A Rare Case of Chronic Lymphoproliferative Disorder of NK Cells with an Unusual Expression of CD57

Abstract Introduction/Objective Natural Killer cells (NK-cells) malignancies are extremely rare and the two NK-cell disorders involving the peripheral blood and bone marrow include chronic lymphoproliferative disorder of NK-cells (CLPDNK) and aggressive NK-cell leukemia (ANKL). ANKL is EBV associated with a prevalence in Asian adults and a fulminant clinical course leading to death within 2 months. Methods A 76-year-old female with a history of iron deficiency anemia presented for evaluation of lymphocytosis (78 TH/mm) and negative EBV with extreme fatigue, weight loss, and worsening weakness from the past 8 months. Results The peripheral and bone marrow aspirate showed an increase in granular lymphocytes with bland nuclei and abundant pale-staining cytoplasm containing fine to coarse azurophilic granules with no evidence of nuclear atypia. Flow cytometry demonstrated an atypical lymphocytic cell population comprising >50% of total marrow and peripheral blood lymphocytes demonstrating loss of CD56 and diminished CD7 expression. The analysis showed atypical lymphocytes with the following immunophenotype: CD2, CD8 (the majority, at least 75%),CD7 (dim), CD38, CD11c (subset) with no expression of CD3, CD5, CD4, CD56, or CD11c. Flow cytometry is important as it helps in the phenotypic detection of aberrancy. A broad panel of immunohistochemical stains revealed scattered positivity for CD3, CD4, CD7, CD8, TIA-1, granzyme, perforin, and tryptase and negative for CD56 and CD57. Our top differential with a similar clinical course included T-cell large granular lymphocytic leukemia which is characterized by a clonal proliferation of mature cytotoxic T cells and also has an indolent course. They show CD3 positivity by flow cytometry along with co-expression of NK cell-associated markers (CD16 and CD57), and variable expression of other pan T cell markers such as CD2, CD5, CD7. It is found incidentally in patients having cytopenias with a persistent increase in circulating mature NK-cells. Of note, the PET scan revealed splenomegaly with no other significant findings.The patient was started on steroids and is currently doing well. Conclusion Thus, overall morphologic, immunophenotypic, and molecular results with negative expression of surface or cytoplasmic CD3 as well as the absence of TCR expression were most consistent with CLPD-NK with an unusual expression of CD57 expression detected in flow cytometry.

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542 Expansion of cytotoxic NK Cells from PBMCs using individualized cytokine combination

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0542 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A578-A578

Author(s):

Andreia Maia ◽

Joana Lerias ◽

Markus Maeurer ◽

Mireia Castillo-Martin

Keyword(s):

Flow Cytometry ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Blood Donors ◽

