Limits...
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.

Show MeSH

Related in: MedlinePlus

Cytotoxicity assay. AAV/GFP/IE1 vectors for DC loading and multiple targets generated using various vectors. Targets were generated by IE1 positive and negative vector loading into PBMC. Resulting CTL killing is shown. IE1 negative PBMCs (no Ag) and K562 cells were not killed, indicating strong antigen specificity for the CTLs generated by AAV/IE1 loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2570669&req=5

Figure 5: Cytotoxicity assay. AAV/GFP/IE1 vectors for DC loading and multiple targets generated using various vectors. Targets were generated by IE1 positive and negative vector loading into PBMC. Resulting CTL killing is shown. IE1 negative PBMCs (no Ag) and K562 cells were not killed, indicating strong antigen specificity for the CTLs generated by AAV/IE1 loading.

Mentions: We analyzed the ability of the AAV/IE1 vectors to generate IE1 specific-CTLs (optimal ratio E:T; 1:20). To analyze CTL activity, we used the following 5 target cell types for the 51Cr release assays (Figures 4, 5, 6): 1) Autologous PBMCs. Because late B cells are only a small percentage of PBMCs, PBMCs served as an autologous, antigen-negative control; 2) PBMCs transfected with AAV/IE1 expression plasmid; 3) PBMCs transfected with AAV only and AAV/GFP, as a negative controls; 4) PBMCs transfected with E6, as a control; 5) PBMCs transfected with IE1 protein.


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)

Cytotoxicity assay. AAV/GFP/IE1 vectors for DC loading and multiple targets generated using various vectors. Targets were generated by IE1 positive and negative vector loading into PBMC. Resulting CTL killing is shown. IE1 negative PBMCs (no Ag) and K562 cells were not killed, indicating strong antigen specificity for the CTLs generated by AAV/IE1 loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Cytotoxicity assay. AAV/GFP/IE1 vectors for DC loading and multiple targets generated using various vectors. Targets were generated by IE1 positive and negative vector loading into PBMC. Resulting CTL killing is shown. IE1 negative PBMCs (no Ag) and K562 cells were not killed, indicating strong antigen specificity for the CTLs generated by AAV/IE1 loading.
Mentions: We analyzed the ability of the AAV/IE1 vectors to generate IE1 specific-CTLs (optimal ratio E:T; 1:20). To analyze CTL activity, we used the following 5 target cell types for the 51Cr release assays (Figures 4, 5, 6): 1) Autologous PBMCs. Because late B cells are only a small percentage of PBMCs, PBMCs served as an autologous, antigen-negative control; 2) PBMCs transfected with AAV/IE1 expression plasmid; 3) PBMCs transfected with AAV only and AAV/GFP, as a negative controls; 4) PBMCs transfected with E6, as a control; 5) PBMCs transfected with IE1 protein.

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