancer cells resulted in decreased sensi- molecules during transendothelial migration (8–11). Human and tivity of tumor cells toward chemotherapeutic drugs (17). Together, murine T cells exchange molecules with DCs in a bidirectional these data implicate that trogocytosis plays an important role in the manner (12). Trogocytosis is a ubiquitous phenomenon as it is modulation of cellular immune responses. The programmed death 1 (PD-1; CD279) receptor that interacts with programmed death ligand 1 (PD-L1; B7-H1, CD274) belongs *Department of Internal Medicine 5, Hematology and Oncology, University of Erlangen-Nuremberg, Erlangen, D-91054, Germany; and †Institute of Clinical Mi- to the coinhibitory molecules that inhibit T cell responses. Ligation crobiology, Immunology and Hygiene, University Clinic of Erlangen, Erlangen, D- of PD-1 impairs the TCR signal cascade, thereby counteracting cell 91054, Germany survival signals triggered by anti-apoptotic molecules as well as Received for publication May 16, 2011. Accepted for publication November 16, effector differentiation initiated by CD28 and IL-2 (22, 23). Fur- 2011. thermore, the balance between PD-L1 and costimulatory mole- This work was supported by the Deutsche Forschungsgemeinschaft (SFB643, the cules such as CD86 on DCs regulates T cell responses in hepatitis graduate program of SFB643, and MA 1351/8-1) and the Bavarian Immunotherapy Network (BayImmuNet). C virus infection. Thus, predominant PD-L1 expression inhibits Address correspondence and reprint requests to Dr. Andreas Mackensen, Depart- the immune response against hepatitis C virus (24). Next, studies ment of Internal Medicine 5, Hematology and Oncology, University of Erlangen- in patients with chronic infections have also revealed an inhibitory Nuremberg, Ulmenweg 18, Erlangen, D-91054, Germany. E-mail address: andreas. role of PD-1–PD-L1 interaction resulting in CD8+ T cell ex- [email protected] haustion (25, 26). Besides impairment of effector T cells, PD-L1 The online version of this article contains supplemental material. also favors the development and maintenance of induced regula- Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; CMA, concana- mycin A; DC, dendritic cell; iDC, immature DC; MCSP, melanoma-associated chon- tory T cells in mice by interference of the mTOR-Akt signaling droitin sulfate proteoglycan; mDC, mature DC; MFI, mean fluorescence intensity; pathway (27). Moreover, human regulatory T cells can induce PD-1, programmed death 1; PD-L1, programmed death ligand 1; RT, room temper- PD-L1 expression on DCs, rendering them to suppressor DCs (28). ature; TM, tetramer; vATPase, vacuolar ATPase. Finally, as PD-L1 is up-regulated on a broad variety of human Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 cancer cells, it is likely that the PD-1/PD-L1 signaling pathway www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101412 The Journal of Immunology 745 may be involved in the immune escape of tumor cells. Multiple For blocking of trogocytosis, T cells or DCs were incubated with 0.1 mg/ clinical studies have demonstrated that PD-L1 expression on ml concanamycin A (CMA; Sigma-Aldrich) overnight, washed, and used tumors correlates with unfavorable prognosis (29, 30). for trogocytosis experiments. CMA revealed no toxic effect on human T cells and DC at the concentration used in our assays (data not shown). In this study, we investigated the Ag-specific transfer of PD-L1 For fixation of APCs, PKH67-labeled, Melan-A–pulsed mDCs were from human APCs onto CD8+ T cells via trogocytosis and found incubated for 30 min with 0.05% glutaraldehyde (Sigma-Aldrich) sup- that acquired PD-L1 strongly impaired T cell responses. PD-L1 plemented with 1% glucose, washed carefully, and used for trogocytosis acquisition may play a role in the limitation of Ag-specific T cell experiments. expansion and may also lead to T cell exhaustion in tumors and MHC peptide TM, mAb, and flow cytometry chronic infections. PE-conjugated HLA-A*0201 TM that had been folded around Melan- A26–35 or gp100209–217 were synthesized by Beckman Coulter (Fullerton, Materials and Methods CA). For phenotypic analyses, the following mAbs were used: anti–CD8- Media and cytokines PerCP, anti–CD8-allophycocyanin, anti–CD8-allophycocyanin-Cy7, anti– CD3-PE-Cy7, anti–CD25-PE, and IgG1-PE (all from BD Pharmingen, T cells were cultured in RPMI 1640 medium (PAN Biotec, Aidenbach, Heidelberg, Germany); anti–PD-L1-PE, anti–PD-1-allophycocyanin, anti– Germany) plus 10% human AB serum (PAN Biotec). The following re- PD-L1-biotin, unconjugated high-purified anti–PD-L1 blocking mAb, combinant human cytokines were used for differentiation of DCs: 500 U/ml and streptavidin-eFluor710 (all from eBioscience/NatuTec, Frankfurt, GM-CSF (Bayer, Leverkusen, Germany), 25 U/ml IL-4 (Promokine, Hei- Germany); anti-CD209 (DC-SIGN)-allophycocyanin (R&D Systems, delberg, Germany), 5 ng/ml TGF-b1 (Peprotech, Hamburg, Germany), 10 Wiesbaden-Nordenstadt, Germany); anti–MCSP-PE (Beckman Coulter); ng/ml IL-1b (Promokine), 1000 U/ml IL-6 (Promokine), 10 ng/ml TNF and anti–CD137-allophycocyanin and IgG2b-allophycocyanin (Caltag, (Promokine), and 1 mg/ml PGE2 (Enzo, Lo¨rrach, Germany). Preparation of T factor was described previously (31). Downloaded from Cells and cell lines Melan-A– and gp100-specific CTL lines were generated, as described previously (32). Briefly, PBMCs from healthy donors were collected by leukapheresis, followed by gradient centrifugation (Pancoll; PAN Biotec). The study was approved by local ethics committee of the University of Erlangen. Informed consent was provided according to the Declaration of Helsinki. CD8+ T cells were enriched from PBMC using a negative iso- http://www.jimmunol.org/ lation strategy (Miltenyi Biotec, Bergisch-Gladbach, Germany). Autolo- gous DCs were generated from monocytes and cultured in complete medium plus 10% FCS, supplemented with GM-CSF, IL-4, and TGF-b. On day 6, GM-CSF, IL-4, TNF, IL-6, IL-1b, and PGE2 were added. The culture was continued for additional 48 h, and mature DCs (mDCs) were pulsed with 30 mg/ml HLA-A2–binding Melan-A26–35 (ELAGIGILTV) or gp100209–217 (ITDQVPFSV) peptide (Bachem, Weil am Rhein, Germany) and 10 mg/ml human b2-microglobulin (Scipac, SitLingbourne, U.K.) for 2 h at 37˚C in serum-free complete medium. Complete medium supple-