Nk Cell ◽

Tumour Cells ◽

List Type ◽

Mhc I ◽

Cytokine Combination

BackgroundAdoptive immunotherapy relies on the use of T-cells to target tumour cells, through Major Histocompatibility Complex (MHC) Class I recognition(1). However, many tumours display alterations in the MHC-I pathway, a well-described immune evasion mechanism(2). Natural Killer (NK) cells recognize transformed cells independently from the presence of MHC-I and may be a reliable therapeutic option for patients with altered tumour MHC-I expression. The source of NK cells may be autologous or allogeneic and NK cells are also clinically relevant recipients of transgenic receptors (TCRs or antibodies) targeting tumour cells. NK cells have been categorized according to their CD56 and CD16 surface expression into different subpopulations: cytotoxic (CD56+CD16+) and regulatory (CD56brightCD16-)(3). Expanding cytotoxic NK cells is challenging, since the frequency of NK cells is low in peripheral blood(4) and there is also – at this point – not an optimal expansion protocol available.The goal of this project is to determine the best cytokine combination that facilitates expansion of cytotoxic NK cells that either target tumor cells directly or serve as recipients for transgenic receptors.MethodsPeripheral Blood Mononuclear Cells (PBMCs) were extracted using Ficoll methodology from blood donors and cultured in T25 flasks with Cell Genix Medium supplemented with 10% human serum and antibiotics. NK cells were expanded supplemented with feeder cells (ratio 1:1) and different cytokine combinations (1000 U/mL of IL-2, 10 U/ml of IL-12, 180 U/mL of IL-15 and/or 1 U/mL of IL-21) during 20 days. The immunophenotype of expanded NK cells was analyzed at days 0, 5, 10, 15 and 20 by flow cytometry. The cytotoxicity of NK cells was measured by a CD107a Assay or by a Total Cytotoxicity and Apoptosis Assay at days 10 and 20. Thirteen different cytokine combinations were tested.Results4/13 cytokine combinations produced a statistically significant increase of the absolute number of NK cells with a higher percentage of cytotoxic NK cells (figure 1). However, induction of cytotoxicity was not associated with a strong NK cell expansion. The regulatory NK cells subset (CD56brightCD16-) showed the highest percentage of CD107a-expressing cells, more than the CD56+CD16+, the most cytotoxic subpopulation of NK cells.Abstract 542 Figure 1Representative percentage of NK cells in total lymphocytes (A), CD56+CD16+ subpopulation in total NK cells (B), and CD56brightCD16- subpopulation amongst total NK cells (C) at different time points (5, 10, 15 and 20 days) expanded from PBMCs* p-value < 0.05ConclusionsThis work shows that we are able to grow and efficiently expand NK cells from PBMCs with different cytokine combinations leading to clinically relevant NK cell numbers as well as cytotoxic functions. This enables to produce NK cell products for therapy and as recipients for transgenic tumor antigen-specific receptors.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank, the Vivarium Facility and the Flow Cytometry Platform of the Champalimaud Centre for the Unknown.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon.ConsentWritten informed consent was obtained from the blood donors to use their samples for research purposes.ReferencesRosenberg SA, Restifo NP, Yang JC, Morgan RA, Mark E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8(4):299–308.Aptsiauri N, Ruiz-Cabello F, Garrido F. The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses. Curr Opin Immunol 2018;51:123–32.Di Vito C, Mikulak J, Mavilio D. On the way to become a natural killer cell. Front Immunol. 2019;10(August):1–15.Zotto G Del, Antonini F, Pesce S, Moretta F, Moretta L. Comprehensive phenotyping of human PB NK Cells by Flow Cytometry. 2020;1–9.

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Production of colony-stimulating activity by human natural killer cells: analysis of the conditions that influence the release and detection of colony-stimulating activity

Blood ◽

10.1182/blood.v74.1.156.bloodjournal741156 ◽

1989 ◽

Vol 74 (1) ◽

pp. 156-164

Author(s):

V Pistoia ◽

S Zupo ◽

A Corcione ◽

S Roncella ◽

L Matera ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Nk Cell ◽

K562 Cells ◽

Cell Suspensions ◽

Normal Peripheral Blood ◽

Colony Stimulating Activity ◽

Culture Supernatants

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony- inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released. However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

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Development and Function of NK and T Cells in AML Patients after Allogeneic Stem Cell Transplantation (SCT).

Blood ◽

10.1182/blood.v104.11.3244.3244 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3244-3244

Author(s):

