A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences

Kropp, Korbinian N.; Schäufele, Tim J.; Fatho, Martina; Volkmar, Michael; Conradi, Roland; Theobald, Matthias; Wölfel, Thomas; Wölfel, Catherine

doi:10.1371/journal.pone.0238875

Cited by 6 publications

(10 citation statements)

References 35 publications

(58 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Next, we employed a tet-on system [ 62 , 63 ] to generate inducible p65 expressing MDA-MB-231 cells (Fig. 5A and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer

Kim

et al. 2022

Cell Death Dis

View full text Add to dashboard Cite

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by poor response to standard therapies and therefore unfavorable clinical outcomes. Better understanding of TNBC and new therapeutic strategies are urgently needed. ROR nuclear receptors are multifunctional transcription factors with important roles in circadian pathways and other processes including immunity and tumorigenesis. Nobiletin (NOB) is a natural compound known to display anticancer effects, and our previous studies showed that NOB activates RORs to enhance circadian rhythms and promote physiological fitness in mice. Here, we identified several TNBC cell lines being sensitive to NOB, by itself or in combination. Cell and xenograft experiments showed that NOB significantly inhibited TNBC cell proliferation and motility in vitro and in vivo. ROR loss- and gain-of-function studies showed concordant effects of the NOB–ROR axis on MDA-MB-231 cell growth. Mechanistically, we found that NOB activates ROR binding to the ROR response elements (RRE) of the IκBα promoter, and NOB strongly inhibited p65 nuclear translocation. Consistent with transcriptomic analysis indicating cancer and NF-κB signaling as major pathways altered by NOB, p65-inducible expression abolished NOB effects, illustrating a requisite role of NF-κB suppression mediating the anti-TNBC effect of NOB. Finally, in vivo mouse xenograft studies showed that NOB enhanced the antitumor efficacy in mammary fat pad implanted TNBC, as a single agent or in combination with the chemotherapy agent Docetaxel. Together, our study highlights an anti-TNBC mechanism of ROR-NOB via suppression of NF-κB signaling, suggesting novel preventive and chemotherapeutic strategies against this devastating disease.

show abstract

“…Next, we employed a tet-on system [ 62 , 63 ] to generate inducible p65 expressing MDA-MB-231 cells (Fig. 5A and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer

Kim

et al. 2022

Cell Death Dis

View full text Add to dashboard Cite

show abstract

“…After sequence verification via Sanger sequencing, 1μg of NotI-linearized plasmid was subjected to in vitro transcription using the HiScribe T7 ARCA mRNA Kit (with tailing) (New England Biolabs, Ipswich, MA, USA). For use in ELISpot, the 29.ct2 TCR was cloned as described in [6].…”

Section: Methodsmentioning

confidence: 99%

“…IFNγ-ELISpot assays were performed as described previously [6, 14]. Briefly, tumor cells were seeded (50,000 cells/well) into ELISpot plates (Millipore, Burlington, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The information in the "all_contig_annotations" files of both experiments enabled us to clone the TCRs for functional testing. To this end we used our previously established TCR cloning system [6,7] that inserts V(D)J segments upstream of murine C segments that contain an additional cysteine residue facilitating a disulfide bridge between the alpha and beta C segments. The amino acid sequences of both recombinant TCRs are provided in Figure S1.…”

Section: Tcrs Are Expressed On the Cell Surface Of T Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Incorporation of TRDV segments into TCR alpha chains

