1. Volume 17, Issue 9, Pages 1616-1625 (September 2009)
Nucleofection of DCs to Generate Multivirus-specific T Cells for Prevention or Treatment of Viral Infections in the Immunocompromised Host 
Ulrike Gerdemann, Anne S Christin, Juan F Vera, Carlos A Ramos, Yuriko Fujita, Hao Liu, Dagmar Dilloo, Helen E Heslop, Malcolm K Brenner, Cliona M Rooney, Ann M Leen 
Molecular Therapy 
Volume 17, Issue 9, Pages (September 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Viability, transgene expression, and maturation state of nucleofected DCs. (a) DC viability, evaluated by Trypan blue exclusion, of nucleofected and unmodified cells in a time course from 0- to 60-hours postnucleofection. Viability was comparable in both DC populations until 24 hours postnucleofection (median 66%; range 58–74%) but thereafter progressively declined in the nucleofected population to median 29%; range 23–38% at the 60-hour time point. (b) Transgene expression (GFP) in nucleofected DCs, which was detectable as early as 6-hour postnucleofection with mean 38 ± 11% SD GFP+ cells, and remained stable until 60-hours postnucleofection in the live DC fraction. (c) The DC maturation state, evaluated by cell surface expression of CD83, CD80, CD86, and HLA-DR on untreated and nucleofected DCs 24 hours postnucleofection. DC, dendritic cell; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Transfection efficiency and stimulatory capacity of DNA plasmids versus adenoviral vectors. (a) GFPpp65 transgene expression (based on %GFP+ cells) in nucleofected (n = 15) or adenovector-transduced DCs (n = 7), 24–48-hours after modification. (b) Representative multimer staining from two HLA-A2 donors using the pp65-NLV pentamer performed 9 days after stimulation. The plasmid-activated CTL are shown in the left panels whereas the adenovector-activated CTL shown on the right-hand side. (c) The frequency of CMV-pp65 reactive T cells in plasmid- (black bars) or adenovector-activated CTL (gray bars) in response to the CMV-pp65 pepmix or a combination of the Hexon and Penton (Adv) pepmixes using IFN-γ ELIspot as a readout (n = 7 donors). Results represent the mean ± SE spot forming cells (SFCs) per 2 × 105 input cells. Control was IFN-γ release in response to stimulation with irrelevant pepmix. CTL, cytotoxic T-lymphocyte; CMV, cytomegalovirus; DC, dendritic cells; ELIspot, enzyme-linked immunospot; GFP, green fluorescent protein; HLA, human leukocyte antigen, IFN-γ, interferon-γ.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Specificity and function of CMVpp65-specific CTL after in vitro expansion. Plasmid- and adenovector-activated CTL were expanded by weekly restimulation using Ad5f35GFPpp65-transduced LCL in the presence of IL-2. On day 23 of culture T-cell expansion, specificity and function was assessed. (a) Overall T-cell expansion, based on cell counting using Trypan blue exclusion. Although the adenovector-activated cells expanded significantly more than the plasmid-activated cells, there was no difference in expansion of antigen-specific T cells based on pentamer analysis. (b) Representative NLV-pentamer staining of CTL lines from two donors on day 23, whereas (c) the expansion of the antigen-specific population of cells, based on HLA-A2 NLV and HLA-B7 TPR pentamer staining. (d) The frequency of CMV-pp65 reactive T cells in plasmid (black bars)- or adenovector (gray bars)-activated CTL in response to pp65 pepmix using IFN-γ ELIspot as readout (n = 7 donors). Results represent the mean ± SE SFC/2 × 105 input cells. Control was IFN-γ release in response to stimulation with irrelevant pepmix. (e) The CTL were functional in a standard 4-hour Cr51 assay and were able to lyse autologous pp65 pepmix-pulsed and antigen-expressing targets, with no recognition of mock or allogeneic targets. Data represent the mean ± SE% of target lysis from seven donors. CTL, cytotoxic T-lymphocyte; CMV, cytomegalovirus; ELIspot, enzyme-linked immunospot; IFN-γ, interferon-γ; LCL, lymphoblastoid cell line.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Generation of CTL against less immunogenic viral antigens. DCs were nucleofected with pShuttle plasmids encoding Adv-Penton Adv-Hexon, BK-Large T, and EBV-LMP2-I-BZLF1. DNA for nucleofection was titrated from 2 to 20 µg DNA and the specificity of the resultant lines was compared to a positive control using DCs pulsed with a pepmix spanning the relevant antigen(s), with IFN-γ ELIspot as readout. Results are expressed as SFC/2 × 105 input cells. Control was IFN-γ release in response to stimulation with irrelevant pepmix. Representative examples from one donor are shown; results were confirmed in at least three donors. DCs, dendritic cells; ELIspot, enzyme-linked immunospot; IFN-γ, interferon-γ; SFC, spot-forming cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Generation of multivirus-specific CTL. (a) IFN-γ ELIspot results from a representative donor comparing the specificity of single (left panel) versus multivirus (right panel) CTL lines reactive against Penton, Hexon, Large T, LMP2, BZLF1, and pp65 generated by T cell reactivation using nucleofected DCs as APCs. Results are expressed as SFC/2 × 105 input cells. Control was IFN-γ release in response to stimulation with irrelevant pepmix. (b) The specificity of multivirus CTL generated from seven donors, who were Adv, BK, and EBV seropositive. Two of seven were CMV seropositive. (c) These CTL should be safe for infusion because in a standard mixed lymphocyte reaction where alloreactive potential is measured by 3H incorporation following incubation with autologous or third party PBMC, the day 9 multivirus CTL proliferated minimally, similar to that of day 23 CTL. The presented results are the mean ± SD of five donors expressed as counts per minute (cpm) after thymidine uptake. Adv, adenovirus; CTL, cytotoxic T-lymphocyte; CMV, cytomegalovirus; ELIspot, enzyme-linked immunospot; EBV, Epstein–Barr virus; IFN-γ, interferon-γ; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Selection of multivirus CTL using the IFN-γ capture assay isolates both central and effector memory T cells. (a) Representative example of the specificity of multivirus CTLs that were selected by IFN-γ capture, and subsequently expanded in vitro to allow functional characterization. Specificity was measured by IFN-γ release in response to relevant and irrelevant pepmixes and results are expressed as SFC/2 × 105 input cells. (b) The selected and expanded cells are functional as measured by Cr51 release assay. Autologous PHA blasts, either alone or pulsed with the relevant pepmixes, were used as targets. Alloreactivity was assessed using allogeneic PHA blasts as a target. (c) shows, by flow cytometric analysis, that both central (CD62L+, CD45RO+) and effector memory (CD62L−, CD45RO+) T cells produce IFN-γ after stimulation with nucleofected DCs. Prior to stimulation PBMCs were selected or depleted for 62L using MACS bead separation. The purity of the selected/depleted fraction was >95% based on flow cytometric analysis. PBMCs were gated on CD3+ and CD45RO+. Selected/depleted CD62L fractions were stimulated with pShuttleGFPpp65 nucleofected DCs and IFN-γ production was evaluated by intracellular IFN-γ staining 18 hours after stimulation. Cells were gated on CD3+ and CD45RO+. Both CD8+ and CD8-(CD4+) T cells derived from central and effector memory T-cell subsets produced IFN-γ. CTL, cytotoxic T-lymphocyte; CMV, cytomegalovirus; IFN-γ, interferon-γ; PHA, phytohemagglutinin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions