1. mTECs produce tissue restricted antigens (TRAs)
Developing T-cells interacts with APCs in the thymus:
mTECs produce tissue restricted antigens:
SAP
CRP
Complement C5
α-fetoprotein
Albumin
Haptoglobin
PLP
MOG
S100β
GAD67
GAD65
Somatostatin
Trypsin2
Elastase
Insulin
Α1-Crystallin
Retinal S-antigen
IRBP
Renin
Erythropoetin
H/K ATPase
Tyrosinase
Gp100
Lactalbumin
Thyroglobulin
iFABP
Amylase1
PTA
MAGE-b1.2.3. - +
-/+
DCs cTECS mTECs
Klein L et al., Nat Rev Immunol, 2014
Derbinski J et al., Nat Immunol, 2001

2. Autoimmune regulator (Aire) induce tissue restricted antigen expression
Expression pattern of Aire:
Expression pattern of tissue restricted antigens:
Aire
β-Actin
cTECs
mTECs
DCs
Macrophages
T-cells
NTC
Klein L et al., Nat Rev Immunol, 2014
Aire is exclusively expressed by mTECs:
Aire regulates espression of tissue restricted antigens
Aire
Derbinski J et al., Nat Immunol, 2001
Anderson MS et al., Science, 2002

3. Aire-reporter expressing cells and CD4 T-cell immune tolerance
Functional experiment:
Donors
Recipient
DO11.10 TCR
CD45.2
Foxp3-hCD2
Rag1-/-
OVA-specific
CD4 T-cell
DO11.10
Aire-HCO
(CD45.1/CD45.2)
Ratio 1:1
Labeling with CFSE
Transfer
Polyclonal CD4 T-cell
CD45.1
Wild type
Wild type
(CD45.1/CD45.2)
CD4+
CD45.2+
Analysis:
0.16
91.1
FCS-W
CD45.1 (PB)
CD45.2 (PE)
0.11
51.6
CD4 (PE-Cy7)
CD45.2 (PE)
KJ (APC)

4. Phenotyping of Aire-reporter positive cellsWT
Adig
Aire-eGFP+
Aire-eGFP+
0.0
0.4
32.1
Aire (eGFP)
Aire (eGFP)
SSC
CD11c (APC-Cy7)
22.2
28.1
MHC II (PE-Cy7)
MHC II (PE-Cy7)
CD45 (PerCP-Cy5.5)
EpCAM (PE)

5. Intracellular FACS detection of Aire
Phenotype:
0.00
0.03
Aire (AF 488)
Thymus
MHCII (PE-Cy5)
CD11c (APC-Cy7)
EpCAM (PE)
Aire -/-
Aire +/+
0.00
0.02
Aire (AF 488)
Lymph nodes
MHCII (PE-Cy5)
CD11c (APC-Cy7)
EpCAM (PE)