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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 expression and signaling capacity in TCRα disrupted Jurkat cells. Detection of pre-TCRα-FL and -D48 by (a) Western blot (WB) (left blot) and (b) flow cytometry 48 hours after transfection, showing that pre-TCRα expression levels correlate with those of CD3 restoration. Nonrelated (NR): cells transfected with a nonrelated plasmid; BFP: cells transfected with a BFP encoding plasmid. The right blot of panel a show the phosphorylation status of CD3ζ chains on Jurkat and in TCRα edited cells (KOx3) 48 hours after transfection with a BFP, pre-TCRα-FL or pre-TCRα–D48 encoding plasmid, and of non electroporated (NEP) cells. (b) Flow data of KOx3 Jurkat cells transfected with the BFP, pre-TCRα-FL or pre-TCRα-D48 expression plasmids and stained with anti-CD3 or anti-CD3 plus anti-pre-TCRα antibodies. The MFIs of CD3 and pre-TCRα signals are shown in the corresponding axes for the global cell population.
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fig2: Pre-TCRα expression and signaling capacity in TCRα disrupted Jurkat cells. Detection of pre-TCRα-FL and -D48 by (a) Western blot (WB) (left blot) and (b) flow cytometry 48 hours after transfection, showing that pre-TCRα expression levels correlate with those of CD3 restoration. Nonrelated (NR): cells transfected with a nonrelated plasmid; BFP: cells transfected with a BFP encoding plasmid. The right blot of panel a show the phosphorylation status of CD3ζ chains on Jurkat and in TCRα edited cells (KOx3) 48 hours after transfection with a BFP, pre-TCRα-FL or pre-TCRα–D48 encoding plasmid, and of non electroporated (NEP) cells. (b) Flow data of KOx3 Jurkat cells transfected with the BFP, pre-TCRα-FL or pre-TCRα-D48 expression plasmids and stained with anti-CD3 or anti-CD3 plus anti-pre-TCRα antibodies. The MFIs of CD3 and pre-TCRα signals are shown in the corresponding axes for the global cell population.

Mentions: Protein expression of pre-TCRα was confirmed by western blot (Figure 2a, left blot) using a polyclonal antibody targeting the extracellular domain of pre-TCRα (residues 61–82). In addition, flow cytometry was carried out to detect pre-TCRα at the cell surface using the K5G3 monoclonal antibody23 (targeting also the extracellular domain), which allowed us to correlate pre-TCRα and CD3 expression upon double staining of the transfected cells (Figure 2b).


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 expression and signaling capacity in TCRα disrupted Jurkat cells. Detection of pre-TCRα-FL and -D48 by (a) Western blot (WB) (left blot) and (b) flow cytometry 48 hours after transfection, showing that pre-TCRα expression levels correlate with those of CD3 restoration. Nonrelated (NR): cells transfected with a nonrelated plasmid; BFP: cells transfected with a BFP encoding plasmid. The right blot of panel a show the phosphorylation status of CD3ζ chains on Jurkat and in TCRα edited cells (KOx3) 48 hours after transfection with a BFP, pre-TCRα-FL or pre-TCRα–D48 encoding plasmid, and of non electroporated (NEP) cells. (b) Flow data of KOx3 Jurkat cells transfected with the BFP, pre-TCRα-FL or pre-TCRα-D48 expression plasmids and stained with anti-CD3 or anti-CD3 plus anti-pre-TCRα antibodies. The MFIs of CD3 and pre-TCRα signals are shown in the corresponding axes for the global cell population.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Pre-TCRα expression and signaling capacity in TCRα disrupted Jurkat cells. Detection of pre-TCRα-FL and -D48 by (a) Western blot (WB) (left blot) and (b) flow cytometry 48 hours after transfection, showing that pre-TCRα expression levels correlate with those of CD3 restoration. Nonrelated (NR): cells transfected with a nonrelated plasmid; BFP: cells transfected with a BFP encoding plasmid. The right blot of panel a show the phosphorylation status of CD3ζ chains on Jurkat and in TCRα edited cells (KOx3) 48 hours after transfection with a BFP, pre-TCRα-FL or pre-TCRα–D48 encoding plasmid, and of non electroporated (NEP) cells. (b) Flow data of KOx3 Jurkat cells transfected with the BFP, pre-TCRα-FL or pre-TCRα-D48 expression plasmids and stained with anti-CD3 or anti-CD3 plus anti-pre-TCRα antibodies. The MFIs of CD3 and pre-TCRα signals are shown in the corresponding axes for the global cell population.
Mentions: Protein expression of pre-TCRα was confirmed by western blot (Figure 2a, left blot) using a polyclonal antibody targeting the extracellular domain of pre-TCRα (residues 61–82). In addition, flow cytometry was carried out to detect pre-TCRα at the cell surface using the K5G3 monoclonal antibody23 (targeting also the extracellular domain), which allowed us to correlate pre-TCRα and CD3 expression upon double staining of the transfected cells (Figure 2b).

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