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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 2. Kinetics of HLA-A2 expression and stimulatory capacity of ivt-RNA-transfected mDC from an HLA-A2- donor. (A) HLA-A2- mDC are electroporated with 25 µg HLA-A2 ivt-RNA prepared by the standard method. Surface staining of HLA-A2 is performed at different time points (1.5 h, 3 h, 6 h, 10 h, 24 h, 48 h and 120 h) after transfection and analyzed by flow cytometry. Stained samples are represented by filled gray curves and corresponding controls by empty curves. Percent positive cells, mean fluorescence intensity (MFI) and x-fold expression are shown in the upper right corner of each histogram. The x-fold expression is calculated by dividing the MFI of the positive sample with the MFI of the control. (B) For comparison, mDC of an HLA-A2- donor are transfected with 25 µg of 5 different species of HLA-A2 ivt-RNA. The quality of the standard poly-A tail (pAAA)-stabilized HLA-A2 ivt-RNA is compared with α-globin-stabilized (1, 2, 3) and β-globin-stabilized HLA-A2 ivt-RNA. The expression at different time points is depicted in a bar histogram as MFI. (C) mDC transfected with the various HLA-A2 ivt-RNA species are used 24 h after electroporation for co-incubation with the HLA-A2 allo-reactive CTL clone JB4 to assess their stimulatory capacity. IFNγ is quantified in culture supernatants by ELISA and presented as pg/ml. Mean values and mean deviations are derived from duplicate measurements.
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Figure 2: Figure 2. Kinetics of HLA-A2 expression and stimulatory capacity of ivt-RNA-transfected mDC from an HLA-A2- donor. (A) HLA-A2- mDC are electroporated with 25 µg HLA-A2 ivt-RNA prepared by the standard method. Surface staining of HLA-A2 is performed at different time points (1.5 h, 3 h, 6 h, 10 h, 24 h, 48 h and 120 h) after transfection and analyzed by flow cytometry. Stained samples are represented by filled gray curves and corresponding controls by empty curves. Percent positive cells, mean fluorescence intensity (MFI) and x-fold expression are shown in the upper right corner of each histogram. The x-fold expression is calculated by dividing the MFI of the positive sample with the MFI of the control. (B) For comparison, mDC of an HLA-A2- donor are transfected with 25 µg of 5 different species of HLA-A2 ivt-RNA. The quality of the standard poly-A tail (pAAA)-stabilized HLA-A2 ivt-RNA is compared with α-globin-stabilized (1, 2, 3) and β-globin-stabilized HLA-A2 ivt-RNA. The expression at different time points is depicted in a bar histogram as MFI. (C) mDC transfected with the various HLA-A2 ivt-RNA species are used 24 h after electroporation for co-incubation with the HLA-A2 allo-reactive CTL clone JB4 to assess their stimulatory capacity. IFNγ is quantified in culture supernatants by ELISA and presented as pg/ml. Mean values and mean deviations are derived from duplicate measurements.

Mentions: In order to stimulate allogeneic T cells, some of which are peptide-specific, the mDC must express allogeneic MHC molecules at the cell surface after transfer of ivt-RNA encoding an HLA allotype. Since mDC display high levels of endogenous MHC class I, the surface expression of transgenic allogeneic MHC must be monitored using HLA allotype-specific monoclonal antibodies to analyze cells by flow cytometry. For example, expression of transgenic HLA-A2 molecules can be studied on mDC prepared from HLA-A2- donors using a primary HLA-A2-specific BB7.2 monoclonal antibody, followed by a secondary PE-conjugated antibody. As an example HLA-A2 surface expression was already detected on the mDC surface at 1.5 h following electroporation of 25 μg ivt-RNA encoding the HLA-A*0201 allele (Fig. 2A). In this case, HLA-A2 expression reached a peak at 10 h, measured as mean fluorescent intensity (MFI), and then decreased over time. Nevertheless, around 50% of the mDC were still positive for HLA-A2 at 120 h. The greatest percentage of HLA-A2+ mDC (63%) was also seen at 10 h.


