Increased Expression of TIGIT And KLRG1 Correlates With Impaired CD56Bright NK Cell Immunity In HPV16-Rrelated Cervical Intraepithelial Neoplasia

Abstract Background: The onset and progression of cervical intraepithelial neoplasia (CIN) are closely associated with the persistent infection of high-risk HPV (especially type16), which is mainly caused by immune escape. Natural killer (NK) cells play an important role against virally infected cells and tumor cells through a fine balance of signals from multiple surface receptors. Overexpression of non-MHC-I specific inhibitory receptors TIGIT, KLRG1, Siglec-7, LAIR-1, and CD300a on NK cells correlates with cellular exhaustion and immune evasion, but these receptors have not been investigated in CIN. The aim of the present study was to examine the potential role of NK cell non-MHC-I specific inhibitory receptors expression in immune escape from HPV16-related CIN patients .Methods: the subset distribution, IFN-γ and TNF-α expression levels and immunophenotype of TIGIT, KLRG1, Siglec-7, LAIR-1, and CD300a of NK cells were investigated in peripheral blood mononuclear cell samples by flow cytometry from 82 women, including HPV16(+) subjects with CIN 0, CIN I, and CIN II-III, and HPV subjects with CIN 0 (control). immunohistochemistry was applied to detect the expression of ligands for NK receptors in the cervical tissues. HPV types were identified by PCR assays.Results: The HPV16(+) subjects with high-grade lesions had an increased number of circulating peripheral blood CD56bright NK cells with reduced functionality and IFN-γ secretion. The expression levels of the inhibitory molecules TIGIT and KLRG1 on CD56bright NK cells increased in parallel with increasing CIN grade. In addition, TIGIT and KLRG1 related ligands, PVR, N-Cadherin and E-Cadherin expression level was also elevated with increasing CIN grade. Conclusions: Our results suggested that the reduced viability of CD56bright NK cells and increased TIGIT and KLRG1 expression represent an escape mechanism associated to persistent HPV16 infection and CIN progression. These results may facilitate the development of early-warning immune predictors and therapeutic strategies for HPV16-associated CIN based on the TIGIT and KLRG1 inhibitory pathways of NK cells.

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542 Expansion of cytotoxic NK Cells from PBMCs using individualized cytokine combination

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0542 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A578-A578

Author(s):

Andreia Maia ◽

Joana Lerias ◽

Markus Maeurer ◽

Mireia Castillo-Martin

Keyword(s):

Flow Cytometry ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Blood Donors ◽

