Trogocytosis Is a Gateway to Characterize Functional Diversity in Melanoma-Specific CD8+ T Cell Clones This information is current as Ronny Uzana, Galit Eisenberg, Yael Sagi, Shoshana of October 2, 2021. Frankenburg, Sharon Merims, Ninette Amariglio, Eitan Yefenof, Tamar Peretz, Arthur Machlenkin and Michal Lotem J Immunol 2012; 188:632-640; Prepublished online 7 December 2011; doi: 10.4049/jimmunol.1101429 Downloaded from http://www.jimmunol.org/content/188/2/632 Supplementary http://www.jimmunol.org/content/suppl/2011/12/07/jimmunol.110142 http://www.jimmunol.org/ Material 9.DC1 References This article cites 50 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/188/2/632.full#ref-list-1 Why The JI? Submit online. by guest on October 2, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Trogocytosis Is a Gateway to Characterize Functional Diversity in Melanoma-Specific CD8+ T Cell Clones Ronny Uzana,* Galit Eisenberg,* Yael Sagi,† Shoshana Frankenburg,* Sharon Merims,* Ninette Amariglio,‡ Eitan Yefenof,x Tamar Peretz,* Arthur Machlenkin,*,1 and Michal Lotem*,1 Trogocytosis, the transfer of membrane patches from target to immune effector cells, is a signature of tumor–T cell interaction. In this study, we used the trogocytosis phenomenon to study functional diversity within tumor-specific T cell clones with identical TCR specificity. MART-126–35–specific CD8 T cell clones, which differed in their trogocytosis capacity (low [2D11], intermediate [2G1], high [2E2]), were generated from melanoma patients. Functional evaluation of the clones showed that the percentage of trogocytosis-capable T cells closely paralleled each clone’s IFN-g and TNF-a production, lysosome degranulation, and lysis of peptide-pulsed targets and unmodified melanoma. The highly cytotoxic 2E2 clone displayed the highest TCR peptide binding Downloaded from affinity, whereas the low-activity 2D11 clone showed TCR binding to peptide-MHC in a CD8-dependent manner. TCR analysis revealed Vb16 for clones 2E2 and 2G1 and Vb14 for 2D11. When peptide-affinity differences were bypassed by nonspecific TCR stimulation, clones 2E2 and 2D11 still manifested distinctive signaling patterns. The high-activity 2E2 clone displayed prolonged phosphorylation of ribosomal protein S6, an integrator of MAPK and AKT activation, whereas the low-activity 2D11 clone generated shorter and weaker phosphorylation. Screening the two clones with identical TCR Vb by immunoreceptor array showed higher phosphorylation of NK, T, and B cell Ag (NTB-A), a SLAM family homophilic receptor, in clone 2E2 compared http://www.jimmunol.org/ with 2G1. Specific blocking of NTB-A on APCs markedly reduced cytokine production by CD8 lymphocytes, pointing to a possible contribution of NTB-A costimulation to T cell functional diversity. This finding identifies NTB-A as a potential target for improving anti-cancer immunotherapy. The Journal of Immunology, 2012, 188: 632–640. ytotoxic CD8+ T cells specific for a single antigenic (pMHC) not only determines the activation capacity of the for- epitope can originate from one or several progenitors, mer, but also that of its clonal progeny (6). Short and loose C distinguishable by their TCR sequences (1). The structure interactions yield subclones of low potency whereas long and tight of the TCR is the major determinant of T cell functional robust- contact results in highly reactive T cells, thus leading to clonal ness, specifying its binding affinity to a cognate peptide presented diversity (2, 7). Although the mechanism underlying the func- by guest on October 2, 2021 by an appropriate MHC class I molecule (2). In vivo, many other tional imprint of T cells is not yet fully elucidated, it is clear that factors affect the actual cytotoxic capacity of T cells, including in addition to TCR affinity, other interactions take place at the their state of differentiation, shifting from naive cells to terminally initial immunological synapse, which shape T cell functional ca- differentiated effectors, length of activation (3), cytokine milieu pacity. Immunological synapse refers to the focal contact between (4), and costimulation (5). Hence, T cell competence reflects the an immune effector cell and APCs. It is a contact-secretion pro- overall outcome of all these variables. cess that leads to effector cell activation by reciprocal molecular Recently, it has been acknowledged that the initial encoun- clustering of ligands and receptors on the cytoplasmic membranes ter between a naive CD8+ T cell with its cognate peptide-MHC of the cellular partners involved (8, 9). The linkage between receptors and ligands leads to the unidirectional transfer of mem- brane fragments from APCs to effector cells, a process termed *Sharett Institute of Oncology, Hadassah Medical Organization, Jerusalem 91120, Israel; †Division of Oncology, School of Medicine, Stanford University, Stanford, CA “trogocytosis” (10–12). It is readily visible by labeling Ag-loaded 94305; ‡Cancer Research Center, Chaim Sheba Medical Center, Ramat Gan 52621, x APCs with a membrane dye that can be detected on cognate Israel; and Lautenberg Center for General and Tumor Immunology, Hebrew T cells by immunofluorescence (13). Thus, trogocytosis provides University of Jerusalem, Jerusalem 91120, Israel a useful signature for T cells that have interacted with APCs. 1A.M. and M.L. share senior authorship of this work. We have previously shown that trogocytosis also occurs between Received for publication May 16, 2011. Accepted for publication November 2, 2011. tumor-reactive CD8+ T cells and melanoma target cells (14), and This work was supported by the Israel Cancer Association, the Chief Scientist of that it is preferentially associated with a highly cytotoxic T cell the Israel Ministry of Health, and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation. population. Notably, TCR specificity was not the only determinant Address correspondence and reprint requests to Dr. Arthur Machlenkin and Dr. of trogocytosis, as a proportion of Ag-specific T cells with the Michal Lotem, Sharett Institute of Oncology, Hadassah Medical Organization, P.O. same TCR specificity did not capture target membranes. In the Box 12000, Jerusalem 91120, Israel. E-mail addresses: [email protected] present study, we aim to elucidate the reasons for the functional (A.M.) and [email protected] (M.L.) diversity within CD8+ T cells with the same TCR specificity. The online version of this article contains supplemental material. Abbreviations used in this article: LCK, lymphocyte-specific protein tyrosine kinase; NTB-A, NK, T, and B cell Ag; PLC, phospholipase C; pMHC, peptide-MHC; rhIL-2, Materials and Methods recombinant human IL-2; SLP-76, Src homology 2 domain-containing leukocyte Tumor cell lines and lymphocyte cultures protein of 76 kDa; TIL, tumor-infiltrating lymphocyte. Melanoma cell line M171 (HLA-A22/MART-1+) was established in the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 Sharett Institute of Oncology, Hadassah Medical Organization (Jerusalem, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101429 The Journal of Immunology 633 Israel) (15). 624mel (HLA-A2+/MART-1+) was a gift of M. Parkhurst cells were then harvested, resuspended in FACS buffer, and surface stained (Surgery Branch, National Institutes of Health, Bethesda, MD). T2 is a with fluorophore-conjugated anti-CD8 mAb for 30 min at 4˚C. Following TAP-2–deficient lymphoblastoid line of HLA-A2 genotype. All cell lines permeabilization with Cytofix/Cytoperm buffer, intracellular proteins were were cultured in RPMI 1640 supplemented with 10% heat-inactivated stained with one of the following mAb: anti–IFN-g-PE or -FITC, anti– FCS, 2 mmol/l L-glutamine, and combined antibiotics. Tumor-infiltrat- TNF-a-PE, anti–perforin-PE, anti-granzyme B-PE (all from eBioscience), ing lymphocyte (TIL) microcultures were initiated and expanded from and anti–TCRz-chain-PE (kind gift from M. Baniyash). Appropriate iso- tumor specimens taken from resected metastases of melanoma patients, as type controls were used. The data were analyzed using FCS Express described (16). Human lymphocytes were cultured in complete medium software (De Novo Software, Los Angeles, CA). consisting of RPMI 1640 supplemented with 10% heat-inactivated hu- man AB serum, 6000 IU/ml recombinant human IL-2 (rhIL-2; Chiron, Cytotoxicity assay Amsterdam, The Netherlands), 2 mmol/l L-glutamine, 1 mmol/l sodium In vitro cytotoxicity assays were performed as described (17). Briefly, pyruvate, 1% nonessential amino acids, 25 mmol/l HEPES (pH 7.4), 50 lymphocytes were washed, resuspended in complete medium, and mixed mmol/l 2-ME, and combined antibiotics (all from Invitrogen