Thymic Selection Determines Ƴƺ T Cell Effector Fate: Antigen-Naive Cells Make Interleukin-17 and Antigen-Experienced Cells Make Interferon g Kirk D.C. Jensen,1 Xiaoqin Su,1 Sunny Shin,1 Luke Li,1 Sawsan Youssef,2,3 Sho Yamasaki,4 Lawrence Steinman,2,3 Takashi Saito,4 Richard M. Locksley,5 Mark M. Davis,1,2,6 Nicole Baumgarth,7 and Yueh-hsiu Chien1,2,* Immunity 29, 90–100, July 18, 2008

Introduction Ƴƺ T Cell: - evolutionarily conserved - found in all jawed vertebrates -patients with bacterial, parasitic, and viral infections: Ƴƺ T Cell↑ in peripheral blood -Overrepresented among infiltrating T cells in the early but not in the late lesions of multiple sclerosis patients -Ƴƺ T Cell-deficient mouse- immune defects 분명히 존재 ( fare worse in neutrophil-dominated inflammatory responses )

Ƴƺ T Cell development in thymus

Ƴƺ T Cell phenotype

Ƴƺ T Cell? Ab T cell과의 공통점: - thymus안에서 발달한 뒤 periphery로 나간다 -V(D)J recombination 함  diverse receptors recognizing antigens Ab T cell는 다르다 : -ligand-driven positive and negative selection 거침  CD4 helper, CD8 cytolytic T cell로

Ƴƺ T Cell 실험들 Thymic selection 후 생기는 Ƴƺ T Cell repertoire? ⇒ KN6 and G8 Ƴƺ T cell receptor (TCR) transgenic mice : two independently derived gd T cell clones - recognize the closely related b2-microglobulin-associated nonclassical MHC class I molecules, T10 and T22 1.crossed to C57BL/6 (B6) mice: express both T10 and T22 2.BALB/c mice: express only T10 3.B2m-/- mice: no cell-surface T10 or T22 expression 특징 :-B6, B2m-/- background mouse 에서는 thymus 와 periphery 에서 transgenic T cell 이 거의 없음 -in vitro stimulation  cytokine secretion↓proliferation↓

 Ƴƺ T Cell 도 ligand-driven positive and negative selection in thymus?  No… G8T cells were able to mature inB2m/ mice Ligand 필요 o? murine skin-dendritic epidermal T cells (DETCs) 발달 : - Ƴƺ T cells express the same TCR - first to appear during fetal thymic development - DETCs are reactive to keratinocytes in a TCR-dependent manner Ligand 필요 x? 1. sizable population (0.1%–1%) of Ƴƺ T cells in normal unimmunized mice recognizes T10 and/or T22 2. T10-and/or-T22-specific Ƴƺ T cells was also found in B2m-/- mice 3. specificity encoded by amino acid residues on V ƺ and D ƺ gene elements  recombination % of the nonselected TCR ƺ sequences contain the T10 and/or T22 recognition motif  repertoire seems to be determined predominantly by gene rearrangement instead of ligand-dependent selection

Major histocompatibility complex class Ib proteins bind various immunoreceptors. Listed are major histocompatibility complex class Ib (MHC Ib) molecules for which receptor binding has been well ch aracterized. Conventional T-cell receptors (TCRs) are those of polyclonal T cells; unconventional T CRs correspond to oligoclonal T-cell subsets, such as natural killer (NK) T cells (for CD1d), T cells ( T10/T22, MICA/B) or gut-associated T cells (MR1). Proposed functional homologues between mice and humans are in the same row. H60, histocompatibility antigen 60; HLA, human leukocyte antige n; MICA/B, MHC class I chain-related peptides A/B; Rae1, retinoic acid early inducible gene 1; ULB P, UL16 binding protein.

Host T10 and T22 Expression Does Little to Affect the Presence of T10- and/or T22-Specific Ƴƺ T Cells To determine the role of ligand recognition in the development of a functional gd T cell repertoire  T22 tetramer staining intensity Frequency TCR surface expression tetramer staining intensity  endogenous ligand expression did little to influence the number, TCR usage, and the recognition motif of T10- and/or T22- specific Ƴƺ T cells. B6: blue B/c: black B2m-/-: red

