T Cells Facilitates Identification of Tumor-Reactive Antigens Acquired

Imprinting of Lymphocytes with Melanoma Antigens Acquired by Trogocytosis Facilitates Identification of Tumor-Reactive T Cells This information is current as of September 25, 2021. Galit Eisenberg, Ronny Uzana, Aviad Pato, Shoshana Frankenburg, Sharon Merims, Eitan Yefenof, Soldano Ferrone, Tamar Peretz, Arthur Machlenkin and Michal Lotem

J Immunol 2013; 190:5856-5865; Prepublished online 26 Downloaded from April 2013; doi: 10.4049/jimmunol.1202879 http://www.jimmunol.org/content/190/11/5856 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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Imprinting of Lymphocytes with Melanoma Antigens Acquired by Trogocytosis Facilitates Identiﬁcation of Tumor-Reactive T Cells

Galit Eisenberg,* Ronny Uzana,* Aviad Pato,* Shoshana Frankenburg,* Sharon Merims,* Eitan Yefenof,† Soldano Ferrone,‡ Tamar Peretz,* Arthur Machlenkin,*,1 and Michal Lotem*,1

Trogocytosis is a contact-dependent intercellular transfer of membrane fragments and associated molecules from APCs to effector lymphocytes. We previously demonstrated that trogocytosis also occurs between tumor target and cognate melanoma Ag- specific cytotoxic T cells (CTL). In this study, we show that, following trogocytosis, immune effector cells acquire molecular components of the tumor, including surface Ags, which are detectable by specific mAbs. We demonstrate that CD8+ and CD4+ T cells Downloaded from from melanoma patients’ PBMC and tumor-infiltrating lymphocytes (TIL) capture melanoma Ags, enabling identification of trogocytosing lymphocytes by staining with Ag-specific Abs. This finding circumvents the necessity of tumor prelabeling, which in the past was mandatory to detect membrane-capturing T cells. Through the detection of melanoma Ags on TIL, we sorted trogocytosing T cells and verified their preferential reactivity and cytotoxicity. Furthermore, tumor Ag–imprinted T cells were detected at low frequency in fresh TIL cultures shortly after extraction from the tumor. Thus, T cell imprinting by tumor Ags may allow the enrichment of melanoma Ag-specific T cells for research and potentially even for the adoptive immunotherapy of http://www.jimmunol.org/ patients with cancer. The Journal of Immunology, 2013, 190: 5856–5865.

he key to successful adoptive cell therapy of metastatic are transferred from the former to the latter. As a result, the re- cancer is the generation of tumor Ag-specific cytotoxic cipient cell acquires membrane fragments containing molecular T T cells (CTL) capable of targeting and destroying the components of the APC. These molecules comprise a diversity of tumor. The major challenge of this approach is the ability to identify cell surface receptors and ligands. For example, the transfer of MHC and isolate a specific CTL population with a spectrum of Ag spe- class II, programmed cell death ligand 1, CD80, and HLA-G from cificities (1). Recently, it became evident that the interaction be- target cells and its subsequent effect on the capturing effector cell by guest on September 25, 2021 tween CTL effector and target cells goes along with an exchange have been documented (6, 10–13). of cell membrane material, a process denoted trogocytosis (2, 3). We and others have described trogocytosis between melanoma- Trogocytosis is a contact-dependent intercellular transfer of mem- specific CTL and their cognate tumor targets (11, 14). Our studies brane fragments and molecules, originally described in lymphocytes have shown that CTL populations integrating melanoma mem- (4). It involves all types of immune cells, including B cells, CD8+, brane patches on their surface have stronger cytotoxic activity CD4+, g-d T cells, and NK cells, while interacting with professional than nontrogocytosing lymphocytes (15). We suggested that the APCs (5–9). During this process, molecules localized in the ad- detection of trogocytosis-capable T cells could be used for the herence plate formed between an APC and an immune effector cell isolation of lymphocytes for potential therapeutic application. Beadling and Slifka (16) were the first to propose that trogocytosis may be used as a tool for recognizing and monitoring Ag- *Sharett Institute of Oncology, Hadassah Medical Organization, Jerusalem 91120, Israel; †Lautenberg Center for General and Tumor Immunology, Hebrew University specific T cells. They named their approach T cell recognition of of Jerusalem, Jerusalem 91120, Israel; and ‡Department of Surgery, Massachusetts APCs by protein transfer. T cell recognition of APCs by protein General Hospital, Boston, MA 02114 transfer is based on prelabeling of target cells prior to their en- 1 A.M. and M.L. share senior authorship of this work. counter with T cells, thus enabling detection of a full range of Received for publication October 16, 2012. Accepted for publication March 21, reactive T cells. However, the assay cannot be performed in the 2013. absence of an antigenic protein loaded onto prelabeled APCs. This work was supported by the Dr. Miriam and Sheldon G. Adelson Medical Re- Similarly, we and others (15, 17) have used tumor cell prelabeling search Foundation, the United States–Israel Binational Science Foundation, the Israel Cancer Association, the Chief Scientist of the Israel Ministry of Health (Grant 4886), to detect cognate T cells regardless of their particular specificity. and a Perlstein Family donation. This work was also supported by Public Health For all these assays, the availability of tumor cells was mandatory. Service Grants RO1 CA110249 and RO1 CA138188 awarded by the National Cancer Institute. An alternative way to monitor tumor-reactive T cells in the absence of tumor is to rely on T cell activation markers (1, 18). However, Address correspondence and reprint requests to Dr. Arthur Machlenkin and Dr. Michal Lotem, Sharett Institute of Oncology, Hadassah Medical Organization, these markers do not only detect tumor-Ag–specific lymphocytes, P.O. Box 12000, Jerusalem 91120, Israel. E-mail addresses: [email protected] but also all other activated effectors. (A.M.) and [email protected] (M.L.) In the current study, we outline a direct approach for identifying The online version of this article contains supplemental material. tumor-reactive T cells. We assumed that CTLs interacting with Abbreviations used in this article: CSPG4, chondroitin sulfate proteoglycan 4; rhIL- melanoma cells would trogocytose a variety of tumor Ags. Acqui- 2, human rIL-2; TIL, tumor-infiltrating lymphocyte. sition of membrane components by CTLs should confer them ex- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 pression of melanoma Ags. Thus, instead of tracing an artificial label www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202879 The Journal of Immunology 5857 acquired from a prestained target, we searched directly for easily phycocyanin-conjugated mouse anti-human NG2/MCSP (CSPG4) (R&D recognized, natural components of the tumor. Systems), mouse anti-human HMB45 (Serotec, Oxford, U.K.), FITC- Our results show that, following trogocytosis, several mela- conjugated mouse anti-human CD146, FITC-conjugated rat anti-human CD8 Ab (Serotec), FITC- or allophycocyanin-conjugated mouse anti- noma Ags were detected on lymphocytes, and that these Ag- human CD8, PE- or FITC-conjugated mouse anti-human CD4, PE-con- imprinted lymphocytes could exert enhanced melanoma-specific jugated mouse anti-human IFN-g, FITC- conjugated mouse anti-human cytotoxicity. We therefore suggest that T cell imprinting with CD107A (LAMP-1) (all from eBioscience, San Diego, CA), Pacific blue– tumor Ags can be used as a tool for the detection and isolation of conjugated mouse anti-human CD107 (BioLegend), allophycocyanin- mouse anti-human CD137 (BD Pharmingen), and allophycocyanin or melanoma Ag-specific CTLs with improved tumor-killing DyLight 649-conjugated donkey anti-mouse IgG (Jackson ImmunoR- capabilities. esearch Laboratories, West Grove, PA) were purchased from the indicated companies. Materials and Methods Ag transfer assay and flow cytometry Tumor cell lines Lymphocytes were cocultured with melanoma cells (1–5 3 105 lymphocytes/ Melanoma cell lines M171 and M579 (HLA-A2 negative) were estab- well in 200 ml final vol) in U-shape 96-well plates in a 1:1 E:T ratio. lished at the Sharett Institute of Oncology, Hadassah Medical Organi- Where specified, peptide-pulsed (1 mg/ml) T2 cells were cocultured zation (Jerusalem, Israel) (19). The M579-A2 clone is a stable HLA-A2+ with the lymphocytes. Following incubation for 2 h at 37˚C, cells were transfectant of M579 cells, which expresses HLA-A2 following transfec- washed twice in FACS buffer consisting of PBS supplemented with 0.5% tion with pcDNA3-HLA-A2 plasmid. The 624mel (HLA-A2+) was a gift of BSA and 0.09% sodium azide. Ag transfer was monitored by concomi- M. Parkhurst (Surgery Branch, National Institutes of Health, Bethesda, tant staining of cells with the indicated Abs for 30 min at 4˚C, followed by washing in FACS buffer. Unconjugated Abs were labeled with a

MD). Downloaded from secondary Ab. The percentage of melanoma Ag imprinting was defined All cell lines were cultured in complete medium consisting of DMEM + + supplemented with 10% FCS, 25 mmol/L HEPES, 2 mmol/L glutamine, as percentage of melanoma-Ag CD8 cells, gated on lymphocytes, fol- and combined antibiotics (all from Invitrogen Life Technologies). Table I lowing doublets exclusion (23). The gating strategy is shown in Supple- mental Fig. 1. summarizes data on the melanoma cell lines used in this work. T2 is 3 5 a TAP-2–deficient lymphoblastoid cell line of HLA-A2 genotype. The In the indicated experiments, 1 10 cells of the four following cell study was approved by the Institutional Review Board, and all patients lines were cocultured in the same wells: MART-126–35–reactive T cells gave their informed consent prior to initiation of melanoma and lymphocyte (2D6), a nonrelevant MUC-163–71–specific T cell line (line 33), mela- cell cultures. noma cells (526mel), and MUC-163–71 peptide-pulsed T2 cells (used for http://www.jimmunol.org/ activation of the MUC-1–specific T cells). Following incubation for 2 h at Cloning of peptide-specific tumor-infiltrating lymphocytes 37˚C, cells were washed twice with FACS buffer and labeled with the indicated Abs. To enable separate gating, the two lymphocyte populations Tumor-infiltrating lymphocyte (TIL) microcultures were initiated and ex- were prelabeled with PKH67 (anti–MART-1, green; Sigma-Aldrich) and panded from tumor specimens taken from resected metastases of melanoma PKH26 (anti-MUC1, red; Sigma-Aldrich), according to the manufacturer’s patients, as described (20). Lymphocytes were cultured in complete me- instructions. dium consisting of RPMI 1640 supplemented with 10% heat-inactivated In other experiments, PKH26-labeled MART-126–35–reactive T cells human AB serum, 6000 IU/ml human rIL-2 (rhIL-2; Chiron, Amsterdam, (2D6) were mixed at different ratios with a nonrelevant MUC-163–71– The Netherlands), 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 25 specific T cell line (line 33) prelabeled with Cell Proliferation Dye eFluor mmol/L HEPES, 50 mmol/L 2-ME, and combined antibiotics (Invitrogen 450 (eBioscience; according to manufacturer’s instructions, 1 3 105 Life Technologies). total lymphocytes in the culture). The cell mix contained a rising per- by guest on September 25, 2021 On day 14 of TIL initiation, the lymphocytes were washed with PBS, centage of MUC-1–specific lymphocytes and a complementary decreasing resuspended in PBS supplemented with 0.5% BSA, and stained with FITC- percentage of MART-1–specific T cells. The lymphocyte mix was cocul- conjugated HLA-A*0201/MART-126–35 dextramer (Immudex, Copenha- tured with melanoma cells (M579-A2) at a 1:1 E:T ratio. Following in- gen, Denmark) for 30 min at 4˚C. The lymphocytes were then incubated cubation for 2 h at 37˚C, cells were washed twice with FACS buffer and with allophycocyanin-conjugated mouse anti-human CD8 (eBioscience) labeled simultaneously with the indicated Abs. for an additional 30 min at 4˚C and washed. CD8+ lymphocytes, positively For intracellular cytokine assay, lymphocyte and melanoma cells were stained by the dextramer (CD8+/dextramer+ cells), were sorted by a BD cocultured 1:1 for 6 h at 37˚C. Cytofix/Cytoperm Plus Kit with Brefeldin A FACSAria (BD Biosciences) and directly cloned at one or two cells/well in (eBioscience) was used according to the manufacturer’s recommendations. 96-well plates in the presence of ortho–anti-CD3 (30 ng/ml; eBioscience), Brefeldin A (10 mg/ml) was added for the last 4 h of coculture to enable rhIL-2 (6000 IU/ml), and 4 Gy-irradiated 5 3 104 allogeneic PBMCs as intracellular proteins to accumulate. Then the cells were harvested, resus- feeder cells. Five days later, rhIL-2 (6000 IU/ml) was added and renewed pended in FACS buffer, and surface stained with fluorophore-conjugated anti- every 2 d thereafter. On day 14, the clones were assayed for IFN-g se- CD8 mAbs for 30 min at 4˚C. Following permeabilization with Cytofix/ cretion in a peptide-specific manner following their coincubation with Cytoperm buffer, intracellular IFN-g or TNF-a was detected with a mouse MART-126–35–pulsed T2 cells (peptides were commercially synthesized anti-human IFN-g or TNF-a mAb, respectively (eBioscience). All samples and purified [.95%] by reverse-phase HPLC by Biomer Technology) were subjected to flow cytometry on a BD LSRII (BD Biosciences). The data using commercially available ELISA reagents (R&D Systems, Minneap- were analyzed using FCS Express software (De Novo Software, Los olis, MN). The MART-126–35–reactive clones were further expanded in Angeles, CA). a second-round exposure to ortho–anti-CD3 (30 ng/ml) and (6000 IU/ml) rhIL-2 in the presence of 50-fold excess irradiated feeder cells. A similar Confocal microscopy protocol was performed to obtain a gp100 -reactive clone. Table II 154–162 Lymphocytes were cocultured with melanoma cells for 2 h at 37˚C and summarizes data on lymphoid cell lines used in this work. labeled as described for the Ag capture assay. Images were obtained by PBMCs a Zeiss LSM 510 META confocal microscope (Zeiss, Jena, Germany) and analyzed using LSM510 software version 3.2 (Zeiss). PBMCs were obtained from melanoma patients by cytopheresis, followed by centrifugation on Ficoll-Paque Plus gradient (Amersham, Uppsala, Cell sorting of melanoma Ag–imprinted TIL Sweden). Cells were cryopreserved until future use. Because melanoma- + reactive T cells in the peripheral blood are of low frequency that is of- Following coincubation with melanoma cells, tumor Ag-positive CD8 + ten beyond detection, a 12-d in vitro stimulation assay was used to increase T cells and tumor Ag-negative CD8 T cells from bulk PBMC or TIL their ratio. PBMCs were cultured for 12 d in the presence of irradiated population were sorted by BD FACSAria (BD Biosciences). Sorted cells autologous melanoma cells. were expanded in vitro with ortho-anti-CD3 (30 ng/ml) and rhIL-2 (6000 IU/ml) in the presence of 50-fold excess irradiated feeder cells. A MUC-163–71–specific T cell line was generated from melanoma patient PBMCs by repeated peptide stimulation, as described (21). Cytotoxicity assay Abs In vitro cytotoxicity assays were performed as described (24). Briefly, The mouse mAb 225.28, which recognizes chondroitin sulfate proteoglycan lymphocytes were washed, resuspended in complete medium, and mixed at 4 (CSPG4), was developed and characterized, as described (22). Allo- different ratios (20:1, 10:1, 5:1, 2.5:1, and 1.25:1 E:T) with 5000 35SL- 5858 FUNCTIONAL CTL IDENTIFICATION BY TUMOR Ag IMPRINTING methionine–labeled melanoma cells. CTL assays were done in U-shaped Results microtiter wells at 37˚C for 5 h, in a 5% CO2 atmosphere. Percentage of Melanoma-specific T cells acquire cognate and noncognate specific lysis was calculated as follows: percentage of lysis = ([cpm in ex- perimental well 2 cpm spontaneous release]/[cpm maximal release 2 cpm tumor Ags during trogocytosis 3 spontaneous release]) 100. During trogocytosis, membrane patches carrying an array of different molecules are transferred from target cells to immune effector Caspase-3 cleavage CTL assay cells. We hypothesized that trogocytosis enables the transfer of Intracellular staining of cleaved caspase 3 was performed, as described (25). both cognate and noncognate tumor Ags. To test this possibility, Briefly, melanoma cells were stained with CellTrace Far Red DDAO-SE (Life we focused on defined proteins known to be melanoma specific. Technologies), according to the manufacturer’s protocol. Following repeated washing with PBS, CellTrace Far Red DDAO-SE–labeled melanoma cells were The melanosomal Ag gp100 (26), CSPG4 (27), and CD146 cocultured with effector lymphocytes for 1 h at a 1:1 ratio. The cells were then (melanoma cell adhesion molecule) (28) were selected because harvested and washed twice with PBS containing 1% BSA. Following fixation they are easily detected on melanoma cells using commercially and permeabilization (according to eBioscience protocol), intracellular cleaved caspase-3 was labeled with rabbit anticleaved caspase-3-PE (BD Pharmingen) available Abs (Fig. 1A). These Ags are normally not detected for 30 min at 4˚C. Appropriate isotype control was used. Cells were washed on T lymphocytes. twice with permeabilization buffer, resuspendedinFACSbuffer,subjectedto To follow the trogocytosis of melanoma Ags, we cocultured flow cytometry, and analyzed, as above. melanoma targets (Table I) with melanoma-specific CTLs (Table II). Fig. 1B shows capture of CSPG4 by gp100 -specific Statistical analysis 154–162 T cells (line 1G2). Note that even though 1G2 cells are gp100

