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Mediators of Inflammation
Volume 2016, Article ID 3605643, 11 pages
http://dx.doi.org/10.1155/2016/3605643
Research Article

A Comparative Study of the T Cell Stimulatory and Polarizing Capacity of Human Primary Blood Dendritic Cell Subsets

1Department of Tumor Immunology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, 6500 HB Nijmegen, Netherlands
2Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, 171 76 Stockholm, Sweden
3Department of Medical Oncology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, 6500 HB Nijmegen, Netherlands

Received 31 August 2015; Revised 21 December 2015; Accepted 4 January 2016

Academic Editor: Amedeo Amedei

Copyright © 2016 Simone P. Sittig et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplementary Figure 1: Gating strategy for the sorting of primary blood DC subsets and purity. Before fluorescence-activated cell sorting (FACS), lineage (CD3, CD14, CD16, CD19, CD20, CD56) positive cells were depleted from PBMCs. The remaining cells were incubated with the antibodies recognizing lineage markers (FITC), HLA-DR (PE-C7), CD1c (BV421), CD141 (APC), and BDCA4 (PE). (a) The cells were sorted from lineage nega-tive, HLR-DR+/high cells into three different populations, based on the expres-sion of CD1c, CD141 or BDCA4 to obtain CD1c+ mDCs, CD141+ mDCs or pDCs, respectively. (b) Purity of isolated cells was assessed by gating on life cells and analyzing expression of HLA-DR and exclusive expression of CD1c, CD141 or BDCA4 (not shown). Shown is HLA-DR with either CD1c, CD141 or BDCA4.

Supplementary Figure 2: Gating strategy for regulatory T cells and effector memory phenotype. (a) The population of regulatory T cells was determined by selecting CD25+ CD127- cells and subsequently gating on the FoxP3+ population. The populations are shown as percentage of live cells in figure 4a. Dead cells were excluded on the basis of the forward-sideward scatter. (b) Central and effector memory T cells were determined on the basis of surface staining of CD45RO (APC), CD197 (CCR7) (+ A488-conjugated secondary Ab) and CD62-L (L-selectin) (+ PE-conjugated secondary Ab). From CD45RO+ cells, central memory T cells (TCM) were determined by further gating on CCR7+/L-selectin+ and effector memory T cells (TEM) were determined by further gating on CCR7- cells; both populations are shown as percentage of live cells in supplementary figure 3.

Supplementary Figure 3: Human DC subsets induce an effector memory pheno-type in naive CD4+ T cells Human blood DCs were incubated with the indicated stimuli. The next day, allogeneic naive CD4 + T cells were added to the DCs together with a low concentration of the superantigen SEB (10 pg/ml) and cultured until resting (11-13 days). The memory phenotype (n=5) was investigated using flow cytometry. The bar graphs show the mean percentage ± SEM of effector (a) and central (b) memory CD4 + T cells gated from live cells (TEM: CD45RO + CCR7 - and TCM: CD45RO+ CCR7 + L-selectin +). Significance was determined by Kruskal-Wallis test followed by Dunns testing comparing the different conditions of the same subset.

Supplementary Figure 4: IL-17 production of re-stimulated CD4+ T cells after co-culture with the DCs Human blood DCs were incubated with the indicated stimuli. The next day, allogeneic naive CD4 + T cells were added to the DCs together with a low concentration of the superantigen SEB (10 pg/ml) and cultured until resting (11-13 days). These CD4+ T cells were re-stimulated for 24 hrs with anti-CD3/CD28-beads. Supernatants were analyzed for IL-17 by sandwich ELISA (n=6 for CD1c+ mDCs and pDCs; n=1-4 for CD141+ mDCs). The graph shows mean cytokine production. Each symbol represents one donor (also across the subsets).

  1. Supplementary Material