mented with 3% T cell growth factor was replenished twice per week. by guest on October 1, 2021 CD8+ T cells were restimulated weekly with Ag-pulsed DCs in 96-well U-bottom plates. T cell clones were isolated from primary CTL lines by single cell sorting (FACSAria; BD Biosciences, San Diego, CA) using appropriate MHC tetramers (TM). CD8+ T cell clones were expanded by repetitive stimulation with allogeneic irradiated EBV-transformed B cells, irradiated allogeneic PBMC, and PHA-L (1 mg/ml; Sigma-Aldrich, Mu- nich, Germany). The melanoma cell line Mel1300 (HLA-A2+, Melan-A+, gp100+) was cultured in complete medium with 10% FCS (PAA Laboratories, Pasching, Austria). The Mel1300 cell line was provided by M. Nishimura (Depart- ment of Surgery, Medical University of South Carolina, Charleston, SC). Expression of HLA-A2, Melan-A, and melanoma-associated chondroitin sulfate proteoglycan (MCSP) on Mel1300 cells was documented via flow cytometry analysis. For the trogocytosis assay, Mel1300 cells were incu- bated in the presence or absence of 200 ng/ml IFN-g (Promokine). The FIGURE 1. Acquisition of membrane fragments, PD-L1, and CD209 lymphoblast cell line T2 (33) was obtained from American Type Culture from mDCs onto T cells by trogocytosis. A, Transfer of membrane frag- Collection (Manassas, VA) and is characterized by an increased expression ments, PD-L1, and CD209 is Ag specific. Melan-A–specific CD8+ T cells of the HLA-A2 Ag. Cells were cultured in complete medium supple- mented with 10% FCS. were coincubated with PKH67-labeled mDCs pulsed with either Melan-A or irrelevant gp100 peptide. Acquisition of PKH67+ membrane fragments, Trogocytosis experiments PD-L1, and CD209 was measured after 2 h of coculture with (upper panel) or without (lower panel) Ag recognition by flow cytometry (solid line). For trogocytosis experiments, APCs (DCs, T2, or Mel1300 cells) were pulsed for 2 h with 10 mg/ml relevant or irrelevant peptide and 10 mg/ml Dashed line in lower panel shows T cells before coculture as negative control. Histograms are gated on CD8+PKH67low lymphocytes. Shown is b2-microglobulin. Next, target cells were stained with lipophilic mem- brane dye PKH67 (Sigma-Aldrich) for 20 min at room temperature (RT) one representative of five independent experiments with similar results. and washed three times with complete medium plus 10% human AB se- Filled histograms represent negative controls for membrane staining and rum. Harvested T cell clones and target cells were placed in 96-well isotype controls for Ab staining. B, Visualization of transferred membrane U-bottom plates (Falcon; BD Biosciences) with an E:T ratio, unless oth- fragments and PD-L1 on T cells by confocal microscopy. mDCs were 3 5 3 5 erwise noted, of 3:1 (7.5 10 T cells plus 2.5 10 target cells in 200 ml pulsed with specific Melan-A or irrelevant peptide, followed by PKH67 complete medium plus 10% human AB serum), incubated at 37˚C, and labeling (green). T cells were stained with PKH26 (red). After a coculture harvested at indicated time. Harvested cocultures were washed with PBS (Life Technologies, Invitrogen, Darmstadt, Germany), resuspended before of 2 h with or without Ag recognition, cells were incubated with anti– staining with mAbs, and analyzed by flow cytometry. PD-L1-biotin, followed by secondary streptavidin-eFluor staining (blue). For the preparation of DC lysate, mDCs were pulsed with peptides and Images were captured with a 363 oil objective and 12-bit sequential scans. labeled with membrane dye, as described before. Lysates were generated by A53 zoom was used for high resolution single-cell images. Pictures are repeated freeze-and-thaw cycles of a defined number of mDCs. enhanced in brightness and contrast for improved printability. 746 TRANSFER OF FUNCTIONAL PD-L1 ONTO HUMAN T CELLS

Buckingham, U.K.). Flow cytometry was performed on a FACSCanto II linear unmixing and a deblurring filter with pixel range of 3. A 53 zoom (BD Biosciences). T cells were gated on CD8+PKH67low cells to be distin- according to Nyquist theorem was used for high resolution single-cell guished from PKH67high-labeled APCs. Doublets were excluded via FSC-W images. gating. FACS data were analyzed with FlowJo software PC version 7.6 (Celeza, Olten, Switzerland). Transwell assay + Cell sorting and apoptosis assay Ag-specific CD8 T cells were added to the bottom chambers of a 24-well tissue Transwell culture plate (Corning/Costar). Into the insert well, Cell sorting of gp100-specific CD3+CD8+ T cell clones for subsequent PKH67-labeled Ag-pulsed mDCs with or without Ag-specific PKH26- analysis after trogocytosis process was performed using a MoFlo cell sorter labeled T cells were placed to the top chamber as donor cells. Insert and (Cytomation, Freiburg, Germany). For apoptosis experiments, cells were bottom chambers were separated by a membrane with 5-mm pores, stained after coculture with Melan-A-TM for 30 min at RT, followed by allowing soluble factors and membrane vesicles, but neither DCs nor staining with surface markers for 10 min at 4˚C. Finally, cells were incu- T cells, to pass through. bated with 5 ml annexin V FITC (Caltag) and 5 ml 7-aminoactinomycin D (7-AAD; BD Pharmingen) in 150 ml annexin-binding buffer (BD Phar- Statistical analysis mingen) at RT for 15 min in the dark, followed by immediate FACS All statistical analyses were performed with two-tailed, paired t test and are analysis. For quantification of apoptotic T cells, the percentage of annexin presented 6SD using GraphPad Prism version 5.02 (GraphPad, La Jolla, V/7-AAD double-positive cells was calculated. Annexin V single-positive CA). T cells cannot be counted as apoptotic cells, as annexin V is also binding to exposed phosphatidylserine on the surface of T cells upon T cell activation (34). For blocking of PD-L1–mediated apoptosis, PD-L1–acquiring cells Results