Statistical analyses were performed with the Instat Biostatistics software speciﬁc, they capture CSPG4 when coincubated with either Downloaded from (GraphPad Software, San Diego, CA). Pearson linear regression analysis 2 624mel or M579-A2, HLA-A2–matched melanoma cells (22 and was used for correlation studies and presented as R values. Paired t test was + + used for group comparisons. All p values are two sided and considered 50% CSPG4 /CD8 cells, respectively). The 1G2 lymphocytes 2 signiﬁcant at the 0.05 level. cocultured with HLA-A2–negative (HLA-A2 ) melanoma cells http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Ag-specific T cell clones and lines acquire a variety of melanoma Ags by trogocytosis. (A) Expression of CSPG4 and gp100 on human melanoma cell lines was detected by flow cytometry using specific mAbs and FITC-conjugated secondary Ab. Expression of CD146 was detected using FITC-conjugated mouse anti-human CD146. Gray lines: secondary Ab alone or isotype control; black lines: anti-CSPG4, gp100, or CD146, as indicated. + + 2 (B) The 1G2 (HLA-A2 /gp100154–162–reactive) T cell line was cocultured with HLA-A2 (M579-A2 and 624mel)orHLA-A2 (M171 and M579) melanoma cells. CSPG4 was detected on CD8+ lymphocytes by staining with specific mAbs, followed by flow cytometry. (C) The 2E2, 2C7, and 1H8 (all

