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Pre-TCRα supports CD3-dependent reactivation and expansion of TCRα-deficient primary human T-cells.

Galetto R, Lebuhotel C, Poirot L, Gouble A, Toribio ML, Smith J, Scharenberg A - Mol Ther Methods Clin Dev (2014)

Bottom Line: Although gene-editing technology can be used to remove the alloreactive potential of third party T-cells through destruction of either the α or β T-cell receptor (TCR) subunit genes, this approach results in the associated loss of surface expression of the CD3 complex.This is nonetheless problematic as it results in the lack of an important trophic signal normally mediated by the CD3 complex at the cell surface, potentially compromising T-cell survival in vivo, and eliminating the potential to expand TCR-knockout cells using stimulatory anti-CD3 antibodies.Thus, heterologous expression of pre-TCRα represents a promising technology for use in the manufacturing of TCR-deficient T-cells for adoptive immunotherapy applications.

View Article: PubMed Central - PubMed

Affiliation: Cellectis Therapeutics , Paris, France.

ABSTRACT
Chimeric antigen receptor technology offers a highly effective means for increasing the anti-tumor effects of autologous adoptive T-cell immunotherapy, and could be made widely available if adapted to the use of allogeneic T-cells. Although gene-editing technology can be used to remove the alloreactive potential of third party T-cells through destruction of either the α or β T-cell receptor (TCR) subunit genes, this approach results in the associated loss of surface expression of the CD3 complex. This is nonetheless problematic as it results in the lack of an important trophic signal normally mediated by the CD3 complex at the cell surface, potentially compromising T-cell survival in vivo, and eliminating the potential to expand TCR-knockout cells using stimulatory anti-CD3 antibodies. Here, we show that pre-TCRα, a TCRα surrogate that pairs with TCRβ chains to signal proper TCRβ folding during T-cell development, can be expressed in TCRα knockout mature T-cells to support CD3 expression at the cell surface. Cells expressing pre-TCR/CD3 complexes can be activated and expanded using standard CD3/CD28 T-cell activation protocols. Thus, heterologous expression of pre-TCRα represents a promising technology for use in the manufacturing of TCR-deficient T-cells for adoptive immunotherapy applications.

No MeSH data available.


Related in: MedlinePlus

Pre-TCRα mediated restoration of surface CD3 expression in TCRα KO primary T-cells. A schema of the lentiviral vectors used for pre-TCRα expression, together with the experimental plan are depicted in panel a. T-cells are purified from buffy-coat and activated at day 0 (D0), followed by transcription activator-like effector nuclease (TALEN) mRNA transfection (D3), lentiviral transduction (D5), and enrichment of TCRα disrupted cells (D7). The bicistronic lentiviral vectors allow the discrimination of pre-TCRα expressing cells by flow cytometry, based on the BFP signal. FACS analysis for BFP and CD3 expression at D7 are shown before and after depletion of CD3 positive cells (panel b, upper and lower rows, respectively). (c) The levels of CD3 and TCRαβ expression in TCRα KO cells transduced with the control BFP or with the BFP-2A-pre-TCRα-FL or –D48 rLV vectors, gated on the BFP(+) (blue histograms) or BFP(-) (red histograms) populations. The light-gray histograms correspond to CD3 and TCRαβ signals from non transduced TCRα deficient T-cells. All the cells that had been transfected with the TRAC TALEN followed the same purification protocol 4 days after transfection. The dark-gray histograms correspond to CD3 and TCRαβ signals obtained in non transfected T-cells from the same donor (WT, wild type). The values showed in each condition represent the MFI data for each population analyzed in a representative experiment. The color code is the same for the histograms and the MFI values. Experiments were carried out at least four times.
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fig3: Pre-TCRα mediated restoration of surface CD3 expression in TCRα KO primary T-cells. A schema of the lentiviral vectors used for pre-TCRα expression, together with the experimental plan are depicted in panel a. T-cells are purified from buffy-coat and activated at day 0 (D0), followed by transcription activator-like effector nuclease (TALEN) mRNA transfection (D3), lentiviral transduction (D5), and enrichment of TCRα disrupted cells (D7). The bicistronic lentiviral vectors allow the discrimination of pre-TCRα expressing cells by flow cytometry, based on the BFP signal. FACS analysis for BFP and CD3 expression at D7 are shown before and after depletion of CD3 positive cells (panel b, upper and lower rows, respectively). (c) The levels of CD3 and TCRαβ expression in TCRα KO cells transduced with the control BFP or with the BFP-2A-pre-TCRα-FL or –D48 rLV vectors, gated on the BFP(+) (blue histograms) or BFP(-) (red histograms) populations. The light-gray histograms correspond to CD3 and TCRαβ signals from non transduced TCRα deficient T-cells. All the cells that had been transfected with the TRAC TALEN followed the same purification protocol 4 days after transfection. The dark-gray histograms correspond to CD3 and TCRαβ signals obtained in non transfected T-cells from the same donor (WT, wild type). The values showed in each condition represent the MFI data for each population analyzed in a representative experiment. The color code is the same for the histograms and the MFI values. Experiments were carried out at least four times.

Mentions: Based on the results from the expression of pre-TCRα variants in TCRα deficient Jurkat T-cells, we developed lentiviral vector constructs driving expression of BFP followed by a 2A ribosomal skip peptide and full length pre-TCRα or pre-TCRα-D48 variants. A schema of the expression vectors is shown in Figure 3a, together with that of the control vector, expressing BFP but not pre-TCRα.


