Project 2: Regulatory T Cells Generated by Gene Transfer to Prevent GVHD

项目2：项目总结 异基因造血干细胞移植(allo-hct)的主要并发症是急性移植物抗宿主 疾病(AGvHD)。AGvHD是一种免疫调节的疾病，它在很大程度上促进了移植- 相关发病率和死亡率。AGvHD的总体发病率在35%至50%之间，这取决于 供受者的组织相容性不同，死亡率约为50%。T调节细胞 (Tregs)，一种表型多样的淋巴细胞亚群，促进和维持免疫耐受， 作为一种治疗aGvHD的细胞疗法已被广泛研究。临床前试验表明， 存在产生IL-10的T调节型1(TR1)细胞或叉头盒P3(FOXP3)T调节性细胞 (Foxp3+Tregs)与异基因造血干细胞移植后aGvHD调节和移植耐受增强相关。T- All10细胞是一种含有供体来源的TR1细胞的细胞产品，该细胞以宿主来源的致耐受树突状细胞为基础 细胞(DC-10)诱导免疫耐受。我们已获得美国食品和药物管理局批准(IND#17292)进行一期临床试验 相亲或不相合的儿童和青壮年患者T-Al10细胞的剂量递增 HCT治疗恶性血液病，2例患者治疗成功。的主要终结点 第一阶段试验是1)在我们的GMP工厂生产T-all10产品的可行性和2)T-all10产品的安全性 T-all10产品的管理。目标1是完成这项试验，包括免疫学评估 T-all10产物的表达及对HCT患者输液持久性的纵向评估 TR1细胞的TCR克隆分析和免疫重建。含有T-all10细胞的TR1提供 巨大的希望，但这是一种混合细胞产品，也含有T效应细胞，可能会减少它们的 有效性。尽管多克隆FOXP3+Treg在提高移植后耐受性方面显示出一定的效果。 移植后，输注的细胞在体内存活受限，纯品FOXP3+Tregs的分离比较困难。 为此，我们建立了携带慢病毒载体(LV)基因的TR1细胞和FOXP3+Treg细胞。 转移到CD4+T效应细胞。LV介导的FOXP3基因转移到CD4+T细胞中 “FOXP3+Treg样”(CD4FOXP3)细胞。同样，可通过表达LV-IL-10获得类TR1(CD4IL-10)细胞 在人类CD4+T效应细胞中。在初步实验中，我们已经证明了第三方多克隆 CD4IL-10和CD4FOXP3T细胞可抑制免疫缺陷小鼠人源化模型GvHD。在AIMS 2中 3我们将比较自体多克隆和同种异体抗原特异性CD4IL-10和CD4FOXP3在预防 AGvHD及其对造血干细胞植入、免疫重建、耐受和GVL的影响 临床前研究的活跃性。这些研究将为开发未来的治疗方法提供临床前数据， 将改善接受allo-HCT的患者的结局。

Project 2: Project Summary A major complication of allogeneic hematopoietic stem cell transplantation (allo-HCT) is acute graft-versus-host disease (aGvHD). aGvHD is an immunologically mediated disease, that contributes substantially to transplant- related morbidity and mortality. The overall incidence of aGvHD is between 35% and 50% depending upon donor-recipient histocompatibility differences and carries approximately a 50% mortality rate. T regulatory cells (Tregs), a phenotypically diverse subset of lymphocytes that promote and maintain immunological tolerance, have been extensively researched as a cellular therapy for aGvHD. Preclinical trials have demonstrated that the presence of IL-10-producing T regulatory type 1 (Tr1) cells or the forkhead box P3 (FOXP3) T regulatory cells (FOXP3+Tregs) correlates with aGvHD modulation and enhanced transplantation tolerance after allo-HCT. T- allo10 cells are a cell product containing donor-derived Tr1 cells primed with host-derived tolerogenic dendritic cells (DC-10) to induce immune tolerance. We have FDA approval (IND# 17292 ) for a Phase 1 clinical trial of escalating doses of T-allo10 cells in pediatric and young adult patients receiving mismatched related or unrelated HCT for hematologic malignancies, and 2 patients have been successfully treated. The primary endpoints of the Phase 1 trial are 1) the feasibility of producing the T-allo10 product in our GMP facility and 2) the safety of the administration of the T-allo10 product. Aim 1 is the completion of this trial including the immunological evaluation of the T-allo10 product and the longitudinal assessment of the HCT patients for the persistence of the infused Tr1 cells using TCR clonotypic analyses and the immune reconstitution. Tr1 containing T-allo10 cells offer enormous promise, but are a mixed cell product also containing T effector cells, that potentially reduce their effectiveness. Although polyclonal FOXP3+Tregs have shown some efficacy in enhancing tolerance post- transplant, the infused cells have limited in vivo survival, and the isolation of pure FOXP3+Tregs is difficult. Therefore, we have developed “second-generation” Tr1 cells and FOXP3+Tregs by lentiviral-vector (LV) gene transfer into CD4+ T effector cells. LV-mediated gene transfer of FOXP3 into CD4+ T cells creates “FOXP3+Treg-like” (CD4FOXP3) cells. Similarly, “Tr1-like” (CD4IL-10) cells can be obtained via LV-IL-10 expression in human CD4+ T effector cells. In preliminary experiments, we have demonstrated that 3rd party polyclonal CD4IL-10 and CD4FOXP3 T cells can suppress GvHD in a humanized model of immune deficient mice. In Aims 2 and 3 we will compare autologous polyclonal and alloAg-specific CD4IL-10 and CD4FOXP3 for their prevention of aGvHD, their impact on hematopoietic stem cell (HSC) engraftment, immune reconstitution, tolerance and GvL activity in preclinical studies. These studies will provide preclinical data toward developing future therapies, that will improve the outcomes of patients receiving allo-HCT.