Induction of T-Cell Responses against Cutaneous T-Cell Lymphomas Ex Vivo by Autologous Dendritic Cells Transfected with Amplified Tumor mRNA  Xiao Ni, Heather M. Richmond, Xingsheng M. Liao, William K. Decker, Lisa H. Shiue, Elizabeth J. Shpall, Madeleine Duvic  Journal of Investigative Dermatology  Volume 128, Issue 11, Pages 2631-2639 (November 2008) DOI: 10.1038/jid.2008.125 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Characterization of monocyte-derived DCs from SzS. (a) The immunophonetypic profile of DCs. DCs generated form CD14+ monocytes from SzS patients in the presence of GM-CSF and IL-4 for 5 days (iDCs) and matured with TNF-α (mDC(TNF-α)) or with the combination of TNF-α, IL-1β, IL-6, and prostaglandin E2 (mDCs(TNF-α, IL-1β, IL-6, and prostaglandin E2)). The diagram shows the upregulated expression of CD40, CD83, HLA-ABC, and CD80 and CD86 over time, downregulated expression of CD14, and no change of CD1a. The results are from patient no. 1. (b) Allogeneic T-cell stimulatory capacities of monocyte-derived DCs from SzS by mixed leukocyte reaction. A total of 5 × 104 allogeneic T cells were incubated with 10,000 and 2,500 fresh immature DCs (iDC), TNF-α matured DCs (mDC(TNF-α)), or ITIP-matured DCs (mDC(ITIP)). Incubation with monocytes was used as negative control. The data presented are the mean±SD of the experiment performed in triplicate in patient no. 1. (c) The production of IL-12p70 by monocyte-derived DCs from SzS; 0, 24, and 48-hour culture supernatants from DCs treated with TNF-α alone, ITIP, and ITIP with IFN-γ, were examined by ELISA specific for IL-12 p70. The graph shows the mean of triplicate values. Data are results from patient no. 2. Journal of Investigative Dermatology 2008 128, 2631-2639DOI: (10.1038/jid.2008.125) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 TWIST, EphA4, and β-actin present in tumor total RNA and amplified RNA. The quality of total RNA (t) and amplified RNA (a) was checked with the Agilent Bioanalyzer and agarose gel (a). Tumor total RNA and paired amplified tumor mRNA were analyzed by conventional two-step RT–PCR for expression of β-actin (positive control, 285bp), Twist (107bp), and EphA4 (96bp). The PCR products were separated on an ethidium bromide-stained 2% agarose gel, and data shown were from patient nos. 9 and 10, respectively (b), and in all total RNA and paired amplified mRNA (c). Journal of Investigative Dermatology 2008 128, 2631-2639DOI: (10.1038/jid.2008.125) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The specificity of CTLs primed by tumor-RNA-transfected DCs. (a) CTLs primed by tumor-mRNA-transfected DCs (CTL-DC(RNA), black bar) and by mock DCs (CTL-DC(−), white bar) were cultured with tumor cells from the same patient at a different ratio of effector (E) to target cells (T) overnight at 37°C. IFN-γ-producing CTLs were shown as red spots by ELISPOT assay. Inhibition of HLA class I was performed by incubation target cells for 1hour with mAb W06/32 at 12:1 of E to T. (b, c) Data were plotted as mean spots per well ±SD from patient no. 8. (d) 51Cr release assay. CTLs were co-cultured with tumor RNA-transfected DCs as targets (-▪-) and empty mock mDCs (-◊-) at a ratio of effector (E) to target cells (T) of 5:1 and 20:1. Data were plotted as mean specific lysis (%)±SD from patient no. 10. Journal of Investigative Dermatology 2008 128, 2631-2639DOI: (10.1038/jid.2008.125) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Induction of antitumor CTLs ex vivo by DCs transfected with amplified tumor mRNA. CTLs primed by tumor mRNA transfected DCs (CTL-DC(RNA), black bar) and by mock DCs (CTL-DC(−), white bar) were cultured with tumor cells from the same patient at a series of ratio of effectors (E) to targets (T) overnight at 37°C. IFN-γ (a) and GrB-producing CTLs (b) were then assayed by ELISPOT, followed by image analysis and spot enumeration in all tested subjects. ND, not done. Journal of Investigative Dermatology 2008 128, 2631-2639DOI: (10.1038/jid.2008.125) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The percentage of CD3+CD8+ T cells and CD4+CD25+ T cells in peripheral blood lymphocytes before and after DC stimulation and expression of foxp3 mRNA. (a) The peripheral blood lymphocytes before and after DC stimulation were stained for CD3+CD8+ T cells and CD4+CD25+ T cells by dual-color immunofluorescent staining. The plots were from patient no. 7. (b) The increase in percentage of CD3+CD8+ T cells were found in four of five pairs of tested samples (patient nos. 4, 7, 8, and 10). (c) The increase in percentage of CD4+CD25+ T cells were found in all five tested pairs. (d) Total RNA was extracted from peripheral blood lymphocytes before and after DC stimulation and the levels of foxp3 mRNA were assessed by real-time quantitative RT–PCR. The relative expression of foxp3 was normalized to glyceraldehye-3-phosphate dehydrogenase. The decrease in expression of foxp3 was found in 2 of 2 tested pairs (patient nos. 7 and 10). *P<0.05. Journal of Investigative Dermatology 2008 128, 2631-2639DOI: (10.1038/jid.2008.125) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions