Thymic Selection Determines T Cell Effector Fate AntigenNaive

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 Chien 1, 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 CD 4 helper, CD 8 cytolytic T cell로

Ƴƺ T Cell 실험들 Thymic selection 후 생기는 Ƴƺ T Cell repertoire? ⇒ KN 6 and G 8 Ƴƺ T cell receptor (TCR) transgenic mice : two independently derived gd T cell clones - recognize the closely related b 2 -microglobulin-associated nonclassical MHC class I molecules, T 10 and T 22 1. crossed to C 57 BL/6 (B 6) mice: express both T 10 and T 22 2. BALB/c mice: express only T 10 3. B 2 m-/- mice: no cell-surface T 10 or T 22 expression 특징: -B 6, B 2 m-/- background mouse 에서는 thymus와 periphery에서 transgenic T cell이 거의 없음 -in vitro stimulation cytokine secretion↓proliferation↓

Ƴƺ T Cell 도 ligand-driven positive and negative selection in thymus? No… G 8 T cells were able to mature in. B 2 m/ mice Ligand 필요o? 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 필요x? 1. sizable population (0. 1%– 1%) of Ƴƺ T cells in normal unimmunized mice recognizes T 10 and/or T 22 2. T 10 -and/or-T 22 -specific Ƴƺ T cells was also found in B 2 m-/- mice 3. specificity encoded by amino acid residues on Vƺ and Dƺ gene elements recombination 4. 0. 85% of the nonselected TCRƺ sequences contain the T 10 and/or T 22 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 characterized. Conventional T-cell receptors (TCRs) are those of polyclonal T cells; unconventional TCRs correspond to oligoclonal T-cell subsets, such as natural killer (NK) T cells (for CD 1 d), T cells (T 10/T 22, MICA/B) or gut-associated T cells (MR 1). Proposed functional homologues between mice and humans are in the same row. H 60, histocompatibility antigen 60; HLA, human leukocyte antigen; MICA/B, MHC class I chainrelated peptides A/B; Rae 1, retinoic acid early inducible gene 1; ULBP, UL 16 binding protein.

Host T 10 and T 22 Expression Does Little to Affect the Presence of T 10 - and/or T 22 -Specific Ƴƺ T Cells To determine the role of ligand recognition in the development of a functional gd T cell repertoire T 22 tetramer staining intensity endogenous ligand expression did little to influence the number, TCR usage, and the recognition motif of T 10 - and/or T 22 specific Ƴƺ T cells. B 6: blue B/c: black B 2 m-/-: red Frequency tetramer staining intensity TCR surface expression

1) mice lacking both b 2 m and class II MHC molecules—spleen, thymus 안의 T-10 and/0 r T 22 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 2 B(PP 2 B) -IL-2 transcription activator -cycloporin, Pimecrolimus (Elidel) Tacrolimus(FK 506)…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, B 2 m associated molecule에 영향 안받음 Develop 할 때 positive, negative selection 영향 안받음

Ligand Expression Is Not Required for Ƴƺ Thymocyte Development Ƴƺ Thymocyte maturation: activation of Mitogenactivated protein(MAP) kinase(Raf- MEKERK) pathway---TCR 의 down stream signal와 연관 됨 T 10, T 22 specific Ƴƺ T cell에서 ERK 1과 ERK 2가 비슷하게 phosphorylation됨 (다른 ligand 발현 환경에서)

Ligand Expression Is Not Required for Ƴƺ Thymocyte Development DP, CD 4 thymocyte에서: Intracellular p. ERK 1, p. ERK 2의 발현 비슷, CD 5발현 비슷 Stable indicator of signaling strength

기존의 설: ligand 없는 상태에서 KN 6 TCR transgenic rd T cell (KN 6+ RAG 2 -/B 2 m -/-)에서 ab T cell fate 따라감 - CD 4, CD 8 발현 - TCR rd T cell 발현 저조 반박 1 : B 2 m -/- mouse의 tetramer + rd T cell는 CD 4 -CD 8 -임 반박 2 : 그림 1 -c: no TCR downregulation 이유: endogenous TCR gene이 prearrange돼서 T 림프구 발달이 skew됨 ⇒ DP KN 6+ 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(S 1 P 1): mature thymocyte이 Thymus에서 나가는데 필요하다… upregulation 그러나 tetramer + or – thymocyte에서 S 1 P 1 의 expression 차이 없다 ⇒ endogenous thymic ligand expression이 rd T cell 발달과 thymic exit 에 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 G 8 rd TCR+ T 22 ligand의 크리스탈 구조를 보면 TCR Vd domain 끼리 dimerized complex를 이루고 있음 Rd TCR에 ligand와 croddlink하지 않고도 dimerize할 수 있는지 알아보자. Human erythropoietin receptor(EPOR)의 Extracellular domain이 TCRd chain의 Extracellular domain으로 대체됨 Baf 3 cell의 TCR r cjain의 Ex domain과 같이 발현됨 *Baf 3 cell: IL-3 dependent from Balb/C EX domain dimer 된 후 EPOR signal가면 IL-3 independent하게 자랄 수 있음 Vd 1 -EPOR Vr 5외에 모두 잘자람 아유: DETC orgin…발달할 때 antigendriven positiv selection받는다고 알 려짐 Rd T cell발달에는 ligand 필요하지 않다. TCR dimerization 만으로도 발달할 수 있을 것이다 (p. ERK 1, 2 high)

A Large Fraction of Lymphoid rd T Cells Exhibit an Antigen-Naive Phenotype B 6, BALB/c, B 2 m-/- 쥐의 T-10, T-22 specific thymocyte이 tetramer 염색 정도가 비슷했지만 Phenotype marker 발현을 보면 앞의 두 쥐의 T cell은 antigen을 만나고, B 2 m-/-쥐는 못 만난 것 알 수 있다 그림 1 -C: TCR down vs TCR up HAS lo VS HAS hi IL-2 & IL-15 receptor common B chain CD 122 high VS CD 122 lo HAS low: ab thymocyte이 ligand 만날 때…ligand 있는 상황에서 G 8 rd TCR tg thymocyte도 HSA low CD 122 up: ab thymocyte의 self-ligand recognitioin…. Dendritic epidermal nurene rd T cell 발달 때도 CD 122 up

Rd T cell이 thymus에서 나올 때 ligand 노출과 상 관 없다! B 2 m-/- mouse의 rd splanocyte은 Ag 만나지 않은 형질을 띰 B 2 m-/1. CD 44 lo and int 2. CD 122 lo and Int B 6 CD 44 hi CD 122 hi NK 1. 1+DX 5+CD 5 lo 그림 1 -B: B 6 -very high tetramer staining rd T cell 적음 T 10, T 22 -high affinity TCR 가진 T cell이 제거 됨 ⇒G 8 tg Tcell이 B 6 mouse spleen에 없는 이유 : 가장 큰 affinity TCR tg이므로 Tetramer – thymocyte, splenicyte(>99%): B 2 m-/- 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 T 10 and/or T 22 -specific rd T cells : turnover rates in vivo by measuring Brd. U in splecnocytes iof each mouse Drink… 0. 8 mg/ml Brd. U for days Intracellular Brd. U staining 7 day때 B 6, BB/c가 더 Brd. U Incorporation 많이 됨 CD 122, CD 44 hi…turnover rate hi 28일 이후에는 차이 안남 ⇒Host T 10, T 22와 만나면 specific rd T cell turnover 증가하지만 repertoire로 specificity가 ‘fix’되지는

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 (B 6 background) 1. YETI mouse의 T 10, 22 specific rd • Rd T cell은 IFNr를 낸다고 알려짐 splenocyte이 대부분 YFP+ 2. CD 122 hi splenocyte이 대부분 YFP + CD 122 lo rd splenocyte은 YFP거의 안 냄

1. CD 122 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. CD 122 lo. B 6 rd splenocyte은 stimulation시켜도 거의 IFNr안냄 Ag안만난 상태에서 cytokine을 낼까? Rd T cell의 주요 cytokine은 IFNr외에도 IL-17. . after stimulation GL-4 stimulated CD 122 lo 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 B 6 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-CD 3로 24시간 in vitro activation IL-17 intracellular staining

MOG specific CD 4+ ab T cell 이 IL-17 내기 전에 (d 3) Rd T cell은 immunize전 후에 이미 Il_17을 50%이상 내기 시작함 B 6 mouse의 spleen과 LN 의 T 10, T 22 specific rd T cell 의 CD 122 틀림 Spleen- CD 122 hi LN- CD 122 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. B 6 에서는 쉽게 일어남 2. Rd T cell-/- mouse에서는 발병도 늦고 Severity떨어짐 Th 17 ab T cell 이 주요 EAE애 기여

Summary • Nonclassical major histocompatibility complex class 1 - T 10, T 22 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 전 에 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