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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.


Expression of activation markers upon treatment with CD3/CD28 beads. (a), flow data of cells stained for detection of the CD69 and CD25 activation markers, measured at 24 or 48 hours after reactivation with CD3/CD28 beads, respectively. Cell size (forward scatter) was also determined 48 hours after reactivation (third row). Blue and red histograms are the signals from TCRα KO T-cells gated on the BFP(+) or BFP(-) cell populations, respectively. The orange histograms correspond to signals obtained upon reactivation of nontransduced TCRα KO T-cells. Green histograms correspond to TCRαβ expressing cells from the same donor (non transfected cells, WT). Black and gray histograms in the first column correspond to TCRαβ(+) or nontransduced TCRα KO cells that were not reactivated with CD3/CD28 beads. The values shown in each of the flow graphs are the MFIs of each of the populations analyzed, using the same color code described before. Data shown correspond to a represantative donor among four different donors analyzed. (b) The MFI for CD69 and CD25 signals on reactivated TCRα KO cells for four independent donors. The orange bars correspond to signals obtained upon reactivation of purified TCRα KO cells, while green bars represent the signals obtained in reactivated TCRα positive cells from the same donor. In the three remaining groups, the red and blue bars correspond to signals obtained from BFP(−) or BFP(+) cells, respectively, upon reactivation. For all the samples that had been transfected with TRAC transcription activator-like effector nuclease (TALEN) mRNA, TCRα knockout cells have been purified by negative selection with CD3 magnetic beads, as depicted in Figure 3. (c) The CD107a mobilization induced upon engagement of effector toward target cells through a soluble antibody. The histograms show the CD107a signal in different populations of viable CD8+ T-cells and following different activation conditions. Basal signals are shown in gray, while those obtained upon incubation with the anti-CD3 (OKT3) antibody alone are shown as a red dashed histogram. Signals corresponding to co-cultures of T-cells with THP-1 are represented as blue dotted lines, and co-cultures of T-cells with THP-1 in the presence of OKT3 are shown as a shaded light-blue histogram. For TCRα KO cells, only the results observed on BFP(+) cells are shown, since no differences were observed on BFP(−) cells, which showed no CD107a mobilization in any of the conditions tested. All the populations yielded the same levels of positive signals upon activation with phorbol myristate acetate and ionomycin, while incubation of any of the cellular populations with Jurkat KOx3 cells (which do not express Fc receptors) showed no degranulation activity in the presence or not of soluble OKT3 (not shown).
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fig4: Expression of activation markers upon treatment with CD3/CD28 beads. (a), flow data of cells stained for detection of the CD69 and CD25 activation markers, measured at 24 or 48 hours after reactivation with CD3/CD28 beads, respectively. Cell size (forward scatter) was also determined 48 hours after reactivation (third row). Blue and red histograms are the signals from TCRα KO T-cells gated on the BFP(+) or BFP(-) cell populations, respectively. The orange histograms correspond to signals obtained upon reactivation of nontransduced TCRα KO T-cells. Green histograms correspond to TCRαβ expressing cells from the same donor (non transfected cells, WT). Black and gray histograms in the first column correspond to TCRαβ(+) or nontransduced TCRα KO cells that were not reactivated with CD3/CD28 beads. The values shown in each of the flow graphs are the MFIs of each of the populations analyzed, using the same color code described before. Data shown correspond to a represantative donor among four different donors analyzed. (b) The MFI for CD69 and CD25 signals on reactivated TCRα KO cells for four independent donors. The orange bars correspond to signals obtained upon reactivation of purified TCRα KO cells, while green bars represent the signals obtained in reactivated TCRα positive cells from the same donor. In the three remaining groups, the red and blue bars correspond to signals obtained from BFP(−) or BFP(+) cells, respectively, upon reactivation. For all the samples that had been transfected with TRAC transcription activator-like effector nuclease (TALEN) mRNA, TCRα knockout cells have been purified by negative selection with CD3 magnetic beads, as depicted in Figure 3. (c) The CD107a mobilization induced upon engagement of effector toward target cells through a soluble antibody. The histograms show the CD107a signal in different populations of viable CD8+ T-cells and following different activation conditions. Basal signals are shown in gray, while those obtained upon incubation with the anti-CD3 (OKT3) antibody alone are shown as a red dashed histogram. Signals corresponding to co-cultures of T-cells with THP-1 are represented as blue dotted lines, and co-cultures of T-cells with THP-1 in the presence of OKT3 are shown as a shaded light-blue histogram. For TCRα KO cells, only the results observed on BFP(+) cells are shown, since no differences were observed on BFP(−) cells, which showed no CD107a mobilization in any of the conditions tested. All the populations yielded the same levels of positive signals upon activation with phorbol myristate acetate and ionomycin, while incubation of any of the cellular populations with Jurkat KOx3 cells (which do not express Fc receptors) showed no degranulation activity in the presence or not of soluble OKT3 (not shown).

