Mechanisms by Which Interleukin-12 Corrects Defective NK Cell Anticryptococcal Activity in HIV-Infected Patients

ABSTRACTCryptococcus neoformansis a pathogenic yeast and a leading cause of life-threatening meningitis in AIDS patients. Natural killer (NK) cells are important immune effector cells that directly recognize and killC. neoformansvia a perforin-dependent cytotoxic mechanism. We previously showed that NK cells from HIV-infected patients have aberrant anticryptococcal killing and that interleukin-12 (IL-12) restores the activity at least partially through restoration of NKp30. However, the mechanisms causing this defect or how IL-12 restores the function was unknown. By examining the sequential steps in NK cell killing ofCryptococcus, we found that NK cells from HIV-infected patients had defective binding of NK cells toC. neoformans. Moreover, those NK cells that bound toC. neoformansfailed to polarize perforin-containing granules to the microbial synapse compared to healthy controls, suggesting that binding was insufficient to restore a defect in perforin polarization. We also identified lower expression of intracellular perforin and defective perforin release from NK cells of HIV-infected patients in response toC. neoformans. Importantly, treatment of NK cells from HIV-infected patients with IL-12 reversed the multiple defects in binding, granule polarization, perforin content, and perforin release and restored anticryptococcal activity. Thus, there are multiple defects in the cytolytic machinery of NK cells from HIV-infected patients, which cumulatively result in defective NK cell anticryptococcal activity, and each of these defects can be reversed with IL-12.IMPORTANCEThe mechanisms by which NK cells bind directly to pathogens and deploy their deadly cytolytic machinery during microbial host defense are only beginning to be elucidated. With the goal of understanding this process, we used NK cells from HIV-infected patients, which were known to have a defect in killing ofCryptococcus neoformans. Taking advantage of previous studies that had shown that IL-12 restored killing, we used the cytokine as a gain-of-function approach to define the relevance of multiple steps in the recognition and cytolytic pathway. We demonstrated that NK cells from HIV-infected patients failed to killCryptococcusdue to defects in perforin expression, granule polarization, and release of perforin. Additionally, IL-12 restored recognition ofC. neoformansthrough binding of the NK-activating receptor NKp30. These observations identify important mechanisms used by NK cells to kill microbes and determine that defects in NK cells from HIV-infected patients are reversible.

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The Advantages and Challenges of Anticancer Dendritic Cell Vaccines and NK Cells in Adoptive Cell Immunotherapy

Vaccines ◽

10.3390/vaccines9111363 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1363

Author(s):

Elena V. Abakushina ◽

Liubov I. Popova ◽

Andrey A. Zamyatnin ◽

Jens Werner ◽

Nikolay V. Mikhailovsky ◽

...

Keyword(s):

Cytotoxic Activity ◽

Nk Cells ◽

Nk Cell ◽

Therapeutic Potential ◽

Ex Vivo ◽

Adoptive Cell Therapy ◽

Immune Effector ◽

Effector Cells ◽

Cell Immunotherapy

In the last decade, an impressive advance was achieved in adoptive cell therapy (ACT), which has improved therapeutic potential and significant value in promising cancer treatment for patients. The ACT is based on the cell transfer of dendritic cells (DCs) and/or immune effector cells. DCs are often used as vaccine carriers or antigen-presenting cells (APCs) to prime naive T cells ex vivo or in vivo. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are used as major tool effector cells for ACT. Despite the fact that NK cell immunotherapy is highly effective and promising against many cancer types, there are still some limitations, including insignificant infiltration, adverse conditions of the microenvironment, the immunosuppressive cellular populations, and the low cytotoxic activity in solid tumors. To overcome these difficulties, novel methods of NK cell isolation, expansion, and stimulation of cytotoxic activity should be designed. In this review, we discuss the basic characteristics of DC vaccines and NK cells as potential adoptive cell preparations in cancer therapy.

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The Tumor Microenvironment—A Metabolic Obstacle to NK Cells’ Activity

Cancers ◽

10.3390/cancers12123542 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3542

Author(s):

Joanna Domagala ◽

Mieszko Lachota ◽

Marta Klopotowska ◽

Agnieszka Graczyk-Jarzynka ◽

Antoni Domagala ◽

...

Keyword(s):

Tumor Microenvironment ◽

Nk Cells ◽

Molecular Mechanisms ◽

Cell Function ◽

Nk Cell ◽

Metabolic Changes ◽

Immune Effector ◽

Effector Cells ◽

Immune Effector Cells ◽

Almost All

NK cells have unique capabilities of recognition and destruction of tumor cells, without the requirement for prior immunization of the host. Maintaining tolerance to healthy cells makes them an attractive therapeutic tool for almost all types of cancer. Unfortunately, metabolic changes associated with malignant transformation and tumor progression lead to immunosuppression within the tumor microenvironment, which in turn limits the efficacy of various immunotherapies. In this review, we provide a brief description of the metabolic changes characteristic for the tumor microenvironment. Both tumor and tumor-associated cells produce and secrete factors that directly or indirectly prevent NK cell cytotoxicity. Here, we depict the molecular mechanisms responsible for the inhibition of immune effector cells by metabolic factors. Finally, we summarize the strategies to enhance NK cell function for the treatment of tumors.

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CXCL12 Inhibition By Nox-A12 (Olaptesed Pegol) Synergizes with the ADCC Activity of CD20 Antibodies By Increasing NK Cell Infiltration in a 3D Lymphoma Model

Blood ◽

10.1182/blood.v128.22.3021.3021 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3021-3021 ◽

Cited By ~ 1

Author(s):

Dirk Zboralski ◽

Anna Kruschinski ◽

Axel Vater

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Hematological Malignancies ◽

Nk Cell ◽

Cell Infiltration ◽

Malignant Cells ◽

Immune Effector ◽

Effector Cells ◽

Immune Effector Cells ◽

Anti Cd20

Abstract Hematological malignancies are characterized by the expansion of malignant cells in the peripheral blood and in stroma-rich niches such as the bone marrow or lymphoid tissues. Anti-CD20 monoclonal antibodies (mAbs) are highly effective in eliminating malignant cells in the peripheral blood with the help of immune effector cells, e.g. NK cells mediating antibody-dependent cellular cytotoxicity (ADCC). However, residual malignant cells often continue to persist in protective stromal niches. These compartments have similarities to the solid tumor microenvironment (TME) where mAb therapy is restricted by poor tissue penetration and low effector cell infiltration. The CXCL12-neutralizing L-RNA aptamer NOX-A12 (olaptesed pegol) has been shown to mobilize malignant cells from the bone marrow into the peripheral blood, thereby sensitizing them to the action of standard therapy such as the anti-CD20 mAb rituximab (Blood. 2014;124(21):1996). In addition to malignant cells, CXCR4 expressing immune cells are effectively mobilized by NOX-A12 (Clin Pharmacol Ther. 2013;94(1):150-157). Recently we have shown that NOX-A12 increases lymphocyte infiltration into solid tumor-stroma spheroids, thereby synergizing with anti-PD-1 checkpoint blockade (Cancer Res 2016;76(14 Suppl): 1473). Here we established 3D lymphoid spheroidal microtissues mimicking the stroma-rich and CXCL12-abundant TME of lymphoid malignancies. We investigated the effect of NOX-A12 on NK effector cell infiltration into lymphoma spheroids and tested the combination with anti-CD20 mAbs. Spheroids were generated by co-culturing of CXCL12-expressing murine stromal MS-5 cells and CD20-expressing lymphoma cells in ultra-low attachment plates for 24 hours. Primary human NK cells, isolated from healthy donors, were added to the spheroids in the presence of various concentrations of NOX-A12 and an anti-CD20 mAb, either rituximab or obinutuzumab. The next day, spheroids were washed and dissociated for NK cell quantification and lymphoma cell viability determination by flow cytometry. We found that the ADCC efficacy of anti-CD20 mAbs is lower in 3D spheroids compared to conventional 2D assays due to low NK cell infiltration into the microtissues. Interestingly, NOX-A12 increased the amount of NK cells in the lymphoma-stroma spheroids up to 8-fold in a dose-dependent manner (Figure A), likely by forming de novo CXCL12 gradients into the dense microtissue due to the particular penetration characteristic of L-RNA aptamers. Of note, the NOX-A12-mediated increase of NK cells in the spheroids synergized with both anti-CD20 mAbs tested in terms of NK cell-mediated killing of lymphoma cells (Figure B). The present work complements the mechanism of action data of NOX-A12 by adding enhancement of NK cell infiltration into stroma-rich tumor compartments to the already established effects of mobilizing malignant and immune effector cells into the peripheral blood. These data as well as the good toxicity profile and the promising data in phase 2a clinical trials in patients with CLL and MM justify further clinical trials in patients with hematological malignancies to verify the greater efficacy of combination treatment using ADCC-inducing mAbs and NOX-A12. Figure Figure. Disclosures Zboralski: NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Vater:NOXXON Pharma AG: Employment.

