Expansion of cytotoxic natural killer cells using irradiated autologous peripheral blood mononuclear cells and anti-CD16 antibody.
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ABSTRACT:
SUBMITTER: Lee HR
PROVIDER: S-EPMC5593981 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Natural killer (NK) cells are becoming valuable tools for cancer therapy because of their cytotoxicity against tumor cells without prior sensitization and their involvement in graft-versus-host disease; however, it is difficult to obtain highly cytotoxic NK cells without adding extra feeder cells. In this study, we developed a new method for obtaining highly cytotoxic NK cells from peripheral blood mononuclear cells (PBMCs) independently of extra feeder cell addition using rituximab not coated on a flask (non-coated rituximab). We found that rituximab could promote both the activation and expansion of NK cells from PBMCs, irrespective of being coated on a flask or not. However, NK cells activated by non-coated rituximab had much greater antitumor activity against cancer cells, and these effects were dependent on autologous living B cells. The antibody-dependent cellular cytotoxicity effect of NK cells activated by non-coated rituximab was also more substantial. Furthermore, these cells expressed higher levels of CD107a, perforin, granzyme B, and IFN-γ. However, there was no difference in the percentage, apoptosis, and cell-cycle progression of NK cells induced by coated and non-coated rituximab. Non-coated rituximab activated NK cells by increasing AKT phosphorylation, further enhancing the abundance of XBP1s. In conclusion, we developed a new method for amplifying NK cells with higher antitumor functions with non-coated rituximab via autologous B cells from PBMCs, and this method more efficiently stimulated NK cell activation than by using coated rituximab.
| S-EPMC8185348 | biostudies-literature
Project description:Hypertrophic scars can cause pain, movement restrictions, and reduction in the quality of life. Despite numerous options to treat hypertrophic scarring, efficient therapies are still scarce, and cellular mechanisms are not well understood. Factors secreted by peripheral blood mononuclear cells (PBMCsec) have been previously described for their beneficial effects on tissue regeneration. In this study, we investigated the effects of PBMCsec on skin scarring in mouse models and human scar explant cultures at single-cell resolution (scRNAseq). Mouse wounds and scars, and human mature scars were treated with PBMCsec intradermally and topically. The topical and intradermal application of PBMCsec regulated the expression of various genes involved in pro-fibrotic processes and tissue remodeling. We identified elastin as a common linchpin of anti-fibrotic action in both mouse and human scars. In vitro, we found that PBMCsec prevents TGFβ-mediated myofibroblast differentiation and attenuates abundant elastin expression with non-canonical signaling inhibition. Furthermore, the TGFβ-induced breakdown of elastic fibers was strongly inhibited by the addition of PBMCsec. In conclusion, we conducted an extensive study with multiple experimental approaches and ample scRNAseq data demonstrating the anti-fibrotic effect of PBMCsec on cutaneous scars in mouse and human experimental settings. These findings point at PBMCsec as a novel therapeutic option to treat skin scarring.
| S-EPMC10143262 | biostudies-literature
Project description:Natural killer (NK) cells are promising tool for cancer treatment. Methods have been developed for large-scale NK cell expansion, including feeder cell-based methods or methods involving stimulation with NK cell activating signals, such as anti-CD16 antibodies. Different clones of anti-CD16 antibodies are available; however, a comprehensive comparison of their differential effects on inducing NK cell activation and expansion has not been conducted among these various clones under the same experimental conditions. Herein, we found that the NK cell expansion rate differed depending on the various anti-CD16 antibodies (CB16, 3G8, B73.1, and MEM-154) coated on microbeads when stimulated with genetically engineered feeder cells, K562‑membrane-bound IL‑18, and mbIL‑21 (K562‑mbIL‑18/-21). Only the CB16 clone combination caused enhanced NK cell expansion over K562‑mbIL‑18/-21 stimulation alone with similar NK cell functionality. Treatment with the CB16 clone once on the initial day of NK cell expansion was sufficient to maximize the combination effect. Overall, we developed a more enhanced NK expansion system by merging a feeder to effectively stimulate CD16 with the CB16 clone.
