The influence of B cells on the therapeutic success of immunotherapy in patients with head and neck cancer

长期以来，肿瘤浸润性B细胞在实体瘤中的作用一直被低估。直到最近几年才清楚的是，B细胞的存在往往与各种实体癌症，如头颈癌(HNC)的良好预后有关。另一方面，B细胞也被分配了促进肿瘤的作用，这一点得到了几个小鼠肿瘤模型的支持。因此，为了进一步的研究，有必要将B细胞群进一步细分为免疫活性效应B细胞和免疫抑制调节性B细胞。使用检查点抑制物(CPI)，如PD1抗体进行免疫治疗的可能性也导致了KHT患者存活率的增加。由于不表达PD-L1配体的肿瘤患者也从这种治疗中受益，PD-1抗体的确切作用机制尚未阐明。同样，这种疗法没有可靠的预后标记物。在目前的应用背景下，我们正在调查不同的B细胞群体如何影响CPI治疗。第一部分研究CPI如何在体外增强免疫系统，特别是B细胞功能。这是使用来自HNC患者或健康对照捐赠者的血液和肿瘤样本来完成的。使用质谱仪、UMAP流式细胞仪和肿瘤有机化合物实验。第二部分重点研究CPI对已建立的原位小鼠肿瘤模型免疫系统的影响。在这个模型中，CPI与其他抗体(抗CD20、抗CD73)结合，或通过基因敲除的方法删除B细胞特异性蛋白。第三部分研究了如何借助肿瘤来源的外切体来改变B细胞的功能。这些研究是在体内和体外进行的。用微型排斥层析法分离外切体，并通过纳米颗粒追踪进行可视化。第四部分将我们的肿瘤患者接受CPI治疗的临床数据与肿瘤中B细胞群和第三淋巴样结构的出现情况进行了关联。现有的肿瘤微阵列允许研究大量的肿瘤样本。利用多重血清学检测患者血清样本的抗原模式。应用的目的是通过操纵HNC患者肿瘤微环境中的B细胞来改进肿瘤治疗。这些结果可以在进一步的过程中转移到其他类型的肿瘤上。两个申请者一方面在临床和另一方面在研究实验室的活动领域是相辅相成的，从而促进了所建议的实验的迅速和成功的实施。

The role of tumor-infiltrating B cells in solid tumors has long been underestimated. Only in the last few years has it become clear that the presence of B cells is often associated with a good prognosis in a variety of solid cancers such as head and neck cancer (HNC). On the other hand, B cells are also assigned a tumor-promoting role, which is supported by several murine tumor models. For further investigations it is therefore necessary to further subdivide the B-cell population into immunocompetent effector B-cells and immunosuppressive regulatory B-cells.The possibilities of immunotherapy with checkpoint inhibitors (CPI), such as PD1 antibodies, have also led to an increase in survival in patients with KHT. Since patients with tumors who do not express the PD-L1 ligand, also benefit from this therapy, the exact mechanism of action of the PD-1 antibodies has not yet been clarified. Likewise, there are no reliable prognostic markers for this therapy.In the context of the present application, we are investigating how different B-cell populations can influence therapy with CPI. The first section examines how CPI can strengthen the immune system and in particular B-cell function in vitro. This is done using blood and tumor samples from patients with HNC or from healthy control donors. Mass spectrometry, UMAP flow cytometry and experiments with tumor organoids are used here.The second section focuses on the influence of CPI on the immune system in an established, orthotopic murine tumor model. In this model, CPI are combined with other antibodies (anti-CD20, anti-CD73) or B-cell-specific proteins are deleted by knockout methods.The third section examines how the function of B cells can be modified with the help of tumor-derived exosomes. These studies are carried out both in vivo and in vitro. The exosomes are isolated using mini-size exclusion chromatography and visualized by nanoparticle tracking.The fourth section correlates the clinical data of our tumor patients treated with CPI with the occurrence of B-cell populations and tertiary lymphoid structures in the tumor. Existing tumor microarrays allow the investigation of a large number of tumor samples. Serum samples from patients are examined for antigen patterns using multiplex serology.The aim of the application is to improve oncological therapy by manipulating B cells in the tumor microenvironment of patients with HNC. These results can be transferred to other types of tumors in the further course. The areas of activity of the two applicants, on the one hand in the clinic and on the other hand in the research laboratory, are complementary and thus promote the rapid and successful implementation of the suggested experiments.