NK Cell Differentiation Research Articles

Glycolipid stimulation of invariant NKT cells mobilizes precursors of mature NK cells and potentiates their participation in immune surveillance against metastatic cancer.

Abstract Precursors of mature natural killer (pre-mNK) cells, initially called IFN-producing killer dendritic cells, have been recently characterized as a novel intermediate in NK cell differentiation. Typified by a unique B220+NK1.1+CD11c+MHCII+ phenotype, pre-mNK cells exhibit prolific anti-tumor cytotoxicity while retaining the ability to present antigens thereby activating neighbouring T cells. Invariant NKT (iNKT) cells are another population highlighted for their potential in anti-cancer immunity. They are activated very early and play an important role in transactivating downstream effectors, such as NK cells. The extent to which iNKT cells can activate pre-mNK cells is unknown. We hypothesized that iNKT cells are able to activate pre-mNK cells and potentiate their anti-tumor properties. Wildtype C57BL/6 mice were injected i.p. with the iNKT cell superagonist, α-galactosylceramide (αGC), and various lymphoid tissues were harvested after 5 days for phenotyping or cytotoxicity assays. We have shown, for the first time that iNKT cell activation via αGC results in robust expansion of pre-mNK cells in the spleen, lung and most notably, in the liver. These cells exhibited cytotoxic activity against YAC-1 thymoma and B16 melanoma cancer cells via the granule exocytosis pathway and significantly contributed to overall NK cytotoxic activity in vivo. The anti-cancer responses due to αGC activation have been well documented; however, the contribution of pre-mNK cells in these responses have never been shown. Our findings demonstrate that pre-mNK cells rapidly expand due to iNKT activation while retaining their capacity to kill tumor targets. This may suggest a novel mechanism of targeting pre-mNK cells in anti-cancer therapies.

HLA-G peptide preferences change in transformed cells: impact on the binding motif

HLA-G is known for its strictly restricted tissue distribution. HLA-G expression could be detected in immune privileged organs and many tumor entities such as leukemia, multiple myeloma, and non-Hodgkin and Hodgkin’s lymphoma. This functional variability from mediation of immune tolerance to facilitation of tumor immune evasion strategies might translate to a differential NK cell inhibition between immune-privileged organs and tumor cells. The biophysical invariability of the HLA-G heavy chain and its contrary diversity in immunity implicates a strong influence of the bound peptides on the pHLA-G structure. The aim was to determine if HLA-G displays a tissue-specific peptide repertoire. Therefore, using soluble sHLA-G technology, we analyzed the K562 and HDLM-2 peptide repertoires. Although both cell lines possess a comparable proteome and recruit HLA-G-restricted peptides through the same peptide-loading pathway, the peptide features appear to be cell specific. HDLM-2 derived HLA-G peptides are anchored by an Arg at p1 and K562-derived peptides are anchored by a Lys. At p2, no anchor motif could be determined while peptides were anchored at pΩ with a Leu and showed an auxiliary anchor motif Pro at p3. To appreciate if the peptide anchor alterations are due to a cell-specific differential peptidome, we performed analysis of peptide availability within the different cell types. Yet, the comparison of the cell-specific proteome and HLA-G-restricted ligandome clearly demonstrates a tissue-specific peptide selection by HLA-G molecules. This exclusive and unexpected observation suggests an exquisite immune function of HLA-G.

T-bet and Eomes govern differentiation and function of mouse and human NK cells and ILC1.

T-bet and Eomes are T-box transcription factors that drive the differentiation and function of cytotoxic lymphocytes such as NK cells. Their DNA-binding domains are highly similar, suggesting redundant transcriptional activity. However, while these transcription factors have different patterns of expression, the phenotype of loss-of-function mouse models suggests that they play distinct roles in the development of NK cells and other innate lymphoid cells (ILCs). Recent technological advances using reporter mice and conditional knockouts were fundamental in defining the regulation and function of these factors at steady state and during pathological conditions such as various types of cancer or infection. Here, we review these recent developments, focusing on NK cells as prototypical cytotoxic lymphocytes and their development, and also discuss parallels between NK cells and T cells. We also examine the role of T-bet and Eomes in human NK cells and ILC1s. Considering divergent findings on mouse and human ILC1s, we propose that NK cells are defined by coexpression of T-bet and Eomes, while ILC1s express only one of these factors, either T-bet or Eomes, depending on the tissue or the species.

Interleukins 12 and 15 induce cytotoxicity and early NK-cell differentiation in type 3 innate lymphoid cells.

Type 3 innate lymphoid cells (ILC3s) fulfill protective functions at mucosal surfaces via cytokine production. Although their plasticity to become ILC1s, the innate counterparts of type 1 helper T cells, has been described previously, we report that they can differentiate into cytotoxic lymphocytes with many characteristics of early differentiated natural killer (NK) cells. This transition is promoted by the proinflammatory cytokines interleukin 12 (IL-12) and IL-15, and correlates with expression of the master transcription factor of cytotoxicity, eomesodermin (Eomes). As revealed by transcriptome analysis and flow cytometric profiling, differentiated ILC3s express CD94, NKG2A, NKG2C, CD56, and CD16 among other NK-cell receptors, and possess all components of the cytotoxic machinery. These characteristics allow them to recognize and kill leukemic cells with perforin and granzymes. Therefore, ILC3s can be harnessed for cytotoxic responses via differentiation under the influence of proinflammatory cytokines.

Low cardiorespiratory fitness is associated with higher differentiation of CD8pos T-cells but not NK-cells in the elderly: Preliminary evidence from the Texas City Stress and Health Study

Aging and latent cytomegalovirus (CMV) infection drives NK-cell and T-cell differentiation, but the effect of cardiorespiratory fitness (VO2max) on this process is unknown. We measured NK-cell (NKG2C/CD57) and T-cell phenotypes (CD28/CD27) by flow cytometry in relation to age, CMV infection, and VO2max in 317 respondents of the Texas City Stress and Health Study (58.4% female) aged 25–91 years. VO2max was estimated using non-exercise equations that incorporate physical activity rating, body-mass index, age and sex. VO2max was associated with the proportions of ‘early’ (CD28pos/CD27pos) and ‘late’ (CD28neg/CD27neg) differentiated CD8pos T-cells in 65plus year olds, but not in those under 65 years. The effect was independent of CMV serostatus and driven by a higher proportion of ‘early’ and lower proportion of ‘late’ differentiated CD8pos T-cells within the middle VO2max tertile compared to those in the lower tertile. There was no effect of VO2max on the proportion of NKG2Cpos, CD57pos or NKG2Cpos/CD57pos NK-cells. There was an age-independent increase in the proportion of ‘late’ and decreased proportion of ‘early’ CD8pos T-cells in CMV-seropositive individuals. CMV seropositivity was also associated with an age-independent increase in the proportion of NKG2Cpos/CD57pos NK-cells. Overall, low cardiorespiratory fitness shifts CD8pos T-cells toward a ‘late’ differentiated phenotype in the elderly that is independent of CMV serostatus, but does not affect NK-cell phenotype. These findings highlight the potential benefits of exercise on the aging immune system.

NK cell heparanase controls tumor invasion and immune surveillance.

NK cells are highly efficient at preventing cancer metastasis but are infrequently found in the core of primary tumors. Here, have we demonstrated that freshly isolated mouse and human NK cells express low levels of the endo-β-D-glucuronidase heparanase that increase upon NK cell activation. Heparanase deficiency did not affect development, differentiation, or tissue localization of NK cells under steady-state conditions. However, mice lacking heparanase specifically in NK cells (Hpsefl/fl NKp46-iCre mice) were highly tumor prone when challenged with the carcinogen methylcholanthrene (MCA). Hpsefl/fl NKp46-iCre mice were also more susceptible to tumor growth than were their littermate controls when challenged with the established mouse lymphoma cell line RMA-S-RAE-1β, which overexpresses the NK cell group 2D (NKG2D) ligand RAE-1β, or when inoculated with metastatic melanoma, prostate carcinoma, or mammary carcinoma cell lines. NK cell invasion of primary tumors and recruitment to the site of metastasis were strictly dependent on the presence of heparanase. Cytokine and immune checkpoint blockade immunotherapy for metastases was compromised when NK cells lacked heparanase. Our data suggest that heparanase plays a critical role in NK cell invasion into tumors and thereby tumor progression and metastases. This should be considered when systemically treating cancer patients with heparanase inhibitors, since the potential adverse effect on NK cell infiltration might limit the antitumor activity of the inhibitors.

Natural killer cell development and maturation is regulated by Sostdc1

Abstract Natural killer (NK) cells are specialized lymphocytes with innate ability to eliminate virally infected and cancerous cells. NK cells stochastically express Ly49 activating and inhibitory surface receptors, through mechanisms that are not fully understood. Ly49 expression during NK cell development is regulated by a combination of external and internal factors, such as major histocompatibility complex class I (MHC-I) ligands and cytokines. Global deletion of sclerostin domain containing-1 (Sostdc1), a secreted protein, leads to altered bone homeostasis via dysregulation of Wnt and BMP signaling, which in turn, could result in altered immune cell development in the bone marrow (BM). Using Sostdc1-knockout (KO) mice, we have discovered a novel role for Sostdc1 in NK cell development. Flow cytometric analysis of the Ly49 repertoire in immature (CD27+CD11b-; iNK), transitional (CD27+CD11b+, tNK) and most mature (CD27-CD11b+, mNK) Sostdc1-KO NK cells demonstrated expression differences in Ly49G2, I, H, and D receptors. We also observed a progressive increase in tNK cell numbers and frequencies as the KOs aged from 12 weeks to 30 weeks, indicating a slowing or partial block in NK cell maturation with aging. Reciprocal BM chimera experiments showed that Sostdc1 regulates NK cells through cellintrinsic and cell-extrinsic manners. Consistent with this, Sostdc1 is expressed in the bone periosteum and we discovered Sostdc1 expression in CD45+ NK1.1-CD11b+ BM cells and iNK cells. Taken together, these data support a role for Sostdc1 in the regulation of NK cell differentiation and identify potential NK cell niches. Current studies are investigating whether Sostdc1-KO NK cells respond to stimulation and lyse MHC-I deficient target cells.

Enhancement of cytokine-driven NK cell IFN-γ production after vaccination of HCMV infected Africans.

Human cytomegalovirus (HCMV) infection drives the phenotypic and functional differentiation of NK cells, thereby influencing the responses of these cells after vaccination. NK cell functional differentiation is particularly advanced in African populations with universal exposure to HCMV. To investigate the impact of advanced differentiation on vaccine‐induced responses, we studied NK‐cell function before and after vaccination with Trivalent Influenza Vaccine (TIV) or diphtheria, tetanus, pertussis, inactivated poliovirus vaccine (DTPiP) in Africans with universal, lifelong HCMV exposure. In contrast to populations with lower prevalence of HCMV infection, no significant enhancement of NK‐cell responses (IFN‐γ, CD107a, CD25) occurred after in vitro re‐stimulation of post‐vaccination NK cells with TIV or DTPiP antigens compared to pre‐vaccination baseline cells. However, both vaccinations resulted in higher frequencies of NK cells producing IFN‐γ in response to exogenous IL‐12 with IL‐18, which persisted for up to 6 months. Enhanced cytokine responsiveness was restricted to less differentiated NK cells, with increased frequencies of IFN‐γ+ cells observed within CD56brightCD57−, CD56dimCD57−NKG2C− and CD56dimCD57−NKG2C+ NK‐cell subsets. These data suggest a common mechanism whereby different vaccines enhance NK cell IFN‐γ function in HCMV infected donors and raise the potential for further exploitation of NK cell “pre‐activation” to improve vaccine effectiveness.

Natural killer cells differentiated in vitro from cord blood CD34+ cells are more advantageous for use as an immunotherapy than peripheral blood and cord blood natural killer cells

Background aimsNatural killer (NK) cells have the potential to become a successful immunotherapy as they can target malignant cells without being direct effectors of graft-versus-host disease. Our group has previously shown that large numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34+ cells. To produce a clinically relevant and effective immunotherapy, we hypothesized that it is essential that the NK cells are able to proliferate and persist in vivo while maintaining an optimal activation status and killing capacity. MethodsWe evaluated the proliferation capacity, telomere length and terminal differentiation markers expressed by NK cells differentiated in vitro. We also determined how their cytotoxicity compared with peripheral blood (PB) NK cells and CBNK cells when targeting patient acute myeloid leukemia (AML) blasts and solid tumor cell lines. ResultsWe found that the differentiated NK cells could respond to interleukin-2 and proliferate in vitro. Telomere length was significantly increased, whereas CD57 expression was significantly reduced compared with PBNK cells. The cytotoxicity of the differentiated NK cells was equivalent to that of the PBNK and CBNK cell controls, and priming consistently led to higher levels of killing of patient leukemic blasts and solid tumor cell lines in vitro. Interestingly, this activation step was not required to observe killing of patient AML blasts in vivo. ConclusionWe are able to generate NK cells from CBCD34+ cells in high numbers, allowing for multiple infusions of highly cytotoxic NK cells that have potential to further proliferate in vivo, making them a desirable product for application as an immunotherapy in the clinic.

Adaptive NK cells can persist in patients with GATA2 mutation depleted of stem and progenitor cells

AbstractHeterozygous GATA2 mutation is associated with immunodeficiency, lymphedema, and myelodysplastic syndrome. Disease presentation is variable, often coinciding with loss of circulating dendritic cells, monocytes, B cells, and natural killer (NK) cells. Nonetheless, in a proportion of patients carrying GATA2 mutation, NK cells persist. We found that peripheral blood NK cells in symptomatic patients uniformly lacked expression of the transcription factor promyelocytic leukemia zinc finger (PLZF), as well as expression of intracellular signaling proteins FcεRγ, spleen tyrosine kinase (SYK), and EWS/FLI1-Activated Transcript 2 (EAT-2) in a variegated manner. Moreover, consistent with an adaptive identity, NK cells from patients with GATA2 mutation displayed altered expression of cytotoxic granule constituents and produced interferon-γ upon Fc-receptor engagement but not following combined interleukin-12 (IL-12) and IL-18 stimulation. Canonical, PLZF-expressing NK cells were retained in asymptomatic carriers of GATA2 mutation. Developmentally, GATA-binding protein-2 (GATA-2) was expressed in hematopoietic stem cells, but not in NK-cell progenitors, CD3−CD56bright, canonical, or adaptive CD3−CD56dim NK cells. Peripheral blood NK cells from individuals with GATA2 mutation proliferated normally in vitro, whereas lineage-negative progenitors displayed impaired NK-cell differentiation. In summary, adaptive NK cells can persist in patients with GATA2 mutation, even after NK-cell progenitors expire. Moreover, our data suggest that adaptive NK cells are more long-lived than canonical, immunoregulatory NK cells.

Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns

The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56hi versus CD56dim defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation stages. Such comprehensive cytotoxic profiling may identify distinct patterns of cytotoxic potential relevant for specific infections, autoimmunity or tumor settings.

Smad3 promotes cancer progression by inhibiting E4BP4-mediated NK cell development

TGF-β is known to influence tumour progression. Here we report an additional role of Smad3 in the tumour microenvironment regulating cancer progression. Deletion or inhibition of Smad3 in the tumour microenvironment suppresses tumour growth, invasion and metastasis in two syngeneic mouse tumour models. Smad3−/− bone marrow gives rise to an expanded NK cell population with enhanced tumour-suppressive activities in vivo, and promotes differentiation of NK cells ex vivo. We identify E4BP4/NFIL3 as a direct Smad3 target gene critical for NK cell differentiation. Smad3 suppresses transcription of IFN-γ via E4BP4 in a T-bet independent manner. Therefore disruption of Smad3 enhances both the E4BP4-mediated NK cell differentiation and anti-cancer effector functions in vivo and in vitro. Furthermore, systemic treatment with a Smad3 inhibitor SIS3 effectively suppresses cancer progression. In summary, suppression of NK cell-mediated immunosurveillance via the Smad3-E4BP4 axis contributes to cancer progression. We propose targeting Smad3-dependent tumour microenvironment may represent an effective anti-cancer strategy.

Establishing High Dimensional Immune Signatures from Peripheral Blood via Mass Cytometry in a Discovery Cohort of Stage IV Melanoma Patients.

The identification of blood-borne biomarkers correlating with melanoma patient survival remains elusive. Novel techniques such as mass cytometry could help to identify melanoma biomarkers, allowing simultaneous detection of up to 100 parameters. However, the evaluation of multiparametric data generated via time-of-flight mass cytometry requires novel analytical techniques because the application of conventional gating strategies currently used in polychromatic flow cytometry is not feasible. In this study, we have employed 38-channel time-of-flight mass cytometry analysis to generate comprehensive immune cell signatures using matrix boolean analysis in a cohort of 28 stage IV melanoma patients and 17 controls. Clusters of parameters were constructed from the abundance of cellular phenotypes significantly different between patients and controls. This approach identified patient-specific combinatorial immune signatures consisting of high-resolution subsets of the T cell, NK cell, B cell, and myeloid compartments. An association with superior survival was characterized by a balanced distribution of myeloid-derived suppressor cell-like and APC-like myeloid phenotypes and differentiated NK cells. The results of this study in a discovery cohort of melanoma patients suggest that multifactorial immune signatures have the potential to allow more accurate prediction of individual patient outcome. Further investigation of the identified immune signatures in a validation cohort is now warranted.

Impact of C-rel inhibition of cord blood-derived B-, T-, and NK cells

The c-Rel transcription factor is a unique member of the nuclear factor (NF)-κB family that has a role in curtailing the proliferation, differentiation, cytokine production, and overall activity of B- and T-cells. In addition, c-Rel is a key regulator of apoptosis in that it influences the expression of anti-apoptotic genes such as Bcl-2 and Bcl-xL; conversely, inhibition of c-Rel increases cell apoptosis. To better understand the relationship between c-Rel expression and effects on B- and T-cell expansion, the current study evaluated c-Rel expression in cord blood mononuclear cells. This particular source was selected as cord blood is an important source of cells used for transplantation and immunotherapy, primarily in treating leukemias. As stem cell factor (SCF) and FLT3 are important agents for hematopoietic stem cell expansion, and cytokines like interleukin (IL)-2, -7, and -15 are essential for T- and B- (and also NK) cell development and proliferation, the current study evaluated c-Rel expression in cord blood mononuclear cells and CD34+ cells, as well as effects on B-, T-, and NK cells associated with alterations in c-Rel expression, using flow cytometry and PCR. The results showed c-Rel expression increased among cells cultured in the presence of SCF and FLT3 but was reduced when IL-2, IL-7, and IL-15 were used all together. Further, inhibition of c-Rel expression by siRNA reduced cord blood-derived B-, T-, and NK cell differentiation and expansion. These results indicated that with cells isolated from cord blood, c-Rel has an important role in B-, T-, and NK cell differentiation and, further, that agents (select cytokines/growth factors) that could impact on its expression might not only affect immune cell profiles in a host but could potentially also limit apoptotic activities in (non-)immune cells in that host. In the context of cancer (immuno)therapy, in particular, when cord blood is used an important source in stem cell transplantation in leukemia patients, such down-regulating changes in c-Rel levels could be counter-productive.

Identification of Human Memory-Like NK Cells.

Our understanding of NK biology is increased dramatically, a product of improved flow-cytometric techniques for analyzing these cells. NK cells undergo significant changes in repertoire during differentiation. A repeating stimulus, such as a cytomegalovirus infection, may result in accumulation of certain types of highly differentiated NK cells designated as memory-like, or adaptive NK cells. Adaptive NK cells are capable of rapid expansion and effective response to the recall stimulus. These cells differ significantly from conventional NK cells both functionally and phenotypically. Here we describe an approach for identification and analysis of adaptive NK cells in human peripheral blood. CD57-positive cells with high expression of activating-receptor NKG2C, increased expression of KIR receptors, lack of co-expression with inhibitory receptor NKG2A, and decreased expression of activating receptor NCR3 (NKp30) all characterize this cell type. The flow-cytometric method described below can identify this NK cell subset on a relatively simple flow cytometer. © 2017 by John Wiley & Sons, Inc.

JAK1 As a Convergent Regulator of Hematopoietic Stem Cell Function and Stress Hematopoiesis

Cytokine-mediated signal transduction is critical to hematopoiesis, immune responses, and other physiological processes. Aberrant production and secretion of pro-inflammatory cytokines disturbs homeostasis and proper immune function and if persistent results in symptoms of chronic inflammation. Previous studies have illustrated the importance of JAK1 as an effector of cytokine signaling, including in immunological and neoplastic diseases such that selective JAK1 inhibition is currently being investigated in clinical trials. However, the role of Jak1 in hematopoietic stem cell (HSC) function has not been delineated. This has led us to investigate the impact of loss of Jak1 signaling on HSC function by developing a novel conditional Jak1 knockout allele (Fig. 1a). Mice with conditional deletion of Jak1 in the hematopoietic system (hereafter referred to as Jak1 KO) are characterized by leukocytosis (Jak1 KO avg. 6.34K/ul, Jak1 WT avg. 10.76K/ul, P<0.01), and reduced spleen (Jak1 KO avg. 73.76mg, Jak1 WT avg. 98.86mg, P<0.01) and thymus weights (Jak1 KO avg. 49.31mg, Jak1 WT avg. 80.82mg, P<0.01). High dimensional single cell analysis of the hematopoietic compartment of these mice using mass cytometry showed that conditional Jak1 loss in hematopoietic cells attenuates B cell and NK cell differentiation in vivo, and results in differentiation towards the myeloid lineage at the expense of lymphoid fate commitment. Further, we observed a significant reduction of lineage-Sca1+cKit+ (LSK) cells in the bone marrow of Jak1 KO mice, including a decrease in CD34-Flk2- long-term HSCs (LT-HSCs) and in CD34+Flk2- short-term HSCs (ST-HSCs) (Fig.1b). Jak1-deficient cells formed fewer colonies in colony formation unit assays, which was also seen when clonogenic assays were performed in the presence of JAK1 inhibitor GLPG0634. Most importantly, Jak1-deficient stem cells exhibited decreased competitiveness in bone marrow transplantation assays. Flow analysis at 4 weeks post transplantation showed a 3-fold reduced blood chimerism in recipients transplanted with Jak1 KO bone marrow cells and at 16 weeks, Jak1KO cells were largely outcompeted by CD45.1-positive WT cells (Fig. 1c). Jak1-deficient stem cells were also unable to rescue hematopoiesis in the setting of myelosuppressive insults leading to a worse survival of Jak1 KO mice when serially injected with 5-fluorouracil (5-FU) (Fig. 1d).Consistent with the stem cell phenotype observed in JAK1 KO mice, we found that a significant larger proportion of Jak1-deficient stem cells lacks expression of the proliferation marker Ki67 and that Jak1-deficient stem cells fail to enter the cell cycle in response to hematopoietic stress. To begin to determine the mechanism by which Jak1 regulates normal stem cell function in vivo, we assessed the impact of loss of Jak1 on transcriptional output. Gene expression profiling of LT-HSCs from Jak1 KO and WT mice identified 259 significant genes, many of which were known to be Jak1 downstream targets. Gene set enrichment analysis (GSEA) revealed that the majority of genes that were altered following deletion of Jak1 corresponded to interferon signaling and inflammatory response pathways. Consistent with these findings, our functional in vitro and in vivo assays demonstrated that Jak1-deficient cells were insensitive to type I interferons as shown by lack of Stat1 and Stat5 activation (Fig. 1e), retained Sca1 surface expression, and an unchanged cell cycle status upon IFN stimulation. Moreover, the HSC defect observed in the setting of Jak1 loss was not fully rescued by expression of a constitutively active Jak2 allele, suggesting there is non-redundant signaling in HSCs within the JAK kinase family. Together, our data suggests that Jak1 functions as a central node for interferon signaling in HSCs and reveals an essential and nonredundant role of Jak1 in HSC homeostasis and stress response. [Display omitted] DisclosuresKoppikar:Amgen: Employment. Nolan:Fluidigm: Consultancy. Levine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Association of Peripheral Regulatory T Cells with Achievement of Deep Molecular Response in Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia Treated with Dasatinib - the Final Results of D-First Study

IntroductionAchievement of deep molecular response (DMR) has become an important treatment goal for patients with chronic phase chronic myeloid leukemia (CP-CML) since it is considered to be necessary for the challenge of stopping tyrosine kinase inhibitor (TKI) treatment. However, prognostic marker for prediction of DMR has not been established. We have previously reported the results of D-First study that shorter having time of BCR-ABL1 transcripts and early cytotoxic lymphocyte expansion were associated with achievement of DMR in newly diagnosed CP-CML patients treated with dasatinib. Here, the long-term follow-up results of the study were analyzed after a minimum 36 months follow-up. In this analysis, we mainly focus on dynamics of regulatory T cells (Treg) influencing patients' clinical course, as well as immunoprofiles during dasatinib treatment.Methods:A total of 52 patients with newly diagnosed CP-CML who were enrolled between June 2011 and June 2012 and treated with dasatinib 100 mg once daily on an open-label, multicenter, prospective phase II clinical trial (NCT01464411). All patients were followed-up for minimum of 36 months. Patients were assessed for molecular response before and 1, 3, 6, 9, 12, 15, 18, 24, and 36 months after starting dasatinib by real-time quantitative polymerase chain reaction analysis of BCR-ABL1 transcripts standardized on an international scale (BCR-ABL1 IS). A DMR was defined as less than 0.01% BCR-ABL1IS (MR4). The analysis of immunophenotyping of lymphocyte fractions in the peripheral blood samples was performed by flow cytometry before and 1, 2, 3, 6, 9, 12, 15, 18, 24, and 36 months after starting dasatinib treatment at a centralized laboratory (BML).Results:Patients’ characteristic at diagnosis has been reported previously. Briefly, the median age was 52 years. High Sokal risk score was seen in 12% patients. With a minimum follow-up of 36 months, 12 (23%) patients have discontinued therapy. Reasons for treatment discontinuation includes: pleural effusion (N=3), pericardial effusion (1), proteinurea and systemic edema (1), pulmonary hypertension (1), malaise (1), elevation of intraocular pressure (1), interstitial pneumonia (1), and patient’s requests (3). A cumulative rate of MMR was 75% by 12 months, 80% by 18 months, 86% by 24 months and 88% by 36 months. A cumulative rate of DMR was 49% by 12 months, 59% by 18 months, 59% by 24 months, and 65% by 36 months. Two patients died because of reasons unrelated to CML. No patients progressed to accelerated or blastic phase. Three-year overall survival was 96%. Flow cytometric analysis of peripheral blood revealed that average number of CD4+ T lymphocytes did not change over the course of 36 months. In contrast, ratio of CD4+CD25+CD127low Treg among CD4+ T cells decreased in a time-dependent manner during the follow-up. The ratio of Treg at 12 months of dasatinib treatment was associated with achievement of DMR, which was especially significant at 18 months (p<0.05). Differentiated NK cell represented a trend of increasing during the period of observation according to the analysis of CD3-57+/CD3-56+ ratio. In addition, differentiation degree in NK cells assessed by CD3-57+/CD3-56+ ratio was negatively associated with the probability of Treg through the treatment period, suggesting a critical role of Treg inhibition by dasatinib for the induction of NK cell differentiation.Conclusion:The long term results from this study of dasatinib as frontline treatment of newly diagnosed CP-CML showed the excellent results of achieving DMR. Inhibition of Treg in peripheral blood, possibly induced by dasatinib, was associated with the achievement of DMR, which could be one of the prognostic markers for predicting the important treatment goal. DisclosuresYoshida:Pfizer: Honoraria, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Iriyama:Bristol-Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Okamoto:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Astellas Pharma Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding. Kumagai:Pfizer: Speakers Bureau; Novartis: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau. Ohyashiki:Bristol-Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Ariad: Consultancy. Morita:Bristol-Myers Squibb: Speakers Bureau. Sakamoto:Takeda Pharmaceutical: Consultancy; Yakult: Other: Remuneration. Inokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

Expansion of Dysfunctional CD56-Negative NK Cells Is a Hallmark of NK Cell Activation in Ph+ Leukemia Patients Treated with Dasatinib

[Background] Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib, are key drugs for the treatment of Philadelphia chromosome-positive (Ph+) leukemia. Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) including NK cells in peripheral blood, which associates with a better prognosis. We previously showed that dasatinib expands CMV-associated highly differentiated NK cells in Ph+ leukemia patients through reactivation of CMV (Ishiyama, et al., Leukemia, 2016). NK cells consist of conventional CD56bright NK cells and CD56dim NK cells, and also rare CD56-negative (CD56neg) NK cells, which have been reported to increase in chronic viral infection such as HIV and HCV. Here, we show that CD56neg NK cells increase in CMV seropositive (CMV+) patients treated with dasatinib (DA), but not in those treated with other TKIs (OT). However, characteristics and clinical implications of CD56neg NK cells in CMV+DA patients remain unknown. Therefore, we sought to examine the phenotypic and functional properties of CD56neg NK cells in these patients.[Methods] We assessed NK cell subsets in 36 DA and 26 OT patients. NK cells were defined as lin-CD16+ or lin-CD56+ cells in peripheral blood. NK-cell marker expression and cytotoxic activity were compared between CD56neg NK cells and CD56dim NK cells in CMV+ DA patients with 5% or more of CD56neg NK cells. In vivo phosphorylation of intracellular signaling molecules in NK cells were evaluated by flow cytometry combined with phospho-specific antibodies (BD PhosflowTM).[Results] CD56neg NK cells exclusively increased in CMV+ DA group, compared to CMV- DA and CMV+ OT groups (median count: 20.9/μl, 1.1/μl, 1.5μl; p < 0.001. Median percentage: 4.5%, 0.8%, 0.4% of total NK cells; p < 0.001). CD56neg NK cell counts strongly correlated with total NK cell counts in CMV+ DA (r = 0.720, p < 0.001). CD56neg NK cells gradually increased over a period of one year after starting dasatinib. When we compared CD56neg NK cells and CD56dim NK cells in CMV+ DA, CD56neg NK cells showed decreased expression of activating NK cell markers including NKG2C, CD57, NKG2D, NKp30 and NKp46. In contrast, expression of PD-1 increased. CD56neg NK cells also showed lower rate of degranulation and IFN-γ production in functional assays. CMV+ DA patients with 4% or more of CD56neg NK cells significantly achieved deep molecular responses at 1 year after starting dasatinib than those with lower CD56neg NK cells (10/14, 3/14; p < 0.05). Phosflow analysis showed enhanced phosphorylation of ZAP70 in NK cells from CMV+ DA compared to CMV+ healthy controls (MFI/Isotype = 24.6 vs. 7.4, p < 0.01).[Discussion] CD56neg NK cells exclusively increased in CMV+ DA, and their increase persisted during dasatinib therapy. This suggests the similarity with the previous findings that CMV-associated differentiation in NK cells is enhanced during dasatinib therapy in CMV+ DA, which reflects the NK cell activation in response to CMV reactivation. In addition, CD56neg NK cells exhibited downregulation of activating receptors, upregulation of PD-1, and lower cytotoxic activity, indicating that these cells are the anergic and exhausted population as seen in chronic viral infection. These findings suggest that CD56neg NK cells accumulate owing to chronic stimulation by reactivated CMV during dasatinib therapy. ZAP70 is an immediate downstream tyrosine kinase of activating NK cell receptors, and is regulated by Src-family kinases, which is potentially inhibited by the kinase inhibitor property of dasatinib. Intriguingly, phosphorylation of ZAP70 in CD56dim NK cells was enhanced in CMV+ DA, although they had taken dasatinib a few hours before collecting blood samples. This finding suggests that strong activation of NK cells by CMV reactivation in CMV+ DA may overcome the direct suppressive effect of dasatinib on NK cell activation.[Conclusion] The accumulation of CD56neg NK cells is a consequence of NK cell activation specifically observed in dasatinib-treated patients. The NK cell activation is likely to be a response to the CMV reactivation induced by dasatinib treatment. Assessing CD56neg NK cells in peripheral blood could be a practical clinical indicator for the immunomodulatory effect of dasatinib, which significantly affects the prognosis. DisclosuresTakaori-Kondo:Chugai Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Merck Sharp and Dohme: Speakers Bureau; Pfizer: Research Funding; Toyama Chemical: Research Funding; Takeda Pharmaceutical: Research Funding; Kyowa Kirin: Research Funding; Eisai: Research Funding; Cognano: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Shionogi: Research Funding; Mochida Pharmaceutical: Research Funding; Alexion Pharmaceuticals: Research Funding; Bristol-Myers Squibb: Speakers Bureau.

Differential pulmonic NK and NKT cell responses in Schistosoma japonicum-infected mice.

Natural killer cells (NK cells) and natural killer T cells (NKT cells) play a role in anti-infection, anti-tumor, transplantation immunity, and autoimmune regulation. However, the role of NK and NKT cells during Schistosoma japonicum (S. japonicum) infection has not been widely reported, especially regarding lung infections. The aim of this study was to research the NK and NKT cell response to S. japonicum infection in the lungs of mice. Using immunofluorescent histological analysis, NK and NKT cells were found near pulmonary granulomas. Moreover, flow cytometry revealed that the percentage and number of pulmonic NK cells in S. japonicum-infected mice were significantly increased (P < 0.05). However, the percentage and cell number of NKT cells were decreased compared to those of normal mice (P < 0.05). The expression of CD69 on pulmonic NK and NKT cells was increased after infection (P < 0.05), and CD25 expression increased only on NKT cells (P < 0.05). Intracellular cytokine staining showed a higher percentage of IFN-γ+ and lower percentage of IL-5+ pulmonic NK cells (P < 0.05) compared to controls. However, the percentage of IL-17+, IL-10+, and IL-5+ pulmonic NKT cells significantly increased (P < 0.05). Additionally, there was a significant decrease in NKG2A/C/E (CD94) expression and an increase of NKG2D (CD314) expression on pulmonic NKT cells (P < 0.05), which might serve as a mechanism for NKT cell activation during S. japonicum infection.

Adaptive features of natural killer cells, lymphocytes of innate immunity

Natural killer cells (NK cells) are traditionally attributed to the innate immune system. It is considered that previously received stimuli have little effect on the functioning of these immune cells. Indeed, NK cells even without prior sensitization provide a rapid effector response against tumor, virus-infected, or otherwise damaged cells. They have a limited repertoire of receptors, the expression of which does not require somatic recombination. However, recent data indicate that NK cells may acquire the properties specific to adaptive immune cells. In particular, NK cells have been shown to possess the features of immunological memory, namely, the ability to more quickly and effectively respond to a repeated stimulus. The mechanisms of memory acquisition in NK cells differ from those implemented in T and B lymphocytes and are still rather vague. Presumably, some of these mechanisms underlie the significant phenotypic and functional NK cell diversity emerging during their differentiation. The recent data accumulated in this area considerably change the existing immunology paradigm, which postulates a clear delineation of the adaptive and innate immune cells. The published data on phenotypic and functional characteristics of NK cells and particular changes in these characteristics during NK cell differentiation and generation of memory-like NK cells are reviewed.