Nk Cell ◽

Tumour Cells ◽

List Type ◽

Mhc I ◽

Cytokine Combination

BackgroundAdoptive immunotherapy relies on the use of T-cells to target tumour cells, through Major Histocompatibility Complex (MHC) Class I recognition(1). However, many tumours display alterations in the MHC-I pathway, a well-described immune evasion mechanism(2). Natural Killer (NK) cells recognize transformed cells independently from the presence of MHC-I and may be a reliable therapeutic option for patients with altered tumour MHC-I expression. The source of NK cells may be autologous or allogeneic and NK cells are also clinically relevant recipients of transgenic receptors (TCRs or antibodies) targeting tumour cells. NK cells have been categorized according to their CD56 and CD16 surface expression into different subpopulations: cytotoxic (CD56+CD16+) and regulatory (CD56brightCD16-)(3). Expanding cytotoxic NK cells is challenging, since the frequency of NK cells is low in peripheral blood(4) and there is also – at this point – not an optimal expansion protocol available.The goal of this project is to determine the best cytokine combination that facilitates expansion of cytotoxic NK cells that either target tumor cells directly or serve as recipients for transgenic receptors.MethodsPeripheral Blood Mononuclear Cells (PBMCs) were extracted using Ficoll methodology from blood donors and cultured in T25 flasks with Cell Genix Medium supplemented with 10% human serum and antibiotics. NK cells were expanded supplemented with feeder cells (ratio 1:1) and different cytokine combinations (1000 U/mL of IL-2, 10 U/ml of IL-12, 180 U/mL of IL-15 and/or 1 U/mL of IL-21) during 20 days. The immunophenotype of expanded NK cells was analyzed at days 0, 5, 10, 15 and 20 by flow cytometry. The cytotoxicity of NK cells was measured by a CD107a Assay or by a Total Cytotoxicity and Apoptosis Assay at days 10 and 20. Thirteen different cytokine combinations were tested.Results4/13 cytokine combinations produced a statistically significant increase of the absolute number of NK cells with a higher percentage of cytotoxic NK cells (figure 1). However, induction of cytotoxicity was not associated with a strong NK cell expansion. The regulatory NK cells subset (CD56brightCD16-) showed the highest percentage of CD107a-expressing cells, more than the CD56+CD16+, the most cytotoxic subpopulation of NK cells.Abstract 542 Figure 1Representative percentage of NK cells in total lymphocytes (A), CD56+CD16+ subpopulation in total NK cells (B), and CD56brightCD16- subpopulation amongst total NK cells (C) at different time points (5, 10, 15 and 20 days) expanded from PBMCs* p-value < 0.05ConclusionsThis work shows that we are able to grow and efficiently expand NK cells from PBMCs with different cytokine combinations leading to clinically relevant NK cell numbers as well as cytotoxic functions. This enables to produce NK cell products for therapy and as recipients for transgenic tumor antigen-specific receptors.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank, the Vivarium Facility and the Flow Cytometry Platform of the Champalimaud Centre for the Unknown.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon.ConsentWritten informed consent was obtained from the blood donors to use their samples for research purposes.ReferencesRosenberg SA, Restifo NP, Yang JC, Morgan RA, Mark E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8(4):299–308.Aptsiauri N, Ruiz-Cabello F, Garrido F. The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses. Curr Opin Immunol 2018;51:123–32.Di Vito C, Mikulak J, Mavilio D. On the way to become a natural killer cell. Front Immunol. 2019;10(August):1–15.Zotto G Del, Antonini F, Pesce S, Moretta F, Moretta L. Comprehensive phenotyping of human PB NK Cells by Flow Cytometry. 2020;1–9.

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Combined Stimulation with Interleukin-18 and Interleukin-12 Potently Induces Interleukin-8 Production by Natural Killer Cells

Journal of Innate Immunity ◽

10.1159/000477172 ◽

2017 ◽

Vol 9 (5) ◽

pp. 511-525 ◽

Cited By ~ 5

Author(s):

Sophie M. Poznanski ◽

Amanda J. Lee ◽

Tina Nham ◽

Evan Lusty ◽

Margaret J. Larché ◽

...

Keyword(s):

Immune Response ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Nk Cell ◽

Critical Role ◽

Interleukin 12 ◽

Tnf Α ◽

Ifn Γ

The combination of interleukin (IL)-18 and IL-12 (IL-18+IL-12) potently stimulates natural killer (NK) cells, triggering an innate immune response to infections and cancers. Strategies exploiting the effects of IL-18+IL-12 have shown promise for cancer immunotherapy. However, studies have primarily characterized the NK cell response to IL-18+IL-12 in terms of interferon (IFN)-γ production, with little focus on other cytokines produced. IL-8 plays a critical role in activating and recruiting immune cells, but it also has tumor-promoting functions. IL-8 is classically produced by regulatory NK cells; however, cytotoxic NK cells do not typically produce IL-8. In this study, we uncover that stimulation with IL-18+IL-12 induces high levels of IL-8 production by ex vivo expanded and freshly isolated NK cells and NK cells in peripheral blood mononuclear cells. We further report that tumor necrosis factor (TNF)-α, produced by NK cells following IL-18+IL-12 stimulation, regulates IL-8 production. The IL-8 produced is in turn required for maximal IFN-γ and TNF-α production. These findings may have important implications for the immune response to infections and cancer immunotherapies. This study broadens our understanding of NK cell function and IL-18+IL-12 synergy by uncovering an unprecedented ability of IL-18+IL-12-activated peripheral blood NK cells to produce elevated levels of IL-8 and identifying the requirement for intermediates induced by IL-18+IL-12 for maximal cytokine production following stimulation.

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Essential Role of c-MYC for Natural Killer Cell Development, Proliferation and Anti-Tumor Activity

Blood ◽

10.1182/blood.v130.suppl_1.786.786 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 786-786

Author(s):

Yuting Tang ◽

Xiaomei Yan ◽

Rui Huang ◽

Yoshihiro Hayashi ◽

Yue Zhang ◽

...

Keyword(s):

Innate Immunity ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Nk Cell ◽

Cell Development ◽

Brdu Incorporation ◽

Dependent Manner ◽

Tumor Surveillance ◽

Ifn Γ

Abstract Natural killer (NK) cells are the major component of innate immunity with both cytotoxicity and cytokine producing effector functions. NK cells also regulate the interplay between innate immunity and adaptive immunity by secreting certain cytokines. Extrinsic regulators of NK cell development and function, including diverse ligands of NK cell receptors and cytokines from the microenvironment, have been extensively studied. However, intrinsic regulators for NK cell biology are still less understood. In our previous study on aggressive NK cell leukemia (ANKL), genomics and transcriptomics analyses indicated that c-MYC was universally upregulated and responsible for the proliferation and survival in ANKL cells (Manuscript in revision). Furthermore, STAT5, as a transcriptional regulator of c-MYC, was found to be essential in the survival and development of NK cells (Eckelhart et al., Blood 2011). In this regard, we want to understand the physiological and oncological roles of c-MYC in NK cells. To achieve our goal, we made two mouse models including c-Myc loss-of-function (LOF) and c-Myc gain-of-function (GOF) in NK cells. Ncr1Cre knock-in mice, in which Cre recombinase was inserted into the Nkp46 locus, was used. We crossed the c-Mycf/f mice with Ncr1Cre mice to generate the NK cell specific c-Myc LOF model c-MycΔ/Δ/Ncr1Cre. To generate the c-Myc GOF model, we crossed the Tg(tetO-MYC) mice with Ncr1Cre and Rosa26-Loxp-Stop-Loxp(LSL)-rtTA-GFP mice to get the Tg(tetO-MYC)/ Ncr1Cre/LSL-rtTA-GFP (iMYC) mice, in which c-Myc expression can be induced in a doxycycline dependent manner in NK cells. c-MycΔ/Δ/Ncr1Cre mice were analyzed between 6 to 14-weeks old. iMYC mice were induced by doxycycline from 6-weeks old for over 2 months and then analyzed. Wild type littermates were used as controls. In both models, mice were born normally and showed no obvious difference in growth compared to their littermates. In c-MycΔ/Δ/Ncr1Cre mice, a significant reduction of NK1.1+/DX5+ NK cell percentages in peripheral blood (3.6 ± 0.4% vs. 0.5 ± 0.1%, P < 0.0001, N=7) and spleen (2.4 ± 0.5% vs. 0.7 ± 0.1%, P < 0.01, N=6) was detected. In addition, the percentage of CD11b+ mature NK cells in the NK1.1+/DX5+ population was also reduced. In bone marrow (BM), although the total percentage of NK1.1+/DX5+ NK cells did not change, an obvious block of NK cell development was seen, as the majority of NK1.1+/DX5+ cells in BM were CD27+/CD11b- cells, which represent an immature pattern. To assess whether the NK cell proliferation is altered in this model, we performed BrdU labeling assays and found that BrdU incorporation rates decreased dramatically both in peripheral NK cells and BM NK progenitors. Functionally, we measured the IFN-γ secretion of splenic NK cells after PMA/Ionomycin stimulation. We found that the percentage of IFN-γ positive NK cells decreased significantly in c-MycΔ/Δ/Ncr1Cre mice (77.1 ± 7.0% vs. 53.8 ± 1.0%, N=3). Consistent with these data, the tumor surveillance was also severely impaired in this LOF model, as the number of lung metastatic sites significantly increased compared to the control mice in a B16F10 transplantation assay. In contrast to the LOF model, in our GOF model, the NK1.1+/DX5+ NK cell number in peripheral blood increased (3.1 ± 0.2% vs. 4.4 ± 0.5%, P < 0.05, N=7). Additionally, a small increase in the percentage of CD27-/CD11b+ population in NK1.1+/DX5+ cells was seen. Interestingly, however, the ability of IFN-γ secretion of splenic NK cells after PMA/Ionomycin stimulation was decreased in iMYC mice (72.8 ± 0.3% vs. 58.7 ± 1.1%, N=3), which showed the same alteration observed in c-MycΔ/Δ/Ncr1Cre mice. This result is probably not due to the impaired maturation, but rather it is the result of the higher percentage of CD27-/CD11b+ cells, which were considered terminally differentiated NK cells with lower cytotoxic functions. In summary, we found that c-Myc is essential for NK cell development, proliferation, and tumor surveillance. NK cell maturation and proliferation were impaired in the c-Myc LOF models and were boosted in the c-Myc GOF models. Our results also provide a mechanism basis for the potential application of targeting c-Myc in NK cells ex vivo or in vivo expansion, and NK-mediated immunotherapy. Future studies are needed to delineate the underlying mechanisms and explore the applications. Disclosures No relevant conflicts of interest to declare.

