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

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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.