A Case of Hypersensitivity to Mosquito Bites with Type III Latency of EB Virus Infection in NK Cells with Prolonged Survival Due to Induction of Specific CD8+ Cytotoxic T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1263-1263
Author(s):  
Shigeru Tsuchiya ◽  
Wei Du ◽  
Hideaki Kikuta ◽  
Toru Uchiyama ◽  
Masaei Onuma ◽  
...  
Keyword(s):  
Nk Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Type Iii ◽  
Ebv Infection ◽  
Eb Virus ◽  
Promoter Usage ◽  
Methylation Patterns

Abstract Hypersensitivity to mosquito bites (HMB) is a rare disease classified into chronic active EB Virus (EBV) infection (CAEBV), characterized by clonal expansion of EBV-infected NK cells. The immunological background of the disease has not been elucidated yet. In this report we extensively studied EBV infected NK cells of a patient with HMB in view of several aspects, including latency, usage of promoter Cp/Wp and Qp of EBNAs, and induction of CD8+ cytotoxic T lymphocytes (CTL) to peptide antigens in order to know the relation between latency of EBV infected NK cells and induction of CTL. Case report: A female patient aged 19 complained of skin symptoms after a mosquito bite. A blister developed at the bite site that subsequently transformed into ulcer. The patient also had general fever and liver dysfunction. Past medical history showed that she suffered from HMB when she was 7 years old. The titers of serum antibodies to EBV were as follows: viral capsid antigen (VCA) IgG, 1280X; IgA, 80X; IgM, 10X; EBNA, 20X; EA DR IgG, 160X and IgA, 10X. These data strongly indicated lytic type of EBV infection. Subset analysis of peripheral blood lymphocytes revealed CD3+ lymphocytes, 50.2%; CD4+, 34.2%; CD8+, 12.7%; CD19, 10.0% and CD56/16+, 22.6%. Real time polymerase chain analysis of her peripheral mononuclear cells showed markedly increased copy number of EBV genome (5 x 10^5 copies/μgDNA). Purification of each lymphocyte fraction by immunomagnetic beads and subsequent PCR analysis showed that EBV infected lymphocytes were NK cells. Latency and Cp and Qp of EBNA promoter usage analyses: In order to determine the latency of EBV infected NK cells RT-PCR was carried out. EBNA1, EBNA2, LMP1, LMP2A, and EBER1 were positive and BZLF was negative. These results indicated that EBV infection in NK cells is associated with type III latency. RT-PCR analyses confirmed that both promoters Cp/Wp and Qp were involved in this disease. Through bisulfite PCR analysis, eighty four % of Cp was found to be methylated, on the other hand, only 6% of Qp was methylated. These methylation patterns were similar to those of hemophagocytic syndrome with latency III, also known in a few patients with CAEBV. Detection and expansion of EBV specific CD8+ CTL: We used HLA-A24 restricted BRLF1, EBNA3A, EBNA3B and LMP2 tetramers for quantification of specific CTL and we found 0.48% of BRLF1 and 0.15% of EBNA3 peptide-specific CTL among CD8+ cells. These cells could be expanded to 6.6% for BRLF1 and 0.22% for EBNA3A peptide-specific CTL after stimulation with an autologous lymphoblastoid cell line (LCL) for 10 days and then with LCL and IL-2 for 7 days. Conclusion: In this study we reported a long-term survivor of HMB with EBV infected NK cell proliferation. Promoter usage of EBNA genes and their methylation patterns might be important to determine latency and host immune responses against EBV infected cells.

CD137 Expression Is Enhanced in EBV-Infected T or NK Cells by Viral Protein LMP1 and Mediates Anti-Apoptotic Intracellular Signaling through NF-ĸB Activation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1929-1929
Author(s):  
Ayako Arai ◽  
Ken-ichi Imadome ◽  
Mayumi Takahashi ◽  
Koichi Naka ◽  
Tetsuya Fukuda ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Intracellular Signaling ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Jurkat Cells ◽  
Rt Pcr ◽  
Infected Cells ◽  
Ebv Dna

Abstract Abstract 1929 Poster Board I-952 Epstein-Barr virus (EBV) can infect not only B cells but also T or NK cells uncommonly and causes lymphoid malignancies, such as extranodal NK/T-cell lymphoma nasal type (ENKL), aggressive NK-cell leukemia, and EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), which is also known as chronic active EBV infection. However, why and how EBV infects T or NK cells and the mechanism of action responsible for these EBV-induced malignancies have not been elucidated to date. To clarify the molecular mechanism underlying development of EBV-T/NK-LPD, we focused on costimulatory receptor CD137, which is expressed on the surface of activated T cells and plays a pivotal role in their proliferation, survival, and differentiation. We investigated CD137 expression on the surface of EBV-infected T/NK cells (EB-T/NK cells) by flow cytometry. First, three EBV-positive T and NK cell lines, SNT8, SNK6, and SNT16, were obtained for examination. These cell lines had been established from primary lesions of ENKL patients (SNT8 and SNK6) and peripheral blood of an EBV-T/NK-LPD patient (SNT16). CD137 expression was confirmed on the cell surface of these cells, whereas the EBV-negative T and NK cell line, Jurkat and KHYG1 cells, respectively, were negative for CD137. Next, we investigated expression on the surface of EB-T/NK cells derived from EBV-T/NK-LPD patients. EBV-T/NK-LPD was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in the peripheral blood (PB), and detection of EBV-infected T or NK cells. To detect the infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. Next, we measured the EBV-DNA titer of each fraction by quantitative RT-PCR. Nine patients (aged 8–41 years; 4 male, 5 female; 4 T and 5 NK cell types) were diagnosed with EBV-T/NK-LPD. Then, we examined surface CD137 expression of the infected cells of each patient. Expression was detected in 7 of 9 patients. Control cells (PB mononuclear cells of a healthy donor, who was negative for EBV-DNA titer in the PB) did not express the molecule. We also examined transcription of CD137 mRNA by RT-PCR assay and detected it in all the 12 EB-T/NK-cell samples described above. From these results we concluded that CD137 expression was induced at the level of both mRNA and protein in EB-T/NK cells. To investigate the molecular mechanism of CD137 overexpression in EBV-T/NK cells, we examined the influence of viral proteins on CD137 expression. EB-T/NK cells express EBV-encoded proteins, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We cotransfected expression plasmids for these proteins with a luciferase reporter plasmid containing the CD137 gene promoter in Jurkat cells and performed a luciferase assay. LMP1 significantly upregulated the CD137 promoter activity, although the other molecules did not. Furthermore, in a transient expression assay of these viral proteins using Jurkat cells, transcription of endogenous mRNA of CD137 was upregulated only in the LMP1 transfectant. These results indicate that LMP1 may transactivate CD137 transcription and expression in EBV-T/NK cells. Next, we investigated the role of CD137 in developing EBV-T/NK-LPD. We cultured the above-mentioned CD137-expressing EBV-T/NK cells on CHO cells that stably express human CD137L on the cell surface. NF-ĸB activation was detected in CD137-positive EBV-T/NK cells that were cocultured with CD137L-expressing CHO cells. We confirmed that both p50 and p52 translocated to the nucleus, indicating that both canonical and non-canonical pathways for NF-ĸB activation were activated downstream of CD137. Finally, we investigated the role of CD137-mediated NF-ĸB activation in the development of EBV-T/NK-LPD. We cocultured EB-T/NK cells on CHO-wt or CHO-CD137L with VP-16 for 48 h and determined apoptosis by measuring DiCO6 uptake. We noted that stimulation of CD137 significantly suppressed VP-16-induced apoptosis of these cells. Together, these results indicate that EBV-infected T/NK-cells express CD137 on the cell surface, which may be induced by LMP1 and activate the anti-apoptotic intracellular signaling pathway through NF-ĸB activation. This pathway may contribute to immortalization of the infected cells and development of EBV-T/NK-LPD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 577
Author(s):  
Adrián Fernández ◽  
Alfonso Navarro-Zapata ◽  
Adela Escudero ◽  
Nerea Matamala ◽  
Beatriz Ruz-Caracuel ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Fold Increase ◽  
Clinical Grade ◽  
Transcriptome Profile ◽  
Expansion Process ◽  
Peripheral Blood Mononuclear ◽  
Activation Status

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation–expansion process and its validation on clinical-scale. Methods: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Conclusions: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

scholarly journals Generation of human natural killer cells from immature progenitors does not require marrow stromal cells

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 841-846 ◽  
Author(s):  
MR Silva ◽  
R Hoffman ◽  
EF Srour ◽  
JL Ascensao
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Mononuclear Cells ◽  
Viral Infections ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Peripheral Blood Mononuclear ◽  
Specific Lysis ◽  
Human Natural Killer Cells

Abstract Human natural killer (NK) cells comprise 10% to 15% of peripheral blood mononuclear cells and have an important role in immune responses against tumors, viral infections, and graft rejection. NK cells originate in bone marrow (BM), but their progenitors and lineage development have not been completely characterized. We studied the generation of NK cells from purified CD34+HLADR- and CD34+HLADR+ BM progenitors and the influence of various cytokines on their production. We show that CD3-CD56+ cytotoxic NK cells can develop from both progenitors populations when interleukin-2 (IL-2) is present in an in vitro suspension culture system containing IL-1 alpha and stem cell factor. Up to 83.8% and 98.6% CD3-CD56+ cells were detected in CD34+HLADR- and CD34+DR+ cultures, respectively, after 5 weeks of culture; significant numbers of NK cells were first detected after 2 weeks. Cytotoxic activity paralleled NK cell numbers; up to 70% specific lysis at an effector:target ratio of 10:1 was observed at 5 weeks. IL-7 also triggered development of CD3-CD56+ cells from these immature progenitors (up to 24% and 55% appeared in CD34+HLADR- and CD34+HLADR+ cultures, respectively). Our data suggest that BM stromas are not necessary for NK cell development and that IL-2 remains essential for this lineage development and differentiation.

scholarly journals Aberrant anti-viral response of natural killer cells in severe asthma

2020 ◽  
Vol 55 (5) ◽  
pp. 1802422
Author(s):  
Justine Devulder ◽  
Cécile Chenivesse ◽  
Valérie Ledroit ◽  
Stéphanie Fry ◽  
Pierre-Emmanuel Lobert ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Severe Asthma ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Asthma Patients ◽  
Ifn Γ

Rhinovirus infections are the main cause of asthma exacerbations. As natural killer (NK) cells are important actors of the antiviral innate response, we aimed at evaluating the functions of NK cells from severe asthma patients in response to rhinovirus-like molecules or rhinoviruses.Peripheral blood mononuclear cells from patients with severe asthma and healthy donors were stimulated with pathogen-like molecules or with the rhinoviruses (RV)-A9 and RV-2. NK cell activation, degranulation and interferon (IFN)-γ expression were analysed.NK cells from severe asthma patients were less cytotoxic than those from healthy donors in response to toll-like receptor (TLR)3, TLR7/8 or RV-A9 but not in response to RV-2 stimulation. Furthermore, when cultured with interleukin (IL)-12+IL-15, cytokines which are produced during viral infections, NK cells from patients with severe asthma were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of the checkpoint molecule Tim-3.Together, our findings indicate that the activation of NK cells from patients with severe asthma may be insufficient during some but not all respiratory infections. The exhausted phenotype may participate in NK cell impairment and aggravation of viral-induced asthma exacerbation in these patients.

Reciprocal Interaction of Monocytes and NK Cells by Activation-Induced Expression of Ligands for the Activating Immunoreceptor NKG2D.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2212-2212
Author(s):  
Matthias Krusch ◽  
Mercedes Kloss ◽  
Andrea Peterfi ◽  
Ingrid Kumbier ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Adaptive Immune System ◽  
Surface Expression ◽  
Pcr Analysis ◽  
High Plasticity ◽  
Blood Monocytes ◽  
Cell Functions ◽  
Adaptive Immune ◽  
Mrna Induction