Gabriele Multhoff ◽

Catharina Gross ◽

Anne Dickinson ◽

Ernst Holler

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Stem Cell ◽

Nk Cells ◽

Nk Cell ◽

Granzyme B ◽

Hla Class I ◽

K562 Cells ◽

Cytolytic Response

Abstract Purpose: Hsp70 was frequently found on the plasma membrane of bone marrow-derived leukemic blasts, but not on normal bone marrow cells. Hsp70 membrane expression could be correlated with protection against therapy-induced apoptosis (Nylandsted et al 2004). In contrast, these tumor cells have been found to be highly sensitive to the cytolytic attack mediated by NK cells. In vitro, Hsp70-activated NK cells efficiently lysed autologous Hsp70 membrane-positive leukemic blasts (Gehrmann et al 2003). Granzyme B release served as a surrogate marker for estimating the cytolytic response of NK cells against Hsp70 membrane-positive tumor target cells (Gross et al 2003). Here, we studied the development of NK and T cells in AML patients (n=6) after allogeneic SCT at different time points (days 14–20, 45, 90, 180, 1 year) after allogeneic stem cell transplantation (SCT). Methods: HLA class I, HLA-E and Hsp70 surface expression was determined on all patient-derived leukemic blasts of the bone marrow by flow cytometry. The amount of NK and T cells was investigated by multicolor flow cytometry using CD3/ CD16 and CD56 and CD94/ CD56 antibody-combinations detecting NK cell specific markers. Effector cell function was tested in a granzyme B ELISPOT assay against patient-derived leukemic blasts and K562 cells. Results: All tested leukemic blasts were positive for HLA class I, HLA-E, and Hsp70. After induction therapy the amount of CD3-negative, CD56/CD94-positive NK cells was 28±16%, that of CD3-positive T cells was 58±3%. On days 14–21 after allogeneic SCT, 58±9% of the donor-derived peripheral blood lymphocytes (PBL) were CD3-negative, CD56/CD94-positive NK cells; the amount of CD3-positive T cells was 26±7.5%. On day 45, the amount of NK cells further increased up to 68±7.9%; that of T cells further decreased down to 16±5.6%. On day 90 and day 180 the amount of NK cells was still 41±10%; that of T cells was 29±12%. Interestingly, high NK cell counts correlated with an increased cytolytic response against leukemic blast and K562 cells. One year after allogeneic SCT, NK (20±1%) and T cell (52±18%) ratios were comparable to that of healthy human individuals. Conclusions: Between days 14 and 180 after allogeneic SCT, the amount of NK cells was significantly elevated if compared to that of T cells. Concomitantly, cytolytic function against leukemic blasts was significantly elevated. Normal levels, in the composition of NK and T cells were reached 1 year after SCT. Project funded by EU-TRANS-EUROPE grant QLK3-CT-2002-01936.

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Platelet Engraftment (PE) in AML Patients Receiving HLA Matched Sibling Donor Allogeneic Bone Marrow Transplant (ABMT) Correlates with Inhibitory HLA Ligand Groups for Killer Immunoglobulin-Like Receptors (KIRs).

Blood ◽

10.1182/blood.v106.11.836.836 ◽

2005 ◽

Vol 106 (11) ◽

pp. 836-836

Author(s):

