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