Targeting dendritic cells for selective modulation of Graft-versus-Host Disease

在aim 1下进行的实验表明，即使是相对轻微的GVHD也可以减少对疫苗接种的定量T细胞免疫反应和对表达疫苗靶向抗原的肿瘤的功能性免疫反应。这项工作已经发表（Capitini等人，Blood, 2009），并证明了如果要将BMT优化为针对恶性肿瘤的免疫治疗方法的平台，预防GVHD的重要性。在目标2下，我们已经确定抑制干扰素γ信号可以阻止GVHD的发展（Capitini et al, Blood 2009）。干扰素γ信号在GVHD中具有重要作用，破坏T细胞上的干扰素γ信号可以降低GVHD的严重程度，这并不奇怪，但这种方法会导致免疫缺陷。这些研究的新发现是，骨髓源性非T细胞上干扰素γ受体的选择性丧失也阻止了由干扰素γ信号完整的T细胞介导的GVHD，并保留了对疫苗的定性和功能性反应。我们还建立了体外光合作用，这是目前用于临床治疗GVHD的一种方式，也通过调节DC人群中IL-10的产生来保留疫苗反应，从而预防GVHD （Capitini等人，《血液和骨髓移植生物学》，2011）。最后，我们已经证明，疫苗反应减弱是由于CD8细胞凋亡增加和CD4和CD8 T细胞增殖减少（已被接受，《免疫学杂志》）。接下来，我们研究干扰素γ通路的其他成分是否可以靶向供体骨髓来预防GVHD。利用缺乏STAT1（干扰素γ信号所必需的转录因子）的骨髓，我们已经证实，在骨髓源性细胞中干扰这一途径可以在保留免疫能力的情况下预防GVHD。为了鉴定相关的骨髓源性细胞群，我们通过产生带有固定STAT1基因（来自Lothar Hennighausen博士）的小鼠，选择性地靶向STAT1，这些小鼠在非t细胞启动子(CD11c （DC表达）、溶菌酶（在所有吞噬细胞上）和CD19 （B细胞表达）下表达Cre重组酶。在这些供者骨髓的接受者中，STAT1基因（因此，干扰素γ信号）可以在选择性细胞群中被切除。所有这些细胞群中STAT1的选择性缺失不足以预防GVHD。有趣的是，对同种异体STAT1缺失骨髓受体DC重建的进一步评估显示浆细胞样DC （pDC）群体扩大。此外，我们已经证实STAT1在迄今为止生成的所有Cre修饰的STAT1小鼠品系中保持完整。耗竭PDCA1+ （pDCs上的一种标记物）证实GVHD的耐药性是由stat1缺失的pDCs介导的。基于这些发现，我们开始分析扩展pDCs介导GVHD抗性的特性。初步研究表明，据报道，CD9阴性的pDCs具有耐受性，其频率有所增加。此外，骨髓细胞中与抑制表型相关的基因S100A8和S100A9的表达在STAT1缺陷骨髓受体的pDCs中增加。使用S100A8/S100A9缺陷小鼠（由Dimitri Gabrilovich博士通过Mackall实验室获得），我们证实供体骨髓中这些基因的缺失会增加GVHD的严重程度。最后，我们证实了STAT3在移植了STAT1缺陷骨髓的小鼠中恢复的pDCs中表达增加，这表明这种转录因子在其他系统中与免疫耐受相关，正在发挥作用。在Aim3下，研究人员已经证明stat1缺陷骨髓受体对GVHD的抵抗保留了T细胞对实体瘤和白血病的免疫。在与Fry实验室发起该项目的Christian Capitini博士合作中，我们正在开发同种异体BMT后靶向STAT1的方法。

The experiments conducted under aim 1 have demonstrated that even relatively mild GVHD can diminish quantitative T cell immune responses to vaccination and functional immune responses to tumors expressing vaccine-targeted antigens. This work has been published (Capitini et al, Blood, 2009) and demonstrated the importance of preventing GVHD if BMT is to be optimized as a platform for immunotherapeutic approaches targeting malignancy. Under aim 2, we have established that inhibition of interferon gamma signaling can prevent the development of GVHD (Capitini et al, Blood 2009). Reduction in GVHD severity with disruption of interferon gamma signaling on T cells was not surprising given the known importance of this cytokine in GVHD but this approach results in immunodeficiency. The novel finding in these studies was that selective loss of interferon gamma receptor on bone marrow-derived non-T cells also prevented GVHD mediated by T cells with intact interferon gamma signaling and did so with preservation of qualitative and functional responses to vaccines. We have also established the extracorporeal photopheresis, a modality currently being used in the clinic to treat GVHD, also prevented GVHD with preserved vaccine response via modulation of IL-10 production in DC populations (Capitini et al, Biology of Blood and Marrow Transplantation, 2011). Finally, we have shown that diminished vaccine reponses is due to increased CD8 apoptosis and reduced proliferation of both CD4 and CD8 T cells (accepted, Journal of Immunology).We next studied whether other components of the interferon gamma pathway could be targeted in donor bone marrow to prevent GVHD. Using bone marrow deficient in STAT1, a transcription factor necessary for interferon gamma signaling, we have confirmed that interference with this pathway in bone marrow-derived cells can prevent GVHD with preserved immune competence. To identify the relevant bone-marrow-derived cell population, we have selectively targeted STAT1 by generating mice with a floxed STAT1 gene (obtained from Dr. Lothar Hennighausen) that express the Cre recombinase under non-T cell promoters (CD11c (DC expression), lysozyme (on all phagocytic cells), and CD19 (B cell expression). In recipients of bone marrow from these donors, the STAT1 gene (and, thus, interferon gamma signaling) can be ablated in selective cell populations. Selective loss of STAT1 in all of these cell populations was not sufficient to prevent GVHD. Interestingly, further assessment of DC reconstitution in recipients of allogeneic STAT1 deficient bone marrow demonstrated expanded plasmacytoid DC (pDC) populations. Further more, we have confirmed that STAT1 remained intact in all of the Cre floxed STAT1 mouse strains generated thus far. Depletion PDCA1+ (a marker on pDCs) has confirmed that resistance to GVHD is mediated by STAT1-deificient pDCs. Based on these findings we have begun to analyze the characteristics of the expanded pDCs mediating GVHD resistance. Preliminary studies indicate that there is an increase in the frequency of CD9 negative pDCs, reported to be tolerogenic. In addition, expression of S100A8 and S100A9, genes associated with a suppressive phenotype in myeloid cells, is increased in pDCs from STAT1 deficient bone marrow recipients. Using S100A8/S100A9 deficient mice (obtained from Dr. Dimitri Gabrilovich through the Mackall laboratory) we have confirmed that loss of these genes in donor bone marrow increases the severity of GVHD. Finally, we have confirmed increased expression of STAT3 in pDCs recovered from mice transplanted with STAT1 deficient bone marrow suggesting that this transcription factor that has been associated in other systems with immune tolerance is playing a role. Under Aim3, when have demonstrated that resistance to GVHD in recipients of STAT1-deficient bone marrow preserves T cell immunity to both solid tumors and leukemia. In collaboration with Dr. Christian Capitini, who initiated this project in the Fry laboratory, we are developing approaches to target STAT1 following allogeneic BMT.