Distinct roles for LFA-1 affinity regulation during T cell adhesion, diapedesis, and inerstital migration in lymph nodes Eun Jeong Park, Antonio Peixoto, Yoichi Imai, Ahmad Goodarzi, Guiying Cheng, Christopher V. Carman, Ulrich H. von Andrian and Motomu Shimaoka Blood Dec 18, 2009 2010. 1. 4. Lee, Hye-Yeong

Introduction Immune Response Innate immune response Adaptive immune response Lymphocyte : B cells, T cells T cell : naïve -> activated T cells Naïve T cells recirculate through secondary lymphoid organs

Introduction Homing cascade Recruitment of circulating T cells to the luminal HEV surface, involving a rolling interaction and its subsequent conversion to firm adhesion upon chemokine activation Intravascular migration of luminally adherent T cells that allows the translocation from the initial attachment site to a suitable exit site Transendothelial diapedesis across HEVs Random migration of T cells within the extravascular compartment in LN parenchyma

Introduction LFA-1 Immunology and Cell Biology 86, 226–231 (1 March 2008)

Introduction LFA-1 : integrin aLb2 Ligand binding inserted domain (I domain) Naïve T : low affinity (LA) domain GPCR stimulated T : intermediate affinity (IA) Fully activated T : high affinity (HA) MIDAS : metal ion-dependent adhesion site Mg2+, Ca2+ : favor the defalut LA state of integrin Mn2+ : induce the HA conformation Hypo. Isoleucine-306 help maintain the mouse LFA-1 I domain in the LA state >> Ile306 into Ala

Figure 1. Generation of αL-I306A mice Mg2+ Integrin alpha L Ligand binding domain : I domain KI : knock in mouse

Transwell migration assay chemokine

>> integrin a4를 통한 migration에는 문제가 없음 Figure 2. Adhesive interactions of WT and αL-I306A (KI) cells with ICAM-1 in vitro Binding of soluble dimeric ICAM-1-Fc to splenocyte a-human Ig-FITC aL-I306a : IA/HA CCL21 : integrin a4 >> integrin a4를 통한 migration에는 문제가 없음 ICAM-1이 있어도 KI의 migration이 억제됨

Figure 2. Adhesive interactions of WT and αL-I306A (KI) cells with ICAM-1 in vitro 2D tracking ICMA-1/CXCL12 Actin Integrin aL

aL-I306A는 intermediate affinity상태로 발현되고 있고 환경에 의해 high affinity 상태로 유도될 수 있다 aL-I306A, KI는 움직임에 있어 WT에 비해 속도나 이동거리 등의 감소가 보인다. 이것은 uropod의 detachment의 결함에 원인이 있을 것이다

Figure 3. In vivo homing of T lymphocytes SP: spleen PBL: peripheral blood lymphocytes PLN: peripheral lymph node MLN: mesenteric lymph node PP: Peyer’s patch SI: small intestine LI: large intestine BM: bone marrow LIV: liver LUN: lung WT-CFSE, KI-CMTMR labeling >> 같은 수의 cell을 섞음 >> C57BL/6J-CD45.1+ mice에 injection : iv LN에서 KI의 homing 감소 > constitutive IA LFA-1 때문에 HEV에 arrest 하는 능력이 감소된 것일까

Epi-fluorescence-IVM imaging of inguinal LN LN vanule tree : I-V I, II : medullary-collecting HEVs III-V : cortical HEVs Only high order venules simultaneously express ligands for L-selectin ligands for LFA-1 and CCR7-activating chemokines >> LFA-1 activation >> bulk of naïve lymphocyte traffic into peripheral LN

>> HEV에 arrest 되는 능력이 사라져서 homing이 줄어든 것은 아니다 Figure 4. Adhesive interactions of T cells with lymph node vessels studied by epifluorescent IVM on inguinal LNs M17/4: aL blocking Ab >> HEV에 arrest 되는 능력이 사라져서 homing이 줄어든 것은 아니다

Figure 5. MP-IVM investigations on T cell interactions with HEV actin promoter driven GFP-transgenic mice BM <- WT or KI T cell popliteal LN HEV에 붙어있는 cell > “frustrated” movement PTX : pertussis toxin : GPCR의 Gi를 inhibition

Figure 5. MP-IVM investigations on T cell interactions with HEV KI가 WT보다 더 짧은 거리를 느린 속도로 움직인다 HEV의 적은 면적만을 scanning한다 TEM이 훨씬 오래 걸린다 crawling, diapedesis와 homing은 어떤 관계인가?

Figure 6. Impact of the delayed LFA-1 inhibition on in vivo T cell homing WT-CFSE, KI-CMTMR labeling >> 같은 수의 cell을 섞음 >> C57BL/6J-CD45.1+ mice에 injection LN으로 들어올 수 있도록 1시간 동안 둠 > LFA-1 blocking Ab injection > Lymphoid organ 관찰 Delayed LFA-1 inhibition : HEV로의 T cell accumulation을 막고 이미 붙어있는 cell 또한 떨어뜨릴 수 있다 > Perturbed intravascular crawling과 diapedesis -> homing과 extravasation에 systemic level에서 영향을 미칠 수 있다

Figure 7. MP-IVM investigations on T cell interstitial motilities LN interstitial environment에서의 migration은 어떠한가 WT, KI 둘다 빠른 움직임을 보임 KI의 displacement는 조금 낮게 나타나고 turning angle은 높게 나타남 >> LFA-1이 interstitial T cell motility에 관계된 ligand binding에는 최소한의 정도로 연관되어있을 것

Figure 7. MP-IVM investigations on T cell interstitial motilities T cell transfer >> 24hr 후 mAb M17/4 넣어줌 >> PLN 꺼내어 FITC-M17-4로 staining >> blocking이 motility 변화에 차이를 주지 않음

> HEV 보다 낮은 ICAM-1, ICAM-2의 density FRC: fibroblastic reticulum cell > HEV 보다 낮은 ICAM-1, ICAM-2의 density > ligand engagement를 막기 때문

Summary LFA-1 upregulation is required for T cell arrest on the luminal surface of HEVs LFA-1 affinity needs to be properly down-regulated to facilitate intraluminal crawling and diapedesis LFA-1 engagement is dispensable for T cell migration in the interstitial space presumably due to the absence of shear and/or reduced LFA-1 ligand expression, failing to convert the IA LFA-1 to the HA form

Further Study Migration에 있어 CD99의 역할 In vivo imaging CD99 KO GFP mouse <- T cell vs. GFP mouse <-T cell LN or spleen에서의 움직임 실험적 기법에 있어 irradiation으로 HEV 주변의 cell을 제거함으로써 IVM을 효율적으로 활용한 점 migration assay in vitro에서 CD99 WT/KO T cell의 움직임 CD99 KO cell이 GVHD를 일으키는 것 Anti-CD99 Ab가 diapepesis를 막기보다는 migration을 막았다 (Muller WA, 2002) CD99 KO은 migration에 오히려 유리할 수 있지 않을까 CD99가 특정 부위에서만 migration에 관계할까 CD99 KO x GFP <- CD99 WT/KO T cell mix >> 장기 별 WT/KO T cell의 % 비교