Ronald Sobecks ◽

Edward J. Ball ◽

Lisa Rybicki ◽

Stacey Brown ◽

Jaroslaw Maciejewski ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

T Cell ◽

Nk Cells ◽

Median Time ◽

Nk Cell ◽

Multivariable Analysis ◽

Sibling Donor ◽

Cell Dose ◽

Hla Ligands

Abstract The interaction of KIRs with target cell HLA class I molecules regulates the activity of NK cells and some T cell populations. KIR interactions are reported to influence allogeneic hematopoietic stem cell transplant outcomes, particularly for AML. We have previously reported that AML pts homozygous for C1 or C2 have superior survival and lower relapse rates than do pts heterozygous for C1/C2. However, the influence of KIR matching on PE has not been well described. We therefore analyzed the KIR ligand phenotypes of 60 AML pts who received HLA identical sibling donor myeloablative ABMT from 4/9/97 – 11/5/03. The median age was 45 years (range, 8–62 yrs). At transplant a minority (40%) were in CR. All pts received a busulfan/cyclophosphamide based preparative regimen and T-cell replete bone marrow as their stem cell source. Patient HLA KIR ligands were categorized as: 1) HLA-Cw group C1- or C2 - homozygous vs C1/C2 heterozygous; and 2) HLA-Bw4 (positive or negative) (reviewed in Farag et al Blood100:1035, 2002). Kaplan Meier estimates of median time to PE >20 K/μL and >50 K/μL were 23 and 30 days, respectively. PE was next assessed in relation to the inhibitory HLA KIR ligand group expressed. PE >20 K/μL was superior for those C1 or C2 homozygous (n=26) compared to C1/C2 heterozygotes (n=34) (median 21 vs 26 days, p=0.049) and 31 Bw4 negative pts had superior PE compared to 29 Bw4+ pts (median 21 vs 30 days, p=0.012); these findings remained significant in multivariable analysis. A similar analysis performed for PE >50 K/μL found that Bw4 negative pts had superior PE compared to Bw4 + pts (median 26 vs 38 days, p=0.015); this remained significant in multivariable analysis. 57 cases had KIR genotyping performed for those KIRs with established HLA ligands and there were no cases in which the donor did not have at least one inhibitory KIR gene specific for expressed HLA ligands. Age at transplant, number of prior chemotherapy regimens, disease status at transplant and CMV status were not predictive of PE. Since both C1/C2 heterozygosity and Bw4+ status correlated with reduced early PE, the possible interaction of these two variables was next investigated. The analysis for PE >20 K/μL and >50 K/μL suggested an additive effect: pts lacking expression of both these variables had the most rapid PE, while those who expressed both variables had the slowest PE. Those who expressed only 1 of the variables had an intermediate time to PE as shown below: HLA KIR Ligand Status N Median CD34+ cell dose (x10^6/kg) Median Total Nucleated Cell Dose (x10^8/kg) Median time to PE>20K/μL Median time to PE >50K/μL C1/C2 and Bw4 negative 14 2.02 2.64 19 days 25 days C1/C2 positive and Bw4 negative OR C1/C2 negative and Bw4 positive 29 1.92 2.62 23 days 29 days C1/C2 and Bw4 positive 17 1.87 2.60 31 days 41 days p= 0.54 p=0.82 p=0.003 p=0.003 These results may suggest that donor NK cells control host effector cells that delay PE. When minimal opportunity for inhibitory KIR engagement exists (C1/C2 negative, Bw4 negative) maximal NK cell control is expected and rapid PE ensues. When maximal opportunity for inhibitory KIR engagement exists (C1/C2 positive, Bw4 positive) donor NK cell controlling activity would be reduced, leading to delayed PE.

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Frequent CD7 Antigen Loss in Aggressive Natural Killer-Cell Leukemia Can Be a Useful Diagnostic Marker.

Blood ◽

10.1182/blood.v110.11.2400.2400 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2400-2400

Author(s):

Sun-Hee Kim ◽

Eun-Hyung Yoo ◽

Hee-Jin Kim ◽

Won Seog Kim

Keyword(s):

Bone Marrow ◽

Natural Killer Cell ◽

Nk Cells ◽

Nk Cell ◽

Diagnostic Marker ◽

Clinical Course ◽

Killer Cell ◽

Cell Leukemia ◽

Laboratory Findings ◽

Antigen Loss

Abstract Aggressive natural killer-cell leukemia (ANKL) is a rare neoplasm characterized by systemic proliferation of NK cells with rapidly progressive clinical course and fatal outcome. Because of the aggressive clinical course, rapid and accurate diagnosis of ANKL is critical. However, the differential diagnosis of NK cell lymphoproliferative disorders including hemophagocytic lymphohistiocytosis is still challenging in the absence of a distinct diagnostic hallmark. Furthermore, cases with a low burden of malignant cell polpuation makes it more difficult. To find any diagnostic markers in ANKL, we analyzed clinical data and laboratory findings from bone marrow studies in Korean patients with bone marrow involvement of ANKL. From January 2000 to July 2007, a total of 20 cases were diagnosed with ANKL based on morphologic and immunophenotypic findings from bone marrow studies. The leukemic cells were surface CD3–CD16/56+ large granular lymphocytes with pale or lightly basophilic cytoplasm containing azurophilic granules. We retrospectively analyzed clinical features and laboratory findings including complete blood count (CBC), Epstein-Barr virus (EBV) status, serum lactate dehydrogenase (LDH) level, immunophenotype, and cytogenetic results from medical records. There were 6 (30%) women and 14 (70%) men with a median age of 44 years (range, 2–70 years). Hepatomegaly (70%), splenomegaly (60%), and lymphadenopathy (30%) were frequently observed. Peripheral blood counts were variable; anemia (hemoglobin <10g/dL) was predominant in 14 patients and thrombocytopenia (platelet <100×109/L) in 16. The proportion of leukemic NK cells ranged 3∼70%. EBV was detected in 15 of 18 cases (83%) by EBV in situ hybridization or EBV quantitative PCR. Cytogenetic studies were performed in 18 cases, and karyotypic abnormalities were observed in 50% (9/18). There were no recurrent cytogenetic abnormalities, except 6q abnormalities observed in 4 cases (4/18, 22%). The immunophenotype of the leukemic NK cells by flow cytometry was cytoplasmic CD3+, surface CD3−, CD16/56+, CD2+, and CD5−. Most cases were CD4− (13/16, 81%) and CD8− (11/14, 79%). Of note, loss of CD7 antigen was observed in 10 patients (10/20; 50%) (normal NK cells: CD2+, CD7+, and CD5−). There were no significant differences in clinical or laboratory parameters between the CD7+ and CD7− groups. All three cases with deletion of 6q revealed absent expression of CD7. When the CD7 loss was combined with cytogenetic abnormalities, clonal markers could be identified in 75% of ANKL cases. We observed frequent CD7 antigen loss in our series of Korean patients with ANKL. This characteristic immunophenotypic finding can provide a reliable and timely information as a diagnostic marker in ANKL along with cytogenetic findings. Therefore, immunophenotypic analysis of the expression of CD7 should be included in the diagnostic workup of NK cell neoplasms.

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The “MarrowMiner”: A Novel, Minimally Invasive Device for the Harvest of Bone Marrow: Initial Human Experience Reveals Safety, Efficacy and Richer Cell Product Than Standard Harvest Methods

Blood ◽

10.1182/blood.v112.11.4130.4130 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4130-4130

Author(s):

Daniel L. Kraft ◽

Vartan Ghazarossian ◽

Mike Crocker ◽

Sergio Najar ◽

Antonio A. Carrasco-YalÃ°n

