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Supplementary figures
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Figure S1:
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A) Gating strategy for the analysis of CD4+CD25high T cells. Using a BD FACSAria sorter,
CD4+CD25- and CD4+CD25high lymphocytes were identified and sorted as described previously
[11], CD4 FITC labelled, CD25 Alexa-Fluor 647 labelled. After 4 days of culture, CD4 labelling
was virtually unchanged (histogram). Thus, in a mixture of previously stained and sorted
responder and unstained stimulator cells (irradiated PBMC), a gate could be set around CD4+
cells to exclude irradiated T cells from the subsequent analysis. In case of cultured (expanded)
CD4+CD25high cells, only one gate was required In case of cultured CD4+CD25- cells, two gates
were placed, defining CD4+CD25- and (induced) CD4+CD25high cells.
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B) Strategy of in vitro induction or expansion of CD4+CD25high T cells.
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Freshly isolated PBMC from healthy or IBH-affected horses were sorted according to their
expression of CD4 and CD25. The CD25high gate was set to incorporate only the top 3% of all
CD4+ T cells with the brightest fluorescence signal for CD25. Sorted CD4+CD25- or CD4+CD25high
cells were cultured with Culicoides-pulsed irradiated autologous-PBMC in the absence or the
presence of two different sets of stimuli (1 or 2). Four days later, resorting was performed.
+
high
were induced in the presence of Culicoides alone; I1CD4+CD25high were induced in
ICD4 CD25
+
high
the presence of Culicoides, rIL-2 and rTGFwere induced in the presence of
I2CD4 CD25
Culicoides, retinoic acid and rapamycin. Similarly expanded CD4+CD25high cells were obtained.
The expanded and induced cells were then used in the suppression assay or in the
determination of FoxP3 and cytokine expression by flow cytometry.
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C) Analysis of cytokine production by Culicoides-stimulated CD4+CD25high T cells
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5x105 CD4+CD25high sorted lymphocytes were cultured with 4x105 irradiated autologous
Culicoides-pulsed PBMC (stimulators). 4 days later, the cells were harvested and triple
intracellular staining for FoxP3, IL-4 and IL-10 or their respective isotype controls (Isc.) was
performed. The percentage of IL-4, FoxP3 and IL-10 was measured using flow cytometry. Data
displayed are from one representative IBH-not suppressing (IBH-N-sup), IBH-suppressing (IBHsup) or healthy Icelandic horse. Lines were set according to the isotype controls to discriminate
negative from positive population
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Figure S2: Comparing the ability of CD4+CD25high T cells to suppress proliferation of
Culicoides-stimulated CD4+CD25- cells at a ratio of 1:1 and 1:0.25
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5x104 CD4+CD25- sorted lymphocytes from freshly isolated peripheral blood mononuclear cells
(PBMC) from 1st generation IBH-affected (n=5) and 2nd generation healthy (n= 5) Icelandic
horses were cultured with 5x104 irradiated autologous Culicoides-pulsed PBMC (stimulator)
alone (CD4+CD25-) or in the presence of freshly isolated CD4+CD25high cells (CD4+CD25- +
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CD4+CD25high) in a ratio of 1:1 or 1:0.25. Proliferation was measured using [3H] thymidine
incorporation. Results are shown as count per minute (CPM). Dots represent individual horses.
*p ≤ 0.05 in the Wilcoxon signed-rank paired t-Test indicate significant differences to
CD4+CD25- cells.
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Figure S3: Inability of CD4+CD25dim T cells to suppress proliferation of Culicoides- or Tetanus
toxoid-stimulated-CD4+CD25- cells.
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5x104 CD4+CD25- sorted lymphocytes from freshly isolated peripheral blood mononuclear cells
(PBMC) from 2nd generation healthy (n= 14) or 1st generation IBH-affected (n=14) Icelandic
horses were cultured with 5x104 irradiated autologous Culicoides-or Tetanus toxoid-pulsed
PBMC (stimulator) alone (CD4+CD25-) or in the presence of freshly isolated CD4+CD25dim cells
(CD4+CD25- + CD4+CD25dim) in a ratio of (1: 0.25). Proliferation was measured using [3H]
thymidine incorporation. Results are shown as count per minute (CPM). Dots represent
individual horses.
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