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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 4. Co-expression of additional combinations of ivt-RNA encoding TAA and MHC. (A) mDC of an HLA-A2- donor are electroporated with four different RNA combinations: (1) 25 µg HLA-A2 and 25 µg melan-A ivt-RNA, (2) 25 µg HLA-A2 and 50 µg melan-A ivt-RNA, (3) 50 µg HLA-A2 and 25 µg melan-A ivt-RNA and (4) 50 µg HLA-A2 and 50 µg melan-A ivt-RNA. mDC of an HLA-A2+ donor are transfected with (5) 25 µg melan-A or (6) 50 µg melan-A ivt-RNA. Surface staining of HLA-A2 and intracellular staining of melan-A is performed 6 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves in the upper panels; the corresponding melan-A expression of the same sample is shown beneath by filled curves. Corresponding controls are depicted in the respective histograms by open curves. (B) mDC of an HLA-A2- donor are electroporated with four different RNA combinations: (1) 25 µg HLA-A2 and 25 µg survivin ivt-RNA, (2) 25 µg HLA-A2 and 50 µg survivin ivt-RNA, (3) 50 µg HLA-A2 and 25 µg survivin ivt-RNA and (4) 50 µg HLA-A2 and 50 µg survivin ivt-RNA. DC of an HLA-A2+ donor are transfected with (5) 25 µg survivin or (6) 50 µg survivin ivt-RNA. Surface staining of HLA-A2 is performed 6 h and intracellular staining of survivin 3 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves; corresponding survivin expression of the same sample is shown beneath by filled curves. Corresponding controls are depicted in the respective histograms by open curves. (C) The percent positive cells, MFI and x-fold expression of all stainings in (A and B) are shown.
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Figure 4: Figure 4. Co-expression of additional combinations of ivt-RNA encoding TAA and MHC. (A) mDC of an HLA-A2- donor are electroporated with four different RNA combinations: (1) 25 µg HLA-A2 and 25 µg melan-A ivt-RNA, (2) 25 µg HLA-A2 and 50 µg melan-A ivt-RNA, (3) 50 µg HLA-A2 and 25 µg melan-A ivt-RNA and (4) 50 µg HLA-A2 and 50 µg melan-A ivt-RNA. mDC of an HLA-A2+ donor are transfected with (5) 25 µg melan-A or (6) 50 µg melan-A ivt-RNA. Surface staining of HLA-A2 and intracellular staining of melan-A is performed 6 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves in the upper panels; the corresponding melan-A expression of the same sample is shown beneath by filled curves. Corresponding controls are depicted in the respective histograms by open curves. (B) mDC of an HLA-A2- donor are electroporated with four different RNA combinations: (1) 25 µg HLA-A2 and 25 µg survivin ivt-RNA, (2) 25 µg HLA-A2 and 50 µg survivin ivt-RNA, (3) 50 µg HLA-A2 and 25 µg survivin ivt-RNA and (4) 50 µg HLA-A2 and 50 µg survivin ivt-RNA. DC of an HLA-A2+ donor are transfected with (5) 25 µg survivin or (6) 50 µg survivin ivt-RNA. Surface staining of HLA-A2 is performed 6 h and intracellular staining of survivin 3 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves; corresponding survivin expression of the same sample is shown beneath by filled curves. Corresponding controls are depicted in the respective histograms by open curves. (C) The percent positive cells, MFI and x-fold expression of all stainings in (A and B) are shown.

Mentions: To establish that other combinations of ivt-RNA also led to strong co-expression of MHC and TAA in mDC, we analyzed melan-A and survivin as TAA, together with HLA-A2. Again, transgenic expression of HLA-A2 was monitored by surface staining and TAA protein expression was detected by intracellular staining. The amounts of ivt-RNA introduced into the mDC were also varied. With both combinations, it was also possible to detect high percentages of HLA-A2+ mDC and TAA+ mDC with substantial levels of protein expression (Fig. 4). These results demonstrated that this approach provides a robust method to efficiently generate mDC that co-express new MHC and TAA. Furthermore, the same method could be easily used to introduce ivt-RNA only encoding a TAA into mDC that naturally expressed the desired HLA restriction element due to presence of the endogenous class I allele, as shown in Figure 4, panels 5 and 6 for both melan-A and survivin (Fig. 4).


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 4. Co-expression of additional combinations of ivt-RNA encoding TAA and MHC. (A) mDC of an HLA-A2- donor are electroporated with four different RNA combinations: (1) 25 µg HLA-A2 and 25 µg melan-A ivt-RNA, (2) 25 µg HLA-A2 and 50 µg melan-A ivt-RNA, (3) 50 µg HLA-A2 and 25 µg melan-A ivt-RNA and (4) 50 µg HLA-A2 and 50 µg melan-A ivt-RNA. mDC of an HLA-A2+ donor are transfected with (5) 25 µg melan-A or (6) 50 µg melan-A ivt-RNA. Surface staining of HLA-A2 and intracellular staining of melan-A is performed 6 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves in the upper panels; the corresponding melan-A expression of the same sample is shown beneath by filled curves. Corresponding controls are depicted in the respective histograms by open curves. (B) mDC of an HLA-A2- donor are electroporated with four different RNA combinations: (1) 25 µg HLA-A2 and 25 µg survivin ivt-RNA, (2) 25 µg HLA-A2 and 50 µg survivin ivt-RNA, (3) 50 µg HLA-A2 and 25 µg survivin ivt-RNA and (4) 50 µg HLA-A2 and 50 µg survivin ivt-RNA. DC of an HLA-A2+ donor are transfected with (5) 25 µg survivin or (6) 50 µg survivin ivt-RNA. Surface staining of HLA-A2 is performed 6 h and intracellular staining of survivin 3 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves; corresponding survivin expression of the same sample is shown beneath by filled curves. Corresponding controls are depicted in the respective histograms by open curves. (C) The percent positive cells, MFI and x-fold expression of all stainings in (A and B) are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Figure 4. Co-expression of additional combinations of ivt-RNA encoding TAA and MHC. (A) mDC of an HLA-A2- donor are electroporated with four different RNA combinations: (1) 25 µg HLA-A2 and 25 µg melan-A ivt-RNA, (2) 25 µg HLA-A2 and 50 µg melan-A ivt-RNA, (3) 50 µg HLA-A2 and 25 µg melan-A ivt-RNA and (4) 50 µg HLA-A2 and 50 µg melan-A ivt-RNA. mDC of an HLA-A2+ donor are transfected with (5) 25 µg melan-A or (6) 50 µg melan-A ivt-RNA. Surface staining of HLA-A2 and intracellular staining of melan-A is performed 6 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves in the upper panels; the corresponding melan-A expression of the same sample is shown beneath by filled curves. Corresponding controls are depicted in the respective histograms by open curves. (B) mDC of an HLA-A2- donor are electroporated with four different RNA combinations: (1) 25 µg HLA-A2 and 25 µg survivin ivt-RNA, (2) 25 µg HLA-A2 and 50 µg survivin ivt-RNA, (3) 50 µg HLA-A2 and 25 µg survivin ivt-RNA and (4) 50 µg HLA-A2 and 50 µg survivin ivt-RNA. DC of an HLA-A2+ donor are transfected with (5) 25 µg survivin or (6) 50 µg survivin ivt-RNA. Surface staining of HLA-A2 is performed 6 h and intracellular staining of survivin 3 h after transfection and analyzed by flow cytometry. HLA-A2 expression is represented by filled curves; corresponding survivin expression of the same sample is shown beneath by filled curves. Corresponding controls are depicted in the respective histograms by open curves. (C) The percent positive cells, MFI and x-fold expression of all stainings in (A and B) are shown.
Mentions: To establish that other combinations of ivt-RNA also led to strong co-expression of MHC and TAA in mDC, we analyzed melan-A and survivin as TAA, together with HLA-A2. Again, transgenic expression of HLA-A2 was monitored by surface staining and TAA protein expression was detected by intracellular staining. The amounts of ivt-RNA introduced into the mDC were also varied. With both combinations, it was also possible to detect high percentages of HLA-A2+ mDC and TAA+ mDC with substantial levels of protein expression (Fig. 4). These results demonstrated that this approach provides a robust method to efficiently generate mDC that co-express new MHC and TAA. Furthermore, the same method could be easily used to introduce ivt-RNA only encoding a TAA into mDC that naturally expressed the desired HLA restriction element due to presence of the endogenous class I allele, as shown in Figure 4, panels 5 and 6 for both melan-A and survivin (Fig. 4).

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