cocultured with Melan-A–specific T cells were incubated with a highly CD8+ T cells acquire PD-L1 and CD209 from APCs after Ag purified anti-PDL1 mAb (10 mg/ml; eBioscience). recognition via trogocytosis Downloaded from Fluorescence microscopy First, we examined whether membrane-bound PD-L1 and CD209 + Melan-A– or gp100-pulsed DCs were labeled with membrane dye PKH67, on mDCs could be acquired by human CD8 T cells in an Ag- as described above, and T cells were labeled analogous with membrane dependent manner via trogocytosis. CD209 has been described as dye PKH26 (Sigma-Aldrich). After coculture for 2 h at 37˚C, cells were a marker exclusively expressed by DC and absent from T cells, harvested, washed twice, and incubated with biotinylated anti–PD-L1 or corresponding IgG Ab for 15 min at 4˚C. Cells were washed and incubated and therefore was chosen as control marker (35). Peptide-pulsed, with streptavidin-eFluor710 nanocrystals for 20 min at 4˚C. After four PKH67-labeled mDCs were cocultured with a Melan-A–specific http://www.jimmunol.org/ additional washing steps, cells were fixed for 15 min at RT with 3% CD8+ T cell clone. As shown in Fig. 1, T cells acquired high paraformaldehyde (Sigma-Adlrich). Subsequently, cells were transferred amounts of membrane patches, PD-L1, and CD209 after Ag rec- on adherent SuperFrost Plus microscope slides (Langenbrinck, Emmen- ognition. In contrast, neither PD-L1, nor CD209, and just low dingen, Germany). After settling and cell adherence, cells were washed with PBS. Almost dried slides were mounted with ProLong Gold Antifade amounts of membrane fragments were acquired when cocultured Reagent (Invitrogen, Darmstadt, Germany). Slides were analyzed utilizing with control peptide-pulsed DCs. Biotinylation and PKH26 la- a laser-scanning microscope (Zeiss LSM 700, Jena, Germany) with the beling of DCs confirmed the results obtained with PKH67 label- respective ZEN software (version 2009). Images were captured with a 363 ing, demonstrating a strong transfer of membrane fragments onto oil objective and in 12-bit mode. All three channels were corrected against bleed through and cross-talk before sequential scan for best separation was T cells after Ag-specific recognition and low Ag-independent performed. Gain was adjusted accordingly and within the same magnitude acquisition of membrane molecules by T cells upon activation by guest on October 1, 2021 for all channels. No signal enhancement was performed afterward, only (data not shown). This Ag-independent transfer seems to be trig-

FIGURE 2. Characterization of the transfer process. A, Kinetics of acquisition of mem- brane fragments, PD-L1, and CD209. Melan- A–specific T cells were cocultured with Melan- A peptide-pulsed, PKH67-labeled mDCs. Ac- quisition of membrane patches and molecules PD-L1 and CD209 was analyzed by flow cy- tometry at indicated times and is displayed as MFI of PKH67, d; PD-L1, :; CD209, n. B, Impact of T cell to mDC ratio on the uptake of membrane fragments and PD-L1 by T cells. mDCs were pulsed with Melan-A peptide, la- beled with PKH67, and cocultured with Melan- A–specific T cells at different ratios, as noted. Data were measured by flow cytometry. The relative MFI expressed as multiple of the MFI of untreated T cells of four independent ex- periments (6SD) is shown for PKH67 and PD-L1 on CD8+PKH67low T cells. The Journal of Immunology 747 gered by preactivation, as nonstimulated T cells do not acquire different time points. As shown in Fig. 2A, transfer of PD-L1, membrane fragments (data not shown). CD209, and membrane patches onto T cells is a process detect- Next, we performed confocal microscopy to visualize the uptake able already at 30 min after coculture. The acquisition of all three of membrane patches and PD-L1 from APCs. PD-L1 expression components peaked at 1.5–2 h and rapidly decreased after 3–4 h, was detected on mDCs, but not on T cells by secondary nanocrystal reaching a plateau for ∼20 h. Between 48 and 72 h of coculture, staining in the far red color spectrum (data not shown). After 2-h PD-L1, CD209, and membrane patches completely disappeared coculture of Melan-A–specific CTL with peptide-pulsed DCs, an from the T cell surface. Of interest, the acquisition of membrane Ag-specific uptake of PD-L1 and membrane fragments by CD8+ fragments, PD-L1, and CD209 revealed the same kinetics, sug- T cells was documented (Fig. 1B). Of importance, PD-L1 and gesting a similar mechanism of capture. In contrast to this ex- membrane patches did not colocalize and could be detected on trinsic PD-L1 signal upon trogocytosis, intrinsic PD-L1 expres- different regions of the T . To verify that PD-L1 is sion peaked on day 4 after Ag-specific stimulation (data not not just upregulated on T cells after Ag-specific stimulation, shown). Melan-A–pulsed, PKH67-labeled PD-L12 T2 cells were used as To determine whether the amount of acquired molecules on APCs for stimulation with Melan-A–specific T cells, demon- T cells was higher when more APCs are targeted by specific T cells, strating an uptake of membrane fragments and no upregulation T cells were cocultured with mDCs at different ratios and analyzed of PD-L1 expression on T cells (Supplemental Fig. 1). Together, for trogocytosis of membrane fragments and PD-L1. As shown in these data show that PD-L1 and CD209 are rapidly acquired after Fig. 2B, an increase in membrane and PD-L1 uptake was observed Ag-specific recognition of mDCs by CD8+ T cells. up to a ratio of 1:1. Saturation was documented at a T cell:APC ratio of 1:3. Characterization of the Ag-specific transfer of PD-L1 and Downloaded from CD209 from APCs onto T cells Role of APCs for Ag-specific trogocytosis To assess the dynamics of the transfer of membrane fragments, PD- To investigate whether PD-L1 can also be acquired from other L1, and CD209, the kinetics of trogocytosis was documented at sources of APCs, Melan-A–pulsed immature DCs (iDCs) and http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Trogocytosis from iDCs and from melanoma cell line Mel1300. A, Expression of PD-L1 and CD209 on iDCs and mDCs. iDCs or mDCs were analyzed for PD-L1 and CD209 expression (solid line) compared with isotype staining (filled histogram) by flow cytometry. B, Acquisition of membrane fragments and surface molecules by T cells from iDCs compared with mDCs. iDCs or mDCs were pulsed with Melan-A peptide, labeled with PKH67, and cocultured with Melan-A–specific T cells for 2 h. Unstained and isotype controls are presented as filled histograms. Trogocytosis was analyzed by flow cytometry. Histograms are gated on CD8+PKH67low lymphocytes. Shown is one representative of three independent experiments with similar results. C, Expression of PD-L1 and MCSP on melanoma cell line Mel1300 before and after IFN-g exposure. PD-L1 and MCSP expression was analyzed on Mel1300 before (upper panel) and 48 h after (lower panel) IFN-g treatment by flow cytometry. Negative and isotype controls are presented as filled histograms. Shown is one representative of three independent experiments. D, Acquisition of PD-L1 and MCSP from melanoma cell line by CD8+ T cells. PKH67-labeled Mel1300 melanoma cells exposed or not to IFN-g were cocultured with Melan-A–specific CD8+ T cells. Acquisition of membrane fragments, PD-L1, and MCSP was analyzed after 2 h by flow cytometry. Negative and isotype controls are presented as filled histograms. Histograms are gated on CD8+PKH67low lymphocytes. Shown is one representative of three independent experiments with similar results. 748 TRANSFER OF FUNCTIONAL PD-L1 ONTO HUMAN T CELLS mDCs were compared in their capacity to induce trogocytosis. As well separated from T cells in the bottom well. In both settings, shown in Fig. 3A, expression of PD-L1 could be documented on transfer of PD-L1 and membrane fragments was absent (Fig. 4A). iDCs, albeit to a lower extent. In contrast, CD209 was expressed Next, we asked whether T cells capture membrane fragments and on iDCs at similar or higher levels than on mDCs. Of interest, molecules out of lysates after Ag-specific killing of APCs. When iDCs were only capable of transferring low amounts of CD209 Melan-A–pulsed, PKH67-labeled mDCs were lysed via repeated and membrane fragments, but no PD-L1, onto T cells. In contrast, freeze-and-thaw cycles, no transfer of PD-L1, but low amounts of T cells cocultured with mDCs captured high amounts of all three membrane fragments were documented (Fig. 4B). These data con- components (Fig. 3B). Of note, Ag-unspecific uptake of mem- firm that Ag-specific trogocytosis requires cell–cell contact and brane fragments from iDCs could not be detected (data not is not induced via soluble proteins from APCs. shown). These results demonstrate that iDCs are poor APCs in Ag-specific trogocytosis requires active interaction between terms of triggering the transfer of surface molecules onto T cells APCs and T cells in an Ag-specific manner. Next, we asked whether tumor Ag-specific T cells are able to Vacuolar ATPases (vATPases) were described to play an important capture PD-L1, as well as membrane fragments from melanoma role in membrane trafficking as well as endo- and exocytosis (36). cells. To address this question, HLA-A2+ Melan-A–expressing We therefore asked whether CMA, a specific inhibitor of vAT- melanoma cells were analyzed for expression of PD-L1 and the Pases, is able to abrogate Ag-specific trogocytosis (37). To address Ag MCSP before and after IFN-g treatment. MCSP was highly this question, T cells or mDCs were exposed to CMA before expressed independent on IFN-g stimulation, whereas PD-L1 was coculture and T cells were analyzed for trogocytosis. Inhibition of only detectable after IFN-g stimulation (Fig. 3C). After coculture vATPases in mDCs resulted in a nonsignificant, slightly reduced of Melan-A–specific T cells with IFN-g–treated melanoma cells, acquisition of membrane patches and PD-L1 by T cells. However, Downloaded from T cells acquired both PD-L1 and MCSP in parallel to membrane when T cells were pretreated with CMA, a strong inhibition of fragments, whereas no PD-L1 could be documented on T cells the transfer of PD-L1 (14.7 6 5.1% mean fluorescence intensity after coculture with untreated melanoma cells (Fig. 3D). These [MFI] of maximal transfer compared with 4.4 6 2.1% background data demonstrate that PD-L1 is not exclusively transferred from MFI; p , 0.001) as well as the transfer of membrane fragments mDCs, but also from tumor cells onto T cells upon Ag recogni- (23.9 6 17.8% MFI of maximal transfer compared with 8.0 6 tion. 7.3% background MFI; p , 0.01) was observed (Fig. 5A). These http://www.jimmunol.org/ data demonstrate an important role of vATPases in the integration Transfer of PD-L1 from APCs onto T cells requires cell–cell of surface molecules on T cells, yet attribute a minor role in the contact and is not mediated by soluble proteins from APCs disruption of molecules out of DCs. Of interest, inhibition of To assure that PD-L1 is transferred in a cell–cell contact-dependent trogocytosis in CMA-pretreated T cells had no impact on the manner, as defined for trogocytosis, in vitro Transwell experi- upregulation of activation markers, such as CD25 and CD137 ments were performed, placing peptide-pulsed DCs in the pres- (Fig. 5B). Next, we asked whether active participation of mDCs is ence or absence of PKH26-labeled Ag-specific T cells in the insert required in the transfer process. To address this question, Ag- by guest on October 1, 2021