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

T Cell ◽

Minimally Invasive ◽

Progenitor Cells ◽

Local Anesthesia ◽

Peripheral Blood ◽

Single Entry

Abstract INTRODUCTION: Bone marrow (BM) contains a rich supply of adult stem and progenitor cells, including hematopoieitic and mesenchymal stem cells which are used in Bone Marrow Transplantation (BMT) and an increasing array of regenerative therapies. Traditional marrow harvest methods utilize percutaneous large bore needle aspiration, result in marrow highly diluted by peripheral blood, and are crude, tedious, labor intensive and expensive, usually requiring general anesthesia, and >100 serial small volume aspirates to obtain adequate cell numbers for BMT. BM is showing increasing long-term advantages over mobilized PBSC for many alloegeneic BMTs, in terms of less cGVHD and in some cases improved survival. Improved BM harvest methods are needed. A novel device, the “MarrowMiner” (MM), was developed for the minimally invasive harvest of BM to enable the rapid, convenient, outpatient harvest of large quantities of BM under local anesthesia for use in allogeneic and autologous BMT and cell therapies utilizing autologous marrow derived cells. The MarrowMiner utilizes a single marrow entry site into the anterior or posterior iliac, through which the flexible, powered, guidable FlexShaft catheter can access the majority of the marrow space and aspirate rich marrow. Extensive testing in human cadavers and porcine models demonstrated a 10X increase in stem cells activity/ml (by CFU) compared to that of traditional needle harvests. The MM recently received both FDA and CE Mark regulatory approved, and ‘First In Human’ trials were successfully completed under local anesthesia, demonstrating safety, efficacy and higher stem cell yields compared to traditional methods. METHODS: In an ongoing prospective study, 10 patients undergoing autologous marrow derived therapy for use in regenerative medicine, had marrow harvested from their anterior or posterior ileac by the MM under local anesthesia on one hip, with direct comparison to standard needle serial marrow aspirates on the patients opposite hip (up to 350 ml per side). Cell viability, counts, CD34+, T cell, and MSC populations were assessed by flow cytometry. RESULTS: The MM successfully harvested marrow from a single entry sites and 2–3 paths under local anesthesia, without complications. Compared to standard harvest in the same patients, MM harvests had significantly number of Total Nucleated Cells ml compared to marrow harvested from the same patient by standard needle ( mean 1.98 fold greater TNC (range 0.87–3.36, p<.05). Viability was equivalent at (>99). In addition to higher TNC/ml, significantly higher levels (mean 3.56 fold) of Aldeflour/ALDH+ cells/ml, CD34+, and phenotypic MSC (CD45−,34−,90+,105+) and endothelial progenitor cells were obtained, as measured by flow cytometry. Mean CD3+ T-cell counts per ml were lower with MM harvests. CONCLUSIONS: The novel FDA approved MarrowMiner system demonstrated safety and efficacy in clinical use, harvesting more stem cells per unit volume in a single entry compared to standard harvest methods. These results suggests the MM may enable improved clinical stem cell harvests in a more rapid and minimally invasive manner in the outpatient setting, while harvesting a richer marrow product with less peripheral blood contamination. Such a system, facilitating convenient, on demand stem cell collection may have significant application for BMT and other marrow based cellular therapies.

