Human NK Cell Subset Functions Are Differentially Affected by Adipokines

Background: Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host’s primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. Methods: Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). Results: FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56dim NK cells. The production of GzmA in CD56bright NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. Conclusion: Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation

Functionality and Cell Senescence of CD4/ CD8-Selected CD20 CAR T Cells Manufactured Using the Automated CliniMACS Prodigy® Platform

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process

Human NK cell subset functions are differentially affected by adipokines.

BACKGROUND: Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host's primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. METHODS: Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). RESULTS: FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56(dim) NK cells. The production of GzmA in CD56(bright) NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. CONCLUSION: Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation

Generation of HLA-deficient platelets from hematopoietic progenitor cells: GENERATION OF HLA-DEFICIENT PLTs

BACKGROUND: Exposure to allogeneic blood products often leads to the development of human leukocyte antigen (HLA) antibodies. Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA Class I antigens constitutes a significant clinical problem. STUDY DESIGN AND METHODS: We developed an RNA interference (RNAi)-based approach to silence the expression of HLA Class I molecules on PLTs derived from CD34+ progenitor cells. A lentiviral-based system was used to express short-hairpin RNA (shRNA) targeting b2-microglobulin (b2m) transcripts in CD34+ progenitor cells. Differentiation to PLTs was performed by incubating progenitor cells in the presence of thrombopoietin and interleukin-3. RESULTS: The transduction of RNAi cassettes containing the sequences for shRNAs targeting b2m caused up to 85% reduction of progenitor cells HLA Class I antigen expression, which was maintained in the culture-derived PLTs. The HLA-deficient PLTs derived from HLA-silenced CD34+ cells proved to be fully functional in in vitro tests when compared to peripheral blood–derived PLTs. CONCLUSIONS: Our data show that in vitro generating HLA Class I–deficient PLTs from hematopoietic progenitor cells prove to be feasible. As malignancy risks associated with insertional mutagenesis are not to be expected in anucleated PLTs, provision of HLA-deficient PLTs from large-scale production units may become reality in the management of patients suffering from PLT transfusion refractoriness

Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma

Background and Aims: Immunocompromised patients are at risk of chronic hepatitis E which can be acquired by blood transfusions. Currently, screening of blood donors (BDs) for HEV RNA with a limit of detection (LOD) of 2,000 IU/ml is required in Germany. However, this may result in up to 440,000 IU of HEV RNA in blood products depending on their plasma volume. We studied the residual risk of transfusion-transmitted (tt) HEV infection when an LOD of 2,000 IU/ml is applied. Methods: Highly sensitive individual donor testing for HEV RNA on the Grifols Procleix Panther system (LOD 7.89 IU/ml) was performed. HEV loads were quantified by real-time PCR. Results: Of 16,236 donors, 31 (0.19%) were HEV RNA positive. Three BDs had viral loads between 710 and 2,000 IU/ml, which pose a significant risk of tt hepatitis E with any type of blood product. Eight BDs had viral loads of >32 to 710 IU/ml, which pose a risk of tt hepatitis E with platelet or plasma transfusions because of their higher plasma volume compared to red blood cell concentrates. Eight of these 11 potentially infectious BDs were seronegative for HEV, indicating a recent infection. Only 8 of 31 donors had viral loads >2,000 IU/ml that would also have been detected by the required screening procedure and 12 had very low HEV loads (<32 IU/ml). Conclusions: Screening of BDs with an LOD of 2,000 IU/ml reduced the risk of tt HEV infection by about 73% for red blood cell concentrates but by just 42% for platelet and fresh frozen plasma transfusions. Single donor screening (LOD <32 IU/ml) should lead to an almost 100% risk reduction. Lay summary: Immunocompromised patients, such as solid organ or hematopoietic stem cell recipients, are at risk of chronic hepatitis E, which can be acquired via blood transfusions. The risk of transfusion-transmitted hepatitis E in these patients may not be sufficiently controlled by (mini-)pool hepatitis E virus RNA screening of blood donors. Single donor screening should be considered to improve the safety of blood products. (C) 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

Distribution of major lymphocyte subsets and memory T-cell subpopulations in healthy adults employing GLP-conforming multicolor flow cytometry

Multiple myeloma remains a largely incurable disease of clonally expanding malignant plasma cells. The bone marrow microenvironment harbors treatment-resistant myeloma cells, which eventually lead to disease relapse in patients. In the bone marrow, CD

TRAIL expression after ACM stimulation.

<p>Human peripheral blood mononuclear cells (PBMCs) were isolated from six leukocyte filters in each case and cultured for 24 hours with R10-medium conditioned with 1% of SGBS ACM harvested on day 7 or day 11 after induction of adipogenesis. PBMCs incubated for 24 hours with medium only served as controls. A detailed description of the incubation procedure can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075703#s2" target="_blank">materials and methods</a> section. A) TRAIL-expressing human blood NK cells as percentage of human blood NK cells; B) TRAIL-expressing human blood CD56^dim and CD56^bright NK cells as percentage of human blood CD56^dim and CD56^bright NK cells. Statistically significant differences between the numbers of TRAIL expressing CD56^dim and CD56^bright NK cells are depicted with an asterisk (*). All data represent means+SEM.</p

IFN-γ and GzmA expression after ACM stimulation.

<p>Human peripheral blood mononuclear cells (PBMCs) isolated from six leukocyte filters in each case were cultured for 24 hours with R10-medium conditioned with 1% of SGBS ACM harvested on day 7 (d7) or day 11 (d11) after induction of adipogenesis. PBMCs incubated for 24 hours with medium only served as controls. A detailed description of the incubation procedure can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075703#s2" target="_blank">materials and methods</a> section. A) IFN-γ-expressing human blood CD56^dim and CD56^bright NK cells as percentage of human blood CD56^dim and CD56^bright NK cells treated with 1% of the ACM harvested on day 7 after induction of adipogenic differentiation; B) GzmA-expressing human blood CD56^dim NK cells as percentage of human blood CD56^dim NK cells treated with 1% of the ACM harvested on day 7 or 11 after induction of adipogenic differentiation; C) GzmA-expressing human blood CD56^bright NK cells as percentage of human blood CD56^bright NK cells treated with 1% of the ACM harvested on day 7 or 11 after induction of adipogenic differentiation; Statistically significant differences between the stimulation with SGBS ACM and the medium control are depicted with an asterisk (*). Statistically significant differences between the stimulation with SGBS ACM harvested on day 7 after induction of adipocyte differentiation and the same dosage of SGBS ACM harvested on day 11 after induction of adipogenic differentiation are depicted with a rhomb (#). Statistically significant differences between the numbers of IFN-γ expressing CD56^dim and CD56^bright NK cells are depicted with a paragraph sign (§). All data represent means+SEM.</p

Ability of human blood NK cells to form conjugates with cells of the K562 erythroleukemia line after ACM stimulation.

<p>Human peripheral blood mononuclear cells (PBMCs) isolated from six leukocyte filters in each case were cultured for 24 hours with R10-medium conditioned with 1% of SGBS ACM harvested on day 7 or day 11 after induction of adipogenic differentiation. PBMCs incubated for 24 hours with medium only served as controls. A detailed description of the incubation procedure can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075703#s2" target="_blank">materials and methods</a> section. A) About 30% of the NK cells (CD3⁻CD56⁺) were able to form conjugates with K562 target cells (upper right quadrant) irrespective of the cultivation conditions. B) Conjugate forming human blood NK cells as percentage of human blood NK cells. All data represent means+SEM.</p