Characterizing anti HCMV immunity in an infection model of explanted human lungs

人类巨细胞病毒(HCMV)感染在新生儿和免疫功能低下的成年人中危及生命。细胞毒性T淋巴细胞(CTL)和自然杀伤细胞(NK)在控制HCMV感染中起着重要作用。巨细胞病毒属于疱疹病毒科，这类病毒以其臭名昭著的物种特异性而闻名。因此，HCMV不会感染实验动物，因此关于CTL和NK细胞如何限制体内三维(3D)器官中的HCMV感染细胞的信息有限。为了克服这些限制，我们开发了一种3D器官培养系统，该系统基于来自HLA单倍型人类器官捐赠者的精密切割肺切片(PCLS)。PLCs可以冷冻保存，解冻后可感染HCMV。同样，从人类白细胞抗原单倍型供者扩增的HCMV特异性CTL也可以被冷冻保存，并用于利用双光子显微镜研究PCLS中HCMV感染细胞的杀伤作用。有了这个创新的实验装置，我们将首先建立一个细胞和组织库，其中包括符合人类白细胞抗原的PCL和CTL。PCLS将被用来通过光谱流式细胞术、组织学和单细胞mRNA测序来深刻地表征肺中对不同株HCMV敏感的感染细胞。然后，我们计划通过双光子显微镜来表征NK细胞和HCMV特异性CTL的不同亚群的杀伤动力学和动力学。最后，我们的目标是研究抗体依赖的细胞毒性(ADCC)在控制HCMV方面的潜在好处，应用针对两种已知抑制Fc-Gamma受体的不同蛋白的新型抗体。

Infection with human cytomegalovirus (HCMV) is life threatening in newborns and in immune compromised adults. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells play an important role in controlling HCMV infections. HCMV belongs to the family of herpesviridae, a class of viruses known for their notorious species specificity. Thus, HCMV does not infect laboratory animals and therefore information is limited about how CTLs and NK cells restrict HCMV-infected cells in vivo in three-dimensional (3D) organs. To overcome these limitations, we developed a 3D organ culture system that is based on precision cut lung slices (PCLS) from HLA-haplotyped human organ donors. PLCS can be cryopreserved and after thawing can be infected with HCMV. Likewise, HCMV-specific CTLs, expanded from HLA-haplotyped donors, can also be cryopreserved and used to study killing of HCMV-infected cells in PCLS applying 2-photon microscopy. With this innovative experimental setup, we will first build a cell and a tissue bank of HLA-matched PCLS and CTLs. PCLS will be used to profoundly characterize infected cells in the lung that are susceptible to different strains of HCMV using spectral flow cytometry, histology and single cell mRNA sequencing. We then plan to characterize killing dynamics and kinetics of various sub-populations of NK cells and HCMV-specific CTLs by 2-photon microscopy. Finally, we aim to study potential benefits of antibody-dependent cytotoxicity (ADCC) in controlling HCMV applying novel antibodies directed against two different proteins of HCMV that are known to inhibit Fc-gamma receptors.