Phenotypic and Functional Effects of Novel Akt Inhibitor Perifosine on Immune System.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1555-1555
Author(s):  
Weihua Song ◽  
Teru Hideshima ◽  
Yu-Tzu Tai ◽  
Kenneth C. Anderson ◽  
Nikhil C. Munshi
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Immune System ◽  
Nk Cells ◽  
Negative Selection ◽  
Akt Inhibitor ◽  
Ifn Gamma ◽  
Alexa Fluor ◽  
Hla Dq ◽  
Dose Dependent

Abstract Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agent which targets cell membranes and inhibits Akt activation. Perifosine inhibits multiple myeloma (MM) cell growth in vitro and in vivo. Currently perifosine is under phase II clinical evaluation in MM. Although perifosine has shown significant direct antitumor effects, its effect on immune system has not yet been clarified. The objective of this study was to investigate the effects of perifosine on immune system including the activity of antigen presenting cells (APCs), T cells and NK cells. Perifosine was used at a clinically relevant concentrations of 2.5 μM, 5 μM and 10 μM. Monocyte-derived dendritic cells (DCs) from healthy donors were used as the APCs. We observed that the perifosine up to 48 hours had no effect on viability (>90%), as assessed by annexin V and PI staining. Alteration of DC phenotype by perifosine was further examined by flow cytometry. Our results demonstrated that perifosine treatment led to a dose-dependent downregulation of surface antigen expression, associated with costimulation (CD40, CD80 and CD86), antigen presentation (HLA-ABC, HLA-DQ) and maturation (CD83) on immature DCs at 24 and 48 hours. The significant downregulation of CD40, CD83, HLA-ABC and HLA-DQ was also observed on mature DCs. Perifosine also inhibited immature DC uptake of antigens (40-kDa Dextran-FITC, 45-kDa protein A-Alexa Fluor 488 and 20-kDa protein G-Alexa Fluor 488) in a dose-dependent manner. Since DCs play a crucial regulatory role via cytokine production, we next determined IL-12p70 and IL-10 secretion by LPS-induced DCs with and without perifosine treatment. Compared to controls, perifosine treatment at 24 hours significantly inhibited LPS-induced-IL-12p70 production by DCs (trt vs. untrt = 166 pg/ml (2.5μM), 111 pg/ml (5μM) and 45 pg/ml (10μM) vs. 192 pg/ml), as well as inhibited IL-10 production (trt vs. untrt = 371 pg/ml (2.5μM), 306 pg/ml (5μM) and 179 pg/ml (10μM) vs. 472 pg/ml). These data suggest that perifosine is able to affect both immature and mature DCs and could contribute to inhibition of DC-mediated immune responses. Furthermore, we evaluated effect of perifosine on T cells obtained from healthy donor peripheral blood mononuclear cells (PBMCs) by negative selection. Although perifosine treatment, up to 10μM, had no effect on T cell survival, it inhibited IFN-gamma production by T cells stimulated with PMA and ionomycin compared to that of the control (intracellular flow cytometry, trt vs. untrt= 11.7% vs. 21.3%). The IFN-gamma inhibition by perifosine treatment (10μM) was further confirmed by ELISA upon stimulation with anti- CD2/CD3/CD28 activation beads (trt vs. untrt =11340 pg/ml vs. 18150 pg/mL). Similarly, viability of NK cells obtained from PBMCs by negative selection was not significantly affected by perifosine; however, decreased cytotoxicities were observed by NK cells treated with perifosine (10μM) against the U266 myeloma cell line (trt vs. untrt=30% vs. 51% (E:T=10:1) and 17% vs 30% (E:T=5:1) respectively). In addition, the IFN-gamma production by IL-2 and IL-12- induced NK cells was significantly inhibited following the perifosine treatment (trt vs. untrt=286 pg/ml (2.5μM), 210 pg/ml (10μM) vs. 439 pg/ml). These studies demonstrate that perifosine treatment significantly affects phenotype and function of human DCs, T cells and NK cells. Our pre-clinical data therefore indicates the need to monitor immune functions in patients under the Akt inhibitor treatment.

scholarly journals Production of hematopoietic colony-stimulating factors by human natural killer cells.

