Therapeutic activity of GARP:TGF-b1 blockade in murine primary myelofibrosis

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by clonal expansion of myeloid cells, notably megakaryocytes (MKs), and aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-b1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-b1, only a few activate the cytokine via cell type-specific mechanisms. The cellular source of the active TGF-b1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-b1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found increased expression of GARP in BM and spleen of mice undergoing PMF and tested the therapeutic potential of a monoclonal antibody that blocks TGF-b1 activation by GARP-expressing cells. GARP:TGF-b1 blockade reduced not only fibrosis, but also clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-b1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-g signals in the spleen, were lost upon CD8 T cell depletion. Our results suggest that selective blockade of TGF-b1 activation by GARP-expressing Tregs increase a CD8 T cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms. RNAseq of spleens collected from non-treated MPLW508A mice on days 13 and 20 were compared to establish signatures of PMF progression. RNAseq of spleens collected on day 34 from MPLW508A mice responding to 58A2 were compared to non-treated controls to analyze effects of treatment with 58A2.

原发性骨髓纤维化（Primary myelofibrosis, PMF）是一类以髓系细胞（尤其是巨核细胞megakaryocytes, MKs）克隆性扩增、异常细胞因子生成，进而引发骨髓（bone marrow, BM）纤维化与功能不全为特征的骨髓增殖性肿瘤。当前临床治疗选择十分有限。转化生长因子-β1（TGF-b1）作为一种促纤维化兼免疫抑制性细胞因子，参与了PMF的发病进程。尽管所有细胞类型均可分泌无活性的潜伏型TGF-b1，但仅少数细胞能通过细胞类型特异性的机制激活该细胞因子。PMF中相关活性TGF-b1的细胞来源目前仍未明确。跨膜蛋白GARP可在调节性T淋巴细胞（regulatory T lymphocytes, Tregs）、巨核细胞或血小板表面结合并激活潜伏型TGF-b1。本研究中，我们在PMF模型小鼠的骨髓与脾脏中检测到GARP表达水平显著上调，并针对一款可阻断表达GARP的细胞激活TGF-b1的单克隆抗体开展了治疗潜力验证实验。阻断GARP:TGF-b1通路不仅能够减轻纤维化程度，还可抑制转化细胞的克隆性扩增。通过分别使用调节性T细胞或巨核细胞中Garp基因敲除的小鼠模型，我们证实，GARP:TGF-b1阻断疗法在PMF模型中的治疗效应依赖于靶向调节性T细胞上的GARP。这类伴随脾脏中干扰素-γ（IFN-g）信号增强的治疗效果，会在CD8 T细胞耗竭后完全消失。本研究结果提示，选择性阻断表达GARP的调节性T细胞对TGF-b1的激活，能够增强CD8 T细胞介导的免疫反应，从而限制转化细胞的扩增，为骨髓增殖性肿瘤患者的治疗提供了一种可供后续验证的全新策略。我们对未接受治疗的MPLW508A小鼠在第13天和第20天采集的脾脏样本进行RNA测序（RNAseq），通过比较两组转录组数据以明确PMF的疾病进展特征谱；将第34天采集的、经58A2治疗后产生应答的MPLW508A小鼠脾脏RNA测序数据与未接受治疗的对照组进行比对，以此分析58A2的治疗效应。