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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

Expansion of TCRα KO BFP(+) cells upon reactivation with CD3/CD28 beads. (a) The percentage of BFP(+) cells when they have been reactivated with CD3/CD28 beads (right panel) or kept in culture in the presence of IL-2 only (left panel). TCRα KO cells transduced with the BFP control vector are shown as circles, while cells expressing pre-TCRα-FL or pre-TCRα-D48 as squares or triangles, respectively. Dataset in gray represents the values observed 48 hours after the reactivation step, while those in black are the values observed at the end of the experiment. Mean ± SD values are represented. The P values obtained when comparing percentages of BFP(+) cells at the beginning and end of experiment are given for each of the reactivated samples. (b) The fold induction in the number of BFP(+) cells at the end of the experiment with respect to those 48 hours after reactivation with CD3/CD28 beads (left panel). The horizontal line represents the median value of each dataset. The mean values are 16.9; 26.9 and 28.8 for the KO/BFP, KO/FL and KO/D48 samples, respectively. The curves in the right panel represent the growth kinetics of the different donors, in terms of fold induction in the number of BFP(+) cells at each timepoint respect to the amount of BFP(+) cells at D2.
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fig5: Expansion of TCRα KO BFP(+) cells upon reactivation with CD3/CD28 beads. (a) The percentage of BFP(+) cells when they have been reactivated with CD3/CD28 beads (right panel) or kept in culture in the presence of IL-2 only (left panel). TCRα KO cells transduced with the BFP control vector are shown as circles, while cells expressing pre-TCRα-FL or pre-TCRα-D48 as squares or triangles, respectively. Dataset in gray represents the values observed 48 hours after the reactivation step, while those in black are the values observed at the end of the experiment. Mean ± SD values are represented. The P values obtained when comparing percentages of BFP(+) cells at the beginning and end of experiment are given for each of the reactivated samples. (b) The fold induction in the number of BFP(+) cells at the end of the experiment with respect to those 48 hours after reactivation with CD3/CD28 beads (left panel). The horizontal line represents the median value of each dataset. The mean values are 16.9; 26.9 and 28.8 for the KO/BFP, KO/FL and KO/D48 samples, respectively. The curves in the right panel represent the growth kinetics of the different donors, in terms of fold induction in the number of BFP(+) cells at each timepoint respect to the amount of BFP(+) cells at D2.

Mentions: As the expansion kinetics of each donor were different, we compared the percentage of BFP(+) cells early after reactivation (48 hours) to those observed at a late stage of the experiment (17–25 days after reactivation, corresponding to the time point at which the maximum percentage of BFP(+) cells were observed). These data are shown in Figure 5a. The two panels correspond to TCRα disrupted cells grown on IL-2 only (left panel) or reactivated with CD3/CD28 beads (right panel), after transduction with either the BFP-only control vector or with the –FL or –D48 pre-TCRα expressing vectors. The dataset in gray corresponds to signals 48 hours after reactivation, while those in black are those at late stages of the experiment. Importantly, we observed a decrease in the percentage of BFP(+) cells over time in all samples when they were maintained on IL-2 alone, demonstrating that pre-T does not generate a constitutive growth signal capable of driving autonomous cell proliferation in a minimally trophic environment, and that such culture conditions lead to a gradual silencing of transgene expression as the cells move into quiescence. However, when cells were reactivated with CD3/CD28 beads, we observed a decrease in the percentage of BFP(+) cells in controls, but an increase in all cases where cells were expressing any of the pre-TCRα variants. These results indicate that TCRα KO cells expressing pre-TCR/CD3 complexes receive enhanced pro-survival signals when exposed to CD3/CD28 beads, and are able to survive and expand to a higher degree than cells transduced with the control BFP vector.


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)

Expansion of TCRα KO BFP(+) cells upon reactivation with CD3/CD28 beads. (a) The percentage of BFP(+) cells when they have been reactivated with CD3/CD28 beads (right panel) or kept in culture in the presence of IL-2 only (left panel). TCRα KO cells transduced with the BFP control vector are shown as circles, while cells expressing pre-TCRα-FL or pre-TCRα-D48 as squares or triangles, respectively. Dataset in gray represents the values observed 48 hours after the reactivation step, while those in black are the values observed at the end of the experiment. Mean ± SD values are represented. The P values obtained when comparing percentages of BFP(+) cells at the beginning and end of experiment are given for each of the reactivated samples. (b) The fold induction in the number of BFP(+) cells at the end of the experiment with respect to those 48 hours after reactivation with CD3/CD28 beads (left panel). The horizontal line represents the median value of each dataset. The mean values are 16.9; 26.9 and 28.8 for the KO/BFP, KO/FL and KO/D48 samples, respectively. The curves in the right panel represent the growth kinetics of the different donors, in terms of fold induction in the number of BFP(+) cells at each timepoint respect to the amount of BFP(+) cells at D2.
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Related In: Results  -  Collection

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fig5: Expansion of TCRα KO BFP(+) cells upon reactivation with CD3/CD28 beads. (a) The percentage of BFP(+) cells when they have been reactivated with CD3/CD28 beads (right panel) or kept in culture in the presence of IL-2 only (left panel). TCRα KO cells transduced with the BFP control vector are shown as circles, while cells expressing pre-TCRα-FL or pre-TCRα-D48 as squares or triangles, respectively. Dataset in gray represents the values observed 48 hours after the reactivation step, while those in black are the values observed at the end of the experiment. Mean ± SD values are represented. The P values obtained when comparing percentages of BFP(+) cells at the beginning and end of experiment are given for each of the reactivated samples. (b) The fold induction in the number of BFP(+) cells at the end of the experiment with respect to those 48 hours after reactivation with CD3/CD28 beads (left panel). The horizontal line represents the median value of each dataset. The mean values are 16.9; 26.9 and 28.8 for the KO/BFP, KO/FL and KO/D48 samples, respectively. The curves in the right panel represent the growth kinetics of the different donors, in terms of fold induction in the number of BFP(+) cells at each timepoint respect to the amount of BFP(+) cells at D2.
Mentions: As the expansion kinetics of each donor were different, we compared the percentage of BFP(+) cells early after reactivation (48 hours) to those observed at a late stage of the experiment (17–25 days after reactivation, corresponding to the time point at which the maximum percentage of BFP(+) cells were observed). These data are shown in Figure 5a. The two panels correspond to TCRα disrupted cells grown on IL-2 only (left panel) or reactivated with CD3/CD28 beads (right panel), after transduction with either the BFP-only control vector or with the –FL or –D48 pre-TCRα expressing vectors. The dataset in gray corresponds to signals 48 hours after reactivation, while those in black are those at late stages of the experiment. Importantly, we observed a decrease in the percentage of BFP(+) cells over time in all samples when they were maintained on IL-2 alone, demonstrating that pre-T does not generate a constitutive growth signal capable of driving autonomous cell proliferation in a minimally trophic environment, and that such culture conditions lead to a gradual silencing of transgene expression as the cells move into quiescence. However, when cells were reactivated with CD3/CD28 beads, we observed a decrease in the percentage of BFP(+) cells in controls, but an increase in all cases where cells were expressing any of the pre-TCRα variants. These results indicate that TCRα KO cells expressing pre-TCR/CD3 complexes receive enhanced pro-survival signals when exposed to CD3/CD28 beads, and are able to survive and expand to a higher degree than cells transduced with the control BFP vector.

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