scholarly journals Phenotypic Heterogeneity of Chronic Lymphoproliferative Disorder of NK Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3876-3876 ◽  
Author(s):  
Barila' Gregorio ◽  
Antonella Teramo ◽  
Giulia Calabretto ◽  
Chiara Ercolin ◽  
Albana Lico ◽  
...  
Keyword(s):  
Nk Cells ◽  
Mutation Analysis ◽  
Lymphoproliferative Disorder ◽  
Conflicts Of Interest ◽  
Flow Analysis ◽  
Population Expansion ◽  
Severe Neutropenia ◽  
Cd56 Expression ◽  
Stat3 Mutation ◽  
Cytotoxic Function

Abstract Background NK cells represent a subset of lymphocytes belonging to the innate immunity branch typically expressing CD16 and CD56 associated to CD3 negativity. Two major subtypes of NK cells can be distinguished through CD16 and CD56 expression: CD56high/CD16dim/neg NK cells with low cytotoxic function and CD56dim/CD16high NK cells with high cytotoxic function. Recently a subtype of NK cells with memory properties characterized by CD56dim/CD16high/CD57+/CD62L- phenotype has been discovered. Chronic Lymphoproliferative Disorders of Granular Lymphocytes are characterized by the clonal expansion of Large Granular Lymphocyte (LGL) that can be CD3 positive (T-LGLL) or CD3 negative (Chronic Lymphoproliferative Disorder of NK Cell, CLPD-NK). The disease generally has indolent course but some patients develop cytopenia, particularly neutropenia, exposing to potentially lethal bacterial infections. Furthermore, NK-CLPD is usually referred as a more indolent disorder with respect to T-LGLL, with lower incidence of cytopenia and treatment need. CLPD-NK therapy does not differ from that of T-LGLL and is usually represented by an immunosuppressive therapy with low dose cyclophosphamide or methotrexate, with cyclosporine A usually being reserved to refractory patients. Somatic STAT3 mutations represent a new diagnostic marker of these disorders, initially reported in T-LGLL in about 40% oh patients, but also present in CLPD-NK in about 30% of cases. Using flow analysis, the aim of the present study was to identify a subset of CLPD NK patients characterized by a more severe disease requiring a shorter follow-up as compared to patients with a more indolent disease. Methods In a cohort of 16 patients affected by CLPD-NK, NK cells were analysed by flow for CD3, CD16, CD56, CD57 and CD62L antigen expression. These patients were studied for the presence of cytopenia and treatment requirement. STAT3 mutation analysis of exon 21 was performed with Sanger sequencing. Finally, p-STAT3 tyr 705 level and total STAT3 level were examined by western blotting. Results In relation to CD16 and CD56 expression, three major NK cells populations can be recognized in CLPD-NK patients: CD56high/CD16neg NK cells, CD56dim/CD16neg NK cells and CD56neg/dim/CD16high. As a consequence, patients can be separated into three groups characterized by the preferentially expansion of one of these populations: 2/16 (13%) with CD56high/CD16neg NK population, 4/16 (25%) with CD56dim/CD16neg NK population and 10/16 (62%) with CD56neg/dim/CD16high NK population. Furthermore, patients with predominance of this last NK cells subset were studied for CD57 and CD62L expression to identify NK cytotoxic subset (CD57-/CD62Llow/neg) and NK memory subset (CD57+/CD62Llow/neg); a NK cytotoxic/memory ratio (C/M ratio) was then calculated. 4 of 10 CD56neg/dim/CD16high patients (40%) were characterized by prevalence of NK cytotoxic cells expansion and high C/M ratio (≥3) while the remaining 6/10 patients were characterized by NK memory cells expansion with low C/M ratio (≤1.6). We then evaluated the presence of cytopenia, in particular neutropenia, in our patients' cohort. Neutropenia was shown in 7/16 (44%) patients with 4/16 (25%) experiencing severe neutropenia. Anemia and thrombocytopenia were less frequent (19% and 6% respectively). Interestingly, 6 out of 7 (86%) neutropenic patients were in the CD56neg/dim/CD16high subset and all patients with severe neutropenia belonged to the high C/M ratio subset. Interestingly, 3 out of 4 patients (75%) of this subset required therapy during the natural history of the disease. Concerning STAT3 mutation analysis, no one mutated patient was found in this setting. By western blot analysis, patients with high C/M ratio presented higher p-STAT3 levels than other patients and normal NK cells. Summary Although CLPD-NK represents an extreme heterogeneous disorder, discrete subtypes of disease characterized by different NK cells population expansion can be identified by flow analysis. Interestingly, this splitting allows to identify a subset of patients with prevalence of CD56neg/dim/CD16high NK cells with high C/M ratio that are characterized by high level of p-STAT3, high frequency of severe neutropenia and treatment requirement. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4335-4335
Author(s):  
Thiago Mantello Bianco ◽  
Diego Antonio Pereira-Martins ◽  
Patrícia Vianna Bonini Palma ◽  
Josiane Lilian Schiavinato ◽  
Cleide Araújo Silva ◽  
...  
Keyword(s):  
Nk Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Critical Role ◽  
Target Cells ◽  
Cell Ratio ◽  
Tumor Surveillance ◽  
And Function ◽  
Cytotoxic Function ◽  
Marker Cd107a

Abstract 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 Disclosures No relevant conflicts of interest to declare.

NK Cell Proliferation and Cytolytic Function Are Compromised In the Hypoxic Tumor Microenvironment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4291-4291 ◽  
Author(s):  
Sonny Ang ◽  
Maria Lima Da Silva ◽  
Margaret Dawson ◽  
Matthew Figliola ◽  
Sourindra Maiti ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cells ◽  
Chronic Hypoxia ◽  
Immune Cell ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Effector Function ◽  
Proliferative Capacity ◽  
Cd56 Expression ◽  
Hypoxic Tumor

Abstract Abstract 4291 Natural Killer (NK) cells have important and potent innate immunoregulatory and immune surveillance functions against tumor. The paradoxical coexistence of tumors and anti-tumor immune cells (“Hellstrom Paradox”) may in part be explained by the pathophysiology of the “hostile” tumor microenvironment which suppress immune-cell function, such as hypoxia, low pH, low tissue glucose, and the presence of immunosuppressive cytokines and metabolites. However, the effect of the malignant environment on the ability of NK cells to infiltrate tumor and exhibit effector function is largely unknown. Therefore, we investigated the ability of NK cells to operate under conditions of hypoxia. Importantly, NK cells showed a 1,000-fold reduction in proliferative capacity when grown under chronic hypoxia (4 weeks of 1% O2). In addition, there was a corresponding decrease in cytotoxicity as revealed by chromium release assay. This was in contrast to autologous T cells which could numerically expand under corresponding growth conditions. Expression profiling uncovers profound upregulation of hypoxia-inducible genes such as EGLN1(9.9x), EGLN3(52x), LDHA(11.5x), SLC2A1(30.5x), PDK1(16.8x), VEGFA(286x) and BNIP3(138x) in hypoxic NK cells. Protein expression confirmed these changes, as NK cells under normoxic culture produced 520 nmoles/million cells of ATP, while those under hypoxic culture managed only 100 nmoles/million cells. This is consistent with a bioenergetic switch from oxidative phosphorylation to glycosis resulting from PDK1 upregulation. NK cells in hypoxia produced 61 pg/mL of VEGF compared to 1480 pg/mL for NK cells in normoxia (20% O2), as determined by ELISA. The inability of NK cells to propagate under conditions of hypoxia may be due to a drop in mitochondrial content we observed when cells were exposed to chronic hypoxia, a potential mitophagic effects of BNIP3 upregulation. In addition to the poor proliferative capacity of NK cells under hypoxia, we also noted the loss of CD56 expression on hypoxic NK cells which is associated with loss of cytotoxicity. Sequence analysis reveals that miR-210 can bind to the 3′UTR of CD56 mRNA, targeting it for degradation. Therefore, we investigated whether miR-210 levels are upregulated in hypoxic NK cells and found that increased presence correlated with loss of CD56 expression. This leads to the conclusion that NK-cell immunotherapy may be improved by downregulating miR-210 levels. Indeed, our findings help shape strategies for obtaining robust and sustained NK-cell effector function for adoptive immunotherapy in the hypoxic tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.