Mentions: To determine if pre-TCR/CD3 complexes were able to transduce cell activation signals in TCRα disrupted primary T-cells, we analyzed the expression of activation markers CD69 and CD25 following exposure of cells to anti-CD3 and anti-CD28 antibodies (Figure 4). As shown in Figure 4a (first and second row), BFP positive cells expressing pre-TCR/CD3 complexes demonstrated upregulation of CD69 and CD25 at 24 and 48 hours, respectively, following stimulation with CD3/CD28 beads, though to a lesser extent than in the control TCRαβ expressing cells (WT cells in the figure). Cell size was also analyzed following exposure to CD3/CD28 beads, as a measure of the competence of the pre-TCR/CD3 complexes to induce “blasting” (Figure 4a, third row). Stimulation with CD3/CD28 beads induced comparable increases in cell size in cells expressing TCRαβ/CD3 complexes versus cells expressing pre-TCR/CD3 complexes. Consistently, the responses tend to correlate with the CD3 restoration levels observed in cells expressing pre-TCRα-D48 or pre-TCRα-FL, as illustrated, for example, via assessment of MFI of activation markers on T-cells isolated from four different donors (Figure 4b).


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)

Expression of activation markers upon treatment with CD3/CD28 beads. (a), flow data of cells stained for detection of the CD69 and CD25 activation markers, measured at 24 or 48 hours after reactivation with CD3/CD28 beads, respectively. Cell size (forward scatter) was also determined 48 hours after reactivation (third row). Blue and red histograms are the signals from TCRα KO T-cells gated on the BFP(+) or BFP(-) cell populations, respectively. The orange histograms correspond to signals obtained upon reactivation of nontransduced TCRα KO T-cells. Green histograms correspond to TCRαβ expressing cells from the same donor (non transfected cells, WT). Black and gray histograms in the first column correspond to TCRαβ(+) or nontransduced TCRα KO cells that were not reactivated with CD3/CD28 beads. The values shown in each of the flow graphs are the MFIs of each of the populations analyzed, using the same color code described before. Data shown correspond to a represantative donor among four different donors analyzed. (b) The MFI for CD69 and CD25 signals on reactivated TCRα KO cells for four independent donors. The orange bars correspond to signals obtained upon reactivation of purified TCRα KO cells, while green bars represent the signals obtained in reactivated TCRα positive cells from the same donor. In the three remaining groups, the red and blue bars correspond to signals obtained from BFP(−) or BFP(+) cells, respectively, upon reactivation. For all the samples that had been transfected with TRAC transcription activator-like effector nuclease (TALEN) mRNA, TCRα knockout cells have been purified by negative selection with CD3 magnetic beads, as depicted in Figure 3. (c) The CD107a mobilization induced upon engagement of effector toward target cells through a soluble antibody. The histograms show the CD107a signal in different populations of viable CD8+ T-cells and following different activation conditions. Basal signals are shown in gray, while those obtained upon incubation with the anti-CD3 (OKT3) antibody alone are shown as a red dashed histogram. Signals corresponding to co-cultures of T-cells with THP-1 are represented as blue dotted lines, and co-cultures of T-cells with THP-1 in the presence of OKT3 are shown as a shaded light-blue histogram. For TCRα KO cells, only the results observed on BFP(+) cells are shown, since no differences were observed on BFP(−) cells, which showed no CD107a mobilization in any of the conditions tested. All the populations yielded the same levels of positive signals upon activation with phorbol myristate acetate and ionomycin, while incubation of any of the cellular populations with Jurkat KOx3 cells (which do not express Fc receptors) showed no degranulation activity in the presence or not of soluble OKT3 (not shown).
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Related In: Results  -  Collection

