CD137 Costimulation Counteracts TGFß Inhibition of NK-cell Antitumor Function.

Enhancing natural killer (NK) cell-based cancer immunotherapy by overcoming immunosuppression is an area of intensive research. Here, we have demonstrated that the anti-CD137 agonist urelumab can overcome TGFß-mediated inhibition of human NK-cell proliferation and antitumor function. Transcriptomic, immunophenotypic, and functional analyses showed that CD137 costimulation modified the transcriptional program induced by TGFß on human NK cells by rescuing their proliferation in response to IL2, preserving their expression of activating receptors (NKG2D) and effector molecules (granzyme B, IFNγ) while allowing the acquisition of tumor-homing/retention features (CXCR3, CD103). Activated NK cells cultured in the presence of TGFß1 and CD137 agonist recovered CCL5 and IFNγ secretion and showed enhanced direct and antibody-dependent cytotoxicity upon restimulation with cancer cells. Trastuzumab treatment of fresh breast carcinoma-derived multicellular cultures induced CD137 expression on tumor-infiltrating CD16+ NK cells, enabling the action of urelumab, which fostered tumor-infiltrating NK cells and recapitulated the enhancement of CCL5 and IFNγ production. Bioinformatic analysis pointed to IFNG as the driver of the association between NK cells and clinical response to trastuzumab in patients with HER2-positive primary breast cancer, highlighting the translational relevance of the CD137 costimulatory axis for enhancing IFNγ production. Our data reveals CD137 as a targetable checkpoint for overturning TGFß constraints on NK-cell antitumor responses.

Long-Term Evolution of the Adaptive NKG2C+ NK Cell Response to Cytomegalovirus Infection in Kidney Transplantation: An Insight on the Diversity of Host-Pathogen Interaction.

Human CMV infection is frequent in kidney transplant recipients (KTR). Pretransplant Ag-specific T cells and adaptive NKG2C+ NK cells associate with reduced incidence of infection in CMV+ KTR. Expansions of adaptive NKG2C+ NK cells were reported in posttransplant CMV-infected KTR. To further explore this issue, NKG2C+ NK, CD8+, and TcRγδ T cells were analyzed pretransplant and at different time points posttransplant for ≥24 mo in a cohort of CMV+ KTR (n = 112), stratified according to CMV viremia detection. In cryopreserved samples from a subgroup (n = 49), adaptive NKG2C+ NK cell markers and T cell subsets were compared after a longer follow-up (median, 56 mo), assessing the frequencies of CMV-specific T cells and viremia at the last time point. Increased proportions of NKG2C+ NK, CD8+, and TcRγδ T cells were detected along posttransplant evolution in viremia(+) KTR. However, the individual magnitude and kinetics of the NKG2C+ NK response was variable and only exceptionally detected among viremia(-) KTR, presumably reflecting subclinical viral replication events. NKG2C+ expansions were independent of KLRC2 zygosity and associated with higher viral loads at diagnosis; no relation with other clinical parameters was perceived. Increased proportions of adaptive NKG2C+ NK cells (CD57+, ILT2+, FcεRIγ-) were observed after resolution of viremia long-term posttransplant, coinciding with increased CD8+ and Vδ2- γδ T cells; at that stage CMV-specific T cells were comparable to viremia(-) cases. These data suggest that adaptive NKG2C+ NK cells participate with T cells to restore CMV replication control, although their relative contribution cannot be discerned.

NK cells eliminate Epstein-Barr virus bound to B cells through a specific antibody-mediated uptake.

Epstein Barr virus (EBV) causes a highly prevalent and lifelong infection contributing to the development of some malignancies. In addition to the key role played by T cells in controlling this pathogen, NK cells mediate cytotoxicity and IFNγ production in response to EBV-infected B cells in lytic cycle, both directly and through antibody (Ab)-dependent activation. We recently described that EBV-specific Ab-dependent NK cell interaction with viral particles (VP) bound to B cells triggered degranulation and TNFα secretion but not B cell lysis nor IFNγ production. In this report we show that NK cell activation under these conditions reduced B cell transformation by EBV. NK cells eliminated VP from the surface of B cells through a specific and active process which required tyrosine kinase activation, actin polymerization and Ca2+, being independent of proteolysis and perforin. VP were displayed at the NK cell surface before being internalized and partially shuttled to early endosomes and lysosomes. VP transfer was encompassed by a trogocytosis process including the EBV receptor CD21, together with CD19 and CD20. Our study reveals a novel facet of the antibody-dependent NK cell mediated response to this viral infection.

Adaptive NKG2C+ natural killer cells are related to exacerbations and nutritional abnormalities in COPD patients.

Chronic obstructive pulmonary disease (COPD) is a chronic and often progressive disorder with a heterogeneous presentation and frequent systemic manifestations. Several aspects like persistence in smoking habit, continuous exacerbations, alpha-1-antitrypsin deficiency and inflammatory-immune response, are involved in the pathophysiology and progression of the disease. However, the role of natural killer (NK) cells remains controversial. Otherwise, human cytomegalovirus (HCMV) infection has been reported to induce an adaptive differentiation and expansion of an NK cell subset which carries the CD94/NKG2C receptor, which may contribute to an upset immune defense. For these reasons, our objective is to assess the distribution of NK cells and their subset in COPD patients and some of its phenotypes. METHODS: Peripheral blood samples were obtained from 66 COPD patients. HCMV serology and the proportions of total NK cells and the NKG2C+ and NKG2A+ subsets were evaluated by flow cytometry. The NKG2C genotype was also assessed. RESULTS: Eighty-eight per cent of COPD patients were HCMV(+), and the proportions of total NK cells were higher in patients with severe-very severe airway obstruction than in those with only mild-moderate involvement. There were no differences in the proportions of NKG2C+ cells between controls and COPD, either among COPD patients classified by severity of the disease. However, the percentage of NKG2C+ cells were higher in COPD patients with frequent exacerbations than in occasional exacerbators, and higher in cases with reduced lean mass (Fat free mass index) than in those with normal nutritional status. CONCLUSION: These results suggest a relationship between levels of NKG2C+ cells in COPD patients and clinical variables closely linked to a poor/worse prognosis.

Pretransplant adaptive NKG2C+ NK cells protect against cytomegalovirus infection in kidney transplant recipients.

Cytomegalovirus (CMV) infection constitutes a complication for kidney transplant recipients (KTR) and CMV-specific T cells reduce the risk of viral replication in seropositive patients. CMV promotes the adaptive differentiation and expansion of an NK cell subset, hallmarked by expression of the CD94/NKG2C receptor with additional characteristic features. We previously reported an association of pretransplant NKG2C+ NK cells with a reduced incidence of CMV infection. We have strengthened the analysis in cryopreserved peripheral blood mononuclear cells from an enlarged KTR cohort (n = 145) with homogeneous immunosuppression, excluding cases at low risk of infection (ie, CMV D-R-) or receiving antiviral prophylaxis. Moreover, adaptive NKG2C+ NK cell-associated markers (ie, NKG2A, CD57, Immunoglobulin-like transcript 2 [LIR1 or LILRB1], FcεRI γ chain, and Prolymphocytic Leukemia Zinc Finger transcription factor) as well as T lymphocyte subsets were assessed by multicolor flow cytometry. The relation of NKG2C+ NK cells with T cells specific for CMV antigens was analyzed in pretransplant patients (n = 29) and healthy controls (n = 28). Multivariate Cox regression and Kaplan-Meier analyses supported that NKG2C+ NK cells bearing adaptive markers were specifically associated with a reduced incidence of posttransplant symptomatic CMV infection; no correlation between NKG2C+ NK cells and CMV-specific T cells was observed. These results support that adaptive NKG2C+ NK cells contribute to control CMV infection in KTR.

Human Cytomegalovirus Antigen Presentation by HLA-DR+ NKG2C+ Adaptive NK Cells Specifically Activates Polyfunctional Effector Memory CD4+ T Lymphocytes.

Natural killer (NK) cells play a dual role in the defense against viral pathogens by directly lysing infected cells as well as by regulating anti-viral T cell immunity. Infection by human cytomegalovirus (HCMV) promotes a persistent expansion of NKG2C+ adaptive NK cells which have been shown to display enhanced antibody-dependent responses against infected targets and associated to viral control in transplanted patients. Based on gene expression data showing increased transcription of CIITA and several genes related to the MHC class II pathway in adaptive NK cells, we explored their putative capacity for antigen presentation to CD4+ T cells. Phenotypic analysis confirmed a preferential steady-state expression of HLA-DR by circulating NKG2C+ adaptive NK cells in healthy individuals. Expression of HLA-DR in NKG2C+ adaptive NK cells was variable and unrelated to the expression of activation (i.e., CD69 and CD25) or differentiation (i.e., FcRγ chain, CD57) markers, remaining stable over time at the individual level. Incubation of purified NK cells with HCMV complexed with serum specific antibodies induced an up-regulation of surface HLA-DR concomitant to CD16 loss whereas no changes in CD80/CD86 co-stimulatory ligands were detected. In addition, surface CX3CR1 decreased upon antigen-loading while HLA-DR+ NK cells maintained a CCR7-, CXCR3low homing profile. Remarkably, HCMV-loaded purified NK cells activated autologous CD4+ T cells in an HLA-DR dependent manner. The fraction of T lymphocytes activated by antigen-loaded NK cells was smaller than that stimulated by monocyte-derived dendritic cells, corresponding to CD28-negative effector-memory CD4+ T cells with cytotoxic potential. Antigen presentation by NK cells activated a polyfunctional CD4+ T cell response characterized by degranulation (CD107a) and the secretion of Th1 cytokines (IFNγ and TNFα). Overall, our data discloses the capacity of NKG2C+ adaptive NK cells to process and present HCMV antigens to memory CD4+ cytotoxic T cells, directly regulating their response to the viral infection.