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1.
Blood Adv ; 8(3): 571-580, 2024 02 13.
Article in English | MEDLINE | ID: mdl-38039514

ABSTRACT

ABSTRACT: ACKR1/DARC-associated neutropenia (NP; ADAN; Online Mendelian Inheritance in Man 611862), caused by a variation in the ACKR1/DARC gene (rs2814778), is common in persons of African or Middle Eastern descent. In a cohort of 66 genetically confirmed subjects with ADAN, we show that absolute neutrophil counts (ANCs) may occasionally be lower than previously recognized (0.1 × 109-0.49 × 109/L for 9% of the subjects), which is similar to ANCs in severe congenital NP (SCNP). ANCs often normalized during inflammation, even mild. Individuals with ADAN (of 327 observed person-years) showed no cases of myelodysplastic syndrome (MDS), which is frequently encountered in SCNP. Unexpectedly, 22% presented with autoantibodies to neutrophils, compared with <1% in controls. Compared with healthy donors, subjects with ADAN demonstrated significantly lower human cationic antimicrobial protein-18/pro-leucin leucin-37 plasma levels; higher levels of nonclassical, proinflammatory, 6-sulfo LacNac-expressing monocytes; and differentially expressed plasma levels of 28 of the 239 analyzed cytokines related to immunity/inflammation, cell signaling, neutrophil activation, and angiogenesis. Collectively, more severe neutropenia in ADAN than previously assumed may complicate differential diagnoses compared with other SCNPs, and various (auto)immune/inflammatory reactions with a distinct profile may be a cause or consequence of this hereditary neutropenia.


Subject(s)
Duffy Blood-Group System , Neutropenia , Receptors, Cell Surface , Humans , Inflammation , Leukocyte Count , Neutropenia/genetics , Neutrophils , Receptors, Cell Surface/genetics , Duffy Blood-Group System/genetics
2.
Sci Signal ; 16(780): eabq0752, 2023 04 11.
Article in English | MEDLINE | ID: mdl-37040441

ABSTRACT

Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I-low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A+ NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A+ NK cells by the MHC-I molecule H2Dd led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6, which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2Dd-educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance.


Subject(s)
Antigens, Ly , Killer Cells, Natural , Mice , Animals , Receptors, NK Cell Lectin-Like/metabolism , Antigens, Ly/metabolism , Histocompatibility Antigens Class I/metabolism , Synapses/metabolism
3.
Front Immunol ; 13: 854312, 2022.
Article in English | MEDLINE | ID: mdl-35757763

ABSTRACT

Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15Rß (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.


Subject(s)
Forkhead Box Protein O1 , Forkhead Box Protein O3 , Killer Cells, Natural , Lymphoid Progenitor Cells , Animals , Cell Differentiation/immunology , Forkhead Box Protein O1/immunology , Forkhead Box Protein O3/immunology , Immunity, Innate , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Lymphoid Progenitor Cells/cytology , Lymphoid Progenitor Cells/immunology , Mice , Mice, Inbred C57BL
4.
Cell Rep Med ; 3(2): 100508, 2022 02 15.
Article in English | MEDLINE | ID: mdl-35243416

ABSTRACT

Few approaches have been made toward exploring autologous NK cells in settings of cancer immunotherapy. Here, we demonstrate the feasibility of infusing multiple doses of ex vivo activated and expanded autologous NK cells in patients with multiple myeloma (MM) post-autologous stem cell transplantation. Infused NK cells were detected in circulation up to 4 weeks after the last infusion. Elevations in plasma granzyme B levels were observed following each consecutive NK cell infusion. Moreover, increased granzyme B levels were detected in bone marrow 4 weeks after the last infusion. All measurable patients had objective, detectable responses after NK cell infusions in terms of reduction in M-component and/or minimal residual disease. The present study demonstrates that autologous NK cell-based immunotherapy is feasible in a setting of MM consolidation therapy. It opens up the possibility for usage of autologous NK cells in clinical settings where patients are not readily eligible for allogeneic NK cell-based immunotherapies.


