Anti-IFNG monoclonal antibody, clone YNH2.3 [FITC]

A population of NK cells expressing the activating receptor NKG2C and the maturation marker CD57 expands in response to human CMV (HCMV) infection. CD3(-)CD56(dim)CD57(+)NKG2C(+) NK cells are similar to CD8(+) memory T cells with rapid and robust effector function upon restimulation, persistence, and epigenetic remodeling of the IFNG locus. Chronic antigen stimulation drives CD8(+) memory T cell proliferation, while also inducing genome-wide epigenetic reprograming and dysfunction. We hypothesized that chronic stimulation could similarly induce epigenetic reprograming and dysfunction in NK cells. Here, we show that chronic stimulation of adaptive NK cells through NKG2C using plate-bound agonistic Abs in combination with IL-15 drove robust proliferation and activation of CD3(-)CD56(dim)CD57(+)NKG2C(+) NK cells, while simultaneously inducing high expression of the checkpoint inhibitory receptors LAG-3 and PO-1. Marked induction of checkpoint inhibitory receptors was also observed on the surface of adaptive NK cells cocultured with HCMV-infected endothelial cells. Chronically stimulated adaptive NK cells were dysfunctional when challenged with tumor targets. These cells exhibited a pattern of epigenetic reprograming, with genome-wide alterations in DNA methylation. We believe our study has important implications for cancer immunotherapy and propose that exhausted NK cells could be targeted with inhibitory checkpoint receptor blockade.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a cattle disease of global importance. M. bovis infects bovine macrophages (Mo) and subverts the host cell response to generate a suitable niche for survival and replication. We investigated the role of the anti-inflammatory cytokine interleukin (IL) 10 during in vitro infection of bovine monocyte-derived Mo (bMDM) with two divergent UK strains of M. bovis, which differentially modulate expression of IL10. The use of IL10-targeting siRNA revealed that IL10 inhibited the production of IL1B, IL6, tumour necrosis factor (TNF) and interferon gamma (IFNG) during infection of bMDM with the M. bovis strain G18. In contrast, IL10 only regulated a subset of these genes; TNF and IFNG, during infection with the M. bovis reference strain AF2122/97. Furthermore, nitric oxide (NO) production was modulated by IL10 during AF2122/97 infection, but not at the nitric oxide synthase 2 (NOS2) mRNA level, as observed during G18 infection. However, IL10 was found to promote survival of both M. bovis strains during early bMDM infection, but this effect disappeared after 24 h. The role of IL10-induced modulation of TNF, IFNG and NO production in M. bovis survival was investigated using siRNA targeting TNF, IFNG receptor 1 (IFNGR1) and NOS2. Knock-down of these genes individually did not promote survival of either M. bovis strain and therefore modulation of these genes does not account for the effect of IL10 on M. bovis survival. However, TNF knock-down was found to be detrimental to the survival of the M. bovis strain G18 during early infection. The results provide further evidence for the importance of IL10 during M. bovis infection of Mo. Furthermore, they highlight M. bovis strain specific differences in the interaction with the infected bMDM, which may influence the course of infection and progression of bovine TB.