Limits...
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

Screening of pre-TCRα constructs in TCRα deficient Jurkat cells. (a) represents the multi-component TCRαβ and pre-TCR complexes as assembled at the cell surface. The TCRα genomic locus (TRA) is shown in the lower part of the panel, with the arrangement of the variable (V), joining (J) and constant (C) gene segments, as well as the position targeted by the transcription activator-like effector nuclease (TALEN) used to generate the TCRα disrupted cells. (b) Flow cytometry data from the generation of TCRα KO Jurkat cells upon three consecutive transfections (EPx1, EPx2, and EPx3) of the plasmids coding for TALEN targeting exon 1 of the TCRα chain constant region. The right-most flow panel (KOx3) shows TCRαβ and CD3 staining of the TCRα KO Jurkat cells after purification of the CD3NEG population. (c) The percentage of cells in which CD3 restoration (CD3LOW) is observed upon transfection of pre-TCRα variants harboring different truncations in the cytoplasmic tail. The name of each construct corresponds to the number of residues eliminated from the C-terminus of the cytoplasmic tail. As a negative control, data of cells transfected with a plasmid encoding blue fluorescent protein (BFP) under the control of the same promoter as the pre-TCRα variants is shown (by using this plasmid, transfection efficiency was determined in each experiment, which was always >90%). Transfections had been carried out in at least three independent experiments for each construct. (d) An example of flow cytometry data from the pre-TCRα-FL and D48 variants. The geometrical mean fluorescence intensity (MFI) of the CD3 signal is shown for the whole cell population in each case, for a single representative experiment, in the lower-right corner of each flow panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362381&req=5

fig1: Screening of pre-TCRα constructs in TCRα deficient Jurkat cells. (a) represents the multi-component TCRαβ and pre-TCR complexes as assembled at the cell surface. The TCRα genomic locus (TRA) is shown in the lower part of the panel, with the arrangement of the variable (V), joining (J) and constant (C) gene segments, as well as the position targeted by the transcription activator-like effector nuclease (TALEN) used to generate the TCRα disrupted cells. (b) Flow cytometry data from the generation of TCRα KO Jurkat cells upon three consecutive transfections (EPx1, EPx2, and EPx3) of the plasmids coding for TALEN targeting exon 1 of the TCRα chain constant region. The right-most flow panel (KOx3) shows TCRαβ and CD3 staining of the TCRα KO Jurkat cells after purification of the CD3NEG population. (c) The percentage of cells in which CD3 restoration (CD3LOW) is observed upon transfection of pre-TCRα variants harboring different truncations in the cytoplasmic tail. The name of each construct corresponds to the number of residues eliminated from the C-terminus of the cytoplasmic tail. As a negative control, data of cells transfected with a plasmid encoding blue fluorescent protein (BFP) under the control of the same promoter as the pre-TCRα variants is shown (by using this plasmid, transfection efficiency was determined in each experiment, which was always >90%). Transfections had been carried out in at least three independent experiments for each construct. (d) An example of flow cytometry data from the pre-TCRα-FL and D48 variants. The geometrical mean fluorescence intensity (MFI) of the CD3 signal is shown for the whole cell population in each case, for a single representative experiment, in the lower-right corner of each flow panel.

Mentions: To address the issues associated with loss of surface CD3 complexes upon TCRα gene disruption, we hypothesized that the expression of pre-TCRα, a surrogate partner for properly folded TCRβ chains during T-cell development,5–7 would be able to pair with all β chains expressed in a T-cell population, and support surface CD3 expression through a pre-TCR/CD3 complex (Figure 1a, upper panel). These complexes are transiently expressed during normal thymocyte development at the β selection step, and are thought to generate a ligand-independent signal.8,9 This signal induces expansion and maturation of developing pre-T-cells into CD4+CD8+ double-positive thymocytes, and TCRα gene rearrangement, resulting in the substitution of pre-TCRα by TCRα chains, subsequent TCRαβ selection, and final differentiation into conventional single-positive thymocytes.10–13


