TwoStage Cooperative T Cell ReceptorPeptide Major Histocompatibility ComplexCD

Two-Stage Cooperative T Cell Receptor-Peptide Major Histocompatibility Complex-CD 8 Trimolecular Interactions Amplify Antigen Discrimination Immunity 34, 13– 23, January 28, 2011 Ning Jiang, Jun Huang, Lindsay J. Edwards, Baoyu Liu, Yan Zhang, Carrie D. Beal, Brian D. Evavold, and Cheng Zhu 2011. 02. 14. Lee, Hye-yeong

Introduction • Antigen recognition by the T cell is central to its development and activation. • Together with its coreceptor, the T cell receptor (TCR) discriminates various peptide-major histocompatibility complex (p. MHC) ligands • These responses are remarkably fast: TCR microclusters (Campi et al. , 2005; Yokosuka et al. , 2005), intracellular calcium fluxes, and phosphorylation of linker for activation of T cells (LAT) are observed within a few seconds upon TCR contact with p. MHC • The coreceptor on cytotoxic T cells is CD 8, which binds the a 3 conservative domain of the MHC without peptide contact. • This binding property is distinct from TCR, which binds the a 1 and a 2 domains as well as the peptide • It is also unclear how p. MHC binds TCR and CD 8, e. g. , concurrently, sequentially, independently, or cooperatively

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Figure 1. Micropipette Adhesion Frequency Assay RBC coating OT-1 - OVA: H-2 Kb F 5 - NP 68: H-2 Db

Figure 2. Two-Stage Kinetics of TCR-p. MHC-CD 8 Trimolecular Interaction MHC Mutant : H-2 Kba 3 A 2 Ø 1 st stage curve: TCR-p. MHC binding에 의한것 Ø 2 nd stage curve : p. MHC-CD 8 binding과 TCR binding에 의한것 이때 TCR binding이 계속적으로 필요 Ø independent concurrent model: 1 st stage에 p. MHC-CD 8 bonds가 추가되어 2 nd stage를 이룬다 (Zhu and Williams, 2000) 이를 부정함 Ø CD 8 -dependent upregulated functional state Ø 1 sec delay : 2 -stage curve가 TCR, CD 8, p. MHC의 reaction kinetics에 의한 것만이 아닐 것

Figure 3. TCR-Induced CD 8 -Dependent Increased Adhesion Can Be Differentially Inhibited PP 2 : Src inhibitor BAPTA-AM : Ca 2+ chelator PD 98059 : MAPKK inhibitor Ø PP 2 single stage : Lc. K (Src family protein kinase) 관여 Ø CD 45 inbition : Lck activation억제 single stage Ø Ca 2+, MKK 관여하지 않음 TCR-CD 8 positive feedback loop

Figure 4. Effect of Anti-CD 8 Clone 53 -6. 7 on T Cell Adhesion to p. MHC and Its Inhibition anti-CD 8 reagents가 T cell의 p. MHC binding 을 증가시킨다 (Luescher 1995) A. anti-CD 8 m. Ab 53 -6. 7 1 st stage에서 adhesion 증가 B. H-2 Kba 3 A 2 lower 1 st stage level C. p. MHC replacing D. Fab same effect PP 2 anti-CD 8 reagents generate intracelluar activation anti-CD 8 m. Ab 53 -6. 7에 의해 p. MHC와 CD 8, TCR의 binding이 증가되었다 이는 2 nd stage에서의 adhesion 증가와 관 련 있을 것.

Figure 5. TCR Engagement with a Single p. MHC Is Sufficient to Induce Cooperation with CD 8 p. MHC가 TCR, CD 8과 direct binding을 한다 2 nd stage에서의 adhesion 증가에 signaling에 의한 TCR-CD 8 cooperation이 일어날 것 RBC에 coating되는 p. MHC의 밀도를 조정하여 TCR, CD 8 -p. MHC binding 관찰 single TCR-p. MHC bond로 인해 CD 8 -dependent upregulated functional state가 유도될 수 있다. trimeric cooperation의 가능성 TCR CD 8 O X O OVA: H-2 Kba 3 A : VSV: H-2 Kb Ø TCR과 cd 8이 같은 p. MHC에 binding해야 upregulated 2 nd stage adhesion이 일어날 수 있다

Figure 6. Second-Stage Binding Does Not Need Accumulation of Repeated Interactions and Is Reversible A. “first-contact” 실험 TCR signaling이 applied force에 의해 일어날 수 있다(Kim, 2009) 반복적인 contact에 의한 TCR pulling으로 2 nd stage를 유도하였을 가능성을 배제하기 위함 control 실험 2 nd stage와 Pa 차이 없음 : TCR signaling이 accumulation되지 않고 바로 종결됨 B. “Long Then Short” 실험 : signaling의 조절 TCR에 의한 signaling이 빠르게 종결되어 제자리 상태로 돌아간다(reversible) C. “alternating” 실험 1. 5초 간격으로 번갈아 0. 25, 5초 contact 1초 delay와 1. 5초 동안의 signaling의 사라짐 signal reulation의 속도, 지속성 등을 의미한다

Figure 7. Synergy between TCR and CD 8 Amplifies T Cell Discrimination OVA : SIINFEKL V-OVA : RGYNYEKL TCR-p. MHC-CD 8 trimolecular interaction synergistic? p. MHC-CD 8 : peptide에 무관 TCR/p. MHC/CD 8의 경우 TCR/p. MHC, p. MHC/CD 8의 단순 adding아님 synergistic. 이러한 synergy는 peptide의 potency에 따라 같은 경향성으로 증가한다. 이것은 T cell proliferation과도 연관성을 갖는다

Conclusion and Discussion TCR-CD 8 positive-feedback loop is involved in initial signaling steps that is sensitive to a single p. MHC rapid, reversible, synergistic, and peptide discriminative