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Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells.

Yu Y, Pilgrim P, Yan J, Zhou W, Jenkins M, Gagliano N, Bumm K, Cannon M, Milzani A, Dalle-Donne I, Kast WM, Cobos E, Chiriva-Internati M - J Transl Med (2008)

Bottom Line: As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV.CTLs were capable to lyse low doses of peptides pulsed into target cells.These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology & Oncology, Texas Tech University Health Sciences Center and Southwest Cancer Treatment and Research Center, Lubbock, TX, USA. yuefei.yu@ttuhsc.edu

ABSTRACT

Background: Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens.

Methods: We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells.

Results: A significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation.

Conclusion: In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

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IE1 expression in infected DCs. Total RNA was isolated from mock-infected and AAV/IE1-infected adherent monocytes at 72 hours after infection. These samples were analyzed by RT-PCR and PCR, as indicated, for the presence of IE1 RNA. PCR product resulting from using the AAV/IE1 vector plasmids as templates was the positive controls. RT-PCR analysis for the cellular TFIIB mRNA was considered as further control. Note that only cDNA from cells infected with AAV/IE1 virus resulted in an appropriate RT-PCR sized product, whereas mock-infected cells did not.
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Figure 3: IE1 expression in infected DCs. Total RNA was isolated from mock-infected and AAV/IE1-infected adherent monocytes at 72 hours after infection. These samples were analyzed by RT-PCR and PCR, as indicated, for the presence of IE1 RNA. PCR product resulting from using the AAV/IE1 vector plasmids as templates was the positive controls. RT-PCR analysis for the cellular TFIIB mRNA was considered as further control. Note that only cDNA from cells infected with AAV/IE1 virus resulted in an appropriate RT-PCR sized product, whereas mock-infected cells did not.

Mentions: Protocols for generating DCs by differentiating PBMCs usually involve the use of GM-CSF and IL-4 during adherent monocyte culturing. We modified this protocol to promote AAV vector transduction in DC precursor monocytes by treating adherent monocytes just after AAV infection with GM-CSF alone, adding IL-4 on day 3. This method allowed higher levels of AAV transduction [34]. Figure 1B shows a schematic diagram of the experimental protocol. Monocyte/DC population transduction was confirmed by measuring polyadenylated RNA expression of the AAV/IE1 transgene. At day 10, polyadenylated RNA was isolated from AAV/IE1-infected and mock-infected DC cultures. The mRNA levels were analyzed by RT-PCR for AAV/IE1 expression. A cellular housekeeping gene, TFIIB, was included as a control. IE1 mRNA expression took place only in the infected DCs (Figure 3). A PCR-only control (no RT step) failed to generate a product, indicating that there was no DNA contamination in our samples.


Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells.

Yu Y, Pilgrim P, Yan J, Zhou W, Jenkins M, Gagliano N, Bumm K, Cannon M, Milzani A, Dalle-Donne I, Kast WM, Cobos E, Chiriva-Internati M - J Transl Med (2008)

IE1 expression in infected DCs. Total RNA was isolated from mock-infected and AAV/IE1-infected adherent monocytes at 72 hours after infection. These samples were analyzed by RT-PCR and PCR, as indicated, for the presence of IE1 RNA. PCR product resulting from using the AAV/IE1 vector plasmids as templates was the positive controls. RT-PCR analysis for the cellular TFIIB mRNA was considered as further control. Note that only cDNA from cells infected with AAV/IE1 virus resulted in an appropriate RT-PCR sized product, whereas mock-infected cells did not.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570669&req=5

Figure 3: IE1 expression in infected DCs. Total RNA was isolated from mock-infected and AAV/IE1-infected adherent monocytes at 72 hours after infection. These samples were analyzed by RT-PCR and PCR, as indicated, for the presence of IE1 RNA. PCR product resulting from using the AAV/IE1 vector plasmids as templates was the positive controls. RT-PCR analysis for the cellular TFIIB mRNA was considered as further control. Note that only cDNA from cells infected with AAV/IE1 virus resulted in an appropriate RT-PCR sized product, whereas mock-infected cells did not.
Mentions: Protocols for generating DCs by differentiating PBMCs usually involve the use of GM-CSF and IL-4 during adherent monocyte culturing. We modified this protocol to promote AAV vector transduction in DC precursor monocytes by treating adherent monocytes just after AAV infection with GM-CSF alone, adding IL-4 on day 3. This method allowed higher levels of AAV transduction [34]. Figure 1B shows a schematic diagram of the experimental protocol. Monocyte/DC population transduction was confirmed by measuring polyadenylated RNA expression of the AAV/IE1 transgene. At day 10, polyadenylated RNA was isolated from AAV/IE1-infected and mock-infected DC cultures. The mRNA levels were analyzed by RT-PCR for AAV/IE1 expression. A cellular housekeeping gene, TFIIB, was included as a control. IE1 mRNA expression took place only in the infected DCs (Figure 3). A PCR-only control (no RT step) failed to generate a product, indicating that there was no DNA contamination in our samples.

Bottom Line: As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV.CTLs were capable to lyse low doses of peptides pulsed into target cells.These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology & Oncology, Texas Tech University Health Sciences Center and Southwest Cancer Treatment and Research Center, Lubbock, TX, USA. yuefei.yu@ttuhsc.edu

ABSTRACT

Background: Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens.

Methods: We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells.

Results: A significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation.

Conclusion: In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

Show MeSH
Related in: MedlinePlus