title: Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells.
abstract: Adoptive T cell therapy of cancer employs a large number of ex-vivo-propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell-extrinsic and intrinsic mechanisms often diminish the in-vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll-like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand-binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin-1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti-tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR-4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR-4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)-γ, triggered the surface expression of CD25, CD69 and 4-1BB and up-regulated a panel of cytokines and chemokines. In tumour-infiltrating lymphocytes prepared from melanoma patients, caTLR-4 induced robust IFN-γ secretion in all samples tested. Furthermore, caTLR-4 enhanced the anti-melanoma cytolytic activity of tumour-infiltrating lymphocytes and augmented the secretion of IFN-γ, tumour necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) for at least 4 days post-transfection. Our results demonstrate that caTLR-4 is capable of exerting multiple T cell-enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy.
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, Kiryat, Shmona.|Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem.
authors.forename: A
authors.initials: A
authors.lastname: Pato
authors.affiliation: Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem.
authors.forename: G
authors.initials: G
authors.lastname: Eisenberg
authors.affiliation: Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem.
authors.forename: A
authors.initials: A
authors.lastname: Machlenkin
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, Kiryat, Shmona.|Department of Biotechnology, Tel-Hai College, Upper, Galilee.
authors.forename: A
authors.initials: A
authors.lastname: Margalit
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, Kiryat, Shmona.|Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: G
authors.initials: G
authors.lastname: Cafri
authors.affiliation: Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem.
authors.forename: S
authors.initials: S
authors.lastname: Frankenburg
authors.affiliation: Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem.
authors.forename: S
authors.initials: S
authors.lastname: Merims
authors.affiliation: Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem.
authors.forename: T
authors.initials: T
authors.lastname: Peretz
authors.affiliation: Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem.
authors.forename: M
authors.initials: M
authors.lastname: Lotem
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, Kiryat, Shmona.|Department of Biotechnology, Tel-Hai College, Upper, Galilee.
authors.forename: G
authors.initials: G
authors.lastname: Gross
mesh_terms: D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018925:Chemokines / Q000276:immunology / Q000378:metabolism; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D018274:Electroporation; D005434:Flow Cytometry; D015870:Gene Expression / Q000276:immunology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology / Q000378:metabolism; D020014:K562 Cells; D037181:Lectins, C-Type / Q000276:immunology / Q000378:metabolism; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014162:Transfection / Q000379:methods; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D018925:Chemokines; D016207:Cytokines; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D007371:Interferon-gamma; D053645:Interleukin-2 Receptor alpha Subunit; D037181:Lectins, C-Type; D012333:RNA, Messenger; D051197:Toll-Like Receptor 4; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D014409:Tumor Necrosis Factor-alpha; D000099192:CD69 Antigens
keywords: CD8 T lymphocytes; constitutively active TLR-4; mRNA electroporation; tumour-infiltrating lymphocytes