Natural Killer Subsets Research Articles

Age-stratified pediatric reference values of lymphocytes in the Moroccan population.

The aim of this study was to establish the age-stratified normal reference values of blood lymphocyte subsets for the pediatric Moroccan population. We measured the concentration of lymphocyte subpopulations by flow cytometry from 83 Moroccan healthy subjects stratified into 5 age groups of 0-1, 1-2, 2-6, 6-12 and > 12-18 (adult). The absolute and relative amounts of the main lymphocyte subsets of T-cells, B cells and Natural Killer (NK) cells were measured and compared to previously described reference values from Cameroonian, Turkish, American and Dutch populations. Additionally, we also observed an age-related decline in the absolute population sizes of lymphocyte subsets within our study group. Relative proportions of CD3+CD4+ helper T lymphocytes decreased with increasing age and by 12 years-adult age, both proportions of CD3+CD4+ helper T lymphocytes and CD3+CD8+ cytotoxic T lymphocytes, as well as CD3-CD19+ B lymphocytes were also decreased. Finally, we compared the median values and range of our Moroccan study group with that of published results from Cameroon, Turkey, USA and Netherlands and observed significant differences in median and mean values of absolute number and relative proportions of lymphocyte subsets especially at 0-1 years and 1-2 years age groups. Above age 12 years, the Moroccan values were lower. For NK cells, the Moroccan values are also lower. The results of this study have a significant impact in improving the threshold values of the references intervals routinely used in the diagnosis of paediatric diseases such as PIDs or mother-to-child transmitted HIV within the Moroccan population.

NK, NKT and Invariant-NKT Cells in Tumor Draining Lymph Nodes of Patients with Breast Cancer.

NK (natural killer) and NKT (natural killer T) cells, as components of innate immune system, play a crucial role in tumor progression and dissemination. To investigate the percentages of NK cells, NKT cells, iNKT (invariant natural killer T) cells, total T lymphocytes as well as activated T lymphocytes, in tumor draining lymph nodes (TDLNs) of patients with breast cancer (BC) and their association with different clinic-pathological features of the patients. Axillary lymph nodes were obtained from 30 Iranian women with breast cancer. After routine pathological evaluations, mononuclear cells were separated from their lymph nodes and incubated with appropriate fluorochrome conjugated monoclonal antibodies specific for CD3, HLA-DR, CD16/56, and Vα24Jα18-TCR. Data were collected on a four-color flow cytometer and analyzed by CellQuest software. The mean percentages of NK (CD3-CD16/56+), NKT (CD3+CD16/56+) and iNKT (Vα24Jα18-TCR+) cells in TDLNs mononuclear cells of BC patients were 2.04%, 2.44% and 0.1%, respectively. A significant decrease in the percentages of NK and iNKT subsets in patients with grade I was observed compared to grade III (p=0.03 and p=0.01, respectively). Moreover, NK cells were increased in patients with grade III of BC compared to grade II (p= 0.003). The increase in the percentage of NK and iNKT cells in TDLNs of patients with higher grade of BC might suggest a suppressive phenotype for these cells in breast cancer, which merit more functional investigation.

MYC Oncogene Abrogates Natural Killer (NK) Cell-Mediated Immune Surveillance of B- and T- Lymphoid Malignancies By Suppressing STAT1/2-Type I IFN Signaling

Background: Many high-risk B- and T- lymphoid malignancies including Acute Lymphoblastic Leukemia (ALL) and lymphomas exhibit hyperactivation of the MYC oncogene and MYC-associated pathways. Experimentally, direct targeting of MYC in mouse models of MYCHigh lymphoid cancers sustains tumor regression. However, the requirement of MYC in normal lymphocyte physiology has impeded the development of MYC inhibitors. Hence, the development of targeted therapies against MYCHigh lymphoid cancers requires the identification of cell-intrinsic and cell-extrinsic (immune microenvironment) processes uniquely regulated by 'oncogenic' MYC (MYCHigh B/T-lymphoblasts) but not by 'normal' MYC (MYCLow B/T-lymphocytes). Approach: We employed an inducible transgenic mouse model of MYC-driven T-ALL (SRα-tTA/Tet-O-hMYC mice; Felsher and Bishop, Molecular Cell, 1999) to study leukemia-intrinsic, and leukemia-extrinsic immune surveillance mechanisms upon MYC activation (MYCHigh/ON, overt T-ALL), and MYC inactivation (MYCLow/OFF, regressed T-ALL). Inducible regulation of the human MYC (hMYC) transgene specifically in T-lymphoblasts enables us to elucidate how T-ALL-intrinsic MYC impacts normal immune cells during leukemogenesis in vivo. Using mass cytometry (CyTOF), and CIBERSORT to profile the immune microenvironment of MYCHigh/ON and MYCLow/OFF T-ALLs in SRα-tTA/Tet-O-hMYC mice, we identified specific anti- and pro-tumorigenic immune subsets that can be modulated to develop targeted immunotherapies against MYC-driven lymphoid cancers. Results: By conducting CyTOF-based immune profiling of lymphoid organs in healthy mice, and mice bearing MYCON or MYCOFF T-ALL, we demonstrated a significant reduction in numbers of Natural Killer (NK) cells, and an increase in the absolute counts of neutrophils and dendritic cells (DCs) in MYCON mice, in comparison to healthy controls and MYCOFF mice. The reduction in NK cell numbers in MYCON mice led us to hypothesize that the NK subset may play an anti-tumorigenic role in MYC-driven T-ALLs. Since anti-tumor immune subsets can be developed as therapies against MYC-driven lymphoid cancers, we decided to focus on how MYC impacts NK cell-mediated immune surveillance. We demonstrated that mature CD3-NKp46+ Natural Killer (NK) cells are specifically 'excluded' from the T-ALL microenvironment, in a MYC-dependent fashion. Residual NK cells in MYCON T-ALL-bearing mice exhibited suppression of the NK cell maturation/cytotoxicity marker, NKp46. Concordant with the suppression of NKp46 on NK cells in MYCON mice, we observed a blockade in early NK cell development from the NK precursor (NKP) to the immature NK (iNK) stage which is marked by the expression of NKp46. Next, we showed that adoptive transfer of mature CD3- NKp46+ syngeneic NK cells alone is sufficient to delay the initiation of MYCON T-ALL, and the recurrence of MYCOFF T-ALL. Further investigation into the molecular mechanism behind blockade of NK cell maturation in MYC-driven B/T-lymphoid cancers revealed that cancer-intrinsic MYC transcriptionally represses STAT1/2-Type I IFN signaling required for early NK cell maturation from NKP to iNK stage. We observed that treating T-ALL-bearing SRα-tTA/Tet-O-hMYC mice (MYCON)with Type I IFN improves survival by rescuing NK cell maturation. We showed that that low expression of both STAT1 and STAT2 in patients with MYCHigh B- and T-lymphoid neoplasms correlates significantly with the absence of activated NK cells, and predicts unfavorable clinical outcomes. Of note, aggressive MYCHigh B/T-lymphoid cancers are often treated with Type I IFNs, but the molecular mechanisms underlying the anti-cancer properties of Type I IFNs are not completely understood. We demonstrate for the first time that MYC-mediated suppression of NK surveillance may in part be responsible for the sensitivity of B/T-lymphoid cancers to Type I IFN therapy. Conclusion: We conclude that subversion of NK cell-mediated immune surveillance is critical for MYC-induced leukemogenesis. Our studies thus provide a rationale for developing targeted NK cell-based therapies as alternatives to direct MYC inhibition for treating refractory MYCHigh B- and T- lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

CD56+CD57+ infiltrates as the most predominant subset of intragraft natural killer cells in renal transplant biopsies with antibody-mediated rejection

Little is known about the characteristics and clinical implications of specific subsets of intragraft natural killer (NK) cells in kidney transplant recipients. We analyzed 39 for-cause renal transplant biopsies performed at our center from May 2015 to July 2017. According to histopathologic reports, 8 patients (20.5%) had no rejection (NR), 11 (28.2%) had T cell-mediated rejections (TCMR) only, and 20 (51.3%) had antibody-mediated rejection (ABMR). NK cells were defined as CD3–CD56+ lymphocytes that are positive for CD57, CD49b, NKG2A, or KIR. The density of NK cells was significantly higher in the ABMR group (2.57 ± 2.58/mm2) than in the NR (0.12 ± 0.22/mm2) or the TCMR (0.25 ± 0.34/mm2) group (P = 0.002). Notably, CD56+CD57+ infiltrates (2.16 ± 1.89) were the most frequently observed compared with CD56+CD49b+ (0.05 ± 0.13), CD56+NKG2A+ (0.21 ± 0.69), and CD56+KIR+ (0.15 ± 0.42) cells in the ABMR group (P < 0.001). Death-censored graft failure was significantly higher in patients with NK cell infiltration than those without (Log-rank test, P = 0.025). In conclusion, CD56+CD57+ infiltrates are a major subset of NK cells in kidney transplant recipients with ABMR and NK cell infiltration is significantly associated with graft failure post-transplant.

Immunomodulatory Activity of Tyrosine Kinase Inhibitors to Elicit Cytotoxicity Against Cancer and Viral Infection.

Tyrosine kinase inhibitors (TKIs) of aberrant tyrosine kinase (TK) activity have been widely used to treat chronic myeloid leukemia (CML) for decades in clinic. An area of growing interest is the reported ability of TKIs to induce immunomodulatory effects with anti-tumor and anti-viral activity, which appears to be mediated by directly or indirectly acting on immune cells. In selected cases of patients with CML, TKI treatment may be interrupted and a non-drug remission may be observed. In these patients, an immune mechanism of increased anti-tumor cytotoxic activity induced by chronic administration of TKIs has been suggested. TKIs increase some populations of natural killer (NK), NK-LGL, and T-LGLs cells especially in dasatinib treated CML patients infected with cytomegalovirus (CMV). In addition, dasatinib increases responses against CMV and is able to inhibit HIV replication in vitro. Recent studies suggest that subclinical reactivation of CMV could drive expansion of specific subsets of NK- and T-cells with both anti-tumoral and anti-viral function. Therefore, the underlying mechanisms implicated in the expansion of this increased anti-tumor and anti-viral cytotoxic activity induced by TKIs could be a new therapeutic approach to take into account against cancer and viral infections such as HIV-1 infection. The present review will briefly summarize the immunomodulatory effects of TKIs on T cells, NKs, and B cells. Therapeutic implications for modulating immunity against cancer and viral infections and critical open questions are also discussed.

Myeloid cells activate iNKT cells to produce IL-4 in the thymic medulla

Interleukin-4 (IL-4) is produced by a unique subset of invariant natural killer T (iNKT) cells (NKT2) in the thymus in the steady state, where it conditions CD8+ T cells to become "memory-like" among other effects. However, the signals that cause NKT2 cells to constitutively produce IL-4 remain poorly defined. Using histocytometry, we observed IL-4-producing NKT2 cells localized to the thymic medulla, suggesting that medullary signals might instruct NKT2 cells to produce IL-4. Moreover, NKT2 cells receive and require T cell receptor (TCR) stimulation for continuous IL-4 production in the steady state, since NKT2 cells lost IL-4 production when intrathymically transferred into CD1d-deficient recipients. In bone marrow chimeric recipients, only hematopoietic, not stromal, antigen-presenting cells (APCs), provided such stimulation. Furthermore, using different Cre-recombinase transgenic mouse strains to specifically target CD1d deficiency to various APCs, together with the use of diphtheria toxin receptor (DTR) transgenic mouse strains to deplete various APCs, we found that macrophages were the predominant cell to stimulate NKT2 IL-4 production. Thus, NKT2 cells appear to encounter and require different activating ligands for selection in the cortex and activation in the medulla.

Impact of CMV Infection on Natural Killer Cell Clonal Repertoire in CMV-Naïve Rhesus Macaques.

Recent functional, gene expression, and epigenetic studies have suggested the presence of a subset of mature natural killer (NK) cells responsible for maintaining NK cell memory. The lack of endogenous clonal markers in NK cells impedes understanding the genesis of these cell populations. In humans, primates, and mice, this phenotype and memory or adaptive functions have been strongly linked to cytomegalovirus or related herpes virus infections. We have used transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPC) to track clonal hematopoiesis in rhesus macaques and previously reported striking oligoclonal expansions of NK-biased barcoded clones within the CD56−CD16+ NK cell subpopulation, clonally distinct from ongoing output of myeloid, B cell, T cell, and CD56+16− NK cells from HSPC. These CD56−CD16+ NK cell clones segregate by expression of specific KIR surface receptors, suggesting clonal expansion in reaction to specific environmental stimuli. We have now used this model to investigate the impact of rhesus CMV(RhCMV) infection on NK clonal dynamics. Following transplantation, RhCMVneg rhesus macaques display less dominant and oligoclonal CD16+ NK cells biased clones compared to RhCMVpos animals, however these populations of cells are still clearly present. Upon RhCMV infection, CD16+ NK cells proliferate, followed by appearance of new groups of expanded NK clones and disappearance of clones present prior to RhCMV infection. A second superinfection with RhCMV resulted in rapid viral clearance without major change in the mature NK cell clonal landscape. Our findings suggest that RhCMV is not the sole driver of clonal expansion and peripheral maintenance of mature NK cells; however, infection of macaques with this herpesvirus does result in selective expansion and persistence of specific NK cell clones, providing further information relevant to adaptive NK cells and the development of NK cell therapies.

1134P - Monalizumab in combination with cetuximab in patients (pts) with recurrent or metastatic (R/M) head and neck cancer (SCCHN) previously treated or not with PD-(L)1 inhibitors (IO): 1-year survival data

BackgroundMonalizumab is a first in class immune checkpoint inhibitor targeting Natural Killer Group 2A (NKG2A), which is expressed as a heterodimer with CD94 on subsets of Natural Killer (NK), γδ T and tumor-infiltrating CD8+ T cells (André et al. Cell 2018). The NKG2A ligand, HLA-E, is upregulated in cancer, including SCCHN. NKG2A blockade promotes innate anti-tumor immunity mediated by NK and CD8+ T cells and enhances human NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) induced by cetuximab. This dual targeting could provide greater antitumor activity than cetuximab alone. MethodsThe multicenter phase II trial tested the combination of monalizumab and cetuximab in pts with R/M SCCHN. Pts received monalizumab 10mg/kg q2weeks and cetuximab according to the label until disease progression or unacceptable toxicity. Pts were required to be progressing after platinum-based chemotherapy and to have received ≤2 prior lines of therapy in the R/M setting. Previous treatment with IO was allowed. The primary endpoint was Overall Response Rate (ORR) per RECIST. ResultsAll 40 pts received prior platinum-based chemotherapy, 18 (45%) prior IO and 5 (13%) prior cetuximab. ORR was 27.5% [95% confidence interval: 16-43] with 11 confirmed responses (1 complete + 10 partial) observed in both IO naive 36% [20-57] and IO pretreated pts 17% [6-39]. As of April 17, 2019, with a median follow-up of 17 months (mo), in all pts, IO naïve and IO pretreated pts respectively, median duration of response was 5.6, 5.3 and 5.6mo, PFS was 4.5, 3.9 and 5.1mo and OS was 8.3, 7.8 and 12.8mo. The 12mo OS estimate is 44% [31-63]. No new safety signal emerged. Pre and post treatment tumor biopsies were collected and RNA seq analyses are ongoing. ConclusionsIn a cohort of 40 patients of heavily pretreated SCCHN patients, monalizumab and cetuximab combination demonstrated high response rate, good duration of response, and promising PFS and OS as well as good safety profile. An additional R/M SCCHN cohort of 40 patients who received both platinum-based chemotherapy and IO is being enrolled in this study. Clinical trial identificationNCT02643550. Legal entity responsible for the studyInnate Pharma. FundingInnate Pharma. DisclosureR.B. Cohen: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Innate Pharma; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Genocea; Research grant / Funding (institution): Celldex; Research grant / Funding (institution): Macrogenics; Research grant / Funding (institution): Merck. M.R. Posner: Advisory / Consultancy: Merck; Advisory / Consultancy: Cel-Sci; Shareholder / Stockholder / Stock options: Promedior; Licensing / Royalties: Bet Isreal-Deaconess Médical Centre, Arizona Stat. J.R. Bauman: Advisory / Consultancy: Pfizer; Advisory / Consultancy: Bayer. C. Even: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Innate Pharma; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Genocea; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Heat; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Alkermes; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Arrys; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Takeda; Honoraria (self), Advisory / Consultancy: Cantargia; Research grant / Funding (institution): Celldex; Research grant / Funding (institution): Xencor. E. Saada-Bouzid: Advisory / Consultancy: AstraZeneca. T. Seiwert: Honoraria (self): Aduro biotech; Honoraria (self): AstraZeneca; Honoraria (self): Bayer; Honoraria (self), Research grant / Funding (institution): BMS; Honoraria (self): Celgene; Honoraria (self): Innate; Honoraria (self): Loxo Oncology; Honoraria (self), Research grant / Funding (institution): Merck; Honoraria (self): Nanobiotix; Research grant / Funding (institution): Jounce. F. Calmels: Shareholder / Stockholder / Stock options, Full / Part-time employment: Innate Pharma. R. Zerbib: Shareholder / Stockholder / Stock options, Full / Part-time employment: Innate Pharma. P. André: Shareholder / Stockholder / Stock options, Full / Part-time employment: Innate Pharma. A. Boyer-Chammard: Shareholder / Stockholder / Stock options, Full / Part-time employment: Innate Pharma. J. Fayette: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy: Merck Serono; Honoraria (self), Advisory / Consultancy: Innate Pharma; Honoraria (self), Advisory / Consultancy: Biogen. All other authors have declared no conflicts of interest.

Enrichment of Cytomegalovirus-induced NKG2C+ Natural Killer Cells in the Lung Allograft.

In lung transplant recipients, immunosuppressive medications result in impaired antiviral immunity and a propensity for cytomegalovirus (CMV) reactivation within the lung allograft. Natural killer (NK) cells play a key role in immunity to CMV, with an increase in the proportion of NK cells expressing activating CD94-NKG2C receptors in the blood being a strong correlate of CMV infection. Whether a similar increase in NKG2C NK cells occurs in lung transplant recipients following CMV reactivation in the allograft and if such cells contribute to viral control remains unclear. In this pilot study, we longitudinally assessed the frequency and phenotype of NKG2C NK cells in the blood and bronchoalveolar lavage (BAL) of lung transplant recipients and stratified recipients based on their risk of developing CMV disease. We observed an increase in the proportion of NKG2C NK cells in the blood and BAL of CMV high-risk patients, coincident with both the cessation of antiviral prophylaxis and subsequent detection of actively replicating CMV in the blood and lung allograft. Additionally, these NKG2C NK cells expressed killer-cell immunoglobulin-like receptors distinct from those of other NK subsets and BAL NKG2C NK cells possessed an activated phenotype. Finally, the frequency of NKG2C NK cells in the BAL may be inversely correlated with CMV blood titers. Monitoring the phenotype of NK cells postlung transplant may be a useful biomarker for monitoring patient levels of CMV immunity.

CD56brightCD16− natural killer cells are shifted toward an IFN-γ-promoting phenotype with reduced regulatory capacity in osteoarthritis

A subset of natural killer (NK) cells with CD56+/brightCD16dim/− expression is recently shown to present critical regulatory functions. Functional characteristics of CD56+/bright NK cells in osteoarthritis (OA) patients remains unknown. Here, we remedied this problem by comparing the NK cells from healthy controls and OA patients. Data showed that the CD56brightCD16− NK subset was significantly enriched in OA patients. These CD56brightCD16− NK cells from OA patients presented significantly higher IFNG transcription and IFN-γ protein secretion than those from healthy controls, both directly ex vivo and after activation via various stimulating reagents, including IL-2/IL-15, K562, and PMA/ionomycin. On the other hand, the transcription and secretion of granzyme A (Gzm-A), Gzm-B, and perforin were significantly lower in CD56brightCD16− NK cells from OA patients than in CD56brightCD16− NK cells from healthy controls. Also, the CD56brightCD16− NK cells from OA patients were less capable of suppressing the proliferation of autologous CD4+ T cells, in a manner that was dependent on the expression of Gzm-B and perforin. Interestingly, CD4+ T cells co-incubated with CD56brightCD16− NK cells were prone to express a higher level of IFNG, and the CD56brightCD16− NK cells from OA patients were more potent at stimulating IFNG than the CD56brightCD16− NK cells from healthy controls. Overall, our investigation demonstrated that CD56brightCD16− NK cells from osteoarthritis patients were shifted toward an IFN-γ-promoting phenotype and with reduced regulatory functions.

Discovery of a novel natural killer cell line with distinct immunostimulatory and proliferative potential as an alternative platform for cancer immunotherapy

BackgroundHuman natural killer (NK) cell lines serve as an attractive source for adoptive immunotherapy, but NK-92 remains the only cell line being assessed in the clinic. Here, we established a novel NK cell line, NK101, from a patient with extra-nodal natural killer/T-cell lymphoma and examined its phenotypic, genomic and functional characteristics.MethodsSingle cell suspensions from lymphoma tissue were expanded with anti-NKp46/anti-CD2-coated beads in the presence of IL-2. A continuously growing CD56+ cell clone was selected and designated as NK101. Flow cytometry and RNA sequencing were used to characterize phenotypic and genomic features of NK101. In vitro cytotoxicity and IFN-γ/TNF-α secretion were measured by flow cytometry-based cytotoxicity assay and enzyme-linked immunosorbent assay, respectively, after direct co-culture with tumor cells. Immunomodulatory potential of NK101 was assessed in an indirect co-culture system using conditioned medium. Finally, in vivo antitumor efficacy was evaluated in an immunocompetent, syngeneic 4T1 mammary tumor model.ResultsNK101 displayed features of CD56dimCD62L+ intermediate stage NK subset with the potential to simultaneously act as a cytokine producer and a cytotoxic effector. Comparative analysis of NK101 and NK-92 revealed that NK101 expressed lower levels of perforin and granzyme B that correlated with weaker cytotoxicity, but produced higher levels of pro-inflammatory cytokines including IFN-γ and TNF-α. Contrarily, NK-92 produced greater amounts of anti-inflammatory cytokines, IL-1 receptor antagonist and IL-10. Genome-wide analysis revealed that genes associated with positive regulation of leukocyte proliferation were overexpressed in NK101, while those with opposite function were highly enriched in NK-92. The consequence of such expressional and functional discrepancies was well-represented in (i) indirect co-culture system where conditioned medium derived from NK101 induced greater proliferation of human peripheral blood mononuclear cells and (ii) immunocompetent 4T1 tumor model where peritumoral injections of NK101 displayed stronger anti-tumor activities by inducing higher tumor-specific immune responses. In a manufacturing context, NK101 not only required shorter recovery time after thawing, but also exhibited faster growth profile than NK-92, yielding more than 200-fold higher cell numbers after 20-day culture.ConclusionNK101 is a unique NK cell line bearing strong immunostimulatory potential and substantial scalability, providing an attractive source for adoptive cancer immunotherapy.

Hematopoietic-Specific Deletion of Foxo1 Promotes NK Cell Specification and Proliferation.

We previously reported that deletion of Foxo1, via Ncr1-iCre mice from the expression of NKp46 onward, led to enhanced natural killer (NK) cell maturation and effector function. In this model, however, the role of Foxo1 in regulating NK cell specification and early development remains exclusive. Herein, we utilized a murine model of hematopoietic-specific deletion of Foxo1 before lymphoid specification, by crossing mice carrying floxed Foxo1 alleles (Foxo1fl/fl) with Vav1-iCre mice, to revisit the role of Foxo1 on NK cell specification and early development. The data showed that hematopoietic-specific deletion of Foxo1 resulted in increased proportion and numbers of common lymphoid progenitors (CLP) (Lin−CD127+c-Kit+Sca-1+), pre-pro NK b cells (Lin−Sca-1+c-Kit−CD135−CD127+), as well as committed Lin−CD122+ cells and CD3−CD19−NKp46+ NK cells in bone marrow. Hematopoietic-specific deletion of Foxo1 also promoted NK cells proliferation in a cell-intrinsic manner, indicated by increased Ki-67 expression and more expansion of NK cell after ex vivo stimulation with IL-15. The reason for Foxo1 suppressing NK cell proliferation might be its direct transcription of the cell-cycle inhibitory genes, such as p21cip1, p27kip1, p130, Gadd45a, and Ccng2 (cyclin G2) in NK cells, supported by the evidence of decreased mRNA expression of p21cip1, p27kip1, p130, Gadd45a, and Ccng2 in Foxo1-deficient NK cells and direct binding of Foxo1 on their promoter region. Furthermore, hematopoietic-specific deletion of Foxo1 resulted in increased ratio of mature NK subsets, such as CD11b+CD27− and CD43+KLRG1+ NK cells, but decreased ratio of immature NK subsets, such as CD27+CD11b− and CD27+CD11b+ NK cells, consistent with the findings in the murine model of Ncr1-iCre mediated Foxo1 deletion. Conclusively, Foxo1 not only acts as a negative checkpoint on NK cell maturation, but also represses NK cell specification and proliferation. The relative higher expression of Foxo1 in CLP and early NK precursors may also contribute to the later NK cell proliferation and responsiveness, which warranties another separate study in the future.

Shaping of CD56bri Natural Killer Cells in Patients With Steroid-Refractory/Resistant Acute Graft-vs.-Host Disease via Extracorporeal Photopheresis

CD56bri natural killer (NK) cells play an important role in the pathogenesis of graft-vs. -host disease (GVHD) and immune defense in the early period after allogeneic hematopoietic stem cell transplantation. Extracorporeal photopheresis (ECP) as an immunomodulating therapy has been widely used for GVHD treatment. However, the mechanism of action of ECP still remains to be elucidated, particularly the influence of ECP on NK cells. Thirty-four patients with steroid-refractory/resistant acute GVHD (aGVHD) ≥ °II and moderate to severe chronic GVHD (cGVHD) received ECP therapy. Patient samples obtained during intensive and long-term treatment were analyzed. Immunomonitoring with respect to cell phenotype and function was performed on rested peripheral blood mononuclear cells (PBMCs) using multiparametric flow cytometry. NK activity in terms of cytokine release was analyzed by intracellular cytokine staining after co-culture with K562 cells. Moreover, the proliferative capacity of NK cells, CD4+, and CD8+ T cells was determined by carboxyfluorescein succinimidyl ester (CFSE) staining. Clinically, 75% of aGVHD and 78% of cGVHD patients responded to ECP therapy. Moreover, our data show that aGVHD, cGVHD patients and healthy donors (HDs) present distinct NK patterns: aGVHD patients have a higher frequency of CD56bri NK subsets with stronger NKG2D and CD62L expression, while CD56−CD16+ NK cells with higher expression of CD57 and CD11b stand out as a signature population for cGVHD. ECP therapy could significantly decrease CD56briCD16− NK cells with shifting the quality from a cytotoxic to a regulatory pattern and additionally mature CD56dim NK cells via upregulation of CD57 in complete responding aGVHD patients. Moreover, ECP could keep the anti-viral and anti-leukemic effects intact via maintaining specialized anti-viral/leukemic CD57+NKG2C+CD56dim NK cells as well as remaining the quality and quantity of cytokine release by NK cells. The proliferative capacity of effector cells remained constant over ECP therapy. In conclusion, ECP represents an attractive option to treat GVHD without compromising anti-viral/leukemic effects. Shaping of CD56bri NK cell compartment by downregulating the cytotoxic subset while upregulating the regulatory subset contributes to the mechanisms of ECP therapy in aGVHD.

Ovalbumin induces natural killer cells to secrete Th2 cytokines IL‑5 and IL‑13 in a mouse model of asthma

Asthma is a chronic lung disease characterized by an imbalance of T-helper (Th)1/Th2 cells and their cytokine profiles. Natural killer (NK) cells constitute a considerable subset of the lymphocyte population in the lungs, and provide protection against respiratory infection by fungi, bacteria and viruses. However, the mechanism by which NK cells are involved in asthma remains to be fully elucidated. The present study analyzed the dynamic changes of NK cells and their subsets during the development of the ovalbumin (OVA)-induced allergic airway response. Lung tissues were histologically examined for cell infiltration and mucus hypersecretion. The number, activity and cytokine-secreting ability of NK cells was determined by flow cytometry. The results showed that the percentage of NK cells in the lung was decreased following OVA sensitization and challenge. However, NK cells exhibited enhanced activity and secreted more Th2 cytokines (IL-5 and IL-13) following OVA challenge. Furthermore, the proportion of CD11b− NK subsets increased with the development of asthma, and CD11b− CD27− NK cells were the primary NK subset producing Th2 cytokines. These findings suggest that, although NK cells are not the crucial type of lymphocytes involved in asthma, OVA induces NK cells to secrete Th2 cytokines that may be involved in the pathogenesis of asthma.

Gene Regulatory Programs Conferring Phenotypic Identities to Human NK Cells

Natural killer (NK) cells develop from common progenitors but diverge into distinct subsets, which differ in cytokine production, cytotoxicity, homing, and memory traits. Given their promise in adoptive cell therapies for cancer, a deeper understanding of regulatory modules controlling clinically beneficial NK phenotypes is of high priority. We report integrated "-omics" analysis of human NK subsets, which revealed super-enhancers associated with gene cohorts that may coordinate NK functions and localization. A transcription factor-based regulatory scheme also emerged, which is evolutionarily conserved and shared by innate and adaptive lymphocytes. For both NK and T lineages, a TCF1-LEF1-MYC axis dominated the regulatory landscape of long-lived, proliferative subsets that traffic to lymph nodes. In contrast, effector populations circulating between blood and peripheral tissues shared aPRDM1-dominant landscape. This resource defines transcriptional modules, regulated by feedback loops, which may be leveraged to enhance phenotypes for NK cell-based therapies.

Early CMV Replication-Associated Anti-Leukemia Effect Is Related to the Reconstitution of Specific Subsets of Natural Killer Cells after Haploidentical Stem Cell Transplantation in Acute Leukemia

Background: Cytomegalovirus (CMV) infection is a major infectious complication after allogeneic hematopoietic cell transplantation. Recently, it has been reproducibly demonstrated that CMV reactivation is associated with decreased relapse rate in patients with acute myeloid leukemia (AML). The aim of this study is to evaluate the impact of early CMV reactivation on the incidence of disease relapse in relation to the reconstitution of subsets of natural killer (NK) cells after haploidentical stem cell transplantation (haploSCT) in acute leukemia.Methods: Clinical data from 47 adult patients diagnosed with acute leukemia who underwent their first haploSCT between September 2009 and December 2017 was retrospectively analyzed. All patients were unable to find a suitable HLA-matched donor in their families or donor registries. Patient blood samples were collected prior to conditioning therapy and following haploSCT at day 30 and day 90. Peripheral blood mononuclear cells obtained from 28 patients abundant cells at every time points were analyzed for flow cytometric immunophenotyping. Expression of specific receptors (NKG2A, NKG2C, NKG2D, DNAM1 and NKp46) on the NK cells (CD56brightCD16- or CD56dim/-CD16+ cells) were serially quantified by multiparametric flow cytometry using appropriate monoclonal antibodies.Results: Median age was 38 years (range, 21-62 years), and 28 (54%) patients were male. Thirty-six (69%) patients received a transplant in their first complete remission (CR) status. Median follow-up duration was 54 months (range, 6.6-83.3 months), and all patients were CMV seropositive before receiving a transplant. Early CMV replication occurred at a median of 23 days after haploSCT in 40 of 47 patients (85%). Among these patients, 14 had more than two episodes of CMV replication throughout the follow-up period. The median peak viral load during CMV replication was 54,000 copies/mL. In univariate analysis, early CMV replication (P < 0.001), older donor age (P = 0.018), high dose of infused T cells (P = 0.022) and chronic graft-versus-host disease (GVHD, P = 0.001) were significantly associated with lower 3-year cumulative incidence of relapse after haploSCT. Early CMV replication was correlated with higher leukemia-free survival (LFS, P < 0.001). In multivariate analysis, early CMV replication (hazard ratio [HR], 0.24; 95% confidence interval [CI], 0.060 to 0.930, P = 0.039) and chronic GHVD (HR, 0.25; CI, 0.089 to 0.695; P = 0.008) were identified as independent factors for higher LFS rate. CMV reactivation was associated with higher overall survival (OS) rates and increased nonrelapse mortality, although there was no statistical significance. The viral load at the initiation of CMV-specific treatment was significantly associated with OS rates. Patients with CMV viral load higher than 45,000 copies/mL had lower OS rate compared to those with lower CMV load (34.5% versus 89.5%, P = 0.022). Longitudinal analysis of NK cell reconstitution after haploSCT showed that the CMV infection was associated with the increased expansion of CD56brightCD16dim/- NK cell, particularly in DNAM1+ NK cell subset. The rate of CD56brightCD16dim/-DNAM1+ NK cells increment was significantly higher in the patients with CMV infection compared with patients without CMV infection (P = 0.022). Importantly, we observed that the cumulative relapse rate was significantly decreased in patients with an increased CD56brightCD16dim/- DNAM1+ NK cells compared to patients with low CD56brightCD16dim/- DNAM1+ NK cells (20.4% versus 63.6% , P = 0.016).Conclusion: Early CMV replication was identified as an independent prognostic factor for LFS in acute leukemia in the haploSCT setting. Increased reconstitution of CD56brightCD16dim/- DNAM1+ NK cells was associated with CMV infection-related reduction in the relapse rate. Further studies are required to elucidate the anti-leukemia effects of these NK cell subsets associated with CMV infection in haploSCT. DisclosuresKim:Novartis Korea: Honoraria.

Recovery of Specific Subsets of Natural Killer and T Cells Highly Associated with Graft-Versus-Host Disease after Haploidentical Stem Cell Transplantation in Acute Leukemia

Background In the allogeneic hematopoietic stem cell transplantation, recent studies showed that T cell and natural killer (NK) cells recovery are implicated in the graft-versus-host disease (GVHD) and graft versus leukemia (GVL) effects. However, the significance of specific subsets of NK and T cell recovery in relation to transplantation outcomes remains to be elucidated in the haploidentical stem cell transplantation (haploSCT).Methods Clinical data of patients with acute myeloid leukemia (n = 21) and acute lymphoblastic leukemia (n = 24) who underwent their first haploSCT between September 2009 and December 2017 were analyzed. Peripheral blood mononuclear cells obtained from 27 patients were examined by multiparametric flow cytometric analysis. PD-1 and Tim-3 expression were examined in CD4+ and CD8+ T-cells and NK cell receptor (NKG2D, NKG2A, NKG2C, DNAM1 and NKp46) expression were analyzed in NK cells, respectively, at the 3 determined times (immediate prior to conditioning therapy, 28 and 90 days after haploSCT).Results Median age at haploSCT was 38 years (range, 21-62) and median follow-up duration was 31.6 months. Myeloablative conditioning was used for 32% and reduced intensity regimen for 68% of patients. GVHD prophylaxis was based on post-transplant cyclophosphamide for 8 (18%) or on anti-thymocyte-globulin for 36 (82%) plus standard prophylaxis. Incidence of grade II-IV acute GVHD was 50%, gastrointestinal tract (GIT) GVHD was 55.6%, non-GIT acute GVHD 35.7%, and chronic GVHD was 52.4%. Longitudinal analysis of immune reconstitution after haploSCT showed that the incidence of acute GVHD was associated with a delayed expansion of the NK cell population and incidence of chronic GVHD was associated with the extent of CD4+ T cell reconstitution. The incidence of acute GVHD was significantly higher in patients with lower counts of CD56bright CD16neg cell (100% for patients with less than 30 cells/uL at day 28 vs 50% for patients with higher counts, P = 0.026), particularly in NKG2A (P = 0.002) and DNAM1 (P = 0.027)-positive NK cell subsets. In univariate analysis, early CMV replication (P < 0.001), chronic GVHD (P = 0.001), donor age ≥ 28years (P = 0.018), CD4/CD8 ratio of product ≥ 2.4 (P = 0.033), and dose of infused T cells ≥ 3.91 x 108 /kg (P = 0.022) were significantly associated with lower 3-year cumulative incidence of relapse after haploSCT. Donor age ≥ 28years was significantly associated with high incidence of chronic GVHD (P = 0.002). Dose of infused T cells ≥ 3.91 x 108 /kg (HR, 0.088; CI, 0.009 to 0.823; P = 0.033) were independent factors for reducing leukemia relapse after adjustment in multivariate analysis. Chronic GVHD was an independent prognostic factor for higher leukemia-free survival rate (72.7% versus 20.1%, P = 0.008). Longitudinal analysis of T cell reconstitution after haploSCT showed that the high dose of infused T cells was associated with the increased expansion of CD4+PD-1- T cells (P = 0.031 at day 28 and P = 0.017 at day 90). Of note, The incidence of chronic GVHD was significantly higher in patients with higher counts of CD4+ T cell at day 28 (100% for patients with over than 150 cells/uL at day 28 vs 38.8% for patients with lower counts, P = 0.008), particularly in CD4+PD-1- subsets (P = 0.008). Among CD4+ T cell, PD-1-/PD-1+ ratio over than 4.5 was significantly associated with increased chronic GVHD (P = 0.005). In 22 patients with chronic GVHD, GIT GVHD was adverse prognostic factor for overall survival (59.5 % in GIT GHVD vs 100% in patients without GIT GVHD, P = 0.063). The incidence of GIT GVHD was significantly higher in patients with lower CD4/CD8 ratio at day 28 (77.8% for patients with less than 1.5 vs 0% for patients with higher ratio, P = 0.045).Conclusions Our findings suggest that high CD56brightCD16neg NK cell count at day 28 after hasploSCT was significantly associated with decreased incidence of acute GVHD. High dose of infused T cells was associated with increased reconstitution of CD4+ PD-1- T cells and high CD4+ T cell counts, particularly in PD-1- subset, are associated with increased development of chronic GVHD. These findings should be further validated for elucidating the roles of these immune effectors cells in the development of GVHD and GVL effect in haploSCT for acute leukemia. DisclosuresKim:Novartis Korea: Honoraria.

MYC Functions As a Master Switch for Natural Killer Cell-Mediated Immune Surveillance of Lymphoid Malignancies

Background: The MYC oncogene drives T and B cell lymphoid malignancies, including Burkitt's lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). Such lymphomas are said to be “oncogene-addicted” to MYC. In order to develop targeted therapies for MYC-driven cancers, it is vital to understand how MYC regulates both cell autonomous and non-cell-autonomous processes, including host immunity.Approach: We have used a particularly tractable approach for studying the role of oncogenic MYC on the host immune system during lymphomagenesis through a tetracycline (tet)-system regulated transgenic mouse model of MYC-driven T cell lymphoma (SRα-tTA/Tet-O-MYC mice; Felsher and Bishop, Molecular Cell, 1999). By delineating the global immunological changes during primary MYC-driven T cell lymphomagenesis in SRα-tTA/Tet-O-MYC mice using mass cytometry (CyTOF) and CIBERSORT, we identified anti-tumor immune subsets that can be developed as therapies to treat MYC-driven lymphomas.Results: Amongst the immune subsets evaluated, our results demonstrated a specific systemic suppression of natural killer (NK) cell-mediated surveillance in SRα-tTA/Tet-O-MYC mice bearing overt MYC-driven T cell lymphomas. Inactivation of lymphoma-intrinsic MYC restores NK cell-mediated immune surveillance, suggesting that the regulation of NK cell-mediated surveillance may be integral to MYC-induced lymphomagenesis. We observed that lymphoma-specific MYC transcriptionally represses STAT1/2 and secretion of Type I Interferons (IFNs) required for normal NK cell homeostasis and NK cell-mediated immune surveillance in the tumor microenvironment. Concordantly, treating T cell lymphoma-bearing SRα-tTA/Tet-O-MYC mice with Type I IFN improves survival by rescuing NK cell production, and in part overriding MYC-mediated suppression of NK surveillance. We showed that human lymphomas with both high levels of MYC and low levels of STAT1/2 have lower NK surveillance, and are associated with poor prognosis. Finally, we established the therapeutic implications of our findings by showing that adoptive transfer of NK cells significantly delays lymphoma initiation, and recurrence after MYC inactivation; suggesting that NK cell-based therapy may be effective against MYC-driven lymphomas.Conclusion: Subversion of NK cell surveillance is integral to MYC-induced lymphomagenesis. Our studies provide a rationale for further developing the NK subset as a cell-based immunotherapy to effectively treat MYC-driven lymphomas in the future. DisclosuresNo relevant conflicts of interest to declare.

Abnormal Distribution and Function of NK Cells Subsets May Lead to Impaired Tumor Surveillance in a JAK2V617F Myeloproliferative Neoplasm Model

Background: The JAK2V617F mutation is the most common molecular abnormality present in Philadelphia chromosome-negative Classical Myeloproliferative Neoplasms (Ph-neg MPNs), present in approximately 95% of patients with Polycythemia Vera and in almost 50% of patients with primary myelofibrosis and essential thrombocythemia. Besides the genetic mutations, different cells in the bone marrow can modulate the microenvironment and contribute to disease initiation and maintenance. Natural killer (NK) cells are part of innate immunity and are divided into subsets, according the expression of CD27 and CD11b. The immature NK cells, CD27-CD11b- (tolerant), CD27+CD11b-, presenting less cytotoxicity, while the mature NK cells, CD27+CD11b+ and CD27-CD11b+ (cytotoxic), have a great cytotoxic function. Considering that the role of NK cells in Ph-neg MPNs is currently unclear, our hypothesis is that a defect of the NK cells may favor HSC malignant transformation and contribute to MPN development. Aim: We aimed to study their distribution and cytotoxic function in murine primary cells from a MPN transgenic model. Methods: NK cells were quantified by flow cytometry from splenocytes of a pre-established conditional vavCre knockin Jak2V617F (Jak2VF) murine model. Polycythemia and splenomegaly were confirmed in Jak2 wt/V617F vavCre+ (Jak2VF) as compared to Jak2 wt/wt vavCre+ (Jak2WT) control animals. Mice (n= 4 per group) were euthanized between 10-16 weeks of age. For immunophenotyping, spleen was isolated, submitted to red cell lysis, and stained with fluorescence-conjugated antibodies against Ter119, CD19, CD4, CD8 CD3, NK1.1, CD27, CD11b, CD69 and CD107a. All the stainings were performed at 4°C for 20 minutes and acquisition of at least ten thousands events per tube was performed in a FACSCanto™II flow cytometer. For the activation and degranulation assays, splenocytes were cultivated and stimulated with PMA (2.5ug/mL) and ionomicyn (0.7ug/mL) for 3 hours and then submitted to the analysis of CD69and CD107a expression, respectively. The cytotoxicity assay was performed by a co-culture of sorted NK cells with YAC-1 cells previously marked with an impermeable cell tracker, for 3 hours. Then, the target cell death percentage was determined by 7-AAD detection. Results: The analysis of the degranulation marker CD107a in activated NK cells (CD69+) showed that, when compared with the Jak2WT group, the Jak2VF mice presented a decreased frequency of CD107a+ NK cells (87.2% vs 74.1% p=.02). Besides, the ability of Jak2VF NK cells to kill the target cells was reduced when compared to the Jak2WT group (80.1%±17.4 vs 65.5%±8.7 at the 5:1 NK:target cell ratio and 84.9%±11.9 vs 75.3%±0.1 at the 10:1 NK:target cell ratio). The maturation profile showed that, when compared to Jak2WT mice, the frequency of immature NK cells was 4.2-fold increase in Jak2VF animals (8% vs 33% p=.009), and the percentage of these cells that expressed the degranulation marker CD107a was reduced when compared with the Jak2WT group (55.5% vs 42.7% p=.02). In agreement, the frequency of mature NK cells was decreased in Jak2-mutated mice (92% vs 67% p=.009) as compared to controls. Among the mature NK subpopulations, CD107+ cells were lower numbered in Jak2VF mice as compared to Jak2WT cells (94.3% vs 88.9% p=.005). We also observed a differential profile of functional NK subsets between Jak2WT and Jak2VF NK cells. There was an increase of CD27-CD11b- and CD27+CD11b- NK cells in Jak2-mutated mice (5.6% vs 18.3% p=.007 and 2.1% vs 14.6% p=.01, respectively). On the other hand, the percentage of CD27-CD11b+ NK cells in Jak2VF mice was lower than in Jak2WT mice (78.1% vs 49.9% p=.01). In summary, when compared to controls, Jak2-mutated mice presented a reduction in the frequency of NK cells able to release their lytic granules, suggesting a lower cytotoxic function. The reduction of cytotoxicity in Jak2VF NK cells may be explained by two factors: (1) the augment of immature tolerant NK cells, and (2) the reduction of cytotoxicity of the mature subset (CD27-CD11b+). To date, although the functional and maturation subsets of NK cells have been studied in solid tumors, they have not been previously associated with MPNs. Conclusion: Considering that mature and functional NK cells play a critical role in tumor surveillance, it is plausible that an impair of their distribution and function can favor stem cell-derived diseases like Ph-neg MPNs DisclosuresNo relevant conflicts of interest to declare.

Corrigendum: Triplebody Mediates Increased Anti-Leukemic Reactivity of IL-2 Activated Donor Natural Killer (NK) Cells and Impairs Viability of Their CD33-Expressing NK Subset.

[This corrects the article DOI: 10.3389/fimmu.2017.01100.].