1)mice lacking both b2m and class II MHC molecules—spleen, thymus 안의 T-10 and/0r T22 specific Ƴƺ T Cells frequency 비슷하다  cyclosporin A (to inhibit ab T cell-positive selection) 처리해도 영향 x 2) normal numbers of Ƴƺ thymocytes were found in calcineurin-deficient animals Calcineurin: -protein phosphatase 2B(PP2B) -IL-2 transcription activator -cycloporin, Pimecrolimus (Elidel) Tacrolimus(FK506)…inhibitors Calcineurin dephosphorylates nuclear facstor of activated T cell, cytoplasmic component(NFATc)  Nuclear transcription of IL-2…  T-10, T-22 specific Ƴƺ T Cells repertoire는 T-10, T-22, MHC class II, B2m associated molecule에 영향 안받음  Develop 할 때 positive, negative selection 영향 안받음

Ƴƺ Thymocyte maturation: activation of Mitogen-activated protein(MAP) kinase(Raf- MEK- ERK) pathway---TCR의 down stream signal와 연관됨  T10, T22 specific Ƴƺ T cell에서 ERK1과 ERK2가 비슷하게 phosphorylation됨 (다른 ligand 발현 환경에서) Ligand Expression Is Not Required for Ƴƺ Thymocyte Development

DP, CD4 thymocyte에서: Intracellular pERK1, pERK 2의 발현 비슷, CD5발현 비슷 Stable indicator of signaling strength

기존의 설: ligand 없는 상태에서 KN6 TCR transgenic rd T cell (KN6+ RAG2-/- B2m -/-)에서 ab T cell fate 따라감 - CD4, CD8 발현 - TCR rd T cell 발현 저조  반박1 : B2m -/- mouse의 tetramer + rd T cell는 CD4-CD8-임  반박2 : 그림 1-c: no TCR downregulation 이유: endogenous TCR gene이 prearrange돼서 T 림프구 발달이 skew됨 ⇒ DP KN6+ T cell 20배 증가해도 DN rd R cell 2배 증가  DP T cell의 증가는 rd T cell의 증가와 관련이 없다

Sphongosine-1-phosphate receptor 1(S1P1): mature thymocyte이 Thymus에서 나가는데 필요하다… upregulation  그러나 tetramer + or – thymocyte에서 S1P1 의 expression 차이 없다 ⇒ endogenous thymic ligand expression이 rd T cell 발달과 thymic exit에 는 영향을 주지 않는다

TCR Dimerization Provides a Possible Ligand- Independent Mechanism for Signaling through the TCR Pre- Ta dimerization에 의한 autonomous signaling: pre-T cell activation  G8rd TCR+ T22 ligand의 크리스탈 구조를 보면 TCR Vd domain 끼리 dimerized complex를 이루고 있음  Rd TCR에 ligand와 croddlink하지 않고도 dimerize할 수 있는지 알아보자.  Rd T cell발달에는 ligand 필요하지 않다. TCR dimerization 만으로도 발달할 수 있을 것이다 (pERK1, 2 high) Human erythropoietin receptor(EPOR)의 Extracellular domain이 TCRd chain의 Extracellular domain으로 대체됨  Baf3 cell의 TCR r cjain의 Ex domain과 같 이 발현됨  EX domain dimer 된 후 EPOR signal가면 IL-3 independent하게 자랄 수 있음  Vd1-EPOR Vr5외에 모두 잘자람 아유: DETC orgin…발달할 때 antigen- driven positiv selection받는다고 알 려짐 *Baf3 cell: IL-3 dependent from Balb/C

A Large Fraction of Lymphoid rd T Cells Exhibit an Antigen-Naive Phenotype B6, BALB/c, B2m-/- 쥐의 T-10, T-22 specific thymocyte이 tetramer 염색 정도가 비슷했지만 Phenotype marker 발현을 보면 앞의 두 쥐의 T cell은 antigen을 만나고, B2m-/-쥐는 못 만난 것 알 수 있다 그림 1-C: TCR down vs TCR up HAS lo VS HAS hi CD122 high VS CD122 lo IL-2 & IL-15 receptor common B chain HAS low: ab thymocyte이 ligand 만날 때…ligand 있는 상황에서 G8 rd TCR tg thymocyte도 HSA low CD122 up: ab thymocyte의 self-ligand recognitioin…. Dendritic epidermal nurene rd T cell 발달 때도 CD122 up

Rd T cell이 thymus에서 나올 때 ligand 노출과 상관 없다!  B2m-/- mouse의 rd splanocyte은 Ag 만나지 않은 형질을 띰 B2m-/- B6 1.CD44 lo and int CD44 hi 2.CD122 lo and Int CD122 hi NK 1.1+DX5+CD5lo 그림 1-B: B6 -very high tetramer staining rd T cell 적음  T10, T22-high affinity TCR 가진 T cell이 제거됨 ⇒G8 tg Tcell이 B6 mouse spleen에 없는 이유 : 가장 큰 affinity TCR tg이므로 Tetramer – thymocyte, splenicyte(>99%): B2m-/- mouse의 phenotye과 비슷  대부분의 rd T cell이 발달 혹은 periphery에서 Ag 안만남

