Natural Killer Lineage Research Articles

Eomes expression identifies the early bone marrow precursor to classical NK cells.

Natural killer (NK) cells traffic through the blood and mount cytolytic and interferon-γ (IFNγ)-focused responses to intracellular pathogens and tumors. Type 1 innate lymphoid cells (ILC1s) also produce type 1 cytokines but reside in tissues and are not cytotoxic. Whether these differences reflect discrete lineages or distinct states of a common cell type is not understood. Using single-cell RNA sequencing and flow cytometry, we focused on populations of TCF7+ cells that contained precursors for NK cells and ILC1s and identified a subset of bone marrow lineage-negative NK receptor-negative cells that expressed the transcription factor Eomes, termed EomeshiNKneg cells. Transfer of EomeshiNKneg cells into Rag2-/-Il2rg-/- recipients generated functional NK cells capable of preventing metastatic disease. By contrast, transfer of PLZF+ ILC precursors generated a mixture of ILC1s, ILC2s and ILC3s that lacked cytotoxic potential. These findings identified EomeshiNKneg cells as the bone marrow precursor to classical NK cells and demonstrated that the NK and ILC1 lineages diverged early during development.

A Genetic Bottleneck of Mitochondrial DNA During Human Lymphocyte Development.

Mitochondria are essential organelles in eukaryotic cells that provide critical support for energetic and metabolic homeostasis. Although the elimination of pathogenic mitochondrial DNA (mtDNA) mutations in somatic cells has been observed, the mechanisms to maintain proper functions despite their mtDNA mutation load are poorly understood. In this study, we analyzed somatic mtDNA mutations in more than 30,000 single human peripheral and bone marrow mononuclear cells. We observed a significant overrepresentation of homoplasmic mtDNA mutations in B, T, and natural killer (NK) lymphocytes. Intriguingly, their overall mutational burden was lower than that in hematopoietic progenitors and myeloid cells. This characteristic mtDNA mutational landscape indicates a genetic bottleneck during lymphoid development, as confirmed with single-cell datasets from multiple platforms and individuals. We further demonstrated that mtDNA replication lags behind cell proliferation in both pro-B and pre-B progenitor cells, thus likely causing the genetic bottleneck by diluting mtDNA copies per cell. Through computational simulations and approximate Bayesian computation (ABC), we recapitulated this lymphocyte-specific mutational landscape and estimated the minimal mtDNA copies as <30 in T, B, and NK lineages. Our integrative analysis revealed a novel process of a lymphoid-specific mtDNA genetic bottleneck, thus illuminating a potential mechanism used by highly metabolically active immune cells to limit their mtDNA mutation load.

Dissecting the NK Cell Population in Hematological Cancers Confirms the Presence of Tumor Cells and Their Impact on NK Population Function.

The lymphocyte lineage natural killer (NK) cell is part of the innate immune system and protects against pathogens and tumor cells. NK cells are the main cell effectors of the monoclonal antibodies (mAbs) that mediates antibody-dependent cell cytotoxicity (ADCC). Hence, it is relevant to understand NK physiology and status to investigate the biological effect of mAbs in the clinic. NK cells are heterogeneous with multiple subsets that may have specific activity against different attacks. The presence of viral-sculpted NK cell populations has already been described, but the presence of cancer-sculpted NK cells remains unknown. Cancer induces a broad NK cell dysfunction, which has not been linked to a specific population. Here, we investigated the NK cell population by Uniform Manifold Approximation and Projection (UMAP) embed maps in Hodgkin lymphoma (HL) and acute myeloid leukemia (AML) patients at diagnosis and at least 30 days after treatment, which correlates with tumor cell clearance. We found that the NK lineage largely responded to the tumor by generating antitumor NK cells and renewing the population with a subset of immature NK cells. However, we failed to identify a specific “memory-like” subset with the NK cell markers used. Moreover, in patients in relapse, we found essentially the same NK populations as those found at diagnosis, suggesting that NK cells equally respond to the first or second tumor rise. Finally, we observed that previous cytomegalovirus (CMV) infection largely affects the tumor-associated changes in NK population, but the CMV-associated CD57+NKG2C+ NK cell population does not appear to play any role in tumor immunity.

Cytoplasmic Expression of CD3ε Heterodimers by Flow Cytometry Rapidly Distinguishes Between Mature T-Cell and Natural Killer-Cell Neoplasms.

Distinguishing between T-cell and natural killer (NK)-cell neoplasms could be difficult given their overlapping immunophenotype. In this study, we investigated whether a flow cytometry assay with cytoplasmic staining for CD3 could be used for this purpose. Flow cytometry immunophenotyping was performed on 19 surface CD3 (sCD3)-negative mature T-cell neoplasms, 10 sCD3-positive mature T-cell neoplasms, 13 mature NK-cell neoplasms, and 19 normal controls. In addition to routine antibody panels (CD2, sCD3, CD4, CD5, CD7, CD8, CD16, CD45, CD56, CD57, CD94, CD158a, CD158b, CD158e, NKG2A TCRγ/δ), cytoplasmic staining for a monoclonal CD3 antibody (clone SK7/Leu-4) was assessed in all cases. A molecular study for T-cell receptor (TCR) gene rearrangement and an immunohistochemical study for TCRβ were performed. Our data showed all T-cell neoplasms were uniformly positive for cytoplasmic CD3 (cCD3) regardless of sCD3 expression, whereas 85% of NK-cell neoplasms completely lacked cCD3 expression. The 2 cases with classic NK-cell immunophenotype but partial cCD3 expression showed no molecular genetic features of T-cell lineage by TCR gene rearrangement studies. Uniform cCD3 positivity and homogeneous cCD3 negativity highly suggest T-cell and NK lineage, respectively. When partial cCD3 expression is encountered, additional confirmatory studies should be pursued for the most accurate lineage assignment.

Dissecting the NK Cell Population in Hematological Cancers Confirms the Presence of Tumor Cells and Their Impact on NK Population Homeostasis

The innate immune lymphocyte lineage natural killer (NK) cell protects against multiple pathogens and tumor cells and contains multiple subsets that could have specific activity against different attacks. The presence of viral-sculpted NK populations has already been described, but the presence of cancer-sculpted NK is unknown. Cancer induces a broad NK cell dysfunction, but this is not related to a specific population. Here we investigate NK cell population by Uniform Manifold Approximation and Projection (UMAP) embed maps in Hodgkin lymphoma (HL) and acute myeloid leukemia (AML) patients at diagnosis and, at least 30 days, after treatment, which correlates with tumor cell clearance. Whereas we identified that the NK lineage largely responded to tumor presence by generating antitumor NK cells and renewing NK cell population with immature subsets, we failed to identify a specific “memory-like” subset with the NK cell markers used. Moreover, in patients in relapse we found basically the same NK populations that those found at diagnostic, suggesting that NK cells equally respond to the first or second tumor rise. Finally, previous CMV infection largely affects the tumor-associated changes in NK population, but the CMV-associated CD57+NKG2C+ NK population does not look like to play any role. Funding Statement: The imaging facility MRI, member of the national infrastructure France BioImaging is supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»). The “Region Languedoc Roussillon” supports the clinical data and samples (HEMODIAG_2020). This work was supported by the PRT-K program 2018 (MV and GC; 2018-021) and Canceropole GSO Emergence (MV; 2018/2019). This work was also supported by La Ligue Regionale contre le Cancer (GC), the “Investissements d’avenir” Grant LabEx MAbImprove: ANR-10-LABX-53 (GC/MV) and the NK 001 projet financed by the “Fonds Europeen de Developpement Regional “ (FEDER-FSE-IEJ 2014/2020) and by “Region Occitanie Pyrenees Mediterranee”. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The use of human specimens for scientific purposes was approved by the French National Ethics Committee. All methods were carried out in accordance with the approved guidelines and regulations of this committee. Written informed consent was obtained from each patient or donor prior to surgery.

CD33 expression on natural killer cells is a potential confounder for residual disease detection in acute myeloid leukemia by flow cytometry.

Detection of minimal/measurable residual disease (MRD) in acute myeloid leukemia (AML) is important for guiding patient-specific clinical management. Natural killer (NK) cells can express various markers not typically associated with NK lineage, potentially confounding the detection of MRD by flow cytometry. We have observed CD33 expression on NK cells when evaluating for AML MRD in routine clinical practice in multiple patient samples. To characterize CD33 expression on NK cells, 40 peripheral blood or bone marrow samples with NK cells present at >5% of lymphocytes were selected for further assessment of NK cell phenotype and CD33 expression. Seven of the 40 samples (17.5%) were found to have CD33 expression on at least 5% of the NK cells. The CD33-positive NK cell population accounted for an average of 11.4% of NK cells (median 11.9%, range 8.0-15.3%) and 2.2% of total white cells (median 1.1%, range 0.1-10.1%). This NK cell subset expressed bright CD2, bright CD56, and dim CD16. On average, CD33 expression on NK cells was dimmer than on monocytes (mean median fluorescence intensity ratio 0.4; range 0.1-1.0). This study characterizes expression of CD33 on NK cells. Recognition of this pattern of antigen expression is critical in evaluating samples for MRD in patients with myeloid neoplasms, particularly AML.

CHARACTERIZATION OF NATURAL KILLER CELL HEMATOPOIETIC PRECURSORS IN HUMAN HEMATOPOIESIS

Natural Killer (NK) cells have been characterized as cytotoxic lymphocytes that are key in regulating viral infections and combatting cancer. In contrast to B and T cells, there has not been a detailed examination of the developmental trajectory of human NK cells. Conflicting reports in literature state that NK cells can develop from both lymphoid and myeloid cell progenitors. Here, we utilized mass cytometry (CyTOF) to perform a multiplexed analysis of NK cell development on healthy human bone marrow. To address the current gap in knowledge, we built a comprehensive CyTOF panel to include lymphoid markers, myeloid markers, and various regulatory factors shown to be important both in mouse and human NK cell development across 4 donor samples. We analyzed this data with multiple supervised and unsupervised single cell trajectory algorithms in order to model NK developmental processes. This approach allows for unbiased observations across developmental lineages and the reconciliation of the conflicting literature. The ability to simultaneously measure multiple cellular features, including phenotypic proteins, transcription factors, and regulatory enzymes allowed us to identify a novel potential NK lineage-restricted progenitor in adult human bone marrow. We have found potential CD34+ NK progenitors with different combinations of surface markers previously used to identify NK progenitors and regulatory markers such as IRF8, NFIL3, ID2 and TdT. Our findings here show that NK cells may be generated through two distinct fate pathways. Our next step is to analyze the capacity of this novel progenitor subpopulation to generate mature and functional CD56+ NK cells in vitro, through co culture with the OP9 and OP9-DL4 cell lines. Ultimately, our studies will shed light on the knowledge of early stages of NK lineage commitment.

Efficacy and mechanism of action of NKTR-255, a polymer-conjugated IL-15 receptor agonist in hematological tumors.

e19512 Background: NKTR-255 is a polymer-conjugated IL-15 receptor agonist that accesses the IL-15 signaling pathway, and enhances the function and numbers of natural killer (NK) cells and CD8 memory T cell populations. Many hematological malignancies are treated with targeted antibodies that utilize effector mechanisms to recruit NK cells and macrophages. Most patients with hematological malignancies experience a progressive decline of NK cell immunity, a phenomenon termed as an “immunological desert”. We evaluated the hypothesis that a combination of NKTR-255 with a targeted antibody such as an anti-CD38 monoclonal antibody (mAb) or an anti-CD20 mAb, when evaluated in a preclinical hematological tumor model, would provide enhanced efficacy. Methods: Human PBMCs were stimulated with NKTR-255 and/or daratumumab for in vitro NK cell characterization. Using a B cell lymphoma model, SCID mice were inoculated i.v. with 107 Daudi cells and treated with a single dose of daratumumab or rituximab two weeks after tumor cell injection. NKTR-255 was administered s.c. concurrently on a q7dx3 dosing schedule. Survival of treated animals was measured using hind limb paralysis and morbidity as surrogate endpoints. Tumor cell depletion and phenotyping of immune cells in bone marrow were conducted by flow cytometry. Results: NKTR-255, when combined with daratumumab, enhanced NK cell degranulation and decreased NK cell death, leading to enhanced antibody-dependent cellular cytotoxicity (ADCC) activity for the daratumumab - NKTR-255 combination over the single agents. NKTR-255 demonstrated synergistic activity and provided a long-term survival benefit when combined with daratumumab or rituximab in the CD38 and CD20-expressing lymphoma mouse models. Mechanistic studies conducted in tumor-bearing mice treated with a combination of NKTR-255 and daratumumab showed effective clearance of lymphoma cells from the bone marrow and increased expansion of effector NK cells and other immune cells. Depletion experiments confirmed the role of NK, CD8 and other monocyte lineage immune cells in the observed anti-myeloma activity. Conclusions: These studies indicate that administration of NKTR-255, when combined with a monoclonal antibody such as daratumumab and rituximab, significantly increases NK cell numbers and activates their tumor killing capacity by virtue of an enhanced ADCC effect. Combinatorial approaches of NKTR-255 with these targeting agents could lead to novel and effective clinical applications in hematology.

Innate lymphocytes-lineage, localization and timing of differentiation.

Innate lymphocytes are a diverse population of cells that carry out specialized functions in steady-state homeostasis and during immune challenge. While circulating cytotoxic natural killer (NK) cells have been studied for decades, tissue-resident innate lymphoid cells (ILCs) have only been characterized and studied over the past few years. As ILCs have been largely viewed in the context of helper T-cell biology, models of ILC lineage and function have been founded within this perspective. Notably, tissue-resident innate lymphocytes with cytotoxic potential have been described in an array of tissues, yet whether they are derived from the NK or ILC lineage is only beginning to be elucidated. In this review, we aim to shed light on the identities of innate lymphocytes through the lenses of cell lineage, localization, and timing of differentiation.

Expression of T-cell receptor signalling pathway components in extranodal NK/T-cell lymphoma.

Although the neoplastic cells of extranodal natural killer (NK)/T-cell lymphoma (ENKTL) usually do not express T-cell antigens, the T-cell receptor (TCR) gene might be rearranged and TCR protein expressed. The aim is to elucidate the expression of the downstream TCR pathway components and their importance in ENKTL. We used formalin-fixed paraffin-embedded tissues from 91 ENKTL samples to immunohistochemically characterise the expression of TCR pathway components. The following proteins were variably expressed: ZAP70 (94%; 83/88), GRAP2/GADS (68%; 60/88), DOK2 (42%; 38/90), LCK (35%; 31/88), and ITK (10%; 9/90). When these tumours were classified as being of T lineage (16%), NK lineage (45%), or indeterminate lineage (38%), the GRAP2/GADS expression rate was higher in T lineage tumours (versus NK, P = 0.0073; versus indeterminate, P = 0.00082). GRAP2/GADS-positive NK lineage tumours more frequently expressed DOK2 (P = 0.0073), and were more often confined to the nasal areas (P = 0.014). Furthermore, when these tumours were immunophenotypically classified into a T signature (42%) or NK signature (58%), the expression rates of GRAP2/GADS and ITK were higher in T signature tumours (P = 0.00074 and P = 0.067, respectively), whereas that of LCK was higher in NK-signature tumours (P = 0.10). Although some ENTKL cases were polyclonal for TCR rearrangement and others lacked TCR expression, we speculate that the TCR pathway might be functioning in ENKTLs. A T signature versus a NK signature might be better for delineating the physiology of ENKTL than cellular lineage. Furthermore, ITK may represent a potential therapeutic target for patients with ITK-expressing tumours.

Murine thymic NK cells are distinct from ILC1s and have unique transcription factor requirements.

Group 1 innate lymphoid cells include natural killer (NK) cells and ILC1s, which mediate the response to intracellular pathogens. Thymic NK (tNK) cells were described with hybrid features of immature NK cells and ILC1 but whether these cells are related to NK cells or ILC1 has not been fully investigated. We report that murine tNK cells expressed the NK-cell associated transcription factor EOMES and developed independent of the essential ILC1 factor TBET, confirming their placement within the NK lineage. Moreover, tNK cells resemble NK cells rather than ILC1 in their requirements for the E protein transcription factor inhibitor ID2. We provide further insight into the mechanisms governing tNK-cell development by showing that the transcription factor ETS1 prevented tNK cell acquisition of the conventional NK-cell maturation markers CD11b and KLRG1. Our data reveal few ILC1 in the thymus and clarify the identity and developmental requirements of tNK cells.

Nasal-type NK/T-cell lymphomas are more frequently T rather than NK lineage based on T-cell receptor gene, RNA, and protein studies: lineage does not predict clinical behavior