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CAR-NK Cell: A New Paradigm in Tumor Immunotherapy

Frontiers in Oncology ◽

10.3389/fonc.2021.673276 ◽

2021 ◽

Vol 11 ◽

Author(s):

Faroogh Marofi ◽

Alaa S. Al-Awad ◽

Heshu Sulaiman Rahman ◽

Alexander Markov ◽

Walid Kamal Abdelbasset ◽

...

Keyword(s):

Nk Cells ◽

Immune Escape ◽

Nk Cell ◽

Tumor Immunotherapy ◽

Antigen Receptors ◽

New Paradigm ◽

Immune Effector ◽

Effector Cells ◽

Therapeutic Competence

The tumor microenvironment (TME) is greatly multifaceted and immune escape is an imperative attribute of tumors fostering tumor progression and metastasis. Based on reports, the restricted achievement attained by T cell immunotherapy reflects the prominence of emerging other innovative immunotherapeutics, in particular, natural killer (NK) cells-based treatments. Human NK cells act as the foremost innate immune effector cells against tumors and are vastly heterogeneous in the TME. Currently, there exists a rapidly evolving interest in the progress of chimeric antigen receptor (CAR)-engineered NK cells for tumor immunotherapy. CAR-NK cells superiorities over CAR-T cells in terms of better safety (e.g., absence or minimal cytokine release syndrome (CRS) and graft-versus-host disease (GVHD), engaging various mechanisms for stimulating cytotoxic function, and high feasibility for ‘off-the-shelf’ manufacturing. These effector cells could be modified to target various antigens, improve proliferation and persistence in vivo, upturn infiltration into tumors, and defeat resistant TME, which in turn, result in a desired anti-tumor response. More importantly, CAR-NK cells represent antigen receptors against tumor-associated antigens (TAAs), thereby redirecting the effector NK cells and supporting tumor-related immunosurveillance. In the current review, we focus on recent progress in the therapeutic competence of CAR-NK cells in solid tumors and offer a concise summary of the present hurdles affecting therapeutic outcomes of CAR-NK cell-based tumor immunotherapies.

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Cryptococcus neoformans Directly Stimulates Perforin Production and Rearms NK Cells for Enhanced Anticryptococcal Microbicidal Activity

Infection and Immunity ◽

10.1128/iai.01232-08 ◽

2009 ◽

Vol 77 (6) ◽

pp. 2436-2446 ◽

Cited By ~ 36

Author(s):

Kaleb J. Marr ◽

Gareth J. Jones ◽

Chunfu Zheng ◽

Shaunna M. Huston ◽

Martina Timm-McCann ◽

...

Keyword(s):

Nk Cells ◽

Cryptococcus Neoformans ◽

Prolonged Exposure ◽

De Novo ◽

Nk Cell ◽

K562 Cells ◽

Strontium Chloride ◽

Mrna Levels ◽

Microbicidal Activity ◽

Anticryptococcal Activity

ABSTRACT NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans.

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An Optimal Control Approach for Enhancing Natural Killer Cells’ Secretion of Cytolytic Molecules

10.1101/2020.08.05.238691 ◽

2020 ◽

Author(s):

Sahak Z. Makaryan ◽

Stacey D. Finley

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Phosphatase Activity ◽

Nk Cell ◽

Mathematical Framework ◽

Theoretic Approach ◽

Immune Effector ◽

Effector Cells ◽

Control Approach ◽

Information Theoretic Approach

ABSTRACTNatural killer (NK) cells are immune effector cells that can detect and lyse cancer cells. However, NK cell exhaustion, a phenotype characterized by reduced secretion of cytolytic models upon serial stimulation, limits the NK cell’s ability to lyse cells. In this work, we investigated in silico strategies that counteract the NK cell’s reduced secretion of cytolytic molecules. To accomplish this goal, we constructed a mathematical model that describes the dynamics of the cytolytic molecules granzyme B (GZMB) and perforin-1 (PRF1) and calibrated the model predictions to published, experimental data using a Bayesian parameter estimation approach. We applied an information-theoretic approach to perform a global sensitivity analysis, from which we found the suppression of phosphatase activity maximizes the secretion of GZMB and PRF1. However, simply reducing the phosphatase activity is shown to deplete the cell’s intracellular pools of GZMB and PRF1. Thus, we added a synthetic Notch (synNotch) signaling circuit to our baseline model as a method for controlling the secretion of GZMB and PRF1 by inhibiting phosphatase activity and increasing production of GZMB and PRF1. We found the optimal synNotch system depends on the frequency of NK cell stimulation. For only a few rounds of stimulation, the model predicts inhibition of phosphatase activity leads to more secreted GZMB and PRF1; however, for many rounds of stimulation, the model reveals that increasing production of the cytolytic molecules is the optimal strategy. In total, we developed a mathematical framework that provides actionable insight into engineering robust NK cells for clinical applications.

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Requirement and Redundancy of the Src Family Kinases Fyn and Lyn in Perforin-Dependent Killing of Cryptococcus neoformans by NK Cells

Infection and Immunity ◽

10.1128/iai.00533-13 ◽

2013 ◽

Vol 81 (10) ◽

pp. 3912-3922 ◽

Cited By ~ 19

Author(s):

Paul Oykhman ◽

Martina Timm-McCann ◽

Richard F. Xiang ◽

Anowara Islam ◽

Shu Shun Li ◽

...

Keyword(s):

Nk Cells ◽

Cryptococcus Neoformans ◽

Kinase Inhibitor ◽

Nk Cell ◽

Pathogenic Fungus ◽

Src Family Kinases ◽

Content Type ◽

Extracellular Signal ◽

Interfering Rna ◽

Anticryptococcal Activity

ABSTRACTNatural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungusCryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase–extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response toC. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blockedC. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse.

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Immunosurveillance of Cancer and Viral Infections with Regard to Alterations of Human NK Cells Originating from Lifestyle and Aging

Biomedicines ◽

10.3390/biomedicines9050557 ◽

2021 ◽

Vol 9 (5) ◽

pp. 557

Author(s):

Xuewen Deng ◽

Hiroshi Terunuma ◽

Mie Nieda

Keyword(s):

Nk Cells ◽

Viral Infections ◽

Nk Cell ◽

Healthy Lifestyle ◽

Cell Activity ◽

Nk Cell Activity ◽

Effector Cells ◽

Cell Dysfunction ◽

Forest Bathing ◽

Infected Cells

Natural killer (NK) cells are cytotoxic immune cells with an innate capacity for eliminating cancer cells and virus- infected cells. NK cells are critical effector cells in the immunosurveillance of cancer and viral infections. Patients with low NK cell activity or NK cell deficiencies are predisposed to increased risks of cancer and severe viral infections. However, functional alterations of human NK cells are associated with lifestyles and aging. Personal lifestyles, such as cigarette smoking, alcohol consumption, stress, obesity, and aging are correlated with NK cell dysfunction, whereas adequate sleep, moderate exercise, forest bathing, and listening to music are associated with functional healthy NK cells. Therefore, adherence to a healthy lifestyle is essential and will be favorable for immunosurveillance of cancer and viral infections with healthy NK cells.

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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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Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization

Journal of Translational Medicine ◽

10.1186/s12967-019-2055-4 ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 4

Author(s):

Allison B. Powell ◽

Sridevi Yadavilli ◽

Devin Saunders ◽

Stacey Van Pelt ◽

Elizabeth Chorvinsky ◽

...

Keyword(s):

Nk Cells ◽

Transforming Growth Factor ◽

Nk Cell ◽

Transforming Growth Factor Β ◽

In Vitro Models ◽

Dominant Negative ◽

Conditioned Media ◽

Retroviral Transduction ◽

Effector Cells

Abstract Background Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor β (TGF-β). Here, we address this challenge in in vitro models of MB. Methods CB-derived NK cells were modified to express a dominant negative TGF-β receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed. Results We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-β-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-β (decreased TGF-β levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008). Conclusions CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.

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