Pre-TCRα supports CD3-dependent reactivation and expansion of TCRα-deficient primary human T-cells.

Galetto R, Lebuhotel C, Poirot L, Gouble A, Toribio ML, Smith J, Scharenberg A - Mol Ther Methods Clin Dev (2014)

Pre-TCRα mediated restoration of surface CD3 expression in TCRα KO primary T-cells. A schema of the lentiviral vectors used for pre-TCRα expression, together with the experimental plan are depicted in panel a. T-cells are purified from buffy-coat and activated at day 0 (D0), followed by transcription activator-like effector nuclease (TALEN) mRNA transfection (D3), lentiviral transduction (D5), and enrichment of TCRα disrupted cells (D7). The bicistronic lentiviral vectors allow the discrimination of pre-TCRα expressing cells by flow cytometry, based on the BFP signal. FACS analysis for BFP and CD3 expression at D7 are shown before and after depletion of CD3 positive cells (panel b, upper and lower rows, respectively). (c) The levels of CD3 and TCRαβ expression in TCRα KO cells transduced with the control BFP or with the BFP-2A-pre-TCRα-FL or –D48 rLV vectors, gated on the BFP(+) (blue histograms) or BFP(-) (red histograms) populations. The light-gray histograms correspond to CD3 and TCRαβ signals from non transduced TCRα deficient T-cells. All the cells that had been transfected with the TRAC TALEN followed the same purification protocol 4 days after transfection. The dark-gray histograms correspond to CD3 and TCRαβ signals obtained in non transfected T-cells from the same donor (WT, wild type). The values showed in each condition represent the MFI data for each population analyzed in a representative experiment. The color code is the same for the histograms and the MFI values. Experiments were carried out at least four times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362381&req=5

fig3: Pre-TCRα mediated restoration of surface CD3 expression in TCRα KO primary T-cells. A schema of the lentiviral vectors used for pre-TCRα expression, together with the experimental plan are depicted in panel a. T-cells are purified from buffy-coat and activated at day 0 (D0), followed by transcription activator-like effector nuclease (TALEN) mRNA transfection (D3), lentiviral transduction (D5), and enrichment of TCRα disrupted cells (D7). The bicistronic lentiviral vectors allow the discrimination of pre-TCRα expressing cells by flow cytometry, based on the BFP signal. FACS analysis for BFP and CD3 expression at D7 are shown before and after depletion of CD3 positive cells (panel b, upper and lower rows, respectively). (c) The levels of CD3 and TCRαβ expression in TCRα KO cells transduced with the control BFP or with the BFP-2A-pre-TCRα-FL or –D48 rLV vectors, gated on the BFP(+) (blue histograms) or BFP(-) (red histograms) populations. The light-gray histograms correspond to CD3 and TCRαβ signals from non transduced TCRα deficient T-cells. All the cells that had been transfected with the TRAC TALEN followed the same purification protocol 4 days after transfection. The dark-gray histograms correspond to CD3 and TCRαβ signals obtained in non transfected T-cells from the same donor (WT, wild type). The values showed in each condition represent the MFI data for each population analyzed in a representative experiment. The color code is the same for the histograms and the MFI values. Experiments were carried out at least four times.
Mentions: Based on the results from the expression of pre-TCRα variants in TCRα deficient Jurkat T-cells, we developed lentiviral vector constructs driving expression of BFP followed by a 2A ribosomal skip peptide and full length pre-TCRα or pre-TCRα-D48 variants. A schema of the expression vectors is shown in Figure 3a, together with that of the control vector, expressing BFP but not pre-TCRα.

Bottom Line: Although gene-editing technology can be used to remove the alloreactive potential of third party T-cells through destruction of either the α or β T-cell receptor (TCR) subunit genes, this approach results in the associated loss of surface expression of the CD3 complex.This is nonetheless problematic as it results in the lack of an important trophic signal normally mediated by the CD3 complex at the cell surface, potentially compromising T-cell survival in vivo, and eliminating the potential to expand TCR-knockout cells using stimulatory anti-CD3 antibodies.Thus, heterologous expression of pre-TCRα represents a promising technology for use in the manufacturing of TCR-deficient T-cells for adoptive immunotherapy applications.

View Article: PubMed Central - PubMed

Affiliation: Cellectis Therapeutics , Paris, France.

ABSTRACT
Chimeric antigen receptor technology offers a highly effective means for increasing the anti-tumor effects of autologous adoptive T-cell immunotherapy, and could be made widely available if adapted to the use of allogeneic T-cells. Although gene-editing technology can be used to remove the alloreactive potential of third party T-cells through destruction of either the α or β T-cell receptor (TCR) subunit genes, this approach results in the associated loss of surface expression of the CD3 complex. This is nonetheless problematic as it results in the lack of an important trophic signal normally mediated by the CD3 complex at the cell surface, potentially compromising T-cell survival in vivo, and eliminating the potential to expand TCR-knockout cells using stimulatory anti-CD3 antibodies. Here, we show that pre-TCRα, a TCRα surrogate that pairs with TCRβ chains to signal proper TCRβ folding during T-cell development, can be expressed in TCRα knockout mature T-cells to support CD3 expression at the cell surface. Cells expressing pre-TCR/CD3 complexes can be activated and expanded using standard CD3/CD28 T-cell activation protocols. Thus, heterologous expression of pre-TCRα represents a promising technology for use in the manufacturing of TCR-deficient T-cells for adoptive immunotherapy applications.

No MeSH data available.


Related in: MedlinePlus