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Morphologic and Phenotypic Features of Concurrent Clonal T-Cell Large Granular Lymphocytic Expansion and Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v118.21.2803.2803 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2803-2803

Author(s):

Xiaohui Zhang ◽

Lynn Moscinski ◽

John M. Bennett ◽

Reza Setoodeh ◽

Deniz Peker ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cell ◽

Peripheral Blood ◽

Low Grade ◽

Gene Rearrangements ◽

High Grade ◽

Bone Marrow Cellularity ◽

Hla Dr ◽

Marrow Cellularity

Abstract Abstract 2803 Myelodysplastic syndrome (MDS) and T-cell large granular (T-LGL) leukemia are both bone marrow failure disorders. It has been reported in a small number of cases that clonal T-LGL proliferation or leukemia can coincidentally occur with MDS. Also, clonal CD8+/CD57+ effector T cells expansion was detected in as many as 50% of MDS bone marrows [Epling-Burnette, 2007]. How clonal LGL cells that reside in the bone marrow interfere with hematopoiesis remains unclear, particularly in the setting of MDS. We analyzed the clinicopathological features of concomitant MDS and T-LGL, and evaluated bone marrow status for lineage or pan-hypoplasia in these patients. Design: Clinical and pathologic data from patients with a diagnosis of MDS and flow cytometry performed on the peripheral blood between 1/2005 and 12/2009 were reviewed. The concurrent bone marrow biopsies from each patient at the time of flow cytometric analysis were reviewed by two hematopathologists. Bone marrow cellularity, lineage hypoplasia (M:E >5: 1 or <1:2) were documented. Peripheral lymphocyte count and CD3+/CD57+ and CD8+/CD57+ populations by flow cytometry were calculated and T cell gene receptor (TCR) rearrangements were correlated. Results: We performed LGL flow cytometry panel on 76 MDS patients (high grade MDS, n=23; low grade, n=54), as well as TCR gene rearrangements, and identified clonal T-LGL cells in peripheral blood of 37 patients (48.7%), including 15 high grade MDS (40.5%, RAEB-I and RAEB-II), and 22 low grade MDS (59.4%), including RCMD(13), RA(1), RS(1), RCMD-RA(1), RCMD-RS (2), 5q- MDS(1), and MDS unclassifiable(3). The immunophenotype of the T-LGL cells was typically CD3+/CD57+/CD7 dim+/CD5 dim+/CD8+ with variable CD11b,CD11c, CD16, CD56 and HLA-DR. A frequent variant in these MDS patients was CD11b-,CD11c -, CD16+/−, CD56+/−, HLA-DR- and CD62L+.The TCRβ or/and TCRγ gene rearrangements were positive in 35 of the 38 cases (92.1%). The peripheral blood lymphocyte counts were 300–3820 cells/μL (1199±799 cells/μL); the CD3+/CD8+/CD57+ T-LGL cell counts were 30–624 cells/μL (229±154 cells/μL). In comparison, the remaining 39 patients with non-clonal T-LGL included 11 high grade MDS cases, and 28 low grade MDS cases. The peripheral blood lymphocyte counts were 308–2210 cells/μL (1030±461 cells/μL). CD3+/CD57+ cells were 1–425 cells/μL (105±98 cells/μL). There was no identifiable phenotypic features suggestive of clonal T-LGL cells such as dim CD5 and/or dim CD7 with aforementioned aberrant expressions on T-cells, although 7 of the 39 cases had TCRβ or/and TCRγ gene rearrangements. Thirty healthy donors were included for controls with absolute lymphocyte counts of 2136±661 cells/μL and baseline CD3+/CD57+ cells of 162±109 cells/μL. All showed no clonal LGL phenotype and negative TCR gene rearrangements. Since the presence of T-LGL cells may impair bone marrow hematopoiesis, we examined if there are bone marrow status differences between these two groups. All the bone marrows were obtained at diagnosis or not on chemotherapy. The bone marrow cellularity of the MDS patients with clonal T-LGL ranged from <3% to almost 100%, averaging 56%, with 8 cases with dramatic hypocellularity (<3%-20%), while the bone marrow cellularity of the MDS patients without clonal T-LGL ranged from 20% to 90%, averaging 62%, with only 2 cases with mild hypocellularity (20% in 73- and 65-year-old). In addition, among MDS patients with clonal T-LGL cells, 14 of 37 (37.8%; 5 high grade, and 9 low grade) bone marrows had certain lineage hypoplasia, including 3 cases of trilineal hypoplasia, 9 cases of erythroid hypoplasia, and 2 cases of myeloid hypoplasia. In contrast, among 39 MDS patients without T-LGL, there were only 1 bone marrow with trilineal hypoplasia and 3 others with erythroid hypoplasia (10.2%). The difference between the two groups was statistically significant (p=0.004, chi square test). In conclusion, our studies indicate that clonal T-LGL cells expansion is a fairly common finding in high grade as well as low grade MDS. The clonal T-LGL cells have more than one variant immunophenotypes and are typically positive for TCR gene rearrangements. Additionally, we observed that the clonal LGL cells present in MDS bone marrows could be associated with lineage hypoplasia, which, in this respect, might impact clinical treatment. Disclosures: No relevant conflicts of interest to declare.

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T Cell Exhaustion and Downregulation of Cytotoxic NK Cells – an Immune Escape Mechanism in Adult Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.3781.3781 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3781-3781

Author(s):

Eolia Brissot ◽

Sawa Ito ◽

Kit Lu ◽

Carly Cantilena ◽

B. Douglas Smith ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Research Funding ◽

Immune Escape ◽

Nk Cell ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Healthy Donors

Abstract Adult acute lymphoblastic leukemia (ALL) remains a therapeutic challenge with less than 40% long term survival. There is growing evidence that malignant diseases exert an immune editing effect which blocks antitumor immunity and permits tumor growth through immune evasion. Such tumor escape represents an obstacle for anticancer immunotherapy. In ALL such immune escape mechanisms are not well characterized. We therefore profiled cellular immunity in ALL, by characterizing the subsets of T cells, regulatory T cells (Treg), natural killers (NK) cells and γd T cells, using various functional markers including T cell exhaustion and NK cell activating or inhibitory molecules. Forty ALL patients were included in the study. The median age was 39 y (range, 18-75). Thirty-six presented with B-lineage ALL and 4 with T-lineage ALL. Mononuclear cells were isolated from blood (n=19) or bone marrow (n=21) at the onset of leukemia or at relapse. The median infiltration of blasts was 85% (range 24-96%). Healthy donor peripheral blood (n=12) and bone marrow (n=9), from age and gender matched population, were simultaneously analyzed as controls. Extra-and intra cellular staining were performed using using antibodies directed against CD3, CD4, CD8, CD45, CD45, CD45RA, CD45RO, CCR7, CD95, CD27, CD19, CD14, CD127, CD25, Foxp3, Helios, αβTCR, HLA-DR, CD117, CD20, CD10, CD22, CD34, LAG3, PD1, PDL1, CD56, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL3, CD57, CD33, CD11b, CD15, CD38 and CD24. Data were acquired on a BD LSRFORTESSA flow cytometer. The expression of programmed cell death 1 (PD-1, CD279) receptor on CD8+T cells was significantly increased in blood and bone marrow of ALL patients compared to healthy donors (p<0.0001 and p=0.004, respectively) (Fig. 1). Focusing on the different subsets, CD8+ effector memory T cells significantly over-expressed PD-1 in blood and bone marrow of ALL patients compared to healthy donors (p=0.008 and p=0.04, respectively). Moreover, there was a significant positive correlation between PD-1 expression on CD8+ effector memory T cells and blast infiltration (R2=0.23, 95%CI 0.026-0.76, p=0.04). Expression of the co-inhibitory receptor lymphocyte-activation gene 3 (LAG-3, CD223) was similar in ALL patients compared to healthy donors. A significantly higher frequency of T regulators (CD25+, CD127 low, Foxp3+) was found in bone marrow microenvironment in ALL patients (4.3% versus 1.6%, p=0.02). Concerning γd T cells, frequency was similar in blood and bone marrow of ALL patients compared with healthy donors. There was a significantly lower frequency of CD56dimNKG2A+KIR-CD57- (p=0.02) in the bone marrow of ALL patients indicating a maturation arrest. Interestingly, expression of the activating receptor NKG2D which plays an important role in triggering the NK cell–mediated tumor cell lysis was significantly reduced in NK cells of ALL patients while no difference in NK cell expression of NKG2C was found(Fig. 2). Adult patients with ALL show evidence of immune-editing of T cells and NK cells. This global immunosuppressive mechanism may contribute to the eventual escape of ALL from immune control. PD-1, overexpression, described in acute myeloid leukemia and chronic myeloid leukemia has been implicated in T-cell exhaustion and subsequent tumor immune evasion. Our data suggests similar immune escape mechanisms pertain in ALL. Effective antileukemia immunotherapy will require targeting one or more of these immunosuppressive pathways to achieve optimum results. Disclosures Fathi: Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

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The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽

10.1182/blood.v126.23.3090.3090 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3090-3090 ◽

Cited By ~ 1

Author(s):

Folashade Otegbeye ◽

Nathan Mackowski ◽

Evelyn Ojo ◽

Marcos De Lima ◽

David N. Wald

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

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Identification of an NK/T cell–restricted progenitor in adult bone marrow contributing to bone marrow– and thymic-dependent NK cells

Blood ◽

10.1182/blood-2009-10-247130 ◽

2010 ◽

Vol 116 (2) ◽

pp. 183-192 ◽

Cited By ~ 31

Author(s):

Hojjatollah Nozad Charoudeh ◽

Yanjuan Tang ◽

Min Cheng ◽

Corrado M. Cilio ◽

Sten Eirik W. Jacobsen ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Nk Cells ◽

Nk Cell ◽

T Cell Development ◽

Cell Development ◽

Cell Potential ◽

Close Link ◽

Nk T Cell

Abstract Although bone marrow (BM) is the main site of natural killer (NK)–cell development in adult mice, recent studies have identified a distinct thymic-dependent NK pathway, implicating a possible close link between NK- and T-cell development in adult hematopoiesis. To investigate whether a potential NK-/T-lineage restriction of multipotent progenitors might take place already in the BM, we tested the full lineage potentials of NK-cell progenitors in adult BM. Notably, although Lin−CD122+NK1.1−DX5− NK-cell progenitors failed to commit to the B and myeloid lineages, they sustained a combined NK- and T-cell potential in vivo and in vitro at the single-cell level. Whereas T-cell development from NK/T progenitors is Notch-dependent, their contribution to thymic and BM NK cells remains Notch-independent. These findings demonstrate the existence of bipotent NK-/T-cell progenitors in adult BM.

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