Download Supplementary Figure Legends (doc 38K)

Supplementary Material
Supplementary Figure 1 Significant Treg depletion in intestines and gut associated
lymphoid tissues (GALT). DEREG mice were gastrically gavaged with C. rodentium strain
ICC 180 and then injected i.p. with DT or PBS day 0 and 1 p.i. At day 2 p.i., mice were
sacrificed for cell isolation from different organs. Isolated cells from different organs were
stained with anti-CD4, anti-CD4 and anti-FoxP3 for FACS analysis of depletion efficiency of
regulatory T cells in spleen, mesenteric lymph nodes (mLN), Peyers Patches (PP), colon (CO)
and small intestines (SI).
Supplementary Figure 2 Frequency of Treg cells after each round of DT- mediated depletion
shows significant reduction in the blood. (a) Schematic picture of protocol of Treg depletion
and C. rodentium infection used in this study. DEREG mice were infected with C. rodentium
strain ICC 180 orally and then injected i.p. with DT on day 0, 1, 7 and 8 p.i.. At day 2 (b) and
9 p.i. (c), mice were bled to collect blood for lysing, and staining with anti-CD4 and antiFoxp3 in order to check depletion efficiency of regulatory T cells. Bar of blood Treg cell
frequency from different groups represents mean ± S.E.M. from one representative of at least
3 individual experiments (n = at least 3 mice per group). *<0.05, **<0.01, ***<0.001.
Supplementary Figure 3 Immune response in the colons at early phase after infection show
impaired IL-17A-producting T cell response but not IL-22-producing innate lymphoid cell
response. Mice were orally infected with C. rodentium strain ICC 180 and treated with DT in
100 μl of PBS on day 0 and 1 p.i.. On day 6 post infection, mice were sacrificed for analysis.
Colonic LPLs were isolated and restimulated at 37 ℃ for 4 h with ionomycin and PMA, and
Brefeldin A within the last 2 h of restimulation. Restimulated cells were further stained with
anti-CD4 and anti-CD3 antibodies for surface staining and anti-RORγt, and anti-IL-22
intracellularly. FACS data show immune responses from ILCs (gated with CD3-RORγt+ for
ILC population). Dot plots represent different colonic LPL subsets. Data are one
representative from 3 individual experiments (n = 3 mice per group) and represent mean ±
S.E.M.. Statistics are analysed according to student’s T test.
Supplementary Figure 4 Treg depletion impairs expression of Th17-associated cytokines or
surface markers. (a) Treg depleted mice showed impaired colonic IL-17A and IL-17F
production by CD4+CD3+TCRβ+ cells (upper panels) but not in CD4-CD3+TCRβ- cells (lower
panels) 10 days post C. rodentium infection. Colonic lamina propria lymphocytes (cLPLs)
were isolated from Treg depleted hosts or non-depleted controls 10 days after C. rodentium
infection and further restimulated at 37 ℃ for 4 h with ionomycin and PMA, and Brefeldin A
within the last 2 h of restimulation. Restimulated cells were further stained with anti-CD4,
anti-CD3 and anti-TCRβ antibodies for surface staining and anti-IL-17A, anti-IL-17F
intracellularly. Data represent one experiment, with 3 mice per group. (b) Treg depletion
decreased total cell number of CCR6 expressing CD4+ T cells in the colon 10 days after C.
rodentium infection. Colonic lamina propria lymphocytes (cLPLs) were isolated from Treg
depleted hosts or non-depleted controls 10 days after C. rodentium infection and further
stained with anti-CD3, anti-CD4, anti-CCR6 antibodies. Data, shown as mean ± S.E.M.,
represent one experiment (n = 3 mice per group). (c) Treg depletion decreased mRNA level of
Il23r, Ccr6 and Ifng in colonic CD4+CD3+ T cells in the colon 10 days after C. rodentium
infection. LP cells were FACS sorted according to the expression of CD4 and CD3, and
pooled from the individual mice of each group. 300,000 cells either from Treg-depleted or
control group were further restimulated with 0.5 µg/ml anti-CD3 mAb for 24 hours and RNA
isolated from these cells was used to analyse gene expression by RT-PCR. Gene expression
was normalized to expression of -actin and is shown as fold difference. Data represent one
experiment, with 2 to 4 mice per group.
2
Supplementary Figure 5 Treg depletion has no impact on systemic Th17 cell response in the
spleen and mesenteric lymph nodes but enhances Th1 cell response 10 days after infection.
Erythrocyte-lysed splenocytes were restimulated in RPMI complete medium containing
phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 h, stained with anti-CD3 and
anti-CD4 mAbs, fixed and permeabilized, and intracellularly labeled with anti-IFN-γ and antiIL17A, and analyzed by FACS. Dot plots of different splenic cell subsets (upper panel). Data
are pooled from 3 individual experiments. Data represents mean. *<0.05, **<0.01. Cells from
mesenteric lymph nodes were restimulated in RPMI complete medium by PMA and
ionomycin for 4 h, stained with surface anti-CD3 and anti-CD4 mAbs, fixed and
permeabilized, and intracellularly labeled with anti-IFN-γ and anti-IL17A, and analyzed by
FACS. Dot plots of different cell subsets (lower panel). Data are pooled from 3 individual
experiments. Data represents mean. *<0.05, **<0.01.
Supplementary Figure 6 Treg depletion in C. rodentium infected mice show no difference in
the production of anti-microbial peptide/protein by colonic epithelial cells (ECs) compared to
non Treg deplete controls 10 days post C. rodentium infection. Colons from Treg-depleted
mice or controls, sacrificed 10 days after C. rodentium infection, were treated with 30mM
EDTA for 30 min and ECs were pooled from the rinsed buffer after vigourously shaking and
several rounds of PBS wash and further collected by centrifugation at 250 x g at 4°C for 10
min. RNA from ECs were isolated by TRIzol, reverse transcribed, and further analyzed by
RT-PCR. Gene expression was normalized to expression of -actin and is shown as fold
difference. Data are pooled from 3 individual experiments and represent mean ± S.E.M. ( n =
3 to 4 mice per group).
3
Supplementary Figure 7 Anti-IL-2 mAbs treatment enhanced systemic Th1 cell response in
the spleen. C. rodentium-infected DT-treated DEREG mice or control groups were
intraperitoneally treated either with anti-IL-2 mAbs or PBS on daily basis for 9 days before
analysis on day 10 p.i.. Splenocytes were collected from each group for FACS analyses.
FACS-plots showing IFN-γ and IL-17A expression of splenocytes. Dot plots of different
splenic cell subsets (upper panel). Data pooled from 3 individual experiments (lower panel).
Data represents mean. *<0.05, **<0.01, ***<0.001.
4