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Establishment of a novel NFAT-GFP reporter platform useful for the functional avidity maturation of HLA class II-restricted TCRs.

ABSTRACT: CD4⁺ T cells that recognize antigenic peptides presented on HLA class II are essential for inducing an optimal anti-tumor immune response, and adoptive transfer of tumor antigen-specific TCR-transduced CD4⁺ T cells with high responsiveness against tumor is a promising strategy for cancer treatment. Whereas a precise evaluation method of functional avidity, an indicator of T cell responsiveness against tumors, has been established for HLA class I-restricted TCRs, it remains unestablished for HLA class II-restricted TCRs. In this study, we generated a novel platform cell line, CD4-2D3, in which GFP reporter was expressed by NFAT activation via TCR signaling, for correctly evaluating functional avidity of HLA class II-restricted TCRs. Furthermore, using this platform cell line, we succeeded in maturating functional avidity of an HLA class II-restricted TCR specific for a WT1-derived helper peptide by substituting amino acids in complementarity determining region 3 (CDR3) of the TCR. Importantly, we demonstrated that transduction of an avidity-maturated TCR conferred strong cytotoxicity against WT1-expressing leukemia cells on CD4⁺ T cells, compared to that of its original TCR. Thus, CD4-2D3 cell line should be useful not only to evaluate TCR functional avidity in HLA class II-restricted TCRs but also to screen appropriate TCRs for clinical applications such as cancer immunotherapy.

SUBMITTER: Fujiki F

PROVIDER: S-EPMC10264488 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Establishment of a novel NFAT-GFP reporter platform useful for the functional avidity maturation of HLA class II-restricted TCRs.

Fujiki Fumihiro F Morimoto Soyoko S Nishida Yuya Y Tanii Satoe S Aoyama Nao N Inatome Miki M Inoue Kento K Katsuhara Akiko A Nakajima Hiroko H Nakata Jun J Nishida Sumiyuki S Tsuboi Akihiro A Oka Yoshihiro Y Oji Yusuke Y Sogo Shinji S Sugiyama Haruo H

Cancer immunology, immunotherapy : CII 20230320 7

CD4<sup>+</sup> T cells that recognize antigenic peptides presented on HLA class II are essential for inducing an optimal anti-tumor immune response, and adoptive transfer of tumor antigen-specific TCR-transduced CD4<sup>+</sup> T cells with high responsiveness against tumor is a promising strategy for cancer treatment. Whereas a precise evaluation method of functional avidity, an indicator of T cell responsiveness against tumors, has been established for HLA class I-restricted TCRs, it remains ...[more]

PMID: 36939853

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Project description:BackgroundMajor histocompatibility complex class-1-related protein (MR1), unlike human leukocyte antigen (HLA) class-1, was until recently considered to be monomorphic. MR1 presents metabolites in the context of host responses to bacterial infection. MR1-restricted TCRs specific to tumor cells have been described, raising interest in their potential therapeutic application for cancer treatment. The diversity of MR1-ligand biology has broadened with the observation that single nucleotide variants (SNVs) exist within MR1 and that allelic variants can impact host immunity.MethodsThe TCR from a MR1-restricted T-cell clone, MC.7.G5, with reported cancer specificity and pan-cancer activity, was cloned and expressed in Jurkat E6.1 TCRαβ- β2M- CD8+ NF-κB:CFP NFAT:eGFP AP-1:mCherry cells or in human donor T cells. Functional activity of 7G5.TCR-T was demonstrated using cytotoxicity assays and by measuring cytokine release after co-culture with cancer cell lines with or without loading of previously described MR1 ligands. MR1 allele sequencing was undertaken after the amplification of the MR1 gene region by PCR. In vivo studies were undertaken at Labcorp Drug Development (Ann Arbor, MI, USA) or Epistem Ltd (Manchester, UK).ResultsThe TCR cloned from MC.7.G5 retained MR1-restricted functional cytotoxicity as 7G5.TCR-T. However, activity was not pan-cancer, as initially reported with the clone MC.7.G5. Recognition was restricted to cells expressing a SNV of MR1 (MR1*04) and was not cancer-specific. 7G5.TCR-T and 7G5-like TCR-T cells reacted to both cancer and healthy cells endogenously expressing MR1*04 SNVs, which encode R9H and H17R substitutions. This allelic specificity could be overcome by expressing supraphysiological levels of the wild-type MR1 (MR1*01) in cell lines.ConclusionsHealthy individuals harbor T cells reactive to MR1 variants displaying self-ligands expressed in cancer and benign tissues. Described "cancer-specific" MR1-restricted TCRs need further validation, covering conserved allomorphs of MR1. Ligands require identification to ensure targeting MR1 is restricted to those specific to cancer and not normal tissues. For the wider field of immunology and transplant biology, the observation that MR1*04 may behave as an alloantigen warrants further study. .

Dataset Information

Establishment of a novel NFAT-GFP reporter platform useful for the functional avidity maturation of HLA class II-restricted TCRs.

Publications

Establishment of a novel NFAT-GFP reporter platform useful for the functional avidity maturation of HLA class II-restricted TCRs.

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