Generation of a Novel Regulatory NK Cell Subset from Peripheral Blood CD34+ Progenitors Promoted by Membrane-Bound IL-15

BACKGROUND: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells

Membrane-bound IL-15 stimulation on peripheral blood natural kiler progenitors leads to the generation of an adherent subset co-expressing dendritic cells and natural kiler functional markers

Human peripheral blood natural killer progenitors represent a flexible, heterogeneous population whose phenotype and function are controlled by their membrane-bound IL-15. Indeed, reciprocal membrane-bond IL-15 trans-presentation commits these cells into NK differentiation, while membrane-bound IL-15 stimulation with its soluble ligand (sIL-15Rα) triggers a reverse signal (pERK1/2 and pFAK) that modifies the developmental program of at least two subsets of PB-NKPs. This treatment generates: i) the expansion of an immature NK subset growing in suspension; ii) the appearance of an unprecedented adherent non-proliferative subset with a dendritic morphology co-expressing marker, cytokines and functions typical of myeloid dendritic cells (CD1a+/BDCA1+/IL-12+) and NK cells (CD3-/NKp46+/ CD56+/IFNγ+). The generation of these putative NK/DCs is associated to the rapid inhibition of negative regulators of myelopoiesis (the transcription factors STAT6 and GATA-3) followed by the transient upregulation of inducers of myeloid development, such as the transcription factors (PU.1, GATA-1) and the anti-apoptotic molecule (MCL-1

Anne sütünde toksik ağır metal varlığı

Amaç: Anne sütüyle beslenen bebeklerde, annenin daha önceden maruz kalmış olduğu toksik kimyasal maddeler bebek için de potansiyel bir risktir. Bu çalışmanın amacı, anne sütünde Ni, Cd, Pb ve Sb varlığının araştırılması ve yayınlanan epidemiyolojik çalışmalar ve bilimsel literatür ışığında toksik ağır metallerin olası potansiyel risklerin ortaya konulmasıdır. Gereç ve Yöntem: Bulunduğu ilde beş yıldan daha uzun süre ikamet eden ve burada doğum yapan, ilk aydan sonra herhangi bir dönemde kontrole gelen, 58 emziren anneden süt örnekleri çalışma amacıyla toplandı. Süt örneklerinin Ni, Cd, Pb ve Sb düzeyleri ICP (Inductively Coupled Plasma) spektroskopi cihazı kullanılarak ölçüldü. Bulgular: İncelenen anne sütlerinin % 53.4’ünde Ni, % 17,2’sinde Cd, %12,1’inde Pb ve % 15,5’inde de Sb saptandı. Çalışmaya dahil olan 58 anneden sadece 13’ünün (%22,4) sütünde bakılan ağır metallerin hiç biri tespit edilmedi, geri kalan annelerin sütlerinde bir veya birden fazla ağır metal bulundu. Sonuç: Kırsal ve kentsel bölgelerde çevre ağır metallerle yaygın olarak kirletilmektedir. Bu toksik maddeler anne sütünde bulunur duruma gelmiştir. Yeryüzünde süt çocuklarının çevresel toksinler ile karşılaşabildikleri başlıca kaynak anne sütüdür. Anne sütüyle maruziyeti azaltabilmek için annelerin toksik ajanlar ile karşılaşma risklerini azaltmak gerekir.Aim: In breast-fed infants, toxic chemicals previously been exposed to the mother are also a potential risk to the baby. The aim of this study is to in- vestigate the presence of Ni, Cd, Pb and Sb in mother’s milk and to highlight the possible potential risks of toxic heavy metals in the light of published epidemiological studies and scientific literature. Material and Method: For the study, milk samples were collected from 58 breast-feeding mothers who were residing in their provinces for more than five years and gave birth here and applied to follow up in any time after the first month period. Ni, Cd, Pb and Sb levels of milk samples were measured by ICP (Inductively Coupled Plasma) spectroscopy device. Results: In examined breast milks, 53.4% Ni, 17.2% Cd, 12.1% Pb and 15.5% Sb were found. Out of 58 mothers enrolled in the study, none of the heavy metals was detected in only 13 (22.4%) mother’s milk, one or more heavy metal was found in the rest of the milks of mothers. Discusssion: In rural and urban areas, the environment is widely contami- nated with heavy metals. This toxic substances come to be found in breast milk. In the earth, the main source of environmental toxins that breast-feed infants faced with is breast milk. To minimize the exposure of toxins with breast milk, the risk of confrontation with mothers and toxic agents should be reduced

Cryopreservation of spleen and lymph nodes as a source of mononuclear cells to be used for the development of monoclonal antibody producing hybridoma cells

In the present study, spleen and lymph nodes of mice were cryopreserved as a whole tissue and after thawing, membrane integrity of mononuclear cells was determined by trypan blue exclusion and PI staining. T and B lymphocytes, macrophages and dendritic cells have been isolated from both cryopreserved tissue and analyzed by Flow cytometry. BALB/c mice were immunized with Hepatitis e antigen (HBeAg) and spleen and lymph nodes of mice were cryopreserved for 3 to 10 months. The cells obtained from both tissue were applied to hybridoma technology to understand if the cells keep their viability and functionality. The cells were isolated and fused with F0 mouse myeloma cells and several antibody producing hybrid cells were developed. Results have shown that cryopreserved spleen and lymph nodes of mice can be efficiently used in hybridoma technology for the successful generation of monoclonal antibody producing hybrid cells

Hepatitis B virus e antigen (HBeAg) may have a negative effect on dendritic cell generation

WOS: 000345541900006PubMed ID: 25150150Hepatitis B virus (HBV) continues to be a serious worldwide health problem despite the use of protective HBV vaccines and therapeutic regimens against chronic HBV infection. Chronic HBV patients cannot induce sufficient immune responses against the virus. HBV and its antigens are believed to suppress immune responses during chronic infection. Hence, studying the role of HBV in immune suppression is very important for the development of alternative therapeutic strategies for HBV infections. In the present study, we investigated the effect of Hepatitis B virus e antigen (HBeAg) on the generation of bone marrow derived dendritic cells (BMDCs) and the stimulation of plasmacytoid DCs (pDCs). In the presence of HBeAg, the ratio of BMDCs was decreased, but the ratio of CD11b(+)Ly6G(+) immature myeloid cells was increased. The expression of 47 proteins was also changed during HBeAg treatment; however, CpG-induced MHC-II expression on pDCs was not affected. Our results indicate that HBeAg may have a negative effect on the generation of DCs from bone morrow precursors. (C) 2014 Elsevier GmbH. All rights reserved.Scientific and Technological Research Council of Turkey (TUBITAK) [108S300, 110S497, 105G0656]This study was supported by grants nos. 108S300, 110S497 and 105G0656 from The Scientific and Technological Research Council of Turkey (TUBITAK). We thank Dr. Betul Guloglu for critically reviewing the manuscript and Duygu OZCELIK for her excellent technical assistance

Follicular CD4 T Cells Tutor CD8 Early Memory Precursors: An Initiatory Journey to the Frontier of B Cell Territory

The early events of CD8 memory generation remain largely unknown. Here we report that as early as 2 days after antigen priming, very earlymemory precursors can be identified by their expression of the chemokine receptor CXCR5. These early precursors, which have an effector phenotype, expand and temporarily migrate to the T-B cell zone junction where they interact with follicular CD4(+) T cells (Tfh). Remarkably, this interaction with Tfh, hitherto considered as exclusive B cell helpers, is required for CD8 memory precursors to become highly competent memory cells. CD40 and interleukin-21 signaling are involved in the help provided to CXCR5(+)CD8memory precursors. This study thus unveils critical early steps in the generation of CD8memory, identifies CXCR5 as the earliest known marker of CD8 memory precursors, suggests a major helper role for Tfh, and points to possible coordination between the pathways of CD8 and B cell memory generation at the T-B-cell zone junction.Institut National de la Sante et de la Recherche Medicale (Inserm)ANRS French National Research Agency (ANR)Government of the Kingdom of Saudi Arabi

Interferon-Alpha Triggers B Cell Effector 1 (Be1) Commitment

B-cells can contribute to the pathogenesis of autoimmune diseases not only through auto-antibody secretion but also via cytokine production. Therapeutic depletion of B-cells influences the functions and maintenance of various T-cell subsets. The mechanisms governing the functional heterogeneity of B-cell subsets as cytokine-producing cells are poorly understood. B-cells can differentiate into two functionally polarized effectors, one (B-effector-1-cells) producing a Th-1-like cytokine pattern and the other (Be2) producing a Th-2-like pattern. IL-12 and IFN-c play a key role in Be1 polarization, but the initial trigger of Be1 commitment is unclear. Type-I-interferons are produced early in the immune response and prime several processes involved in innate and adaptive responses. Here, we report that IFN-a triggers a signaling cascade in resting human naive B-cells, involving STAT4 and T-bet, two key IFN-c gene imprinting factors. IFN-a primed naive B-cells for IFN-c production and increased IFN-c gene responsiveness to IL-12. IFN-c continues this polarization by re-inducing T-bet and up-regulating IL-12Rb2 expression. IFN-a and IFN-c therefore pave the way for the action of IL-12. These results point to a coordinated action of IFN-a, IFN-c and IL-12 in Be1 polarization of naive B-cells, and may provide new insights into th