Severe chronic autoimmune thrombocytopenic purpura is associated with an expansion of CD56+ CD3- natural killer cells subset [see comments]

Recent studies have indicated that autoimmune thrombocytopenic purpura (ATP) patients show immune system alterations that are not restricted to the B-cell compartment, but that also affect T lymphocytes. This report studies the phenotypic characteristics of natural killer (NK) cells in the peripheral blood of ATP patients, as well as their clinical significance in 33 ATP patients with active disease. Ten patients had stable disease (sustained platelet counts > 50,000/microL without the need for treatment), whereas 23 patients had therapy- dependent disease (platelet counts < 50,000/microL). A significant increase in both CD56+ CD3- NK cells and CD56+ CD3+ cytotoxic T lymphocytes was observed in peripheral blood mononuclear cells and in purified CD2+ cells from therapy-dependent ATP patients as compared with ATP patients with stable disease and healthy controls. Moreover, there were more major histocompatibility complex (MHC) class II molecules in the CD56+ CD3- cells from the therapy-dependent patients' peripheral blood preparations than there were in the stable ATP patients' and healthy controls' peripheral blood preparations. This growth in the number of CD56+ CD3- NK cells was statistically higher in patients whose disease was refractory to conventional therapy (corticosteroids and splenectomy). In addition to the CD56+ CD3- NK cells, the percentage of CD3+ T lymphocytes and their proliferative response to phytohemagglutinin (PHA) stimulation were studied in fresh CD2+ preparations from nine patients with stable disease, 22 patients with therapy-dependent disease, and 26 healthy controls. The proliferative response of CD2+ lymphocytes from both groups was similar and significantly defective with respect to that found in healthy controls. In conclusion, clinically severe ATP (therapy-dependent disease) is associated with a significant increase of CD56+ CD3- NK cells, which is particularly marked in patients whose disease is refractory to therapy.

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Severe chronic autoimmune thrombocytopenic purpura is associated with an expansion of CD56+ CD3- natural killer cells subset [see comments]

Blood ◽

10.1182/blood.v82.5.1538.1538 ◽

1993 ◽

Vol 82 (5) ◽

pp. 1538-1545 ◽

Cited By ~ 35

Author(s):

J Garcia-Suarez ◽

A Prieto ◽

E Reyes ◽

L Manzano ◽

JL Merino ◽

...

Keyword(s):

T Lymphocytes ◽

Nk Cells ◽

Natural Killer ◽

Stable Disease ◽

Peripheral Blood ◽

Proliferative Response ◽

Healthy Controls ◽

Thrombocytopenic Purpura ◽

Autoimmune Thrombocytopenic Purpura ◽

Platelet Counts

Abstract Recent studies have indicated that autoimmune thrombocytopenic purpura (ATP) patients show immune system alterations that are not restricted to the B-cell compartment, but that also affect T lymphocytes. This report studies the phenotypic characteristics of natural killer (NK) cells in the peripheral blood of ATP patients, as well as their clinical significance in 33 ATP patients with active disease. Ten patients had stable disease (sustained platelet counts > 50,000/microL without the need for treatment), whereas 23 patients had therapy- dependent disease (platelet counts < 50,000/microL). A significant increase in both CD56+ CD3- NK cells and CD56+ CD3+ cytotoxic T lymphocytes was observed in peripheral blood mononuclear cells and in purified CD2+ cells from therapy-dependent ATP patients as compared with ATP patients with stable disease and healthy controls. Moreover, there were more major histocompatibility complex (MHC) class II molecules in the CD56+ CD3- cells from the therapy-dependent patients' peripheral blood preparations than there were in the stable ATP patients' and healthy controls' peripheral blood preparations. This growth in the number of CD56+ CD3- NK cells was statistically higher in patients whose disease was refractory to conventional therapy (corticosteroids and splenectomy). In addition to the CD56+ CD3- NK cells, the percentage of CD3+ T lymphocytes and their proliferative response to phytohemagglutinin (PHA) stimulation were studied in fresh CD2+ preparations from nine patients with stable disease, 22 patients with therapy-dependent disease, and 26 healthy controls. The proliferative response of CD2+ lymphocytes from both groups was similar and significantly defective with respect to that found in healthy controls. In conclusion, clinically severe ATP (therapy-dependent disease) is associated with a significant increase of CD56+ CD3- NK cells, which is particularly marked in patients whose disease is refractory to therapy.

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Cytotoxic T lymphocytes and natural killer cells display impaired cytotoxic functions and reduced activation in patients with alcoholic hepatitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00200.2014 ◽

2015 ◽

Vol 308 (4) ◽

pp. G269-G276 ◽

Cited By ~ 17

Author(s):

Sidsel Støy ◽

Anders Dige ◽

Thomas Damgaard Sandahl ◽

Tea Lund Laursen ◽

Christian Buus ◽

...

Keyword(s):

T Lymphocytes ◽

Nk Cells ◽

Natural Killer ◽

Cytotoxic T Lymphocytes ◽

Alcoholic Hepatitis ◽

Nk Cell ◽

Alcoholic Cirrhosis ◽

Nkt Cells ◽

Receptor Expression ◽

Healthy Controls

The dynamics and role of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and NKT cells in the life-threatening inflammatory disease alcoholic hepatitis is largely unknown. These cells directly kill infected and damaged cells through, e.g., degranulation and interferon-γ (IFNγ) production, but cause tissue damage if overactivated. They also assist tissue repair via IL-22 production. We, therefore, aimed to investigate the frequency, functionality, and activation state of such cells in alcoholic hepatitis. We analyzed blood samples from 24 severe alcoholic hepatitis patients followed for 30 days after diagnosis. Ten healthy abstinent volunteers and 10 stable abstinent alcoholic cirrhosis patients were controls. Using flow cytometry we assessed cell frequencies, NK cell degranulation capacity following K562 cell stimulation, activation by natural killer group 2 D (NKG2D) expression, and IL-22 and IFNγ production. In alcoholic hepatitis we found a decreased frequency of CTLs compared with healthy controls ( P < 0.001) and a similar trend for NK cells ( P = 0.089). The NK cell degranulation capacity was reduced by 25% compared with healthy controls ( P = 0.02) and by 50% compared with cirrhosis patients ( P = 0.04). Accordingly, the NKG2D receptor expression was markedly decreased on NK cells, CTLs, and NKT cells ( P < 0.05, all). The frequencies of IL-22-producing CTLs and NK cells were doubled compared with healthy controls ( P < 0.05, all) but not different from cirrhosis patients. This exploratory study for the first time showed impaired cellular cytotoxicity and activation in alcoholic hepatitis. This is unlikely to cause hepatocyte death but may contribute toward the severe immune incompetence. The results warrant detailed and mechanistic studies.

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Phenotypic and Functional Changes in Peripheral Blood Natural Killer Cells in Crohn Disease Patients

Mediators of Inflammation ◽

10.1155/2020/6401969 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15

Author(s):

Suzanne Samarani ◽

Patrick Sagala ◽

Prevost Jantchou ◽

Guy Grimard ◽

Christophe Faure ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Crohn Disease ◽

Peripheral Blood ◽

Nk Cell ◽

Healthy Controls ◽

Inhibitory Receptors ◽

Color Flow ◽

Functional Changes ◽

Treatment Naïve

We investigated activation status, cytotoxic potential, and gut homing ability of the peripheral blood Natural Killer (NK) cells in Crohn disease (CD) patients. For this purpose, we compared the expression of different activating and inhibitory receptors (KIR and non-KIR) and integrins on NK cells as well as their recent degranulation history between the patients and age-matched healthy controls. The study was conducted using freshly obtained peripheral blood samples from the study participants. Multiple color flow cytometry was used for these determinations. Our results show that NK cells from treatment-naïve CD patients expressed higher levels of activating KIR as well as other non-KIR activating receptors vis-à-vis healthy controls. They also showed increased frequencies of the cells expressing these receptors. The expression of several KIR and non-KIR inhibitory receptors tended to decrease compared with the cells from healthy donors. NK cells from the patients also expressed increased levels of different gut-homing integrin molecules and showed a history of increased recent degranulation events both constitutively and in response to their in vitro stimulation. Furthermore, treatment of the patients tended to reverse these NK cell changes. Our results demonstrate unequivocally, for the first time, that peripheral blood NK cells in treatment-naïve CD patients are more activated and are more poised to migrate to the gut compared to their counterpart cells from healthy individuals. Moreover, they show that treatment of the patients tends to normalize their NK cells. The results suggest that NK cells are very likely to play a role in the immunopathogenesis of Crohn disease.

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The Utility of Detection Populations of T-Lymphocytes, T-Killer Cells and Natural Killer Cells by CD2 Expression In White Blood Cell Differential Using Flow Cytometry

Blood ◽

10.1182/blood.v116.21.4879.4879 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4879-4879

Author(s):

Ekaterina B. Rusanova ◽

Margarita V. Gorchakova ◽

Konstantin U. Slobodnyuk ◽

Denis V. Cherednichenko ◽

Yekaterina E. Zueva ◽

...

Keyword(s):

Natural Killer Cells ◽

T Lymphocytes ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Wide Spectrum ◽

Mean Value ◽

Killer Cells ◽

Normal Peripheral Blood ◽

T Killer Cells

Abstract Abstract 4879 Introduction. The differential leukocyte count using Cytodiff™ reagent from Beckman Coulter (BEC) allows to detect a wide spectrum of normal and pathological cells in peripheral blood and is useful as a screening test. The CytoDiff™ tube is a 5-color/6-marker panel that provides an extended cytometric differential for whole blood specimens and comprises of: CD36-FITC, (CD2+CD294)-PE, CD19-ECD, CD16-PC5, and CD45-PC7. This provides clinicians with a diagnostic tool to determine appropriate therapies for a variety of blood-related diseases rapidly. CytoDiff™ allows to detect T&NK lymphocytes in the mutual gate using the gating strategy. The aim of this study was to analyze the CD2 expression on T-lymphocytes, T killer cells and natural killer cells in order to separate T&NK lymphocytes, as it was published by Catovsky (Catovsky et al., 1996) and to investigate if CytoDiff™ reagent can be useful to differentiate these types of lymphocytes. Materials and methods. 18 normal peripheral blood (PB) samples in K2EDTA were enrolled into this study. Each sample was stained with the CytoDiff™ reagent and with the reference mixture of monoclonals to detect T-killer, NK-cells and T-Lymphocytes (CD3-FITC, CD(16+56)-PE, CD45-ECD, CD2-PC7, all BEC). The PB was lyzed using VersaLyse (BEC) to prevent selective loss of cells and to conserve light scatter characteristics. PB leukocytes were analyzed according to their immunofluorescence reactivity. At least 20,000 CD45+ events per tube were acquired on FC500 Cytomics flow cytometer (BEC) and analyzed using CXP software (BEC). In the reference tube we identified three populations using the CD45brightSSlow lymphocyte gate in the CD3-FITC/CD(16+56)-PE plot: CD3+CD(16+56)- T lymphocytes, CD3+CD(16+56)+ T killer cells and CD3-CD(16+56)+ natural killer cells. Each population after detecting was applied to CD2/SS plot and X-mean value of CD2 population was calculated. For the CytoDiff tube we analyzed the expression of CD2 and CD16 on the different sub-populations of the T&NK lymphocytes, The X-mean values of CD2 expression and percentages of sub-populations of T&NK lymphocytes in the reference tube and in the CytoDiff™ tube were calculated. For statistic analysis we used Statistica 6.0. The study was approved by The Pavlov State Medical University's Institutional Review Board. Results. The data, obtained with reference tube, shows that the populations of T-lymphocytes, T killer cells and natural killer cells are different by X-mean of CD2 expression (p<0.001). The natural killer cells have the lower intensity the CD2 antigen compared with CD3+CD(16+56)- T lymphocytes (p<0.001), while the highest CD2 expression was found in the CD3+CD(16+56)+ T killer cells (p<0.001). According to Mann-Whithey test the percentages and X-mean value of CD2 of the described populations are not different in the reference and in the CytoDiff™ tubes. Conclusion. These findings show that different populations of T&NK cells can be differentiated by CD2 expression and flow cytometric WBC differential can provide more wide possibilities for screening of pathology, especially in cases of viral infection and T-cell leukemia. Disclosures: Sukhacheva: Beckman Coulter: Employment. Simon-Lopez: Beckman Coulter: Employment.

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Characterization of natural killer cells with antileukemia activity following allogeneic bone marrow transplantation

Blood ◽

10.1182/blood.v67.3.722.722 ◽

1986 ◽

Vol 67 (3) ◽

pp. 722-728 ◽

Cited By ~ 93

Author(s):

T Hercend ◽

T Takvorian ◽

A Nowill ◽

R Tantravahi ◽

P Moingeon ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

T Lymphocytes ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Marrow Transplantation ◽

Phenotypic Characterization ◽

Allogeneic Bone

Abstract To identify cells with potential antileukemia activity following bone marrow transplantation, we have monitored immunologic reconstitution in a patient with acute lymphocytic leukemia in second remission who received intensive chemotherapy and total body irradiation followed by infusion of allogeneic histocompatible marrow. Prior to transplantation, donor bone marrow cells were depleted of T lymphocytes by in vitro treatment with anti-T12 monoclonal antibody and rabbit complement. In the first 3 weeks following bone marrow transplantation, the predominant regenerating mononuclear cell population in peripheral blood exhibited a phenotype characteristic of natural killer (NK) cells. After 4 weeks, T lymphocytes became predominant, but NK cells persisted. Cultured peripheral blood lymphocytes obtained 12 weeks posttransplant were able to display significant cytotoxicity against leukemic blasts that had been cryopreserved at the time of relapse 5 months prior to bone marrow transplantation. To further characterize those cells with antileukemia activity, we used in vitro cloning techniques to identify four monoclonal populations, termed TC12, -48, - 50, and -59, with strong antitumor activity. Cytogenetic analysis demonstrated that each clone was of donor origin. Phenotypic characterization showed that the four clones expressed NKH1A but did not express T3, T4, or T8 antigens. Three of the four clones expressed T11/E rosette antigen. Each clone exhibited strong cytotoxicity against genetically unrelated hematopoietic tumor cell lines such as K562, Molt- 4, JM, and U937. In addition, we found that these patient clones were similar to cloned NK cells previously derived from normal individuals. Taken together, these results suggest that at least some clones with antileukemia activity following bone marrow transplantation are cells with NK-like function and phenotype. Functional analysis of these cytolytic cells in larger numbers of patients will be necessary to determine the clinical significance of this finding.

