1. Volume 26, Issue 1, Pages 184-198 (January 2018)
Self-Transducible Bimodal PDX1-FOXP3 Protein Lifts Insulin Secretion and Curbs Autoimmunity, Boosting Tregs in Type 1 Diabetic Mice 
Christina Amatya, Ilian A. Radichev, Jacob Ellefson, Mark Williams, Alexei Y. Savinov 
Molecular Therapy 
Volume 26, Issue 1, Pages (January 2018)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Figure 1 Generation of Cell-Permeable PDX1-FOXP3-TAT Fusion Protein
(A) Schematic representation of the three fusion protein (FP) variants (FP, FP-1, and FP-2). (B) Coomassie staining of the isolated recombinant proteins. Purity was calculated to be higher than 85%. (C) Western blotting analysis with His6 antibodies of FP variants isolated from E. coli inclusion bodies and purified by Ni-HiTrap. (D) FACS analysis of the recombinant proteins’ translocation into T cells by FOXP3 staining. Naive CD4+CD25− T cells isolated from NOD spleens were incubated for 1 hr with the indicated concentrations of FP, FP-1, or FP-2 and then rested for 24 hr before being fixed, permeabilized, stained, and analyzed by FACS. (E) Immunofluorescent analysis of FP internalization into WBF344 cells. Hepatocyte precursor (stem-like) WBF344 cells were treated with 0.108 μM FP, 0.160 μM FP-1, or 0.160 μM FP-2 for 1 hr before being fixed, permeabilized, and stained with anti-PDX1 and Alexa Fluor 594-conjugated secondary antibody. DNA was stained with DAPI. Scale bar, 25 μm
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 2
Naive CD4+CD25− T Cells Acquire Suppressor Treg-like Phenotype after In Vitro Treatment with FP
(A) qRT-PCR analysis of IL-2, IFN-gamma, IL-10, and TGF-beta mRNAs in T cells after treatment with FP. CD4+CD25− T cells were incubated for 1 hr with FP and rested for 24 hr before RNA extraction. The mRNA expression was normalized using the housekeeping genes Gapdh and beta-actin. (B) qRT-PCR analysis of Tregs marker genes (FoxP3, CD25, and CD127) in T cells after treatment with FP as described in (A). (C) ELISA analysis of cytokines levels in conditioned media from FP-treated T cells. CD4+CD25− T cells were incubated with FP as in (A) and then activated for 48 hr with anti-CD3/CD28 beads before collection of the conditioned medium (n = 6). (D–F) FP induces functional Treg-like cells. (D) Schematic representation of suppressor assay designed to determine the suppressive activity of FP-treated T cells (described in detail in Materials and Methods). Briefly, diabetogenic monoclonal G9C8 T cells labeled with CFSE cytosolic dye were mixed in a 1:1 ratio with either FP- or vehicle-pretreated naive T cells, or conventional Tregs, all pre-activated with CD3 and CD28 antibodies, and then co-cultured with NOD macrophages loaded with or without insulin peptide (InsB12–23). Both controls −InsB and +InsB contained vehicle treated pre-activated CD4+ T cells. The co-cultures were incubated for 5 days, and then proliferation of G9C8 cells was evaluated. (E) FACS analysis of G9C8 T cell proliferation after co-culturing with FP-treated Treg-like cells. (F) ELISA analysis of IL-2 and IFN-γ in conditioned media collected on the third day of the suppressor assay. (G) Quantification of FACS analyses for intracellular levels of IL-2, IFN-γ, Granzyme B, and LAMP1 in CD8+ G9C8 T cells after suppressor assay. Data are presented as average fold change ± SEM. *p < 0.05, **p < 0.01, and ***p < compared with non-treated control T cells, unless otherwise stated.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 3
FP Internalizes into WBF344 Cells and Promotes β Cell Reprogramming
(A) Immunofluorescence of WB-F344 cells incubated with 0.108 μM of FP for 1 or 24 hr and then analyzed for intracellular cytosolic and nuclear location of the FP with Pdx1 antibody. White arrowheads point at visible nuclear localization of the recombinant protein. (B) Higher glucose concentration promotes the nuclear translocation of FP. Immunofluorescence analysis of permeabilized WBF344 cells treated with 0.108 μM of FP for 24 hr in the presence of either 5 or 20 mM glucose. White arrowheads point at the areas of nuclear FP localization. Scale bars, 25 μm. (C) qRT-PCR analysis of the expression of β cell and hepatocyte lineage-specific genes in WB-F344 cells 48 hr after 1-hr-long treatment with 0.108 μM of FP. Data are normalized to rat β-actin and presented as average fold change ± SEM (n = 6); **p < 0.01 and ***p < compared with vehicle-treated WB-F344 cells.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 4
