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•Largest and most diverse genetic study of plasma fibrinogen identifies 54 regions (18 novel), housing 69 distinct variants (20 novel).•Links to (1) liver enzyme, blood cell, and lipid genetic signals, (2) liver regulatory elements, and (3) thrombotic and inflammatory disease.

Neutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.

Neutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56brightNK cells and a lack of mature CD56dimNK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56brightsignatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.

Analysis of measurable residual disease (MRD) is a powerful tool for assessment of treatment response in acute myeloid leukemia (AML). In patients with mutation in NPM1, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is recommended by European Leukemia Net for refinement of risk stratification and monitoring after treatment. However, since RT-qPCR is mutation-specific and requires stringent quality control, many clinical laboratories restrict NPM1monitoring to the type A mutation (c.860_863dupTCTG) although >50 mutations have been reported in exon 12. Deep sequencing is an alternative method, performed on DNA, that covers all NPM1exon 12variants in the same assay. Guidelines for its clinical use are however lacking. We here performed a retrospective analysis in a population-based cohort of AML patients to evaluate if deep sequencing MRD analysis of NPM1during chemotherapy provides prognostic information.

Human natural killer (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 virus infections. By determining the human “KIR-ome” at a single-cell level in more than 200 donors, we were able to analyze the magnitude of NK cell adaptation to virus 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 the diversity of KIR repertoire and its adaptation to virus infection, suggesting a role for both activating and inhibitory KIRs in immunity to CMV infection.

Human natural killer (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 virus infections. By determining the human “KIR-ome” at a single-cell level in more than 200 donors, we were able to analyze the magnitude of NK cell adaptation to virus 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 the diversity of KIR repertoire and its adaptation to virus infection, suggesting a role for both activating and inhibitory KIRs in immunity to CMV infection.

Inhibitory killer cell immunoglobulin-like receptors (KIRs) preserve tolerance to self and shape the functional response of human natural killer (NK) cells. Here, we have evaluated the influence of selection processes in the formation of inhibitory KIR repertoires in a cohort of 44 donors homozygous for the group A KIRhaplotype. Coexpression of multiple KIRs was more frequent than expected by the product rule that describes random association of independent events. In line with this observation, the probability of KIR acquisition increased with the cellular expression of KIRs. Three types of KIR repertoires were distinguished that differed in frequencies of KIR- and NKG2A-positive cells but showed no dependency on the number of self-HLA class I ligands. Furthermore, the distribution of self- and nonself-KIRs at the cell surface reflected a random combination of receptors rather than a selection process conferred by cognate HLA class I molecules. Finally, NKG2A was found to buffer overall functional responses in KIR repertoires characterized by low-KIR expression frequencies. The results provide new insights into the formation of inhibitory KIR repertoires on human NK cells and support a model in which variegated KIR repertoires are generated through sequential and random acquisition of KIRs in the absence of selection.

Inhibitory killer cell immunoglobulin-like receptors (KIRs) preserve tolerance to self and shape the functional response of human natural killer (NK) cells. Here, we have evaluated the influence of selection processes in the formation of inhibitory KIR repertoires in a cohort of 44 donors homozygous for the group A KIR haplotype. Coexpression of multiple KIRs was more frequent than expected by the product rule that describes random association of independent events. In line with this observation, the probability of KIR acquisition increased with the cellular expression of KIRs. Three types of KIR repertoires were distinguished that differed in frequencies of KIR- and NKG2A-positive cells but showed no dependency on the number of self-HLA class I ligands. Furthermore, the distribution of self- and nonself-KIRs at the cell surface reflected a random combination of receptors rather than a selection process conferred by cognate HLA class I molecules. Finally, NKG2A was found to buffer overall functional responses in KIR repertoires characterized by low-KIR expression frequencies. The results provide new insights into the formation of inhibitory KIR repertoires on human NK cells and support a model in which variegated KIR repertoires are generated through sequential and random acquisition of KIRs in the absence of selection.

A growing body of evidence suggests that the human natural killer (NK)-cell compartment is phenotypically and functionally heterogeneous and is composed of several differentiation stages. Moreover, NK-cell subsets have been shown to exhibit adaptive immune features during herpes virus infection in experimental mice and to expand preferentially 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 CD56dim NKG2A+ immunoglobulin-like receptor- NK cells. Moreover, this NK-cell subset exhibits features of terminal differentiation and persists at higher frequency during at least the first 6 months after acute IM. Finally, we demonstrate that this NK-cell subset preferentially degranulates and proliferates on 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.

A growing body of evidence suggests that the human natural killer (NK)-cell compartment is phenotypically and functionally heterogeneous and is composed of several differentiation stages. Moreover, NK-cell subsets have been shown to exhibit adaptive immune features during herpes virus infection in experimental mice and to expand preferentially 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 CD56dimNKG2A+immunoglobulin-like receptor-NK cells. Moreover, this NK-cell subset exhibits features of terminal differentiation and persists at higher frequency during at least the first 6 months after acute IM. Finally, we demonstrate that this NK-cell subset preferentially degranulates and proliferates on 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.

Allogeneic hematopoietic stem cell transplantation (HSCT) is widely used to treat hematopoietic cell disorders but is often complicated by graft-versus-host disease (GVHD), which causes severe epithelial damage. Here we have investigated longitudinally the effects of induction chemotherapy, conditioning radiochemotherapy, and allogeneic HSCT on composition, phenotype, and recovery of circulating innate lymphoid cells (ILCs) in 51 acute leukemia patients. We found that reconstitution of ILC1, ILC2, and NCR−ILC3 was slow compared with that of neutrophils and monocytes. NCR+ ILC3 cells, which are not present in the circulation of healthy persons, appeared both after induction chemotherapy and after allogeneic HSCT. Circulating patient ILCs before transplantation, as well as donor ILCs after transplantation, expressed activation (CD69), proliferation (Ki-67), and tissue homing markers for gut (α4β7, CCR6) and skin (CCR10 and CLA). The proportion of ILCs 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.

Allogeneic hematopoietic stem cell transplantation (HSCT) is widely used to treat hematopoietic cell disorders but is often complicated by graft-versus-host disease (GVHD), which causes severe epithelial damage. Here we have investigated longitudinally the effects of induction chemotherapy, conditioning radiochemotherapy, and allogeneic HSCT on composition, phenotype, and recovery of circulating innate lymphoid cells (ILCs) in 51 acute leukemia patients. We found that reconstitution of ILC1, ILC2, and NCR−ILC3 was slow compared with that of neutrophils and monocytes. NCR+ILC3 cells, which are not present in the circulation of healthy persons, appeared both after induction chemotherapy and after allogeneic HSCT. Circulating patient ILCs before transplantation, as well as donor ILCs after transplantation, expressed activation (CD69), proliferation (Ki-67), and tissue homing markers for gut (α4β7, CCR6) and skin (CCR10 and CLA). The proportion of ILCs 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.

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.

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.

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.

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.

Epistatic interactions between killer cell immunoglobulin-like receptors (KIRs) and their cognate HLA class I ligands have important implications for reproductive success, antiviral immunity, susceptibility to autoimmune conditions and cancer, as well as for graft-versus-leukemia reactions in settings of allogeneic stem cell transplantation. Although CD8 T cells are known to acquire KIRs when maturing from naive to terminally differentiated cells, little information is available about the constitution of KIR repertoires on human CD8 T cells. Here, we have performed a high-resolution analysis of KIR expression on CD8 T cells. The results show that most CD8 T cells possess a restricted KIR expression pattern, often dominated by a single activating or inhibitory KIR. Furthermore, the expression of KIR, and its modulation of CD8 T-cell function, was independent of expression of self-HLA class I ligands. Finally, despite similarities in the stochastic regulation of KIRs by the bidirectional proximal promoter, the specificity of inhibitory KIRs on CD8 T cells was often distinct from that of natural killer cells in the same individual. The results provide new insight into the formation of KIR repertoires on human T cells.

Epistatic interactions between killer cell immunoglobulin-like receptors (KIRs) and their cognate HLA class I ligands have important implications for reproductive success, antiviral immunity, susceptibility to autoimmune conditions and cancer, as well as for graft-versus-leukemia reactions in settings of allogeneic stem cell transplantation. Although CD8 T cells are known to acquire KIRs when maturing from naive to terminally differentiated cells, little information is available about the constitution of KIR repertoires on human CD8 T cells. Here, we have performed a high-resolution analysis of KIR expression on CD8 T cells. The results show that most CD8 T cells possess a restricted KIR expression pattern, often dominated by a single activating or inhibitory KIR. Furthermore, the expression of KIR, and its modulation of CD8 T-cell function, was independent of expression of self-HLA class I ligands. Finally, despite similarities in the stochastic regulation of KIRs by the bidirectional proximal promoter, the specificity of inhibitory KIRs on CD8 T cells was often distinct from that of natural killer cells in the same individual. The results provide new insight into the formation of KIR repertoires on human T cells.

Natural killer (NK) cells are lymphocytes of the innate immune system that, following differentiation from CD56brightto CD56dimcells, have been thought to retain fixed functional and phenotypic properties throughout their lifespan. In contrast to this notion, we here show that CD56dimNK cells continue to differentiate. During this process, they lose expression of NKG2A, sequentially acquire inhibitory killer cell inhibitory immunoglobulin-like receptors and CD57, change their expression patterns of homing molecules, and display a gradual decline in proliferative capacity. All cellular intermediates of this process are represented in varying proportions at steady state and appear, over time, during the reconstitution of the immune system, as demonstrated in humanized mice and in patients undergoing hematopoietic stem cell transplantation. CD56dimNK-cell differentiation, and the associated functional imprint, occurs independently of NK-cell education by interactions with self–human leukocyte antigen class I ligands and is an essential part of the formation of human NK-cell repertoires.

Natural killer (NK) cells are lymphocytes of the innate immune system that, following differentiation from CD56bright to CD56dim cells, have been thought to retain fixed functional and phenotypic properties throughout their lifespan. In contrast to this notion, we here show that CD56dim NK cells continue to differentiate. During this process, they lose expression of NKG2A, sequentially acquire inhibitory killer cell inhibitory immunoglobulin-like receptors and CD57, change their expression patterns of homing molecules, and display a gradual decline in proliferative capacity. All cellular intermediates of this process are represented in varying proportions at steady state and appear, over time, during the reconstitution of the immune system, as demonstrated in humanized mice and in patients undergoing hematopoietic stem cell transplantation. CD56dim NK-cell differentiation, and the associated functional imprint, occurs independently of NK-cell education by interactions with self–human leukocyte antigen class I ligands and is an essential part of the formation of human NK-cell repertoires.

Natural killer (NK)–cell alloreactivity in recipients of hematopoietic stem cell grafts from HLA-identical siblings is intriguing and has suggested breaking of NK-cell tolerance during the posttransplantation period. To examine this possibility, we analyzed clinical outcomes in a cohort of 105 patients with myeloid malignancies who received T cell–replete grafts from HLA-matched sibling donors. Presence of inhibitory killer cell immunoglobulin-like receptors (KIRs) for nonself HLA class I ligands had no effect on disease-free survival, incidence of relapse, or graft-versus-host disease. A longitudinal analysis of the NK-cell repertoire and function revealed a global hyporesponsiveness of NK cells early after transplantation. Functional responses recovered at approximately 6 months after transplantation. Importantly, NKG2A− NK cells expressing KIRs for nonself HLA class I ligands remained tolerant at all time points. Furthermore, a direct comparison of NK-cell reconstitution in T cell–replete and T cell–depleted HLA-matched sibling stem cell transplantation (SCT) revealed that NKG2A+ NK cells dominated the functional repertoire early after transplantation, with intact tolerance of NKG2A− NK cells expressing KIRs for nonself ligands in both settings. Our results provide evidence against the emergence of alloreactive NK cells in HLA-identical allogeneic SCT.

Natural killer (NK)–cell alloreactivity in recipients of hematopoietic stem cell grafts from HLA-identical siblings is intriguing and has suggested breaking of NK-cell tolerance during the posttransplantation period. To examine this possibility, we analyzed clinical outcomes in a cohort of 105 patients with myeloid malignancies who received T cell–replete grafts from HLA-matched sibling donors. Presence of inhibitory killer cell immunoglobulin-like receptors (KIRs) for nonself HLA class I ligands had no effect on disease-free survival, incidence of relapse, or graft-versus-host disease. A longitudinal analysis of the NK-cell repertoire and function revealed a global hyporesponsiveness of NK cells early after transplantation. Functional responses recovered at approximately 6 months after transplantation. Importantly, NKG2A−NK cells expressing KIRs for nonself HLA class I ligands remained tolerant at all time points. Furthermore, a direct comparison of NK-cell reconstitution in T cell–replete and T cell–depleted HLA-matched sibling stem cell transplantation (SCT) revealed that NKG2A+NK cells dominated the functional repertoire early after transplantation, with intact tolerance of NKG2A−NK cells expressing KIRs for nonself ligands in both settings. Our results provide evidence against the emergence of alloreactive NK cells in HLA-identical allogeneic SCT.

Expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self–major histocompatibility complex (MHC) class I molecules provides an educational signal that generates functional natural killer (NK) cells. However, the effects of activating KIRs specific for self-MHC class I on NK-cell education remain elusive. Here, we provide evidence that the activating receptor KIR2DS1 tunes down the responsiveness of freshly isolated human NK cells to target cell stimulation in donors homozygous for human leukocyte antigen (HLA)–C2, the ligand of KIR2DS1. The tuning was apparent in KIR2DS1+ NK cells lacking expression of inhibitory KIRs and CD94/NKG2A, as well as in KIR2DS1+ NK cells coexpressing the inhibitory MHC class I–specific receptors CD94/NKG2A and KIR2DL3, but not KIR2DL1. However, the tuning of responsiveness was restricted to target cell recognition because KIR2DS1+ NK cells responded well to stimulation with exogenous cytokines. Our results provide the first example of human NK-cell education by an activating KIR and suggest that the education of NK cells via activating KIRs is a mechanism to secure tolerance that complements education via inhibitory KIRs.

Expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self–major histocompatibility complex (MHC) class I molecules provides an educational signal that generates functional natural killer (NK) cells. However, the effects of activating KIRs specific for self-MHC class I on NK-cell education remain elusive. Here, we provide evidence that the activating receptor KIR2DS1 tunes down the responsiveness of freshly isolated human NK cells to target cell stimulation in donors homozygous for human leukocyte antigen (HLA)–C2, the ligand of KIR2DS1. The tuning was apparent in KIR2DS1+NK cells lacking expression of inhibitory KIRs and CD94/NKG2A, as well as in KIR2DS1+NK cells coexpressing the inhibitory MHC class I–specific receptors CD94/NKG2A and KIR2DL3, but not KIR2DL1. However, the tuning of responsiveness was restricted to target cell recognition because KIR2DS1+NK cells responded well to stimulation with exogenous cytokines. Our results provide the first example of human NK-cell education by an activating KIR and suggest that the education of NK cells via activating KIRs is a mechanism to secure tolerance that complements education via inhibitory KIRs.

Malmberg: Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.

Goodridge: Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Mahmood:Fate Therapeutics, Inc: Current Employment. Reiser:FATE THERAPEUTICS: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Blum:Fate Therapeutics: Current Employment. Cichocki:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Chu:Fate Therapeutics, Inc: Current Employment. Bonello:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Groff:Fate Therapeutics, Inc: Current Employment. Meza:Fate Therapeutics, Inc: Current Employment. Chu:Roche Holding AG: Current equity holder in publicly-traded company; Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Vycellix: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Patents & Royalties. Miller:Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Neutrophils are innate cells that have been suggested to play a critical role in terminal differentiation of NK cells. Whether this is a direct effect or a consequence of global immune changes with effects on NK cell homeostasis remains unknown. Here, we used high-resolution flow and mass cytometry to examine NK cell repertoires in 64 neutropenic patients and 27 healthy age- and gender-matched controls. A subgroup of neutropenic patients had lower frequencies and absolute numbers of NK cells, yet increased frequencies of CD56brightamong NK cells (Figure 1A-C). Moreover, their CD56dimcompartment was characterized by a block in differentiation, with a relative lack of NKG2A-CD57+KIR+NK cells. In line with the differentiation arrest, no expansion of adaptive NK cells could be detected in CMV-seropositive patients from this subgroup. Furthermore, CD56dimNK cells showed increased frequencies of Ki-67+, Tim-3+and TIGIT+cells suggestive of activation and exhaustion (Figure 1D). The systemic imprint in the NK cell repertoire was associated with a blunted tumor target cell response with inefficient killing and a lower proportion of degranulating CD56dimcells (Figure 1E). RNA sequencing of the NK cell compartment further revealed that the differentiation arrest was linked to increased expression of transcription factors and genes involved in proliferation and cytokine signaling (Figure 1F). Serum protein profiling of 264 proteins showed upregulation of pathways related to apoptosis and cell turnover, as well as immune regulation and inflammation including higher levels of IL-10, IL-18 and IL-27 in these patients (Figure 1G-H). Notably, the majority of patients with perturbed NK cell compartment exhibited high-grade neutropenia, overall suggesting that the profoundly altered NK cell homeostasis was tightly connected to the severity of their underlying etiology (Figure 1I).

Natural Killer (NK) cells play an essential role in cancer surveillance and have a unique capability of spontaneous cytotoxicity against cancer cells. The human NK cell repertoire is functionally diversified through a tightly regulated differentiation process characterized by an early transition from CD56bright to CD56dim NK cells, followed by coordinated changes in expression of inhibitory receptors, including NKG2A and killer cell immunoglobulin-like receptors (KIR). The acquisition of self HLA class I binding KIRs during NK cell differentiation tunes the cytotoxic potential of NK cells in a process termed education, characterized by increased loading of granzyme B in dense core granules. Although NK cell differentiation and education are critical determinants of the functional potential of the cell, little is known about how these events shape the migratory behavior of NK cells.

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against cancer. A unique benefit of iPSC-derived immune effector cells is the possibility to perform multiple precision editing steps at the single cell level to achieve a homogenous effector cell population tailored to target a desired cancer type and equipped with selected functional properties. These functional edits are superimposed on the innate reactivity of NK cells to stress ligands and MHC downregulation (missing self). The ability of NK cells to sense missing self is based on a functional calibration to self MHC during a process termed NK cell education, the latter being critically dependent on signaling through inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). Whereas the process of NK cell differentiation into mature effector cells from iPSCs has been well characterized, the role of natural variation in inhibitory receptor expression and NK cell education remains poorly defined in iNK cells.

Multiple redundancy within the spectrum of an immune response is required to prevent antigen escape or adaptation of the targeted population to host defenses. As adoptive cell therapies continue to evolve, multi-modal engineering of effector cells offers the prospect of tackling increasingly complex disease settings such as multiple myeloma (MM), where targeting of a single tumor associated antigen is frequently confounded by antigen shedding and escape variation resulting in the inability to develop a curative therapy. There are multiple advantages in expanding treatment options beyond autologous primary T and NK cells, including the use of induced pluripotent stem cells (iPSC) to derive effector cells that can be uniformly manufactured at scale from renewable starting cellular material and where precision genetic engineering can be achieved at the clonal level which can be applied sequentially in order to build multiple specificities and functional modalities. To create a platform targeted toward MM, a multiplexed edited base iPSC-derived NK (iNK) cell configuration consisting of a CD38 KO iPSC modified to overexpress a recombinant IL-15 signaling complex (IL15RF) for autonomous persistence and a functionally enhanced high-affinity, non-cleavable CD16 (hnCD16) was developed. Introduction of IL15RF enabled expansion of iNK cells without additional exogenous cytokine support during the manufacturing process and greatly improved functional persistence of iNK cells both in vitroand in various xenograft mouse models (Figure 1).

Close surveillance of measurable residual disease (MRD) following completion of therapy for acute myeloid leukemia (AML) enables early detection of relapse in time for pre-emptive treatment. Today this is available only for the fraction of patients with recurrent targets quantifiable with standardized RT-qPCR. Patient-tailored MRD monitoring based on leukemia-specific mutations may be an attractive option in patients lacking such aberrations. In this population-based study, we used whole exome sequencing (WES) to identify leukemia-specific mutations suitable for MRD monitoring, and targeted deep sequencing (deep seq) and droplet digital PCR (ddPCR) for quantitative monitoring in children with AML. We investigated the clinical applicability of patient-tailored deep seq and ddPCR for early detection of AML relapse in peripheral blood (PB).

Allogeneic off-the-shelf cell therapies offer distinct advantages over conventional autologous cell therapies in terms of scaled manufacturing, on-demand availability and optimization of cellular starting material. A unique consideration in the use of allogeneic cell therapies is the potential for immune cell-mediated recognition of the allogeneic cell product by the patient's immune system. CAR T-cell therapies are commonly combined with conditioning chemotherapies that suppress a patient's immune system, creating a suitable window of activity to elicit clinical response. However, protracted lympho-conditioning also affects immune reconstitution and can negatively impact the rate of infection. Alternative approaches to prevent allorejection may therefore help to enhance the efficacy of the therapy while preserving the immune system of the patient.

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies, however, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility to patients. Many of these challenges can be overcome with an allogeneic cellular product that is available off-the-shelf, and overcoming immune cell-mediated rejection of allogeneic cell therapy is an area of significant research. Conditioning chemotherapies, which are commonly administered with CAR T-cell therapy, suppress a patient's immune system and may create a suitable window of activity for allogeneic cell therapies to elicit clinical response. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Deletion of human leukocyte antigen (HLA) surface expression is known to abrogate T-cell alloreactivity, but deletion of class I HLA must be combined with other immune-modulating strategies to avoid NK cell-mediated recognition.

Williams: Fate Therapeutics: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Lee: Fate Therapeutics, Inc.: Current Employment. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Shouval: Medexus: Consultancy. Kroeger: AOP Pharma: Honoraria; Gilead/Kite: Honoraria; Riemser: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Sanofi: Honoraria; Neovii: Honoraria, Research Funding; Novartis: Honoraria. Horowitz: Daiicho Sankyo: Research Funding; Allovir: Consultancy; Miltenyi Biotech: Research Funding; Medac: Research Funding; Kite/Gilead: Research Funding; Genentech: Research Funding; Jazz Pharmaceuticals: Research Funding; Janssen: Research Funding; Kiadis: Research Funding; CSL Behring: Research Funding; Gamida Cell: Research Funding; bluebird bio: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Research Funding; Astellas: Research Funding; Chimerix: Research Funding; GlaxoSmithKline: Research Funding; Novartis: Research Funding; Magenta: Consultancy, Research Funding; Actinium: Research Funding; Mesoblast: Research Funding; Omeros: Research Funding; Orca Biosystems: Research Funding; Pfizer, Inc: Research Funding; Pharmacyclics: Research Funding; Regeneron: Research Funding; Sanofi: Research Funding; Seattle Genetics: Research Funding; Shire: Research Funding; Sobi: Research Funding; Stemcyte: Research Funding; Takeda: Research Funding; Tscan: Research Funding; Vertex: Research Funding; Vor Biopharma: Research Funding; Xenikos: Research Funding. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Miller: Sanofi: Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Wugen: Membership on an entity's Board of Directors or advisory committees. Mohty: Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Jazz: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria; Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Adaptive Biotechnologies: Honoraria. Romee: Crispr Therapeutics: Research Funding; Glycostem: Membership on an entity's Board of Directors or advisory committees. Schetelig: Roche: Honoraria, Other: lecture fees; Novartis: Honoraria, Other: lecture fees; BMS: Honoraria, Other: lecture fees; Abbvie: Honoraria, Other: lecture fees; AstraZeneca: Honoraria, Other: lecture fees; Gilead: Honoraria, Other: lecture fees; Janssen: Honoraria, Other: lecture fees . Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding. Koreth: Biolojic Design: Other: Scientific Advisory Board; Mallinckrodt: Other: Scientific Advisory Board; Cugene: Other: Scientific Advisory Board; Moderna: Consultancy; Amgen: Consultancy; EMD Serono/Merck: Consultancy; Gentibio Inc.: Consultancy; Miltenyi Biotec: Research Funding; BMS: Research Funding; Clinigen Labs: Research Funding; Regeneron: Research Funding; Equillium: Research Funding.

Williams: Fate Therapeutics: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Mamonkin: Beam Therapeutics: Other: Licensing payments; Fate Therapeutics: Other: Licensing payments; Allogene Therapeutics: Consultancy, Other: Licensing payments; Xenetic Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Abrahamsson: wedish Children´s Cancer Foundation. Research grants and 50% senior research position for clinical research on pediatric leukemia: Research Funding.

Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Fong:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:CytoSen: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

Olweus: Gilead Kite: Research Funding; Intellia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Wahlin:Roche and Gilead: Consultancy. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse). Holte:Novartis: Honoraria, Other: Advisory board. Kolstad:Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Global gene expression profiling of the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL) has revealed broad innate immune signatures that distinguish the heterogeneous disease subtypes and correlate with good treatment outcome. However, we still lack tools to identify the relatively large group of patients that are refractory to initial therapy and have a dismal prognosis. Here, we used mass cytometry and serum profiling in a systems-level approach to analyze immune responses in 36 patients with aggressive B cell lymphoma and age- and sex-matched healthy controls. Stochastic neighbor embedding (t-SNE) analysis of protein profiles divided patients into two distinct clusters, with cluster 2 representing patients with a more severe deviation in their protein expression compared to healthy controls. Patients in cluster 2 showed a more dramatic perturbation of their immune cell repertoires with expansion of myeloid-derived suppressor cells (MDSCs), increased T cell differentiation and significantly higher expression of metabolic markers such as GLUT-1 and activation markers, including Ki67, CD38 and PD-1. An extended analysis of serum protein profiles in two independent cohorts (n=69 and n=80 patients, respectively) revealed that that the identified systemic immune signatures were linked to poor progression free survival (PFS) and inferior overall survival (OS). Immune monitoring during chemo-immunotherapy showed that most patients normalized their serum protein profiles. Notably, non-responding patients retained higher than normal expression of several proteins, including PD-L1, CD70, IL-18, granzyme A and CD83. These studies demonstrate distinct patterns of disease-driven alterations in the systemic immune response of DLBCL patients that are associated with poor survival and persist in patients who are refractory to therapy.

Goodridge: FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Bjordahl:Fate Therapeutics, Inc.: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Chu:FATE THERAPEUTICS: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Groff:FATE THERAPEUTICS: Employment. Meza:FATE THERAPEUTICS: Employment. Malmberg:Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Consultancy, Research Funding. Miller:Moderna: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; CytoSen: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.