1989 ◽  
Vol 169 (2) ◽  
pp. 569-583 ◽  
Author(s):  
M C Cuturi ◽  
I Anegón ◽  
F Sherman ◽  
R Loudon ◽  
S C Clark ◽  
...  
Keyword(s):  
T Cells ◽  
Biological Activity ◽  
Nk Cells ◽  
Negative Selection ◽  
Lymphoblastoid Cell Lines ◽  
Ionophore A23187 ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Human Natural Killer Cells ◽  
Kinetics Of

We have analyzed the ability of highly purified preparations of human NK cells to produce CSF. NK cells, purified by negative selection from 10-d cultures of PBMC incubated with irradiated B-lymphoblastoid cell lines, were stimulated with rIL-2, FcR(CD16) ligands (particulate immune complexes or anti-CD16 antibodies bound to Sepharose), a combination of CD16 ligands and rIL-2, or the phorbol diester phorbol dibutyrate (PDBu) together with the Ca2+ ionophore A23187. Both rIL-2 and CD16 ligands induce accumulation of GM-CSF mRNA in NK cells and the combined effect of the two stimuli is synergistic. Maximal accumulation of GM-CSF mRNA is observed after PDBu/A23187 stimulation. The participation of contaminant T cells in the observed expression of the GM-CSF gene is excluded because CD16 ligands do not stimulate T cells and CD3 ligands, powerful stimulators of T cells, are inactive on NK cells. Accumulation of CSF-1 mRNA is observed only in NK cells stimulated with both CD16 ligands and rIL-2, whereas accumulation of IL-3 mRNA is observed only in NK cells stimulated with PDBu/A23187. Transcripts of the G-CSF, IL-1 alpha, and IL-1 beta genes were never detected in NK cells in these experiments. The kinetics of accumulation of GM-CSF and CSF-1 mRNA in NK cells stimulated with CD16 ligands and rIL-2 peaked at 2-4 h and was slower than that of TNF and IFN-gamma mRNA, which peak at 1 h. GM-CSF was precipitated from the supernatant fluids of NK cells stimulated with PDBu/A23187 and its biological activity was demonstrated by the ability of the supernatants to sustain proliferation of the TALL-101 cell line or CML blasts. Biological activity of IL-3 and CSF-1 was demonstrable in supernatant fluids of NK cells stimulated with PDBu/A23187 and CD16 ligands/rIL-2, respectively.

scholarly journals Pharmacokinetic and Pharmacodynamic Study of NKTR-255, a Polymer-Conjugated Human IL-15, in Cynomolgus Monkey

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2952-2952
Author(s):  
Takahiro Miyazaki ◽  
Peiwen Kuo ◽  
Mekhala Maiti ◽  
Palakshi Obalapur ◽  
Murali Addepalli ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cynomolgus Monkey ◽  
Memory T Cells ◽  
Granzyme B ◽  
Employment Equity ◽  
Equity Ownership ◽  
Dose Dependent

Abstract Introduction IL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey. Methods In vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry. Results NKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis. Conclusions Nektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

scholarly journals Flow cytometry and targeted immune transcriptomics identify distinct profiles in patients with chronic myeloid leukemia receiving tyrosine kinase inhibitors with or without interferon-α

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Raquel Alves ◽  
Stephanie E. B. McArdle ◽  
Jayakumar Vadakekolathu ◽  
Ana Cristina Gonçalves ◽  
Paulo Freitas-Tavares ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Immune System ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Nk Cells ◽  
Tumor Cells ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Immune Surveillance