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Phenotypic and Functional Changes in Peripheral Blood Natural Killer Cells in Crohn Disease Patients

Mediators of Inflammation ◽

10.1155/2020/6401969 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15

Author(s):

Suzanne Samarani ◽

Patrick Sagala ◽

Prevost Jantchou ◽

Guy Grimard ◽

Christophe Faure ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Crohn Disease ◽

Peripheral Blood ◽

Nk Cell ◽

Healthy Controls ◽

Inhibitory Receptors ◽

Color Flow ◽

Functional Changes ◽

Treatment Naïve

We investigated activation status, cytotoxic potential, and gut homing ability of the peripheral blood Natural Killer (NK) cells in Crohn disease (CD) patients. For this purpose, we compared the expression of different activating and inhibitory receptors (KIR and non-KIR) and integrins on NK cells as well as their recent degranulation history between the patients and age-matched healthy controls. The study was conducted using freshly obtained peripheral blood samples from the study participants. Multiple color flow cytometry was used for these determinations. Our results show that NK cells from treatment-naïve CD patients expressed higher levels of activating KIR as well as other non-KIR activating receptors vis-à-vis healthy controls. They also showed increased frequencies of the cells expressing these receptors. The expression of several KIR and non-KIR inhibitory receptors tended to decrease compared with the cells from healthy donors. NK cells from the patients also expressed increased levels of different gut-homing integrin molecules and showed a history of increased recent degranulation events both constitutively and in response to their in vitro stimulation. Furthermore, treatment of the patients tended to reverse these NK cell changes. Our results demonstrate unequivocally, for the first time, that peripheral blood NK cells in treatment-naïve CD patients are more activated and are more poised to migrate to the gut compared to their counterpart cells from healthy individuals. Moreover, they show that treatment of the patients tends to normalize their NK cells. The results suggest that NK cells are very likely to play a role in the immunopathogenesis of Crohn disease.

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Inhibitory receptors specific for MHC class I educate murine NK cells but not CD8αα intestinal intraepithelial T lymphocytes

Blood ◽

10.1182/blood-2011-01-331124 ◽

2011 ◽

Vol 118 (2) ◽

pp. 339-347 ◽

Cited By ~ 11

Author(s):

Sylvie Taveirne ◽

Jessica Filtjens ◽

Els Van Ammel ◽

Veerle De Colvenaer ◽

Tessa Kerre ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Cell Types ◽

Cell Receptor ◽

Intraepithelial Lymphocytes ◽

Class I ◽

Inhibitory Receptors ◽

Class I Mhc ◽

Mhc I ◽

Inhibitory Function

Abstract The engagement of inhibitory receptors specific for major histocompatibility complex class I (MHC-I) molecules educates natural killer (NK) cells, meaning the improvement of the response of activation receptors to subsequent stimulation. It is not known whether inhibitory MHC-I receptors educate only NK cells or whether they improve the responsiveness of all cell types, which express them. To address this issue, we analyzed the expression of inhibitory MHC-I receptors on intestinal intraepithelial lymphocytes (iIELs) and show that T-cell receptor (TCR)-αβ CD8αα iIELs express multiple inhibitory receptors specific for MHC-I molecules, including CD94/NKG2A, Ly49A, and Ly49G2. However, the presence of MHC-I ligand for these receptors did not improve the response of iIELs to activation via the TCR. The absence of iIEL education by MHC-I receptors was not related to a lack of inhibitory function of these receptors in iIELs and a failure of these receptors to couple to the TCR. Thus, unlike NK cells, iIELs do not undergo an MHC-I–guided education process. These data suggest that education is an NK cell–specific function of inhibitory MHC-I receptors.

