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

在AIM 1下进行的实验表明，即使相对轻微的GVHD也会降低对疫苗接种的数量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，布拉德，2009)，并证明了如果骨髓移植要优化为针对恶性肿瘤的免疫治疗方法的平台，预防移植物抗宿主病的重要性。在目标2下，我们已经确定抑制干扰素伽马信号可以防止移植物抗宿主病的发展(Capitini等人，血液2009)。鉴于干扰素在GVHD中的重要性，干扰T细胞上的干扰素-伽马信号，GVHD严重程度的降低并不令人惊讶，但这种方法会导致免疫缺陷。这些研究中的新发现是，选择性丢失骨髓来源的非T细胞上的干扰素伽马受体也可以防止由干扰素伽马信号完整的T细胞介导的GVHD，并在保留对疫苗的定性和功能反应的情况下做到这一点。我们还建立了体外光分离，这是一种目前在临床上用于治疗GVHD的方法，也通过调节DC人群中IL-10的产生来通过保存疫苗反应来预防GVHD(Capitini等人，血液和骨髓移植生物学，2011年)。最后，我们已经证明，疫苗应答的减少是由于CD8细胞凋亡增加以及CD4和CD8T细胞的增殖减少(已接受，《免疫学杂志》)。接下来，我们研究了干扰素-伽马途径的其他成分是否可以在捐赠者的骨髓中靶向，以预防移植物抗宿主病。利用干扰素-γ信号转导所必需的转录因子STAT1的骨髓缺陷，我们已经证实，在骨髓来源的细胞中干扰这一途径可以在保留免疫功能的情况下预防GVHD。为了确定相关的骨髓源性细胞群，我们选择性地以STAT1为靶标，方法是将STAT1基因(从Lothar Hennighausen博士那里获得)培养成在非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重组酶的小鼠。在接受这些捐赠者的骨髓的受者中，STAT1基因(因此，干扰素伽马信号)可以在选择性细胞群中被去除。在所有这些细胞群中选择性缺失STAT1不足以预防移植物抗宿主病。有趣的是，对STAT1基因缺陷的异基因骨髓受者DC重建的进一步评估显示了扩大的浆细胞样树突状细胞(PDC)群体。此外，我们已经证实，在迄今为止产生的所有Cre Flosed STAT1小鼠品系中，STAT1保持完整。耗竭PDCA1+(pDCs上的标志物)已经证实对GVHD的抵抗是由缺乏STAT1的pDCs介导的。基于这些发现，我们已经开始分析扩增的pDC介导GVHD抵抗的特征。初步研究表明，CD9阴性pDC的频率增加，据报道是耐受性的。此外，在STAT1缺陷的骨髓受者的pDC中，与髓系细胞抑制表型相关的基因S100A8和S100A9的表达增加。使用S100A8/S100A9缺陷小鼠(通过Mackall实验室从Dimitri Gabrilovich博士那里获得)，我们证实了供者骨髓中这些基因的丢失会增加GVHD的严重程度。最后，我们证实了STAT3在移植STAT1缺陷骨髓的小鼠pDC中的表达增加，这表明这种在其他免疫耐受系统中被关联的转录因子正在发挥作用。根据Aim3，当已经证明STAT1缺陷骨髓的接受者对GVHD的抵抗力保持了对实体瘤和白血病的T细胞免疫。与在南联盟实验室发起这一项目的克里斯蒂安·卡皮蒂尼博士合作，我们正在开发针对异基因骨髓移植后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.