FIGURE 4. Mechanism of trogocytosis of PD-L1. A, Membrane fragments and PD-L1 are transferred in a cell–cell contact-dependent way. Melan-A peptide-pulsed, PKH67-labeled mDCs and PKH26- labeled, Melan-A–specific T cells were set into Transwell insert separated by a membrane with 5-mm pores from bottom well. Melan-A–specific CD8+ T cells set in bottom well were analyzed after 2 h by flow cytometry (Transwell mDC + T cells). In the second setting, only Melan-A–pulsed, PKH67-labeled mDCs were set into insert well separated from Melan-A–specific CD8+ T cells in bottom well (Transwell mDC). Data were analyzed by flow cytometry. In bar graphs, MFI of Ag-spe- cific maximum transfer was set to 100% (transfer). Inhibited transfer and no transfer are shown as percentage of maximum uptake of membrane frag- ments and PD-L1 of three independent experi- ments 6 SD (***p , 0.001, **p , 0.01 paired t test). B, No impact of lysis of mDCs on acquisition of surface molecules. Melan-A–pulsed, PKH67-labeled mDCs were lysed by freeze-thaw cycles and incu- bated with Melan-A–specific CD8+ T cells for 2 h (lysate of mDC). As control, Melan-A–specific CD8+ T cells were cocultured with Melan-A pep- tide-pulsed, PKH67-labeled mDCs (vital mDC). In bar graphs, MFI of Ag-specific transfer was set to 100% as maximum. Inhibited transfer and no trans- fer are shown as percentage of maximum uptake of membrane fragments and PD-L1 of three inde- pendent experiments 6 SD (ns p . 0.05, **p , 0.01 paired t test). The Journal of Immunology 749 Downloaded from http://www.jimmunol.org/

FIGURE 5. Inhibition of trogocytosis. A, Impairment of trogocytosis of membrane fragments and PD-L1 by vATPase-inhibitor CMA pretreatment of T cells and mDCs. CD8+ Melan-A–specific T cells or mDCs were pretreated overnight with CMA, which was washed out before coculture. mDCs were pulsed with Melan-A peptide and labeled with PKH67. After coculture of 2 h, uptake of membrane fragments and PD-L1 was analyzed by flow cytometry. MFI data of Ag-specific transfer were set to 100% as maximum transfer. Inhibited transfer of coculture of mDCs with CMA-pretreated mDCs [mDC by guest on October 1, 2021 (CMA)], CMA-pretreated T cells [T cells (CMA)], and no transfer is shown as percentage of maximum uptake of membrane fragments and PD-L1 of four independent experiments 6 SD (**p , 0.01, ***p , 0.001, ns p . 0.05 paired t test). B, T cell activation despite CMA pretreatment after coculture with mDCs. Melan-A–specific T cells with or without CMA pretreatment were cocultured with Melan-A–pulsed, PKH67-labeled mDCs. After 22 h, expression of activation marker CD25 and CD137 was analyzed by flow cytometry. Dot plots are gated on CD8+PKH67low lymphocytes. Shown is one representative of three independent experiments with similar results. C, Impairment of trogocytosis by fixation of APCs. Before coculture, mDCs were pulsed with Melan-A peptide, labeled with PKH67, and fixed with glutaraldehyde solution. After 2 h, transfer with fixed mDCs was analyzed by flow cytometry and presented as histograms. Coculture of unfixed mDCs with T cells served as a positive control. Negative and isotype controls are presented as filled histograms. Ac- tivation of CD8+ T cells by fixed mDCs. Melan-A–specific T cells were cocultured with Melan-A–pulsed, PKH67-labeled mDCs, fixed or unfixed. After 22 h, expression of activation markers CD25 and CD137 was analyzed by flow cytometry compared with unstimulated T cells shown in dot plots. Histograms are gated on CD8+PKH67low lymphocytes. Shown is one representative of three independent experiments with similar results. pulsed, PKH67-labeled APCs were fixed with glutaraldehyde and upregulated on T cells after anti-CD3/CD28 mAb stimulation. cocultured with Ag-specific T cells. As shown in Fig. 5C, fixation Subsequently, sorted and Melan-A–pulsed gp100-specific T cells of mDCs resulted in a strong inhibition of the transfer of mem- were cocultured with Melan-A–specific, PD-1–expressing T cells. brane fragments and PD-L1. Of importance, DC–T cell interaction The PD-1 expression on Melan-A–specific CTL before using the was still functional because CD25 and CD137 were upregulated cells in the experiment is shown in Supplemental Fig. 2. Of im- on T cells after coculture with glutaraldehyde-fixed mDCs. These portance, annexin V and 7-AAD staining of PD-1+, Melan-A– data clearly indicate that acquisition is not merely induced by specific T cells, which were cocultured with PD-L1–acquiring disruption of molecules out of the membrane, but depends on gp100-specific T cells, revealed an increase in apoptotic cells of active interaction of APCs with T cells. 28% compared with 12% in the control setting (coculture with PD-L12 gp100-specific T cells; p = 0.0251). To confirm these Acquired PD-L1 triggers apoptosis in neighboring T cells findings, a blocking anti–PD-L1 mAb was added to the cocul- + To explore whether acquired PD-L1 on human CD8 T cells is of ture, resulting in a significant reduction of the apoptosis to 15.3% functional relevance, gp100-specific T cells were stimulated with (p = 0.0357). Together, these data clearly emphasize that acquired acq+ gp100 peptide-pulsed mDCs (PD-L1 ) or with anti-CD3/CD28 PD-L1 on Ag-specific T cells is functional and can interact with 2 beads (PD-L1 ) according to the experimental setup depicted in its receptor PD-1 on neighboring T cells to induce apoptosis. Fig. 6A. Next, CD3+CD8+ cells were sorted out of both cultures and subsequently pulsed with Melan-A peptide to ensure inter- Discussion action with Melan-A–specific neighboring T cells. Expression of Our study demonstrates a new role of trogocytosis in immune acquired PD-L1 on gp100-specific T cells remained stable during regulation via Ag-specific transfer of the functionally active coin- the experimental procedure (Fig. 6B). Of note, PD-L1 was not hibitory molecule PD-L1 onto human CD8+ T cells. Compared 750 TRANSFER OF FUNCTIONAL PD-L1 ONTO HUMAN T CELLS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. PD-L1 is still functional to trigger apoptosis after transfer. A, Experimental setup. For these experiments, T cells of a gp100-specific T cell clone were either cocultured with gp100-pulsed mDCs or stimulated with anti-CD3/CD28 beads for 2 h. Afterward, CD3+CD8+ T cells were sorted out of both cultures, the one population gp100(mDC)PD-L1acq+, and the other population gp100(beads)PD-L12. These sorted T cells were pulsed with Melan-A peptide. Afterward, either gp100(mDC)PD-L1acq+ or gp100(beads)PD-L12 sorted T cells were cocultured with Melan-A–specific T cells from a CD8+ T cell culture (CTL) expressing PD-1. After 6 h, apoptosis rate was analyzed by annexin V and 7-AAD staining in CD3+CD8+ Melan-A TM+ T cells via flow cytometry. B, PD-L1 maintenance of gp100 (mDC)PD-L1acq+ T