| S-EPMC10258201 | biostudies-literature
Project description:Clinical studies with adoptive immunotherapy using allogeneic natural killer (NK) cells showed feasibility, but also limitation regarding the transfused absolute cell numbers. First promising results with peripheral blood mononuclear cells (PBMCs) as feeder cells to improve the final cell number need further optimization and investigation of the unknown controlling mechanism in the cross-talk to NK cells. We investigated the influence of irradiated autologous PBMCs to boost NK cell proliferation in the presence of OKT3 and IL-2. Our findings demonstrate a requirement for receptor-ligand interactions between feeders and NK cells to produce soluble factors that can sustain NK cell proliferation. Thus, both physical contact between feeder and NK cells, and soluble factors produced in consequence, are required to fully enhance NK cell ex vivo proliferation. This occurred with an indispensable role of the cross-talk between T cells, monocytes, and NK cells, while B cells had no further influence in supporting NK cell proliferation under these co-culture conditions. Moreover, gene expression analysis of highly proliferating and non-proliferating NK cells revealed important phenotypic changes on 5-day cultured NK cells. Actively proliferating NK cells have reduced Siglec-7 and -9 expression compared with non-proliferating and resting NK cells (day 0), independently of the presence of feeder cells. Interestingly, proliferating NK cells cultured with feeder cells contained increased frequencies of cells expressing RANKL, B7-H3, and HLA class II molecules, particularly HLA-DR, compared with resting NK cells or expanded with IL-2 only. A subset of HLA-DR expressing NK cells, co-expressing RANKL, and B7-H3 corresponded to the most proliferative population under the established co-culture conditions. Our results highlight the importance of the crosstalk between T cells, monocytes, and NK cells in autologous feeder cell-based ex vivo NK cell expansion protocols, and reveal the appearance of a highly proliferative subpopulation of NK cells (HLA-DR+RANKL+B7-H3+) with promising characteristics to extend the therapeutic potential of NK cells.
| S-EPMC5522833 | biostudies-literature
Project description:BackgroundIgE-mediated hypersensitivity is becoming increasingly prevalent and activation of mast cells and basophils represent key events in the pathophysiology of allergy. We have previously reported that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) exerts beneficial anti-inflammatory effects. Yet, its ability to alleviate allergic symptoms has not been investigated so far.MethodsSeveral experimental in vitro and in vivo models have been used in this basic research study. A murine ear swelling model was used to study the effects of PBMCsec on 48/80-induced mast cell degranulation in vivo. The transcriptional profile of murine mast cells was analysed by single cell RNA sequencing (scRNAseq). Mast cell activation was studied in vitro using primary skin mast cells. Basophils from individuals allergic to birch pollens were used to investigate basophile activation by allergens. Transcriptomic and lipidomic analyses were used to identify mRNA expression and lipid species present in PBMCsec, respectively.FindingsTopical application of PBMCsec on mouse ears (C57BL/6) significantly reduced tissue swelling following intradermal injection of compound 48/80, an inducer of mast cell degranulation. Single cell RNA sequencing of PBMCsec-treated murine dermal mast cells (Balb/c) revealed a downregulation of genes involved in immune cell degranulation and Fc-receptor signalling. In addition, treatment of primary human dermal mast cells with PBMCsec strongly inhibited compound 48/80- and α-IgE-induced mediator release in vitro. Furthermore, PBMCsec remarkably attenuated allergen driven activation of basophils from allergic individuals. Transcriptomic analysis of these basophils showed that PBMCsec downregulated a distinct gene battery involved in immune cell degranulation and Fc-receptor signalling, corroborating results obtained from dermal mast cells. Finally, we identified the lipid fraction of PBMCsec as the major active ingredient involved in effector cell inhibition.InterpretationCollectively, our data demonstrate that PBMCsec is able to reduce activation of mast cells and basophils, encouraging further studies on the potential use of PBMCsec for treating allergy.FundingAustrian Research Promotion Agency (852748 and 862068, 2015-2019), Vienna Business Agency (2343727, 2018-2020), Aposcience AG, Austrian Federal Ministry of Education, Science and Research (SPA06/055), Danube Allergy Research Cluster, Austrian Science Fund (I4437 and P32953).
