Capture of tumor cell membranes by trogocytosis facilitates detection and isolation of tumor-specific functional CTLs.

The success of adoptive cell transfer in the treatment of metastatic cancer in humans is dependent on the selection of highly active tumor-specific cytotoxic T cells. We report here that CTLs capture membrane fragments from their targets while exerting cytotoxic activity and thus gain a detectable functional signature by which they can be identified. Fluorochrome labeling or biotinylation was used to tag tumor cells. CD8(+) T cells were coincubated with the tagged targets, sorted, and functionally evaluated. Our results show that membrane capture by CD8(+) lymphocytes is T-cell receptor dependent, epitope specific, and preferentially associated with highly cytotoxic clonal subsets. CTLs that captured membranes from unmodified melanoma exhibited enhanced cytotoxic activity against tumor cell lines and autologous melanoma. In a human melanoma in vivo model, adoptive transfer of membrane-capturing, peptide-specific T cells, but not noncapturing or bulk CD8(+) T cells, inhibits tumor progression. Membrane capture is therefore a signature of antigen-specific CTLs endowed with high functional avidity and may have direct relevance in the clinical application of adoptive immunotherapy.

[1]  M. Mannie,et al.  Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition. , 1999, Journal of immunology.

[2]  D. Maric,et al.  Detection of virus-specific T cells and CD8+ T-cell epitopes by acquisition of peptide–HLA-GFP complexes: analysis of T-cell phenotype and function in chronic viral infections , 2003, Nature Medicine.

[3]  S. Rosenberg,et al.  Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[4]  F. Marincola,et al.  Expansion of Tumor-T Cell Pairs from Fine Needle Aspirates of Melanoma Metastases , 2000, The Journal of Immunology.

[5]  T. Peretz,et al.  Autologous cell vaccine as a post operative adjuvant treatment for high-risk melanoma patients (AJCC stages III and IV) , 2002, British Journal of Cancer.

[6]  E. Carosella,et al.  Immune regulation by pretenders: cell-to-cell transfers of HLA-G make effector T cells act as regulatory cells. , 2006, Blood.

[7]  P. Moreau,et al.  Trogocytosis‐based generation of suppressive NK cells , 2007, The EMBO journal.

[8]  Michael Loran Dustin,et al.  A dynamic view of the immunological synapse. , 2005, Seminars in immunology.

[9]  E. Joly,et al.  What is trogocytosis and what is its purpose? , 2003, Nature Immunology.

[10]  M. Jackson,et al.  T Cells Can Use Either T Cell Receptor or Cd28 Receptors to Absorb and Internalize Cell Surface Molecules Derived from Antigen-Presenting Cells , 2000, The Journal of experimental medicine.

[11]  M. Jackson,et al.  TCR-Mediated internalization of peptide-MHC complexes acquired by T cells. , 1999, Science.

[12]  B. Tirosh,et al.  Combined Dendritic Cell Cryotherapy of Tumor Induces Systemic Antimetastatic Immunity , 2005, Clinical Cancer Research.

[13]  Michael Loran Dustin,et al.  Cytoskeletal polarization and redistribution of cell-surface molecules during T cell antigen recognition. , 2000, Seminars in immunology.

[14]  L. Tuosto,et al.  Quantitative Contribution of CD4 and CD8 to T Cell Antigen Receptor Serial Triggering , 1997, The Journal of experimental medicine.

[15]  S Miltenyi,et al.  Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. Rosenberg,et al.  Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. , 1989, Journal of immunology.

[17]  Yoshimasa Tanaka,et al.  Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[18]  G. Ogg,et al.  Cutting edge: rapid cloning of tumor-specific CTL suitable for adoptive immunotherapy of melanoma. , 1999, Journal of immunology.

[19]  M. Raffeld,et al.  Cancer Regression and Autoimmunity in Patients After Clonal Repopulation with Antitumor Lymphocytes , 2002, Science.

[20]  F. Lemonnier,et al.  Human CTL epitopes prostatic acid phosphatase-3 and six-transmembrane epithelial antigen of prostate-3 as candidates for prostate cancer immunotherapy. , 2005, Cancer research.

[21]  S. Rosenberg,et al.  Gene Transfer of Tumor-Reactive TCR Confers Both High Avidity and Tumor Reactivity to Nonreactive Peripheral Blood Mononuclear Cells and Tumor-Infiltrating Lymphocytes1 , 2006, The Journal of Immunology.

[22]  N. Restifo,et al.  Natural selection of tumor variants in the generation of “tumor escape” phenotypes , 2002, Nature Immunology.

[23]  S. Rosenberg,et al.  Stimulation of tumor-reactive T lymphocytes using mixtures of synthetic peptides derived from tumor-associated antigens with diverse MHC binding affinities. , 2003, Journal of immunological methods.

[24]  T. Blankenstein,et al.  Adoptive tumor therapy with T lymphocytes enriched through an IFN-γ capture assay , 2001, Nature Medicine.

[25]  H. Hoogenboom,et al.  Direct detection and quantitation of a distinct T-cell epitope derived from tumor-specific epithelial cell-associated mucin using human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells. , 2002, Cancer research.

[26]  E. Joly,et al.  A very rapid and simple assay based on trogocytosis to detect and measure specific T and B cell reactivity by flow cytometry , 2006, European journal of immunology.

[27]  J. Gairin,et al.  Capture of membrane components via trogocytosis occurs in vivo during both dendritic cells and target cells encounter by CD8+ T cells , 2007, Scandinavian journal of immunology.

[28]  T. McKeithan,et al.  Peptide-Specific Intercellular Transfer of MHC Class II to CD4+ T Cells Directly from the Immunological Synapse upon Cellular Dissociation1 , 2005, The Journal of Immunology.

[29]  J. Fournié,et al.  Profiling Blood Lymphocyte Interactions with Cancer Cells Uncovers the Innate Reactivity of Human γδ T Cells to Anaplastic Large Cell Lymphoma1 , 2005, The Journal of Immunology.

[30]  T. Mosmann,et al.  Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing , 2003, Nature Medicine.

[31]  Mario Roederer,et al.  Ex vivo identification, isolation and analysis of tumor-cytolytic T cells , 2003, Nature Medicine.

[32]  C. Beadling,et al.  Quantifying viable virus-specific T cells without a priori knowledge of fine epitope specificity , 2006, Nature Medicine.

[33]  G. Griffiths,et al.  The immunological synapse of CTL contains a secretory domain and membrane bridges. , 2001, Immunity.

[34]  E. Joly,et al.  Cutting Edge: CTLs Rapidly Capture Membrane Fragments from Target Cells in a TCR Signaling-Dependent Manner1 , 2001, The Journal of Immunology.

[35]  T. Mosmann,et al.  Functions of CD8 T-cell subsets secreting different cytokine patterns. , 1997, Seminars in immunology.

[36]  C. Craddock,et al.  Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA–peptide tetramers , 2005, The Journal of experimental medicine.

[37]  P. Greenberg,et al.  Activation-induced expression of CD137 permits detection, isolation, and expansion of the full repertoire of CD8+ T cells responding to antigen without requiring knowledge of epitope specificities. , 2007, Blood.

[38]  E. Joly,et al.  A simple trogocytosis-based method to detect, quantify, characterize and purify antigen-specific live lymphocytes by flow cytometry, via their capture of membrane fragments from antigen-presenting cells , 2006, Nature Protocols.