Systemic and Intra-Nodal Activation of NK Cells After Rituximab Monotherapy for Follicular Lymphoma.

Monotherapy with the anti-CD20 monoclonal antibody rituximab can induce complete responses (CR) in patients with follicular lymphoma (FL). Resting FcRγIII+ (CD16+) natural killer (NK) cells respond strongly to rituximab-coated target cells in vitro. Yet, the contribution of NK cells in the therapeutic effect in vivo remains unknown. Here, we followed the NK cell repertoire dynamics in the lymph node and systemically during rituximab monotherapy in patients with FL. At baseline, NK cells in the tumor lymph node had a naïve phenotype albeit they were more differentiated than NK cells derived from control tonsils as determined by the frequency of CD56dim NK cells and the expression of killer cell immunoglobulin-like receptors (KIR), CD57 and CD16. Rituximab therapy induced a rapid drop in NK cell numbers coinciding with a relative increase in the frequency of Ki67+ NK cells both in the lymph node and peripheral blood. The Ki67+ NK cells had slightly increased expression of CD16, CD57 and higher levels of granzyme A and perforin. The in vivo activation of NK cells was paralleled by a temporary loss of in vitro functionality, primarily manifested as decreased IFNγ production in response to rituximab-coated targets. However, patients with pre-existing NKG2C+ adaptive NK cell subsets showed less Ki67 upregulation and were refractory to the loss of functionality. These data reveal variable imprints of rituximab monotherapy on the NK cell repertoire, which may depend on pre-existing repertoire diversity.

NK Cell Responses to Human Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV) is a flavivirus that is transferred to humans by infected ticks. The virus causes tick-borne encephalitis, a severe infection of the CNS with a high risk for long-lasting sequelae. Currently, no treatment exists for the disease. Understanding the cellular immune response to this infection is important to gain further understanding into the pathogenesis, treatment, and prevention of the disease. NK cells are known to participate in the control of viral infections. We performed a longitudinal analysis of the human NK cell response to TBEV infection in a cohort of infected individuals from the onset of severe clinical symptoms to the convalescence phase. NK cell activation, as measured by expression of Ki67, was apparent at the time of hospitalization. By 3 wk after hospitalization, it decreased to levels seen in healthy controls. Concomitant with the increase in NK cell activation, augmented levels of IL-12, IL-15, IL-18, IFN-γ, and TNF were detected in patient plasma. This TBEV-induced NK cell activation was restricted predominantly to differentiated CD57(+)CD56(dim) NK cells. Functionally, CD56(dim) NK cells responded poorly to target cells at the time of hospitalization, but they recovered functional capacity to control levels during the convalescent phase. In contrast, the responsiveness of NK cells to cytokine stimulation remained intact throughout the disease. These findings demonstrate that NK cells respond to TBEV infection with characteristics that are distinct from those of other human viral infections and provide insights into the NK cell response to clinical TBEV infection.

Critical Role of CD2 Co-stimulation in Adaptive Natural Killer Cell Responses Revealed in NKG2C-Deficient Humans.

Infection by human cytomegalovirus (HCMV) leads to NKG2C-driven expansion of adaptive natural killer (NK) cells, contributing to host defense. However, approximately 4% of all humans carry a homozygous deletion of the gene that encodes NKG2C (NKG2C(-/-)). Assessment of NK cell repertoires in 60 NKG2C(-/-) donors revealed a broad range of NK cell populations displaying characteristic footprints of adaptive NK cells, including a terminally differentiated phenotype, functional reprogramming, and epigenetic remodeling of the interferon (IFN)-γ promoter. We found that both NKG2C(-) and NKG2C(+) adaptive NK cells expressed high levels of CD2, which synergistically enhanced ERK and S6RP phosphorylation following CD16 ligation. Notably, CD2 co-stimulation was critical for the ability of adaptive NK cells to respond to antibody-coated target cells. These results reveal an unexpected redundancy in the human NK cell response to HCMV and suggest that CD2 provides "signal 2" in antibody-driven adaptive NK cell responses.

Microchip-Based Single-Cell Imaging Reveals That CD56dimCD57-KIR-NKG2A+ NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56dimCD57-KIR-NKG2A- NK Cells.

NK cells are functionally educated by self-MHC specific receptors, including the inhibitory killer cell Ig-like receptors (KIRs) and the lectin-like CD94/NKG2A heterodimer. Little is known about how NK cell education influences qualitative aspects of cytotoxicity such as migration behavior and efficacy of activation and killing at the single-cell level. In this study, we have compared the behavior of FACS-sorted CD56(dim)CD57(-)KIR(-)NKG2A(+) (NKG2A(+)) and CD56(dim)CD57(-)KIR(-)NKG2A(-) (lacking inhibitory receptors; IR(-)) human NK cells by quantifying migration, cytotoxicity, and contact dynamics using microchip-based live cell imaging. NKG2A(+) NK cells displayed a more dynamic migration behavior and made more contacts with target cells than IR(-) NK cells. NKG2A(+) NK cells also more frequently killed the target cells once a conjugate had been formed. NK cells with serial killing capacity were primarily found among NKG2A(+) NK cells. Conjugates involving IR(-) NK cells were generally more short-lived and IR(-) NK cells did not become activated to the same extent as NKG2A(+) NK cells when in contact with target cells, as evident by their reduced spreading response. In contrast, NKG2A(+) and IR(-) NK cells showed similar dynamics in terms of duration of conjugation periods and NK cell spreading response in conjugates that led to killing. Taken together, these observations suggest that the high killing capacity of NKG2A(+) NK cells is linked to processes regulating events in the recognition phase of NK-target cell contact rather than events after cytotoxicity has been triggered.

Coordinated expression of DNAM-1 and LFA-1 in educated NK cells.

The functional capacity of NK cells is dynamically tuned by integrated signals from inhibitory and activating cell surface receptors in a process termed NK cell education. However, the understanding of the cellular and molecular mechanisms behind this functional tuning is limited. In this study, we show that the expression of the adhesion molecule and activation receptor DNAX accessory molecule 1 (DNAM-1) correlates with the quantity and quality of the inhibitory input by HLA class I-specific killer cell Ig-like receptors and CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell recognition, the conformational state of LFA-1 changed in educated NK cells, associated with rapid colocalization of both active LFA-1 and DNAM-1 at the immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a potential mechanism for controlling cytotoxicity by functionally mature NK cells.

Influence of KIR gene copy number on natural killer cell education.

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 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, coexpression of multiple copies from a single locus, or duplicated loci, was infrequent, which is in line with independent transcriptional regulation of each allele or copy. Intriguingly, coexpression of 2 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.

Classification of human natural killer cells based on migration behavior and cytotoxic response.

Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas 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 5 distinct classes. A small but particularly active subclass 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 in which a small fraction of NK cells drives tumor elimination and inflammation.

Selenite induces posttranscriptional blockade of HLA-E expression and sensitizes tumor cells to CD94/NKG2A-positive NK cells.

CD94/NKG2A is an inhibitory receptor that controls the activity of a large proportion of human NK cells following interactions with the nonclassical HLA class Ib molecule HLA-E expressed on target cells. In this study, we show that selenite (SeO(3)(2-)), an inorganic selenium compound, induces an almost complete loss of cell surface expression of HLA-E on tumor cells of various origins. Selenite abrogated the HLA-E expression at a posttranscriptional level, since selenite exposure led to a dose-dependent decrease in cellular HLA-E protein expression whereas the mRNA levels remained intact. The loss of HLA-E expression following selenite treatment was associated with decreased levels of intracellular free thiols in the tumor cells, suggesting that the reduced HLA-E protein synthesis was caused by oxidative stress. Indeed, HLA-E expression and the level of free thiols remained intact following treatment with selenomethionine, a selenium compound that does not generate oxidative stress. Loss of HLA-E expression, but not of total HLA class I expression, on tumor cells resulted in increased susceptibility to CD94/NK group 2A-positive NK cells. Our results suggest that selenite may be used to potentiate the anti-tumor cytotoxicity in settings of NK cell-based immunotherapies.