1. Volume 15, Issue 4, Pages 818-824 (April 2007)
Lentivirally Transduced Recipient-derived Dendritic Cells Serve to Ex Vivo Expand Functional FcRγ-sufficient Double-negative Regulatory T Cells 
Christopher W Thomson, Miriam E Mossoba, Christopher Siatskas, Wenhao Chen, April Sung, Jeffrey A Medin, Li Zhang 
Molecular Therapy 
Volume 15, Issue 4, Pages (April 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
BMDCs were successfully transduced with lentirvirus expressing MHC class I antigens. (a) Schematic representation of plasmid used to create lentiviral vector expressing MHC class I Ld. Gene transfer vector (pHR'EF-Ld-W-SIN), packaging vector (pCMV ΔR8.91), and envelope construct (pMD.G) were transfected into 293T cells to produce a replication-deficient lentiviral vector expressing Ld. (b) BMDCs were transduced with a lentiviral vector expressing Ld or eGFP.C57BL/6 (Ld−). BM was isolated and cultured with IL-4 and GM-CSF to produce BMDCs. Day 3 BMDC cultures were transduced with lentiviral vectors expressing MHC class I antigen Ld or eGFP and transduction efficiency was between 50 and 60% at 3 days after transduction.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Mature BMDCs transduced with lentirvirus expressing MHC class I antigen Ld expand DN Treg cells. BMDCs were transduced with lentivirus vector expressing Ld or eGFP as described in Figure 1 and used to stimulate the proliferation of (a) FcRγ+/+ or (b) FcRγ−/− DN T cells. At day 5, 500 U/ml TNF-α was added to BMDC cultures to create mDCs, or no additional supplements were added to keep the DCs in an immature state (iDCs). DN T cells were isolated as described in Materials and Methods. The viability and purity of cells were monitored by flow cytometry and were more than 95%. The purified DN T cells were stimulated with LV-Ld-transduced, LV-eGFP-transduced, or untransduced mDCs or iDCs in α-MEM supplemented with 10% fetal bovine serum. rIL-2 (50 U/ml) and rIL-4 (30 U/ml) were added to the cultures. After 4 days, cells were labeled with 1 μCi (0.037 MBq)/well tritium-thymidine, harvested 18 h later, and counted in a scintillation counter (TopCount; Packard, Meriden, CT). All experiments were repeated three times and similar results were obtained. Graph shows mean of triplicate samples (error bars represent SEM).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Phenotype of mDC clone DC2.4 that is transduced with lentirvirus expressing MHC class I antigen. The mDC clone DC2.4 was trasnduced with a lentiviral vector expressing Ld or eGFP and cell sorting was performed to increase transgene expression to >95%. (a) The expression level of eGFP (LV-eGFP-transduced), and Ld or IgG2a control antibody (LV-Ld transduced) by flow cytometry is shown. (b) The mature phenotype of DC2.4-Ld DC clones was confirmed by staining with MHC II-FITC, CD40-FITC, B71-PE, and B72-PE (gray line shows isotype controls).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
DC2.4 cells transduced with lentivirus expressing MHC class I antigen expand DN Treg cells. (a) FcRγ+/+ or (b) FcRγ−/− DN T cells were purified as described in Figure 2 and stimulated with DC2.4 cells. The purified DN T cells were stimulated with irradiated Ld+ spleen cells, or irradiated DC2.4 cells that were untransduced (DC2.4) or transduced with either LV-Ld (DC2.4-Ld) or LV-eGFP (DC2.4-eGFP) in α-MEM supplemented with 10% FCS. rIL-2 (50 U/ml) and rIL-4 (30 U/ml) were added to the cultures. After 4 days, cells were labeled with 1 μCi (0.037 MBq)/well tritium-thymidine, harvested 18 h later, and counted in a scintillation counter (TopCount; Packard, Meriden, CT). All experiments were repeated three times and similar results were obtained. Graph shows mean of triplicate samples (error bars represent SEM).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
DN T cells expanded by DC2.4 cells transduced with lentivirus expressing MHC class I antigen retain regulatory function in vitro. Expanded (a) FcRγ+/+ or (b) FcRγ−/− DN T cells were assessed for their ability to suppress CD8+ T cells. DN T cells and CD8+ T cells were purified as described in Materials and Methods. The purified DN T cells were expanded with DC2.4-Ld, DC2.4-eGFP, or untransduced DC2.4 cells as described in Figure 4 and used as putative suppressors. Cell proliferation was measured by [3H] incorporation. The data are expressed as percent inhibition of proliferation as compared to controls to which no putative suppressor cells were added in α-MEM supplemented with 10% FCS, 50 U/ml rIL-2 and 30 U/ml rIL-4. All experiments were repeated three times and similar results were obtained. Graph shows mean of triplicate samples (error bars represent SEM).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
DN T cells expanded by DC2.4 cells tranduced with LV-Ld prolong donor-specific skin allograft survival. (B6×dm2)F1 recipients were either left untreated or infused with 5×106 (1×) or 15×106 (3×) DN T cells from syngeneic 2CF1.FcRγ+/+ (circles and triangles, respectively) or 5×106 DN T cells from 2CF1.FcRγ−/− mice (squares) that were expanded by coculture with DC2.4-Ld (open symbols) or DC2.4-eGFP (filled symbols) as described previously in Figure 4. After 1 day, recipient mice were given (B6×BALB/c)F1 (donor-specific) and bm1 (third-party) skin allografts. Data show skin graft survival of eight mice for each group. Third-party grafts in all groups were rejected in a similar rate.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions