Strategic use of an adenoviral vector for rapid and efficient ex vivo‐generation of cytomegalovirus pp65‐reactive cytolytic and helper T cells

Abstract

+T cells. This implies that sufficient number of pp65-specific cytolytic and helper T cells for adoptive transfer may be obtained in only 2 weeks.

Adenovirus Serotype 5 Vectors with Tat-PTD Modified Hexon and Serotype 35 Fiber Show Greatly Enhanced Transduction Capacity of Primary Cell Cultures

“…We have previously shown that a population of CMV pp65 495–503 -specific T cells can be significantly expanded if T cells from CMV-seropositive, HLA-A2-positive blood donors are stimulated by Ad5(pp65)-transduced autologous DCs [16], [22]. However, large amounts of viral vector need to be used to obtain efficient transduction of DCs.…”

“…We therefore constructed a non-modified Ad5(pp65) and a double-modified Ad5PTDf35(pp65), which both express the full-length CMV pp65 transgene. We transduced monocytes with Ad5(pp65) or Ad5PTDf35(pp65) at a relatively low dosage (100 evg/cell), differentiated them into DCs [16], [22] and used them to stimulate and expand autologous pp65-specific T cells. The Ad5PTDf35(pp65)-transduced DC stimulation increased the pp65 495–503 -reactive T cells population for all donors, approximately 50–100 fold, while Ad5(pp65)-transduced DC stimulation only increased the pp65 495–503 -reactive T cell population 2–8 fold (Figure 5).…”

“…T cells were activated for 72 hours in culture medium supplemented with OKT-3 (100 ng/ml, BioLegend, San Diego, CA) and interleukin (IL)-2 (50 IU/ml, Proleukin, Novartis, Basel, Switzerland). Monocytes were isolated from PBMCs by plastic adhesion for 90 min [22]. Immature dendritic cells (DCs) were obtained by differentiating monocytes in culture medium supplemented with GM-CSF (50 ng/mL, Gentaur, Brussels, Belgium) and IL-4 (25 ng/mL, Gentaur) for 7 days with fresh medium every second day.…”

“…The Ad-transduced monocytes were then differentiated to fast DCs by growing them in culture medium supplemented with GM-CSF (50 ng/mL, Gentaur) and IL-4 (25 ng/mL, Gentaur) for 3 days with fresh medium every day. DCs were then maturated by adding TNF-α (40 ng/ml, Invitrogen) and poly(I:C) (30 µg/mL, Sigma-Aldrich, St Louis, MO) at day 3 and cultured for 24 hours as previously described [22]. The transduced and matured DCs were then mixed with autologous T cells at a 1∶10 ratio and cultured for 11 days to stimulate and expand antigen-specific T cells.…”

The HDAC Inhibitor FK228 Enhances Adenoviral Transgene Expression by a Transduction-Independent Mechanism but Does Not Increase Adenovirus Replication

“…Monocytes are precursors of dendritic cells and as such, highly interesting as cancer vaccines after being modified to express tumor-associated antigens, often in the form of an adenoviral vector carrying the transgene coding for the tumor-associated antigen. For example, efficient adenoviral transduction of monocytes is highly warranted for ex vivo generation of T cells against specific antigens [35]. Such T cells could then be used for adoptive transfer to cancer patients.…”