Marion Lambert

Hepatitis C virus (HCV) evades NKG2D-dependent NK cell responses through NS5A-mediated imbalance of inflammatory cytokines.

Understanding how hepatitis C virus (HCV) induces and circumvents the hosts natural killer (NK) cell-mediated immunity is of critical importance in efforts to design effective therapeutics. We report here the decreased expression of the NKG2D activating receptor as a novel strategy adopted by HCV to evade NK-cell mediated responses. We show that chronic HCV infection is associated with expression of ligands for NKG2D, the MHC class I-related Chain (MIC) molecules, on hepatocytes. However, NKG2D expression is downmodulated on circulating NK cells, and consequently NK cell-mediated cytotoxic capacity and interferon-γ production are impaired. Using an endotoxin-free recombinant NS5A protein, we show that NS5A stimulation of monocytes through Toll-like Receptor 4 (TLR4) promotes p38- and PI3 kinase-dependent IL-10 production, while inhibiting IL-12 production. In turn, IL-10 triggers secretion of TGFβ which downmodulates NKG2D expression on NK cells, leading to their impaired effector functions. Moreover, culture supernatants of HCV JFH1 replicating Huh-7.5.1 cells reproduce the effect of recombinant NS5A on NKG2D downmodulation. Exogenous IL-15 can antagonize the TGFβ effect and restore normal NKG2D expression on NK cells. We conclude that NKG2D-dependent NK cell functions are modulated during chronic HCV infection, and demonstrate that this alteration can be prevented by exogenous IL-15, which could represent a meaningful adjuvant for therapeutic intervention.

21-Hydroxylase epitopes are targeted by CD8 T cells in autoimmune Addison’s disease

In autoimmune adrenal deficiency, autoantibodies target the 21-hydroxylase (21OH) protein. However, it is presumed that autoreactive T cells, rather than antibodies, are the main effectors of adrenal gland destruction, but their identification is still lacking. We performed a T-cell epitope mapping study using 49 overlapping 20mer peptides covering the 21OH sequence in patients with isolated Addisons disease, Autoimmune Polyendocrine Syndrome 1 and 2. IFNγ ELISPOT responses against these peptides were stronger, broader and more prevalent among patients than in controls, whatever the disease presentation. Five peptides elicited T-cell responses in patients only (68% sensitivity, 100% specificity). Blocking experiments identified IFNγ-producing cells as CD8 T lymphocytes, with two peptides frequently recognized in HLA-B8+ patients and a third one targeted in HLA-B35+ subjects. In particular, the 21OH(431-450) peptide was highly immunodominant, as it was recognized in more than 30% of patients, all carrying the HLA-B8 restriction element. This 21OH(431-450) region contained an EPLARLEL octamer (21OH(431-438)) predicted to bind to HLA-B8 with high affinity. Indeed, circulating EPLARLEL-specific CD8 T cells were detected at significant frequencies in HLA-B8+ patients but not in controls by HLA tetramer staining. This report enlightens disease-specific T-cell biomarkers and epitopes targeted in autoimmune adrenal deficiency.

Human Herpesvirus 8 (HHV8) Sequentially Shapes the NK Cell Repertoire during the Course of Asymptomatic Infection and Kaposi Sarcoma

The contribution of innate immunity to immunosurveillance of the oncogenic Human Herpes Virus 8 (HHV8) has not been studied in depth. We investigated NK cell phenotype and function in 70 HHV8-infected subjects, either asymptomatic carriers or having developed Kaposis sarcoma (KS). Our results revealed substantial alterations of the NK cell receptor repertoire in healthy HHV8 carriers, with reduced expression of NKp30, NKp46 and CD161 receptors. In addition, down-modulation of the activating NKG2D receptor, associated with impaired NK-cell lytic capacity, was observed in patients with active KS. Resolution of KS after treatment was accompanied with restoration of NKG2D levels and NK cell activity. HHV8-latently infected endothelial cells overexpressed ligands of several NK cell receptors, including NKG2D ligands. The strong expression of NKG2D ligands by tumor cells was confirmed in situ by immunohistochemical staining of KS biopsies. However, no tumor-infiltrating NK cells were detected, suggesting a defect in NK cell homing or survival in the KS microenvironment. Among the known KS-derived immunoregulatory factors, we identified prostaglandin E2 (PGE2) as a critical element responsible for the down-modulation of NKG2D expression on resting NK cells. Moreover, PGE2 prevented up-regulation of the NKG2D and NKp30 receptors on IL-15-activated NK cells, and inhibited the IL-15-induced proliferation and survival of NK cells. Altogether, our observations are consistent with distinct immunoevasion mechanisms that allow HHV8 to escape NK cell responses stepwise, first at early stages of infection to facilitate the maintenance of viral latency, and later to promote tumor cell growth through suppression of NKG2D-mediated functions. Importantly, our results provide additional support to the use of PGE2 inhibitors as an attractive approach to treat aggressive KS, as they could restore activation and survival of tumoricidal NK cells.

Tissue-Specific Microvascular Endothelial Cells Show Distinct Capacity To Activate NK Cells: Implications for the Pathophysiology of Granulomatosis with Polyangiitis

The relevance of tissue specificity of microvascular endothelial cells (MECs) in the response to inflammatory stimuli and sensitivity to immune cell–mediated injury is not well defined. We hypothesized that such MEC characteristics might shape their interaction with NK cells through the use of different adhesion molecules and NK cell receptor ligands or the release of different soluble factors and render them more or less vulnerable to NK cell injury during autoimmune vasculitis, such as granulomatosis with polyangiitis (GPA). To generate a comprehensive expression profile of human MECs of renal, lung, and dermal tissue origin, we characterized, in detail, their response to inflammatory cytokines and to proteinase 3, a major autoantigen in GPA, and analyzed the effects on NK cell activation. In this study, we show that renal MECs were more susceptible than lung and dermal MECs to the effect of inflammatory signals, showing upregulation of ICAM-1 and VCAM-1 on their surface, as well as release of CCL2, soluble fractalkine, and soluble VCAM-1. Proteinase 3–stimulated renal and lung MECs triggered CD107a degranulation in control NK cell. Notably, NK cells from GPA patients expressed markers of recent in vivo activation (CD69, CD107a), degranulated more efficiently than did control NK cells in the presence of renal MECs, and induced direct killing of renal MECs in vitro. These results suggest that, upon inflammatory conditions in GPA, renal MECs may contribute to the recruitment and activation of NK cells in the target vessel wall, which may participate in the necrotizing vasculitis of the kidney during this disease.

NKp30 isoforms and NKp30 ligands are predictive biomarkers of response to imatinib mesylate in metastatic GIST patients

ABSTRACT Despite effective targeted therapy acting on KIT and PDGFRA tyrosine kinases, gastrointestinal stromal tumors (GIST) escape treatment by acquiring mutations conveying resistance to imatinib mesylate (IM). Following the identification of NKp30-based immunosurveillance of GIST and the off-target effects of IM on NK cell functions, we investigated the predictive value of NKp30 isoforms and NKp30 soluble ligands in blood for the clinical response to IM. The relative expression and the proportions of NKp30 isoforms markedly impacted both event-free and overall survival, in two independent cohorts of metastatic GIST. Phenotypes based on disbalanced NKp30B/NKp30C ratio (ΔBClow) and low expression levels of NKp30A were identified in one third of patients with dismal prognosis across molecular subtypes. This ΔBClow blood phenotype was associated with a pro-inflammatory and immunosuppressive tumor microenvironment. In addition, detectable levels of the NKp30 ligand sB7-H6 predicted a worse prognosis in metastatic GIST. Soluble BAG6, an alternate ligand for NKp30 was associated with low NKp30 transcription and had additional predictive value in GIST patients with high NKp30 expression. Such GIST microenvironments could be rescued by therapy based on rIFN-α and anti-TRAIL mAb which reinstated innate immunity.

Bidirectional intragraft alloreactivity drives the repopulation of human intestinal allografts and correlates with clinical outcome

Clinical transplant outcomes reflect the balance between host-versus-graft and graft-versus-host reactivities. Transplantation stalemate Successful organ transplantation depends on both preventing rejection of the graft by host cells [host versus graft (HvG)] and blocking graft cells that attack the host (GvH). Zuber et al. demonstrated that a balance in this two-way alloreactivity affected clinical outcomes in organ transplant recipients. They examined gut-resident T cell turnover kinetics in human intestinal allografts and found that HvG-reactive cells persisted long-term in the graft, acquiring a tissue-resident phenotype. However, GvH-reactive cells expanded within the graft in the absence of rejection. The GvH-reactive cells may balance out the HvG-reactive cells, preventing rejection. One paradigm in transplantation is that graft-infiltrating T cells are largely nonalloreactive “bystander” cells. However, the origin and specificity of allograft T cells over time have not been investigated in detail in animals or humans. We used polychromatic flow cytometry and high-throughput T cell receptor sequencing of serial biopsies to show that gut-resident T cell turnover kinetics in human intestinal allografts are correlated with the balance between intragraft host-versus-graft (HvG) and graft-versus-host (GvH) reactivities and with clinical outcomes. In the absence of rejection, donor T cells were enriched for GvH-reactive clones that persisted in the long term in the graft. Early expansion of GvH clones in the graft correlated with the rapid replacement of donor antigen-presenting cells by the recipient. Rejection was associated with transient infiltration by blood-like recipient CD28+ NKG2DHi CD8+ αβ T cells, marked predominance of HvG clones, and accelerated T cell turnover in the graft. Ultimately, these recipient T cells acquired a steady-state tissue-resident phenotype but regained CD28 expression during rejections. Increased ratios of GvH to HvG clones were seen in nonrejectors, potentially mitigating the constant threat of rejection posed by HvG clones persisting within the tissue-resident graft T cell population.