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Soluble and Exosome-Bound α-Galactosylceramide Mediate Preferential Proliferation of Educated NK Cells with Increased Anti-Tumor Capacity

Cancers ◽

10.3390/cancers13020298 ◽

2021 ◽

Vol 13 (2) ◽

pp. 298

Author(s):

Arnika K. Wagner ◽

Ulf Gehrmann ◽

Stefanie Hiltbrunner ◽

Valentina Carannante ◽

Thuy T. Luu ◽

...

Keyword(s):

Nk Cells ◽

Cancer Immunotherapy ◽

Nk Cell ◽

Mouse Strains ◽

Target Cells ◽

Class I Mhc ◽

Mhc I ◽

Cell Responses ◽

Missing Self

Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response.

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Production of colony-stimulating activity by human natural killer cells: analysis of the conditions that influence the release and detection of colony-stimulating activity

Blood ◽

10.1182/blood.v74.1.156.bloodjournal741156 ◽

1989 ◽

Vol 74 (1) ◽

pp. 156-164

Author(s):

V Pistoia ◽

S Zupo ◽

A Corcione ◽

S Roncella ◽

L Matera ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Nk Cell ◽

K562 Cells ◽

Cell Suspensions ◽

Normal Peripheral Blood ◽

Colony Stimulating Activity ◽

Culture Supernatants

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony- inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released. However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

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584 Powerful synergistic effects of a STING agonist and the IL-2 superkine, H9, in eliciting NK and T cell responses against MHC I- and MHC I+ tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.584 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A614-A614

Author(s):

Natalie Wolf ◽

Cristina Blaj ◽

Lora Picton ◽

Gail Snyder ◽

Li Zhang ◽

...

Keyword(s):

T Cell ◽

Combination Therapy ◽

Nk Cells ◽

Nk Cell ◽

Tumor Regression ◽

T Cell Responses ◽

Cd8 T Cell ◽

Mhc I ◽

Therapy Regimen ◽

Cell Responses

BackgroundMost current cancer immunotherapies are based on mobilizing CD8 T cell responses. However, many types of tumors evade CD8 T cell recognition by displaying few or no antigens, or losing expression of MHC I. These considerations underlie the need for complementary therapies that mobilize other antitumor effector cells, such as NK cells, which preferentially kill MHC I-deficient cells. Cyclic dinucleotides (CDNs) activate the cGAS-STING pathway of the innate immune system and are candidates as immunotherapy agents. Intratumoral CDN injections induce type I IFNs and other mediators that amplify the CD8 T cell response and induce tumor regression [1]. CDN therapy also induces long-term tumor regressions in some MHC I-deficient tumor models, mediated primarily by NK cells [2].MethodsTo extend the efficacy of CDN therapy, we combined the IL-2 superkine, H9, or half-life extended H9, with CDNs to target and activate NK cells in the tumor microenvironment and prevent or delay the onset of NK cell desensitization [3,4]. In these studies, we utilized B16-F10 and MC38 tumor cells lacking B2m to examine effects of the combination therapy on MHC I-deficient tumor growth as well as to examine the activation of NK cells by flow cytometry and cytotoxicity assays. We also utilized B16-F10 WT and the spontaneous tumor model, MCA, to assess the effect of the combination therapy on MHC I+ tumors.ResultsHere we show that H9 synergized with CDN therapy to mobilize much more powerful antitumor responses against MHC I-deficient tumors than CDN alone. The responses were mediated by NK cells and in some cases CD4 T cells, and were accompanied by increased recruitment to and sustained activation of NK cells in the tumor. This combination therapy regimen activated NK cells systemically, as shown by antitumor effects distant from the site of CDN injection and enhanced cytolytic activity of splenic NK cells against tumor cell targets ex vivo. Finally, the same combination therapy regimen synergistically mobilized powerful CD8 T cell responses in the case of MHC I+ tumor cells, suggesting the generality of the approach. The approach was effective against primary sarcomas, as well, especially when combined with checkpoint therapy, leading to tumor regressions and long-term survival of many mice with MCA-induced sarcoma.ConclusionsOverall, our work demonstrates the impact of a novel combination therapy in mobilizing powerful NK and T cell-mediated antitumor activity, providing important justification for evaluating this approach for treating cancers that are refractory to available treatment options.ReferencesCorrales, L., Glickman, L.H., McWhirter, S.M., Kanne, D.B., Sivick, K.E., Katibah, G.E., Woo, S.R., Lemmens, E., Banda, T., Leong, J.J., et al. (2015). Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep 11, 1018–1030.Nicolai, C.J., Wolf, N., Chang, I.C., Kirn, G., Marcus, A., Ndubaku, C.O., McWhirter, S.M., and Raulet, D.H. (2020). NK cells mediate clearance of CD8(+) T cell-resistant tumors in response to STING agonists. Science immunology 5, eaaz2738.Levin, A.M., Bates, D.L., Ring, A.M., Krieg, C., Lin, J.T., Su, L., Moraga, I., Raeber, M.E., Bowman, G.R., Novick, P., et al. (2012). Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. Nature 484, 529–533.Ardolino, M., Azimi, C.S., Iannello, A., Trevino, T.N., Horan, L., Zhang, L., Deng, W., Ring, A.M., Fischer, S., Garcia, K.C., and Raulet, D.H. (2014). Cytokine therapy reverses NK cell anergy in MHC-deficient tumors. J Clin Invest 124, 4781–4794.

