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）由人乳头状瘤病毒（HPV）6和11引起。这些HPV在气道中产生良性肿瘤，通过需要每两周进行一次手术来保持气道开放来降低“生活质量”，并且它们可以变成恶性并导致死亡。然而，控制疾病易感性和RRP变异的免疫机制仍不清楚。我们发现RRP是一种TH 2样疾病。细胞因子/趋化因子环境富含IL-4、IL-10，但不富含IFN-g，并且在HPV-11 E6暴露后，T细胞和非T细胞均表达IL-10。此外，由交替激活的巨噬细胞（AAMP）和未成熟树突状细胞（DC）表达的趋化因子CCL 18在RRP血浆中增加。我们假设HPV特异性、TH 2样记忆和调节性T细胞（Tr 1）表达IL-4和/或IL-10，其使静息巨噬细胞（MP）变成也表达IL- 10的AAMP。这使免疫抑制细胞的周期得以延续，这些细胞抑制对HPV蛋白的TH 1样T细胞应答。我们的长期目标是设计一种治疗性疫苗来中断这一周期，并支持HPV特异性的、对HPV的TH 1样反应。具体目标是：1）表征/定量响应于HPV蛋白表达TH 2细胞因子和IL-10的HPV特异性T细胞，并将其与疾病严重程度相关联。将通过流式细胞术鉴定对E6/E2的T细胞和细胞因子应答，以及HPV-11 E6/ E2内的T细胞表位（使用E6/E2肽加载的II类MHC四聚体作图）。2)使用流式细胞术确定Tr 1细胞是否可以从应答E6/E2的naTve T细胞产生。我们将使用朗格汉斯细胞、E6/ E2转导的角质形成细胞或DC作为APC来探索这种转化。3)表征E6/E2暴露的PBMC中的MP极化，并通过CCL 17、CCL 18和一氧化氮表达鉴定AAMPs与经典活化的MP。4)确定E6/E2暴露的APC和TH 2细胞是否可以抑制同种异体反应性T细胞杀伤。我们将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检测中检测T细胞杀伤。我们将确定是否加入IFN-g或抗IL-10阻止这种抑制。5)确定IL-10启动子单核苷酸多态性（SNP）是否可预测疾病严重程度。我们将鉴定和比较IL-10 SNP在重度和轻度/中度疾病中的作用。这些研究将确定对HPV不适当反应的免疫系统细胞，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.