Magnitude of Type I Interferon Responses by Plasmacytoid Dendritic Cells After TLR7 Stimulation Is Associated With Human Immunodeficiency Virus Type 1 (HIV-1) Reservoir Sizes in Cisgender Women With HIV-1 on Antiretroviral Therapy.

Human immunodeficiency virus type 1 (HIV-1) disease manifestations differ between cisgender women and men, including better control of viral replication during primary infection and less frequent residual HIV-1 replication on antiretroviral therapy (ART) in cisgender women with HIV-1 (WWH). Investigating plasmacytoid dendritic cell (pDC) functions and HIV-1 reservoir sizes in 20 WWH on stable ART, we observed inverse correlations between interferon-α and tumor necrosis factor responses of pDCs to Toll-like receptor 7/8 stimulation and intact/total proviral HIV-1 DNA levels. Additionally, ISG15 mRNA levels in peripheral blood mononuclear cells correlated with cytokine responses of pDCs. These findings demonstrate an association between higher type I interferon responses and lower HIV-1 reservoir sizes in WWH on ART, warranting studies to identify the underlying mechanisms.

The co-inhibitory receptor TIGIT regulates NK cell function and is upregulated in human intrahepatic CD56bright NK cells.

The crosstalk between NK cells and their surrounding environment is enabled through activating and inhibitory receptors, which tightly control NK cell activity. The co-inhibitory receptor TIGIT decreases NK cell cytotoxicity and is involved in NK cell exhaustion, but has also been associated with liver regeneration, highlighting that the contribution of human intrahepatic CD56bright NK cells in regulating tissue homeostasis remains incompletely understood. A targeted single-cell mRNA analysis revealed distinct transcriptional differences between matched human peripheral blood and intrahepatic CD56bright NK cells. Multiparameter flow cytometry identified a cluster of intrahepatic NK cells with overlapping high expression of CD56, CD69, CXCR6, TIGIT and CD96. Intrahepatic CD56bright NK cells also expressed significantly higher protein surface levels of TIGIT, and significantly lower levels of DNAM-1 compared to matched peripheral blood CD56bright NK cells. TIGIT+ CD56bright NK cells showed diminished degranulation and TNF-α production following stimulation. Co-incubation of peripheral blood CD56bright NK cells with human hepatoma cells or primary human hepatocyte organoids resulted in migration of NK cells into hepatocyte organoids and upregulation of TIGIT and downregulation of DNAM-1 expression, in line with the phenotype of intrahepatic CD56bright NK cells. Intrahepatic CD56bright NK cells represent a transcriptionally, phenotypically, and functionally distinct population of NK cells that expresses higher levels of TIGIT and lower levels of DNAM-1 than matched peripheral blood CD56bright NK cells. Increased expression of inhibitory receptors by NK cells within the liver environment can contribute to tissue homeostasis and reduction of liver inflammation.

Ku70 and Rad51 vary in their importance for the repair of doxorubicin- versus etoposide-induced DNA damage.

For DNA targeting anticancer drugs, cellular DNA repair mechanisms may cause resistance and hamper the therapeutic outcome. DNA damage induced by topoisomerase IIα inhibitors like etoposide and anthracyclines, which are a mainstay of cancer therapy, is also repaired in many cell types, but the impact and precise mechanisms of this repair are still obscure. To investigate the DNA damage response of human adenocarcinoma HT29-cells to doxorubicin and to compare the involvement of Ku70 and Rad51 in the repair of doxorubicin- versus etoposide-induced DNA damage, we assessed cell cycle distribution and cell death, DNA damage, proteins relevant for repair by homologous recombination and non-homologous end-joining, and clonogenicity following exposure to doxorubicin at clinically achievable concentrations. Also, we assessed changes in the repair kinetics after siRNA-mediated attenuation of Ku70 or Rad51 expression. We found that exposure to doxorubicin for 24 h induced a substantial amount of DNA damage that was largely repaired when doxorubicin was removed and the cells were maintained in drug-free medium. Nevertheless, a pronounced G(2)/M arrest occurred at times when repair was maximal. This was followed by a distinct increase in cell death and loss of clonogenicity. In this regard, responses to doxorubicin and etoposide were similar. However, distinct differences in the repair process following doxorubicin versus etoposide were seen in concentration dependency, time-course and requirement of Ku70 and Rad51 proteins. In spite of the shared molecular target of doxorubicin and etoposide, DNA lesions induced by these compounds are repaired differently.

Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage.

The topoisomerase IIalpha inhibitor etoposide is a 'broad spectrum' anticancer agent and a potent inducer of DNA double strand breaks. DNA damage response of mammalian cells usually involves cell cycle arrest and DNA repair or, if unsuccessful, cell death. We investigated these processes in the human colon cancer cell line HT-29 treated with three different etoposide regimens mimicking clinically relevant plasma concentrations of cancer patients. Each involved a period of drug-free incubation following etoposide exposure to imitate the decline of plasma levels between the cycles of chemotherapy. We found a massive induction of double strand breaks that were rapidly and nearly completely fixed long before the majority of cells underwent apoptosis or necrosis. An even greater percentage of cells lost clonogenicity. The occurrence of double strand breaks was accompanied by a decrease in the levels of Ku70, Ku86 and DNA-PK(cs) as well as an increase in the level of Rad51 protein. Twenty-four hours after the first contact with etoposide we found a pronounced G(2)/M arrest, regardless of the duration of drug exposure, the level of double strand breaks and the extent of their repair. During the subsequent drug-free incubation period, the loss of clonogenicity correlated well with the preceding G(2)/M arrest as well as with the amount of cell death found several days after exposure. However, it correlated neither with early apoptosis or necrosis nor with any of the other investigated parameters. These results suggest that the G(2)/M arrest is an important determinant in the cytostatic action of etoposide and that the removal of DNA double strand breaks is not sufficient to ensure cell survival.