Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi’s sarcoma–associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning and viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected.

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.

Patients with immunodeficiencies and older age are at an increased risk of anal cancer. Transgenic K14E6/E7 mice with established high-grade anal dysplasia were treated topically at the anus with the protease inhibitor saquinavir (SQV) in the setting of CD4+ T-cell depletion to mimic immunodeficiency. To ensure tumor development, specific groups were treated with a topical carcinogen (7,12-Dimethylbenz[a]anthracene (DMBA)). The treatment groups included the vehicle (control), DMBA only, topical SQV, and topical SQV with DMBA, as well as the same four groups with CD4 depletion. The mice were monitored weekly for tumor development. Upon reaching 20 weeks of treatment, the mice were sacrificed, and their anal tissue was harvested for histological analysis. None of the mice in the SQV or control groups developed overt anal tumors, except three mice that were CD4-depleted. The CD4-depleted mice treated with DMBA had significantly increased tumor-free survival and overall survival as well as decreased tumor-volume growth over time when treated with SQV. These data suggest that topical SQV, in the setting of CD4 depletion and high-grade anal dysplasia, can increase tumor-free and overall survival; thus, it may represent a viable topical therapy to decrease the risk of progression of anal dysplasia to anal cancer.

Companion canines need advances in therapeutic options for solid tumour malignancies. Natural killer (NK) cell infusions offer a promising treatment modality in canines with solid tumours. Autologous natural killer products are limited by dysfunctional immunity and a manufacturing process that delays care whereas allogeneic NK cells offer the possibility of ‘off‐the‐shelf’ therapy to be administered from healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy canine donors via density gradient separation. NK cells were expanded with recombinant human IL‐2 and canine IL‐21 with the addition of K562 feeder cells transfected with CD137 ligand and membrane bound human IL‐15. Additional experiments included IL‐12 during expansion. In vitro potency was assessed via co‐culture with the D17‐mKate2 canine osteosarcoma cell line. Three canines were enrolled in a phase 1 trial infusing ex vivo expanded allogeneic NK cells after lymphodepletion. Flow cytometric analysis confirmed successful expansion of canine NK cells with up to 50% of cells demonstrating NKp46+ after 14 days. Residual T cell numbers varied based on donor. The addition of IL‐12 led to increased NK cell expansion. Incucyte demonstrated potency with increasing osteosarcoma cell death at higher effector to target ratios. Three canines with metastatic/refractory solid tumours were successfully lymphodepleted and infused with allogeneic NK cell products. The canines tolerated the infusions well. Canine allogeneic NK cells were successfully expanded and activated ex vivo, demonstrated potency in vitro and safety in vivo. Further studies will optimise the NK cell product and escalate dosing to reach the maximal tolerable dose.