Thymic Selection Determines Ƴƺ T Cell Effector Fate: Antigen-Naive Cells Make Interleukin-17 and Antigen-Experienced Cells Make Interferon g Kirk D.C.

Presentation on theme: "Thymic Selection Determines Ƴƺ T Cell Effector Fate: Antigen-Naive Cells Make Interleukin-17 and Antigen-Experienced Cells Make Interferon g Kirk D.C."— Presentation transcript:

1 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

2 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 )

3 Ƴƺ T Cell development in thymus

4 Ƴƺ T Cell phenotype

5 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

6 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↓

7  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 4. 0.85% 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

8 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.

9 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

10 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

11 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

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

13 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

14 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

15 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

16 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

17 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

18 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

19 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

20 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

21 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

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23 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

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25 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