Volkmar

Fakhr

Zens

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

In human tumor models we tried to identify and clone the TCR of tumor-reactive T cells enriched in mixed lymphocyte tumor-cell cultures (MLTC). In a particular MLTC, we identified a predominant TCR beta chain using the Beta Mark Vbeta Kit, but a corresponding alpha chain could not be amplified via RT-PCR using TRAV-specific forward primers. Therefore, we applied 5'RACE to obtain the TCR alpha chain sequences. The 5'RACE product revealed an alpha chain that encompassed 89bp of the TRDV1 5'UTR, followed by the TRDV1 coding sequence joined in frame to TRAJ24. The ORF reaching from the TRDV1 start codon to the TRAC segment was intact, suggesting a functional TCR. To analyze this MLTC population in greater depth we conducted 10X VDJ sequencing. CellRanger identified the beta chain known from the Beta Mark analysis, but no corresponding alpha chain in the filtered results. The corresponding TRDV-containing TCR alpha chain could, however, be detected in the "all_contig_annotations" files. In a separate project, we performed TCR sequencing of tumor-infiltrating lymphocytes (TILs) in a murine tumor model. Also here, a predominant clonotype contained a TCR alpha chain joining Trdv2-2 in frame to Traj49. Transfection of both TCR cDNAs resulted in cell surface localization of TCR and CD3 as validated by FACS. Tumor recognition of the human, TRDV1-containing TCR could be demonstrated by IFNgamma ELISpot whereas the murine TCR did not recognize a tumor-derived cell line. TRDV-containing alpha chains have been reported in the literature for two HLA I-restricted TCRs against HIV peptides (Ueno et al., Eur J Immunol, 2003). To determine whether such TDRV-containing TCRs are unique events or whether Vdelta segments are commonly incorporated into TCR alpha chains, we queried the NCBI Sequence Read Archive (SRA) for 10X VDJ data and analyzed 21 human and 23 murine datasets. We found that especially TRDV1, Trdv1 and to some extent Trdv2-2 are more commonly incorporated into TCR alpha chains than some TRAV genes, making the TRDV segments a relevant contribution to TCR alpha diversity. For apparently solitary beta chains in 10X VDJ datasets, we suggest to scrutinize the "all_contig_annotations" files as these may contain an accompanying, TRDV-containing alpha chain.

show abstract

“…TCR expression constructs were generated as previously described ( 21 ). HLA-C*07:01 , HLA-C*07:02 cDNA, as well as truncated CSPG4 cDNA ending with codon 562 were cloned into pcDNA3.1/V5-His TOPO (Invitrogen, Waltham, MA, USA) using standard cloning techniques as previously described ( 22 ).…”

Section: Methodsmentioning

confidence: 99%

Targeting the melanoma-associated antigen CSPG4 with HLA-C*07:01-restricted T-cell receptors

Kropp,

Fatho,

Huduti

et al. 2023

Front. Immunol.

Self Cite

View full text Add to dashboard Cite

IntorductionChondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen, is expressed in melanoma but also other tumor entities and constitutes an attractive target for immunotherapeutic approaches. While recent preclinical reports focused on anti-CSPG4 chimeric antigen receptors (CAR), we here explore T-cell receptor (TCR)-based approaches targeting CSPG4.MethodsThe TCRs of two CSPG4-reactive T-cell clones (11C/73 and 2C/165) restricted by the highly prevalent HLA-C*07:01 allele were isolated and the respective αβTCR pairs were retrovirally expressed in CRISPR/Cas9-edited TCR-knockout T cells for functional testing. We also combined alpha and beta TCR chains derived from 11C/73 and 2C/165 in a cross-over fashion to assess for hemichain dominance. CSPG4+ melanoma, glioblastoma and lung cancer cell lines were identified and, if negative, retrovirally transduced with HLA-C*07:01.ResultsFunctional tests confirmed specific recognition of CSPG4+HLA-C*07:01+ target cells by the αβTCR retrieved from the parental T-cell clones and in part also by the cross-over TCR construct 2Cα-11Cβ. Despite high surface expression, the 11Cα-2Cβ combination, however, was not functional.DiscussionCollectively, 11C/73- and 2C/165-expressing T cells specifically and efficiently recognized CSPG4+HLA-C*07:01+ cancer cells which warrants further preclinical and clinical evaluation of these TCRs.

show abstract

A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences

Cited by 6 publications

References 35 publications

ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer

ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer

Incorporation of TRDV segments into TCR alpha chains

Targeting the melanoma-associated antigen CSPG4 with HLA-C*07:01-restricted T-cell receptors

Contact Info

Product

Resources

About