cells during experimental procedures. Populations of both cocultures were monitored for PD-L1 after coculture, after cell sort, and after Ag pulsing. Histograms are gated on CD8+ lymphocytes. Shown is one representative of three inde- pendent experiments. C, PD-L1 triggering apoptosis in neighboring T cells. After 6-h coculture of Melan-A–specific CTL with either gp100 PD-L1acq+ T cells or gp100 PD-L12 T cells, CD8+ Melan-A TM+ CTL were analyzed for 7-AAD and annexin V double-positive cells. Shown are dot plots from one representative of three independent experiments with similar results. Bar graph shows the x-fold increase of percental apoptosis rate compared with unstimulated CD8+ Melan-A TM+ CTL with data of three independent experiments 6 SD (*p , 0.05, paired t test). with previous reports on trogocytosis focusing mostly on the This phenomenon of trogocytosis has been described for a variety transfer of stimulatory molecules, we report here on acquisition of of molecules, such as costimulatory and coinhibitory molecules, a relevant T cell-inhibitory molecule. HLA-G was described to lineage markers as CD4, and adhesion molecules (2). The reason render NK and T cells temporary to regulatory cells after acqui- for the transfer of these membrane fragments and molecules re- sition of these nonclassical MHC class I molecules (15, 21). HLA- mains unclear. One reason for the transfer of PD-L1 may be recy- G is characterized by limited polymorphism and a restricted ex- cling of the molecule, as discussed by Hudrisier and Bongrand pression to immune-privileged sites such as fetal-maternal inter- (2). Neither PD-L1 nor CD209 is located at the immunological face (38). In contrast, PD-L1 and its receptor PD-1 are expressed synapse; thus, it remains to be investigated which mechanisms on a wide variety of human cells, indicating that acquisition of regulate and trigger the transfer of molecules via trogocytosis. PD-L1 could have important regulatory function. Of note, trogocytosis was described as a phenomenon of very fast PD-L1, CD209, and membrane fragments are transferred after exchange of cell components compared with transfer via exosomes Ag recognition. The Ag-independent acquisition of low amounts of and nanotubes (39). This is in accordance with our kinetic studies membrane fragments by T cells is exclusively found on activated of the uptake of membrane patches, PD-L1, and CD209. Kinetic T cells, suggesting trogocytosis could be tested as marker for T cell studies indicate a reduction of transferred membrane molecules activation. Although human T cells are described to express PD-L1 over time. The cause of this reduction could be internalization by upon activation, expression of mRNA and translation into protein the acceptor cells as it has been described for pMHC complexes take much longer than acquisition from APCs via trogocytosis. and rituximab–Ab complexes (4, 40). The Journal of Immunology 751

We also show that T cells express surface PD-L1 neither after process is abolished by inhibition of vATPases, T cells still be- coculture with Ag-pulsed PD-L12 APCs, nor after stimulation come activated. Of note, fixation of mDCs strongly inhibited the with anti-CD3/CD28 beads, demonstrating that PD-L1 is not en- transfer process, supporting the idea of an active process between dogenously upregulated on T cells after Ag-specific activation DCs and T cells and the prerequisite of a nonstatic surface of DCs with APCs in this time frame. The acquisition of molecules is for trogocytosis. Although inhibition of trogocytosis does not restricted on the one side by the quantity of interactions with APC, impair the upregulation of activation markers, resting T cells can and on the other side presumably by saturation of the integration be triggered to perform trogocytosis by prestimulation probably of additional membrane fragments into the T cell’s surface. In due to an increase in avidity of adhesion molecules upon activa- summary, trogocytosis induces human CD8+ T cells with transient tion (21). regulatory activity via acquired PD-L1 that triggers apoptosis in The acquisition of PD-L1 by tumor-infiltrating T cells may neighboring PD-1–expressing T cells. spread the inhibitory function to the tumor environment. Many Of interest, membrane fragments and surface molecules such as melanoma cell lines are described to upregulate PD-L1 upon IFN-g PD-L1 and CD209 are not equally acquired from immature and exposition (45), hence in the case of an inflammatory immune mature DCs, which might be explained by the higher expression of response. As PD-L1 is described to play a role in chronic infec- costimulatory molecules on mDCs than on iDCs (data not shown), tions, acquired PD-L1 may contribute to exhaustion of T cell mediating a stronger activation of T cells and therefore triggering responses. trogocytosis. In accordance with this hypothesis, tumor cells, In conclusion, this study shows that coinhibitory PD-L1 can be missing costimulatory molecules, transfer less surface molecules transferred between immune cells that may influence the outcome than mDCs. A very high expression of costimulatory molecules on of immune responses during infections and malignant diseases. mDCs suppresses the inhibitory effect of the PD-L1. In contrast, Further understanding of the mechanism of trogocytosis and the Downloaded from a shift causing imbalance with a predominant PD-L1 expression on physiologic relevance in immune regulation may have important mDCs results in an overweighing inhibitory signaling of PD-L1, as implications for novel immune-based strategies. described in chronic infections (24). In case of PD-L1 acquisition by T cells, the inhibitory signaling of PD-L1 is dominant, leading Acknowledgments to apoptosis of neighboring T cells. We gratefully thank D. Gebhardt for technical support and U. Appelt for Triggering apoptosis in neighboring PD-1–expressing T cells, support in cell sorting. http://www.jimmunol.org/ trogocytosis of PD-L1 may represent a new immunoregulatory mechanism. PD-1 signaling is described to result in either anergy Disclosures or T cell deletion, depending on the amount of Ag levels: low Ag The authors have no financial conflicts of interest. levels cause deletion, and high Ag levels end up in anergy of T cells (41–43). Trogocytosis of pMHC complexes has been de- scribed in mice and humans (4, 5, 10); thus, T cells may present References acquired pMHC complexes and PD-L1 to neighboring T cells in 1. Rechavi, O., I. Goldstein, and Y. Kloog. 2009. Intercellular exchange of proteins: the immune cell habit of sharing. FEBS Lett. 583: 1792–1799. parallel. After clearance of Ag-presenting DCs and/or infected

