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1.
Figure 2

Figure 2. Proliferation and phenotype of IL-21 transduced TAPC. From: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes.

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.

Anjum S. Kaka, et al. J Immunother. ;32(7):726-736.
2.
Figure 5

Figure 5. Generation of gp100 and RHAMM-specific CTL using TAPC-IL21. From: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes.

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.

Anjum S. Kaka, et al. J Immunother. ;32(7):726-736.
3.
Figure 4

Figure 4. Cytotoxicity of ELA-specific CTL generated by TAPC-IL21. From: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes.

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.

Anjum S. Kaka, et al. J Immunother. ;32(7):726-736.
4.
Figure 1

Figure 1. Gene-modification of TAPC with IL-21. From: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes.

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.

Anjum S. Kaka, et al. J Immunother. ;32(7):726-736.
5.
Figure 7

Figure 7. Unique gene expression program and functional characteristics of ELA-specific CTL generated with TAPC-IL21. From: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes.

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).

Anjum S. Kaka, et al. J Immunother. ;32(7):726-736.
6.
Figure 3

Figure 3. Generation of ELA-specific CD8+ CTL using TAPC-IL21. From: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes.

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.

Anjum S. Kaka, et al. J Immunother. ;32(7):726-736.
7.
Figure 6

Figure 6. Central memory phenotype in ELA-specific CTL stimulated with TAPC-IL21. From: Genetic Modification of T Cells with IL-21 Enhances Antigen Presentation and Generation of Central Memory Tumor-Specific Cytotoxic T Lymphocytes.

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).

Anjum S. Kaka, et al. J Immunother. ;32(7):726-736.

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