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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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Flow cytometric characterization. Shown are the results of FACS analysis for the antigen delivery. Note that the use of AAV/GFP/IE1 loading DC resulted in a higher delivery effect (80%) than IE1 protein lipofected DC did (15%).
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Figure 7: Flow cytometric characterization. Shown are the results of FACS analysis for the antigen delivery. Note that the use of AAV/GFP/IE1 loading DC resulted in a higher delivery effect (80%) than IE1 protein lipofected DC did (15%).

Mentions: To determine the ability of AAV/IE1-transduced DCs to stimulate IE1-specific CTLs, we performed a standard 6-hour51Cr assay on day 7 using a 1:20 (ratio: Effector:Target) (Figure 5) using the T-cell populations primed in co-culture with the rAAV-transduced DCs [30]. We generated autologous targets by infecting donor PBMCs with AAV/IE1 virus 4 days before the CTL assay. AAV/IE1-infected PBMCs were found to express IE1 by RT-PCR analysis, whereas unaltered PBMCs and K562 cells did not express IE1 (data not shown). T-cells incubated with AAV/IE1-loaded DCs were able to kill the IE1-positive autologous target cells. These data are consistent with a strong antigen-specific CTL response. Figure 7 shows that CTL killing activity was dose-dependent and MHC class I restricted. In this experiment, 2 different doses of AAV/IE1 vector were used for DC loading and a zero virus control (PBMC only). The cytotoxicity of the stimulated T-cells directly correlated with the amount of AAV/IE1 used to load the DCs at day 0. Alternately, the addition of anti-class I antibodies significantly inhibited the killing activity (P < 0.05), suggesting that CTLs were MHC class I restricted. The CTL stimulation performed by AAV/IE1 loaded DCs was superior to the one performed by IE1 protein lipofection (P < 0.05). The negative controls (K562 and the targets pre-incubated with anti-MHC class I antibodies) did not induce significant killing activity. These data showed CTLs to be highly AAV/IE1 specific and MHC class I restricted. Figure 7 demonstrates that the use of AAV/GFP/IE1 loading DCs resulted in a higher delivery effect (80%) than IE1 protein lipofected DCs did (15%).


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)

Flow cytometric characterization. Shown are the results of FACS analysis for the antigen delivery. Note that the use of AAV/GFP/IE1 loading DC resulted in a higher delivery effect (80%) than IE1 protein lipofected DC did (15%).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Flow cytometric characterization. Shown are the results of FACS analysis for the antigen delivery. Note that the use of AAV/GFP/IE1 loading DC resulted in a higher delivery effect (80%) than IE1 protein lipofected DC did (15%).
Mentions: To determine the ability of AAV/IE1-transduced DCs to stimulate IE1-specific CTLs, we performed a standard 6-hour51Cr assay on day 7 using a 1:20 (ratio: Effector:Target) (Figure 5) using the T-cell populations primed in co-culture with the rAAV-transduced DCs [30]. We generated autologous targets by infecting donor PBMCs with AAV/IE1 virus 4 days before the CTL assay. AAV/IE1-infected PBMCs were found to express IE1 by RT-PCR analysis, whereas unaltered PBMCs and K562 cells did not express IE1 (data not shown). T-cells incubated with AAV/IE1-loaded DCs were able to kill the IE1-positive autologous target cells. These data are consistent with a strong antigen-specific CTL response. Figure 7 shows that CTL killing activity was dose-dependent and MHC class I restricted. In this experiment, 2 different doses of AAV/IE1 vector were used for DC loading and a zero virus control (PBMC only). The cytotoxicity of the stimulated T-cells directly correlated with the amount of AAV/IE1 used to load the DCs at day 0. Alternately, the addition of anti-class I antibodies significantly inhibited the killing activity (P < 0.05), suggesting that CTLs were MHC class I restricted. The CTL stimulation performed by AAV/IE1 loaded DCs was superior to the one performed by IE1 protein lipofection (P < 0.05). The negative controls (K562 and the targets pre-incubated with anti-MHC class I antibodies) did not induce significant killing activity. These data showed CTLs to be highly AAV/IE1 specific and MHC class I restricted. Figure 7 demonstrates that the use of AAV/GFP/IE1 loading DCs resulted in a higher delivery effect (80%) than IE1 protein lipofected DCs did (15%).

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