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Show All Figures
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fig4: Expression of activation markers upon treatment with CD3/CD28 beads. (a), flow data of cells stained for detection of the CD69 and CD25 activation markers, measured at 24 or 48 hours after reactivation with CD3/CD28 beads, respectively. Cell size (forward scatter) was also determined 48 hours after reactivation (third row). Blue and red histograms are the signals from TCRα KO T-cells gated on the BFP(+) or BFP(-) cell populations, respectively. The orange histograms correspond to signals obtained upon reactivation of nontransduced TCRα KO T-cells. Green histograms correspond to TCRαβ expressing cells from the same donor (non transfected cells, WT). Black and gray histograms in the first column correspond to TCRαβ(+) or nontransduced TCRα KO cells that were not reactivated with CD3/CD28 beads. The values shown in each of the flow graphs are the MFIs of each of the populations analyzed, using the same color code described before. Data shown correspond to a represantative donor among four different donors analyzed. (b) The MFI for CD69 and CD25 signals on reactivated TCRα KO cells for four independent donors. The orange bars correspond to signals obtained upon reactivation of purified TCRα KO cells, while green bars represent the signals obtained in reactivated TCRα positive cells from the same donor. In the three remaining groups, the red and blue bars correspond to signals obtained from BFP(−) or BFP(+) cells, respectively, upon reactivation. For all the samples that had been transfected with TRAC transcription activator-like effector nuclease (TALEN) mRNA, TCRα knockout cells have been purified by negative selection with CD3 magnetic beads, as depicted in Figure 3. (c) The CD107a mobilization induced upon engagement of effector toward target cells through a soluble antibody. The histograms show the CD107a signal in different populations of viable CD8+ T-cells and following different activation conditions. Basal signals are shown in gray, while those obtained upon incubation with the anti-CD3 (OKT3) antibody alone are shown as a red dashed histogram. Signals corresponding to co-cultures of T-cells with THP-1 are represented as blue dotted lines, and co-cultures of T-cells with THP-1 in the presence of OKT3 are shown as a shaded light-blue histogram. For TCRα KO cells, only the results observed on BFP(+) cells are shown, since no differences were observed on BFP(−) cells, which showed no CD107a mobilization in any of the conditions tested. All the populations yielded the same levels of positive signals upon activation with phorbol myristate acetate and ionomycin, while incubation of any of the cellular populations with Jurkat KOx3 cells (which do not express Fc receptors) showed no degranulation activity in the presence or not of soluble OKT3 (not shown).
Mentions: To determine if pre-TCR/CD3 complexes were able to transduce cell activation signals in TCRα disrupted primary T-cells, we analyzed the expression of activation markers CD69 and CD25 following exposure of cells to anti-CD3 and anti-CD28 antibodies (Figure 4). As shown in Figure 4a (first and second row), BFP positive cells expressing pre-TCR/CD3 complexes demonstrated upregulation of CD69 and CD25 at 24 and 48 hours, respectively, following stimulation with CD3/CD28 beads, though to a lesser extent than in the control TCRαβ expressing cells (WT cells in the figure). Cell size was also analyzed following exposure to CD3/CD28 beads, as a measure of the competence of the pre-TCR/CD3 complexes to induce “blasting” (Figure 4a, third row). Stimulation with CD3/CD28 beads induced comparable increases in cell size in cells expressing TCRαβ/CD3 complexes versus cells expressing pre-TCR/CD3 complexes. Consistently, the responses tend to correlate with the CD3 restoration levels observed in cells expressing pre-TCRα-D48 or pre-TCRα-FL, as illustrated, for example, via assessment of MFI of activation markers on T-cells isolated from four different donors (Figure 4b).

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.