Antigen-Naive Ƴƺ T Cells Can Turn over In Vivo To assess the functional properties of T10 and/or T22-specific rd T cells : turnover rates in vivo by measuring BrdU in splecnocytes iof each mouse Drink…0.8mg/ml BrdU for days  Intracellular BrdU staining 7 day때 B6, BB/c가 더 BrdU Incorporation 많이 됨  CD122, CD44 hi…turnover rate hi 28일 이후에는 차이 안남 ⇒Host T10, T22와 만나면 specific rd T cell turnover 증가하지만 repertoire로 specificity가 ‘fix’되지는 않는다

Antigen Recognition during Development Defines Ƴƺ T Cell Effector Function To test whether encointering ligand during development affects the ability of rd T cells to make Cytokines  YETI mouse: IFNr-yellow fluorescence protein bicistronic reporter 발현 (B6 background) Rd T cell은 IFNr를 낸다고 알려짐  1. YETI mouse의 T10,22 specific rd splenocyte이 대부분 YFP+ 2. CD122hi splenocyte이 대부분 YFP +  CD122lo rd splenocyte은 YFP거의 안냄

1. CD122 hi rd splenocytes stimulated with plate- bound TCRd crosslingking Ab GL-4  IFNr를 매우 많아 냄  Ag있는 상태로 발달한 rd T cell은 기능적으로 anergic하거나 inactive하지 않음 2. CD122loB6 rd splenocyte은 stimulation시켜도 거의 IFNr안냄  Ag안만난 상태에서 cytokine을 낼까?  Rd T cell의 주요 cytokine은 IFNr외에도 IL-17.. after stimulation  GL-4 stimulated CD122lo rd splanocyte이 IL-17을 많이 냄! …thymus와 lymph node도 마찬가지 ⇒rd T cell subtype존재…Trd-17, Trd-IFNr

IL-17-Producing gd T Cells Appear Early after Peptide-CFA Immunization IL-17 regulates expansion and recruitment of neutrophils and monocytes to iniciate the inflammatory Response…acute inflammation에서 Ag 노출 없어도 IL-17 response일어나야 함  rd T cell이 적당  B6 mouse에 myelin oligodendrocyteglycoprotein(MOG) peptide (35-55) 주사 in CFA MOG: surrogate for induction of acute inflammation and to prime antigen-specific ab T cells induces autoantibody production and relapsing-remitting neurological disease causing extensive plague-like demyelination. Antoantibody responses to MOG(35-55) are present in multiple sclerosis(MS) patients and Mog induced experimental autoimmune encephalomyelitis (EAE) mice  LN 에서 T cell분리  Anti-CD3로 24시간 in vitro activation  IL-17 intracellular staining

MOG specific CD4+ ab T cell 이 IL-17 내기 전에 (d3) Rd T cell은 immunize전 후에 이미 Il_17을 50%이상 내기 시작함 B6 mouse의 spleen과 LN 의 T10, T22 specific rd T cell 의 CD122 틀림 Spleen- CD122 hi LN- CD122 lo  CFA immunize한 뒤 두 cell 다 IL-17 잘냄  rd T cell이 발달동안 ligand 만나는 것은 IL-17내는 것과 다른 문제이다. Ag-specific ab T cell은 감염 후 neutrophil influx, 다른 림프구 recruit  local cytokine milieu 형성  Ag specific B cell, T cell response일어남 EAE: after MOG+ CFA immunization 1.B6 에서는 쉽게 일어남 2.Rd T cell-/- mouse에서는 발병도 늦고 Severity떨어짐  Th17 ab T cell 이 주요 EAE애 기여

Summary Nonclassical major histocompatibility complex class 1- T10, T22 1.Thymus에서 Ag 만나는 것은 발달에 필요x 저해x 2.TCR trigering 후에는 ligand-naïve lymphoid rd T cell이 IL-17내는 반면, ligand-experienced cell은 IFNr 낸다 3.Immunize 직후 IL-17+ rd T cell이 IL-17+ab T cell나타나기 며칠 전에 drining lymph node에 나타남  Thymic selection은 rd T cell의 ag specificity를 정하기 보다 effector fate을 결정함 4. Ag-naïve rd T cell이 IL-17 swift하는 것은 새로운 Ag에 대해 acute inflammatory respose를 onset할 수 있는 의미가 있다