Supplementary Material for: Presence of Tertiary Lymphoid Organ in Nasal Inverted Papilloma Is Correlated with Eosinophil Infiltration and Local Immunoglobulin Production

<b><i>Introduction:</i></b> Nasal inverted papilloma (NIP) is a benign tumour with multiple inflammatory cell infiltration. Tertiary lymphoid organs (TLOs) support local antibody production and play important roles in airway inflammation. However, the evidence of TLOs and local immunoglobulins in NIP has not been reported yet. We investigated the presence of TLOs and immunoglobulins in NIP tissues and their association with the clinical-pathological characteristics of NIPs. <b><i>Methods:</i></b> We analyzed the occurrence and composition of TLOs and local immunoglobulins by immunohistochemistry and evaluated the lymph organogenesis associated genes and cytokines by quantitative qPCR and Luminex assays, respectively, in papilloma tissues from 84 NIP cases. <b><i>Results:</i></b> TLOs were present in 54% (45/84) of the NIP patients but not in control subjects. TLOs were composed of T cells, B cells, follicular dendritic cells, macrophages, and natural killer cells. Compared to NIP tissues without TLOs, tissues with TLOs showed significantly higher eosinophil infiltration levels (3.5-fold), elevation of lymphorganogenic genes (CXCL12, CXCL13, CCL20, CCL21, CD21L, and lymphotoxin alpha and beta), and increased Th17 (IL-21, IL-22, and GM-CSF) and Th2 (IL-5 and IL-13) cytokine production. Moreover, NIP with TLOs demonstrated a higher number of follicular T helper cells and immunoglobulin-producing plasma cells (CD138+ IgA+, CD138+ IgM+, CD138+ IgE+, and CD138+ IgG+) than those without TLOs, and these antibody-producing cells were positively correlated with the eosinophil number. <b><i>Conclusion:</i></b> The high frequency of TLOs and excess local immunoglobulin production are associated with an eosinophilic and Th2 skew microenvironment in the NIP mucosa, which would contribute to an important immunopathogenic response during NIP pathogenesis.

引言：鼻内翻性乳头状瘤（Nasal inverted papilloma, NIP）是一种伴多种炎症细胞浸润的良性肿瘤。三级淋巴器官（Tertiary lymphoid organs, TLOs）可支持局部抗体产生，并在气道炎症中发挥重要作用。然而，目前尚未见关于NIP中TLOs及局部免疫球蛋白的相关研究报道。本研究旨在探究NIP组织中TLOs与免疫球蛋白的存在情况，及其与NIP临床病理特征的关联。
方法：本研究纳入84例NIP患者的乳头状瘤组织，通过免疫组化法（immunohistochemistry）分析TLOs的发生与组成及局部免疫球蛋白表达，分别采用定量qPCR（quantitative qPCR）与Luminex检测技术评估淋巴器官发生相关基因及细胞因子的表达水平。
结果：54%（45/84）的NIP患者组织中可检测到TLOs，而对照组样本中未发现TLOs。TLOs由T细胞、B细胞、滤泡树突状细胞、巨噬细胞及自然杀伤细胞组成。与无TLOs的NIP组织相比，携带TLOs的组织中嗜酸性粒细胞浸润水平显著升高（达3.5倍），淋巴器官发生相关基因（CXCL12、CXCL13、CCL20、CCL21、CD21L以及淋巴毒素α和β）的表达量上调，同时Th17型细胞因子（IL-21、IL-22及GM-CSF）与Th2型细胞因子（IL-5、IL-13）的分泌量增加。此外，携带TLOs的NIP组织中滤泡辅助T细胞及分泌免疫球蛋白的浆细胞（CD138+ IgA+、CD138+ IgM+、CD138+ IgE+及CD138+ IgG+）数量显著高于无TLOs的组织，且此类抗体分泌细胞数量与嗜酸性粒细胞数呈正相关。
结论：NIP黏膜中TLOs的高发生率与过量的局部免疫球蛋白产生，与以嗜酸性粒细胞浸润为主的Th2偏态微环境密切相关，这可能在NIP的发病过程中发挥重要的免疫致病作用。