1. Volume 21, Issue 5, Pages 1044-1054 (May 2013)
Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells 
Francesca Giannoni, Cinnamon L Hardee, Jennifer Wherley, Eric Gschweng, Shantha Senadheera, Michael L Kaufman, Rebecca Chan, Ingrid Bahner, Vivian Gersuk, Xiaoyan Wang, David Gjertson, David Baltimore, Owen N Witte, James S Economou, Antoni Ribas, Donald B Kohn 
Molecular Therapy 
Volume 21, Issue 5, Pages (May 2013)
DOI: /mt
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
CCLc-MND-F5 lentiviral vector map and experimental layout. The CCLc-MND-F5 lentiviral vector (LV) has the self-inactivating (SIN) deletion of the LTR enhancers (indicated by X's), the HIV-1 packaging sequence (ψ), central polypurine tract (cP) and rev-responsive element (R). It uses the MND retroviral LTR U3 region (MNDU3)28 to drive expression of the cDNA encoding the TCRα and TCRβ chains of the F5 TCR linked for cotranslation by a 2A sequence and followed by the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Human CD34+ hematopoietic stem/progenitor cells (HSPC) from umbilical cord blood were transduced with the CCLc-MND-F5 LV (or mock transduced without addition of vector) by overnight culture (O/N) in medium supplemented with recombinant cytokines stem cell factor (SCF), Flt-3 ligand (F3L), and thrombopoietin (TPO) on a recombinant fibronectin (rFBN) extracellular matrix support. An aliquot of the cells was grown for 2 weeks in vitro culture in Iscove's modified Dulbecco's medium (IMDM) with 20% fetal calf serum (FCS) and recombinant cytokines interleukin-3, interleukin-6, and SCF (3/6/S) for subsequent measurement of vector copy number (VCN) using quantitative PCR (qPCR). The majority of the cells were used for xenotransplant into NOD/SCID/γc−/− (NSG) mice or NSG mice with the human HLA-A*0201 allele (NSG-A2). Cells were transplanted by intrahepatic injection (IH). After 4–5 months, mice were euthanized and marrow, spleen, thymus, and blood were analyzed for cell counts, fluorescence-activated cell sorting (FACS), VCN by qPCR, antigen-specific interferon-γ (IFN-γ) production by ELISA assay, and FACS sorting to isolate tetramer-positive and -negative T cells to extract genomic DNA for TCR sequence analysis.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Engraftment of human cells in bone marrow and peripheral blood of transplanted NSG mice. (a) Engraftment of human leukocytes in murine bone marrow measured as the percentage of total bone marrow cells that were positive for human CD45, comparing mock mice (filled circles, n = 52) and F5 mice (open squares, n = 64). Each symbol refers to an individual mouse and the horizontal black bars indicate the average engraftment for all mice in that group, and SEMs. Dashed horizontal line indicates the 5% engraftment value used as the lower-limit threshold for further analyses. (b) Human T and B cells in bone marrow of mock mice and F5 mice. Bars indicate mean ± SEM of the total human CD45+ cells in the bone marrow of mice engrafted with ≥5% human CD45+ cells and the percentage of human CD45 that were positive for CD3 or CD19 in mock mice (gray bars, n = 37) and in F5 mice (black bars, n = 46). (c) Human T and B cells in peripheral blood of mock mice and F5 mice. Bars indicate mean ± SEM of the total human CD45+ cells in the peripheral blood and the percentage of human CD45 that were positive for CD3 or CD19 in mock mice (gray bars, n = 35) and in F5 mice (black bars, n = 41).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Analysis of human thymocytes produced in NSG mice transplanted with F5 TCR-transduced and mock-transduced human CD34+ cells. (a) Flow cytometric analyses of human thymocyte populations and expression of F5 TCR in representative NSG mice transplanted with human CD34+ HSPC. Thymocytes from mock mouse (top) or F5 mouse (bottom) were isolated and analyzed by flow cytometry by gating on the CD4 or CD8 single-positive populations and measuring binding of the MART-1 peptide/A*A201 tetramer. (b) Human thymocyte populations in transplanted mice. Human CD3+ thymocytes were isolated from mock mice and F5 mice from the four transplant groups and analyzed by flow cytometry for CD4 and CD8 subpopulations: CD4/CD8 double positive (4–8 DP), CD4 single positive (4 SP), and CD8 single positive (8 SP). Transplant groups were as follows: mock mice: HSC−/NSG− (n = 14, open bars), HSC+/NSG− (n = 6, light gray bars), HSC−/NSG+ (n = 10, dark gray bars), and HSC+/NSG+ (n = 9, black bars); F5 mice: HSC−/NSG− (n = 19, open bars), HSC+/NSG− (n = 6, light gray bars), HSC−/NSG+ (n = 6, dark gray bars), and HSC+/NSG+ (n = 9, black bars). (c) Thymocytes from the F5 mice that stained positive with the MART/A2 tetramer positive (+) or negative (−) were analyzed by flow cytometry for CD4 and CD8 subpopulations: CD4/CD8 double positive (4–8 DP), CD4 single positive (4 SP), and CD8 single positive (8 SP). Transplant groups were HSC−/NSG− (n = 17, open bars), HSC+/NSG− (n = 5, light gray bars), HSC−/NSG+ (n = 6, dark gray bars), and HSC+/NSG+ (n = 10, black bars). The same thymocyte samples from the F5 mice were interrogated for (d) the percentage and (e) the median fluorescent intensity of MART-1 tetramer-positive cells expressing the F5 TCR in thymocyte populations: CD4/CD8 double negative (4–8 DN), CD4/CD8 double positive (4–8 DP), CD4 single positive (4 SP), and CD8 single positive (8 SP). (f) Expression of CD7 and CD1a was analyzed on MART tetramer positive (open bars) and MART tetramer-negative (filled bars) human CD3+ thymocytes from F5 mice (n = 22).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Analysis of human splenocytes produced in NSG mice transplanted with F5 TCR-transduced and mock-transduced human CD34+ cells. (a) Human splenocytes populations by transplant group. The percentage of human CD45 cells in the spleens of mock and F5 mice that were positive for CD3 or CD19 are shown. Transplant groups were HSC−/NSG− (n = 19, open bars), HSC+/NSG− (n = 5, light gray bars), HSC−/NSG+ (n = 9, dark gray bars), and HSC+/NSG+ (n = 8, black bars). (b) MART-1 tetramer-positive human CD45+ splenocytes in F5 mice. The percentage of human CD45+ cells (CD3+, CD4+, or CD8+) in the spleens of transplanted mice that were expressing the F5 TCR, based upon binding of the MART-1/A*A0201, are shown. Transplant groups were HSC−/NSG− (n = 19, open bars), HSC+/NSG− (n = 6, light gray bars), HSC−/NSG+ (n = 10, dark gray bars), and HSC+/NSG+ (n = 10, black bars). (c) MART-1 peptide-induced IFN-γ production by human splenocytes from NSG. IFN-γ produced by splenocytes of mock mice (n = 3) or F5 mice (n = 5) stimulated ex vivo for 72 with an irrelevant negative control peptide (NC) or with the MART-127–35 peptide (MART) was determined by ELISA. Bars show averages for the groups ±SEM.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Sequence analysis of TCR Vβ CDR3 VDJ sequences. The relative abundance of each unique T-cell receptor (TCR) sequence among the total unique sequences identified for the sample is represented by a pie slice. (a) Human CD3+ cells from NSG spleens. Samples were from a mock mouse (upper left) or from an F5 mouse that were stained with the MART-1/HLA-A*0201 tetramer and were sorted as MART tetramer negative (upper right) or MART tetramer positive (bottom center). (b) Human peripheral blood mononuclear cell (PBMC) mock and F5 transduced. Samples were from mock-transduced PBMC (upper left) or from PBMC that were transduced with the F5 vector and were stained with the MART-1/HLA-A*0201 tetramer and sorted as MART tetramer negative (upper right) or MART tetramer positive (bottom center).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions