A Distinct Subset of Self-Renewing Human Memory CD8+ T Cells Survives Cytotoxic Chemotherapy Lee, Hye-Yeong

Introduction A hallmark of adaptive immunity to pathogens is the establishment of long- lived memory T cells The durability of T cell memory under normal homeostasis is due in part to slow cell division mediated by cytokines such as IL-15 CMV(cytomegalovirus), EBV(Epstein Barr virus) 에 감염이 되지 않기 위해서 는 이들에 specific 한 CD8+ T cell 이 있어야 한다. 그런데 AML(acute myeloid leukemia) 로 chemotherapy 를 반복적으로 받는 환자들은 lymphocytopenia 상태임에도 불구하고 CMV 나 EBV 에 감염되지 않 는다. –chemotherapy 동안에도 virus-specific memory T cell 이 살아남아 있음을 시사한다. memory T cell 의 subset –Tcm(central memory T cell) : CM Tem(effector memory T cell) : EM –heterogeneity : 서로 다른 역할을 할 memory cell 집단으로서의 가능성

Introduction 어떤 mechanism 으로 살아남는가 ? HSCs(Hematopoietic Stem Cells) 이 chemotherapy 에 살아남는 mechanism –cell quiescence –over expression of ATP-binding cassette (ABC)-superfamily ABC protein –multi-drug efflux protein –drug resistance Why stem cell? –self renewal –differentiation ATP-binding cassette

exp. healthy man  CD8+ T cells AML patient  chemotherapy  after recovery  CD8+ T cells

Figure 1. CD8+ Virus-Specific Memory T Cells Persist through Profound Chemotherapy-Induced Lymphocytopenia patient PBMC brief in vitro co-culture w/ DC pulsed with CMV, EBV, influenza-derived peptides(CEF) IFN-  positive >> chemotherapy 에 resistant 한 memory T cell subset 이 실제로 존재하는가 ?

exclude CD4+ T cells,  T cells, NKT cells, NK cells >> CD4/TcR  /Va24/CD16 negative exclude naïve T >> CD95 positive distinct Tcm, Tem >> CD62L hi or lo

Figure 2. Subsets of Tcm and Tem Cells Rapidly Efflux Rh123 and DiOC2(3) Vbl : vinblastine : ABCB1, ABCC1 specific CyA : cyclosporine : ABCB1 PK11195 : ABCB1 Rh123 : fluorescent, substrate of ABC protein

CM EM 중에서도 Rh123 의 efflux 가 높은 cell type 을 구별할 수 있는 marker? >> memory cell 과 HSC 의 microarray data 를 기초로 찾아냄 IL-18Ra, CD161 이 Rh123 efflux 가 많 은 cell 에서 많이 expression 됨을 찾아 냄 >> CM hi EM hi 로 명명 Rh123 efflux 적은 group : Cm lo EM lo

CD161+ IL-18Ra+ 에서 Rh123 efflux 확인

specificity of the efflux pathway? DiOC 2 (3) : high specificity for ABCB1, minimal specificity for ABCG2, no specificity for ABCC1

Subsets of Tcm and Tem were identified by high expression of IL-18Ra and CD161 rapidly efflux Rh123 through the ABCB1 multidrug transporter

Figure 3. CM hi and EM hi Efflux Daunorubicin and Are Protected from Cytotoxicity during Exposure to Daunorubicin CM hi EM hi 는 다른 drug 에 대해서도 resistance 를 보이는가 ? w/ PK11195 w/o PK11195

Figure 4. CM hi and EM hi Are Absent from Cord Blood and Exhibit a Memory Phenotype CD8+ CD161hi

Figure 4. CM hi and EM hi Are Absent from Cord Blood and Exhibit a Memory Phenotype surface molecule expression : 두 그룹이 homogeneous CMhi EMhi : CD45R0+ CD127 hi CD28 hi CD27+ CD122+ CD45RA lo perforin lo granzyme A lo

CMhi EMhi T cells 이 chemotherapy 이후 antiviral immunity 를 recovery 시 키는가 ? 만약 그렇다면, 이들 subset 으로부터 polyclonal virus-specific T cells 이 생 겨야 한다.

Figure 5. CM hi and EM hi Cells Proliferate to Anti-CD3 and Costimulation and Harbor Virus-Specific CTLs CMhi EMhi Proliferation assay CMhi, EMhi < culture for 3 days with plate-bound anti- CD3 w/ or w/o IL-18 or aCD28 >> thymidine incorporation OKT3: CD3 monoclonal Ab tetramer staining CMhi, EMhi < stimulated with DC pulsed with Flu or EBV culture for 8 days with IL-2, 7, 15

Figure 6. CM hi and EM hi Are bcl-2 hi, bclxL hi, and Quiescent in Healthy Individuals, but Proliferate and Are Enriched in Lymphocytopenia drug 에 대한 susceptibility 를 결정하는 것은 efflux 말고도 다른 요인이 존재할 것이다. >> anti-apoptotic molecule (bcl-2, bcl-xL) expression 정도 확인

Figure 6. CM hi and EM hi Are bcl-2 hi, bclxL hi, and Quiescent in Healthy Individuals, but Proliferate and Are Enriched in Lymphocytopenia >> resistace to chemotherapy drug efflux higher levels of antiapoptotic molecules lower cell-cycle fraction >> IL-7, IL-15 같은 homeostatic cytokines 에 대해서는 반응하여 proliferation 한다.

Figure 6. CM hi and EM hi Are bcl-2 hi, bclxL hi, and Quiescent in Healthy Individuals, but Proliferate and Are Enriched in Lymphocytopenia how about in vivo situation? AML patient  chemotherapy  PBMC : CD8+ T cells >> percent of Ki67+ >> fold increase between healthy individuals and AML patient proportion in AML patient

Figure 7. CM hi and EM hi Differentiate into CD161lo Subsets after Antigen Stimulation CMhi EMhi 즉, CD161hi 가 증가하는 것이 CD161lo cell 이 CD161hi phenotype 을 획득한 것은 아닐까 ? CFSE labeling culture with IL-7 and IL-15 day11 CMhi >> CD62Llo phenotype : EMhi 로의 conversion

Figure 7. CM hi and EM hi Differentiate into CD161lo Subsets after Antigen Stimulation acquisition of CD161 ne-int phenotype CD161 hi CD161 lo plate-bound aCD3, aCD28, IL-7 + Rh123 culture with IL-7 and IL-15 >stimulation with fibroblast infected with CMV CD161hi memory CD8+ T proliferate to homeostatic cytokines respond to antigen differentiate into CD161lo RV798 : CMV peptide

Summary human memory CD8+ T cells 의 subset 인 Tcm 과 Tem 에서 hematopoietic stem cell 과 drug resistance mechanism 을 공유하는 compartment 를 정의 하 였다. –ABCB1 mediated drug efflux capacity –high expression of c-Kit, IL-18Ra, CD161, bcl-2, CD28, CD127 and bcl- xL –low proportion that are Ki-67+ : cells are quiescent in normal state after antigen stimulation –differentiate into CD161lo population –loss of drug efflux capacity

Discussion