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151 Potentiating the Large-Scale Expansion and Engineering of Peripheral Blood-Derived CAR NK Cells for Off-the-Shelf Application

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.151 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A159-A159

Author(s):

Michael Whang ◽

Ming-Hong Xie ◽

Kate Jamboretz ◽

Hadia Lemar ◽

Chao Guo ◽

...

Keyword(s):

T Cells ◽

Cell Therapy ◽

Nk Cells ◽

Peripheral Blood ◽

Nk Cell ◽

Adoptive Cell Therapy ◽

Therapeutic Window ◽

Target Cells ◽

Healthy Donors ◽

Custom Made

BackgroundPeripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population.MethodsUsing the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte.ResultsIsolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay.ConclusionsIn this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics.Ethics ApprovalHuman samples were collected with written informed consent by an approved vendor.

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Tissue Trafficking Kinetics of Rhesus Macaque Natural Killer Cells Measured by Serial Intravascular Staining

Frontiers in Immunology ◽

10.3389/fimmu.2021.772332 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ryland D. Mortlock ◽

Chuanfeng Wu ◽

E. Lake Potter ◽

Diana M. Abraham ◽

David S. J. Allan ◽

...

Keyword(s):

Steady State ◽

Nk Cells ◽

Tissue Distribution ◽

Natural Killer ◽

Rhesus Macaque ◽

Peripheral Blood ◽

Cell Biology ◽

Nk Cell ◽

Lymphoid Tissues ◽

Intravascular Staining

The in vivo tissue distribution and trafficking patterns of natural killer (NK) cells remain understudied. Animal models can help bridge the gap, and rhesus macaque (RM) primates faithfully recapitulate key elements of human NK cell biology. Here, we profiled the tissue distribution and localization patterns of three NK cell subsets across various RM tissues. We utilized serial intravascular staining (SIVS) to investigate the tissue trafficking kinetics at steady state and during recovery from CD16 depletion. We found that at steady state, CD16+ NK cells were selectively retained in the vasculature while CD56+ NK cells had a shorter residence time in peripheral blood. We also found that different subsets of NK cells had distinct trafficking kinetics to and from the lymph node as well as other lymphoid and non-lymphoid tissues. Lastly, we found that following administration of CD16-depleting antibody, CD16+ NK cells and their putative precursors retained a high proportion of continuously circulating cells, suggesting that regeneration of the CD16 NK compartment may take place in peripheral blood or the perivascular compartments of tissues.

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