HPV-Specific, Immune Suppression in Patients with RRP

描述（由申请人提供）：复发性呼吸道乳头状瘤病（RRP）是由人乳头状瘤病毒（HPV） 6和11引起的。这些人乳头瘤病毒在气道中产生良性肿瘤，每两周就需要进行手术以保持气道畅通，从而降低“生活质量”，而且它们可能变成恶性肿瘤并导致死亡。然而，控制疾病易感性和RRP变异的免疫机制尚不清楚。我们发现RRP是一种th2样疾病。细胞因子/趋化因子环境富集IL-4、IL-10，但不富集IFN-g，暴露于hpv - 11e6后，T细胞和非T细胞均表达IL-10。此外，由选择性活化巨噬细胞（aamp）和未成熟树突状细胞（DCs）表达的趋化因子CCL18在RRP血浆中升高。我们假设hpv特异性，th2样记忆和调节性t细胞（Tr1）表达IL-4和/或IL-10，使静息巨噬细胞（MP）极化成为也表达IL-10的aamp。这延续了免疫抑制细胞的循环，抑制th1样t细胞对HPV蛋白的反应。我们的长期目标是设计一种治疗性疫苗来中断这种循环，并支持HPV特异性的th1样应答。具体目标是：1)表征/定量HPV特异性t细胞，表达TH2细胞因子和IL-10响应HPV蛋白，并将其与疾病严重程度相关联。t细胞和细胞因子对E6/E2的反应，以及HPV-11 E6/E2中的t细胞表位（使用装载E6/E2肽的ⅱ类MHC四聚体绘制）将通过流式细胞术进行鉴定。2)利用流式细胞术确定原生t细胞对E6/E2的反应是否能产生Tr1细胞。我们将使用朗格汉斯细胞，E6/ E2转导的角质形成细胞（dc）作为apc来探索这种转化。3)表征E6/ e2暴露的PBMC的MP极化，并通过CCL17、CCL18和一氧化氮的表达来鉴定aamp与经典激活的MPs。4)确定暴露于E6/ e2的apc和TH2细胞是否能抑制同种异体反应性t细胞杀伤。我们将使PBMC暴露于E6/E2和同种异体抗原，并在MLR中检测同种异体反应性，在CML试验中检测t细胞杀伤。我们将确定加入IFN-g或抗il -10是否可以阻止这种抑制。5)确定IL-10启动子单核苷酸多态性（snp）是否预测疾病严重程度。我们将确定并比较重度疾病与轻度/中度疾病的IL-10 snp。这些研究将识别对HPV反应不适当的免疫系统细胞，帮助开发治疗性疫苗，以及治疗RRP患者的新策略。

DESCRIPTION (provided by applicant): Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus (HPV) 6 and 11. These HPVs generate benign tumors in the airway, reduce "quality of life" by requiring surgery as often as every two weeks to keep the airway open, and they can become malignant and cause mortality. However the immunologic mechanism(s) that governs disease predisposition and variation in RRP remains unknown. We have found RRP to be a TH2-like disease. The cytokine/chemokine milieu is enriched with IL-4, IL-10, but not IFN-g, and both T and non-T-cells express IL-10 after HPV-11 E6 exposure. In addition, the chemokine CCL18, expressed by alternatively activated macrophages (AAMPs) and immature dendritic cells (DCs), is increased in RRP plasma. We hypothesize that HPV-specific, TH2-like memory and regulatory T-cells (Tr1) express IL-4 and/or IL-10, that polarize resting macrophages (MP) to become AAMPs which also express IL- 10. This perpetuates a cycle of immunosuppressive cells that inhibit TH1-like T-cell responses to HPV proteins. Our long-term goal is to design a therapeutic vaccine to interrupt this cycle and support HPV- specific,TH1-like responses to HPV. Specific aims are: 1) Characterize/quantitate HPV-specific, T-cells that express TH2 cytokines and IL-10 in response to HPV proteins, and correlate this with disease severity. T-cell and cytokine responses to E6/E2, and T-cell epitopes within HPV-11 E6/ E2 (mapped using E6/E2 peptide- loaded, class II MHC tetramers), will be identified by flow cytometry. 2) Determine if Tr1 cells can be generated from naTve T-cells in response to E6/E2 using flow cytometry. We will use Langerhans cells, E6/ E2 transduced keratinocytes, or DCs, as APCs to explore this conversion. 3) Characterize MP polarization in E6/E2-exposed PBMC, and identify AAMPs vs. classically activated MPs by CCL17, CCL18, and nitric oxide expression. 4) Determine if E6/E2-exposed APCs and TH2 cells can inhibit alloreactive T-cell killing. We will expose PBMC to E6/E2 and alloantigens, and test for alloreactivity in a MLR, and T-cell killing in a CML assay. We will determine if adding IFN-g or anti-IL-10 prevents this inhibition. 5) Determine if IL-10 promoter single nucleotide polymorphisms (SNPs) predict disease severity. We will identify and compare IL-10 SNPs in severe, vs. mild/moderate disease. These studies will identify immune system cells that inappropriately respond to HPV, help develop a therapeutic vaccine, and new strategies to treat patients with RRP.