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 2. Kinetics of HLA-A2 expression and stimulatory capacity of ivt-RNA-transfected mDC from an HLA-A2- donor. (A) HLA-A2- mDC are electroporated with 25 µg HLA-A2 ivt-RNA prepared by the standard method. Surface staining of HLA-A2 is performed at different time points (1.5 h, 3 h, 6 h, 10 h, 24 h, 48 h and 120 h) after transfection and analyzed by flow cytometry. Stained samples are represented by filled gray curves and corresponding controls by empty curves. Percent positive cells, mean fluorescence intensity (MFI) and x-fold expression are shown in the upper right corner of each histogram. The x-fold expression is calculated by dividing the MFI of the positive sample with the MFI of the control. (B) For comparison, mDC of an HLA-A2- donor are transfected with 25 µg of 5 different species of HLA-A2 ivt-RNA. The quality of the standard poly-A tail (pAAA)-stabilized HLA-A2 ivt-RNA is compared with α-globin-stabilized (1, 2, 3) and β-globin-stabilized HLA-A2 ivt-RNA. The expression at different time points is depicted in a bar histogram as MFI. (C) mDC transfected with the various HLA-A2 ivt-RNA species are used 24 h after electroporation for co-incubation with the HLA-A2 allo-reactive CTL clone JB4 to assess their stimulatory capacity. IFNγ is quantified in culture supernatants by ELISA and presented as pg/ml. Mean values and mean deviations are derived from duplicate measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376998&req=5

Figure 2: Figure 2. Kinetics of HLA-A2 expression and stimulatory capacity of ivt-RNA-transfected mDC from an HLA-A2- donor. (A) HLA-A2- mDC are electroporated with 25 µg HLA-A2 ivt-RNA prepared by the standard method. Surface staining of HLA-A2 is performed at different time points (1.5 h, 3 h, 6 h, 10 h, 24 h, 48 h and 120 h) after transfection and analyzed by flow cytometry. Stained samples are represented by filled gray curves and corresponding controls by empty curves. Percent positive cells, mean fluorescence intensity (MFI) and x-fold expression are shown in the upper right corner of each histogram. The x-fold expression is calculated by dividing the MFI of the positive sample with the MFI of the control. (B) For comparison, mDC of an HLA-A2- donor are transfected with 25 µg of 5 different species of HLA-A2 ivt-RNA. The quality of the standard poly-A tail (pAAA)-stabilized HLA-A2 ivt-RNA is compared with α-globin-stabilized (1, 2, 3) and β-globin-stabilized HLA-A2 ivt-RNA. The expression at different time points is depicted in a bar histogram as MFI. (C) mDC transfected with the various HLA-A2 ivt-RNA species are used 24 h after electroporation for co-incubation with the HLA-A2 allo-reactive CTL clone JB4 to assess their stimulatory capacity. IFNγ is quantified in culture supernatants by ELISA and presented as pg/ml. Mean values and mean deviations are derived from duplicate measurements.
Mentions: In order to stimulate allogeneic T cells, some of which are peptide-specific, the mDC must express allogeneic MHC molecules at the cell surface after transfer of ivt-RNA encoding an HLA allotype. Since mDC display high levels of endogenous MHC class I, the surface expression of transgenic allogeneic MHC must be monitored using HLA allotype-specific monoclonal antibodies to analyze cells by flow cytometry. For example, expression of transgenic HLA-A2 molecules can be studied on mDC prepared from HLA-A2- donors using a primary HLA-A2-specific BB7.2 monoclonal antibody, followed by a secondary PE-conjugated antibody. As an example HLA-A2 surface expression was already detected on the mDC surface at 1.5 h following electroporation of 25 μg ivt-RNA encoding the HLA-A*0201 allele (Fig. 2A). In this case, HLA-A2 expression reached a peak at 10 h, measured as mean fluorescent intensity (MFI), and then decreased over time. Nevertheless, around 50% of the mDC were still positive for HLA-A2 at 120 h. The greatest percentage of HLA-A2+ mDC (63%) was also seen at 10 h.

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