预防和治疗移植物抗宿主病(GVHD)的新策略 Simrit Parmar, MD Stem Cell Transplant & Cellular Therapy BTG2013, Hong Kong
急性 GVHD的危险因素 HLA 配型不合 年龄增加 供受者性别不同 所患疾病类型和状态 放疗数量和移植预处理方案的强度 氨甲蝶林和环孢素或他克莫司的剂量
急性 GVHD: 病理生理 1. 受者状况 3. 细胞和 炎症因子效应器 2. 供者 T 性别激活
急性 GVHD 急性 GVHD 主要发生于植活前后. 以往错误定义为发生于移植后100天内的GVHD. 3个主要受累器官: 皮肤: 皮疹 胃肠系统: 恶心 / 呕吐和腹泻 肝功异常: 典型的是於胆 (黄疸). 同胞移植发生率为 9-50%. Vigorito et al. Blood 2009
急性 GVHD: 生存与复发 Grade 0 acute GVHD — hazard ratio (HR) for TRM: 1.0 T H Grade 0 acute GVHD — hazard ratio (HR) for TRM: 1.0 Grade I — HR 1.5 (95% CI 1.2-2.0) Grade II — HR 2.5 (95% CI 2.0-3.1) Grade III — HR 5.8 (95% CI 4.4-7.5) Grade IV — HR 14.7 (95% CI 11-20) Grade 0 acute GVHD — hazard ratio (HR) for relapse 1.0 Grade I — HR 0.94 (95% CI 0.8-1.2) Grade II — HR 0.60 (95% CI 0.5-0.8) Grade III — HR 0.48 (95% CI 0.3-0.8) Grade IV — HR 0.14 (95% CI 0.02-0.99) R E L A P S E
“你想接受小剂量 vidaza 或全身皮肤脱落?” “很好，否则我将送你去做移植” “我告诉你，你做完后，就会带上尿布” “你想接受小剂量 vidaza 或全身皮肤脱落?”
GVHD 的预防
“没有免费午餐” 原则 GVHD GVHD 复发 排斥 延迟免疫重建
HCT 中的免疫功能 临床医学中免疫功能异常很多见 疾病控制的主要机制是依赖 GVT 反应, 然而异基因移植中的 GVHD是主要障碍
GVHD 在2个时代的风险 Gooley et al. N. Engl. J Med 363:2091, 2010
Absolute light emission 体内供者细胞光发射追踪 CD4+ CD8+ B220+ NK1.1+ Gr-1/Mac-1+ 2x105 cells/well Absolute light emission 0.00 0.05 0.10 0.15 Luciferase 2A eGFP bAct luc+ reporter mouse 异基因 HCT B T M BM Bone Marrow Splenocytes FVB/N WT luc+ Balb/c H-2q/Thy1.1H-2d/Thy1.2
急性移植物抗宿主病的发生 Beilhack, A. et al. Blood. 2005. 106:1113
急性 GVHD 的演变
预防 GVHD 的方法 药物 移植物来源 T 细胞去除 免疫调节 CNI/MTX CNI/MTX vs Rapa/MTX BM vs PBPC MRD vs URD vs UCB T 细胞去除 CD34 选择 ATG, Campath 免疫调节
免疫功能的调节 对健康和疾病是最重要的 免疫反应的划分 细胞因子 调节T 细胞 (Treg, NK-T, iTreg, others) CD4+ T Cell Subsets 调节 反应
CD4+CD25+ 调节 T 细胞 调节免疫反应的主要细胞群 表达转录因子FoxP3 混合淋巴反应(MLR)中，细胞接触依赖的异体反应抑制 动物模型中，预防器官特异的自身免疫疾病 (如 IBD, 糖尿病) 体外，IL-10和 TGF- 涉及介导抑制效应
调节 T 细胞 Treg 频率随着aGVHD的严重性而降低. aGVHD 发生时 Treg 频率预示疗效. 伴有aGVHD的异基因 HCT 受者 的 Treg 频率低于无aGVHD者的40%. Treg 频率随着aGVHD的严重性而降低. aGVHD 发生时 Treg 频率预示疗效. Magenau et al. BBMT. 2010.
循环中 Tregs 预示OS 63% 38% Magenau et al. BBMT. 2010.
以保留GVL来控制 GVHD d5 Death from d15 GVHD Survival [%] TCD BM only, n = 14 TCD BM + Tcon, n = 15 TCD BM + Tcon + Treg n = 9 Time [d] post BMT Relative Signal Intensity Tcon BM only Tcon + Treg 500 5000 d5 d15 Death from GVHD 100 5000 1000 20000 Edinger et al. Nature Medicine 9:1144, 2003
临床移植Treg的挑战 Treg 是细胞群 缺乏临床分级药物分离和可能应用的独立标志 人体临界功能分析 调节需要
扩增CB Tregs 显示 FOXP3 去甲基化并抑制 alloMLR
3rd Party CB Tregs 防止 GVHD
通过GFP和荧光素进行体内跟踪 Treg
dorsal Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10
dorsal Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10
dorsal Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10
dorsal Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10
ventral Treg Treg+PBPC Day 3 Day 10
Treg Doses to be Studied Dose Cohort Treg Dose Dose Level 1 1 × 105 Tregs/kg Dose Level 2 5 × 105 Tregs/kg Dose Level 3 1 × 106 Tregs/kg Dose Level 4 5 × 106 Tregs/kg Dose Level 5 1 × 107 Tregs/kg
下一步: 应用Treg的过继免疫治疗 MMF+Sirolimus Infusion of Ex-vivo Expanded Tregs Day -6 -5 -4 -3 -2 Day-1 MEL BU Infusion of Ex-vivo Expanded Tregs BMT FLU 40 mg/m2 Day -8 -7 -6 -5 -4 -3 -2 Day-1 +1 +2 +3 +4 +6 BU Test Dose 32mg/m2 Rest BMT Infusion of Ex-vivo Expanded Tregs FLU 40 mg/m2 CY** 50 mg/kg
患者接受Treg 剂量 > 30x105/kg 临床结果
CD34+ cell selected graft 半相合移植计划 (Stanford) Endpoints: Chimerism Immune reconstitution Acute and chronic GVHD EFS, OS CD34+ cell selected graft CD4+CD25+ Treg CD4+/CD8+ Tcon Mel, TT, Flu + Thymoglobulin@ Day -10 +14 +16 5-10 x 106/kg 105/kg 3x105/kg 106/kg Cell Dose BB IND13923
选择 CD4+CD25+ Tregs (U. Perugia) 1st step: Depletion of CD8+/CD19+cells Immunomagnetic Selection of CD4+CD25+Cells 2ndstep: Enrichment of CD25+ cells CD25 CD127 CD4 FoxP3 Gate on CD4CD25+high Gate on CD4CD25+ Cells (x109) 1060 (540-1370) 280 (202- 390) %CD4CD25 3.0 (1.5-7.45) 92.4 (90-97.1) N° cells (x 106) 330 (221-1020) 256 (185.6-365.4) %CD4CD25high 0.3 (0.12- 0.89) 33.6 (14.4-39.6) N° cells (x 106) 36.12 (19.98 - 84) 68.6 (20.9-143) Starting fraction Final fraction . We employed a fully automated immunomagnetic procedure to select the naturally occurring CD4+cd25+ REGULATORY T CELLS . Starting from a leukophoresis product we first performed depletion of CD8+/cd19+ cells and then positive immunoselection of CD 25 +cells. The final fraction contained between 2 and 400 hundred million cells. Purity in terms of CD4CD25+was over 90% and about one third were 25 high. In the final fraction percentage of FOX P3+ cells was about 70% .WE also observed a profound depletion of CD127 expression.. Fox P3+ cells 71.9 ± 15 % 38
免疫重建模型 Recovery of CD4+ and CD8+ T cell subpopulations Spectratyping Donors In our patients the pattern of immunoreconstitution was very different to what we usually see in the haplo setting. First of all there was a rapid increase in peripheral blood T cell subpopulations. Indeed the CD4 count reached 50 x microliter at median of 34 days and rapidily rose to 200 at median of 70 days. A similar behaviour was observed for the CD8 counts. Spectratyping showed the rapid development of a wide T cell repertoire , which approached donor scores in a few months, Complexity score Months after transplant
CMV 再激活情况 Tregs Group Control Group p<0.05 Evaluable Patients Days after transplant Patients with CMV reactivation CMV reactivation episodes are significantly lower after “Treg-haplo transplant” as compared to our control group p<0.05 Control Group Days after transplant 40 40
结果 – U. of Perugia 无事件生存 12/26 (46%) 相关毒性的治疗: 急性 GVHD III-IV级 (2) 静脉阻塞病 (3) 多器官衰竭 (1) 急性 GVHD III-IV级 (2) 严重感染 (7) 复发 (AML 1) 中位追踪 18.5 个月 (范围 16.1-27.6) However the bad new is the high TRM. -The 50% transplant related mortality needs to be viewed in light of the clinical characteristics of this cohort of patients: 4 deaths were due to regimen related toxicity and 3 of these patients have been heavily pretreated with several lines of chemotherapy. The 4 patients who died of aspergillosis had fungal disease in the lung and in the brain before the transplant. On the other hand no fatal infections occurred after the first 2 months post-transplant, confirming the good recovery of immune response against pathogens One patient with AML relapsed 6 months after transplant. At median follow-up of ???? months, 46% of patients are alive and disease free. D’Ianni et al. Blood 2011
结论 GVHD 仍是异基因HCT后最重要的并发症 鼠的研究已证实，免疫调节在控制免疫反应功能异常（包括GVHD ）中起重要作用 临床移植正在进行中，并已经有了有前景的早期结果