An Historical Overview: The Discovery of How NK Cells Can Kill Enemies, Recruit Defense Troops, and More

Natural killer (NK) cells were originally defined as effector lymphocytes of innate immunity characterized by the unique ability of killing tumor and virally infected cells without any prior priming and expansion of specific clones. The \u201cmissing-self\u201d theory, proposed by Klas Karre, the seminal discovery of the first prototypic HLA class I-specific inhibitory receptors, and, later, of the Natural Cytotoxicity Receptors (NCRs) by Alessandro Moretta, provided the bases to understand the puzzling behavior of NK cells. Actually, those discoveries proved crucial also for many of the achievements that, along the years, have contributed to the modern view of these cells. Indeed, NK cells, besides killing susceptible targets, are now known to functionally interact with different immune cells, sense pathogens using TLR, adapt their responses to the local environment, and, even, mount a sort of immunological memory. In this review, we will specifically focus on the main activating NK receptors and on their crucial role in the ever-increasing number of functions assigned to NK cells and other innate lymphoid cells (ILCs)

Haploidentical Haematopoietic Stem Cell Transplantation: Role of NK Cells and Effect of Cytomegalovirus Infections

Natural killer cells play an important role in the immune responses against cancer and viral infections. In addition, NK cells have been shown to exert a key role in haploidentical hematopoietic stem cell (HSC) transplantation for the therapy of high-risk leukemias. The anti-leukemia effect is mostly related to the presence of "alloreactive" NK cells, i.e., mature KIR(+) NK cells that express inhibitory KIR mismatched with HLA class I (KIR-L) of the patient. In addition, an important role is played by certain activating KIR (primarily, but not only, KIR2DS1) upon interaction with their HLA class I ligand (C2 alleles). In general, the presence of activating KIR correlates with a better prognosis. Beside the infusion of "pure" CD34(+) cells, a novel protocol has been recently developed in which depletion of \u3b1\u3b2 T cells and CD19(+) B cells makes it possible to infuse into the patient, together with donor CD34(+) HSCs, important effector cells including mature PB NK cells and \u3b3\u3b4 T cells. Recent studies revealed that cytomegalovirus (CMV) infection/reactivation may induce rapid NK cell maturation and greatly influence the NK receptor repertoire. The remarkable expansion of a subset expressing the activating receptor NKG2C, together with a more efficient virus-specific effector response after rechallenge with CMV (i.e., antigen specificity), and the longevity of the expanded population are all features consistent with an adaptive type of response and support the notion of a memory-like activity of NK cells

Late development of Fc\u3b5R\u3b3neg adaptive natural killer cells upon human cytomegalovirus reactivation in umbilical cord blood transplantation recipients

In human natural killer (NK) cells, human cytomegalovirus (HCMV) has been shown to be a driving force capable of inducing the expansion of a highly differentiated NKG2C+CD57+subset, persisting over time in both HCMV+healthy subjects and umbilical cord blood transplantation (UCBT) recipients experiencing HCMV viral reactivation. In HCMV+healthy subjects, such expanded NK-cells are characterized by epigenetic modifications that modulate their phenotypic and functional characteristics. In particular, an enhanced ADCC activity is detectable in NK cells lacking the signaling protein Fc\u3b5R\u3b3. Timing and mechanisms involved in the acquisition of HCMV-induced, adaptive-like features by NK cells are currently unknown. In this study, we investigated the de novo acquisition of several adaptive features in NK cells developing after UCBT by monitoring NK-cell differentiation for at least 2 years after transplant. In UCBT recipients experiencing HCMV reactivation, a rapid phenotypic reconfiguration occurred resulting in the expected expansion of CD56dimNKG2C+CD57+NK cells. However, while certain HCMV-driven adaptive hallmarks, including high KIR, LILRB1, CD2 and low/negative NKG2A, Siglec-7, and CD161 expression, were acquired early after UCBT (namely by month 6), downregulation of the signaling protein Fc\u3b5R\u3b3 was detected at a later time interval (i.e., by month 12). This feature characterized only a minor fraction of the HCMV-imprinted NKG2C+CD57+CD56dimNK cell subset, while it was detectable in higher proportions of CD57+NK cells lacking NKG2C. Interestingly, in patients developing a hyporesponsive CD56-CD16brightNK-cell subset, Fc\u3b5R\u3b3 downregulation occurred in these cells earlier than in CD56dimNK cells. Our data suggest that the acquisition of a fully "adaptive" profile requires signals that may lack in UCBT recipients and/or longer time is needed to obtain a stable epigenetic reprogramming. On the other hand, we found that both HCMV-induced Fc\u3b5R\u3b3negand Fc\u3b5R\u3b3+NK cells from these patients, display similar CD107a degranulation and IFN-\u3b3 production capabilities in response to different stimuli, thus indicating that the acquisition of specialized effector functions can be achieved before the "adaptation" to HCMV is completed. Our study provides new insights in the process leading to the generation of different adaptive NK-cell subsets and may contribute to develop new approaches for their employment as novel immunotherapeutic tools

Features of memory-like and PD-1+ human NK cell subsets

Human NK cells are distinguished into CD56brightCD16- cells and CD56dimCD16+ cells. These two subsets are conventionally associated with differential functional outcomes and are heterogeneous with respect to the expression of KIR and CD94/NKG2 heterodimers that represent the two major types of HLA class I-specific receptors. Recent studies indicated that immature CD56bright NK cells, homogeneously expressing the inhibitory CD94/NKG2A receptor, are precursors of CD56dim NK cells that in turn during their process of differentiation lose expression of CD94/NKG2A, and subsequentially acquire inhibitory KIRs and LIR-1. The terminally differentiated phenotype of CD56dim cells is marked by the expression of the CD57 molecule that is associated with poor responsiveness to cytokine stimulation, but retained cytolytic capacity.Remarkably, this CD56dimNKG2A-KIR+LIR-1+CD57+ NK cell subset when derived from individuals previously exposed to pathogens, such as HCMV, may contain memory-like NK cells. These cells are generally characterized by an up-regulation of the activating receptor CD94/NKG2C and a down-regulation of the inhibitory receptor Siglec-7. The memory-like NK cells are persistent over-time and display some hallmarks of adaptive immunity, i.e. clonal expansion, more effective anti-tumor and anti-viral immune responses, longevity as well as given epigenetic modifications. Interestingly, unknown co-factors associated to HCMV infection may induce the onset of a recently identified fully mature NK cell subset, characterized by marked downregulation of the activating receptors NKp30 and NKp46 and by the unexpected expression of the inhibitory PD-1 receptor. This phenotype correlates with an impaired anti-tumor NK cell activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction

Analysis of memory-like natural killer cells in human cytomegalovirus-infected children undergoing αβ+T and B cell-depleted hematopoietic stem cell transplantation for hematological malignancies.

We analyzed the impact of human cytomegalovirus infection on the development of natural killer cells in 27 pediatric patients affected by hematological malignancies, who had received a HLA-haploidentical hematopoietic stem cell transplantation, depleted of both α/β+ T cells and B cells. In line with previous studies in adult recipients of umbilical cord blood transplantation, we found that human cytomegalovirus reactivation accelerated the emergence of mature natural killer cells. Thus, most children displayed a progressive expansion of a memory-like natural killer cell subset expressing NKG2C, a putative receptor for human cytomegalovirus, and CD57, a marker of terminal natural killer cell differentiation. NKG2C(+)CD57(+) natural killer cells were detectable by month 3 following hematopoietic stem cell transplantation and expanded until at least month 12. These cells were characterized by high killer Ig-like receptors (KIRs) and leukocyte inhibitory receptor 1 (LIR-1) and low Siglec-7, NKG2A and Interleukin-18Rα expression, killed tumor targets and responded to cells expressing HLA-E (a NKG2C ligand). In addition, they were poor Interferon-γ producers in response to Interleukin-12 and Interleukin-18. The impaired response to these cytokines, together with their highly differentiated profile, may reflect their skewing toward an adaptive condition specialized in controlling human cytomegalovirus. In conclusion, in pediatric patients receiving a type of allograft different from umbilical cord blood transplantation, human cytomegalovirus also induced memory-like natural killer cells, possibly contributing to controlling infections and reinforcing anti-leukemia effects.Investigator Grants n. 15704 (A.M.), 15283 (L.M.), 15925 (A.B.) and Special Project 5×1000 n. 9962 (A.M., L.M. and F.L.) from Associazione Italiana Ricerca sul Cancro; PRIN 2010 (Progetto di Rilevante Interesse Nazionale to F.L., A.M.) and RF-2010-2316606 (L.M., F.L., D.P.) from The Ministry of Health; progetto Cordon de Vie (F.L.) and Progetto Ricerca Ateneo 2013 (M.D.C.)

In depth characterization of human CMV-driven adaptive NK cells through mass spectrometry and flow cytometry analyses

Human Natural killer (NK) cells are a population of innate lymphocytes, usually short-lived and rapidly responding against viral infections and tumors. However, upon cytomegalovirus (CMV) infection, NK cells reveal unexpected adaptive traits typical of T cells, such as enhanced effector function and longevity. CMV-driven adaptive NKs show high NKG2C (i.e. a HLA-E-specific activating receptor) levels, a mature phenotype (CD57+KIR+NKG2A-), epigenetic modifications and could display anti-tumor properties. In view of their longevity and specialized functional features, adaptive NK cells are suitable candidates in cellular immunotherapy. Adaptive NK cells have been investigated so far mainly through cytofluorimetric approaches and transcriptomic studies. Thus, to better characterize and possibly identify novel functional pathways associated to this NK cell subset, we have set up a mass spectrometry proteome analysis on FACS-sorted adaptive NK cell subsets isolated from six CMV-seropositive healthy donors. In particular we compared the proteomic signature of adaptive NKG2C+CD57+ NK cells with conventional NKG2C-CD57- NK cells, i.e. more immature NK cells lacking CMV-driven imprinting. We identified 4618 proteins from NK cell subsets lysates, representing the largest protein catalogue reported so far for NK cells, and observed a sharply different proteome profile in adaptive compared to conventional NK cells. Indeed, we found 165 proteins that were significantly more expressed and 263 proteins that were significantly down-regulated on NKG2C+CD57+ NK cells as compared to NKG2C-CD57- NK cells. The different proteome profile shown by adaptive NK cells confirmed previous data obtained by cytofluorimetric studies, such as higher expression of LILRB1 and lower expression of CD161. On the other hand, our proteome analysis confirmed at the protein level data from transcriptomic studies, such as higher expression of the antiapoptotic protein Bcl-2, possibly responsible for adaptive NK cell longevity, and of the signaling protein CD3e. Based on the hints from mass spectrometry data, we further investigated the most significant differentially expressed proteins through intracellular flow cytometry to validate our results in a larger donors cohort and to explore in detail NK cell subsets not included in the proteomic analysis. We could thus reveal that high Bcl-2 expression mainly characterizes NKG2C+ NK cells including not only adaptive NKG2C+CD57+ but also the less differentiated NKG2C+CD57- NK cells. In addition, we confirmed that cytoplasmic CD3e expression marks adaptive NKG2C+CD57+ NK cells highlighting the phenotypic proximity that this NK cell subset displays with T lymphocytes. Thus, mass spectrometry proteome profiling of adaptive NK cells, combined to cytofluorimetric analyses, has provided further insights in their biology and revealed novel features that could be relevant also to better harness this NK cell subset for immunotherapeutic purposes

Proteomic characterization of human CMV-driven adaptive NK cells

Human Natural killer (NK) cells are a lymphocyte population of innate immunity, usually short-living and capable of providing rapid responses against viral infections and tumors without prior sensitization. However, upon cytomegalovirus (CMV) infection, human NK cells reveal unexpected adaptive traits, sharing features typical of T cells, such as enhanced effector function and long-term survival in response to a viral challenge (1). These peculiar CMV-driven NK cells, indicated as adaptive or memory NK cells, can be found to varying degrees in both healthy CMV-seropositive individuals and transplanted patients undergoing CMV infection/reactivation (2). CMV-driven adaptive NK cells are characterized by the expression of the specific HLA-E activating receptor NKG2C, a mature phenotype (CD57+KIR+NKG2A-), epigenetic modifications and could display anti-tumor properties (3). In view of their longevity and specialized functional features, adaptive NK cells represent suitable candidates to be exploited in cellular immunotherapy against cancer and viral infections. Adaptive NK cells have been investigated so far mainly through cytofluorimetric approaches and transcriptomic studies. Thus, to better characterize and possibly identify novel functional pathways associated to this CMV-driven NK cell population, we have set up a mass spectrometry proteome analysis on FACS-sorted adaptive NK cell subsets isolated from six CMV-seropositive healthy donors. In particular we compared the proteomic signature of adaptive NKG2C+CD57+ NK cells with conventional NKG2C-CD57- NK cells, i.e. more immature NK cells lacking CMV-driven imprinting. We identified 4618 proteins from NK cell subsets lysates, representing the largest protein catalogue reported so far for NK cells, and observed a sharply different proteome profile in adaptive compared to conventional NK cells. Indeed, we found 165 proteins that were significantly more expressed in adaptive NKG2C+ CD57+ NK cells as compared to conventional NKG2C-CD57- NK cells, with 75 proteins showing a remarkably high fold change. Interestingly, 263 proteins were significantly down-regulated on NKG2C+ CD57+ NK cells as compared to NKG2C-CD57- NK cells. The different proteome profile shown by adaptive NK cells confirmed previous data obtained by cytofluorimetric studies, such as higher expression of the inhibitory receptor LILRB1 and lower expression of CD161 characterizing adaptive NK cells. On the other hand, our proteome analysis confirmed the expression of molecules at the protein level that had been described only by transcriptomic studies, such as higher expression levels of the antiapoptotic protein Bcl-2 that is possibly responsible for long-term persistence of adaptive NK cells. Interestingly, several differentially expressed proteins have not been described in NK cell biology so far. Indeed, further analyses through the “Human Base tissue-specific network-based functional interpretation of genes and gene sets” tool revealed a significant enrichment in adaptive NK cells, compared to conventional NK cells, of proteins belonging to pathways related to regulating cell activation and proliferation (e.g. CCL5, LGALS3, LILRB1, CD3E, MNDA), involved in endosomal trafficking and cytoskeletal organization (e.g. CHMP2B, ARFGEF1, ATL1), regulating RNA transport from the nucleus to cytoplasm (e.g. FYTTD1, ATXN1), regulating viral processes/responses (e.g. TRIM14, OASL2, RELA), involved in autophagy (e.g. ATG13, GOLGA2) and also in ribosome biogenesis (e.g. ZNHIT6,EMG1). Thus, mass spectrometry proteome profiling of adaptive NK cells, combined to cytofluorimetric analyses and compared to transcriptomic data, has provided further insights in their biology and revealed novel features that could be relevant also to better harness this NK cell subset for immunotherapeutic purposes

Late Development of FcεRγneg Adaptive Natural Killer Cells Upon Human Cytomegalovirus Reactivation in Umbilical Cord Blood Transplantation Recipients

In human natural killer (NK) cells, human cytomegalovirus (HCMV) has been shown to be a driving force capable of inducing the expansion of a highly differentiated NKG2C+CD57+ subset, persisting over time in both HCMV+ healthy subjects and umbilical cord blood transplantation (UCBT) recipients experiencing HCMV viral reactivation. In HCMV+ healthy subjects, such expanded NK-cells are characterized by epigenetic modifications that modulate their phenotypic and functional characteristics. In particular, an enhanced ADCC activity is detectable in NK cells lacking the signaling protein FcεRγ. Timing and mechanisms involved in the acquisition of HCMV-induced, adaptive-like features by NK cells are currently unknown. In this study, we investigated the de novo acquisition of several adaptive features in NK cells developing after UCBT by monitoring NK-cell differentiation for at least 2 years after transplant. In UCBT recipients experiencing HCMV reactivation, a rapid phenotypic reconfiguration occurred resulting in the expected expansion of CD56dim NKG2C+CD57+ NK cells. However, while certain HCMV-driven adaptive hallmarks, including high KIR, LILRB1, CD2 and low/negative NKG2A, Siglec-7, and CD161 expression, were acquired early after UCBT (namely by month 6), downregulation of the signaling protein FcεRγ was detected at a later time interval (i.e., by month 12). This feature characterized only a minor fraction of the HCMV-imprinted NKG2C+CD57+ CD56dim NK cell subset, while it was detectable in higher proportions of CD57+ NK cells lacking NKG2C. Interestingly, in patients developing a hyporesponsive CD56−CD16bright NK-cell subset, FcεRγ downregulation occurred in these cells earlier than in CD56dim NK cells. Our data suggest that the acquisition of a fully “adaptive” profile requires signals that may lack in UCBT recipients and/or longer time is needed to obtain a stable epigenetic reprogramming. On the other hand, we found that both HCMV-induced FcεRγneg and FcεRγ+ NK cells from these patients, display similar CD107a degranulation and IFN-γ production capabilities in response to different stimuli, thus indicating that the acquisition of specialized effector functions can be achieved before the “adaptation” to HCMV is completed. Our study provides new insights in the process leading to the generation of different adaptive NK-cell subsets and may contribute to develop new approaches for their employment as novel immunotherapeutic tools

data_sheet_1_Late Development of FcεRγneg Adaptive Natural Killer Cells Upon Human Cytomegalovirus Reactivation in Umbilical Cord Blood Transplantation Recipients.PDF

<p>In human natural killer (NK) cells, human cytomegalovirus (HCMV) has been shown to be a driving force capable of inducing the expansion of a highly differentiated NKG2C⁺CD57⁺ subset, persisting over time in both HCMV⁺ healthy subjects and umbilical cord blood transplantation (UCBT) recipients experiencing HCMV viral reactivation. In HCMV⁺ healthy subjects, such expanded NK-cells are characterized by epigenetic modifications that modulate their phenotypic and functional characteristics. In particular, an enhanced ADCC activity is detectable in NK cells lacking the signaling protein FcεRγ. Timing and mechanisms involved in the acquisition of HCMV-induced, adaptive-like features by NK cells are currently unknown. In this study, we investigated the de novo acquisition of several adaptive features in NK cells developing after UCBT by monitoring NK-cell differentiation for at least 2 years after transplant. In UCBT recipients experiencing HCMV reactivation, a rapid phenotypic reconfiguration occurred resulting in the expected expansion of CD56^dim NKG2C⁺CD57⁺ NK cells. However, while certain HCMV-driven adaptive hallmarks, including high KIR, LILRB1, CD2 and low/negative NKG2A, Siglec-7, and CD161 expression, were acquired early after UCBT (namely by month 6), downregulation of the signaling protein FcεRγ was detected at a later time interval (i.e., by month 12). This feature characterized only a minor fraction of the HCMV-imprinted NKG2C⁺CD57⁺ CD56^dim NK cell subset, while it was detectable in higher proportions of CD57⁺ NK cells lacking NKG2C. Interestingly, in patients developing a hyporesponsive CD56⁻CD16^bright NK-cell subset, FcεRγ downregulation occurred in these cells earlier than in CD56^dim NK cells. Our data suggest that the acquisition of a fully “adaptive” profile requires signals that may lack in UCBT recipients and/or longer time is needed to obtain a stable epigenetic reprogramming. On the other hand, we found that both HCMV-induced FcεRγ^neg and FcεRγ⁺ NK cells from these patients, display similar CD107a degranulation and IFN-γ production capabilities in response to different stimuli, thus indicating that the acquisition of specialized effector functions can be achieved before the “adaptation” to HCMV is completed. Our study provides new insights in the process leading to the generation of different adaptive NK-cell subsets and may contribute to develop new approaches for their employment as novel immunotherapeutic tools.</p