Figure 1.
Schematic representation of the APC-dependent assay to monitor DC-induced naive CD4+ T cell polarization.
Monocytes were isolated from leukapheresis products obtained from healthy volunteers by counter flow centrifugal elutriation. These enriched monocytes (purity 87.9%±5.2) were frozen and upon thawing differentiated into iDC using IL-4 and GM-CSF. After 7 days, iDC were harvested and plated in a round bottom 96-well plate (2×104 cells/well) in presence of maturation stimuli (A–C). The following day, autologous naive CD4+ T cells were isolated from fresh PBMC obtained from 500 ml of blood by negative immunomagnetic separation (purity of 99.9% of total CD4+ T cells) and 5×104 T cells were added to the different wells, preceded by a 1-hour incubation with PADRE, a pan HLA-DR-restricted peptide. The co-culture was maintained over a period of 7 days and each day supernatant and cells of a single well of each condition were harvested to analyze the secretion of Th-specific cytokines (CBA) and the expression of Th-related transcription factors and cytokines (qPCR).
Figure 2.
FMKp/IFN-γ-matured DC polarize a subpopulation of naive T cells into the Th1 lineage.
iDC or FMKp/IFN-γ-matured DC were pulsed with PADRE for 1 h before the addition of autologous CD4+CD45RA+ T cells to the 7-day co-culture. (A) Expression of T-bet and IFN-γ and IFN-γ production of naive CD4+ T cells co-cultured with iDC (□) or FMKp/IFN-γ-matured DC (▪) are compared. CD3ε was used as housekeeping gene and the expression data were normalized to the relative mRNA content of naive T cells on day 0. IFN-γ production by naive T cells co-cultured with differently matured DC was determined in the supernatant of the co-culture on day 1, 3, 5 and 7 by CBA. Graphs are representative of 11 independent experiments. (B) On day 7, T cells were stained for expression of CD45RO and CD25 and analyzed by flow cytometry. Cells are gated in FSC/SSC on lymphocyte gate, excluding dead cells and doublets (light gray dots), and selected for CD4+ cells (dark gray and black dots). Percentages of CD45RO and CD25 positive populations are indicated in the plots. Dot plots are representative of 5 independent experiments. (C) On day 6 of the co-culture, GolgiPlug and GolgiStop were added to T cells with or without PMA/ionomycin restimulation. The next day, intracellular IFN-γ staining was performed and cells were analysed by flow cytometry. Representative dot plots of CD45RO and IFN-γ expression of CD4+ T cells co-cultured with FMKp/IFN-γ-matured DC are shown. Data are representative of 5 independent experiments.
Figure 3.
Dependency of DC-induced Th1 polarization on DC-derived soluble factors.
(A) FMKp/IFN-γ-matured DC were either extensively washed (○) or not (•) 6 h after induction of maturation and co-cultured for 7 days with autologous CD4+CD45RA+ T cells. Expression of T-bet and IFN-γ and total IFN-γ production are shown. Graphs are representative of 5 independent experiments. (B) APC-independent assay using immobilized anti-CD3 allows studying the influence of DC-derived soluble factors on T cell polarization. 5×104 cells CD4+CD45RA+ T cells were cultured for 5 days without (Δ) or with (▴) washed FMKp/IFN-γ-matured DC-derived supernatant in presence of plate-bound α-CD3 (0.25 µg/ml) in a round bottom 96-well plate. Transcriptional induction of T-bet and IFN-γ and secretion of IFN-γ were determined. Data shown are representative data of 3 independent experiments. (C) FMKp/IFN-γ- and HKLM/IFN-γ-matured DC induce Th1 polarization. iDC were matured with HKLM/IFN-γ (□) or FMKp/IFN-γ (▪) and co-cultured with CD4+CD45RA+ T cells. Expression of T-bet and IFN-γ and total IFN-γ production are shown. Data are representative of 4 independent experiments.
Figure 4.
Presence of CD4+CD45RO+ T cells in the DC-CD4+CD45RA+ T cell co-culture influences Th1 read-out parameters.
(A) CD4+, CD4+CD45RO+, CD4+CD45RA+ populations have been isolated by negative immunomagnetic separation from freshly isolated PBMC. IFN-γ production of total CD4+ (black square), CD4+CD45RA+ (light gray circle), and CD4+CD45RO+ (dark gray triangle) T cells co-cultured with FMKp/IFN-γ-matured DC for 7 days as measured by CBA. (B) Contribution of contaminating CD4+CD45RO+ T cells (2.5, 5 or 10%) to CD4+CD45RA+-derived IFN-γ-production compared with pure (>99.9%) CD4+CD45RA+ T cell populations. Data shown are representative of 2 independent experiments.
Figure 5.
Differential Th1 polarizing capacity of differently matured DC.
Naive CD4+ T cells were co-cultured for 7 days with PGE2/TNF-α (dark gray triangle), LPS/IFN-γ (light gray circle) or FMKp/IFN-γ-(black square) matured DC. (A) Expression of T-bet and IFN-γ and production of IFN-γ were monitored. Graphs are representative of 11 independent experiments. (B) Comparison of the expression of T-bet and IFN-γ on day 5 and IFN-γ production on day 7 of differently matured DC cultured with naive CD4+ T cells. 11 independent experiments and their median levels are shown. Wilcoxon signed-rank test significance **P≤0.01, ***P<0.001 (C) Correlation between T-bet and IFN-γ expression on day 5 and IFN-γ secretion on day 7 of FMKp/IFN-γ-matured DC in co-culture with naive CD4+ T cells. Nonparametric Spearman correlation test significance indicated in the graphs. (D) Expression of CD45RO, CD25, and intracellular IFN-γ of naive CD4+ T cells cultured for 7 days with differently matured DC as indicated above the graphs. On day 6 GolgiPlug and GolgiStop were added to the co-culture and the staining was performed on day 7. Cells shown in the plots represent living singlet cells gated on CD3+CD4+ (dark gray population). T cells positive for IFN-γ are shown in black. Dot plots are representative graphs of 3 independent experiments.
Figure 6.
Differential Th2 polarizing capacities of differently matured moDC.
Naive CD4+ T cells were co-cultured for 7 days with PGE2/TNF-α (dark gray triangle), LPS/IFN-γ (light gray circle) or FMKp/IFN-γ (black square) matured DC. Expression of GATA3, IL-4, IL-5 and IL-13 and production of IL-5 and IL-13 were monitored. IL-5 and IL-13 protein production by naive T cells co-cultured with differently matured DC was determined in the supernatant of the co-culture. Graphs are representative of 5 independent experiments.
Figure 7.
Differential Th1 polarizing capacity of differently matured plasmacytoid DC.
pDC, isolated from fresh blood, were stimulated overnight with different cocktails and 24 h later autologous naive CD4+ T cells were added and the expression of T-bet and IFN-γ and secretion of IFN-γ were monitored during 7 days. (A) pDC were stimulated overnight with IL-3 (full gray line), ODN2216 (dashed black line) or with a combination of both (full black line). (B) Comparison of the capacity of differently matured pDC to induce Th1 polarization. pDC were incubated with PGE2/TNF-α (dark gray triangle), LPS/IFN-γ (light gray circle) or FMKp/IFN-γ (black square) cocktail in the presence of IL-3. Representative data from 2 independent experiments are shown.