A2/MART-126–35–reactive) T cell clones were cocultured and analyzed, as described in (B). (D) The 1H8 T cell clone was cocultured with melanoma cell lines, as described in (B). Presence of gp100 and CD146 on CD8+ cells was monitored by ﬂow cytometry. One representative experiment of ﬁve performed is shown. (E)

The four following cell lines were cocultured: MART-126–35–reactive T cells (2D6), a nonrelevant MUC-163–71–specific T cell line (line 33), melanoma cells + (526mel), and MUC-163–71 peptide-pulsed T2 cells. CSPG4 and CD107Awere detected on CD8 lymphocytes by staining with specific mAbs, followed by flow cytometry. To enable separate gating, the two lymphocyte populations, 2D6 and line33, were prelabeled with PKH67 and PKH26, respectively. The Journal of Immunology 5859

Table I. Characterization of melanoma cell lines Table II. Characterization of lymphoid cell lines

Melanoma Cell Lines T Cell Lines (all HLA-A2+)

Name HLA-A2 Melanoma Ag Expression Name Peptide Speciﬁcity

+ + M171 No MART-1 , gp100 1G2 gp100154–162 + + M579 No MART-1 , gp100 2E2 MART-126–35 + + M579-A2 Yes MART-1 , gp100 2C7 MART-126–35 + + 624mel Yes MART-1 , gp100 1H8 MART-126–35 + + 526mel Yes MART-1 , gp100 2D6 MART-126–35 + + 888mel No MART-1 , gp100 Line 33 MUC-163–71

(M171 and M579) did not stain for the Ag. Similar results were and MHC-matched melanoma cells. The results (Fig. 1E) dem- obtained when Ag transfer was monitored by using three TIL- onstrate that, whereas melanoma-specific T cells capture CSPG4, activated MUC-1–specific T cells do not acquire the derived clones specific for MART-126–35 (2E2, 2C7, and 1H8). A considerable percentage of CSPG4+-imprinted CD8+ lym- tumor Ag. These results confirm that only the melanoma-specific phocytes was detected following incubation with M579-A2 lymphocytes in the mixture acquire Ag. melanoma cells (ranging from 10 to 70%) (Fig. 1C). We con-

ﬁrmed that melanoma Ag detection on T cells is not unique to Downloaded from CSPG4 by analyzing two additional melanoma Ags: gp100 and CD146 (Fig. 1D). Because CD146 has been previously detected on a small subset of effector memory lymphocytes in the peripheral blood (29), we veriﬁed that this Ag was not endoge- nously expressed on resting or activated lymphocytes (data not

shown). http://www.jimmunol.org/ To ascertain that trogocytosis is not the result of cell activation per se, we evaluated trogocytosis by activated nonmelanoma- speciﬁc T cells that were mixed with melanoma-speciﬁc T cells by guest on September 25, 2021

