Natural Cell-Mediated Resistance Against Cryptococcus neoformans: A Possible Role for Natural Killer (NK) Cells

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.

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

Pathogens ◽

10.3390/pathogens10070866 ◽

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.

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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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-002193 ◽

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.

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Natural Killer–Dendritic Cell Interactions in Liver Cancer: Implications for Immunotherapy

Cancers ◽

10.3390/cancers13092184 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2184

Author(s):

Valentina Cazzetta ◽

Sara Franzese ◽

Claudia Carenza ◽

Silvia Della Bella ◽

Joanna Mikulak ◽

...

Keyword(s):

Liver Cancer ◽

Immune Responses ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Primary Liver Cancer ◽

Circulating Antigens ◽

Direct Cytotoxicity ◽

Immune Changes

Natural killer (NK) and dendritic cells (DCs) are innate immune cells that play a crucial role in anti-tumor immunity. NK cells kill tumor cells through direct cytotoxicity and cytokine secretion. DCs are needed for the activation of adaptive immune responses against tumor cells. Both NK cells and DCs are subdivided in several subsets endowed with specialized effector functions. Crosstalk between NK cells and DCs leads to the reciprocal control of their activation and polarization of immune responses. In this review, we describe the role of NK cells and DCs in liver cancer, focusing on the mechanisms involved in their reciprocal control and activation. In this context, intrahepatic NK cells and DCs present unique immunological features, due to the constant exposure to non-self-circulating antigens. These interactions might play a fundamental role in the pathology of primary liver cancer, namely hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Additionally, the implications of these immune changes are relevant from the perspective of improving the cancer immunotherapy strategies in HCC and ICC patients.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22073489 ◽

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.

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