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)

Screening of pre-TCRα constructs in TCRα deficient Jurkat cells. (a) represents the multi-component TCRαβ and pre-TCR complexes as assembled at the cell surface. The TCRα genomic locus (TRA) is shown in the lower part of the panel, with the arrangement of the variable (V), joining (J) and constant (C) gene segments, as well as the position targeted by the transcription activator-like effector nuclease (TALEN) used to generate the TCRα disrupted cells. (b) Flow cytometry data from the generation of TCRα KO Jurkat cells upon three consecutive transfections (EPx1, EPx2, and EPx3) of the plasmids coding for TALEN targeting exon 1 of the TCRα chain constant region. The right-most flow panel (KOx3) shows TCRαβ and CD3 staining of the TCRα KO Jurkat cells after purification of the CD3NEG population. (c) The percentage of cells in which CD3 restoration (CD3LOW) is observed upon transfection of pre-TCRα variants harboring different truncations in the cytoplasmic tail. The name of each construct corresponds to the number of residues eliminated from the C-terminus of the cytoplasmic tail. As a negative control, data of cells transfected with a plasmid encoding blue fluorescent protein (BFP) under the control of the same promoter as the pre-TCRα variants is shown (by using this plasmid, transfection efficiency was determined in each experiment, which was always >90%). Transfections had been carried out in at least three independent experiments for each construct. (d) An example of flow cytometry data from the pre-TCRα-FL and D48 variants. The geometrical mean fluorescence intensity (MFI) of the CD3 signal is shown for the whole cell population in each case, for a single representative experiment, in the lower-right corner of each flow panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Screening of pre-TCRα constructs in TCRα deficient Jurkat cells. (a) represents the multi-component TCRαβ and pre-TCR complexes as assembled at the cell surface. The TCRα genomic locus (TRA) is shown in the lower part of the panel, with the arrangement of the variable (V), joining (J) and constant (C) gene segments, as well as the position targeted by the transcription activator-like effector nuclease (TALEN) used to generate the TCRα disrupted cells. (b) Flow cytometry data from the generation of TCRα KO Jurkat cells upon three consecutive transfections (EPx1, EPx2, and EPx3) of the plasmids coding for TALEN targeting exon 1 of the TCRα chain constant region. The right-most flow panel (KOx3) shows TCRαβ and CD3 staining of the TCRα KO Jurkat cells after purification of the CD3NEG population. (c) The percentage of cells in which CD3 restoration (CD3LOW) is observed upon transfection of pre-TCRα variants harboring different truncations in the cytoplasmic tail. The name of each construct corresponds to the number of residues eliminated from the C-terminus of the cytoplasmic tail. As a negative control, data of cells transfected with a plasmid encoding blue fluorescent protein (BFP) under the control of the same promoter as the pre-TCRα variants is shown (by using this plasmid, transfection efficiency was determined in each experiment, which was always >90%). Transfections had been carried out in at least three independent experiments for each construct. (d) An example of flow cytometry data from the pre-TCRα-FL and D48 variants. The geometrical mean fluorescence intensity (MFI) of the CD3 signal is shown for the whole cell population in each case, for a single representative experiment, in the lower-right corner of each flow panel.
Mentions: To address the issues associated with loss of surface CD3 complexes upon TCRα gene disruption, we hypothesized that the expression of pre-TCRα, a surrogate partner for properly folded TCRβ chains during T-cell development,5–7 would be able to pair with all β chains expressed in a T-cell population, and support surface CD3 expression through a pre-TCR/CD3 complex (Figure 1a, upper panel). These complexes are transiently expressed during normal thymocyte development at the β selection step, and are thought to generate a ligand-independent signal.8,9 This signal induces expansion and maturation of developing pre-T-cells into CD4+CD8+ double-positive thymocytes, and TCRα gene rearrangement, resulting in the substitution of pre-TCRα by TCRα chains, subsequent TCRαβ selection, and final differentiation into conventional single-positive thymocytes.10–13

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