Journal:

Article Title: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes

doi: 10.1097/CJI.0b013e3181ad4071

Figure Lengend Snippet: A) IL-21 was cloned by PCR and subcloned into the pLenti6.2/C-Lumio/DEST vector by LR recombination, which allows a IL-21 fusion protein expressing the Lumio tag. The plasmid pLenti6.2/LacZ-V5 was used as a control plasmid. B) TAPC were transduced with pLenti-IL21-Lumio or mock transduced labeled with 1 μM Lumio Green and analyzed by fluorescent microscopy or (C) by flow cytometry, where >50% of pLenti-IL21-Lumio TAPC (black line) were positive for Lumio Green compared to mock transduced (gray line). D) TAPC transduced with either IL-21 or lacZ lentivirus were assayed by ELISA for IL-21 production following CD3/CD28 stimulation. E) TAPC cultured for 10 days were reactivated with CD3/CD28 for 24 hours and tested for IL-21 by ELISA demonstrating that IL-21 production can be restored in pLenti-IL21-Lumio transduced T cells.

Article Snippet: Plates were incubated for 24 hours at 4°C, washed and then incubated with biotinylated mouse anti-IL-21 antibody (BD Pharmingen) for 2 hours at room temperature.

Techniques: Clone Assay, Plasmid Preparation, Expressing, Transduction, Labeling, Microscopy, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal:

Article Title: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes

doi: 10.1097/CJI.0b013e3181ad4071

Figure Lengend Snippet: A) TAPC transduced with IL-21 or lacZ were enumerated for 14 days following CD3/CD28 stimulation demonstrating that IL-21 did not affect proliferation of non-specific T cell expansion. B) The phenotype of IL-21 and control TAPC was analyzed by flow cytometry for the expression of CD28, CD80, CD83, CD86, HLA-ABC and HLA-DR demonstrating increased CD28 expression in TAPC-IL21.

Article Snippet: Plates were incubated for 24 hours at 4°C, washed and then incubated with biotinylated mouse anti-IL-21 antibody (BD Pharmingen) for 2 hours at room temperature.

Techniques: Transduction, Flow Cytometry, Expressing

Journal:

Article Title: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes

doi: 10.1097/CJI.0b013e3181ad4071

Figure Lengend Snippet: A) CTL cultures stimulated weekly with ELA peptide-pulsed TAPC-IL21 or TAPC-lacZ were assayed for ELA frequency by pentamer analysis. B) ELA-pentamer frequency was measured on day 14 for 6 donors demonstrating a significant increase in ELA-specific CTL when stimulated with TAPC-IL21 compared to TAPC-lacZ (*P<.05). C) Analysis of CTL cultures generated from IL21 or control TAPC was assessed by IFN-γ ELIspot showing a signficant increase in ELA specificity in IL-21 stimulated cultures (*P<.05). D) Expansion of ELA-specific CTL was calculated by multiplying the frequency of ELA pentamer-specific T cells by overall culture growth. Fold-expansion was significantly greater in IL-21 stimulated cultures (*P<.05). E) IL-21 alone was insufficient for the generation of ELA-specific CTL, and required the presence of IL-7 and IL-12 during priming. Frequency of ELA-specific CTL as determined by pentamer staining shown at day 0 (top) compared to TAPC-IL21 alone, or TAPC-LacZ plus rhIL-7 and rhIL-12, or with TAPC-IL21 plus rhIL-7 and rhIL-12.

Article Snippet: Plates were incubated for 24 hours at 4°C, washed and then incubated with biotinylated mouse anti-IL-21 antibody (BD Pharmingen) for 2 hours at room temperature.

Techniques: Generated, Enzyme-linked Immunospot, Staining

Journal:

Article Title: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes

doi: 10.1097/CJI.0b013e3181ad4071

Figure Lengend Snippet: A) ELA-specific CTL generated by both IL-21 and lacZ transduced TAPC showed specific killing of HLA-A2+ (matched), MART-1+ tumor cells (Senma) but not HLA-A2−, MART-1+ tumor cells (Mel-1143) demonstrating that cytotoxic effector function was not adversely affected by IL-21. B) To determine if IL-21 enhanced cytotoxicity on a cell per cell basis, ELA-specific CTL were FACS sorted after ELA pentamer labeling (>95%) and used as effector cells against CEM.T2 (T2) cells pulsed with ELA peptide. Both CTL generated in the presence of IL-7 and IL-12, and stimulated with either TAPC-LacZ or TAPC-IL21 showed equivalent killing to peptide-bearing target cells.

Article Snippet: Plates were incubated for 24 hours at 4°C, washed and then incubated with biotinylated mouse anti-IL-21 antibody (BD Pharmingen) for 2 hours at room temperature.

Techniques: Generated, Labeling

Journal:

Article Title: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes

doi: 10.1097/CJI.0b013e3181ad4071

Figure Lengend Snippet: A) To demonstrate that TAPC-IL21 have the capacity to stimulate CTL specific for other tumor antigens, CTL cultures were initiated using IMD (gp100) peptide-pulsed TAPC and subsequently analyzed for IMD-specific T cell frequency using pentamer analysis. B) IFN-γ ELIspot analysis for IMD frequency was elevated in 3 normal donors when TAPC-IL21 were used as APC compared to TAPC-lacZ. C) Similarly, CTL generated with RHAMM peptide-pulsed TAPC-IL21 showed increased RHAMM-specific CTL when compared to cultures stimulated with TAPC-lacZ.

Article Snippet: Plates were incubated for 24 hours at 4°C, washed and then incubated with biotinylated mouse anti-IL-21 antibody (BD Pharmingen) for 2 hours at room temperature.

Techniques: Enzyme-linked Immunospot, Generated

Journal:

Article Title: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes

doi: 10.1097/CJI.0b013e3181ad4071

Figure Lengend Snippet: A) Flow cytometric analysis of bulk culture CTL stimulated with IL-21 TAPC showed increased expression of CD27, CD28, CD62L and CCR7 compared to control TAPC. IL-21 producing TAPC also enhanced the generation of CD27highCD28high CTL (inset box). B) To examine the phenotype of ELA-specific CTL, cultures stimulated with either IL-21 or control TAPC were labeled with ELA pentamer APC, and subsequently labeled with CD62L FITC and IL-7Rα. During analysis, CTL cultures were gated on ELA pentamer positive cells and analyzed for CD62L and IL-7Rα expression, which showed a marked increase in IL-7Rα expression in IL-21 treated CTL cultures. C) ELA pentamer gated CTL were analyzed for expression of CD27, CD28 and CD62L following stimulation with control of IL-21 producing TAPC. This analysis showed that IL-21 treatment enhanced expression of all 3 receptors whereas control TAPC produced a large number of cells (bimodal expression) that lacked CD27, CD28 and CD62L expression. D) Analysis of ELA-specific CTL lines generated from 6 donors showed significantly higher expression of CD27+, CD27+CD28+ and CD62L+ CTL with IL-21 compared to control TAPC (*P<.05).

Article Snippet: Plates were incubated for 24 hours at 4°C, washed and then incubated with biotinylated mouse anti-IL-21 antibody (BD Pharmingen) for 2 hours at room temperature.

Techniques: Expressing, Labeling, Produced, Generated

Journal:

Article Title: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes

doi: 10.1097/CJI.0b013e3181ad4071

Figure Lengend Snippet: A) Gene expression of Tcf7 and Lef1 were compared by RT-PCR from ELA-specific (MART-1) CTL generated from 3 donors with either IL-21 or control TAPC. Tcf7 was upregulated in 3/3 CTL generated from IL-21 producing TAPC, while Lef1 was upregulated in 2/3 CTL lines, when compared to the internal housekeeping control GAPDH. B) Survival of IL-21 or control treated CTL was analyzed by Annexin V/PI staining following stimulation with CD3/CD28 antibodies and subsequent culture in IL-2 containing media, or media alone. IL-21 stimulated CTL lines showed increased survival during IL-2 withdrawal. C) IL-21 enhanced the frequency of IFN-γ producing CTL. CTL stimulated with and without IL-21 were subsequently activated with PMA/ionomycin. In 5 donors, IFN-γ production was significantly increased in IL-21 treated CTL (*P<.05).

Article Snippet: Plates were incubated for 24 hours at 4°C, washed and then incubated with biotinylated mouse anti-IL-21 antibody (BD Pharmingen) for 2 hours at room temperature.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Staining

Journal: Frontiers in Immunology

Article Title: The Number of Donor-Specific IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection

doi: 10.3389/fimmu.2019.00748

Figure Lengend Snippet: Number of post-transplant donor-specific IL-21 producing PBMC in patients who will or will not develop rejection in pre-transplant cohort ( A : n = 20 without rejection, n = 15 with rejection) and 6 months post-transplant cohort ( B : n = 33 without rejection, n = 13 with rejection).

