maria hinterberger, martin aichinger, olivia prazeres da ... · wt c2takd mhcii cd80 60.8 ± 7 55.8...
TRANSCRIPT
Autonomous role of medullary thymic epithelial cells in central CD4+ T cell tolerance
Maria Hinterberger, Martin Aichinger, Olivia Prazeres da Costa, David Voehringer,
Reinhard Hoffmann and Ludger Klein
Supplementary Information
Nature Immunology: doi:10.1038/ni.1874
WT C2TAkd
MHCII
CD
80
60.8 ± 7 55.8 ± 5
100
101
104
102
103
100 101 104102 103
Supplementary Figure 1. Normal frequency, but reduced MHCII expression, of CD80hi mTECs in C2TAkd mice. Expression of CD80 versus MHCII on gated total (CD45–EpCAM+Ly51–) mTECs from wild-type or C2TAkd mice. Data summarize at least 22 independent cell preparations from each genotype.
Nature Immunology: doi:10.1038/ni.1874
WT C2TAkd
Keratin 5
200μm
Keratin 8
Supplementary Figure 2. Thymi of C2TAkd mice are properly organized into cortical and medullary regions. Cryosections of wild-type or C2TAkd thymi were stained for medullary (Keratin 5; green) and cortical (Keratin 8; red) areas. The scale bar represents 200 μm. Images are representative of at least three organs of each genotype.
Nature Immunology: doi:10.1038/ni.1874
salivarygland
lacrimalgland
WT C2TAkd
Supplementary Figure 3. Sporadic incidence of lymphocytic infiltrates in peripheral organs of wild-type and C2TAkd mice. Salivary and lacrimal glands of wild-type or C2TAkd mice (5 months of age) were fixed, sectioned and stained with haematoxilin and eosin. Arrows indicate infiltrates. Scale bars are 200 μm. The incidence of infiltrates was: Salivary gland 1 / 4 (wild-type) versus 4 / 4 (C2TAkd) in Experiment (Exp.) 1 and 0 / 6 versus 0 / 5 in Exp. 2; Lacrimal gland 0 / 4 versus 3 / 4 in Exp. 1 and 3 / 6 versus 3 / 5 in Exp. 2.
Nature Immunology: doi:10.1038/ni.1874
0
10
20
30
40
50
WT C2TAkd WT
TCR-HA × Pgk-HATCR-HA
TCR
-HA
+ (%
of C
D4S
P ce
lls)
a
b
TCR-HA × Pgk-HATCR-HA
WT WTC2TAkd C2TAkd
TCR-HA × Pgk-HA
WT C2TAkd
20.5 ± 5.4 25.4 ± 3.9 16.8 ± 2.3 20.9 ± 3.8
Gated on CD4SP cells30.5 ± 3.5 32.9 ± 4.9 19.5 ± 8.8 32.2 ± 8.9
P = 0.00001
NS
6.0 ± 3.0
9.4± 3.8
c TCR-HA
WT0.2 ± 0.1
CD
4
CD8
TCR-HA (6.5)
100 101 104102 103100
101
104
102
103
100 101 104102 103
Foxp
3
TCR-HA (6.5)
100
101
104
102
103
100 101 104102 103
Supplementary Figure 4. Rescue from clonal deletion and increased generation of HA-specific Treg cells in TCR-HA × Pgk-HA × C2TAkd thymi. (a) Thymocyte subset composition in TCR-HA or TCR-HA × Pgk-HA mice in the absence or presence of mTEC-specific C2TA silencing. Average frequencies of CD4SP cells (dot plots) and TCR-HA+ cells among gated CD4SP thymocytes (histograms) are indicated. (b) Summary of the frequencies of TCR-HA+ cells among gated CD4 SP thymocytes depicted in (c). P-values are indicated, NS = not significant. (Total n: TCR-HA = 7; TCR-HA × Pgk-HA = 20; TCR-HA × Pgk-HA × C2TAkd = 19). (c) Expression of Foxp3 in CD4SP cells. The average frequencies (± SD) of TCR-HA+Foxp3+ cells among CD4SP thymo-cytes are indicated. Expression of Foxp3 was assessed by intracellular staining.
100
020406080
Eve
nts
(% m
ax)
Nature Immunology: doi:10.1038/ni.1874
32.6 ± 1.2
TCR-HA × ∆DCTCR-HA
WT Aire-HA
6.2 ± 1.7 4.3 ± 1.1 15.3 ± 3.6
21.1 ± 3.3 16.7 ± 2.7 8.8 ± 2.5 20.1 ± 7.8
Gated on CD4SP cells
BM:
Recipient: Aire-HA
TCR-HA × ∆DC
a
b
1.1± 0.5
5.8± 1.8
Aire-HA × C2TAkd
Aire-HA Aire-HA × C2TAkd
BM:
Recipient:
19.2 ± 5.2 22.8 ± 3.9
P = 0.007
TCR
-HA
+ Fo
xp3+
cel
ls (×
106 )
TCR-HA × ∆DC
Aire-HAAire-HA
×C2TAkd
c
0
0.5
1
1.5
2
CD
4
CD8100 101 104102 103
100
101
104
102
103
TCR-HA (6.5)100 101 104102 103
Foxp
3
TCR-HA (6.5)
100
101
104
102
103
100 101 104102 103
Supplementary Figure 5. The C2TAkd mediated ‘cell fate conversion’ of TCR-HA × Aire-HA thymocytes is independent of cross-presentation by DCs. (a) Thymocyte subsets in wild-type, Aire-HA or Aire-HA × C2TAkd recipients 6 to 8 weeks after reconstitution with TCR-HA or TCR-HA × ΔDC bone marrow cells. Frequencies of CD4SP cells (dot plots) and TCR-HA+ cells among gated CD4SP thymocytes (histograms) ± SD are indicated. The absence or presence of DCs did not affect the extent of negative selection (middle two dot plots and histograms). Irrespective of the absence or presence of DCs (compare Fig. 7b), silencing of C2TA in mTECs promoted a significant (P = 0.00003) increase in the fraction of TCR-HA+ CD4SP cells that escape negative selection. Among TCR-HA+ CD4SP cells, the percentage (b) (P = 0.001) and absolute number (c) of Foxp3+ cells was significantly increased. Data are representative of at least 3 chimeras in two independent experiments.
100
020406080
Eve
nts
(% m
ax)
Nature Immunology: doi:10.1038/ni.1874