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Generation of allo-restricted peptide-specific T cells using RNA-pulsed dendritic cells: A three phase experimental procedure.

Wilde S, Geiger C, Milosevic S, Mosetter B, Eichenlaub S, Schendel DJ - Oncoimmunology (2012)

Bottom Line: Designer T cells expressing transgenic T cell receptors (TCR) with anti-tumor specificity offer new treatment options for cancer patients.Autologous dendritic cells (DC) are co-transfected with ivt-RNA encoding an allogeneic MHC molecule and a selected antigen to allow them to express allogeneic MHC-peptide complexes that activate allo-restricted peptide-specific T cells.This approach provides great flexibility for obtaining high-avidity T cells as potential sources of TCR for adoptive T cell therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Immunology; Helmholtz Zentrum München; German Research Center for Environmental Health; Munich, Germany.

ABSTRACT
Designer T cells expressing transgenic T cell receptors (TCR) with anti-tumor specificity offer new treatment options for cancer patients. We developed a three phase procedure to identify T cells of high avidity based on the fact that T cells recognizing peptides presented by allogeneic MHC efficiently kill tumor cells. Autologous dendritic cells (DC) are co-transfected with ivt-RNA encoding an allogeneic MHC molecule and a selected antigen to allow them to express allogeneic MHC-peptide complexes that activate allo-restricted peptide-specific T cells. This approach provides great flexibility for obtaining high-avidity T cells as potential sources of TCR for adoptive T cell therapy.

No MeSH data available.


Related in: MedlinePlus

Figure 3. Co-expression of two different ivt-RNA species encoding HLA-A2 and tyrosinase. mDC are electroporated with four different ivt-RNA combinations: (1) 24 µg HLA-A2 and 24 µg tyrosinase ivt-RNA, (2) 24 µg HLA-A2 and 48 µg tyrosinase ivt-RNA, (3) 48 µg HLA-A2 and 24 µg tyrosinase ivt-RNA and (4) 48 µg HLA-A2 and 48 µg tyrosinase ivt-RNA are mixed and co-transfected into mDC of an HLA-A2- donor. (A) Surface staining of HLA-A2 is performed at 6 h and intracellular staining of tyrosinase at 3 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves, corresponding tyrosinase expression of the same sample is shown beneath by filled curves. Mock-control mDC are included in the respective histograms by open curves. Percent positive cells, MFI and x-fold expression are shown in the upper right corner of each histogram. (B) Stimulatory capacity of the mDC loaded with different concentrations of HLA-A2 and tyrosinase ivt-RNA is analyzed by induction of IFNγ secretion of specific T cell clones. Columns represent the amount of IFNγ (pg/ml) secreted by a tyrosinase-independent HLA-A2 allo-reactive CTL (JB4) and an HLA-A2-restricted tyrosinase peptide-specific CTL (Tyr-F8) after co-incubation with ivt-RNA-pulsed DC, 24 h after electroporation. IFNγ is quantified in culture supernatants by ELISA. Mean values and mean deviations are derived from duplicate measurements. IFNγ secretion of T cells alone and in co-culture with non-transfected mock mDC serve as negative controls. Maximal IFNγ release of the CTL is observed in co-cultures with the tumor cell line Mel-93.04A12 as the positive control.
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Figure 3: Figure 3. Co-expression of two different ivt-RNA species encoding HLA-A2 and tyrosinase. mDC are electroporated with four different ivt-RNA combinations: (1) 24 µg HLA-A2 and 24 µg tyrosinase ivt-RNA, (2) 24 µg HLA-A2 and 48 µg tyrosinase ivt-RNA, (3) 48 µg HLA-A2 and 24 µg tyrosinase ivt-RNA and (4) 48 µg HLA-A2 and 48 µg tyrosinase ivt-RNA are mixed and co-transfected into mDC of an HLA-A2- donor. (A) Surface staining of HLA-A2 is performed at 6 h and intracellular staining of tyrosinase at 3 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves, corresponding tyrosinase expression of the same sample is shown beneath by filled curves. Mock-control mDC are included in the respective histograms by open curves. Percent positive cells, MFI and x-fold expression are shown in the upper right corner of each histogram. (B) Stimulatory capacity of the mDC loaded with different concentrations of HLA-A2 and tyrosinase ivt-RNA is analyzed by induction of IFNγ secretion of specific T cell clones. Columns represent the amount of IFNγ (pg/ml) secreted by a tyrosinase-independent HLA-A2 allo-reactive CTL (JB4) and an HLA-A2-restricted tyrosinase peptide-specific CTL (Tyr-F8) after co-incubation with ivt-RNA-pulsed DC, 24 h after electroporation. IFNγ is quantified in culture supernatants by ELISA. Mean values and mean deviations are derived from duplicate measurements. IFNγ secretion of T cells alone and in co-culture with non-transfected mock mDC serve as negative controls. Maximal IFNγ release of the CTL is observed in co-cultures with the tumor cell line Mel-93.04A12 as the positive control.