Article Snippet: Thereafter, the wells were firmly shaked-out and washed with PBS, and 100 μl/well of an appropriately diluted biotinylated anti-human IL-21 detection antibody (U-CyTech Biosciences) was added for a period of 2 h. After washing, the wells were incubated with streptavidin-HRP conjugate (U-CyTech Biosciences) for 1 h followed by AEC substrate (U-CyTech Biosciences) until distinct spots emerged within 30 min. Color development was stopped by washing extensively with water.

Techniques:

Journal: Frontiers in Immunology

Article Title: The Number of Donor-Specific IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection

doi: 10.3389/fimmu.2019.00748

Figure Lengend Snippet: Receiver operating characteristic (ROC) analysis was performed to define the cut-off number of donor-specific IL-21 producing cells (pc) ( A : PBMC samples taken from patients prior to transplantation; C : PBMC samples taken from patient at 6 months after transplantation), and discriminated between patients with and without rejection. A cut-off of 18 spots per 300.000 PBMCs was determined with a specificity of 80% and a sensitivity of 73% in the pre-transplant cohort (A) , and a cut-off of 62 spots per 300.000 PBMCs was determined with a specificity of 88% and a sensitivity of 54% in the 6 months post-transplant cohort (C) . Thereafter, the percentage of patients with high and low numbers (cut-off values) of IL-21 pc free from rejection were determined in the pre-transplant (B) and post-transplant cohort (D) . AUC, area under the curve.

Article Snippet: Thereafter, the wells were firmly shaked-out and washed with PBS, and 100 μl/well of an appropriately diluted biotinylated anti-human IL-21 detection antibody (U-CyTech Biosciences) was added for a period of 2 h. After washing, the wells were incubated with streptavidin-HRP conjugate (U-CyTech Biosciences) for 1 h followed by AEC substrate (U-CyTech Biosciences) until distinct spots emerged within 30 min. Color development was stopped by washing extensively with water.

Techniques: Transplantation Assay

Journal: Frontiers in Immunology

Article Title: The Number of Donor-Specific IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection

doi: 10.3389/fimmu.2019.00748

Figure Lengend Snippet: The donor-specific IL-21 producing cell frequency determined in PBMC, CD4 + and CD8 + T cells in PBMCs of four transplant recipients. The IL-21 response in PBMC was defined as 100%. Mean with SEM is presented.

Article Snippet: Thereafter, the wells were firmly shaked-out and washed with PBS, and 100 μl/well of an appropriately diluted biotinylated anti-human IL-21 detection antibody (U-CyTech Biosciences) was added for a period of 2 h. After washing, the wells were incubated with streptavidin-HRP conjugate (U-CyTech Biosciences) for 1 h followed by AEC substrate (U-CyTech Biosciences) until distinct spots emerged within 30 min. Color development was stopped by washing extensively with water.

Techniques:

Journal: Frontiers in Immunology

Article Title: The Number of Donor-Specific IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection

doi: 10.3389/fimmu.2019.00748

Figure Lengend Snippet: A typical example is depicted for intracellular IL-21 production after stimulation of patient's PBMCs with irradiated donor PBMCs depleted for CD3 (A) . The proportion of IL-21 producing cells was determined after 3 days within the CD8 + (B) and CD4 + T cells (C) . Tfh cells (CXCR5 + PD1 + ) were determined within the CD8 + (D) and CD4 + (E) T cell population. The percentage of CD8 + Tfh cells was too low to be analyzed for IL-21. Within the CD4 + Tfh cells 31.22% produced IL-21 (F) . The proportion of IL-21 producing cells was determined in PBMC samples from 5 kidney transplant recipients (G) . The percentage donor-reactive IL-21 was determined in CD4 + and CD8 + T cells, and Tfh cells (CD4 + CXCR5 + PD1 + ). IgG1 isotypes are presented of each T cell subpopulations.

Article Snippet: Thereafter, the wells were firmly shaked-out and washed with PBS, and 100 μl/well of an appropriately diluted biotinylated anti-human IL-21 detection antibody (U-CyTech Biosciences) was added for a period of 2 h. After washing, the wells were incubated with streptavidin-HRP conjugate (U-CyTech Biosciences) for 1 h followed by AEC substrate (U-CyTech Biosciences) until distinct spots emerged within 30 min. Color development was stopped by washing extensively with water.

Techniques: Irradiation, Produced