Abstract Background Tumor cells have evolved complex strategies to escape immune surveillance, a process which involves NK cells and T lymphocytes, and various immunological factors. Indeed, tumor cells recruit immunosuppressive cells [including regulatory T-cells (Treg), myeloid-derived suppressor cells (MDSC)] and express factors such as PD-L1. Molecularly targeted therapies, such as imatinib, have off-target effects that may influence immune function. Imatinib has been shown to modulate multiple cell types involved in anti-cancer immune surveillance, with potentially detrimental or favorable outcomes. Imatinib and other tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) have dramatically changed disease course. Our study aimed to characterize the different populations of the immune system in patients with CML affected by their treatment. Methods Forty-one patients with CML [33 treated with TKIs and 8 with TKIs plus interferon (IFN)-α] and 20 controls were enrolled in the present study. Peripheral blood populations of the immune system [referred to as the overview of immune system (OVIS) panel, Treg cells and MDSCs] and PD-1 expression were evaluated by flow cytometry. The immunological profile was assessed using the mRNA Pan-Cancer Immune Profiling Panel and a NanoString nCounter FLEX platform. Results Patients receiving combination therapy (TKIs + IFN-α) had lower numbers of lymphocytes, particularly T cells [838/µL (95% CI 594–1182)] compared with healthy controls [1500/µL (95% CI 1207 – 1865), p = 0.017]. These patients also had a higher percentage of Treg (9.1%) and CD4+PD-1+ cells (1.65%) compared with controls [Treg (6.1%) and CD4+/PD-1+(0.8%); p ≤ 0.05]. Moreover, patients treated with TKIs had more Mo-MDSCs (12.7%) whereas those treated with TKIs + IFN-α had more Gr-MDSC (21.3%) compared to controls [Mo-MDSC (11.4%) and Gr-MDSC (8.48%); p ≤ 0.05]. CD56bright NK cells, a cell subset endowed with immune-regulatory properties, were increased in patients receiving TKIs plus IFN-α compared with those treated with TKIs alone. Interestingly, serum IL-21 was significantly lower in the TKIs plus IFN-α cohort. Within the group of patients treated with TKI monotherapy, we observed that individuals receiving 2nd generation TKIs had lower percentages of CD4+ Treg (3.63%) and Gr-MDSC (4.2%) compared to patients under imatinib treatment (CD4+ Treg 6.18% and Gr-MDSC 8.2%), but higher levels of PD-1-co-expressing CD4+ cells (1.92%). Conclusions Our results suggest that TKIs in combination with IFN-α may promote an enhanced immune suppressive state.

scholarly journals Cytotoxicity of Environmentally Relevant Concentrations of Aluminum in Murine Thymocytes and Lymphocytes

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Jamal Kamalov ◽  
David O. Carpenter ◽  
Irina Birman
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Immune System ◽  
Dna Binding ◽  
Aluminum Chloride ◽  
Cell Swelling ◽  
Cytotoxic Effects ◽  
Minute Exposure ◽  
Low Concentrations ◽  
Dose Dependent

The effects of low concentrations of aluminum chloride on thymocytes and lymphocytes acutely dissociated from young mice were studied using flow cytometry with a DNA-binding dye. We demonstrate a rapid and dose-dependent injury in murine thymocytes and lymphocytes resulting from exposure to aluminum, as indicated by an increase in the entry into the cell of the DNA-binding dye, propidium iodine. A 60-minute exposure to 10 μM AlCl3caused damage of about 5% of thymocytes, while 50% were injured after 10 minutes at 20 μM. Nearly all thymocytes showed evidence of damage at 30 μM AlCl3after only 5 minutes of incubation. In lymphocytes, injury was observed at 15 μM AlCl3and less than 50% of cells were injured after a 60-minute exposure to 20 μM. Injury only rarely proceeded to rapid cell death and was associated with cell swelling. These results suggest that aluminum has cytotoxic effects on cells of the immune system.

scholarly journals PD-1 expression on NK cells can be related to cytokine stimulation and tissue residency

2021 ◽  
Author(s):  
Arnika K Wagner ◽  
Nadir Kadri ◽  
Chris Tibbitt ◽  
Koen van de Ven ◽  
Sunitha Bagawath-Singh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Mhc Class I ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Cytokine Stimulation ◽  
Deficient Mice

ABSTRACTAlthough PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells which in part could be attributed to a decrease in tumor-infiltrating NK cells in PD-1-deficient mice. NK cells from PD-1-deficient mice exhibited a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Finally, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wildtype mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells.

scholarly journals 0031 Photoperiodic Effects on Diurnal Rhythms in the Immune System, Rest-Activity Behavior, and Cortisol Revealed in a Diurnally Active Large Animal Model - The Domestic Pig

SLEEP ◽  
2020 ◽  
Vol 43 (Supplement_1) ◽  
pp. A12-A13
Author(s):  
L C Engert ◽  
U Weiler ◽  
B Pfaffinger ◽  
V Stefanski ◽  
S S Schmucker
Keyword(s):  
T Cells ◽  
Immune System ◽  
Nk Cells ◽  
Cortisol Concentration ◽  
Diurnal Rhythms ◽  
Domestic Pig ◽  
Cell Numbers ◽  
Peripheral Leukocyte ◽  
Activity Behavior ◽  
Rest Activity

Abstract Introduction Diurnal variations in immune cell number and function are regarded important for immune competence and are thought to be mediated by rest-activity rhythms and hormones. Moreover, the photoperiod is also known to modulate the immune system and considered to affect seasonal disease susceptibility. Whereas few studies investigated seasonal effects, the present study is the first investigating the specific effect of the photoperiod on diurnal rhythms in cell numbers of peripheral leukocyte types in any species. Methods Domestic pigs were held either under long day (LD, 16L:8D, lights-on 07:00-23:00, n=9) or short day conditions (SD, 8L:16D, lights-on 07:00-15:00, n=11) and fed concentrate at 07:30 + 14:00 (ad libitum hay/water). Blood samples were taken every 2 h over periods of 50 h via indwelling vein catheters. Rest-activity behavior of the pigs was analyzed using continuous camera recordings. Results Cosinor analyses (p<.05) revealed photoperiodic differences in diurnal rhythms of cell numbers of various peripheral leukocyte subtypes, rest-activity behavior, and cortisol concentration. Cell numbers of total leukocytes, NK cells, T cells, and eosinophils in blood, rest-activity behavior, and cortisol concentration peaked earlier relative to lights-on under SD (p<.05). Relative amplitudes in rest-activity behavior and cell counts of total leukocytes, NK cells, T cells, and monocytes were higher under SD (p<.05). However, there was no photoperiodic effect on diurnal rhythms in neutrophil counts and mesor in any leukocyte type. Generalized linear mixed models revealed associations of leukocyte counts with rest-activity behavior and cortisol concentration in most cell types (p<.05). Moreover, the found photoperiodic effects on diurnal rhythms in rest-activity behavior and cortisol concentration are in agreement with research in humans and primates. Conclusion The present study revealed photoperiodic effects on diurnal rhythms in the immune system, rest-activity behavior, and cortisol concentration in pigs and strengthens the importance of the domestic pig as suitable model for chronoimmunology. Support German Research Foundation DFG (grant provided to SS, grant number SCHM3162/1-1), Federal Ministry of Education and Research, Germany (grant number 01PL16003), Faculty of Agricultural Sciences, University of Hohenheim (scholarship provided to LE).

scholarly journals Sinonasal T-Cell Expression of Cytotoxic Mediators Granzyme B and Perforin is Reduced in Patients with Chronic Rhinosinusitis

2017 ◽  
Vol 31 (6) ◽  
pp. 352-356 ◽  
Author(s):  
Sarah E. Smith ◽  
Rodney J. Schlosser ◽  
James R. Yawn ◽  
Jose L. Mattos ◽  
Zachary M. Soler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyp ◽  
Granzyme B ◽  
Cytotoxic Cells ◽  
Sinonasal Mucosa ◽  
Perforin Expression

Background CD8+ T cells and natural killer (NK) cells are cytotoxic cells that use granzyme B (GrB) and perforin. Defective cytotoxic function is known to play a role in dysregulated immune response as seen in chronic sinusitis, also referred to as chronic rhinosinusitis (CRS). However, to our knowledge, in the United States, neither GrB or perforin expression has been reported in patients with CRS. Objective The aim of this study was to investigate sinonasal cytotoxic cells, their mediators, and cell-specific distribution of these mediators in patients with CRS with nasal polyp (CRSwNP) and in patients with CRS without nasal polyp (CRSsNP). Methods Blood and sinus tissue samples were taken from patients with CRSsNP (n = 8) and CRSwNP (n = 8) at the time of surgery. Control subjects (n = 8) underwent surgery for cerebrospinal fluid leak repair or to remove non-hormone-secreting pituitary tumors. The cells were analyzed via flow cytometry by using CD8 expression to identify cytotoxic T cells and CD56 expression to identify NK cells. Intracellular GrB and perforin expression were analyzed with flow cytometry. Results We observed no significant differences in plasma or peripheral blood immune cell numbers or specific levels of GrB or perforin among the groups. In the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP, there was a significant decrease in GrB and perforin levels (p <0.05) despite similar or increased numbers of cytotoxic cells when compared with the controls. The overall decrease in GrB and perforin in the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP was due to decreased T cell production. There was no difference in total NK cell count or expression of perforin or GrB among all the groups. Conclusion Total levels of sinonasal GrB and perforin were decreased in the sinonasal mucosa of both the patients with CRSwNP and the patients with CRSsNP compared with the controls, whereas sinonasal CD8+ T cells, (but not NK cells,), intracellular stores of GrB and perforin were reduced in the patients with CRSwNP compared with the controls.

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

Blood ◽  
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.

Long Lasting Alteration of Natural Killer Cells Post-Chemotherapy in Elderly Patients with Acute Myeloid Leukemia (AML).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1653-1653
Author(s):  
Daniel Olive ◽  
Nicolas Anfossi ◽  
Pascale Andre ◽  
Jerome Rey ◽  
Florence Orlanducci ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
Elderly Patients ◽  
Nk Cells ◽  
Natural Killer ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Time Points ◽  
Early Stages ◽  
Cell Counts