Abstract Reciprocal Interactions of NK cells with Dendritic Cells (DC) can induce activation of NK cells, maturation or lysis of DC and influence subsequent adaptive immune responses. However, little is known about the interaction of peripheral blood monocytes with NK cells, especially regarding involved immunoregulatory surface molecules. Here we report that monocytes express ligands for the activating immunoreceptor NKG2D expressed on NK cells upon treatment with various stimuli. Incubation of monocytes with TNF, GM-CSF, IFN-g and various TLR ligands (LPS, Pam3Cys, R848, PolyI:C) induced surface expression of the NKG2D ligands (NKG2DL) MICA and to a lesser extent MICB, but no relevant changes of ULBP molecules, as determined by FACS analysis. Expression was confirmed by quantitative PCR analysis of NKG2DL mRNA induction. To elucidate the functional consequences of NKG2DL expression on monocytes for NK cell functions we performed coculture assays of monocytes and autologous NK cells. NKG2DL expression on stimulated monocytes lead to a significant induction of IFN-g release into the culture supernatant by NK cells as determined by ELISA. This IFN-g release was blocked by addition of a NKG2D-Ig fusionprotein, but not by isotype control demonstrating that the induction of NK cell IFN-g production was in fact specifically due to NKG2DL expression on monocytes. Since both monocytes and NK cells rapidly migrate to sites of inflammation, and monocytes display a high plasticity regarding their function and maturation which is influenced by IFN-g, our data indicate that NKG2DL expression on monocytes might not only mediate reciprocal activation of NK cells and monocytes but also might influence other components of the innate and adaptive immune system and thereby determine the course of subsequent immune reactions.

CD69 Is Required for Activated NK Cell-Mediated Killing of Resistant Targets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3322-3322 ◽  
Author(s):  
Chloe M. Marden ◽  
Janet North ◽  
Robert Anderson ◽  
Ismail A. Bakhsh ◽  
Elena Addison ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Nk Cells ◽  
Myeloid Leukaemia ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Autologous Bone Marrow Transplantation ◽  
Target Cells ◽  
Monomeric Form ◽  
Normal Peripheral Blood ◽  
Raji Cells

Abstract The C-type lectin receptor CD69 is expressed on a range of haematopoietic cells following activation. On natural killer (NK) cells CD69 may play a direct role in mediating cytotoxicity of tumour targets. We have previously shown that remission following chemotherapy or autologous bone marrow transplantation (BMT) for acute myeloid leukaemia (AML) is dependent on NK cell cytotoxicity and have observed CD69 capping the immune synapse between autologous NK cells and conjugated AML cells (Lowdell et al, Br J Haematol, 2002; 117(4):821–7). Tumour cells which are resistant to NK-mediated lysis are often susceptible to lysis by activated NK cells (e.g. after stimulation with IL-2); thus we hypothesized that the interaction between CD69 on activated NK cells and its unidentified ligand (CD69L) on target cells is required for target cell lysis. Here we use soluble recombinant CD69 (rCD69) to investigate the role of CD69-CD69L interaction in mediating activated-NK cytotoxicity of NK-resistant and NK-sensitive target cells. The extracellular domain of CD69 (amino acids 65–199) fused with an N-terminal biotinylation sequence (Avidity) was expressed in Escherichia coli and a multivalent rCD69 reagent was created by binding biotinylated CD69 protein to avidin coated fluorescent beads (Spherotech Inc). Binding of rCD69 to NK-resistant Raji and Daudi Burkitt’s lymphoma cell line targets was determined by flow cytometry (11.9%, 12.4% positive respectively) and confocal microscopy; rCD69 did not bind to 293 kidney epithelial cells, K562 chronic myeloid leukaemia (an NK-sensitive target) or normal peripheral blood mononuclear cells. rCD69 was used to block the interaction between activated-NK cells and target cells; pre-incubation of Raji target cells with rCD69 reduced specific cytotoxicity to the level of unactivated NK cells (31.2 +/−1.6% to 8.0 +/−0.7%, Figure 1). Furthermore, activation of the intracellular tyrosine kinase Syk, which is selectively phosphorylated following CD69 signalling on activated NK cells (Pisegna et al, JI, 2002; 169: 68–74), was abrogated by rCD69 pre-incubation as determined by confocal microscopy. These data show that rCD69 binds NK-resistant target cells and blocks the killing of these cells by activated NK cells. We conclude that CD69 is required for activated NK-cell-mediated killing of resistant targets and that CD69L may be a tumour-restricted marker. Screening of primary tumours for CD69L is ongoing. Figure 1. rCD69 fractions block lysis of Raji cells by activated NK cells. Killing of Raji target cells by activated NK cells (aNK) is reduced to that of unactivated NK cells by pre-incubating Raji with HPLC purified fractions of rCD69. F3 contains rCD69 in dimeric and monomeric form, F2 in monomeric form only. Figure 1. rCD69 fractions block lysis of Raji cells by activated NK cells. Killing of Raji target cells by activated NK cells (aNK) is reduced to that of unactivated NK cells by pre-incubating Raji with HPLC purified fractions of rCD69. F3 contains rCD69 in dimeric and monomeric form, F2 in monomeric form only.

NK-Cell Reconstitution after HLA-Identical Blood and Marrow Transplantations for CML: The Recovery of NKG2D Is Inversely Correlated with NKG2A and It Promotes the GVL Effect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4879-4879
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Liangquan Geng ◽  
Zimin Sun ◽  
Baolin Tang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Linear Regression Analysis ◽  
Cell Activity ◽  
Target Cells ◽  
Peripheral Blood Stem Cells ◽  
Peripheral Blood Mononuclear ◽  
Blood Stem Cells

Abstract Natural killer (NK) cell alloreactivity is reported to mediate strong graft versus leukemia (GVL) effect in patients after allogeneic stem-cell transplantation. NKG2D receptors recognize human MHC class Ichain related A and B (MICA/B) and UL16-binding protein 1∼4(ULBP 1∼4) on target cells, thereby regulating NK cell activity. To examine the recovery of NKG2D, NKG2A and other receptors expression by NK cells, we used flow cytometry to evaluate samples from 11 chronic myeloid leukemia patients and their donors in the year following unmanipulated HLA completely matched peripheral blood stem cells plus bone marrow transplantation. Peripheral blood mononuclear cells from patients and their donors were tested in standard 51Cr release assays against cultured K562 targets to determine the cytotoxicity of the NK cells in the same intervals. There is no mismatched immunoglobulin-like receptor (KIR) ligand in both GVH and HVG direction. The reconstitution of KIR2DL1 (CD158a) after this transplantation protocol was very slow and these receptors didn’t reach normal value in the year and KIR2DL2 (CD158b) was much better. The NKG2D increased and the NKG2A decreased quickly at the same time after engraftment, and used linear regression analysis we demonstrated that NKG2A recovery was inversely correlated with NKG2D recovery in the year following transplantation. The ratio of NKG2D/NKG2A was directly associated with the capacity of NK-cell cytotoxicity. Thus, the reconstitution of NKG2D makes contribution to the recovery of the NK cytotoxicity. These results reveals that the NK cells generated after HLA matched blood plus bone morrow transplantation of CML patients are promoted at an immature state characterized by specific phenotypic features and enhanced functioning, having potential impact for immune responsiveness and transplantation outcome.

Chronic NK Cell Leukemia with Haemophagocytic syndrome: Case Report and Literature Review

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4970-4970
Author(s):  
Hua Lu ◽  
Wenyi Shen ◽  
Jianfu Zhang ◽  
Yujie Wu ◽  
Jianyong Li
Keyword(s):  
Renal Failure ◽  
Nk Cells ◽  
Nk Cell ◽  
Lower Limbs ◽  
High Density Lipoproteins ◽  
Cell Leukemia ◽  
Blood Calcium ◽  
Haemophagocytic Syndrome ◽  
Ebv Infection ◽  
Fcm Analysis

