CD200R1 Distinguishes Uncommitted Precursors from Functionally Mature NK Cells within the Human Tonsil Stage 4A NK Cell Population

Abstract Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) whose development and anti-tumor functions can be critical for the successful treatment and long-term disease-free survival of patients with hematologic malignancies. In humans, NK cells derive from bone marrow resident hematopoietic progenitor cells that traffic to secondary lymphoid tissues (SLTs) where they complete their terminal differentiation and maturation through a series of developmental stages before returning to the blood as mature NK cells. Although major stages of human NK cell development in SLTs have been clearly defined according to the differential surface expression of CD34, CD117, CD94, NKp80, CD16, and CD57 among lineage antigen (Lin) negative lymphocytes, continued investigation has revealed additional phenotypic and functional heterogeneity at each developmental stage. Through extensive ex vivo single-cell RNA sequencing and flow cytometry analyses we have identified two subsets of tonsil-resident Lin -CD34 -CD117 +/-CD94 +NKp80 -CD16 -CD57 - stage 4A NK cells. These two subsets differ in their expression of the inhibitory receptor, CD200R1, which is not expressed by mature NK cells in the peripheral blood from healthy individuals. The majority of stage 4A cells expressed high amounts of surface CD200R1, which correlated with low gene and undetectable protein expression of intracellular cytolytic granules (perforin and granzymes A, B, K, and M), killer immunoglobulin-like receptors (KIRs), and transcription factors required for terminal NK cell maturation (EOMES, T-BET). In addition, upon ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, CD200R1 + stage 4A NKDIs did not produce interferon-gamma (IFN-g), a hallmark feature of mature NK cells. In contrast, many CD200R1 - stage 4A cells constitutively expressed perforin, granzymes, EOMES, and/or T-BET; many expressed KIRs; and many produced IFN-g upon ex vivo stimulation. Furthermore, the frequency of KIR + cells among CD200R1 - stage 4A cells was significantly higher than that among autologous tonsil stage 4B NK cells (Lin -CD34 -CD117 +/-CD94 +NKp80 +CD16 -CD57 -) (20.8 ± 1.65 vs. 8.12 ± 1.66; p < 0.01; n = 14), suggesting that as a population CD200R1 - stage 4A cells are potentially out of sequence in terms of the linear NK cell developmental pathway. Based on these ex vivo findings, we hypothesized that CD200R1 + stage 4A cells represent NK cell precursors, whereas the CD200R1 - stage 4A population contains more mature NK cells that lack NKp80, CD16, and CD57. To further address this hypothesis and to determine their ex vivo potentials for NK cell and non-NK ILC differentiation, we cultured CD200R1 + and CD200R1 - stage 4A cells in vitro in the presence of OP9-DL1 stroma and recombinant human IL-7 and IL-15, conditions previously shown to support all human ILC and NK cell subset differentiation. Under these conditions, both stage 4A populations generated NKp80 + NK cells in bulk and single-cell clonal assays, whereas neither population gave rise to ILC2s (CD294 +) which precede stage 4A NK cells in the developmental scheme. However, while the majority of cultures derived from CD200R1 + stage 4A clones contained ILC3s (CD94 -NKp44 +), significantly fewer clones from CD200R1 - stage 4A cells produced ILC3s (7 of 26 CD200R1 - clones vs. 20 of 23 CD200R1 + clones; p = 0.000587). Moreover, none of the CD200R1 - stage 4A-derived clonal cultures that contained KIR + NK cells contained ILC3s, suggesting that the majority of CD200R1 - stage 4A cells are lineage committed NK cells. Collectively, these data further characterize the heterogeneity of the human tonsil stage 4A NK cell population and identify CD200R1 as a marker distinguishing uncommitted precursor cells from a minor population of cells with otherwise mature NK-associated phenotype and function. In light of the role of CD200R1 in regulating lymphocyte functions in the setting of cancer, further research is warranted to determine its potential role(s) in regulating human NK cell development. Disclosures Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

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HIV-1-induced cytokines deplete homeostatic ILCs and expand TCF7-dependent memory NK cells: Supplemental Figures and Tables

10.1101/221010 ◽

2017 ◽

Cited By ~ 1

Author(s):

Yetao Wang ◽

Kyle Gellatly ◽

Sean McCauley ◽

Pranitha Vangala ◽

Kyusik Kim ◽

...

