Rapid induction of cytotoxic T-cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector

Abstract

We have shown that the pulsing of dendritic cells ( DCs ) with human papillomavirus type 16 ( HPV -16 ) antigen proteins by lipofection stimulates class I -restricted cytotoxic T lymphocyte ( CTL ) response against primary cervical cancer cells. Also, we have shown that adeno -associated virus ( AAV ) was able to effectively deliver a cytokine gene into DCs. It has been our hypothesis that the delivery of antigen genes into DCs, resulting in endogenous and continuous antigen protein expression, may result in an improvement in T -cell priming by DCs. Here, DCs are pulsed ( infected ) with an AAV vector containing the HPV -16 E6 gene. After infection, transduced E6 gene mRNA expression and vector chromosomal integration could be identified in infected DCs. Furthermore, priming rosettes formed at early times when the AAV / E6 vector was used. Most importantly, AAV / E6 vector pulsing of DCs induced, after only 7 days of priming, a strong CTL response against primary cervical cancer cell lines, compared to bacterial E6 protein lipofection. Killing was significantly blocked by the addition of anti -MHC class I antibodies. Fluorescence -activated cell sorter ( FACS ) analysis of resulting primed cell populations revealed higher levels of CD8 + T cells by AAV -based pulsing, with little evidence of CD56 ( NK ). FACS analysis of the DC populations revealed that AAV / E6 vector -pulsed DCs had higher levels of CD80 and lower levels of CD86 than protein -pulsed DCs. These data suggest that rAAV may be appropriate for antigen pulsing of DCs for immunotherapy protocols. Finally, our protocol represents an advance in regards to the time needed for generating a CTL response compared to other techniques. Cancer Gene Therapy ( 2001 ) 8, 948 -957

Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination

“…Rapid induction of CTL response from naïve T-cell precursors after stimulation with neo-or self-antigen-loaded DC is not a new finding and has been demonstrated previously in several studies. [53][54][55] We believe it likely that there are multiple reasons for the rapid induction of CTL response Immune responses to gene-modified dendritic cells N Chinnasamy et al against eGFP + DCs in our DC culture system. One reason is the high transduction frequency of DCs and transgene expression we have observed (B90%) with Lenti-eGFP vector.…”

Immune responses to gene therapy vectors: influence on vector function and effector mechanisms

“…The vector dose, the route of administration, the nature of the transgene, and host-related factors 3 Activation of innate immunity by adenoviruses varies with the vector dose, as established in vitro with cultured peripheral blood mononuclear cells. 4 PBMC infection by adenoviruses induces cytokine production in a dosedependent manner up to a threshold adenovirus dose, above which no further cytokine increase occurs. This plateau effect is probably ascribable to saturation of adenovirus receptors such as CAR.…”

“…has been used to generate cytotoxic responses to tumor or virus antigens in antitumor vaccination strategies, 4,22 or anti-HIV vaccination. 23 A T-cell response specific of the transgene and responsible for elimination of the infected cells can occur, however, in some situations.…”

T-cell responses to human papillomavirus type 16 among women with different grades of cervical neoplasia

“…Studies using whole HPV proteins to restimulate CTL responses, either in a soluble form (Nakagawa et al, 1997) or expressed by recombinant viral vectors (Nimako et al, 1997), where the whole protein is naturally processed, have been more successful. Autologous dendritic cells (DCs) presenting HPV16 peptides or whole proteins have also been used to restimulate E6-and E7-specific CTL responses from both normal donors and women with cervical carcinoma in vitro (Murakami et al, 1999;Schoell et al, 1999;Thornberg et al, 2000;Davidson et al, 2001;Liu et al, 2001;Chiriva-Internati et al, 2002;Valdespino et al, 2005), although it is not clear whether these represent primary or memory T-cell responses. Overall, the results suggest that naturally occurring HPV E6-and E7-specific CTLs do exist in patients with HPV-associated disease, although their detection may depend on the methods used to reactivate them in vitro.…”