| S-EPMC9168057 | biostudies-literature
Project description:One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.
| S-EPMC8008054 | biostudies-literature
Project description:Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin (IL)-15 and 41BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Seven-day coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56(+)CD3(-) NK cells from peripheral blood (range, 5.1- to 86.6-fold; n = 50), which was considerably superior to that produced by stimulation with IL-2, IL-12, IL-15, and/or IL-21 and caused no proliferation of CD3(+) lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and their unstimulated or IL-2-stimulated counterparts showed marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells against acute myeloid leukemia cells in vitro. They could be detected for >1 month when injected into immunodeficient mice and could eradicate leukemia in murine models of acute myeloid leukemia. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer.
| S-EPMC2716664 | biostudies-literature
Project description:Periodontal inflammation is associated with dying cells that potentially release metabolites helping to promote inflammatory resolution. We had shown earlier that the secretome of irradiated, dying peripheral blood mononuclear cells support in vitro angiogenesis. However, the ability of the secretome to promote inflammatory resolution remains unknown. Here, we determined the expression changes of inflammatory cytokines in murine bone marrow macrophages, RAW264.7 cells, and gingival fibroblasts exposed to the secretome obtained from γ-irradiated peripheral blood mononuclear cells in vitro by RT-PCR and immunoassays. Nuclear translocation of p65 was detected by immunofluorescence staining. Phosphorylation of p65 and degradation of IκB was determined by Western blot. The secretome of irradiated peripheral blood mononuclear cells significantly decreased the expression of IL1 and IL6 in primary macrophages and RAW264.7 cells when exposed to LPS or saliva, and of IL1, IL6, and IL8 in gingival fibroblasts when exposed to IL-1β and TNFα. These changes were associated with decreased phosphorylation and nuclear translocation of p65 but not degradation of IκB in macrophages. We also show that the lipid fraction of the secretome lowered the inflammatory response of macrophages exposed to the inflammatory cues. These results demonstrate that the secretome of irradiated peripheral blood mononuclear cells can lower an in vitro simulated inflammatory response, supporting the overall concept that the secretome of dying cells promotes inflammatory resolution.
| S-EPMC7370068 | biostudies-literature
Project description:Osteoclastogenesis required for bone remodeling is also a key pathologic mechanism of inflammatory osteolysis being controlled by paracrine factors released from dying cells. The secretome of irradiated, dying peripheral blood mononuclear cells (PBMCs) has a major impact on the differentiation of myeloid cells into dendritic cells, and macrophage polarization. The impact on osteoclastogenesis, however, has not been reported. For this aim, we used murine bone marrow macrophages exposed to RANKL and M-CSF to initiate osteoclastogenesis, with and without the secretome obtained from γ-irradiated PBMCs. We reported that the secretome significantly enhanced in vitro osteoclastogenesis as determined by means of histochemical staining of the tartrate-resistant acid phosphatase (TRAP), as well as the expression of the respective target genes, including TRAP and cathepsin K. Considering that TGF-β enhanced osteoclastogenesis, we confirmed the TGF-β activity in the secretome with a bioassay that was based on the increased expression of IL11 in fibroblasts. Neutralizing TGF-β by an antibody decreased the ability of the secretome to support osteoclastogenesis. These findings suggested that TGF-β released by irradiated PBMCs could enhance the process of osteoclastogenesis in vitro.
| S-EPMC7696998 | biostudies-literature
Project description:Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.
| S-EPMC10197019 | biostudies-literature