2. Hudrisier, D., and P. Bongrand. 2002. Intercellular transfer of antigen-presenting by guest on October 1, 2021 cells, the presence of just low amounts of Ag favors apoptosis cell determinants onto T cells: molecular mechanisms and biological signifi- mediated by PD-L1 and not anergy, which is triggered in the cance. FASEB J. 16: 477–486. 3. Bona, C., R. Robineaux, A. Anteunis, C. Heuclin, and A. Astesano. 1973. presence of high amounts of Ag. Moreover, transferred adhesion Transfer of antigen from macrophages to lymphocytes. II. Immunological sig- molecules such as CD209 onto T cells may temporarily support nificance of the transfer of lipopolysaccharide. Immunology 24: 831–840. 4. Huang, J. F., Y. Yang, H. Sepulveda, W. Shi, I. Hwang, P. A. Peterson, the interaction and cross-talk with neighboring T cells. M. R. Jackson, J. Sprent, and Z. Cai. 1999. TCR-mediated internalization of PD-L1 is acquired by T cells in a cell–cell contact-dependent peptide-MHC complexes acquired by T cells. Science 286: 952–954. manner and is not induced by an uptake of floating cell fragments 5. Tsang, J. Y., J. G. Chai, and R. Lechler. 2003. by mouse CD4+ T cells involving acquired MHC class II:peptide complexes: another after lysis of targeted DCs, suggesting that this is not a random mechanism to limit clonal expansion? Blood 101: 2704–2710. process taking up surrounding molecules, but a directed transfer 6. Mostbo¨ck, S., M. Catalfamo, Y. Tagaya, J. Schlom, and H. Sabzevari. 2007. triggered by Ag recognition. Acquisition of antigen presentasome (APS), an MHC/costimulatory complex, is a checkpoint of memory T-cell homeostasis. Blood 109: 2488–2495. Together, PD-L1 and CD209 transfer matches the characteristics 7. Sabzevari, H., J. Kantor, A. Jaigirdar, Y. Tagaya, M. Naramura, J. Hodge, of trogocytosis: the transfer is fast and dependent on cell–cell J. Bernon, and J. Schlom. 2001. Acquisition of CD80 (B7-1) by T cells. J. Immunol. 166: 2505–2513. contact and on Ag recognition. Of note, bystander T cells are not 8. Game, D. S., N. J. Rogers, and R. I. Lechler. 2005. Acquisition of HLA-DR and triggered by neighboring T cells recognizing the APCs to acquire costimulatory molecules by T cells from allogeneic antigen presenting cells. Am. surface molecules (data not shown), demonstrating that trogocy- J. Transplant. 5: 1614–1625. 9. Tatari-Calderone, Z., R. T. Semnani, T. B. Nutman, J. Schlom, and H. Sabzevari. tosis is an Ag-specific process and thereby a selective tool to or- 2002. Acquisition of CD80 by human T cells at early stages of activation: chestrate immune responses. Moreover, we demonstrated that both functional involvement of CD80 acquisition in T cell to T cell interaction. J. inhibition of vATPases in T cells and fixation of APCs impair Immunol. 169: 6162–6169. 10. Fischer, K., S. Voelkl, J. Heymann, G. K. Przybylski, K. Mondal, M. Laumer, trogocytosis, suggesting that the trogocytosis process is controlled L. Kunz-Schughart, C. A. Schmidt, R. Andreesen, and A. Mackensen. 2005. by membrane dynamics. This hypothesis is supported by Hudrisier Isolation and characterization of human antigen-specific TCR alpha beta+ CD4 (2)CD82 double-negative regulatory T cells. Blood 105: 2828–2835. et al. (44), who demonstrated that trogocytosis in T cells, but not 11. Brezinschek, R. I., N. Oppenheimer-Marks, and P. E. Lipsky. 1999. Activated in B cells, is dependent on an active actin cytoskeleton. vATPases T cells acquire endothelial cell surface determinants during transendothelial are described to play a role in endocytosis, exocytosis, and mem- migration. J. Immunol. 162: 1677–1684. 12. Busch, A., T. Quast, S. Keller, W. Kolanus, P. Knolle, P. Altevogt, and A. Limmer. brane trafficking (reviewed in Ref. 36). Of interest, impairment 2008. Transfer of T cell surface molecules to dendritic cells upon CD4+ T cell of vATPases in mDCs has only a minor impact on the transfer of priming involves two distinct mechanisms. J. Immunol. 181: 3965–3973. cell components from mDCs onto T cells, whereas blockade of 13. Aucher, A., E. Magdeleine, E. Joly, and D. Hudrisier. 2008. Capture of plasma membrane fragments from target cells by trogocytosis requires signaling in vATPases in T cells results in a very strong inhibition of the T cells but not in B cells. Blood 111: 5621–5628. trogocytosis process. These results indicate that vATPases 14. Quah, B. J., V. P. Barlow, V. McPhun, K. I. Matthaei, M. D. Hulett, and C. R. Parish. 2008. Bystander B cells rapidly acquire antigen receptors from play a role in the integration of disrupted molecules from neigh- activated B cells by membrane transfer. Proc. Natl. Acad. Sci. USA 105: 4259– boring cells into the plasma membrane. Although the transfer 4264. 752 TRANSFER OF FUNCTIONAL PD-L1 ONTO HUMAN T CELLS