575 Regression by hetIL-15 monotherapy in different mouse breast cancer models correlates with intratumoral infiltration of a novel population of dendritic cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A609-A609
Author(s):  
Sevasti Karaliota ◽  
Dimitris Stellas ◽  
Vasiliki Stravokefalou ◽  
Bethany Nagy ◽  
Cristina Bergamaschi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dendritic Cells ◽  
Nk Cells ◽  
Flow Analysis ◽  
Laboratory Animals ◽  
Tumor Infiltration ◽  
The Novel ◽  
Phenotypic Profile ◽  
Conventional Type

BackgroundIL-15 is a cytokine which stimulates the proliferation and cytotoxic function of CD8+ T and NK cells. We have produced and applied the native heterodimeric IL-15 (hetIL-15) on several preclinical models, which have supported the anti-tumor activity of hetIL-15. Based on these results, hetIL-15 has advanced to clinical trials. The objectives of this study were to explore how hetIL-15 shapes the tumor microenviroment and to characterize the interactions between tumor-infiltrating lymphoid and myeloid cells.MethodsWe studied the efficacy of locoregional administration of heterodimeric IL-15 (hetIL-15) in two different orthotopic triple-negative breast cancer (TNBC) mouse models, syngeneic for C57BL/6 and Balb/c, respectively. The effects of hetIL-15 on immune cells were analyzed by flow cytometry, immunohistochemistry (IHC) and gene expression profiling. The profile of the novel infiltrated dendritic cell populations was further explored by bulk and single cell RNAseq.Results hetIL-15 resulted in tumor eradication in 40% of treated mice and reduction of metastasis. Subsequent rechallenges with the same cell line failed to generate tumor regrowth, suggesting the development of immunological memory in hetIL-15 treated mice. hetIL-15 promoted tumor accumulation of proliferating and cytotoxic CD8+ T and NK cells. Additionally, peritumoral hetIL-15 administration resulted in an increased tumor infiltration of both conventional type 1 dendritic cells (cDC1s) and of a novel DC population found only in the hetIL-15 treated animals. Phenotypic profile analysis confirmed the expression of several cDC1 specific markers, including CD103 and IRF8 on this DC population.Transcriptomics and flow analysis of intratumoral dendritic cells indicate that the new hetIL-15 induced cells reside preferentially in the tumors and are distinct from cDC1 and cDC2 populations. Both cDC1s and the novel DC population were inversely correlated with the tumor size.ConclusionsLocoregional administration of hetIL-15 results in complete eradication of EO771 and significant reduction of 4T1 primary breast cancer tumors, prolonged survival and long-lasting specific anti-tumor immunity. hetIL-15 increases the tumor infiltration of activated T and NK cells and intensifies the tumor infiltration of conventional type 1 dendritic cells (cDC1) and a new population of dendritic cells. We propose that the anti-cancer activity of hetIL-15 in primary EO771 tumors is orchestrated by the interplay of NK, CD8+T cells, cDC1 and a novel subset of DCs with a distinct phenotypic profile. These findings suggest a role for hetIL-15 in the treatment of breast cancer.Ethics ApprovalThe study was approved by the National Cancer Institute-Frederick Animal Care and Use Committee, approval number 19–324 and was conducted in accordance with the ACUC guidelines and the NIH Guide for the Care and Use of Laboratory Animals.

Poster: CLL-156: Novel CCL22 Mutations Drive Chronic Lymphoproliferative Disorder of NK Cells Through Biased GPCR Signaling

2021 ◽  
Vol 21 ◽  
pp. S222
Author(s):  
Shunsuke Kimura ◽  
Constance Baer ◽  
Mitra S Rana ◽  
Andrew Kleist ◽  
David J. Feith ◽  
...  
Keyword(s):  
Nk Cells ◽  
Lymphoproliferative Disorder ◽  
Gpcr Signaling

Natural Killer Cell Cytolytic Function in Korean Patients with Adult-onset Still’s Disease

2012 ◽  
Vol 39 (10) ◽  
pp. 2000-2007 ◽  
Author(s):  
JEONG HA PARK ◽  
HEE-SUN KIM ◽  
JIN SOOK LEE ◽  
JIN JU KIM ◽  
KYONG-HEE JUNG ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Adult Onset Still’S Disease ◽  
Adult Onset ◽  
Still’S Disease ◽  
Cytolytic Function ◽  
Cell Proportion ◽  
Cytotoxic Function ◽  
Adult Onset Still's Disease

Objective.To investigate natural killer (NK) cell proportions, NK cell cytotoxicity, and interleukin 18 (IL-18) expression, in patients with adult-onset Still’s disease (AOSD).Methods.Forty-five patients with AOSD (active = 22, inactive = 23) and 32 healthy controls were included. The proportions of NK cells among peripheral blood mononuclear cells were assessed by flow cytometry. IL-18 and IL-18-binding protein (IL-18BP) concentrations were measured by ELISA. Twenty-four patients with AOSD and 18 controls were examined for cytotoxic activity of NK cells by co-incubating NK cells with NK-sensitive K562 cells. The association of NK cell function with clinical and laboratory measures was investigated.Results.The proportions of NK cells were significantly lower in patients with active AOSD than in patients with inactive disease and controls. NK cell cytotoxic function was significantly lower in patients with AOSD than in controls. NK cell proportions and cytotoxic functions were reexamined in 11 and 6 patients, respectively, after treatment. Low NK cell proportion and cytotoxic dysfunction were improved with clinical improvements of the patients. IL-18 and IL-18BP levels were much higher in patients with active AOSD than in controls. NK cell cytotoxic functions were consistently low and IL-18 and IL-18BP levels were constantly high in patients with AOSD, regardless of disease activity.Conclusion.Low NK cell proportion, defective cytotoxic function, and elevated IL-18 levels may be significant features of AOSD. After resolution of the acute phase, low NK cell proportion was recovered and NK cell cytolytic function was restored along with clinical improvement. These findings possibly contribute to immunologic abnormalities in AOSD.

scholarly journals Cytokine-Mediated Natural Killer Cells Effects Impair Hematopoietic Stem Cell Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2641-2641
Author(s):  
Lorena Lobo Figueiredo-Pontes ◽  
Robert S. Welner ◽  
Miroslava Kardosova ◽  
Hong Zhang ◽  
Meritxell Alberich-Jorda ◽  
...  
Keyword(s):  
Stem Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Cytokine Production ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Nk Cytotoxicity

Abstract Natural killer (NK) cells participate in innate and adaptive immune responses, and upon activation rapidly produce cytokines, chemokines, and growth factors, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of other hematopoietic cells. Considering the recent evidences that hematopoietic stem cells (HSCs) respond to cytokine signaling, we hypothesized that NK cell-mediated cytokine production could mediate HSC function. By the use of co-cultures of purified Ly5.1 murine NK cells and congenic Ly5.2 HSCs, we concluded that NK activity affects HSC frequency in vitro as well as hematopoietic reconstitution in vivo. Sorted NK cells (CD3- NK1.1+) and HSCs (Lin-, Sca1+, ckithi, CD48-, CD150+) were co-cultured in the presence or absence of IL2 over an OP9 stromal cells layer for 14 to 28 days. After 14 days, the addition of NK cells to HSC cultures resulted in an approximate 2-fold reduction of lineage negative cells (Lin-) recovered cells, as compared to control HSC cultures, as determined by flow cytometry analysis. Lin- counts were even lower in HSC+NK long-term cultures when compared to HSC only cultures. Ly5.1 HSCs and/or Ly5.2 NK cells were injected into sublethally irradiated Ly5.1/2 chimeric mice in a ratio of 105 NK to 103 HSCs per mouse. The addition of IL2-stimulated NK to injected HSCs reduced engraftment from 15.7% to 1.82% when the 16 weeks bone marrow (BM) chimerism was analyzed. In agreement, donor CD45.1 cells contribution to the LSK and HSC subpopulations was reduced in the HSC+NK transplanted mice. To test whether NK depletion from BM grafts would affect HSC function, we performed limiting dilution transplantation assays where whole BM from Ly5.2 mice was submitted to immunonagnetic NK1.1 or IgG depletion and injected into lethally irradiated Ly5.1 animals. Donor chimerism after 8 and 16 weeks of transplant showed that depleting NK cells improves the engraftment ability of HSC in a cell dose-dependent manner. When 25 x104 BM cells were injected, chimerism increased from 40 to more than 90% in NK depleted group. Of note, HSC frequency was 1 in 1595 in the control and 1 in 95 in the NK depleted group. In order to understand the mechanisms by which NK cells could regulate HSCs, we took advantage of a CCAAT/enhancer-binding protein gamma (C/ebpg) knockout (KO) conditional mouse model generated in our laboratory, considering that C/ebpg had been previously shown to regulate NK cytotoxicity. Using similar culture conditions, HSCs and NK cells isolated from control (CT) or Cebpg KO mice were injected into congenic sublethally irradiated recipients. Results showed that Cebpg-deficient NK cells do not harm HSC engraftment as CT NK cells do. For instance, after 8 weeks, the addition of CT non-stimulated and IL-2-stimulated NK cells to normal transplanted HSCs reduced the engraftment from 40% to 20% and 10%, respectively. In contrast, chimerism was not different when HSCs only or HSCs + stimulated KO NK cells were transplanted. Gene expression and cytokine profiles of deficient and normal NK cells revealed the potential players of this HSC-NK regulation. Of these, interferon gamma (IFNg), was lower produced by the C/ebpg deficient NK cells. Therefore, besides controlling NK cytotoxicity, we showed here that C/ebpg also plays a role in the regulation of HSCs by NK-mediated cytokine production. Next, we investigated whether depletion of NK cells from human BM samples would improve transplantation efficiency. NK cells were removed using CD56 antibody and transplanted into sublethally irradiated NSG mice. Sixteen weeks after transplantation, animals were sacrificed and the percentage of human CD45 cells in blood, BM, and spleen demonstrated that NK depletion from human BM favors engraftment. Altogether, these findings provide new insights to the knowledge of HSC regulation by NK cells, which are present in BM transplantation (BMT) grafts. Although the alloreactive effect of NK cells against non-identical tumor cells from BMT recipients is well known, its cytokine-mediated effects over identical progenitor cells from the graft were not previously explored. We show that NK-secreted cytokines harm stem cell function, thus suggesting that depletion of NK cells from BM donor cells preparations can improve stem cell engraftment, particularly in the setting of alternative transplants with limiting cell numbers or non-myeloablative conditioning regimens. Disclosures No relevant conflicts of interest to declare.

scholarly journals Prospective, Multicentric Phase II Randomized Trial Comparing the Efficacy of Methotrexate or Cyclophosphamide in Large Granular Lymphocytic Leukemia: A French National Study. Report on the Interim Analysis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1545-1545 ◽  
Author(s):  
Thierry Lamy ◽  
Cedric Pastoret ◽  
Roch Houot ◽  
Loic Ysebaert ◽  
Mathilde Hunault ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Research Funding ◽  
Sequential Analysis ◽  
Severe Neutropenia ◽  
National Study ◽  
Sufficient Information ◽  
Transfusion Requirements ◽  
Stat3 Mutation ◽  
To Receive ◽  
Lgl Leukemia

Introduction Large granular lymphocyte (LGL) leukemia is characterized by a clonal expansion of CD3+ cytotoxic T or CD3- NK cells. Prominent clinical features include neutropenia, anemia and autoimmune-associated diseases such as rheumatoid arthritis (RA). Although the disease is usually chronic and indolent, some patients may be symptomatic and require treatment. No standard therapy has been established due to the absence of prospective clinical trials. So far, low dose methotrexate, oral cyclophosphamide, and cyclosporine represent the 3 main options for initial therapy. In 2014, we launched a prospective clinical trial comparing methotrexate to cyclophosphamide in previously-untreated patients with LGL leukemia in need of treatment. Patients and methods The study was designed as a multicentric, national, open label, randomized, controlled trial on two parallel groups, comparing methotrexate and cyclophosphamide. Patients were included if they had at least one of the following indications of treatment: isolated severe neutropenia (ANC <0.5x109/L) or neutropenia (ANC <1.5x109/L) with infections, anemia requiring transfusions or symptomatic anemia, associated complications such as systemic diseases or auto-immune diseases resistant to steroids and/or immunomodulating agents (colchicin, disulone, hydrochloroquine). They were randomly assigned to receive either methotrexate (10 mg/m²/w) or cyclophosphamide (100 mg/d) for 4 months. Responders at M4 continued with the same treatment until M12 (cyclophosphamide was then delivered at 50 mg/d). Non-responders at M4 were randomly assigned to receive either cyclosporine (3 mg/kg/d) or the drug which had not been administered at the first randomization (methotrexate or cyclophosphamide). Response was assessed using previously published criteria (Lamy T, Blood 123:1182, 2014). Complete response (CR) was defined as a normalization of clinical exam (disappearance of splenomegaly or associated autoimmune symptoms) and a complete normalization of blood counts. Partial response (PR) was defined as an improvement in blood counts which did not meet criteria for complete remission (e.g., ANC >0.5x109/L or decrease of transfusion requirements). Treatment failure was defined as no response or any response which did not meet the above-mentioned criteria within four months after the beginning of treatment. To stop the trial as soon as sufficient information was collected, a sequential analysis was planned each time 20 patients were included and evaluated using the triangular test (Sébille V, Bellissant E. Fundam Clin Pharmacol. 2003;17(5):505-16). The primary endpoint was the hematological CR rate evaluated at M4 (binary endpoint). Secondary endpoints were overall response rate (ORR) at M4, M8 and M12, time to relapse. For non-responders at M4, cyclosporine was compared to the treatment which had not been administered during the first phase. Results From Nov 2013 to July 2019, 99 patients met inclusion criteria among which 96 were randomized. The baseline characteristics of these patients are shown in Table1. STAT3 mutation was observed 52% of cases. After the 4th sequential analysis performed on the first 80 patients evaluable for response at M4, the sample path remained in the continuation region of the triangular test. Thus, the trial has to be continued. At M4, 13 patients were in CR (16.3%) and 29 patients were in PR (36.3%), ORR was 52.6%, 36 patients were considered as refractory and underwent a second randomization: 18 patients received cyclosporine and 17 received methotrexate or cyclophosphamide. Conclusions This first prospective randomized clinical trial in LGL leukemia shows that the CR after first line therapy using either methotrexate or cyclophosphamide is relatively low (< 20%). Recruitment is still ongoing to assess if there is a difference in terms of response between the two drugs. Predictive biomarkers of response will be presented at the meeting. Regarding a 52% of incidence of Stat3 mutation (higher than that previously published), Jak/Stat targeted therapy should be prospectively evaluated in this disease. Disclosures Houot: Bristol Myers Squibb: Honoraria; Merck Sharp Dohme: Honoraria. Gyan:Pfizer: Honoraria. Feugier:janssen: Honoraria, Research Funding, Speakers Bureau; abbvie: Honoraria, Research Funding, Speakers Bureau; gilead: Honoraria, Research Funding, Speakers Bureau; roche: Honoraria, Research Funding, Speakers Bureau.

scholarly journals A Novel Method to Study the in Vivo Trafficking and Homing of Adoptively Transferred NK Cells in Rhesus Macaques and Humans

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 659-659 ◽  
Author(s):  
Jan Davidson-Moncada ◽  
Noriko Sato ◽  
Robert F Hoyt ◽  
Robert N Reger ◽  
Marvin Thomas ◽  
...  
Keyword(s):  
Nk Cells ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Raji Cell ◽  
K562 Cells ◽  
Ct Imaging ◽  
Pet Ct ◽  
Hematological Cancers

Abstract Adoptive transfer of allogeneic or autologous natural killer (NK) cells is now being developed for therapy of both hematological and solid malignancies. The efficacy of NK immunotherapy to mediate anti-tumor effects will ultimately be dependent on their ability to traffic and home to the tumor microenvironment. Recent data suggest expanded NK cells are ineffective at homing to the bone marrow (BM) and lymph nodes (LN) where hematological malignancies reside. A variety of techniques to maintain and/or enforce expression of homing receptors in NK cells are now being explored in preclinical models to improve their localization to the BM and LN. Historically, xenogeneic human into mouse or mouse into mouse models have been utilized for preclinical development of adoptive NK transfer. These experiments often use fluorescent dye-labeled NK cells and require repeated invasive biopsies, which can be confounded by sampling error, or the requirement for post mortem analysis. Here we present a method to track in real time and in vivo adoptively infused zirconium-89 (89Zr) labelled NK cells by PET imaging. A rhesus macaque (RM) model was used for these preclinical experiments as RM and human NK cells have similar expansion kinetics, and have greater similarity than mice in their phenotype, function, and homing receptors and ligands. PBMCs collected from the PB of 13 RMs were enriched for NK cells by CD3+ T-cell depletion and were then expanded for 14 days by culturing with irradiated human EBV-LCL cells in X-VIVO 20 media containing 10% human AB serum and 500 IU/μl of human IL-2. RM NK cells expanded a mean 145±41 fold and contained >99% pure CD3- and CD56+ cells. The phenotype and tumor cytotoxicity of RM NK cells were similar to NK cells expanded from humans (n=3) using similar expansion cultures; at a 10:1 E:T ratio, 67% and 73% of K562 cells were lysed by RM and human NK cell respectively. To label NK cells, 89Zr was conjugated to oxine, which readily permeabilized the cellular membrane and was retained in the cells. Expanded NK cells from both humans and RM showed no changes in CD16 or CD56 expression for up to 6 days following radiolabeling. Human and RM NK cell viability 0 to 24 hours following radiolabelling was 60-100% then declined to 20-30% after 6 days. 89Zr retention by both human and RM NK cells was 75-80% in the first 24 hours of culture but gradually declined with time, decreasing to 20-30% after 7 days of culture. Culturing radiolabeled human NK cells for 24-36 hours with different cellular populations including Ramos and Raji cell lines and normal human PBMCs revealed no significant transfer of radioactivity (max 2% above baseline), establishing that 89Zr was not transferred from labeled to unlabeled cells. Oxine labeling did not alter the cytotoxicity of human or RM NK cells vs K562 cells compared to unlabeled controls. 89Zr-oxine labeling of expanded RM NK cells is currently being used to quantify NK cell trafficking and survival following adoptive transfer in autologous macaques. In these experiments, RM recipients of adoptively infused 89Zr labeled NK cells receive concurrent deferoxamine to chelate and then enhance renal excretion of any free 89Zr that is released from dead cells. In the experiments shown below, 13 x 107 autologous ex vivo expanded 89Zr-labeled RM NK cells were injected IV into a 5.7 kg RM and tracked by sequential PET/CT imaging for 7 days. Up to 1-hour post infusion, most NK cell activity was restricted to the lungs. By 4 hours, NK cells began to traffic from the lungs to the liver and spleen. By 2 days, NK cells were no longer detectable in the lungs and resided largely in the liver and spleen, where they remained for the remainder of the 7 day imaging period. During the entire observation period, little to no NK cell radioactivity was detected in the LN or BM. In conclusion, 89Zr oxine labelling of NK cells followed by PET/CT imaging represents a powerful tool to track the in vivo fate of adoptively transferred NK cells. The RM model presented here provides a method to evaluate and optimize various strategies aimed at altering the phenotype of NK cells, with the goal of improving their homing to the BM and LN where hematological cancers reside. These preclinical in vitro and in vivo data suggest this technology could be safely extended to humans and could be applied to other cellular populations besides NK cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Seroreactivity to an Envelope Protein of Human T-Cell Leukemia/Lymphoma Virus in Patients With CD3− (Natural Killer) Lymphoproliferative Disease of Granular Lymphocytes

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1977-1981 ◽  
Author(s):  
Thomas P. Loughran ◽  
Kenneth G. Hadlock ◽  
Qing Yang ◽  
Raisa Perzova ◽  
Renato Zambello ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Lymphoproliferative Disorder ◽  
Enzyme Linked Immunosorbent Assay ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Htlv Infection ◽  
Human T Cell ◽  
Granular Lymphocytes

Abstract Natural killer (NK) cells are CD3− large granular lymphocytes (LGL) responsible for immunity against viral infections. A chronic lymphoproliferative disorder of NK cells has been described in which the expanded NK cells display a restricted phenotype and cytotoxic activity. These data raise the hypothesis that proliferating LGL in these patients result from discrete expansions of NK cells responding to an unknown, perhaps viral, antigen. Recently, it was found that mice transgenic for the tax gene of human T-cell leukemia/lymphoma virus (HTLV) develop NK leukemia. Therefore, we studied 15 patients with chronic NK lymphoproliferative disorder for evidence of HTLV infection. Sera were tested using an HTLV-I/II-enzyme linked immunosorbent assay and a modified Western blot assay containing recombinant env proteins. None of the sera met conventional criteria for HTLV seroreactivity. However, sera from 11 patients (73%) reacted with the recombinant HTLV env protein p21E. The anti-p21E reactivity of these sera was then mapped employing the recombinant proteins GD21 and BA21. No reactivity to the immunodominant HTLV epitope GD21 was observed, suggesting that prototypical HTLV infection is unlikely in these patients. This was confirmed by finding no evidence for HTLV nucleic acids by PCR analyses employing primers specific for conserved regions in the env, pol, and pX genes. In contrast, 10 of the 15 sera reacted with the epitope BA21, documenting for the first time an association between a unique seroreactivity and disease. The high incidence of BA21 seroreactivity in these patients suggests that exposure to a protein containing homology to BA21 may be important in the pathogenesis of this lymphoproliferative disorder.

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