Supplementary Material
Supplementary figure 1: High purity of FACS-sorted Foxp3+/RORγt+/ T cells using
Foxp3RFPRORγtGFP reporter mice. Foxp3+/-RORγt+/- T cells were sorted from pooled spleen
and lymph nodes of Foxp3RFPRORγtGFP reporter mice using the displayed gating strategy.
Samples with purity over 99.5% were used for further experiments.
Supplementary figure 2: Phenotypic characterization of Foxp3+RORγt+ T cells from
mLN and the colon. Cells were isolate from mLN and the cLP of Foxp3RFPRORγtGFP
reporter mice containing a complex microbiota and analyzed for the indicated marker. (a)
Representative FACS plots for CD62L and CD44 expression ofof live CD3+CD4+
Foxp3+/RORγt+/ T cell population). Numbers indicate the quadrant frequency. (b) Frequency
of naïve, effector and memory Foxp3+/RORγt+/ T cells. Data are shown as mean ± SD. (c)
Surface expression levels of ICOS and CD25 on live CD3+CD4+ Foxp3+/RORγt+/ T cells
shown as histogram (upper panel) and summarized as geometric mean for ICOS expression
(lower left) or percentage of CD25 positive cells (lower right). Data are shown as mean ± SD.
Supplementary figure 3: Foxp3+RORγt+ T cells display an increased repertoire of gut
specific homing molecules. Foxp3+/RORγt+/ T cells were sorted from the spleen, pLN,
mLN and the colon from Foxp3RFPRORγtGFP reporter mice. Surface expression levels of
CCR4, CCR6, and CCR9 were assessed by FACS. Data obtained from the mLN are
displayed. Data are representative of 3 independent experiments with 1-3 samples per group.
Data are shown as mean ± SD.
Supplementary figure 4: Migration behavior of Foxp3+ RORγt+ T cells is similar to
Foxp3+ T cells. Ex vivo-isolated and sorted CD4+ Foxp3+/-RORγt+/- subsets were labeled with
111
In (Indiumoxin; Mallinckrodt, Germany; 1 × 108 cells/mL; 10 μCi/mL [0.37 MBq]) for
20 minutes at room temperature, followed by 1 hour of incubation at 37°C in fresh medium
and removal of dead cells on Nycodenz (17.1 % isotonic Nycodenz; Nyegaard, Norway) as
previously described
63
. Labeled cells were injected intravenously into C57BL/6 mice. In all
experiments a minimum of 104 cpm was injected. The distribution of radioactivity in lung,
small intestine, pLN, PP, mLN, colon and the remaining body was measured 24 hours after
transfer of labeled cells in a Wallac γ counter. Percentage of total recovered radioactivity is
shown (n=7-10; mean ± SD; data pooled from two independent experiments; *P<0.05).
Supplementary figure 5: Foxp3+ RORγt+ T cells show enhanced frequency in Ki-67+
dividing cells. Cells were isolated from the spleen, pLN, mLN and the colon (cLP) of
Foxp3RFPRORγtGFP reporter mice containing a complex microbiota and stained for the
proliferation marker Ki-67. Frequency of Ki-67 expression in live CD3+CD4+
Foxp3+/RORγt+/ T cells is shown as mean ± SD.
Supplementary figure 6: Foxp3+ Treg, RORγt+ and Foxp3+ RORγt+ T cells show minimal
overlap in their TCR repertoire. Foxp3+/ RORγt+/T cells were sorted from pooled spleen
and lymph node cells of Foxp3RFPRORγtGFP reporter mice for RNA extraction. Analysis of the
TRAV12 TCR diversity and overlap of Foxp3+/RORγt+/ T cells was performed by next
generation sequencing. (a) Correlation between the product of cloneset sizes and the number
of shared individual sequences (R² = 0.91) of the indicated cell subsets of one mouse. Dashed
lines indicate the 90% prediction interval for the regression line. (b) Heatmap of the 25 most
abundant productive CDR3 amino acid sequences of Foxp3+ RORγt+ T cells and the
corresponding overlap in all other T cell subsets.
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Supplementary figure 7: Location-dependent loss of Foxp3/RORγt expression in
Foxp3+RORγt+
T
cells.
Sorted
CD90.2+CD4+
Foxp3+RORγt+
T
cells
from
Foxp3RFPRORγtGFP mice were co-transferred with CD90.1+CD4+CD45RBhigh T cells from
CD90.1 mice into RAG2-deficient mice in a ratio of 1:4. The mice were sacrificed at week 47 after transfers. Cells from spleen, pLN, mLN and colon were analyzed for Foxp3 and
RORγt expression in CD4+CD90.2+ cells. The Foxp3+/RORγt+/ frequencies of transferred
Foxp3+RORγt+ T cells were averaged and are depicted for different organs. The graph
represents pooled data from four independent experiments with 3-5 mice per group.
Supplementary figure 8: Frequency and number of IL-17 producing Foxp3+RORγt+ T
cells in different organs after transfer. Sorted CD90.2+CD4+ Foxp3+RORγt+ or
Foxp3+RORγt
T
cells
from
Foxp3RFPRORγtGFP
mice
were
co-transferred
with
CD90.1+CD4+CD45RBhigh T cells from CD90.1 mice into RAG2-deficient mice in a ratio of
1:4. The mice were sacrificed at week 4-7 after transfers. Graphs represent the (a) frequency
and (b) numbers of IL-17 producing CD3+CD4+CD90.2+Foxp3+ or Foxp3+RORγt+ T cells.
Data are shown as mean ± SD. Graphs are pooled data from 3 independent experiments with
3-5 mice per experimental group.
Supplementary figure 9: Foxp3+ and Foxp3+RORγt+ T cells proliferate to a similar
extend in spleen and pLN at early time points after transfer. Sorted CD90.2+ CD4+
Foxp3+RORγt+
T
cells
from
Foxp3RFPRORγtGFP
mice
were
co-transferred
with
CD4+CD45RBhigh T cells from CD90.1+ mice into RAG2-deficient mice in a ratio of 1:4. The
mice were sacrificed at week 2 after transfers. (a) Number of Foxp3+ CD90.2+ T cells in the
spleen and pLN. (b) Ratio of transferred CD90.2+ and CD90.1+ T cells in the spleen and pLN.
(c) Frequency of CD90.2+ Foxp3+Ki67+ T cells in the spleen and pLN. Graphs are pooled data
from 8 individual mice from 2 independent experiments.
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Supplementary figure 10: Total numbers of CD90.1+ and CD90.2+Foxp3+RORγt+ T cells
in different organs. Sorted CD90.2+CD4+ Foxp3+RORγt+ or Foxp3+RORγt T cells from
Foxp3RFPRORγtGFP mice were co-transferred with CD90.1+CD4+CD45RBhigh T cells (Tn)
from CD90.1 mice into RAG2-deficient mice in a ratio of 1:4. The mice were sacrificed at
week 4-7 after transfers. The absolute numbers of CD3+CD4+CD90.1+ and CD90.2+ T cells in
different organs are depicted for the three experimental groups. Data are shown as mean ±
SD. Graphs are pooled data from 3 independent experiments with 3-5 mice per experimental
group.
Supplementary figure 11: Co-transfer of Foxp3+RORγt+ T cells results in a reduced
number of cytokine producing Teff cells. Sorted CD90.2+CD4+ Foxp3+RORγt+ or
Foxp3+RORγt
T
cells
from
Foxp3RFPRORγtGFP
mice
were
co-transferred
with
CD90.1+CD4+CD45RBhigh T cells from CD90.1 mice into RAG2-deficient mice in a ratio of
1:4. The mice were sacrificed at week 4-7 after transfers. Absolute number of cytokineproducing CD3+CD4+CD90.1+ T cells isolated from the colon. Data are shown as mean ± SD.
Graphs are pooled data from 3independent experiments with 3-5 mice per experimental
group.
Supplementary references
63.
Siegmund K, Feuerer M, Siewert C, Ghani S, Haubold U, Dankof A et al. Migration
matters: regulatory T-cell compartmentalization determines suppressive activity in
vivo. Blood 2005; 106(9): 3097-3104.
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