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Characterization of natural killer cells with antileukemia activity following allogeneic bone marrow transplantation

Blood ◽

10.1182/blood.v67.3.722.bloodjournal673722 ◽

1986 ◽

Vol 67 (3) ◽

pp. 722-728 ◽

Cited By ~ 1

Author(s):

T Hercend ◽

T Takvorian ◽

A Nowill ◽

R Tantravahi ◽

P Moingeon ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

T Lymphocytes ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Marrow Transplantation ◽

Phenotypic Characterization ◽

Allogeneic Bone

To identify cells with potential antileukemia activity following bone marrow transplantation, we have monitored immunologic reconstitution in a patient with acute lymphocytic leukemia in second remission who received intensive chemotherapy and total body irradiation followed by infusion of allogeneic histocompatible marrow. Prior to transplantation, donor bone marrow cells were depleted of T lymphocytes by in vitro treatment with anti-T12 monoclonal antibody and rabbit complement. In the first 3 weeks following bone marrow transplantation, the predominant regenerating mononuclear cell population in peripheral blood exhibited a phenotype characteristic of natural killer (NK) cells. After 4 weeks, T lymphocytes became predominant, but NK cells persisted. Cultured peripheral blood lymphocytes obtained 12 weeks posttransplant were able to display significant cytotoxicity against leukemic blasts that had been cryopreserved at the time of relapse 5 months prior to bone marrow transplantation. To further characterize those cells with antileukemia activity, we used in vitro cloning techniques to identify four monoclonal populations, termed TC12, -48, - 50, and -59, with strong antitumor activity. Cytogenetic analysis demonstrated that each clone was of donor origin. Phenotypic characterization showed that the four clones expressed NKH1A but did not express T3, T4, or T8 antigens. Three of the four clones expressed T11/E rosette antigen. Each clone exhibited strong cytotoxicity against genetically unrelated hematopoietic tumor cell lines such as K562, Molt- 4, JM, and U937. In addition, we found that these patient clones were similar to cloned NK cells previously derived from normal individuals. Taken together, these results suggest that at least some clones with antileukemia activity following bone marrow transplantation are cells with NK-like function and phenotype. Functional analysis of these cytolytic cells in larger numbers of patients will be necessary to determine the clinical significance of this finding.

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Treated HIV Infection Alters Phenotype But Not HIV-specific Function of Peripheral Blood Natural Killer Cells

10.1101/2020.04.12.038604 ◽

2020 ◽

Author(s):

Nancy Q. Zhao ◽

Anne-Maud Ferreira ◽

Philip M. Grant ◽

Susan Holmes ◽

Catherine A. Blish

Keyword(s):

Hiv Infection ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Nk Cell ◽

Cell Phenotype ◽

Healthy Controls ◽

Functional Responses ◽

Cell Repertoire

ABSTRACTNatural killer (NK) cells are the predominant antiviral cells of the innate immune system, and may play an important role in acquisition and disease progression of HIV. While untreated HIV infection is associated with distinct alterations in the peripheral blood NK cell repertoire, less is known about how NK phenotype is altered in the setting of long-term viral suppression with antiretroviral therapy (ART), as well as how NK memory can impact functional responses. As such, we sought to identify changes in NK cell phenotype and function using high-dimensional mass cytometry to simultaneously analyze both surface and functional marker expression of peripheral blood NK cells in a cohort of ART-suppressed, HIV+ patients and HIV-healthy controls. We found that the NK cell repertoire following IL-2 treatment was altered in individuals with treated HIV infection compared to healthy controls, with increased expression of markers including NKG2C and CD2, and decreased expression of CD244 and NKp30. Using co-culture assays with autologous, in vitro HIV-infected CD4 T cells, we identified a subset of NK cells with enhanced responsiveness to HIV-1-infected cells, but no differences in the magnitude of anti-HIV NK cell responses between the HIV+ and HIV-groups. In addition, by profiling of NK cell receptors on responding cells, we found similar phenotypes of HIV-responsive NK cell subsets in both groups. Lastly, we identified clusters of NK cells that are altered in individuals with treated HIV infection compared to healthy controls, but found that these clusters are distinct from those that respond to HIV in vitro. As such, we conclude that while chronic, treated HIV infection induces a reshaping of the IL-2-stimulated peripheral blood NK cell repertoire, it does so in a way that does not make the repertoire more HIV-specific.

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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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