Subject(s)
Hematopoietic Stem Cell Transplantation , Multiple Myeloma , Consolidation Chemotherapy , Granzymes , Humans , Killer Cells, Natural , Multiple Myeloma/therapy , Stem Cell Transplantation , Transplantation, Autologous
5.
Br J Haematol ; 196(1): 224-233, 2022 01.
Article in English | MEDLINE | ID: mdl-34528253

ABSTRACT

Prophylactic platelet (PLT) transfusion is a common practice in severely thrombocytopenic patients that reduces mortality, but responses to platelet transfusions are variable and difficult to predict in individual patients. In this prospective study, we evaluated the outcome of PLT transfusions in 40 patients with haematological malignancies, linking corrected count increment (CCI) to clot formation and agonist-induced platelet activation after transfusion. The CCI was highly variable between patients and 34% showed no response (1-h CCI < 7,5). Short time since the last PLT transfusion and extended storage time of the PLT product were linked to poor transfusion response, while patient sex, C-reactive protein or the number of chemotherapy cycles prior to transfusion did not influence transfusion outcome. High CCI and good PLT responsiveness to agonist stimulation predicted efficient clot formation in rotational thromboelastometry, but transfusion did not restore poor PLT function in patients to the level of healthy controls. Our study provides new insights into factors affecting PLT transfusion outcome in haematology patients with severe thrombocytopenia, and suggests that the thrombocytopenic environment, or disease-associated factors, may hamper platelet responsiveness.


Subject(s)
Blood Coagulation , Platelet Transfusion , Thrombocytopenia/blood , Thrombocytopenia/therapy , Adult , Aged , Aged, 80 and over , Blood Coagulation Tests , Case-Control Studies , Female , Flow Cytometry , Humans , Male , Middle Aged , Platelet Count , Platelet Function Tests , Platelet Transfusion/methods , Thrombelastography , Thrombocytopenia/diagnosis , Thrombocytopenia/etiology , Treatment Outcome
6.
Blood ; 139(5): 704-716, 2022 02 03.
Article in English | MEDLINE | ID: mdl-34699594

ABSTRACT

Neutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.


Subject(s)
Killer Cells, Natural/pathology , Neutropenia/pathology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Hepatitis A Virus Cellular Receptor 2/analysis , Homeostasis , Humans , Infant , Ki-67 Antigen/analysis , Male , Middle Aged , Receptors, Immunologic/analysis , Severity of Illness Index , Young Adult
7.
Hemasphere ; 5(5): e564, 2021 May.
Article in English | MEDLINE | ID: mdl-33912812

ABSTRACT

Ibrutinib is a covalently binding inhibitor of the B-cell receptor signaling-mediator Bruton's tyrosine kinase (BTK) with great efficacy in chronic lymphocytic leukemia (CLL). Common side effects like atrial fibrillation (AF), bleeding and infections might be caused by ibrutinib's inhibition of other kinases in non-B cells. Five-year follow-up of plasma biomarkers by proximity extension assay and immune cell numbers by flow cytometry during ibrutinib treatment revealed that 86 of the 265 investigated plasma biomarkers significantly changed during treatment, 74 of which decreased. Among the 12 markers that increased, 6 are associated with cardiovascular diseases and therefore potentially involved in ibrutinib-induced AF. Comparison between healthy donors and X-linked agammaglobulinemia (XLA) patients, who have nonfunctional BTK and essentially lack B cells, showed indicative changes in 53 of the 265 biomarkers while none differed significantly. Hence, neither B cells nor BTK-dependent pathways in other cells seem to influence the levels of the studied plasma biomarkers in healthy donors. Regarding immune cells, the absolute number of T cells, including subsets, decreased, paralleling the decreasing tumor burden. T helper 1 (Th1) cell numbers dropped strongly, while Th2 cells remained relatively stable, causing Th2-skewing. Thus, long-term ibrutinib treatment has a profound impact on the plasma proteome and immune cells in patients with CLL.

8.
Front Immunol ; 12: 640578, 2021.
Article in English | MEDLINE | ID: mdl-33777033

ABSTRACT

The growing insights in the complex interactions between metastatic cancer-cells and platelets have revealed that platelet tumor cell interactions in the blood stream are an important factor supporting tumor metastasis. An increased coagulability of platelets facilitates the vascular evasion and establishment of solid tumor metastasis. Furthermore, platelets can support an immunosuppressive tumor microenvironment or shield tumor cells directly from engagement of cytotoxic lymphocytes as e.g., natural killer (NK) cells. Platelets are both in the tumor microenvironment and systemically the quantitatively most important source of TGF-ß, which is a key cytokine for immunosuppression in the tumor microenvironment. If similar platelet-tumor interactions are of physiological relevance in hematological malignancies remains less well-studied. This might be important, as T- and NK cell mediated graft vs. leukemia effects (GvL) are well-documented and malignant hematological cells have a high exposure to platelets compared to solid tumors. As NK cell-based immunotherapies gain increasing attention as a therapeutic option for patients suffering from hematological and other malignancies, we review the known interactions between platelets and NK cells in the solid tumor setting and discuss how these could also apply to hematological cancers. We furthermore explore the possible implications for NK cell therapy in patients with solid tumors and patients who depend on frequent platelet transfusions. As platelets have a protective and supportive effect on cancer cells, the impact of platelet transfusion on immunotherapy and the combination of immunotherapy with platelet inhibitors needs to be evaluated.


Subject(s)
Blood Platelets/immunology , Immunologic Surveillance/physiology , Neoplasms/immunology , Tumor Escape/physiology , Animals , Humans , Immunotherapy , Killer Cells, Natural/immunology , Neoplasms/therapy , Tumor Microenvironment/immunology
9.
Transfusion ; 61(4): 1222-1234, 2021 04.
Article in English | MEDLINE | ID: mdl-33580979

ABSTRACT

BACKGROUND: Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. METHODS AND MATERIALS: Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). RESULTS: Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. CONCLUSION: The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.


Subject(s)
Citric Acid/adverse effects , Histocompatibility Antigens Class I/drug effects , Platelet Transfusion/methods , Severe Combined Immunodeficiency/chemically induced , Antibodies/immunology , Blood Grouping and Crossmatching/methods , Blood Platelets/radiation effects , Female , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class I/radiation effects , Histocompatibility Testing/economics , Histocompatibility Testing/methods , Humans , P-Selectin/metabolism , Platelet Transfusion/adverse effects , Plateletpheresis/methods , Tetraspanin 30/metabolism , Thrombelastography/methods , Thrombocytopenia/therapy , Up-Regulation/genetics
10.
Life Sci Alliance ; 4(4)2021 04.
Article in English | MEDLINE | ID: mdl-33593878

ABSTRACT

IL-15 priming of NK cells is a broadly accepted concept, but the dynamics and underlying molecular mechanisms remain poorly understood. We show that as little as 5 min of IL-15 treatment in vitro, followed by removal of excess cytokines, results in a long-lasting, but reversible, augmentation of NK cell responsiveness upon activating receptor cross-linking. In contrast to long-term stimulation, improved NK cell function after short-term IL-15 priming was not associated with enhanced metabolism but was based on the increased steady-state phosphorylation level of signalling molecules downstream of activating receptors. Inhibition of JAK3 eliminated this priming effect, suggesting a cross talk between the IL-15 receptor and ITAM-dependent activating receptors. Increased signalling molecule phosphorylation levels, calcium flux, and IFN-γ secretion lasted for up to 3 h after IL-15 stimulation before returning to baseline. We conclude that IL-15 rapidly and reversibly primes NK cell function by modulating activating receptor signalling. Our findings suggest a mechanism by which NK cell reactivity can potentially be maintained in vivo based on only brief encounters with IL-15 trans-presenting cells.


Subject(s)
Energy Metabolism , Interleukin-15/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Signal Transduction , Animals , Biomarkers , Cytokines/metabolism , Immunophenotyping , Interferon-gamma/metabolism , Interleukin-15/pharmacology , Interleukin-2/metabolism , Interleukin-2/pharmacology , Killer Cells, Natural/drug effects , Lymphocyte Activation , Mice , Phosphorylation , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects
11.
Eur J Immunol ; 50(4): 494-504, 2020 04.
Article in English | MEDLINE | ID: mdl-31834938

ABSTRACT

NK cells are innate immune cells characterized by their ability to spontaneously lyse tumor and virally infected cells. We have recently demonstrated that IL-15-sufficient DC regulate NK cell effector functions in mice. Here, we established that among ITAM-proximal signaling molecules, the expression levels of the scaffold molecule Linker for Activation of T cells (LAT) and its transcription factor ELF-1 were reduced 4 days after in vivo depletion of DC. Addition of IL-15, a cytokine presented by DC to NK cells, regulates LAT expression in NK cells with a significant effect on the DNAM1+ subset compared to DNAM1- cells. We also found that LAT expression is regulated via interaction of the DNAM1 receptor with its ligand CD155 in both immature and mature NK cells, independently of NK cell education. Finally, we found that LAT expression within DNAM1+ NK cells might be responsible for enhanced calcium mobilization following the triggering of activating receptors on NK cells. Altogether, we found that LAT expression is tightly regulated in DNAM1+ NK cells, via interaction(s) with DC, which express CD155 and IL-15, resulting in rapid activation of the DNAM1+ subset during activating receptor triggering.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Interleukin-15/metabolism , Killer Cells, Natural/immunology , Large Neutral Amino Acid-Transporter 1/metabolism , Receptors, Virus/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Antigens, Differentiation, T-Lymphocyte/metabolism , Calcium Signaling , Cells, Cultured , Cytotoxicity, Immunologic , DNA-Binding Proteins/genetics , Interleukin-15/genetics , Large Neutral Amino Acid-Transporter 1/genetics , Lymphocyte Activation , Lymphocyte Depletion , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , Receptors, Virus/genetics , Transcription Factors/genetics , Transcriptional Activation
12.
Transfus Med Rev ; 33(4): 231-235, 2019 10.
Article in English | MEDLINE | ID: mdl-31679761

ABSTRACT

Patients with hematological cancers often have low platelet counts because of progressing bone marrow failure or cytostatic therapy. A large fraction of those patients need platelet transfusions, which can be life-saving if bleedings occur and also allow diagnostic and therapeutic interventions. The outcomes of platelet transfusions are not always easy to predict in terms of bleeding control or increase in platelet count. Reasons could be disease-specific factors, fever, or infections leading to platelet consumption, but the immune system may also be involved, in particular, in patients previously immunized against foreign human leukocyte antigens (HLA). Mechanisms underlying immune-mediated platelet destruction in the presence of antibodies again HLA are not well understood in clinical situations. This review discusses the role of complement in platelet refractoriness, with a focus on HLA antibody-mediated platelet refractoriness. We summarize recent work in this area, discuss complement-platelet interactions in general terms, and a suggest a possible role of complement in platelet transfusion in general.


Subject(s)
Blood Platelets/immunology , Complement System Proteins/immunology , Platelet Transfusion , Thrombocytopenia/therapy , Treatment Failure , HLA Antigens/immunology , Hemorrhage/therapy , Humans , Platelet Count
13.
Front Immunol ; 10: 2249, 2019.
Article in English | MEDLINE | ID: mdl-31616424

ABSTRACT

Mesenchymal stromal cell (MSC) therapy is a promising tool in the treatment of chronic inflammatory diseases. This has been ascribed to the capacity of MSC to release a large variety of immune-modulatory factors. However, all aspects of the mode of therapeutic MSC action in different diseases remain unresolved, mainly because most of the infused MSC are undetectable in the circulation within hours after infusion. The aim of this study was to elucidate the fate of MSC after contact with plasma. We found that upon contact with blood, complement proteins including C3b/iC3b are deposited on MSC. Importantly, we also found that complement bound to MSC enhanced their phagocytosis by classical and intermediate monocytes via a mechanism that involves C3 but not C5. Thus, we describe for the first time a mechanism which might explain, at least partly, why MSC are not found in the blood circulation after infusion. Our results indicate that MSC immune-modulatory effects could be mediated by monocytes that have phagocytosed them.


Subject(s)
Complement System Proteins/immunology , Mesenchymal Stem Cells/immunology , Monocytes/immunology , Phagocytosis/immunology , Complement C3b/immunology , Humans
14.
Transfusion ; 58(11): 2657-2668, 2018 11.
Article in English | MEDLINE | ID: mdl-30281156

ABSTRACT

BACKGROUND: Cryopreserved platelets (CPPs) are considered a promising approach for extended platelet storage, bridging inventory shortages of conventionally stored platelets. It is unknown if platelet concentrates exposed to photochemical treatment (PCT) with amotosalen and ultraviolet A (UVA) light, to inactivate pathogens, are suitable for freezing. The objective of this study was to analyze potential effects of PCT on CPPs as compared with untreated CPPs. STUDY DESIGN AND METHODS: A total of 12 PCT-treated and 12 untreated platelet units from buffy coats were cryopreserved at -80°C in 5% dimethyl sulfoxide. CPPs of both types were rapidly thawed at 37°C and resuspended in 200 mL fresh plasma. In vitro properties were analyzed prefreezing, postfreezing and thawing, and on Day 1 after thawing. RESULTS: Directly after thawing, no major differences in platelet content, lactase hydrogenase, adenosine triphosphate, mitochondrial membrane potential, CD62P, CD42b, and platelet endothelial cell adhesion molecule were seen between PCT-CPPs and conventional CPPs. Agonist-induced PAC-1 expression and contribution of CPPs to blood coagulation in an experimental rotational thromboelastometry setup were also similar between the groups. On Day 1 after thawing, the CPPs of both types performed less well. The PCT-CPPs tended to be more affected by the freezing process than the conventional CPPs. CONCLUSIONS: PCT-CPPs appeared slightly more susceptible to lesion effects by freezing than conventional CPPs, in particular in assays on Day 1 after thawing, but these differences were small relative to the dramatic effects of the freezing process itself.


Subject(s)
Blood Buffy Coat/cytology , Blood Platelets/drug effects , Blood Platelets/radiation effects , Furocoumarins/pharmacology , Ultraviolet Rays , Apoptosis/physiology , Blood Platelets/cytology , Cryopreservation , Humans , Microscopy, Electrochemical, Scanning , Photosensitizing Agents/pharmacology , Platelet Aggregation/drug effects , Platelet Aggregation/radiation effects
15.
Br J Haematol ; 183(2): 212-224, 2018 10.
Article in English | MEDLINE | ID: mdl-30125946

ABSTRACT

In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19+ circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.


Subject(s)
Antineoplastic Agents/pharmacology , Down-Regulation/drug effects , Inflammation Mediators/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Adenine/analogs & derivatives , Aged , Antineoplastic Agents/therapeutic use , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Leukemic/drug effects , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Lymph Nodes/pathology , Lymphocyte Activation/drug effects , Male , Middle Aged , Piperidines , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , RNA, Neoplasm/genetics , Transcription, Genetic/drug effects
16.
Sci Rep ; 6: 37996, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27905484

ABSTRACT

During infection and inflammation, dendritic cells (DC) provide priming signals for natural killer (NK) cells via mechanisms distinct from their antigen processing and presentation functions. The influence of DC on resting NK cells, i.e. at steady-state, is less well studied. We here demonstrate that as early as 1 day after DC depletion, NK cells in naïve mice downregulated the NKG2D receptor and showed decreased constitutive phosphorylation of AKT and mTOR. Subsequently, apoptotic NK cells appeared in the spleen concomitant with reduced NK cell numbers. At 4 days after the onset of DC depletion, increased NK cell proliferation was seen in the spleen resulting in an accumulation of Ly49 receptor-negative NK cells. In parallel, NK cell responsiveness to ITAM-mediated triggering and cytokine stimulation dropped across maturation stages, suggestive of a functional deficiency independent from the homeostatic effect. A role for IL-15 in maintaining NK cell function was supported by a gene signature analysis of NK cell from DC-depleted mice as well as by in vivo DC transfer experiments. We propose that DC, by means of IL-15 transpresentation, are required to maintain not only homeostasis, but also function, at steady-state. These processes appear to be regulated independently from each other.


Subject(s)
CD11c Antigen/metabolism , Dendritic Cells/immunology , Killer Cells, Natural/immunology , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Animals , Cell Line , Cell Proliferation , Dendritic Cells/cytology , Homeostasis , Humans , Killer Cells, Natural/cytology , Lymphocyte Activation , Mice , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism
17.
J Immunol Methods ; 434: 9-15, 2016 07.
Article in English | MEDLINE | ID: mdl-27059653

ABSTRACT

We have developed a modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) with enhanced sensitivity in detecting antibodies against human platelet antigens (HPA), using biotinylated monoclonal antibodies, streptavidin-coated beads and detection by flow cytometry. The beads-MAIPA gave superior signal-to-noise resolution (>10-fold higher) for detection of anti-HPA-1a and anti-HPA-5b compared with the in-house standard MAIPA. Also, efficient and reproducible detection of anti-HPA-15 (CD109) was shown. The enhanced sensitivity was confirmed using WHO International Reference Reagents for anti-HPA-1a, anti-HPA-3a and anti-HPA-5b, which allowed comparison of detection endpoints with other laboratories. Finally, the beads-MAIPA was validated for quantification of anti-HPA-1a. The lower limit of quantification was 0.4IU/mL for beads-MAIPA, compared to 1IU/mL previously reported for standard MAIPA. Based on improved performance against all HPA-antibodies tested, the beads-MAIPA has replaced the standard MAIPA in our laboratory in diagnostics of conditions due to HPA-immunization, such as fetal and neonatal alloimmune thrombocytopenia (FNAIT).


Subject(s)
Antigens, Human Platelet/blood , Immunoassay/methods , Antibodies, Monoclonal/chemistry , Antigens, Human Platelet/classification , Biotinylation , Blood Platelets/chemistry , Flow Cytometry , Humans , Linear Models , Microspheres , Sensitivity and Specificity , Streptavidin/chemistry , Thrombocytopenia, Neonatal Alloimmune/diagnosis
18.
Transfusion ; 56(2): 370-82; quiz 369, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26442787

ABSTRACT

BACKGROUND: The presence of antibodies against HLA Class I can lead to platelet (PLT) transfusion refractoriness, that is, the repeated failure to achieve adequate posttransfusion PLT count increments. PLT refractoriness can be overcome by transfusion of HLA-matched donor PLTs. A different approach is to remove HLA from the PLT surface using low pH. Previous case studies using HLA-stripped PLTs showed encouraging but inconsistent results and lacked information on the biologic effects of acid treatment on PLT function as well as sensitivity to PLT destruction in the presence of HLA antibodies. STUDY DESIGN AND METHODS: PLTs prepared from buffy coats were stripped from HLA Class I using a brief incubation at pH 2.9. Kinetics of acid stripping, viability, phenotypic alterations, and sensitivity to complement-mediated lysis and phagocytosis were determined by flow cytometry. Functional potential was evaluated using a multiplate analyzer. RESULTS: Acid-treated PLTs were viable, upregulated activation markers normally and aggregated to a similar extent as untreated PLTs in response to stimulation with three natural agonists. Acid treatment removed 70% to 90% of HLA Class I complexes from the PLT surface, which led to complete protection from HLA antibody-mediated complement lysis and reduced monocyte-mediated phagocytosis in the presence of anti-HLA in vitro. CONCLUSION: Our study fills an important knowledge gap in how acid treatment affects PLT function and interactions with immune cells, paving the way for controlled clinical trials to evaluate acid-treated PLTs as an alternative to HLA-matched donors in PLT refractoriness.