Abstract Abstract 1653 Poster Board I-679 Background The immune system is involved in AML control and Natural Killer (NK) cells are among the most promising effectors. The therapeutic potential of NK cells has been revealed by the Killer Immunoglobulin Receptor (KIR) mismatch allogeneic transplant model where the anti-leukemic effect of the graft, is due to unleashed NK cells towards AML blasts, as suggested by enhanced in vitro lytic activity of KIR-HLA mismatched donor NK cells against recipient blasts (Miller et al. 2005; Ruggeri et al. 2002). Receptors involved in the function of NK cells against AML blasts have been identified (Pende et al., 2005). Some of these receptors are altered in AML patients at diagnosis and might be involved in the immune escape of AML blasts (Costello et al., 2002). However, the status of NK cells during early stages of patient's chemotherapy (CT) treatment is unknown. The present study monitored status of NK cells during early stages following patient's remission after CT that may be critical for their long lasting clinical response, and results might provide new targets for immunotherapy. Methods We enrolled 20 elderly patients (60 to 80 years old) with non promyelocytic AML in first CR following induction and pre-consolidation CT with normal renal and hepatic functions. Patient peripheral NK, gd T and CD8 T cells were analyzed before consolidation CT and every other week after treatment for 8 weeks. 6-colors flow cytometry was performed to investigate the expression of MHC receptors (CD158a, b, e, i, CD85j and NKG2A), activating receptors (NKp30, NKp46, NKG2D, CD16, DNAM-1, 2B4) as well as their differentiation status (perforin and granzyme expression). Their function, as determined by cytotoxicity (51Cr release and CD107 expression) and cytokine production (intracellular staining of IFN-g), was analyzed using purified NK cells stimulated by K562, or in redirected assays using NKp30, NKp46 and CD16 mAbs. Results NK cell counts were depressed away from the induction and pre-consolidation CT as compared to NK cell counts of age-matched controls (ctl) (95±107 NK/μL vs 229±91 NK/μL respectively); they were further depressed during the first 2 weeks post-consolidation CT (55±57 NK/μL), but were back to pre-consolidation CT level at 4 weeks (105±102 NK/μL). In contrast, CD8 T cells and gd T cells counts were normal even at early times post-CT. Expression of 2B4 was depressed at all time points. In contrast, NKp30 expression was lower at diagnosis and close to ctl level post-consolidation CT (p=0.0003) and NKp46 expression increased after CT (p<0.0001). Sizes of NK cell subsets expressing CD158a or CD158b in patients post-induction and consolidation CT were smaller than those of ctl (% CD158a+: p=0.003; % CD158b+: p=0.014). In contrast the NKG2A or CD85j positive NK cell subsets were either unchanged or slightly increased respectively at all time points (p=0.0015 for CD85j+). Moreover, sizes of perforin or granzyme positive NK cell subsets were increased in treated AML patients (% Granzyme+: p= 0.0125 and % Perforin+: p=0.0268). In addition, we observed an important heterogeneity in the expression of the surface receptors among patients that is currently analyzed with respect to the duration of the CR. Finally, NK cell cytoxicity was comparable at all time points to the one of age-matched ctl. In contrast, IFN-g secretion was decreased, at all time points, against K562 or in redirected assays using CD16 mAb and almost abolished using redirected assay with NKp30 mAb. Conclusions This study demonstrates that in elderly AML patients in CR after CT (1) several alterations are detected at all time points, (2) NK cell number is lower and (3) IFN-g secretion is impaired. However NK cytotoxic function is comparable to age-matched controls. The likely basis of the complex pattern of modifications might rely on an interplay between the direct and indirect effects of chemotherapy, activation of immune system, NK cell differentiation and its interaction with AML blasts. Altogether this study indicates that new immunotherapeutic approaches might be used to increase NK cell numbers and functions (cytotoxicity and IFN-g secretion) at early times post-CT in elderly patients with AML. Disclosures Romagne: Innate Pharma: Employment.

Multidimensional Flow Cytometric (MFC) Analysis of the Immune System of Multiple Myeloma (MM) Patients Achieving Long Term Disease Control

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 810-810
Author(s):  
Roberto J. Pessoa Magalhaes ◽  
María-Belén Vidriales ◽  
Bruno Paiva ◽  
Maria-Victoria Mateos ◽  
Norma C. Gutierrez ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Disease Control ◽  
B Cell ◽  
Nk Cells ◽  
Cell Populations ◽  
High Dose ◽  
Newly Diagnosed