Abstract Objective To study the bionomics of chronic NK-cell leukemia (CNKL), comprehend this disease deeply and identify it’s clinical diagnosis and therapy correctly. Methods The clinical features, laboratory examinations, treatment and prognosis of a very rare case of CNKL were reported, and the related literature was reviewed. Results The CNKL patient was diagnosed by a persistent high level of lymphocytes in the peripheral blood for 7 years. Flow cytometry (FCM) analysis showed the NK cells had a proportion of about 25%, which were positive for CD2,CD7,CD16,CD56. Chromosome analysis displayed a result of 47,XY,+5. While EB virus detection, TCR-γand IgH gene rearrangement analysis through polymerase chain reaction (PCR) were all negative. The blood smear showed a typical morphology of large granular lymphocytes. The total-body CT scan didn’t show any lymphadenopathy or splenohepatomegaly when the diagnosis was given. The patient got an acute renal failure in February 2007. After the possibility of splenohepatomegaly caused by hepatitis virus infection was excluded, liver and spleen infiltration of NK-cells became the most possible reason and this can also explain why the man got a renal failure in such a short time. When the therapy of liver conservation, diuresis, anticoagulation, hematodialysis, and oral use of prednisolone (60 mg/d) were given, the patient got renal function gradually recoverd and puffiness disappeared. In August 2007, the patient was admitted again because of fever (body temperature waved from 38°C to 39°C), palmus, manifested night sweat and a weight loss of 7 Kg in the past six months. He appeared depressed, and was bloodless, there was hepatosplenomegaly and lymphadenopathy on right cervical part, gentle puffiness of the lower limbs. Laboratory investigations showed a pancytopenia, alanine aminotransferase was 72.2 U/L, aspartate aminotransferase was 120U/l, triglyceride was 2.68 mmol/L,high-density lipoproteins was 0.22 mmol/L, low density lipoprotein was 0.56 mmol/L, total protein was 59.3 g/L, albumn was 24.1 g/L, total bilirubin was 25.3 mmol/L, direct bilirubin was 11.7 mmol/L, blood urea nitrogen was 13.6 mmol/L, b2-microglobulin was 10.7 mg/L, serum ferritin was 10800 mg/L, serum potassium was 2.92 mmol/L, blood calcium was 1.91 mmol/L, APTT was 63 second, PT was14.4 second,Fib was 1.64 g/L,D-Dimer was 3.89 ng/mL. The bone marrow slides examination showed an active proliferation of marrow with a high proportion of abnormal lymphocyte with more granules in the endochylema and hemophagocytosis. The neutrophil alkaline phosphatase was strongly positive. The FCM analysis of marrow shown that the lymphocyte cells had a proportion of about 86%, which were positive for CD2,CD7,CD16,CD56, and negative for CD3,CD5,CD19. The blood film examination showed that the total white blood cells were decreased, the proportion of lymphocyte was increased. Haemophagocytic syndrome (HPS) was established based on all of these. The therapy of etoposide, dexamethasone and ciclosporin A were given to the patient,but he was died of an abrupt onset serious hemoptysis. Conclusion CNKL was a very rare disease which can display as a chronic course, some precipitating factor like the EBV infection can make it progress and aggravate quickly. In the case we reported, the patient had a chronic and indolent course of nearly six years with typical presentation of CNKL,but in final stage of disease he got a progress from CNKL to ANKL based on EBV infection and eventually proceeded to HPS. Since similar cases haven’t been seen, more cases were needed to confirm that ANKL could be a turnover of CNKL.

In Vivo Modulation of T Cell and Monocyte Function Following Infusion of Mesenchymal Stromal Cells (MSC)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4739-4739
Author(s):  
Cristina Castilla-LLorente ◽  
Mineo Iwata ◽  
Marco Mielcarek ◽  
V. Kraig Abrams ◽  
Billanna Hwang ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Lymph Nodes ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Blood Mononuclear Cells ◽  
Post Infusion

Abstract Mesenchymal stromal cells (MSCs) expanded ex vivo from aspirated marrow, have been used clinically with variable success to facilitate repair of infarcted hearts, treat graft versus host disease, and facilitate marrow reconstitution after radiation damage. While it is now generally acknowledged that these benefits are not the result of engraftment and differentiation of MSC into the target tissues, the mechanism by which these beneficial effects are achieved is not clear. We hypothesize that MSCs mediate their effect by activating an endogenous cell population which in turn modulates the immune response and/or homes to damaged tissue and participates in repair. To begin to test this hypothesis immortalized and cloned populations of canine MSC were generated to provide a consistent product for in vivo testing. One line, designated DS-1, has been evaluated in vivo by infusion into two normal dogs. Blood samples were taken pre infusion, immediately following infusion and at 1, 6, 24, 48, 72, 96 hours, and 7, 14, 21, and 28 days post infusion. Following infusion there was no consistent change in the number of WBC, however by day 3 there was a marked decrease in the % of CD3+ cells expressing FOXP3 and TGFβ in the blood, which did not recover to pre-infusion levels during the period of observation. At autopsy there was an increased number of these cells in the lymph nodes and spleen, whereas there was an overall decrease in the number of TH1 cells in these tissues. Quantitative RT- PCR analysis of cDNA prepared from blood mononuclear cells indicated an upregulation in the expression of CD133, Tie-2, and MARCO between 1–24 hours post infusion, and an increase in LOX1/OLR1 between 2–4 days. However the % of monocytes and the expression levels of CD14, CD68, CD45, and CD105/Endoglin were constant at all time points. Samples taken at 6 hours, 4 and 7 days post infusion were also analyzed for the presence of DS-1 cells by PCR and in vitro out growth assays. Results indicated that the DS-1cells were detectable up to 6 hours post infusion, but not thereafter. Adherent cells grown from blood mononuclear cells at days 4 and 7, displayed macrophage and endothelial cell morphologies. RT-PCR analysis of these cultures detected expression of macrophage associated markers CD14+/CD68+/MARCO+/LOX1+, as well as endothelial cell associated markers CD34+/CD144/VECAD+. These data indicate that a single infusion of DS-1 cells results in activation of circulating monocytes and a shift of regulatory T cells from the periphery to lymph nodes and spleen which persists for at least 28 days. We speculate that these changes may contribute to the immunomodulatory effects reported for some preparations of MSC.

Synergistic Action of the Human Inhibitory KIR Antibody IPH2102, and the Human CD38 Antibody Daratumumab to Enhance the Lysis of Primary Multiple Myeloma (MM) Cells in the Bone Marrow Mononuclear Cells (MNCs) From Myeloma Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1865-1865
Author(s):  
Inger S. Nijhof ◽  
Michel de Weers ◽  
Pascale Andre ◽  
Berris van Kessel ◽  
Henk M. Lokhorst ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Human Monoclonal Antibody ◽  
Cell Activity

Abstract Abstract 1865 Despite significant improvements in the treatment of multiple myeloma (MM), this progressive malignancy of antibody-producing clonal plasma cells is still considered incurable. New innovative treatments need to be developed to improve long term outcomes. Recent successes of CD20 antibodies in the clinical lymphoma management indicate that targeted immunotherapy can represent a powerful therapeutical strategy for hematological malignancies. Towards developing a similar strategy for MM, we have recently generated a novel human monoclonal antibody, daratumumab (DARA), which targets the CD38 molecule expressed at high levels on MM cells. We have demonstrated that DARA mediates the lysis of CD38+ MM cells via direct apoptosis, complement mediated lysis and antibody-dependent cell mediated cytotoxicity (ADCC). Natural killer (NK) cells appeared important effector cells mediating the ADCC effect. Since NK cell activity against tumor cells is regulated by the balance of signals generated by inhibitory or activating receptors of NK cells (KIRs), we now explored whether blocking the inhibitory KIRs would improve the NK cell mediated DARA dependent lysis of MM cells. Thus, we evaluated the potential benefits of combining DARA with a novel human anti KIR monoclonal antibody, IPH2102, which blocks the inhibitory KIR2DL1/2/3 receptors (HLA-C specific KIRs), and has been shown to augment NK cell function against MM cells. We recently developed FACS-based ex vivo MM cell lysis assays, in which DARA-dependent NK cell-mediated lysis of MM cells can be directly measured in bone marrow MNCs, thus without separating the malignant cells from autologous NK cells and other accessory cells. Using these, we investigated whether the addition of IPH2102 would augment the DARA dependent lysis of MM cells. As expected, DARA induced lysis of MM cells in bone marrow MNCs isolated from MM patients (n=10). Mean lysis at 10 μg/ml DARA was 27.6% (range 11.3–48.1%). IPH2102 showed little or no lysis of MM cells (at 0.3, 1, 3 and 10 μg/ml) in this setting. The combination of 10 μg/ml IPH2102 with 3 and 10 μg/ml DARA significantly enhanced cytotoxicity against primary MM tumor cells compared to DARA alone (p=0.013 and p=0.028 respectively). Mean lysis of MM tumor cells at 10 μg/ml DARA and 10 μg/ml IPH2102 was 38%. These data confirm our previous findings that NK-cell mediated killing is an important mechanism of action of DARA. We demonstrate a clear synergy between DARA and IPH2102 to achieve effective lysis of MM cells directly in the bone marrow MNC of MM patients, indicating that complementary effects may be achieved by combining IPH2102 and DARA in clinical MM management. Disclosures: Weers: Genmab: Employment. Andre:Innate Pharma: Employment. Lokhorst:Genmab: Research Funding. Parren:Genmab: Employment. Morel:Innate Pharma: Employment. Mutis:Genmab: Research Funding.

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