Subject(s)
Blood Platelets/chemistry , Citric Acid/chemistry , Complement System Proteins/chemistry , HLA Antigens/chemistry , Phagocytes , Platelet Aggregation , Blood Platelets/cytology , Female , Humans , Hydrogen-Ion Concentration , Male
19.
Eur J Immunol ; 45(7): 2134-42, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25824372

ABSTRACT

Carcinoembryonicantigen-related cell adhesion molecule 1 (CEACAM1) is a receptor involved in the regulation of NK-cell function. In most species, the CEACAM1 cytoplasmic tail possesses a membrane-proximal ITIM paired with a membrane-distal immunoreceptor tyrosine-based switch motif (ITSM) signaling motif. Human CEACAM1 has phylogenetically relatively recently acquired a second ITIM instead of the ITSM and was shown to inhibit NKG2D-mediated NK-cell activation. Here, we compare the function of bovine and human CEACAM1. We show that in addition to NKG2D, human CEACAM1 can inhibit NK-cell activation via NKp30 or 2B4. Bovine CEACAM1, possessing an ITIM and an ITSM signaling motif, is also inhibitory. However, bovine CEACAM1 inhibition of NKp30-mediated lysis is less pronounced compared with its human counterpart. Bovine CEACAM1 inhibition is dependent on the membrane-proximal ITIM and our data suggest that also the membrane distal ITSM motif contributes to inhibitory signaling. Biochemically, human and bovine CEACAM1 can recruit the phosphatases SHP-1 and SHP-2 after receptor phosphorylation to a similar extend. Bovine CEACAM1 can additionally recruit the adapter molecule Ewing's sarcoma virus-activated transcript-2 (EAT-2), but not SLAM-associated protein (SAP). Taken together, we show that although human and bovine CEACAM1 are differentially equipped with ITIM and ITSM motifs, both receptors can inhibit NKp30 and 2B4 activation of NK cells.


Subject(s)
Antigens, CD/immunology , Cell Adhesion Molecules/immunology , Evolution, Molecular , Killer Cells, Natural/immunology , Lymphocyte Activation/immunology , Natural Cytotoxicity Triggering Receptor 3/immunology , Receptors, Immunologic/immunology , Animals , Blotting, Western , Cattle , Cell Line , Humans , Immunoprecipitation , Signaling Lymphocytic Activation Molecule Family , Transfection
20.
Transfusion ; 54(3): 602-12, 2014 Mar.
Article in English | MEDLINE | ID: mdl-23889505

ABSTRACT

BACKGROUND: In connection with platelet (PLT) production, random but transient aggregates sometimes form in the newly produced units. The underlying mechanisms as well as the impact on cellular level of this phenomenon are unknown. Hypothetically, random occurrence of aggregates may induce biochemical changes leading to PLT activation and release of immunomodulatory factors from the PLTs. STUDY DESIGN AND METHODS: PLTs were aliquoted and prepared with an automated system for PLT pooling (OrbiSac, Terumo BCT) for a three-arm nonpaired study design (n=8). Initially aggregated PLT units in SSP+ were selected by visual inspection and compared to unaffected PLT units stored in SSP+ or 100% plasma. Cellular, metabolic, and functional variables were analyzed, including the concentrations of RANTES, sCD40L, and sTWEAK in the bags during a 9-day storage period. RESULTS: Isolated aggregated PLTs show signs of spontaneous activation and respond less efficiently to TRAP stimulation. RANTES, sCD40L, and sTWEAK accumulated in the various PLT units during storage but sCD40L and RANTES accumulated in the initially aggregated PLT units to higher concentrations than the reference units and PLTs stored in 100% plasma (p<0.001). Over time, the levels of sTWEAK increased more in the plasma storage environment compared with PLT units stored in SSP+ (p<0.001). CONCLUSION: Our data indicate that random occurrence of aggregates may lead to higher activation level and increased release of immunomodulatory factors from the PLTs.


Subject(s)
Blood Platelets/metabolism , CD40 Ligand/metabolism , Chemokine CCL5/metabolism , Platelet Activation/physiology , Humans , Immunomodulation/physiology
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