A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies.

Sandra Rosskopf,Peter Steinberger,Renate S Hagedoorn,Laura T Morton,Mirjam H.M Heemskerk,Judith Leitner,Wolfgang Paster

doi:10.18632/oncotarget.24807

Sandra Rosskopf, Peter Steinberger + Show 5 more

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https://doi.org/10.18632/oncotarget.24807

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Abstract

Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.

Highlights

Adoptive transfer of T cell receptor-transgenic (TCR-tg) autologous T cells holds great promise for the treatment of tumor patients [1, 2]
To generate a cellular platform for analysis of TCRs, our previously described transcriptional reporter constructs for nuclear factor kappalight-chain-enhancer of activated B-cells (NF-κB), nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1) were introduced into the Jurkat 76 (J76) cell line [16]
The resulting J76 triple parameter reporter (TPR) were screened with phorbol 12-myristate 13-acetate (PMA) and Ionomycin and a single cell clone showing high induction of all three reporter genes (NFAT-eGFP, NF-κB-CFP and AP-1mCherry) was selected for further use (Figure 1A)

Summary

Introduction

Adoptive transfer of T cell receptor-transgenic (TCR-tg) autologous T cells holds great promise for the treatment of tumor patients [1, 2]. Since it was observed that CD58, the ligand for CD2, and CD112 and CD155, which function as ligands for CD226, are ubiquitously expressed in different cancer cell lines (Supplementary Figure 1A), we investigated whether introducing these molecules in our TCR-transgenic reporters will increase www.oncotarget.com their sensitivity for their cognate antigens. We used the J76 CMV TPR to overexpress CD2 or CD226 or both molecules (Figure 3A) and tested the reporter reactivity with the K562 HLA-A2+ and K562 HLAA2+CD80+ cells loaded with antigenic peptide (Figure 3B) Both accessory molecules significantly increased the reporter activation and an additive effect was seen when using the CD2+CD226+ J76 CMV reporter. While it was not possible to detect PRAME-specific responses against the

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