Mentions: In this analysis, two concentrations (24 μg and 48 μg) of ivt-RNA encoding HLA-A2 and tyrosinase were introduced simultaneously by electroporation into mDC prepared from an HLA-A2- donor (Fig. 3A). The intracellular expression of tyrosinase was analyzed after 3 h and surface expression of HLA-A2 was assessed at 6 h, based on preliminary studies of each individual protein (data not shown). The percentages of HLA-A2+ mDC increased from around 30% using 24 μg ivt-RNA (Fig. 3A, panels 1 and 2) to around 60% using 48 μg ivt-RNA (Fig. 3A, panels 3 and 4). Only a small but non-significant increase in the percentages of mDC expressing intracellular tyrosinase protein was observed when the concentration of tyrosinase ivt-RNA was doubled (Fig. 3A, panels 1 and 3 vs. 2 and 4). A paradoxical decrease in HLA-A2 expression was seen when the concentration of tyrosinase ivt-RNA was increased, leading to a decrease in MFI from 101 when 24 μg of tyrosinase ivt-RNA (Fig. 3A, panel 3) was used to a MFI of only 53 when 48 μg of tyrosinase ivt-RNA was co-expressed in the mDC (Fig. 3A, panel 4). This suggested that the total amount of ivt-RNA expressed in the mDC might be inhibitory. This observation was similar to effects we observed when multiple sources of ivt-RNA that encode different TAA were introduced into mDC.


Generation of allo-restricted peptide-specific T cells using RNA-pulsed dendritic cells: A three phase experimental procedure.

Wilde S, Geiger C, Milosevic S, Mosetter B, Eichenlaub S, Schendel DJ - Oncoimmunology (2012)