Extranodal natural killer (NK)/T-cell lymphoma (ENKTL), nasal type, comprises NK or cytotoxic T cells. We evaluated the clinical impact of cell type and the usefulness of T-cell receptor (TCR) gene transcripts in distinguishing cell lineage. One hundred and eight cases of ENKTL were analyzed for TCR gene rearrangements using the BIOMED-2 protocol and for TCR gene expression using immunohistochemistry for TCR-βF1 and TCR-cγM1, and RNA in situ hybridization for TCR gene transcripts. Prognostic factors were analyzed. Among the 108 cases, 44 were monoclonal for a TCR rearrangement (40%) while 64 (60%) were undefinable. The monoclonal cases expressed TCR-βF1 in 14 out of 40 cases (35%) and TCR-cγM1 in 1 out of 44 cases (2%). The 64 undetermined cases expressed TCR-βF1 in 15 cases (23%) and TCR-cγM1 in 1 (2%). Thirteen of 40 TCR-β constant gene transcript-positive cases (33%) expressed TCR-βF1 and one of nine TCR-γ constant gene transcript-positive cases (11%) expressed TCR-cγM1. TCR gene transcripts were not useful in the distinction of cell lineages. TCR gene transcripts were positive in ENKTLs as well as in normal B cells and aggressive NK-cell leukemia. Based on gene rearrangements and immunohistochemistry for TCR, there were 60 T-cell type cases (56%), 32 NK-cell type cases (30%), and 16 cases with an undetermined cell type (14%). TCR protein was expressed in 30/60 T-ENKTLs (50%) in a variable fraction of tumor cells. There were no significant differences in clinical findings or overall patient survival between T- or NK-cell types of ENKTL, although those with a T-cell type tended to show a better prognosis for those with localized nasal lymphomas. Univariate and multivariate analysis showed that a non-nasal ENKTL, age >60 years, high level of lactate dehydrogenase, bone marrow involvement, and the absence of radiotherapy were independent prognostic factors.

Ezh2 regulates differentiation and function of natural killer cells through histone methyltransferase activity

Changes of histone modification status at critical lineage-specifying gene loci in multipotent precursors can influence cell fate commitment. The contribution of these epigenetic mechanisms to natural killer (NK) cell lineage determination from common lymphoid precursors is not understood. Here we investigate the impact of histone methylation repressive marks (H3 Lys27 trimethylation; H3K27(me3)) on early NK cell differentiation. We demonstrate that selective loss of the histone-lysine N-methyltransferase Ezh2 (enhancer of zeste homolog 2) or inhibition of its enzymatic activity with small molecules unexpectedly increased generation of the IL-15 receptor (IL-15R) CD122(+) NK precursors and mature NK progeny from both mouse and human hematopoietic stem and progenitor cells. Mechanistic studies revealed that enhanced NK cell expansion and cytotoxicity against tumor cells were associated with up-regulation of CD122 and the C-type lectin receptor NKG2D. Moreover, NKG2D deficiency diminished the positive effects of Ezh2 inhibitors on NK cell commitment. Identification of the contribution of Ezh2 to NK lineage specification and function reveals an epigenetic-based mechanism that regulates NK cell development and provides insight into the clinical application of Ezh2 inhibitors in NK-based cancer immunotherapies.

Indolent CD56-Positive Clonal T-Cell Lymphoproliferative Disease of the Stomach Mimicking Lymphomatoid Gastropathy.

Natural killer (NK)- or T-cell lymphomas of the gastrointestinal (GI) tract are uncommon, accounting for less than 10% of all malignant lymphomas arising in the GI tract [1]. According to the third national survey by the Hematopathology Study Group of the Korean Society of Pathologists, peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), is the most common type, followed by extranodal NK/T-cell lymphoma, nasaltype (ENKL). Enteropathy-associated T-cell lymphoma (EATL) is rare [1]. Malignant lymphoma of T and NK lineage is an aggressive disease with poor prognosis [2]. Recently, two indolent forms of mature T- or NK-cell lymphoproliferative diseases (LPD) of the GI tract have drawn the attention of pathologists. One is indolent NK cell proliferative lesion of the stomach and intestine reported under the name of lymphomatoid gastropathy [3] or NK-cell enteropathy [4]. The lesion is often self-limited, even when left untreated. Histological findings have been described as dense infiltration of lymphoid cells with mild to moderate atypia and peculiar eosinophilic cytoplasmic granules [3]. Frequently, there are lymphoepithelial lesion-like changes with or without necrosis. All cases published previously have been positive for both cytoplasmic CD3 and CD56 and negative for CD4 and CD8. T-cell receptor (TCR) gene rearrangement is polyclonal [3-5], and unlike ENKL, infiltrating cells were negative for Epstein-Barr virus (EBV). The other form of indolent T- or NK-cell LPD of the GI tract is a clonal T-cell proliferative disease involving the oral cavity, esophagus, stomach, small intestine, and colon [6]. The lesion shows a superficial and nondestructive lymphoid infiltrate that occasionally extends into the muscularis mucosae and submucosa. Infiltrating cells are small T-cells, which are CD4+ or CD8+ or uncommonly CD4–/CD8–phenotype [6-9]. CD56 and EBV-encoded RNA (EBER) are negative, and TCR gene rearrangement has been monoclonal in all cases reported so far. Unlike indolent NK-cell proliferative lesions, indolent T-cell LPD does not spontaneously regress, but is persistent without progression [6,9]. These two indolent entities can be easily mistaken as aggressive T- or NK-cell lymphoma; therefore, recognition of these entities is important to avoid unnecessary aggressive chemotherapy. Herein we report, to our knowledge, the first Korean case of indolent T-cell LPD of the stomach with expression of CD56.

Extranodal natural killer/T-cell lymphoma, nasal type in Taiwan: a relatively higher frequency of T-cell lineage and poor survival for extranasal tumors.

Extranodal natural killer (NK)/T-cell lymphoma, nasal type, is a predominantly extranodal lymphoma associated with Epstein-Barr virus occurring most frequently in the upper aerodigestive tract. There are limited reports on cellular origin and prognostic factors. We retrospectively investigated 73 cases with a median age of 54 years and a male-female ratio of 2.0:1. The upper aerodigestive tract (nasal group) was the most common site of involvement (51 cases; 70%). The other organs (n = 22; extranasal group) included the skin (12 cases; 16%) and gastrointestinal tract (5; 7%). Of the 70 cases with complete staging, 71% had stage I/II disease. All cases were positive for Epstein-Barr virus by in situ hybridization. Using immunohistochemistry and clonality assay for T-cell receptor gene rearrangement, these tumors were classified into NK (n = 39; 53%), T (n = 13; 18%), and indeterminate lineage (n = 21; 29%). The only clinicopathological difference among these 3 groups was rare CD5 expression in the NK-cell group. Nasal tumors were more frequently of NK-cell origin, and extranasal tumors were equally of either T- or NK-cell origin. The 5-year overall survival rate was 35.6%. The overall survival time was shorter in the extranasal group, although there was no statistical difference in age, sex, and histologic or immunophenotypic features between the 2 groups. Excluding the cases with indeterminate lineage, 75% of cases were of NK lineage; and 25%, T lineage. Extranasal tumors were more aggressive than their nasal counterparts. A prospective national study is warranted for a better understanding of the clinicopathological and genetic features of this uncommon tumor and the prognostic factors.

The transcription factor E4bp4/Nfil3 controls commitment to the NK lineage and directly regulates Eomes and Id2 expression

The transcription factor E4bp4 (Nfil3) is essential for natural killer (NK) cell production. Here, we show that E4bp4 is required at the NK lineage commitment point when NK progenitors develop from common lymphoid progenitors (CLPs) and that E4bp4 must be expressed at the CLP stage for differentiation toward the NK lineage to occur. To elucidate the mechanism by which E4bp4 promotes NK development, we identified a central core of transcription factors that can rescue NK production from E4bp4(-/-) progenitors, suggesting that they act downstream of E4bp4. Among these were Eomes and Id2, which are expressed later in development than E4bp4. E4bp4 binds directly to the regulatory regions of both Eomes and Id2, promoting their transcription. We propose that E4bp4 is required for commitment to the NK lineage and promotes NK development by directly regulating the expression of the downstream transcription factors Eomes and Id2.

A committed precursor to innate lymphoid cells

Innate lymphoid cells (ILC) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers.1–9 Their diversity and similarities with previously characterized NK cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 based solely on cytokine properties,10 but their developmental pathways and lineage relationships remain elusive. Using lineage tracing and transfer studies, we identified and characterized a novel subset of lymphoid precursors in fetal liver and adult bone marrow that transiently expressed high amounts of PLZF, a transcription factor previously associated with NKT cell development.11,12 PLZFhigh cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.1+DX5− ‘NK-like’ cells residing in the liver. Deletion of PLZF markedly altered the development of several ILC subsets, but not LTi or NK cells. PLZFhigh precursors also expressed high amounts of Id2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages.13 These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILC, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.

The Unique Ontogeny Of Natural Killer Cells As Revealed By Genetic Barcoding In The Nonhuman Primate Model

Natural killer (NK) cells are cytotoxic leukocytes defined in humans and rhesus macaques by the expression of CD56 and/or CD16, and the absence of T, B, and myeloid markers. Functionally, they are defined by lysis of tumor targets in a major histocompatibility complex (MHC)–unrestricted fashion, and production of cytokines critical to innate immunity. Ex vivo expanded NK cells are being developed as adoptive transfer therapeutics for a variety of malignancies. However, the ontogeny and hematopoietic lineage relationships of human NK cells have been difficult to study, given differences in phenotype and function between human and murine NK cells, poor NK engraftment and development in human xenografts, and restricted development of NK cells from hematopoietic stem and progenitor cells (HSPC) in vitro. In addition, several phenotypic subsets of NK cells have been described, and their relationships and relative locations within the hematopoietic hierarchy are supported by limited in vivo data. We “barcoded” individual CD34+ HSPC from 3 macaques using lentiviral vectors carrying highly diverse oligonucleotides, allowing precise quantitation of clonal contributions via low cycle PCR amplification followed by high-throughput sequencing and computational analysis (Lu et al, Nat Biotech). We quantitated barcode levels, and thus individual HSPC clonal contributions, over time and between lineages, following TBI and infusion of autologous barcoded CD34+ cells. Reconstitution of the NK compartment was clonally distinct from T, B and myeloid (My) lineages. Following reconstitution of NK, T, B and My from uni-lineage progenitors at 1-2 months(m), by 3-4 m, as expected from murine and in vitro models, multi-lineage clones began to contribute and dominate, first B/My, then B/T/My. However, NK cells remained clonally distinct through 9 m, despite overall clonal diversity and marking levels in NK lineage similar to B/T and My. Even the most abundant clones in the NK lineage were not found contributing in any significant way to B/T or My lineages. Shared B/T/My/NK clones finally began to contribute more extensively at 9-14 m. We fractionated peripheral blood NK cells into 3 NK subsets and compared them to overall PB and lymph node NK cells at 4.5-6.5 m. The majority (75-90%) of PB rhesus NK cells are found in the CD16+/CD56- PB subset (corresponding to human CD16+/CD56dim cytotoxic NK), and this subset accounted for the clonal pattern in overall NK cells (Pearson r=0.98 vs overall NK cells), and the disparity from B/T/My, with r values all <0.17 for CD16+/CD56- vs B/T or My lineages at the same time point. The minor PB (but dominant lymph node) cytokine-producing CD16-/CD56+ or CD16+/CD56+ NK subsets, previously hypothesized to be precursors for CD16+/CD56- NK cells, had clonal patterns that were more closely correlated with T/B/My lineages (r=0.37-0.62) than the CD16+/CD56- cells. The clonal correlations between the putative precursor CD16-/CD56+ cells and mature cytotoxic CD16+/CD56- cells was very low, only r=0.08. Furthermore, we expanded purified NK cells from barcoded macaques in vitro in the presence of IL-2 and irradiated EBV-LCL cells and assessed NK phenotype, function, and clonal diversity over time. 40-90X expansion was achieved by 15 days and was highly polyclonal, with 90% of the starting number of barcodes still present at the end of expansion. Clonal contribution levels between pre and post-expanded NK were highly correlated (r=0.73). CD16+/CD56+ cells became more dominant in post-exp NK cells, in contrast to the majority CD16+CD56- pre-exp. The clonal contributions in the post-exp CD16+/CD56- and CD16+/CD56+ cells correlated with each other (r=0.66), and with the starting CD16+/CD56- cells (r=0.75), but not with the starting CD16-/CD56+ (r=0.15) nor the post-exp CD16-/CD56+ cells (r=0.18). Our in vivo and in vitro results call into question the hypothesis that CD16-/CD56+ NK cells are precursors of circulating cytotoxic CD16+/CD56- NK cells, as does the complete deficiency of CD16-/CD56+ but not CD16+/CD56dim cells in GATA2 mutant patients. Our results also reveal that the dominant blood rhesus NK cell population has a distinct ontogeny in macaques, and thus potentially in humans. NK cells expanded ex vivo using EBV-LCL cells are polyclonal, and barcoding allows dissection of events during expansion, and will permit tracking studies of expanded cells following adoptive transfer. Disclosures:No relevant conflicts of interest to declare.

Identification and Characterization Of Human NK Lineage Restricted Progenitor (NKP)

Natural Killer (NK) cells, the third lymphoid lineage after T and B cells, are large granular lymphocytes belonging to innate immune system, classically defined as CD3-CD56+CD16+ in human. NK cells can kill target cells without prior activation, based on the missing self-hypothesis, either due to the lack of MHC-class I molecule or up-regulation of NKG2D ligands, two properties commonly associated with transformed cells (D.H. Raulet, N. Guerra, 2009). Moreover, by secreting cytokines (such as TNF-α and IFN-γ), NK cells can also activate the response of the adaptive immune system. Experiments in mice have shown that NK cells are primarily responsible for the in vivo elimination of transplanted tumor cells (J. Wu, L. L. Lanier, 2003) and, in human, a direct correlation between low NK cytotoxicity and elevated cancer incidence was reported in a large cohort of Japanese aged over 40 (K. Imai et al., 2000). The important anti-tumor role for alloreactive NK cells has been shown in patients with acute myeloid leukemia where transplanted donor NK cells, expressing mismatch inhibitory receptors, provided graft versus leukemia effect in the absence of graft versus host disease (L. Ruggeri et al., 2006). These and many other findings are the basis of a large number of ongoing studies and clinical trials for NK cell immunotherapy against cancer (M. Terme et al., 2008). However, in contrast to B and T cell development where both cellular and regulatory pathways are well studied, the development of NK cells from hematopoietic stem cells (HSCs) is not well understood and the identity of restricted NK cell progenitor (NKP) is unknown (S. Doulatov et al., 2012).To identify human NKP, we applied the expression of known early lymphoid markers (CD45RA, CD10, CD7) and cytokine receptors (IL-7R: CD127) important for lymphoid development. Using multicolor flow cytometry, Lin-CD34+CD38+CD123-CD45RA+CD7+CD10+/-CD127+/- populations were sorted from human bone marrow (hBM) and umbilical cord blood (hUCB). Purified candidate NKPs were cultured on OP9, OP9DL1 stroma and in Terasaki cultures in the presence of specific cytokines and generation of CD3-CD56+CD16+NKp46+ NK cells, CD19+ B, CD3+ CD4+ CD8+ T and CD33+CD14+ myeloid cells was investigated. To study the ability of NKP candidate to generate NK cells after transplantation, purified NKPs were injected into new-born NOD/SCID γcnull (NSG) mice and at 11 weeks after transplantation the phenotype of generated progeny was evaluated. In addition, NK cells generated in stroma cultures and after transplantation were tested for their cytotoxic activity against K562 leukemic cell line in degranulation assay, where the surface expression of membrane glycoprotein LAMP-1 (CD107a) is measured after activation of specific killing receptors.The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate population sorted from hUCB has a robust NK cell potential in vitro at the single cell level and lacked the ability to generate T, B and myeloid cells. Furthermore, NK cells generated from this candidate NKP were functionally mature: show cytotoxic activity against K562 cells and produce cytokines: IFNγ and TNFα after activation in vitro. Further phenotypic characterization showed that the candidate NKP is highly positive for lymphoid markers CD244 and CD62L, lacks the expression of mature NK cell markers NKp46 and NKG2D, and 50% of NKPs express c-kit and Flt3 receptors. At 11 weeks after transplantation, 9 out of 17 NSG mice injected with 600 Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKPs had a significant human cells engraftment and only CD3-CD56+NKp46+CD16+/- NK cells were found in the peripheral blood, bone marrow and spleen; whereas 15 out 17 NSG mice injected with CD34+ cells were positive for the presence of human B, T, NK and myeloid cells in these tissues. Human NK cells generated after transplantation into NSG mice were functionally active and able to kill K562 leukemic cells. The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate was also found in adult hBM and showed NK-lineage restriction.Our results indicate that the Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- cells, found in human BM and UCB, represent NK-lineage restricted progenitors that generate fully mature functional NK cells. Disclosures:No relevant conflicts of interest to declare.

RAG-1 and Ly6D Independently Reflect Progression in the B Lymphoid Lineage

Common lymphoid progenitors (CLPs) are thought to represent major intermediates in the transition of hematopoietic stem cells (HSCs) to B lineage lymphocytes. However, it has been obvious for some time that CLPs are heterogeneous, and there has been controversy concerning their differentiation potential. We have now resolved four Flt3+ CLP subsets that are relatively homogenous and capable of forming B cells. Differentiation potential and gene expression patterns suggest Flt3+ CLPs lacking both Ly6D and RAG-1 are the least differentiated. In addition to B cells, they generate natural killer (NK) and dendritic cells (DCs). At the other extreme is a subset of the recently described Flt3+ Ly6D+ CLPs that have a history of RAG-1 expression and are B lineage restricted. These relatively abundant and potent CLPs were depleted within 48 hours of acute in vivo estrogen elevation, suggesting they descend from hormone regulated progenitors. This contrasts with the hormone insensitivity of other CLP subsets that include NK lineage progenitors. This progenitor heterogeneity and differentiation complexity may add flexibility in response to environmental changes. Expression of RAG-1 and display of Ly6D are both milestone events, but they are neither synchronized nor dependent on each other.