FIGURE 3. CD8+ and CD4+ bulk lymphocyte populations acquire melanoma Ag by trogocytosis. (A) PBMCs from melanoma patient 171 were cultured for 12 d in the presence of irradiated autologous melanoma (M171). Afterward, the cells were cocultured with autologous (M171) or irrelevant (624mel) melanoma cells. CSPG4 was monitored on CD8+ lymphocytes by staining with specific mAbs, followed by flow cytometry. (B) PBMCs from melanoma patient 579 were cultured for 12 d in the presence of irradiated FIGURE 2. Ag-specific T cells acquire melanoma Ags by trogocytosis. autologous melanoma (M579). Afterward, the cells were cocultured with (A–C) Confocal microscopy of melanoma Ag–imprinted CD8+ T cell autologous (M579) or irrelevant (M171) melanoma cells. IFN-g secretion of clones after coincubation with HLA-A2–matched melanoma. (A) Capture nonstimulated and 12-d–stimulated lymphocytes after restimulation with of gp100; (B, C) capture of CSPG4. (D) Coculture of T cell clones with 624mel cells or autologous melanoma (M579, filled bar) was evaluated by HLA-A2–mismatched melanoma. Green, CD8+ lymphocytes; red, gp100 ELISA. (C) Presence of CSPG4 was monitored on CD4+ lymphocytes de- or CSPG4 on melanoma cells and imprinted lymphocytes. Arrows point to rived from PBMCs (patient 579) 12 d after stimulation with autologous tumor Ag on lymphocytes. Scale bars for (A–C), 10 mM; (D), 20 mM. melanoma by staining with specific mAbs, followed by flow cytometry. 5860 FUNCTIONAL CTL IDENTIFICATION BY TUMOR Ag IMPRINTING

The observation that melanoma-imprinted lymphocytes could be anoma Ags (Fig. 3A, 3C). CSPG4 was detected on 19% of in vitro directly detected by double staining was also confirmed by confocal expanded CD8+ T cells (Fig. 3A). This relatively high proportion microscopy, using anti-CSPG4 and anti-gp100 Abs in conjunction of CSPG4-imprinted T cells postexpansion reflects a 20-fold in- with anti-CD8 (Fig. 2). Coculturing with HLA-A2–matched mela- crease in their frequency. This outcome demonstrates the efficacy noma cells (Fig. 2A–C), but not with HLA-A2–mismatched mela- of a single melanoma Ag to expose the diverse population of noma cells (Fig. 2D), revealed a red-dotted staining of melanoma melanoma-expanded T cells. In another experiment with a different Ag–imprinted CTLs. patient, the 12-d coculture assay resulted in preferential expansion These results demonstrate that T cells with different antigenic of CD4+ T cells (Fig. 3B, 3C), thus providing an oppor- specificities decorate themselves with an array of melanoma Ags as tunity to evaluate trogocytosis of tumor Ag by CD4 lymphocytes. the result of T cell–tumor interaction. The latter cells exhibited hyperreactivity against the tumor as measured by IFN-g secretion (Fig. 3B), and 38% of them were im- PBLs of melanoma patients contain melanoma-reactive T cells printed with CSPG4 (Fig. 3C). Class II expression in melanoma that can be detected by trogocytosis in vitro M579 was confirmed by flow cytometry (Supplemental Fig. 2). In the aforementioned experiments,weusedmelanomaAg-specific These findings indicate that patient’s PBLs contain tumor Ag- T cell clones that are homogenous in their antigenic specificity specific CD8 and CD4 effectors that could be identified by and therefore an easier system to demonstrate a subtle phenom- staining of the acquired tumor Ags and expanded for potential therapeutic use. enon. However, PBLs of melanoma patients are often characterized

by low frequency of melanoma-reactive cells. We therefore asked Transfer of melanoma Ags detected in bulk TIL Downloaded from whether Ag transfer could be detected in PBLs following trogo- Detection of melanoma Ag-specific T cells in patient’s PBLs by cytosis with autologous melanoma cells. To ensure that melanoma- trogocytosis requires their preincubation with melanoma cells. reactive T cells go beyond the detection threshold, they were Unlike PBLs, TIL reside in the tumor and are therefore in close stimulated in vitro with autologous melanoma cells for 12 d. Under physical contact with cancer cells. Hence, lymphocytes derived such circumstances, a patient’s PBL acquired expression of mel- from TIL may already carry melanoma Ags and be detectable http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 4. TIL acquire melanoma Ags from tumor cells. (A) TIL from melanoma patient 412 were cultured for 12 d in the presence of irradiated autologous melanoma. Expanded cells were cocultured with HLA-A2–matched melanoma (624mel, M579-A2) or HLA-A2– mismatched melanoma cells (M171, M579). CSPG4 was monitored on CD8+ lymphocytes by staining with specific mAbs, followed by flow cytometry. One representative experiment of three performed is shown. (B) Melanoma Ags gp100 (in patients 1 and 2) and CSPG4 (in patient 3) were monitored on CD4+ and CD8+ TIL 4 d after tumor resection from the patient (the minimal time required for TIL expansion in the culture medium). Cells were stained with specific mAbs, followed by flow cytometry. Dot plots are the results of tumor Ag and background staining for each individual patient. Bars summarize the data from all three patients. Gray bars represent background staining with secondary Ab only (patients 1 and 2) or with isotype (patient 3). Black bars represent specific staining with anti-melanoma Ag-specific Ab. The Journal of Immunology 5861 as the most valuable cell population for clinical utility. To test Melanoma Ag imprinting of CD8+ T cells correlates with whether TIL from melanoma patients can trogocytose melanoma antitumor T cell function + Ags, we incubated archived TIL from two HLA-A2 patients (412 To assess the functional relevance of Ag imprinting of tumor- and 209) with HLA-A2–matched or nonmatched melanoma cells. reactive lymphocytes, Ag capture was compared with antitumor Fig. 4A shows that 22 and 9% of CD8+ T cells from TIL 412 reactivity of a given T cell clone. Reactivity was measured by + stained positively for CSPG4 following incubation with HLA-A2+ surface expression of the degranulation marker CD107A on CD8 M579-A2 and 624mel melanomas, respectively, compared to cells. For this purpose, MART-126–35–reactive T cells (clone 2D6) #2% with the control melanomas. Similarly, gp100 was detected were mixed, at different ratios, with nonrelevant MUC-163–71– specific T cells (line 33) and cocultured with melanoma cells. To on 3.7 and 2.8% of TIL 412 and 209, in comparison with ,1% distinguish between melanoma-specific and nonspecific T cells, with nonmatched melanoma cells (Supplemental Fig. 3). The each T cell population was prelabeled with a different cell tracker, different frequencies of CSPG4 and gp100 detected could be due as specified in Materials and Methods. Tumor Ag capture and to their different densities on the melanoma cell lines or to lymphocyte cytolytic activity were analyzed based on concomi- a shorter window of expression on the lymphocytes. Using freshly tant staining of CSPG4 and CD107A on CD8+ T cells. Fig. 5A acquired TIL, we could detect melanoma Ags on unexpanded and 5B shows results of CSPG4 imprinting and CD107A ex- lymphocytes as early as 4 d postsurgery, the time frame required to pression (respectively) in melanoma-Ag–specific CD8 cells. A 2 , release an adequate amount of lymphocytes from the meshed tumor linear correlation (R = 0.99, p 0.0001) was recognized between the percentage of Ag-imprinted cells and the percentage of mel- (Fig. 4B). There was no need to incubate fresh TIL with melanoma Downloaded from anoma Ag-specific cells in the culture (Fig. 5A, left panel). A dot cells because the resected tumor tissue harbored both TIL and plot showing results from a representative lymphocyte concen- melanoma Ags. Fig. 4B summarizes data of gp100 and CSPG4 tration is shown (Fig. 5A, right panel). A similar result was ob- expression on CD8+ and CD4+ TIL in early cultures generated from tained for CD107A expression (R2 = 0.96, p , 0.0001; Fig. 5B). fresh metastatic lesions removed from three melanoma patients. These data demonstrate that the percentage of CSPG4+/CD8+ and http://www.jimmunol.org/

FIGURE 5. Melanoma Ag imprinting of CD8+ cells by guest on September 25, 2021 correlates with antitumor T cell function. MART-126–35– specific T cells (clone 2D6) were mixed with melanoma-nonrelevant, MUC-163–71–specific T cells (line 33) at the indicated ratios and cocultured with HLA-A2–matched melanoma cells (526mel) or irrelevant melanoma cells (888mel). (A) The percentage of CSPG4+/CD8+ melanoma Ag (MA)–imprinted cells of total CD8+ cells was correlated to the proportion of melanoma Ag-specific cells in the culture (MA-specific cells, R2 = 0.99, p , 0.0001) (left panel). A representative dot plot of a cell mixture consisting of 60% 2D6 clone and 40% line 33 cocultured with the indicated melanoma is presented (right panel). (B) The percentage of CD107A+/CD8+ cells of total CD8+ cells was correlated to the proportion of melanoma Ag-specific cells in the culture (R2 = 0.96, p , 0.0001) (left panel). A representative dot plot of a cell mixture consisting of 60% 2D6 clone and 40% line 33 cocultured with the indicated melanoma is shown (right panel). (C) The percentage of CSPG4+/CD8+ cells was correlated to the percentage of CD107A+/CD8+ cells (R2 = 0.98, p , 0.0001) (left panel). A representative dot blot of a cell mixture consisting of 60% 2D6 clone and 40% line 33 cocultured with the indicated melanoma is presented (right panel). One representative experiment of three is shown. 5862 FUNCTIONAL CTL IDENTIFICATION BY TUMOR Ag IMPRINTING

CD107A+/CD8+ cells correlates with the frequency of Ag-speciﬁc with only 3.5% among the CSPG4-negative/CD8+ population. CD8+ T cells. When the percentage of CSPG4+/CD8+ cells was Enrichment of cells expressing TNF-a in the CSPG4-imprinted/ compared with the percentage of CD107A+/CD8+ cells by re- CD8+ population was also detected (10 and 2% for imprinted and gression analysis, a strong linear correlation (R2 =0.98,p , negative cells, respectively; Fig. 6A, right panel). Similar results 0.0001) between the parameters was found (Fig. 5C, left panel). A were obtained for two additional melanoma patients (Fig. 6B, 6C). dot plot showing results from a representative lymphocyte con- To examine whether comparable enrichment could be achieved centration is shown (Fig. 5C, right panel). These results show that using bulk PBMC, lymphocytes from a melanoma patient were tumor Ag imprinting of CD8+ T cells correlates with cytolytic sorted to obtain CSPG4-imprinted/CD8+ and CSPG4-negative/ activity of melanoma-reactive T cells. CD8+ cells. Sorted cells were expanded as above, cocultured with autologous (M171) or irrelevant (624mel) melanoma cells, and Melanoma Ag imprinting identiﬁes T cells with increased analyzed for CD137 expression and CD107A mobilization. As antitumor reactivity shown in Supplemental Fig. 4A (left panel), 63% of CSPG4+/CD8+ To examine whether Ag imprinting can identify tumor Ag–reactive cells expressed CD137 when stimulated with the autologous mela- 2 T cells, TIL from three melanoma patients were sorted to obtain noma cells, compared with 37% among the CSPG4 /CD8+ CSPG4-imprinted CD8-positive (CSPG4+/CD8+) and CSPG42/CD8+ population. Similarly, enrichment of cells expressing the degran- cells. Sorted cells were expanded, cocultured with HLA-A2– ulation marker CD107A in the CSPG4+/CD8+ population was matched (526mel) or irrelevant (888mel) melanoma cells, and observed (Supplemental Fig. 4A, right panel). In parallel with the analyzed for intracellular cytokine production. As shown in Fig. activation markers, percentage of IFN-g and TNF-a–producing 6A (left panel), 21% of CSPG4-imprinted CTL produced IFN-g CTL increased from 17 to 41% and from 6 to 12%, respectively Downloaded from when stimulated with HLA-matched melanoma cells, compared (Supplemental Fig. 4B). http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Melanoma Ag imprinting is associated with increased production of cytokines. TIL from three melanoma patients, M431, M412, and M209 (A, B,andC, respectively), were stimulated for 12 d with HLA-A2–matched melanoma cells and sorted to CSPG4 imprinted (CSPG4+/CD8+) and CSPG4 negative (CSPG42/CD8+)byﬂow cytometry. Sorted cells were expanded in the presence of anti-CD3 Ab, cocultured with HLA- A2–matched melanoma (526mel)orirrelevant (888mel) melanoma cells, and analyzed for IFN-g and TNF-a secretion by intracellular staining. The results of one of three experiments are shown. The Journal of Immunology 5863

FIGURE 7. Lymphocytes isolated on the basis of melanoma Ag imprinting have improved antitumor cytotoxic activity. (A) TIL from melanoma patient 209 (M209) were sorted to gp100-positive and gp100- negative CD8+ T cells by flow cytometry. Sorted cells were expanded and cocultured with HLA-A2–matched (624mel) or irrelevant (M171) melanoma cells. Per- centage of specific lysis of target melanoma cells was measured using a [35S] release-based cytotoxicity assay (p = 0.016, left panel). TIL from M209 were sorted to CSPG4 imprinted (CSPG4+/CD8+)and CSPG4 negative (CSPG42/CD8+)byflowcytom- etry. Cells were cocultured with HLA-A2–matched (526mel,624mel) or irrelevant (M171) melanoma Downloaded from cells. The level of intracellular cleaved caspase-3 in melanoma cells was measured by flow cytometry (right panel). (B) As for (A), level of cleaved caspase-3 in melanomas was measured after incubation with sorted TIL M431 or (C) with sorted TIL M412. The results of one of three experiments are shown. http://www.jimmunol.org/

To determine whether tumor Ag imprinting is useful for iden- with their cognate tumor. In this manner, tumor-reactive T cells are tifying highly functional lymphocytes, we evaluated the dif- temporarily imprinted with tumor components that can be easily ferential antitumor cytotoxic activity of trogocytosing versus detected by specific Abs. The question ensuing is whether this by guest on September 25, 2021 nontrogocytosing lymphocytes. To this end, TIL from a melanoma biological phenomenon can be exploited for T cell–based immu- patient were sorted to yield gp100-imprinted (gp100+/CD8+) and notherapy. To this end, we identified and isolated melanoma- gp100-negative (gp1002/CD8+) cells. Following cell expansion, reactive T cells on the basis of tumor-Ag imprinting. This de- the two lymphocyte populations were coincubated with HLA-A2– tection method was applicable to lymphocytes of various sources, matched melanoma cells (624mel) and tested for their cytotoxic including T cell clones, PBMC, and archived and fresh TIL activity. The results of a [35S]methionine release assay showed populations. Both CD4 and CD8 T cells were imprinted with augmented killing of HLA-A2–matched melanoma by gp100- melanoma Ags following engagement with tumor targets. Most imprinted compared with gp100-negative lymphocytes (Fig. 7A, importantly, tumor-imprinted lymphocytes were functionally su- left panel). TIL from the same melanoma patient were sorted perior in comparison with their nonimprinted counterparts as de- based on imprinting by another melanoma Ag, CSPG4. Cyto- termined by cytokine secretion and antimelanoma cytotoxicity. toxic activity of CSPG4-imprinted versus CSPG4-negative CD8+ In this regard, our observation that imprinted T cells, albeit in lymphocytes was assessed based on the expression of cleaved low numbers, were identified in TIL shortly after extraction from caspase-3 in target cells (25). As shown in Fig. 7A (right panel), the tumor is encouraging, because these are the T cells most likely CSPG4-imprinted CTL exhibited superior killing of 526mel and to possess therapeutic potential for adoptive cell transfer. 624mel, compared with CSPG4-negative lymphocytes. Further- The prototypical method to produce TIL for clinical adminis- more, we performed trogocytosis-based sorting of TIL from two tration relies on the selection of tumor-reactive T cells based on IFN-g secretion, followed by further expansion. This process is additional melanoma patients, followed by comparative analy- time consuming, and does not guarantee that IFN-g secretion sis of antimelanoma CTL activity. In line with the results shown originates from the majority of the selected cells. If it originates above, sorting of T cells on the basis of their imprinting by tumor only from a few of them, a portion of the expanded cells is not Ag resulted in selection of highly functional CTLs with augmented significant for the treatment success. To shorten the time required antimelanoma cytotoxicity (Fig. 7B, 7C). Taken together, these for TIL growth, Tran et al. (30, 31) used young TIL without results demonstrate that detection of tumor compounds on TIL screening for specific tumor recognition in a markedly simplified and/or PBMC enables identification of highly reactive antimel- process and with improved phenotypic attributes. Although min- anoma T cells and could be exploited for their sorting and in vitro imally cultured young TIL survive longer, the low proportion of expansion. T cells capable of tumor recognition offsets the benefit of their improved viability. In the present work, we show that tumor- Discussion imprinted fresh TIL could be identified at day 4 posttumor re- In the current study, we show that melanoma-reactive T cells section. Because we show that trogocytosing T cells constitute a acquire unprocessed tumor Ags by trogocytosis when interacting subset with upgraded antitumor properties, isolation and expan- 5864 FUNCTIONAL CTL IDENTIFICATION BY TUMOR Ag IMPRINTING sion of TIL obtained by trogocytosis from early tumor resections 2. Huang, J. F., Y. Yang, H. Sepulveda, W. Shi, I. Hwang, P. A. Peterson, M. R. Jackson, J. Sprent, and Z. Cai. 1999. TCR-mediated internalization of could prove to be a valuable tool to enhance the efficacy of adop- peptide-MHC complexes acquired by T cells. Science 286: 952–954. tive cell transfer. 3. Stinchcombe, J. C., G. Bossi, S. Booth, and G. M. Griffiths. 2001. The immu- Previous studies documenting some aspects of trogocytosis nological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15: 751–761. entailed the labeling or modification of target cells. Lipophilic dyes 4. Bona, C., R. Robineaux, A. Anteunis, C. Heuclin, and A. Astesano. 1973. (8, 15, 32, 33), biotin (15, 16, 23), and genetic engineering of the Transfer of antigen from macrophages to lymphocytes. II. Immunological sig- target cell to produce fluorescent peptide–MHC complexes were nificance of the transfer of lipopolysaccharide. Immunology 24: 831–840. 5.LeMaoult,J.,J.Caumartin,M.Daouya, B. Favier, S. Le Rond, A. Gonzalez, used to study trogocytosis (34–36). As an alternative to these and E. D. Carosella. 2007. Immune regulation by pretenders: cell-to-cell manipulations, we propose to rely on the pool of Abs against transfers of HLA-G make effector T cells act as regulatory cells. Blood 109: tumor Ags, which are familiar, available, and easy to use for most 2040–2048. 6. Caumartin, J., B. Favier, M. Daouya, C. Guillard, P. Moreau, E. D. Carosella, immunologists. The advantage of skipping the coculture step in and J. LeMaoult. 2007. Trogocytosis-based generation of suppressive NK cells. the lymphocyte-imprinting process is unique to TIL, with the out- EMBO J. 26: 1423–1433. 7. Espinosa, E., J. Tabiasco, D. Hudrisier, and J. J. Fournie´. 2002. Synaptic transfer come being that young TIL assays could be further improved. by human gamma delta T cells stimulated with soluble or cellular antigens. J. Whereas this work deals with the early identification of tumor- Immunol. 168: 6336–6343. reactive T cells capitalizing on their ability to perform trogocytosis, 8. Poupot, M., F. Pont, and J. J. Fournie´. 2005. Profiling blood lymphocyte inter- actions with cancer cells uncovers the innate reactivity of human gamma delta Ag imprinting adds a new power to T cells: Ag presentation. In T cells to anaplastic large cell lymphoma. J. Immunol. 174: 1717–1722. addition to mechanisms used by professional APCs, that is, direct 9. Puaux, A. L., J. Campanaud, A. Salles, X. Pre´ville, B. Timmerman, E. Joly, and and cross-presentation, trogocytosis and its resultant Ag imprinting D. Hudrisier. 2006. A very rapid and simple assay based on trogocytosis to detect and measure specific T and B cell reactivity by flow cytometry. Eur. J. Immunol. is a novel avenue of Ag presentation (37). Because trogocytosis 36: 779–788. Downloaded from is typically performed by immune effector cells, it expands the 10. Zhou, G., Z. C. Ding, J. Fu, and H. I. Levitsky. 2011. Presentation of acquired repertoire of cells that can function as APCs. peptide-MHC class II ligands by CD4+ regulatory T cells or helper cells dif- ferentially regulates antigen-specific CD4+ T cell response. J. Immunol. 186: The implications of trogocytosis extend beyond Ag imprinting 2148–2155. and include the acquisition of new functions endowed by the 11. Gary, R., S. Voelkl, R. Palmisano, E. Ullrich, J. J. Bosch, and A. Mackensen. snatched biomolecules. In line with previous studies, which fo- 2012. Antigen-specific transfer of functional programmed death ligand 1 from human APCs onto CD8+ T cells via trogocytosis. J. Immunol. 188: 744–752. cused on immune-modulating molecules, Gary et al. (11) showed 12. Tatari-Calderone, Z., R. T. Semnani, T. B. Nutman, J. Schlom, and H. Sabzevari. http://www.jimmunol.org/ that Ag-specific CD8+ T cells acquire the coinhibitory pro- 2002. Acquisition of CD80 by human T cells at early stages of activation: functional involvement of CD80 acquisition in T cell to T cell interaction. J. grammed death ligand 1 from mature dendritic and tumor cells. Immunol. 169: 6162–6169. While doing so, the lymphocytes induce apoptosis on neighboring 13. Brown, R., K. Kabani, J. Favaloro, S. Yang, P. J. Ho, J. Gibson, P. Fromm, H. programmed cell death 1–expressing CD8+ T cells. Similarly, the Suen, N. Woodland, N. Nassif, et al. 2012. CD86+ or HLA-G+ can be transferred via trogocytosis from myeloma cells to T cells and are associated with tolerogenic molecule HLA-G, when acquired by T and NK cells, poor prognosis. Blood 120: 2055–2063. provides them with regulatory functions (35). Interestingly, tro- 14. Alegre, E., K. Y. Howangyin, B. Favier, J. Baudhuin, E. Lesport, M. Daouya, gocytosis was exploited in a transient protein–transfer assay to A. Gonzalez, E. D. Carosella, and J. Lemaoult. 2010. 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