Figure 3. Co-expression of two different ivt-RNA species encoding HLA-A2 and tyrosinase. mDC are electroporated with four different ivt-RNA combinations: (1) 24 µg HLA-A2 and 24 µg tyrosinase ivt-RNA, (2) 24 µg HLA-A2 and 48 µg tyrosinase ivt-RNA, (3) 48 µg HLA-A2 and 24 µg tyrosinase ivt-RNA and (4) 48 µg HLA-A2 and 48 µg tyrosinase ivt-RNA are mixed and co-transfected into mDC of an HLA-A2- donor. (A) Surface staining of HLA-A2 is performed at 6 h and intracellular staining of tyrosinase at 3 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves, corresponding tyrosinase expression of the same sample is shown beneath by filled curves. Mock-control mDC are included in the respective histograms by open curves. Percent positive cells, MFI and x-fold expression are shown in the upper right corner of each histogram. (B) Stimulatory capacity of the mDC loaded with different concentrations of HLA-A2 and tyrosinase ivt-RNA is analyzed by induction of IFNγ secretion of specific T cell clones. Columns represent the amount of IFNγ (pg/ml) secreted by a tyrosinase-independent HLA-A2 allo-reactive CTL (JB4) and an HLA-A2-restricted tyrosinase peptide-specific CTL (Tyr-F8) after co-incubation with ivt-RNA-pulsed DC, 24 h after electroporation. IFNγ is quantified in culture supernatants by ELISA. Mean values and mean deviations are derived from duplicate measurements. IFNγ secretion of T cells alone and in co-culture with non-transfected mock mDC serve as negative controls. Maximal IFNγ release of the CTL is observed in co-cultures with the tumor cell line Mel-93.04A12 as the positive control.
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Figure 3: Figure 3. Co-expression of two different ivt-RNA species encoding HLA-A2 and tyrosinase. mDC are electroporated with four different ivt-RNA combinations: (1) 24 µg HLA-A2 and 24 µg tyrosinase ivt-RNA, (2) 24 µg HLA-A2 and 48 µg tyrosinase ivt-RNA, (3) 48 µg HLA-A2 and 24 µg tyrosinase ivt-RNA and (4) 48 µg HLA-A2 and 48 µg tyrosinase ivt-RNA are mixed and co-transfected into mDC of an HLA-A2- donor. (A) Surface staining of HLA-A2 is performed at 6 h and intracellular staining of tyrosinase at 3 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves, corresponding tyrosinase expression of the same sample is shown beneath by filled curves. Mock-control mDC are included in the respective histograms by open curves. Percent positive cells, MFI and x-fold expression are shown in the upper right corner of each histogram. (B) Stimulatory capacity of the mDC loaded with different concentrations of HLA-A2 and tyrosinase ivt-RNA is analyzed by induction of IFNγ secretion of specific T cell clones. Columns represent the amount of IFNγ (pg/ml) secreted by a tyrosinase-independent HLA-A2 allo-reactive CTL (JB4) and an HLA-A2-restricted tyrosinase peptide-specific CTL (Tyr-F8) after co-incubation with ivt-RNA-pulsed DC, 24 h after electroporation. IFNγ is quantified in culture supernatants by ELISA. Mean values and mean deviations are derived from duplicate measurements. IFNγ secretion of T cells alone and in co-culture with non-transfected mock mDC serve as negative controls. Maximal IFNγ release of the CTL is observed in co-cultures with the tumor cell line Mel-93.04A12 as the positive control.
Mentions: In this analysis, two concentrations (24 μg and 48 μg) of ivt-RNA encoding HLA-A2 and tyrosinase were introduced simultaneously by electroporation into mDC prepared from an HLA-A2- donor (Fig. 3A). The intracellular expression of tyrosinase was analyzed after 3 h and surface expression of HLA-A2 was assessed at 6 h, based on preliminary studies of each individual protein (data not shown). The percentages of HLA-A2+ mDC increased from around 30% using 24 μg ivt-RNA (Fig. 3A, panels 1 and 2) to around 60% using 48 μg ivt-RNA (Fig. 3A, panels 3 and 4). Only a small but non-significant increase in the percentages of mDC expressing intracellular tyrosinase protein was observed when the concentration of tyrosinase ivt-RNA was doubled (Fig. 3A, panels 1 and 3 vs. 2 and 4). A paradoxical decrease in HLA-A2 expression was seen when the concentration of tyrosinase ivt-RNA was increased, leading to a decrease in MFI from 101 when 24 μg of tyrosinase ivt-RNA (Fig. 3A, panel 3) was used to a MFI of only 53 when 48 μg of tyrosinase ivt-RNA was co-expressed in the mDC (Fig. 3A, panel 4). This suggested that the total amount of ivt-RNA expressed in the mDC might be inhibitory. This observation was similar to effects we observed when multiple sources of ivt-RNA that encode different TAA were introduced into mDC.

Bottom Line: Designer T cells expressing transgenic T cell receptors (TCR) with anti-tumor specificity offer new treatment options for cancer patients.Autologous dendritic cells (DC) are co-transfected with ivt-RNA encoding an allogeneic MHC molecule and a selected antigen to allow them to express allogeneic MHC-peptide complexes that activate allo-restricted peptide-specific T cells.This approach provides great flexibility for obtaining high-avidity T cells as potential sources of TCR for adoptive T cell therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Immunology; Helmholtz Zentrum München; German Research Center for Environmental Health; Munich, Germany.

ABSTRACT
Designer T cells expressing transgenic T cell receptors (TCR) with anti-tumor specificity offer new treatment options for cancer patients. We developed a three phase procedure to identify T cells of high avidity based on the fact that T cells recognizing peptides presented by allogeneic MHC efficiently kill tumor cells. Autologous dendritic cells (DC) are co-transfected with ivt-RNA encoding an allogeneic MHC molecule and a selected antigen to allow them to express allogeneic MHC-peptide complexes that activate allo-restricted peptide-specific T cells. This approach provides great flexibility for obtaining high-avidity T cells as potential sources of TCR for adoptive T cell therapy.

No MeSH data available.


Related in: MedlinePlus