Abstract Abstract 810FN2 Increasing evidence shows that a small fraction of MM patients (pts) treated with high-dose therapy followed by autologous stem cell transplantation achieve long-term remission. Interestingly, this is not restricted to pts in complete response (CR), since those that revert to a monoclonal gammopathy of undetermined significance (MGUS) profile may also achieve long-term remission, despite the persistence of residual myeloma plasma cells (PCs). These results suggest that in addition to the anti-myeloma therapy, other factors may play a role in the control of the disease. Herein, we used 8-color MFC for detailed characterization of the structural components of the immune system and hematopoietic precursor cells (HPC) in paired bone marrow (BM) and peripheral blood (PB) samples from 26 MM patients in long-term disease control (LTDC): 9 in continuous CR and 17 who reverted to an MGUS profile and that subsequently showed stable disease without treatment for ≥5 years (median of 9 years; range, 5–19). As controls, paired BM and PB samples from 23 newly-diagnosed MGUS and 16 MM pts, together with 10 healthy adults (HA), were studied in parallel. In all BM and PB samples the distribution of the major T- (CD4, CD8, Tregs and γδ), NK- (CD56dim and CD56bright) and B-cell subsets (Pro-B, Pre-B, naïve and memory), in addition to normal PCs, dendritic cell (DC) subsets (plasmacytoid, myeloid and monocytic), monocytes, and CD34+ HPC (myeloid and lymphoid), were studied. The percentage and absolute count of each cell population was analysed in the BM and PB, respectively. Comparison of the two groups of MM pts with LTDC (9 CR vs. 17 MGUS-like) showed similar (p>.05) cellular profiles in PB and BM, except for an increased number of BM and PB normal PCs in CR patients (P≤.04). Consequently, for all subsequent analyses, LTDC myeloma pts were pooled together. When compared to HA, patients with LTDC had increased numbers of CD8 T-cells and CD56dim NK-cells in both the BM and PB (p≤.03 and p≤.01, respectively). Despite this, the distribution of BM and PB CD4, CD8 and γδ T-cells among LTDC patients was similar (p>.05) to that of both newly-diagnosed MM and MGUS cases; in contrast, BM and PB Tregs were significantly decreased vs newly-diagnosed MM (P=.03) and MGUS (P=.04). Regarding B-cells and normal PCs, LTDC patients showed increased numbers of BM B-cell precursors (both Pro-B and Pre-B cells) and normal PCs vs. newly diagnosed MM (P≤.05), but not MGUS, together with increased numbers of naïve B-cells vs. both MM and MGUS pts (P≤.01); all such cell populations returned to levels similar (p>.05) to those of HA. As expected, this also included the number of CD34+ B-cell HPC which was increased among patients who achieved LTDC vs MM (p=.02), at levels similar (p>.05) to those of MGUS and HA. Regarding DC, LTDC patients showed normal DC numbers in PB (but with higher PB myeloid-DC numbers vs. MM; p=.02), in association with decreased numbers of plasmacytoid DC and increased monocytic-DC in the BM vs. HA (p≤.04). No differences were found for the numbers of BM and PB monocytes. In summary, here we investigated for the first time the immune cell profile of MM patients who achieve long-term disease control. Our results show that, as newly-diagnosed MM, patients that achieve long-term disease control also show increased numbers of cytotoxic T-cells and CD56dim NK-cells; however, in contrast to newly-diagnosed MM, among LTDC patients such increase is associated with lower numbers of T-regs and an almost complete recovery of the normal PC, B-cell precursor and naïve B-cell compartments both in BM and PB. Further investigations on the activation and functional status of these cell populations are warranted.MO (%)/SP (cels./μl)HA N= 10MGUS N= 23MM N= 16LTDC-MM N= 26T cells9.588110.6117313113711926    CD4+4.85004.6624^6*5085463    CD8+3.7∼216∼4.63865.32645.3431    TCR γδ.2426.3230.2428.3421    Treg.4137.4141^.54*38.3432NK cells.7∼87∼1.51982.11721.6212    CD56 dim.65∼79∼1.41922.21681.6202B cells2.81471.8104.97*68*1.9160    Pro B.11—.06—.02*—.07—    Pre B.6—.4—.08—.23—    Naive SP—80—57^—36*—118    Normal-PCS.18.9.11.7.008.72*.11.84DCs.3449.3653.6848.558    Monocytes2.22472.42853.43023.1315    m-DC SP—11—14—8*—12    MO-DC.11∼29.2036.434.2837    p-DC.2∼4.1.145.112.8.123.8CD34+.9∼1.46.61.1.261.4.431.4    Mie-HPC.8∼—.53—.26—.36—    Linfo-HPC.1—.07—.03*—.05—*p≤.05 LTDC vs MM: ^ p≤.05 LTDC vs MGUS; ∼ p≤.05 LTDC vs HA Disclosures: Paiva: Jansen-Cillag: Honoraria; Celgene: Honoraria. Martinez:Janssen: Honoraria; Celgene: Honoraria. Maiolino:Centocor Ortho Biotech Research & Development: Research Funding.

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