FP Treatment of Chronically Diabetic NOD Mice Improves β Cell Function
(A) Scheme of in vivo treatment regimen. (B) Dynamics of blood glucose levels in mice during the treatment protocol described in (A). Fasting blood glucose was measured 2 hr post-FP injection. (C) Dot blot analysis for the presence of antibodies against FP in the sera collected from animals at the end of the treatment protocol. Left: representative blots; right: quantification of relative intensity. Data are presented as average relative intensity (R.I.) ± SEM; *p < (D) ELISA analysis of fasting insulin and C-peptide levels in blood collected from animals at the end of the treatment protocol. Data are presented as average concentrations ± SEM; **p < (E) Immunofluorescence of liver cryosections from control and FP-treated NOD mice stained with insulin antibody (red) and DAPI (blue). White dotted regions indicate hepatic lobular veins. Scale bars, 100 μm. (F) Liver cryosections from FP-treated NOD mice stained with either insulin (red), C-peptide (green), and Mafa (yellow) (left) or insulin and Pdx1 (yellow) (right). Scale bars, 50 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 5
FP Treatment of Chronically Diabetic NOD Mice Induces Systemic Treg-Dependent Anti-inflammatory Shift
(A) FACS analysis of CD4+ and CD8+ T cells in peripheral blood of mice mid-way through FP treatment. PBMCs were isolated after 12 days of FP treatment and stained for CD4 and CD8. Left: representative histograms; right: quantification of percentages of CD8+ cells. (B) FACS analysis of CD4+CD25+Foxp3+ T cells in different lymphoid organs at the end of the FP treatment protocol. Lymphocytes from spleen, mesenteric lymph nodes (MLNs), and pancreatic lymph nodes (PLNs) were isolated from FP-treated and vehicle-treated control NOD mice and stained for Tregs and activation markers. Left: representative dot plots of live CD4+ T cells stained for CD25 and Foxp3. Right bar graphs: quantification of percentages of CD25+Foxp3+ and CD8+CD44+ cells in spleen (n = 5), MLNs (n = 5), and PLNs (n = 3). (C and D) ELISPOT analysis of IFN-γ-producing (C) and IL-10-producing (D) cells. Splenocytes and PLN-resident cells were isolated from FP- or vehicle-treated NOD mice. Cells were plated in IFN-γ and IL-10 coated ELISPOT plates for 48 hr before the detection of cytokine-specific spots. Data are presented as average ± SEM. *p < 0.05 and **p < 0.01 compared with control.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. Figure 6
Short-Term i.v. Treatment with FP Improves Its Transduction into Lymphocytes
(A) Outline of the study timeline. (B–D) Lymphocytes from spleen, mesenteric lymph nodes (MLNs), and pancreatic lymph nodes (PLNs) were isolated from FP-treated and vehicle-treated control NOD mice and stained for CD4, CD8, CD19, and Foxp3. (B) FACS analysis of CD3+ Foxp3+ T cells in different lymphoid organs at the end of the FP treatment protocol. Left: representative FACS plots of Foxp3 and CD3-positive T cells. Right: quantification of percentages of CD3+Foxp3+ cells in spleen, MLNs, PLNs, and PBMCs. (C) FACS analysis of CD4+Foxp3+ and CD8+Foxp3+ T cells in PBMCs. Left: representative FACS plots; right bar graphs: quantification of the data. (D) FACS analysis of CD19+Foxp3+ B cells in PBMCs and spleen. Left: representative FACS plots and the quantification of CD19+Foxp3+ cells. Right bar graphs: qRT-PCR analysis of CD22 (B cell marker), CD80, CD86 (activation maker), and Bax (apoptotic marker) mRNAs in B cells after treatment with FP. mRNA expression was normalized using the housekeeping genes Gapdh and beta-actin. Data are presented as average ± SEM. *p < 0.05 and **p < 0.01 compared with control. (E) Liver cryosections from NOD mice treated with FP for 3 consecutive days, stained with either insulin (red), C-peptide (green), and MafA (yellow) (left) or insulin and Pdx1 (yellow) (right). Scale bars, 50 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions