scholarly journals Enhanced mesenchymal stromal cell recruitment via natural killer cells by incorporation of inflammatory signals in biomaterials

2011 ◽  
Vol 9 (67) ◽  
pp. 261-271 ◽  
Author(s):  
Catarina R. Almeida ◽  
Daniela P. Vasconcelos ◽  
Raquel M. Gonçalves ◽  
Mário A. Barbosa
Keyword(s):  
Inflammatory Response ◽  
Nk Cells ◽  
Natural Killer ◽  
Tissue Repair ◽  
Bone Repair ◽  
Nk Cell ◽  
Fold Increase ◽  
Cell Recruitment ◽  
Inflammatory Signals ◽  
Differentiation Marker

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an ‘ideal’ inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell–MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies.

Assessment of PD-1 expression in human resting and activated natural killer cells and murine tumor models.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 36-36
Author(s):  
Sean J. Judge ◽  
Cordelia Dunai ◽  
Ian R. Sturgill ◽  
Kevin M. Stoffel ◽  
William J. Murphy ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Fold Increase ◽  
Wild Type ◽  
Immunodeficient Mice

36 Background: Blockade of the PD-1/PD-L1/2 axis has revolutionized cancer therapy. Although reinvigorated PD-1+ T cells are the main effectors in the response to checkpoint blockade, the contribution of Natural Killer (NK) cells to PD-1/PD-L1 inhibition is under debate. While PD-1 has been identified on NK cells, this appears to be restricted to small populations under limited conditions. We sought to evaluate the extent of PD-1 expression in mouse and human resting and activated NK cells. Methods: Human NK cells were isolated from healthy donor PBMCs and cancer patients. Ex vivo activation and proliferation techniques included recombinant human cytokine and feeder line co-culture. Murine NK cells were isolated from splenocytes, and PBMCs from wild type and immunodeficient mice. We assessed NK cell surface markers and intracellular cytokine by flow cytometry, and gene expression by quantitative RT-PCR. Results: Over 21-days of ex vivo expansion, expression of PD-1 or PD-L1 on human NK cells was < 1% at all time points, while TIGIT+ expression increased to > 85%. Conversely, ConA stimulation of T cells increased PD-1 expression with no change in TIGIT expression. QRT-PCR demonstrated absent PD-1 expression in purified NK cells compared to a 5-fold increase in PD-1 gene expression in ConA stimulated PBMCs. PD-1/PD-L1 was also < 1% in the NK92 cell line and < 2.5% in peripheral CD56+CD3- NK cells from patients with soft tissue sarcoma (STS). NK cells from digested freshly resected STS show variable PD-1 ( < 10%) and minimal PD-L1 ( < 1%) expression with a small, but measurable population of intra-tumoral NK cells (1% of immune cells). In vivo mouse studies showed < 5% PD-1+ NK cells in spleen and tumor of CT26 tumor-bearing mice, while PD-L1+ NK cells increased in frequency from spleen (5-35%) to tumor (40-95%) in both wild type BALB/C and SCID mice. Conclusions: In contrast to prior studies, we did not observe a substantial PD-1+ population on human or murine NK cells after multiple activation strategies compared to T cells. Contrary to its application in T cells, our data suggest that PD-1 is not a useful marker for NK cell exhaustion/dysfunction. PD-L1 on NK cells may represent an important link between NK and T cell immunotherapy.

scholarly journals Suppression of tumor formation in lymph nodes by L-selectin–mediated natural killer cell recruitment

2005 ◽  
Vol 202 (12) ◽  
pp. 1679-1689 ◽  
Author(s):  
Shihao Chen ◽  
Hiroto Kawashima ◽  
John B. Lowe ◽  
Lewis L. Lanier ◽  
Minoru Fukuda
Keyword(s):  
Lymph Nodes ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Metastasis ◽  
Nk Cell ◽  
Wild Type ◽  
Cell Recruitment ◽  
Selectin Ligands ◽  
Secondary Lymphoid Organs ◽  
Deficient Mice

Natural killer (NK) cells are known to reject certain tumors in vivo; however, the ability of NK cells to prevent metastasis of tumors into secondary lymphoid organs has not been addressed. Here, we report that in tumor-bearing hosts, NK cells are recruited to regional lymph nodes in wild-type mice, but not in mice deficient for L-selectin or L-selectin ligands. By adoptive transfer and complete Freund's adjuvant stimulation experiments, we demonstrated that L-selectin on NK cells and L-selectin ligands on endothelial cells are essential for NK cell recruitment to lymph nodes. Furthermore, freshly isolated resident lymph node NK cells lysed tumors efficiently, and metastasis of B16 melanoma cells to draining lymph nodes was suppressed in wild-type or Rag-1–deficient mice, but not when NK cells were depleted. Although L-selectin–deficient NK cells efficiently lysed tumor cells in vitro, NK cell–dependent suppression of tumor metastasis was diminished in mice deficient for L-selectin or L-selectin ligands because of insufficient NK cell recruitment to lymph nodes. Moreover, tumor metastasis was substantially inhibited in L-selectin–deficient mice reconstituted with wild-type NK cells. These findings indicate that L-selectin–mediated NK cell recruitment plays a crucial role in the control of tumor metastasis into secondary lymphoid organs.

scholarly journals Interleukin-12 is chemotactic for natural killer cells and stimulates their interaction with vascular endothelium

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2261-2268
Author(s):  
P Allavena ◽  
C Paganin ◽  
D Zhou ◽  
G Bianchi ◽  
S Sozzani ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Vascular Endothelium ◽  
Nk Cell ◽  
Interleukin 12 ◽  
Polymorphonuclear Cells ◽  
Activated T Cells ◽  
Cell Recruitment

We investigated the chemotactic activity of interleukin (IL)-12 on human natural killer (NK) cells and other leukocyte subsets. It was found that IL-12 induced directional migration of highly enriched preparations of NK cells (> 80% CD16+ and CD56+) and CD3-activated T cells (both of CD4 and CD8 subset), but not resting T cells and monocytes. On the contrary, purified polymorphonuclear cells (PMN) showed significant and reproducible chemotactic response to IL-12. The effects of IL-12 on leukocyte migration were observed in a narrow concentration range with a peak at approximately 7.5 ng/mL, and were abrogated by monoclonal antibody (MoAb) anti-IL-12 or after cytokine boiling. We also investigated the interaction of NK cells with vascular endothelium in vitro. Overnight treatment of NK cells with IL-12 augmented their binding to cultured endothelial cells (EC) obtained from umbilical veins. IL-12-increased binding was better observed when resting rather than IL-1-activated EC were used as substratum of adhesion. IL-12-augmented binding of NK cells to resting or IL-1- activated EC involved the LFA-1/ICAM-1 and VLA-4/VCAM-1 pathways. Thus, by inducing migration and interaction with EC, IL-12 regulates crucial determinants of NK-cell recruitment in tissues.

scholarly journals Interleukin-12 is chemotactic for natural killer cells and stimulates their interaction with vascular endothelium

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2261-2268 ◽  
Author(s):  
P Allavena ◽  
C Paganin ◽  
D Zhou ◽  
G Bianchi ◽  
S Sozzani ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Vascular Endothelium ◽  
Nk Cell ◽  
Interleukin 12 ◽  
Polymorphonuclear Cells ◽  
Activated T Cells ◽  
Cell Recruitment

Abstract We investigated the chemotactic activity of interleukin (IL)-12 on human natural killer (NK) cells and other leukocyte subsets. It was found that IL-12 induced directional migration of highly enriched preparations of NK cells (> 80% CD16+ and CD56+) and CD3-activated T cells (both of CD4 and CD8 subset), but not resting T cells and monocytes. On the contrary, purified polymorphonuclear cells (PMN) showed significant and reproducible chemotactic response to IL-12. The effects of IL-12 on leukocyte migration were observed in a narrow concentration range with a peak at approximately 7.5 ng/mL, and were abrogated by monoclonal antibody (MoAb) anti-IL-12 or after cytokine boiling. We also investigated the interaction of NK cells with vascular endothelium in vitro. Overnight treatment of NK cells with IL-12 augmented their binding to cultured endothelial cells (EC) obtained from umbilical veins. IL-12-increased binding was better observed when resting rather than IL-1-activated EC were used as substratum of adhesion. IL-12-augmented binding of NK cells to resting or IL-1- activated EC involved the LFA-1/ICAM-1 and VLA-4/VCAM-1 pathways. Thus, by inducing migration and interaction with EC, IL-12 regulates crucial determinants of NK-cell recruitment in tissues.

scholarly journals Natural Killer Cells Are Present in Rag1−/− Mice and Promote Tissue Damage During the Acute Phase of Ischemic Stroke

2021 ◽  
Author(s):  
Leoni Rolfes ◽  
Tobias Ruck ◽  
Christina David ◽  
Stine Mencl ◽  
Stefanie Bock ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Immune Cell ◽  
Nk Cell ◽  
Cell Subset ◽  
Neurological Deficits ◽  
In Vitro Assays ◽  
Experimental Stroke ◽  
Transfer Model

AbstractRag1−/− mice, lacking functional B and T cells, have been extensively used as an adoptive transfer model to evaluate neuroinflammation in stroke research. However, it remains unknown whether natural killer (NK) cell development and functions are altered in Rag1−/− mice as well. This connection has been rarely discussed in previous studies but might have important implications for data interpretation. In contrast, the NOD-Rag1nullIL2rgnull (NRG) mouse model is devoid of NK cells and might therefore eliminate this potential shortcoming. Here, we compare immune-cell frequencies as well as phenotype and effector functions of NK cells in Rag1−/− and wildtype (WT) mice using flow cytometry and functional in vitro assays. Further, we investigate the effect of Rag1−/− NK cells in the transient middle cerebral artery occlusion (tMCAO) model using antibody-mediated depletion of NK cells and adoptive transfer to NRG mice in vivo. NK cells in Rag1−/− were comparable in number and function to those in WT mice. Rag1−/− mice treated with an anti-NK1.1 antibody developed significantly smaller infarctions and improved behavioral scores. Correspondingly, NRG mice supplemented with NK cells were more susceptible to tMCAO, developing infarctions and neurological deficits similar to Rag1−/− controls. Our results indicate that NK cells from Rag1−/− mice are fully functional and should therefore be considered in the interpretation of immune-cell transfer models in experimental stroke. Fortunately, we identified the NRG mice, as a potentially better-suited transfer model to characterize individual cell subset-mediated neuroinflammation in stroke.

scholarly journals The m15 Locus of Murine Cytomegalovirus Modulates Natural Killer Cell Responses to Promote Dissemination to the Salivary Glands and Viral Shedding

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 866
Author(s):  
Baca Chan ◽  
Maja Arapović ◽  
Laura Masters ◽  
Francois Rwandamuiye ◽  
Stipan Jonjić ◽  
...  
Keyword(s):  
Nk Cells ◽  
Salivary Glands ◽  
Natural Killer ◽  
Nk Cell ◽  
Acute Infection ◽  
Killer Cell ◽  
Viral Escape ◽  
Murine Cytomegalovirus ◽  
Viral Shedding ◽  
Cell Responses

As the largest herpesviruses, the 230 kb genomes of cytomegaloviruses (CMVs) have increased our understanding of host immunity and viral escape mechanisms, although many of the annotated genes remain as yet uncharacterised. Here we identify the m15 locus of murine CMV (MCMV) as a viral modulator of natural killer (NK) cell immunity. We show that, rather than discrete transcripts from the m14, m15 and m16 genes as annotated, there are five 3′-coterminal transcripts expressed over this region, all utilising a consensus polyA tail at the end of the m16 gene. Functional inactivation of any one of these genes had no measurable impact on viral replication. However, disruption of all five transcripts led to significantly attenuated dissemination to, and replication in, the salivary glands of multiple strains of mice, but normal growth during acute infection. Disruption of the m15 locus was associated with heightened NK cell responses, including enhanced proliferation and IFNγ production. Depletion of NK cells, but not T cells, rescued salivary gland replication and viral shedding. These data demonstrate the identification of multiple transcripts expressed by a single locus which modulate, perhaps in a concerted fashion, the function of anti-viral NK cells.

scholarly journals Short-course IL-15 given as a continuous infusion led to a massive expansion of effective NK cells: implications for combination therapy with antitumor antibodies

2021 ◽  
Vol 9 (4) ◽  
pp. e002193
Author(s):  
Sigrid P Dubois ◽  
Milos D Miljkovic ◽  
Thomas A Fleisher ◽  
Stefania Pittaluga ◽  
Jennifer Hsu-Albert ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Intravenous Infusion ◽  
Nk Cell ◽  
Continuous Intravenous Infusion ◽  
Dose Escalation Study ◽  
Link Type ◽  
Best Response ◽  
Antitumor Antibodies ◽  
Interleukin 15

BackgroundFull application of cytokines as oncoimmunotherapeutics requires identification of optimal regimens. Our initial effort with intravenous bolus recombinant human interleukin-15 (rhIL-15) was limited by postinfusional reactions. Subcutaneous injection and continuous intravenous infusion for 10 days (CIV-10) provided rhIL-15 with less toxicity with CIV-10 giving the best increases in CD8+ lymphocytes and natural killer (NK) cells. To ease rhIL-15 administration, we shortened time of infusion. Treatment with rhIL-15 at a dose of 3–5 µg/kg as a 5-day continuous intravenous infusion (CIV-5) had no dose-limiting toxicities while effector cell stimulation was comparable to the CIV-10 regimen.MethodsEleven patients with metastatic cancers were treated with rhIL-15 CIV-5, 3 µg (n=4), 4 µg (n=3), and 5 µg/kg/day (n=4) in a phase I dose-escalation study (April 6, 2012).ResultsImpressive expansions of NK cells were seen at all dose levels (mean 34-fold), including CD56bright NK cells (mean 144-fold for 4 µg/kg), as well as an increase in CD8+ T cells (mean 3.38-fold). At 5 µg/kg/day, there were no dose-limiting toxicities but pulmonary capillary leak and slower patient recovery. This led to our choice of the 4 µg/kg as CIV-5 dose for further testing. Cytolytic capacity of CD56bright and CD56dim NK cells was increased by interleukin-15 assayed by antibody-dependent cellular cytotoxicity (ADCC), natural cytotoxicity and natural killer group 2D-mediated cytotoxicity. The best response was stable disease.ConclusionsIL-15 administered as CIV-5 substantially expanded NK cells with increased cytotoxic functions. Tumor-targeting monoclonal antibodies dependent on ADCC as their mechanism of action including alemtuzumab, obinutuzumab, avelumab, and mogamulizumab could benefit from those NK cell expansions and provide a promising therapeutic strategy.Trial registration numbersNCT01572493, NCT03759184, NCT03905135, NCT04185220 and NCT02689453.

scholarly journals CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3489
Author(s):  
Takayuki Morimoto ◽  
Tsutomu Nakazawa ◽  
Ryosuke Matsuda ◽  
Fumihiko Nishimura ◽  
Mitsutoshi Nakamura ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Protein Complexes ◽  
Lymphocyte Activation ◽  
Exon 2 ◽  
Effector Cells ◽  
Guide Rna ◽  
Human Natural Killer Cells

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.

scholarly journals α-Pinene Enhances the Anticancer Activity of Natural Killer Cells via ERK/AKT Pathway

2021 ◽  
Vol 22 (2) ◽  
pp. 656
Author(s):  
Hantae Jo ◽  
Byungsun Cha ◽  
Haneul Kim ◽  
Sofia Brito ◽  
Byeong Mun Kwak ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Granzyme B ◽  
Akt Pathway ◽  
Cell Cytotoxicity ◽  
Anticancer Effects ◽  
Nk Cell Cytotoxicity

Natural killer (NK) cells are lymphocytes that can directly destroy cancer cells. When NK cells are activated, CD56 and CD107a markers are able to recognize cancer cells and release perforin and granzyme B proteins that induce apoptosis in the targeted cells. In this study, we focused on the role of phytoncides in activating NK cells and promoting anticancer effects. We tested the effects of several phytoncide compounds on NK-92mi cells and demonstrated that α-pinene treatment exhibited higher anticancer effects, as observed by the increased levels of perforin, granzyme B, CD56 and CD107a. Furthermore, α-pinene treatment in NK-92mi cells increased NK cell cytotoxicity in two different cell lines, and immunoblot assays revealed that the ERK/AKT pathway is involved in NK cell cytotoxicity in response to phytoncides. Furthermore, CT-26 colon cancer cells were allografted subcutaneously into BALB/c mice, and α-pinene treatment then inhibited allografted tumor growth. Our findings demonstrate that α-pinene activates NK cells and increases NK cell cytotoxicity, suggesting it is a potential compound for cancer immunotherapy.

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