Autonomous role of medullary thymic epithelial cells in central CD4 + T cell tolerance Maria Hinterberger1, Martin Aichinger1, Olivia Prazeres da Costa2, David Voehringer1, Reinhard Hoffmann2 & Ludger Klein Ju, Ji Min

Introduction

mTECs serve an essential function in central tolerance by expressing PTA PTA expression is to a considerable degree under control of the autoimmune regulator protein Aire mTECs serve antigen reservoir and function as APC Diminished but did not fully eliminate MHC class II on mTECs –By using tissue-specific expression of miRNA that silences the gene encoding CIITA –CIITA : the master regulator of MHC class II expression

'Designer' miRNA–mediated silencing of Ciita in vitro Figure 1: Lower MHC class II expression through Ciita silencing in vitro. FACS Immunoblot RNA polymerase II promoters -> tissue-specific or temporally controlled gene silencing in vivo Mir30-backbone in the retroviral vector LMP

Transgenic silencing of Ciita in mTECs Figure 2: Transgenic expression of the C2TA-specific synthetic miRNA in mTECs. The C2TAkd BAC transgene Small RNA TaqMan assayQuantitative RT-PCR analysis mature (CD80 hi Aire-GFP + ) immature (CD80 lo Aire-GFP − )

Transgenic silencing of Ciita in mTECs Figure 3: Silencing of Ciita and MHC class II genes in C2TAkd mTECs. Quantitative RT-PCR C2TA target, H2-Aa Microarray expression profiling of C2TA target genes (MHC II ) positively correlated with mTEC maturation MHC class I expression is largely C2TA independent

Phenotype and APC function of C2TAkd mTECs Figure 4: Phenotype, APC-function and promiscuous PTA expression of C2TAkd mTECs. mature mTECs less surface MHC II Sirpα − autochthonous Sirpα + migratory mature C2TAkd mTECs had 10% of H-2E a protein in WT c. Frequency of GFP + cells among HA-specific GFP reporter hybridoma cells (A5 cells) ->cultured for 17 h with mTECs d. Frequency of GFP + cells among total mTECs from Aire-GFP reporter (Adig) mice with or without C2TAkd transgene Aire + mTECs were normally distributed f. Degree of decrease in MHC class II achieved in C2TAkd mTECs does not interfere with their normal development.

Enlarged CD4SP compartment in C2TAkd mice Figure 5: Nonredundant contribution of mTECs and hematopoietic APCs to the deletional tolerance of CD4 + T cells. Enlarged CD4SP compartment in C2TAkd thymi relative to that of wild- type controls No change in the abundance of CD8 + single-positive cells Bone marrow chimeras H2-Ab1 −/− ( MHC class II–deficient ) Additive, nonredundant contribution of DCs and mTECs to the negative selection of MHC class II–restricted thymocytes 1.mTECs act as APCs 2.Distinct spectra of MHC class II– restricted TCR specificities of DCs and mTECs.

Altered fate of autoreactive CD4 + T cells Figure 6: Rescue from clonal deletion and enhanced generation of OVA-specific T reg cells in DO11.10 × Aire-OVA × C2TAkd thymi. DO11.10 × Aire-OVA double-transgenic mice -> two-thirds of DO CD4SP cells are deleted -> increase in Foxp3 + T reg cells a. Quantitative RT-PCR for expression of the Aire-OVA transgene b,c. the loss of OVA-specific cells was much lower in DO11.10 × Aire-OVA × C2TAkd mice d-f. DO11.10 × Aire-OVA × C2TAkd thymi -> more DO T reg cells -> more OVA-specific Foxp3 − CD4SP cells (naïve)

Altered fate of autoreactive CD4 + T cells Figure 7: Rescue from clonal deletion and enhanced generation of HA-specific T reg cells in TCR-HA × Aire-HA × C2TAkd thymi. a-e reproduced in a related model of different antigen specificity, Diminishing the avidity of autoantigen recognition on mTECs 1. escape of autoreactive CD4 + T cells from clonal deletion 2. greater emergence of antigen-specific T reg cells  avidity model of deletion versus T reg cell differentiation.

Induction of clonal deletion and T reg differentiation by mTECs Figure 8: The C2TAkd-mediated cell-fate conversion of DO11.10 × Aire-OVA thymocytes is independent of cross-presentation by DCs. mice expressing diphtheria toxin in CD11c + cells (ΔDC mice) 1. mTEC-specific silencing of Ciita 2. diminution of MHC class II expression 3. attenuation of the autonomous APC function 4. lower negative selection & greater T reg cell induction => This process is independent of DC BM transfer -> WT, Aire-OVA, Aire-OVA x C2TAkd

Discussion mTECs 과의 cognate interaction 에 의해 polyclonal thymocytes 의 deletion 은 DCs 의 역할과 유사 mTECs 과 DC 에 의해 present 되는 autoantigen-derived MHC class II–bound epitopes 의 spectra 에 차이 존재 antigen recognition on mTECs 양상은 negative selection or T reg cell differentiation 으로 진행

Further study GVHD 와 autoimmune disease 에 mTECs 의 관련성 ? Tolerance study 에서 mTECs 과 DC 의 작용 비교 ( different spectra )