Keyword(s):

Nk Cells ◽

Inflammatory Cytokines ◽

Nk Cell ◽

Ex Vivo ◽

Developmental Trajectory ◽

Lymphoid Cells ◽

Infected People ◽

Cell Induction ◽

Hiv 1 ◽

Memory Nk Cells

HIV-1-infected people who take medications that suppress viremia, preserve CD4+ T cells, and prevent AIDS, have chronic inflammation with increased cardiovascular mortality. To investigate the etiology of this inflammation, the effect of HIV-1 on innate lymphoid cells (ILCs) and NK cells was examined. Homeostatic ILCs in blood and intestine were depleted permanently. NK cells were skewed towards a memory subset. Cytokines that are elevated during HIV-1 infection reproduced both abnormalities ex vivo. Pseudotime analysis of single NK cell transcriptomes revealed a developmental trajectory towards a subset with expression profile, chromatin state, and biological function like memory T lymphocytes. Expression of TCF7, a WNT transcription factor, increased over the course of the trajectory. TCF7 disruption, or WNT inhibition, prevented memory NK cell induction by inflammatory cytokines. These results demonstrate that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt homeostatic ILCs and cause developmental shift towards TCF7+ memory NK cells.

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KIR Ligation During Ex Vivo Expansion Allows Molding Of The KIR Repertoire

Blood ◽

10.1182/blood.v122.21.2288.2288 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2288-2288

Author(s):

Dean A. Lee ◽

Vladimir V Senyukov ◽

Jerome R Trembley

Keyword(s):

Nk Cells ◽

Cell Population ◽

Nk Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Cell Populations ◽

Hematopoietic Stem ◽

Hla Ligand ◽

Cell Subpopulations ◽

Hla Haplotypes

Abstract NK Cell subpopulations express tremendous diversity through polymorphisms, haplotypes, differential expression, and licensing of the Killer Immunoglobulin-like Receptors (KIR). KIR diversity affects both the predisposition to cancer, and the response to therapies such as hematopoietic stem cell transplantation. Clinical trials that take advantage of the anti-cancer properties of NK cells have been limited to choosing donors on the basis of KIR genotypes and/or HLA haplotypes. Moreover, adoptive immunotherapy approaches have been limited by low NK cell doses. The latter hurdle has been recently mitigated by methods for expanding clinical grade NK cells ex vivo. These approaches for growing large numbers of cells now enable investigation into selecting more potent NK cell subsets for increased therapeutic efficacy. We hypothesized that the desired KIR repertoire could be molded through inhibition of undesirable KIR populations by crosslinking with relevant anti-KIR antibodies during expansion with our previously described method, which produces a mean 30,000-fold expansion of NK cells in 3 weeks. First, we determined that maximum inhibition was obtained when anti-KIR antibodies were applied to previously activated NK cells, crosslinked with secondary antibody, and then restimulated for proliferation. Robust reduction of targeted KIR-positive populations could be achieved for each inhibitory KIR (Fig. 1). When pre-activated with anti-KIR2DL1 for one stimulation cycle, NK cells expressing this KIR were decreased by a median of 70.4% ± 19.3%. Similarly, KIR2DL2/3+ NK cells could be reduced by 56% ± 17.5%, and KIR3DL1+ NK cells could be reduced by 53.5% ± 16.3%. When anti-KIR antibodies were combined, similar suppression of multiple-KIR subpopulations was observed. Other NK cell receptors were not significantly affected during targeted KIR inhibition. We then assessed the resulting NK cell populations for degranulation responses to targets with selected HLA as KIR ligands. Inhibition of KIR-expressing subpopulations during expansion resulted in NK cell populations with enhanced degranulation against tumor cells expressing the HLA ligand of the targeted KIR. Importantly, the cytotoxicity of the bulk NK cell population against HLA-negative targets remained. These results indicate that KIR crosslinking during NK cell propagation enables significant reduction in the targeted KIR subpopulations, resulting in an NK cell population with a selective decrease in KIR inhibition. By utilizing antibody-controlled expansion for molding of the KIR repertoire according to patient HLA type, a personalized NK cell product may be produced with enhanced potency, improving NK cell immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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A Novel CD94+ Immature NK Cell Population Based on NKp80 Expression with ILC3-like Features in Human Secondary Lymphoid Tissue

Blood ◽

10.1182/blood.v124.21.751.751 ◽

2014 ◽

Vol 124 (21) ◽

pp. 751-751

Author(s):

Steven D Scoville ◽

Karen Keller ◽

Bethany Mundy-Bosse ◽

Michael A. Caligiuri ◽

Aharon G. Freud

Keyword(s):

Protein Expression ◽

Nk Cells ◽

Cell Population ◽

Lymphoid Tissue ◽

Nk Cell ◽

Ex Vivo ◽

Differentiation Potential ◽

Secondary Lymphoid Tissue ◽

Stage 4 ◽

Mrna And Protein Expression

Abstract Natural killer (NK) cells are important for their innate ability to identify and kill cancer cells without previous stimulation. However, many of the mechanisms concerning their differentiation from immature precursor cells are still unknown. Elucidation of these pathways are critical to improving targeted therapies that can both activate as well as overcome inhibitory pathways that prevent an effective response. NK cells are thought to develop in secondary lymphoid tissue (SLT) through five stages distinguished by the expression of CD34, CD117, CD94, and CD16. While virtually all NK cells in blood express the pan-NK cell activating receptor, NKp80, we have discovered a distinct Lin CD94+NKp80-CD16- population that is selectively enriched in SLT. The NKp80- population appears to comprise those cells “in transit” between stage 3 (CD117+CD94-) and stage 4 (CD117loCD94+) when evaluating CD94 and CD117 expression among Lin-CD34- cells by flow cytometry. These ex vivo data suggest that this NKp80- population may represent a novel NK cell developmental intermediate (NKDI). SLT-derived NKp80- cells lack cytolytic granules, show minimal cytotoxicity, and produce negligible amounts of interferon-gamma when compared to NKp80+ NK cells in SLT and blood. In addition, NKp80- cells have lower mRNA and protein expression of TBET and EOMES compared to NKp80+ NK, while expressing these factors at higher levels compared to stage 3 cells. Unexpectedly, the NKp80- population also demonstrates Group 3 innate lymphoid cell (ILC3)-like features ex vivo with expression of surface interleukin-1 (IL-1) receptor 1 and CD127 and mRNA and protein expression of the ILC3-associated transcription factors, AHR and RORC. Moreover, following overnight stimulation with IL-1-beta and IL-23, NKp80- cells produce IL-22, albeit at lower concentrations compared to stage 3 cells. Collectively, these data identify NKp80 as a surrogate marker of functional competence during in vivo human NK cell development and provide evidence for the existence of at least two distinct maturation steps within the previously described stage 4 NK cell population in SLT. Since CD94 expression is currently considered to be NK-restricted, these findings raise new questions regarding the developmental relationship between NK cells and ILC3 in humans. In addition, as ILC3s are primarily used to stimulate an anti-microbial defense while NK cells are functional anti-cancer effector cells, defining the differentiation patterns of these cells can have impacts in both of these areas of research. Studies are ongoing to investigate the in vitro differentiation potential(s) of this novel SLT-derived Lin-CD94+NKp80-CD16- population. Disclosures No relevant conflicts of interest to declare.

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Effect of Aging on NK Cell Population and Their Proliferation at Ex Vivo Culture Condition

Analytical Cellular Pathology ◽

10.1155/2018/7871814 ◽

2018 ◽

Vol 2018 ◽

pp. 1-6 ◽

Cited By ~ 13

Author(s):

Sellamuthu Subbanna Gounder ◽

Basri Johan Jeet Abdullah ◽

Nur Ezzati Izyan Binti Mohd Radzuanb ◽

Farah Dalila Binti Mohd Zain ◽

Nurhidayah Bt Mohamad Sait ◽

...

Keyword(s):

Nk Cells ◽

Cell Population ◽

Cytokine Production ◽

Nk Cell ◽

Ex Vivo ◽

Age Groups ◽

Proliferation Ability ◽

Males And Females ◽

Ex Vivo Culture ◽

Proliferation Potential

Age-associated changes in natural killer (NK) cell population, phenotype, and functions are directly attributed to the risk of several diseases and infections. It is predicted to be the major cause of the increase in mortality. Based on the surface density of CD56, NK cells are subdivided into two types, such as CD56brightand CD56dimcells, which represent cytokine production and cytotoxicity. In our study, we have examined the age-associated changes in the NK cell population and their subsets at different age groups of males and females (at a range from 41 to 80 years). We found that the total lymphocyte count significantly dropped upon aging in both genders. Although, the level of total immune cells also dropped on aging, and surprisingly the total NK cell population was remarkably increased with the majority of NK cells being CD56dim. Subsequently, we evaluated the proliferation potential of NK cells and our results showed that the NK cell proliferation ability declines with age. Overall, our findings prove that there is an increase in the circulating NK cell population upon aging. However, the proliferation rate upon aging declines when compared to the young age group (<41 yrs).

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Phenotypic and functional heterogeneity of human NK cells developing after umbilical cord blood transplantation: a role for human cytomegalovirus?

Blood ◽

10.1182/blood-2011-08-372003 ◽

2012 ◽

Vol 119 (2) ◽

pp. 399-410 ◽

Cited By ~ 163

Author(s):

Mariella Della Chiesa ◽

Michela Falco ◽

Marina Podestà ◽

Franco Locatelli ◽

Lorenzo Moretta ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Nk Cell ◽

Cord Blood Transplantation ◽

Cell Subset ◽

Hematologic Malignancies ◽

Cell Development ◽

Hematopoietic Stem

Abstract Natural killer (NK) cells play a crucial role in early immunity after hematopoietic stem cell transplantation because they are the first lymphocyte subset recovering after the allograft. In this study, we analyzed the development of NK cells after intrabone umbilical cord blood (CB) transplantation in 18 adult patients with hematologic malignancies. Our data indicate that, also in this transplantation setting, NK cells are the first lymphoid population detectable in peripheral blood. However, different patterns of NK-cell development could be identified. Indeed, in a group of patients, a relevant fraction of NK cells expressed a mature phenotype characterized by the KIR+NKG2A− signature 3-6 months after transplantation. In other patients, most NK cells maintained an immature phenotype even after 12 months. A possible role for cytomegalovirus in the promotion of NK-cell development was suggested by the observation that a more rapid NK-cell maturation together with expansion of NKG2C+ NK cells was confined to patients experiencing cytomegalovirus reactivation. In a fraction of these patients, an aberrant and hyporesponsive CD56−CD16+p75/AIRM1− NK-cell subset (mostly KIR+NKG2A−) reminiscent of that described in patients with viremic HIV was detected. Our data support the concept that cytomegalovirus infection may drive NK-cell development after umbilical CB transplantation.

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Development and function of murine B220+CD11c+NK1.1+ cells identify them as a subset of NK cells

Journal of Experimental Medicine ◽

10.1084/jem.20070991 ◽

2007 ◽

Vol 204 (11) ◽

pp. 2561-2568 ◽

Cited By ~ 118

Author(s):

Amanda L. Blasius ◽

Winfried Barchet ◽

Marina Cella ◽

Marco Colonna

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Cell Subset ◽

Lymphoid Organs ◽

Surface Receptors ◽

Histocompatibility Complex ◽

Function Expression ◽

Interleukin 15 ◽

And Function

Lymphoid organs contain a B220+CD11c+NK1.1+ cell population that was recently characterized as a novel dendritic cell (DC) subset that functionally overlaps with natural killer (NK) cells and plasmacytoid DCs (PDCs). Using Siglec-H and NK1.1 markers, we unambiguously dissected B220+CD11c+ cells and found that PDCs are the only professional interferon (IFN)-α–producing cells within this heterogeneous population. In contrast, B220+CD11c+NK1.1+ cells are a discrete NK cell subset capable of producing higher levels of IFN-γ than conventional NK cells. Unlike DCs, only a minute fraction of B220+CD11c+NK1.1+ cells in the spleen expressed major histocompatibility complex class II ex vivo or after stimulation with CpG. Consistent with being a NK cell subset, B220+CD11c+NK1.1+ cells depended primarily on interleukin 15 and common cytokine receptor γ chain signaling for their development. In terms of function, expression of distinctive cell surface receptors, and location in lymphoid organs, NK1.1+B220+CD11c+ appear to be the murine equivalent of human CD56bright NK cells.

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Current Perspectives on “Off-The-Shelf” Allogeneic NK and CAR-NK Cell Therapies

Frontiers in Immunology ◽

10.3389/fimmu.2021.732135 ◽

2021 ◽

Vol 12 ◽

Author(s):

Erica L. Heipertz ◽

Evan R. Zynda ◽

Tor Espen Stav-Noraas ◽

Andrew D. Hungler ◽

Shayne E. Boucher ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Peripheral Circulation ◽

Immune Defense ◽

Innate Immune ◽

Lymphoid Tissues ◽

First Line ◽

Cell Therapies ◽

Vector Delivery

Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo. NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for “off-the-shelf” allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy.

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The Dual Role of Innate Lymphoid and Natural Killer Cells in Cancer. From Phenotype to Single-Cell Transcriptomics, Functions and Clinical Uses

Cancers ◽

10.3390/cancers13205042 ◽

2021 ◽

Vol 13 (20) ◽

pp. 5042

Author(s):

Stefania Roma ◽

Laura Carpen ◽

Alessandro Raveane ◽

Francesco Bertolini

Keyword(s):

Nk Cells ◽

Single Cell ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Cell Activity ◽

Lymphoid Cells ◽

Cell Populations ◽

Transcriptional Level

The role of innate lymphoid cells (ILCs), including natural killer (NK) cells, is pivotal in inflammatory modulation and cancer. Natural killer cell activity and count have been demonstrated to be regulated by the expression of activating and inhibitory receptors together with and as a consequence of different stimuli. The great majority of NK cell populations have an anti-tumor activity due to their cytotoxicity, and for this reason have been used for cellular therapies in cancer patients. On the other hand, the recently classified helper ILCs are fundamentally involved in inflammation and they can be either helpful or harmful in cancer development and progression. Tissue niche seems to play an important role in modulating ILC function and conversion, as observed at the transcriptional level. In the past, these cell populations have been classified by the presence of specific cellular receptor markers; more recently, due to the advent of single-cell RNA sequencing (scRNA-seq), it has been possible to also explore them at the transcriptomic level. In this article we review studies on ILC (and NK cell) classification, function and their involvement in cancer. We also summarize the potential application of NK cells in cancer therapy and give an overview of the most recent studies involving ILCs and NKs at scRNA-seq, focusing on cancer. Finally, we provide a resource for those who wish to start single-cell transcriptomic analysis on the context of these innate lymphoid cell populations.

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Involvement of NFAT Signaling in Function and Anti-Tumor Reactivity of NK Cells

Blood ◽

10.1182/blood.v120.21.1887.1887 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1887-1887

Author(s):

Julia S Wild ◽

Benjamin J Schmiedel ◽

Melanie Märklin ◽

Lothar Kanz ◽

Martin R Müller ◽

...

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Conflicts Of Interest ◽

Clinical Success ◽

Immunosuppressive Treatment ◽

Cell Development ◽

Lymphoid Cells ◽

Inosine Monophosphate Dehydrogenase ◽

And Function

Abstract Abstract 1887 NK cells play an important role in anti-tumor immunity. They significantly contribute to the clinical success of allogeneic stem cell transplantation (SCT). Their reactivity as a consequence of an integrative response mediated by various activating and inhibitory surface receptors results in the induction of yet only partially defined signal transduction pathways. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells). While its role in T cell development and function is meanwhile well defined, surprisingly little is known on its function in NK cells. NFAT seems to be dispensable for NK cell development, but several lines of evidence clearly point to its involvement in NK reactivity and function. Cyclosporin A (CsA) and tacrolimus are immunosuppressive drugs that are widely used in transplant medicine. They mediate their immunosuppressive effects through inhibition of the serine/threonine phosphatase calci-neurin, which dephosphorylates and thereby activates NFAT. Here we studied the role of NFAT in NK cells and found that all five NFAT family members are expressed in NK cells with their levels being dependent on NK cell activation state. CsA and tacrolimus, but not mycophenolic acid which mediates its immunosuppressive effects by inhibiting inosine monophosphate dehydrogenase, reduced activation and degranulation of NK cells, resulting in impaired cytotoxicity and IFN-γ production in response to leukemia targets. NK reactivity was also suppressed by the specific NFAT inhibitors VIVIT and INCA-6, indicating that the calcineurin inhibitors CsA and tacrolimus in fact modulate NK reactivity by inhibition of NFAT proteins and not by potential “off target”-effects. These results provide evidence for the critical involvement of the transcription factor NFAT in NK cell reactivity and also indicate that potential effects on NK cell immunosurveillance should be considered upon choice and dosing of immunosuppressive treatment regimens after SCT. Disclosures: No relevant conflicts of interest to declare.

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CD69 and CXCR6 Identify a Distinct Population of Human Bone Marrow and Spleen Resident NK Cells

Blood ◽

10.1182/blood.v126.23.1018.1018 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1018-1018

Author(s):

Gertjan Lugthart ◽

Carly Vervat ◽

Janine E Melsen ◽

Monique M Van Ostaijen-ten Dam ◽

Dave L Roelen ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Cell Population ◽

Nk Cell ◽

Cell Subset ◽

Blood Stream ◽

Human Bone ◽

Human Bone Marrow ◽

Cell Populations ◽

Effector Cells

Abstract In recent years, evidence has been provided that natural killer (NK) cells can function as anti-leukemic effector cells. In humans, two NK cell populations are usually distinguished: CD56dimCD16+ NK cells form the predominant population in blood while the CD56brightCD16- NK cell population is more prominent in tissues. However, little data regarding tissue specific characteristics of human NK cells are available, especially in bone marrow as an important localization of leukemic cells. Therefore, we evaluated the expression of chemokine receptors and adhesion molecules on NK cells in healthy donor blood, bone marrow and spleen by flow cytometry. Besides the two conventional NK cell subsets, a major third NK cell population was identified in bone marrow and spleen based on the combined expression of CD69 and CXCR6 (Figure 1). CD69+CXCR6+ NK cells represented 9-50% (mean 30%) of NK cells in marrow (n=15) and 26-57% (mean 43%) of NK cells in spleen (n=7). This CD69+CXCR6+ population was not detected in blood (n=15) nor in cord blood, neither mobilized into the blood stream after G-CSF treatment. The ratio and phenotype of the remaining conventional CD56bright and CD56dim NK cells in bone marrow and spleen were comparable to blood. Early after pediatric hematopoietic stem cell transplantation, CD69+CXCR6+ NK cells were absent in bone marrow, but gradually reached normal levels within the first year after transplantation. CD56 was expressed on marrow and spleen CD69+CXCR6+ NK cells at slightly lower levels than the conventional CD56bright NK cells and CD16 was expressed by 7-30% of CD69+CXCR6+ NK cells. CD69+CXCR6+ NK cells expressed high levels of the adhesion molecule CD54 (ICAM-1) as well as natural cytotoxicity triggering receptor NKp46 compared to the conventional CD56bright and CD56dim NK cell populations. CD69+CXCR6+ NK cells did not express the early differentiation markers CD117 (c-kit) and CD127 (IL7Rα), which are expressed by immature NK cells, type III innate lymphoid cells and, to some extent, by conventional CD56bright NK cells. The inhibitory receptor NKG2A, which is acquired early in maturation but lost during the differentiation of CD56dim NK cells, was expressed by 60% of CD69+CXCR6+ NK cells. Furthermore, CD69+CXCR6+ NK cells did not express markers acquired late in differentiation (KIRs, CD57, KLRG1, NKG2C). In functional experiments assessing cytokine producing capacity, CD69+CXCR6+ NK cells were comparable to the CD56dim subset, requiring the combined stimulation with IL12+IL15+IL18 to produce IFN-γ. However, with respect to cytotoxic potential, CD69+CXCR6+ NK cells more resembled the CD56bright NK cell subset; in resting state, CD69+CXCR6+ NK cells expressed perforin but not granzyme B, which was upregulated during overnight IL12+IL15 stimulation. Upon co-culture with K562 tumor cells, CD69+CXCR6+ NK cells degranulated (CD107a) at levels comparable to the conventional CD56bright and CD56dim NK cells. In summary, we identified a distinct NK cell population in human bone marrow and spleen. These cells were a) absent in blood, b) expressed tissue retention marker CD69, and c) were present alongside the conventional NK cell subsets in marrow and spleen. Together, these findings indicate that NK cells with the discriminative CD69+CXCR6+ phenotype constitute a tissue resident NK cell subset. Furthermore, CD69+CXCR6+ NK cells did not express CD49a+, distinguishing them from the recently described liver resident NK cells (Marquardt et al, 2015). Based on their surface receptor expression profile and functional characteristics, CD69+CXCR6+ NK cells have a mature signature that differs from both conventional NK cell subsets. Additional studies in healthy individuals and patients with immuno-hematological diseases are needed to investigate the immunological function of CD69+CXCR6+ NK cells. For example, these cells could have a local immunoregulatory or effector function. Alternatively, bone marrow and spleen may form a reservoir for effector cells waiting to be released into the blood stream. Figure 1. Identification of bone marrow and spleen resident NK cells Figure 1. Identification of bone marrow and spleen resident NK cells Disclosures No relevant conflicts of interest to declare.

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