15. Caumartin, J., B. Favier, M. Daouya, C. Guillard, P. Moreau, E. D. Carosella, 29. Hino, R., K. Kabashima, Y. Kato, H. Yagi, M. Nakamura, T. Honjo, T. Okazaki, and J. LeMaoult. 2007. Trogocytosis-based generation of suppressive NK cells. and Y. Tokura. 2010. Tumor cell expression of programmed cell death-1 ligand 1 EMBO J. 26: 1423–1433. is a prognostic factor for malignant melanoma. Cancer 116: 1757–1766. 16. McCann, F. E., P. Eissmann, B. Onfelt, R. Leung, and D. M. Davis. 2007. The 30. Thompson, R. H., S. M. Kuntz, B. C. Leibovich, H. Dong, C. M. Lohse, activating NKG2D ligand MHC class I-related chain A transfers from target cells W. S. Webster, S. Sengupta, I. Frank, A. S. Parker, H. Zincke, et al. 2006. Tumor to NK cells in a manner that allows functional consequences. J. Immunol. 178: B7-H1 is associated with poor prognosis in renal cell carcinoma patients with 3418–3426. long-term follow-up. Cancer Res. 66: 3381–3385. 17. Rafii, A., P. Mirshahi, M. Poupot, A. M. Faussat, A. Simon, E. Ducros, E. Mery, 31. Mackensen, A., G. Carcelain, S. Viel, M. C. Raynal, H. Michalaki, F. Triebel, B. Couderc, R. Lis, J. Capdet, et al. 2008. Oncologic trogocytosis of an original J. Bosq, and T. Hercend. 1994. Direct evidence to support the immunosurveillance stromal cell induces chemoresistance of ovarian tumours. PLoS One 3: e3894. concept in a human regressive melanoma. J. Clin. Invest. 93: 1397–1402. 18. Beum, P. V., D. A. Mack, A. W. Pawluczkowycz, M. A. Lindorfer, and 32. Meidenbauer, N., J. Marienhagen, M. Laumer, S. Vogl, J. Heymann, R. Andreesen, R. P. Taylor. 2008. Binding of rituximab, trastuzumab, cetuximab, or mAb T101 and A. Mackensen. 2003. Survival and tumor localization of adoptively transferred to cancer cells promotes trogocytosis mediated by THP-1 cells and monocytes. J. Melan-A-specific T cells in melanoma patients. J. Immunol. 170: 2161–2169. Immunol. 181: 8120–8132. 33. Salter, R. D., D. N. Howell, and P. Cresswell. 1985. Genes regulating HLA class 19. Riond, J., J. Elhmouzi, D. Hudrisier, and J. E. Gairin. 2007. Capture of mem- I antigen expression in T-B lymphoblast hybrids. Immunogenetics 21: 235–246. brane components via trogocytosis occurs in vivo during both dendritic cells and 34. Fischer, K., S. Voelkl, J. Berger, R. Andreesen, T. Pomorski, and A. Mackensen. target cells encounter by CD8(+) T cells. Scand. J. Immunol. 66: 441–450. 2006. Antigen recognition induces phosphatidylserine exposure on the cell 20. Machlenkin, A., R. Uzana, S. Frankenburg, G. Eisenberg, L. Eisenbach, surface of human CD8+ T cells. Blood 108: 4094–4101. J. Pitcovski, R. Gorodetsky, A. Nissan, T. Peretz, and M. Lotem. 2008. Capture 35. Geijtenbeek, T. B., R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, of tumor cell membranes by trogocytosis facilitates detection and isolation of G. J. Adema, Y. van Kooyk, and C. G. Figdor. 2000. Identification of DC-SIGN, tumor-specific functional CTLs. Cancer Res. 68: 2006–2013. a novel dendritic cell-specific ICAM-3 receptor that supports primary immune 21. LeMaoult, J., J. Caumartin, M. Daouya, B. Favier, S. Le Rond, A. Gonzalez, and responses. Cell 100: 575–585. E. D. Carosella. 2007. Immune regulation by pretenders: cell-to-cell transfers of 36. Marshansky, V., and M. Futai. 2008. The V-type H+-ATPase in vesicular traf- HLA-G make effector T cells act as regulatory cells. Blood 109: 2040–2048. ficking: targeting, regulation and function. Curr. Opin. Cell Biol. 20: 415–426. 22. Chemnitz, J. M., R. V. Parry, K. E. Nichols, C. H. June, and J. L. Riley. 2004. 37. Huss, M., and H. Wieczorek. 2009. Inhibitors of V-ATPases: old and new SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of players. J. Exp. Biol. 212: 341–346. programmed death 1 upon primary human T cell stimulation, but only receptor 38. Carosella, E. D., P. Moreau, J. Le Maoult, M. Le Discorde, J. Dausset, and Downloaded from ligation prevents T cell activation. J. Immunol. 173: 945–954. N. Rouas-Freiss. 2003. HLA-G molecules: from maternal-fetal tolerance to 23. Carter, L., L. A. Fouser, J. Jussif, L. Fitz, B. Deng, C. R. Wood, M. Collins, tissue acceptance. Adv. Immunol. 81: 199–252. T. Honjo, G. J. Freeman, and B. M. Carreno. 2002. PD-1:PD-L inhibitory 39. Davis, D. M. 2007. Intercellular transfer of cell-surface proteins is common and pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2. Eur. can affect many stages of an immune response. Nat. Rev. Immunol. 7: 238–243. J. Immunol. 32: 634–643. 40. Pham, T., P. Mero, and J. W. Booth. 2011. Dynamics of macrophage trogocytosis 24. Shen, T., X. Chen, Y. Chen, Q. Xu, F. Lu, and S. Liu. 2010. Increased PD-L1 of rituximab-coated B cells. PLoS One 6: e14498. expression and PD-L1/CD86 ratio on dendritic cells were associated with im- 41. Tsushima, F., S. Yao, T. Shin, A. Flies, S. Flies, H. Xu, K. Tamada, D. M. Pardoll,

paired dendritic cells function in HCV infection. J. Med. Virol. 82: 1152–1159. and L. Chen. 2007. Interaction between B7-H1 and PD-1 determines initiation http://www.jimmunol.org/ 25. Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe, and reversal of T-cell anergy. Blood 110: 180–185. G. J. Freeman, and R. Ahmed. 2006. Restoring function in exhausted CD8 42. Goldberg, M. V., C. H. Maris, E. L. Hipkiss, A. S. Flies, L. Zhen, R. M. Tuder, T cells during chronic viral infection. Nature 439: 682–687. J. F. Grosso, T. J. Harris, D. Getnet, K. A. Whartenby, et al. 2007. Role of PD-1 26. Freeman, G. J., E. J. Wherry, R. Ahmed, and A. H. Sharpe. 2006. Reinvigorating and its ligand, B7-H1, in early fate decisions of CD8 T cells. Blood 110: 186– exhausted HIV-specific T cells via PD-1-PD-1 ligand blockade. J. Exp. Med. 192. 203: 2223–2227. 43. Parish, I. A., and S. M. Kaech. 2009. Diversity in CD8(+) T cell differentiation. 27. Francisco, L. M., V. H. Salinas, K. E. Brown, V. K. Vanguri, G. J. Freeman, Curr. Opin. Immunol. 21: 291–297. V. K. Kuchroo, and A. H. Sharpe. 2009. PD-L1 regulates the development, 44. Hudrisier, D., A. Aucher, A. L. Puaux, C. Bordier, and E. Joly. 2007. Capture of maintenance, and function of induced regulatory T cells. J. Exp. Med. 206: target cell membrane components via trogocytosis is triggered by a selected set 3015–3029. of surface molecules on T or B cells. J. Immunol. 178: 3637–3647. 28. Amarnath, S., C. M. Costanzo, J. Mariotti, J. L. Ullman, W. G. Telford, 45. He, Y. F., X. H. Wang, G. M. Zhang, H. T. Chen, H. Zhang, and Z. H. Feng.

V. Kapoor, J. L. Riley, B. L. Levine, C. H. June, T. Fong, et al. 2010. Regulatory 2005. Sustained low-level expression of interferon-gamma promotes tumor de- by guest on October 1, 2021 T cells and human myeloid dendritic cells promote tolerance via programmed velopment: potential insights in tumor prevention and tumor immunotherapy. death ligand-1. PLoS Biol. 8: e1000302. Cancer Immunol. Immunother. 54: 891–897.