[Show abstract][Hide abstract] ABSTRACT: Acute and latent human CMV cause profound changes in the NK cell repertoire, with expansion and differentiation of educated NK cells expressing self-specific inhibitory killer cell Ig-like receptors. In this study, we addressed whether such CMV-induced imprints on the donor NK cell repertoire influenced the outcome of allogeneic stem cell transplantation. Hierarchical clustering of high-resolution immunophenotyping data covering key NK cell parameters, including frequencies of CD56(bright), NKG2A(+), NKG2C(+), and CD57(+) NK cell subsets, as well as the size of the educated NK cell subset, was linked to clinical outcomes. Clusters defining naive (NKG2A(+)CD57(-)NKG2C(-)) NK cell repertoires in the donor were associated with decreased risk for relapse in recipients with acute myeloid leukemia and myelodysplastic syndrome (hazard ratio [HR], 0.09; 95% confidence interval [CI]: 0.03-0.27; p < 0.001). Furthermore, recipients with naive repertoires at 9-12 mo after hematopoietic stem cell transplantation had increased disease-free survival (HR, 7.2; 95% CI: 1.6-33; p = 0.01) and increased overall survival (HR, 9.3; 95% CI: 1.1-77, p = 0.04). Conversely, patients with a relative increase in differentiated NK cells at 9-12 mo displayed a higher rate of late relapses (HR, 8.41; 95% CI: 6.7-11; p = 0.02), reduced disease-free survival (HR, 0.12; 95% CI: 0.12-0.74; p = 0.02), and reduced overall survival (HR, 0.07; 95% CI: 0.01-0.69; p = 0.02). Thus, our data suggest that naive donor NK cell repertoires are associated with protection against leukemia relapse after allogeneic HSCT.
Full-text · Article · Jan 2016 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: 5-azacytidine (5-aza) is a hypomethylating agent approved for the treatment of high-risk myelodysplastic syndrome (MDS). It is assumed to act by demethylating tumor suppressor genes and via direct cytotoxic effects on malignant cells. In vitro treatment with hypomethylating agents has profound effects on the expression of killer-cell immunoglobulin-like (KIR) receptors on natural killer (NK) cells, as these receptors are epigenetically regulated via methylation of the promoters. Here we investigated the influence of 5-aza on the NK-cell repertoire during cytokine-induced proliferation in vitro and homeostatic proliferation in vivo in patients with high-risk MDS. In vitro treatment of NK cells from both healthy donors and MDS patients with low doses of 5-aza led to a significant increase in expression of multiple KIRs, but only in cells that had undergone several rounds of cell division. Proliferating 5-aza exposed NK cells exhibited increased IFN-γ production and degranulation towards tumor target cells. MDS patients had lower proportions of educated KIR-expressing NK cells than healthy controls but after systemic treatment with 5-aza, an increased proportion of Ki-67+ NK cells expressed multiple KIRs suggesting uptake of 5-aza in cycling cells in vivo. Hence, these results suggest that systemic treatment with 5-aza may shape the NK cell repertoire, in particular during homeostatic proliferation, thereby boosting NK cell-mediated recognition of malignant cells.
[Show abstract][Hide abstract] ABSTRACT: Adaptive natural killer (NK) cell responses to human cytomegalovirus infection are characterized by the expansion of NKG2C+ NK cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs). Here, we set out to study the HLA class I dependency of such NKG2C+ NK cell expansions. We demonstrate the expansion of NKG2C+ NK cells in patients with transporter associated with antigen presentation (TAP) deficiency, who express less than 10% of normal HLA class I levels. In contrast to normal individuals, expanded NKG2C+ NK cell populations in TAP-deficient patients display a polyclonal KIR profile and remain hyporesponsive to HLA class I-negative target cells. Nonetheless, agonistic stimulation of NKG2C on NK cells from TAP-deficient patients yielded significant responses in terms of degranulation and cytokine production. Thus, while interactions with self-HLA class I molecules likely shape the KIR repertoire of expanding NKG2C+ NK cells during adaptive NK cell responses in normal individuals, they are not a prerequisite for NKG2C+ NK cell expansions to occur. The emergence of NKG2C-responsive adaptive NK cells in TAP-deficient patients may contribute to antiviral immunity and potentially explain these patients’ low incidence of severe viral infections.
Full-text · Article · Oct 2015 · Frontiers in Immunology
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are innate lymphocytes with a refined ability to recognize transformed cells through a broad array of activating receptors in combination with stochastically expressed inhibitory receptors that recognize MHC-class I. Recent advances in NK cell biology have revealed a high degree of functional plasticity that can be attributed to dynamic cell-to-cell interactions in concert with transcriptional and epigenetic reprogramming. Here, we discuss how new insights into the adaptive behavior of NK cells pave the way for next generation cell therapy based on guided differentiation and selective expansion of particularly cytotoxic NK cell subsets.
Full-text · Article · Oct 2015 · Molecular oncology
[Show abstract][Hide abstract] ABSTRACT: A growing body of evidence suggests that the human natural killer (NK) cell compartment is phenotypically and functionally heterogeneous and composed of several differentiation stages. Moreover, NK cell subsets have recently been shown to exhibit adaptive immune features during herpesvirus infection in experimental mice and to preferentially expand during viral infections in humans. However, both phenotype and role of NK cells during acute symptomatic Epstein-Barr virus (EBV) infection, termed infectious mononucleosis (IM), remain unclear. Here, we longitudinally assessed the kinetics, the differentiation and the proliferation of subsets of NK cells in pediatric IM patients. Our results indicate that acute IM is characterized by the preferential proliferation of early-differentiated CD56(dim) NKG2A(+) KIR(-) NK cells. Moreover, this NK cell subset exhibits features of terminal differentiation and persists at higher frequency over at least the first 6 months after acute IM. Finally, we demonstrate that this NK cell subset preferentially degranulates and proliferates upon exposure to EBV-infected B cells expressing lytic antigens. Thus, early-differentiated NK cells might play a key role in the immune control of primary infection with this persistent tumor-associated virus.
[Show abstract][Hide abstract] ABSTRACT: Allogeneic hematopoietic stem cell transplantation (HSCT) is widely used to treat hematopoietic cell disorders, but is often complicated by graft versus host disease (GvHD) that causes severe epithelial damage. Here we have investigated longitudinally the effects of induction chemotherapy, conditioning radio-chemotherapy and allogeneic HSCT on composition, phenotype and recovery of circulating innate lymphoid cells (ILC) in 51 acute leukemia patients. We found that reconstitution of ILC1, ILC2 and NCR(-) ILC3 was slow compared to that of neutrophils and monocytes. NCR(+) ILC3 which are not present in the circulation of healthy individuals appeared both after induction chemotherapy and after allogeneic HSCT. Circulating patient ILC before transplantation as well as donor ILC after transplantation expressed activation (CD69), proliferation (Ki-67) and tissue homing markers for gut (α4β7, CCR6) and skin (CCR10 and CLA). The proportion ILC expressing these markers was associated with a decreased susceptibility to therapy-induced mucositis and acute GvHD. Taken together, these data suggest that ILC recovery and treatment-related tissue damage are interrelated and affect the development of GvHD.
[Show abstract][Hide abstract] ABSTRACT: Although hepatitis delta is considered an immune-mediated disease, adaptive immune responses to hepatitis delta virus (HDV) are hardly detectable. Thus, the role of other immune responses, including those mediated by natural killer (NK) cells, must be considered in HDV pathogenesis and in treatments with immune-stimulating agents such as interferon (IFN)α. However, the phenotype and function of NK cells in chronic HDV infection, or in HDV-infected individuals undergoing IFNα treatment, have not been extensively studied.
We performed an extensive analysis of NK cells in chronically HDV-infected patients before and during treatment with IFNα, and compared the results with those for patients with HBV mono-infection as well as healthy controls.
In untreated HDV-infected patients, a higher than normal frequency of NK cells was observed in peripheral blood with unaltered phenotypic NK cell differentiation status. In contrast, long-term IFNα treatment of HDV-infected patients caused a significant change in NK cell differentiation status, with selective loss of terminally differentiated NK cells and, in parallel, a relative enrichment in immature NK cell subsets. Treatment was associated with marked functional impairment of the NK cells, which was independent of the changes in NK cell differentiation status. Furthermore, treatment polarised NK cell IFN signalling from STAT4 towards STAT1 dependency. Strikingly, a high frequency of CD56(dim) NK cells at baseline was positively associated with IFNα treatment outcome in the patients.
We describe in detail how HDV infection, and IFNα treatment of this infection, affects the NK cell compartment and what consequences this has for the functional capacity of NK cells.
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are functionally tuned by education via killer cell immunoglobulin receptors (KIRs) interacting with HLA class I molecules. We examined the effect of KIR gene copy number variation (CNV) on the education of human NK cells. The frequency of NK cells expressing a given KIR correlated with the copy number of that gene. However, co-expression of multiple copies from a single locus, or duplicated loci, was infrequent, in line with independent transcriptional regulation of each allele or copy. Intriguingly, co-expression of two KIR alleles, resulting in higher surface expression, did not lead to enhanced functional responses in vitro or to selective advantages during in vivo responses to cytomegalovirus infection, suggesting that receptor density does not influence NK education at the single cell level. However, individuals with multiple KIR gene copies had higher frequencies of responding cells, consistent with heightened overall responsiveness.
[Show abstract][Hide abstract] ABSTRACT: The Natural Killer (NK) cell population is composed of subsets of varying sizes expressing different combinations of inhibitory receptors for MHC class I molecules. Genes within the NK gene complex, including the inhibitory receptors themselves, seem to be the primary intrinsic regulators of inhibitory receptor expression, but the MHC class I background is an additional Modulating factor. In this paper, we have performed a parallel study of the inhibitory receptor repertoire in inbred mice of the C57Bl/6 background and in a cohort of 44 humans. Deviations of subset frequencies from the "product rule (PR)," i.e., differences between observed and expected frequencies of NK cells, were used to identify MHC-independent and MHC-dependent control of receptor expression frequencies. Some deviations from the PR were similar in mice and humans, such as the decreased presence of NK cell subset lacking inhibitory receptors. Others were different, including a role for NKG2A in determining over- or under-representation of specific subsets in humans but not in mice. Thus, while human and murine inhibitory receptor repertoires differed in details, there may also be shared principles governing NK cell repertoire formation in these two species.
Full-text · Article · Mar 2013 · Frontiers in Immunology
[Show abstract][Hide abstract] ABSTRACT: Human NK cells are functionally regulated by killer cell immunoglobulin-like receptors (KIRs) and their interactions with HLA class I molecules. As KIR expression in a given NK cell is genetically hard-wired, we hypothesized that KIR repertoire perturbations reflect expansions of unique NK cell subsets and may be used to trace adaptation of the NK cell compartment to viral infections. By determining the human "KIR-ome" at a single cell level in over 200 donors, we were able to analyze the magnitude of NK cell adaptation to viral infections in healthy individuals. Strikingly, infection with human cytomegalovirus (CMV), but not with other common herpesviruses, induced expansion and differentiation of KIR-expressing NK cells, visible as stable imprints in the repertoire. Education by inhibitory KIRs promoted the clonal-like expansion of NK cells, causing a bias for self-specific inhibitory KIRs. Furthermore, our data revealed a unique contribution of activating KIRs (KIR2DS4, KIR2DS2, or KIR3DS1), in addition to NKG2C, in the expansion of human NK cells. These results provide new insight into KIR repertoire diversity and its adaptation to viral infection, suggesting a role for both activating and inhibitory KIRs in immunity to CMV infection.
[Show abstract][Hide abstract] ABSTRACT: Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of basic functional heterogeneity in individual NK cell behavior. By using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, we could quantify how the cytotoxic response varies between individual NK cells. Strikingly, about half of the NK cells did not kill any target cells at all, while a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into five distinct classes. A small but particularly active sub-class of NK cells killed several target cells in a consecutive fashion. These 'serial killers' delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model where a small fraction of NK cells drives tumor elimination and inflammation.