Immature NK Cells Research Articles

Old‐onset Energy Restriction is Detrimental to NK Cell Maturation

Even though young adult‐onset energy restriction (ER) has been shown to increase lifespan, we have previously reported increased susceptibility to influenza infection due to impaired NK cells in young mice. Other research suggests that there may be a benefit to young adult‐onset vs. old‐onset ER in preventing T cell senescence in rhesus monkeys. The aims of this research were to determine if aging exacerbates the impaired NK cell phenotype observed in ER mice, as well as determine if there is an optimal age of ER onset to prevent NK cell defects associated with aging. Male C57Bl/6 mice were analyzed at 22 mo, 40% ER began at either 4 mo (young adult‐onset, standard NIA protocol) or 20 mo (old‐onset). Aged ER mice had significantly increased immature (CD127+/CD27+CD11b−) NK cells in the lung and spleen, and significantly decreased mature (CD27‐CD11b+) NK cells in the lung, spleen, liver, and bone marrow compared to aged AL mice. Additionally, old‐onset ER is more detrimental to NK cells than young‐onset ER, as evidenced by increased immature (TRAIL‐DX5+) and decreased mature (TRAIL+DX5‐) NK cells in the bone marrow, lung, and spleen. Here we show that in aging, ER results in less mature NK cells, especially with old‐onset. Because NK cells are crucial to innate protection against viruses such as influenza, ER may lead to decreased protection from these immune challenges.Grant Funding Source: R01AG034949 (E.G.)

A case report on a patient who presented with myelofibrosis, developed breast cancer with myeloid metaplasia, and finally showed immature NK-cell leukemia or AML without maturation with CD56 expression transformation in the pleura with extramedullary hematopoiesis: A 25-year course

This study reports a female patient who suffered from primary myelofibrosis at 38 years of age, breast cancer with myeloid metaplasia at 49 years of age, and pleural effusion and multiple subcutaneous nodules at 62 years of age. She was finally diagnosed with immature NK-cell leukemia or AML without maturation with CD56 expression transformation of extramedullary hematopoiesis that developed in the pleura, and died 11 months later. Atypical cells in the pleural effusion had surface markers of CD13, CD33, CD34, and CD56 using a fluorescence-activated cell sorter analysis, and were positive for myeloperoxidase, CD34, CD43, and CD56 in a cell block material using an immunohistochemical method. Megakaryocytic and erythroblastic cells were also seen in the pleural effusion.

Peripheral Blood NK Cells from Breast Cancer Patients Are Tumor-Induced Composite Subsets

Human NK lymphocytes are involved in antitumor immunity. The therapeutic potential of this population against cancers has stimulated their study and led to the discovery of several NK cell subsets, each of which is endowed with different immunoregulatory functions. We have previously reported that NK cell functions are profoundly altered in advanced breast cancer patients. In this study, we show that these tumor-mediated alterations also variably affect NK cell subsets. We found that in addition to the known human CD56(dim)CD16(+), CD56(bright)CD16(-), and CD56(-)CD16(+) NK cell subsets, two additional subsets, namely the CD56(bright)CD16(+) and CD56(dim)CD16(-) subsets, were increased in the peripheral blood of patients with advanced invasive breast cancers. These subsets corresponded to the main two subsets found at the tumor site. The extensive phenotype of these subsets revealed an "à la carte" pattern of expression for the various NK receptors, functional molecules, adhesion molecules, and chemokine receptors, depending on the subset. We next compared these subsets to known NK cell populations endowed with specific phenotypic characteristics, but also with functional properties. Our data show that advanced breast cancer patients have an increased proportion of more immature and noncytotoxic NK cell subsets in their peripheral blood, which might account for at least part of the low cytotoxic functions observed in these patients. They reveal a major heterogeneity and plasticity of the NK cell compartment, which are both tightly linked to the microenvironment. The identification of NK cell subsets endowed with particular functional capabilities might help monitor residual antitumor NK cell-mediated responses in breast cancer patients.

Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Abstract 580Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer.Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product.The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures:Wunderlich:Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

Myeloid-Derived Suppressor Phenotype of Leukemic Blasts in Association with Increased Suppressor T Cell Subsets in Acute Myelogenous Leukemia– Evidence for Immunoediting.

AbstractAbstract 2528The quantitative lymphocyte recovery after remission induction is known to correlate with the outcome of the patients with acute myeloid leukemia (AML), suggesting the immune-surveillance is at least partly responsible for maintained remissions. However defects in lymphocyte function and increased regulatory T cell numbers suggest a permissive immune environment for leukemia cell growth. Understanding the interplay between the leukemia and the immune system may provide insight into the mechanisms governing leukemia cure or relapse after induction chemotherapy and lead to better remission treatments to prevent relapse. To look for evidence of immune escape and possible immunoediting of lymphocyte function by the leukemia, we undertook an extensive phenotypic characterization of lymphocytes and AML blasts in blood and marrow at presentation, through remission to full hematological recovery.Twenty patients (mean age 57 years; range 31–69) with newly diagnosed AML were enrolled in a protocol permitting blood and marrow collection for investigational purposes (VICCHEM 1073). Mononuclear cells were obtained from blood and marrow samples collected at onset, 14–21 days and 28–35 days after start of induction chemotherapy. Mononuclear cells were analyzed for lymphocyte phenotyping by flow cytometry and subset frequencies were compared with 5 healthy controls (mean age 31). Leukemic blast cells (Lineage CD2/3/19 negative, CD45+ cells) were characterized in blood and marrow. T cells showed several, largely persisting, abnormalities: (1) A decrease in central memory CD197+, CD27+, CD45RO+ CD8+ (p=0.03) and CD4+ T cells (p=0.1) in the blood at presentation with a corresponding decrease in CD197+CD27+ CD45RO-naïve subsets (2) A significant increase in PD1+ high expression suppressor phenotype for CD8+ (p=0.001) and CD4+ (p=0.01) cells at recovery (days 28–35). (3) At presentation FoxP3+ CD4+ regulatory T cells (Treg) were increased significantly at presentation (p=0.03) and remained elevated. This increase was due mainly to augmentation in the HeliosloFoxP3hi induced Treg (p=0.04) and CD45RAloFoxP3hi effector Treg fraction (p=0.002). These changes persisted into remission. The leukemic blast population included a variable population of cells with the CD11b+CD124+HLA-DRlo myeloid derived suppressor (MDSC) phenotype. The frequency of MDSC was higher in bone marrow (median 6.1% vs 3.1% in blood; p=0.44) and a significantly higher proportion of marrow MDSC expressed the lymphocyte suppressor molecule PDL-1 (54.5% vs 28.5%; p=0.0189). NK cell subsets showed significantly lower frequencies of immature CD56bright NK cells (p=0.02), and correspondingly decreased more mature CD56dim CD57-KIR-NKG2A+ subsets at presentation (p=0.039). NK cell subsets trend to recover during remission but with reduced frequency of mature CD57+, KIR+ CD56dim NK cells in recovery (p=0.018).This study confirms the occurrence of significant alterations in T cell phenotype associated with increased regulatory and suppressor phenotype. The occurrence of the MDSC phenotype within the leukemia blast population suggests that myeloid leukemia cells promote a permissive immune environment for leukemia growth and persistence through immunoediting. These changes as well as abnormalities in NK cells tend to persist into remission and may contribute to leukemic relapse. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Bone Marrow Functions as the Central Site of Proliferation for Long-Lived NK Cells

NK cells play an important role in the early defense against invading pathogens. Although it is well established that infection leads to a substantial, local increase in NK cell numbers, little is known about the mechanisms that trigger their proliferation and migration. In this study, we investigated the dynamics of NK cell responses after intranasal respiratory virus infection. We show that NK cell numbers increased in the airways after influenza virus infection but find no evidence of proliferation either at the site of infection or in the draining lymph nodes. Instead, we find that the bone marrow (BM) is the primary site of proliferation of both immature and mature NK cells during infection. Using an adoptive transfer model, we demonstrate that peripheral, long-lived and phenotypically mature NK cells migrate back to the BM and proliferate there, both homeostatically and in response to infection. Thus, the BM is not only a site of NK cell development but also an important site for proliferation of long-lived mature NK cells.

Receptor modulation and functional activation of human CD34+Lin−‐derived immature NK cells in vitro by Mycobacterium bovisBacillus Calmette‐Guerin (BCG)

It is not yet clear whether immature NK (iNK) cells are bystanders to or rather participate in immune responses to pathogens that may colocalize in areas of NK-cell maturation such as bone marrow or lymph nodes. Mycobacteria, including Bacillus Calmette-Guerin (BCG), have been shown to interact with peripheral NK cells and in vivo may colocalize in areas of iNK-cell development. We studied infection with BCG of human cord blood CD34(+) Lin(-)-derived cultures containing myelomonocytes and iNK cells in vitro. Increased iNK-cell DNAM-1 expression, transient natural cytotoxicity receptor modulation, and production of IFN-γ were observed. Transcriptional receptor modulation was associated to BCG challenge, which determined increased iNK-cell cytotoxic activity against tumor cell lines and also increased killing of immature dendritic cells (iDCs). No requirement for cell contact was recorded for BCG-induced iNK-cell activation, while cytokine production including IL-18, IL-10, GM-CSF, and TGF-β contributed to the observed effects. Thus, iNK cells are affected by mycobacteria in vitro and may contribute to shaping of adaptive mature innate responses through iDC-iNK cross-talk. In addition, iNK-cell activation by BCG may represent a novel additional mechanism contributing to the effects observed upon BCG administration in vivo.

Resident Peritoneal NK Cells

In this study, we describe a new population of NK cells that reside in the normal, uninflamed peritoneal cavity. Phenotypically, they share some similarities with the small population of CD49b(-), CD27(+) immature splenic NK cells, as well as liver NK cells, but they differ in their expression of CD62L, TRAIL, and EOMES. Functionally, the peritoneal NK cells resemble the immature splenic NK cells in their production of IFN-γ, GM-CSF, and TNF-α and in the killing of YAC-1 target cells. We also found that the peritoneum induces different behavior in mature and immature splenic NK cells. When transferred i.v. into RAGγc knockout mice, both populations undergo homeostatic proliferation in the spleen, but only the immature splenic NK cells are able to reach the peritoneum. When transferred directly into the peritoneum, the mature NK cells survive but do not divide, whereas the immature NK cells proliferate profusely. These data suggest that the peritoneum is not only home to a new subset of tissue-resident NK cells, but that it differentially regulates the migration and homeostatic proliferation of immature versus mature NK cells.

IL-15–High-Responder Developing NK Cells Bearing Ly49 Receptors in IL-15−/− Mice

In mice lacking IL-15, NK cell development is arrested at immature stages, providing an opportunity to investigate the earliest developing NK cells that would respond to IL-15. We show in this study that immature NK cells were present in the spleen as well as bone marrow (BM) and contained IL-15-high-responder cells. Thus, mature NK cells were generated more efficiently from IL-15(-/-) than from control donor cells in radiation BM chimeras, and the rate of IL-15-induced cell division in vitro was higher in NK cells in the spleen and BM from IL-15(-/-) mice than in those from wild-type mice. Phenotypically, NK cells developed in IL-15(-/-) mice up to the minor but discrete CD11b(-)CD27(+)DX5(hi)CD51(dull)CD127(dull)CD122(hi) stage, which contained the majority of Ly49G2(+) and D(+) NK cells both in the spleen and BM. Even among wild-type splenic NK cells, IL-15-induced proliferation was most prominent in CD11b(-)DX5(hi) cells. Notably, IL-15-mediated preferential expansion (but not conversion from Ly49(-) cells) of Ly49(+) NK cells was observed in vitro only for NK cells in the spleen. These observations indicated the uneven distribution of NK cells of different developing stages with variable IL-15 responsiveness in these lymphoid organs. Immature NK cells in the spleen may contribute, as auxiliaries to those in BM, to the mature NK cell compartment through IL-15-driven extramarrow expansion under steady-state or inflammatory conditions.

Major Remodelling of the Murine Stem Cell Kinome Following Differentiation in the Hematopoietic Compartment

The changes in signal transduction associated with the acquisition of specific cell fates remain poorly understood. We performed massive parallel assessment of kinase signatures of the radiations of the hematopoietic system, including long-term repopulating hematopoietic stem cells (LT-HSC), short-term repopulating HSC (ST-HSC), immature natural killer (iNK) cells, NK cells, B cells, T cells, and myeloid cells. The LT-HSC kinome is characterized by noncanonical Wnt, Ca(2+) and classical protein kinase C (PKC)-driven signaling, which is lost upon the transition to ST-HSC, whose kinome signature prominently features receptor tyrosine kinase (RTK) activation of the Ras/MAPK signaling cassette. Further differentiation to iNK maintains signaling through this cassette but simultaneously leads to activation of a PI3K/PKB/Rac signaling, which becomes the dominant trait in the kinase signature following full differentiation toward NK cells. Differentiation along the myeloid and B cell lineages is accompanied by hyperactivation of both the Ras/MAPK and PI3K/PKB/Rac signaling cassette. T cells, however, deactivate signaling and only display residual G protein-coupled pathways. Thus, differentiation along the hematopoietic lineage is associated with major remodelling of cellular kinase signature.

CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adult mice

AbstractNatural killer (NK) cells are granular lymphocytes that are generated from hematopoietic stem cells and play vital roles in the innate immune response against tumors and viral infection. Generation of NK cells is known to require several cytokines, including interleukin-15 (IL-15) and Fms-like tyrosine kinase 3 ligand, but not IL-2 or IL-7. Here we investigated the in vivo role of CXC chemokine ligand-12 (CXCL12) and its primary receptor CXCR4 in NK-cell development. The numbers of NK cells appeared normal in embryos lacking CXCL12 or CXCR4; however, the numbers of functional NK cells were severely reduced in the bone marrow, spleen, and peripheral blood from adult CXCR4 conditionally deficient mice compared with control animals, probably resulting from cell-intrinsic CXCR4 deficiency. In culture, CXCL12 enhanced the generation of NK cells from lymphoid-primed multipotent progenitors and immature NK cells. In the bone marrow, expression of IL-15 mRNA was considerably higher in CXCL12-abundant reticular (CAR) cells than in other marrow cells, and most NK cells were in contact with the processes of CAR cells. Thus, CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adults, and CAR cells might function as a niche for NK cells in bone marrow.

In Vitro Generation of NK22 Cells From Hematopoietic Stem Cells

AbstractAbstract 108NK cells are the first lymphocytes to recover after allogeneic hematopotiec cell transplantation (allo-HCT). Rapid NK recovery after allo-HCT is associated with reduced treatment related mortality. Because NK cells elaborate inflammatory cytokines (IFN-g) and mediate cytotoxic killing of malignant cells, they are also implicated in graft vs. leukemia reactions. Curiously early after transplant, donor-derived NK cells are hypofunctional and immature. Over the past year, investigators identified a new category of NK cells, called NK22 cells. These cells are present in secondary lymphoid tissue, such as tonsils, lymph nodes and Peyer's patches. Previous investigators have not been able to identify NK22 cells in adult blood or UCB, likely due to lymphoid tissue homing receptor expression (CCR6 and CCR7). NK22 cells are CD56+/−CD117highCD94−IL-1bR+, a phenotype which overlaps with one previously used to describe NK progenitors (i.e., stage III immature NK cells). At present, it is not known whether NK22 cells are a distinct branch of the NK lineage or are NK developmental intermediates. NK22 cells are present in secondary lymphoid tissue at vanishingly small quantities (<1% of all mononuclear cells), thereby making the study of these cells challenging. Functionally, NK22 cells lack of “classical” NK functions (cytotoxicity and IFN-g production) and instead elaborate IL-22 in response to dendritic cell derived IL-1 and/or IL-23. IL-22 does not act on hematopoietic cells, but rather on mucosal tissues to induce proliferation, anti-apoptotic functions and the production of antimicrobial proteins (b defensins). NK22 cells also increase the expression of adhesion molecules on MSCs after co-culture, suggesting a role in secondary lymphoid generation and homeostasis. We have previously used a stromal cell based culture system to study NK development from hematopoietic stem cells. Briefly, CD34+ cells are cultured in the presence of IL-3 (for the first week), FLT-3L, SCF, IL-7 and IL-15 for ~4-5 weeks. At the end of this culture period, functional mature NK cells are obtained. Because this system closely recapitulates ontongeny, we hypothesized that it could be used to study NK22 development. At D28 of culture, we found that 90% (range=88-94%) of cells expressed CD56. Approximately 22% (range=16-28%) had a stage III immature NK cell phenotype (i.e., CD56+CD117highCD94−), of which ~87% (range=77-93%) also expressed IL-bR, a phenotype consistent with NK22 cells (n=5). We next purified CD56− and CD56+ cell populations in these cultures and neither showed IL-22 expression at rest. Following IL-1 and/or IL-23 stimulation, the CD56+ fraction made IL-22 transcripts (by qPCR) and protein by ELISA. We next purified the stage III (CD56+CD117highCD94−) and stage IV (CD56+CD117lowCD94+) fractions and found that only the stage III cells were capable of IL-22 production following IL-1/23 stimulation. Co-culture of NK22 cells (or their supernatant) with MSCs resulted in a >2 log increase in ICAM. Likewise, the supernatant of from activated stage III cells induced keritinocyte proliferation and production of antimicrobial compounds. In vitro derived NK22 cells were compared to freshly isolated NK22 cells from human tonsils and nearly identical staining patterns for ROR-gt, Acyl hydrocarbon receptor, NKp44, NKp46, CD127, CD161, CCR6 and CCR7 were observed. Lastly, purified CD56+CD117highCD94− cells could acquire IL-bR and then further differentiate into stage IV cells (CD56+CD117lowCD94+) in the presence of IL-15. However, this was less likely in the presence of IL-15 and IL-1b, suggesting that NK22 cells are developmental intermediates with specific functions (SLT homeostasis and maintenance of mucosal surfaces and immunity). Depending upon the environmental stimuli, these cells will either maintain their IL-22 producing capacity or develop into cytotoxic lymphocytes. These studies are the first studies to describe the generation of NK22 cells from hematopoietic stem cells. They also allow a better understanding of the developmental requirements and functions of these rare cells. Lastly, this simple culture system creates a new opportunity to use NK22 cells therapeutically to enhance SLT tissue repair and mucosal immunity after allo-HCT. Disclosures:No relevant conflicts of interest to declare.

Immature NK cells, capable of producing IL-22, are present in human uterine mucosa.

NK cells are the dominant population of immune cells in the endometrium in the secretory phase of the menstrual cycle and in the decidua in early pregnancy. The possibility that this is a site of NK cell development is of particular interest because of the cyclical death and regeneration of the NK population during the menstrual cycle. To investigate this, we searched for NK developmental stages 1-4, based on expression of CD34, CD117, and CD94. In this study, we report that a heterogeneous population of stage 3 NK precursor (CD34(-)CD117(+)CD94(-)) and mature stage 4 NK (CD34(-)CD117(-/+)CD94(+)) cells, but not multipotent stages 1 and 2 (CD34(+)), are present in the uterine mucosa. Cells within the uterine stage 3 population are able to give rise to mature stage 4-like cells in vitro but also produce IL-22 and express RORC and LTA. We also found stage 3 cells with NK progenitor potential in peripheral blood. We propose that stage 3 cells are recruited from the blood to the uterus and mature in the uterine microenvironment to become distinctive uterine NK cells. IL-22 producers in this population might have a physiological role in this specialist mucosa dedicated to reproduction.

Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages.

Thymocyte selection-associated HMG box factor (TOX) is a DNA-binding factor required for development of CD4 T cells, natural killer T cells, and T regulatory cells. Here we document that both NK cell development and lymphoid tissue organogenesis are inhibited in the absence of TOX. We find that development of lymphoid tissue inducer cells, a rare subset of specialized cells that plays an integral role in lymphoid tissue organogenesis, requires TOX. Tox is highly upregulated in immature NK cells in the bone marrow, consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus, multiple cell lineages in the immune system share a TOX-dependent step for development.

Continuous CD27 triggering in vivo strongly reduces NK cell numbers

NK cells are important mediators of the early defense. In mice, immature and mature NK (mNK) cells constitutively express the TNF receptor family member CD27; however, mNK cells eventually lose CD27 expression and become resting NK cells. Interaction of CD27 with its ligand, CD70, enhances proliferation and effector functions of NK cells. We used mice that constitutively express CD70 on B cells (CD70-Tg) to study the in vivo effects of continuous triggering of CD27 on NK cells. Continuous CD70-CD27 interaction resulted in strongly down-modulated CD27 expression on NK cells and gradually reduced absolute NK cell numbers. This reduction was most prominent in the mNK cell subpopulation and was at least partially due to increased apoptosis. Residual NK cells showed lower expression of activating Ly49 receptors and normal (liver) or decreased (spleen) IFN-gamma production. Nevertheless, NK cells from CD70-Tg mice displayed higher YAC-1 killing capacities. CD70-Tg NK cells exhibited up-regulated expression of NKG2D, which is in accordance with the increased YAC-1 lysis, as this is mainly NKG2D-dependent. Taken together, this study is the first to demonstrate that continuous CD70 triggering of CD27 on NK cells in vivo results in a severe reduction of NK cells. On a single cell basis, however, residual NK cells display enhanced cytotoxicity.

Salivary gland NK cells are phenotypically and functionally unique (89.20)

Abstract NK cells and CD8+ T cells play vital roles in containing and eliminating systemic cytomegalovirus. However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Notably, naïve salivary gland NK cells have a reduced expression of CD27, are nearly absent of the inhibitory receptor KLRG1, yet are mostly CD11b positive. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer (LTi) NK-like cells and are not thymic derived. During the course of MCMV infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited effector functions in vivo. Salivary gland NK cell effector functions are not regulated by iNKT or Treg cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that latency in SMG may result from both the presence of unfit NK cells and the lack of recruitment of peripheral NK cells.

Pediatric myeloid/NK cell precursor lymphoma/leukemia expressing T/NK immunophenotype markers

Myeloid/NK cell precursor lymphoma/leukemia has been suggested to be of precursor NK origin. We report a 1-year-old boy with myeloid/NK cell precursor lymphoma/leukemia who presented with a skin nodule. Biopsy of the skin nodule specimen revealed CD45(+), CD56(+), myeloid antigen(+), CD7(-), CD3(-), CD19(-), CD34(-), CD4(-) by flow cytometry and myeloperoxidase (MPO)(+), TdT(+), cytoplasmic CD3(+) by immunohistochemistry. He received acute myeloid leukemia-oriented combination chemotherapy. Although his skin nodule disappeared, regrowth was observed; a biopsy was performed again, showing that the immunophenotype was CD56(+), myeloid antigen(+), HLA-DR(+), MPO(-), CD3(+) and TCRgammadelta(+). Being refractory to chemotherapy, he underwent stem cell transplantation from his father. However, he relapsed again and died from septic shock. Although myeloid/NK cell precursor acute leukemia is characterized by CD7(+), CD56(+), CD3(-), CD34(+) and myeloid antigen(+) phenotype, the blast cells of our patients lacked CD34 and CD7 expression while expressing myeloid antigens. Furthermore, CD94 1A transcripts were predominantly expressed rather than CD94 1B, implying their origin in immature NK cells. After acute myeloid leukemia-oriented combination chemotherapy, his blasts acquired stable CD3 expression and TCRgammadelta rearrangement at recurrence. The blast cells possessed features overlapping both myeloid/NK precursor acute leukemia and blastic NK/precursor acute lymphoma/leukemia. These observations suggest that two subclones originated from common precursors.

Mouse ChemR23 Is Expressed in Dendritic Cell Subsets and Macrophages, and Mediates an Anti-Inflammatory Activity of Chemerin in a Lung Disease Model

Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.

Leptin Signaling Protects NK Cells from Apoptosis During Development in Mouse Bone Marrow

Increasing evidence indicates a role of leptin in immune response, but it remains largely unclear whether leptin signaling is involved in regulating NK cell development in the bone marrow (BM). In this study, we have characterized NK cell differentiation and maturation in the BM of leptin-receptor deficient db/db mice at a prediabetic stage. Although the BM cellularity was similar to the control value, the total number of NK cells was severely reduced in mutant mice. Flow cytometric analysis of db/db BM cells revealed significantly decreased frequencies of developing NK cells at various stages of differentiation. BM db/db NK cells displayed markedly increased apoptosis but maintained normal cell cycling status and proliferative capacity. Moreover, recombinant leptin could significantly enhance the survival of NK cells from wild-type mice in cultures. Further examination on NK cell functional activity showed that db/db NK cells exhibited normal intrinsic cytotoxicity with significantly increased IL-10 production. Taken together, our findings suggest that leptin signaling regulates NK cell development via enhancing the survival of immature NK cells in mouse BM.

Characterization of natural killer cells in nonobese diabetic/severely compromised immunodeficient mice during pregnancy

To characterize uterine natural killer (uNK) cells in nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice and investigate the potential role of these cells in pregnancy tolerance. An animal model-based study. Academic research center in a university. Syngeneic pregnant NOD/SCID mice were compared with non-immunodeficient BALB/c mice. Induction of Toll-like receptor (TLR) agonists. Flow cytometric analysis was performed to detect the percentage of cell subsets, and standard (51)Cr release assay was performed to determine cytotoxicity. The dominant subset of uNK cells in NOD/SCID mice is DX5 (CD49b)(+), asialo ganglio-N-tetraosylceramide(+), CD25(+), CD122(+), Thy-1 (CD90)(hi), c-kit (CD117)(hi), and interleukin-10(+). In addition, the percentage of interferon-gamma(+) subset was slightly increased in response to selected TLR agonists in the NOD/SCID mice, whereas the corresponding percentage in BALB/c mice could be increased dramatically. Such an effect could be abrogated by inhibitors, including LY294002, SP600125, and PD98059. The significant increase of interferon-gamma(+) NK cell percentage in BALB/c mice was concomitant with the increase of the embryo resorption rate. In contrast, the resorption rate in NOD/SCID mice was not significantly increased upon the induction of polyinosinic polycytidylic acid or lipopolysaccharide. As expected, the NK cells from NOD/SCID mice display a detectable but lower cytotoxicity than BALB/c, as determined by standard (51)Cr release assay. In addition, the uNK cells from NOD/SCID mice also display a hyposensitivity to lipopolysaccharide-induced production of inducible nitric oxide synthase. A considerable percentage of immature NK cells were detected at the fetomaternal interface in NOD/SCID mice. These cells were hyposensitive to the stimulation of selected TLR agonists. Such a status seemed to be beneficial for the maintenance of pregnancy.