Metabolic changes of Interleukin‑12/15/18‑stimulated human NK cells

Natural Killer (NK) cells acquire memory-like properties following a brief stimulation with IL-12, IL-15 and IL-18. These IL-12/15/18-preactivated NK cells, also known as cytokine-induced memorylike (CIML) NK cells, have been revealed as a powerful tool in cancer immunotherapy due to their persistence in the host and their increased effector functions. Several studies have shown that NK cells modulate their metabolism in response to cytokine-stimulation and other stimuli, suggesting that there is a link between metabolism and cellular functions. In this paper, we have analyzed metabolic changes associated to IL-12/15/18-stimulation and the relevance of glycolytic pathway for NK cell effector functions. We have found CIML NK cells are able to retain a metabolic profile shifted towards glycolysis seven days after cytokine withdrawal. Furthermore, we found that treatment with 2-DG differently affects distinct NK cell effector functions and is stimuli-dependent. These findings may have implications in the design of NK cell-based cancer immunotherapies.This work was supported by the following grants: AECC-Spanish Association Against Cancer (PROYE16074BORR) and Health Department, Basque Government (2019222027 and 2020333024). I.T. is recipient of a fellowship from the Jesús de Gangoiti Barrera Foundation (FJGB18/002) and a predoctoral contract funded by the Department of Education, Basque Government (PRE_2020_2_0007). A.O. is recipient of a fellowship from the Jesús de Gangoiti Barrera Foundation (FJGB19/002). O.Z. is recipient of a postdoctoral contract funded by “Instituto de Salud Carlos III-Contratos Sara Borrell 2017 (CD17/00128)” and the European Social Fund (ESF)-The ESF invests in your future. F.B. is an Ikerbasque Research Professor, Ikerbasque, Basque Foundation for Science

Increased expression levels of CD300c on basophils from allergic individuals

CD300c is a co-stimulatory receptor for IgE-mediated signals on human basophils. Basophils from allergic individuals exhibit higher expression levels of CD300c. This receptor could serve as a potential therapeutic target for the treatment of IgEdependent allergy.This study was supported by a grant from “Instituto de Salud Carlos III” through the project PI16/01223 (co-funded by European Regional Development Fund “A way to make Europe”)

Implication of Interleukin-12/15/18 and Ruxolitinib in the Phenotype, Proliferation, and Polyfunctionality of Human Cytokine-Preactivated Natural Killer Cells

A brief in vitro stimulation of natural killer (NK) cells with interleukin (IL)-12, IL-15, and IL-18 endow them a memory-like behavior, characterized by higher effector responses when they are restimulated after a resting period of time. These preactivated NK cells, also known as cytokine-induced memory-like (CIML) NK cells, have several properties that make them a promising tool in cancer immunotherapy. In the present study, we have described the effect that different combinations of IL-12, IL-15, and IL-18 have on the generation of human CIML NK cells. Our data points to a major contribution of IL-15 to CIML NK cell-mediated cytotoxicity against target cells. However, the synergistic effect of the three cytokines grant them the best polyfunctional profile, that is, cells that simultaneously degranulate (CD107a) and produce multiple cytokines and chemokines such as interferon γ, tumor necrosis factor α, and C-C motif chemokine ligand 3. We have also analyzed the involvement of each cytokine and their combinations in the expression of homing receptors CXCR4 and CD62L, as well as the expression of CD25 and IL-2-induced proliferation. Furthermore, we have tested the effects of the Jak1/2 inhibitor ruxolitinib in the generation of CIML NK cells. We found that ruxolitinib-treated CIML NK cells expressed lower levels of CD25 than non-treated CIML NK cells, but exhibited similar proliferation in response to IL-2. In addition, we have also found that ruxolitinib-treated NK cells displayed reduced effector functions after the preactivation, which can be recovered after a 4 days expansion phase in the presence of low doses of IL-2. Altogether, our results describe the impact that each cytokine and the Jak1/2 pathway have in the phenotype, IL-2-induced proliferation, and effector functions of human CIML NK cells.This study was supported by grants from Health Department, Basque Government (2013111034 and 2017222005), Basque Foundation for Research and Innovation- EiTB Maratoia (BIO14/TP/003) and AECC-Spanish Association Against Cancer (PROYE16074BORR)

Increased Frequency of CTLA-4 and PD-1 Expressing Regulatory T Cells and Basophils With an Activating Profile in Infants With Moderate-to-Severe Atopic Dermatitis Hypersensitized to Food Allergens

Background: Infants with severe atopic dermatitis (AD) may be sensitized to foods that have not been introduced into their diet, posing a risk for developing an immediate hypersensitivity reaction on the first exposure to the food to which they are sensitized. The aim of this work was to perform an analysis of the sensitization profile in infants with moderate-to-severe AD and to identify cellular and molecular markers for food allergy (FA). Methods: Blood samples from healthy donors and children with moderate-to-severe AD were studied. Specific IgE to several allergens were determined using ImmunoCAP FEIA system and ISAC technology. Furthermore, using flow cytometry-based studies, basophils and regulatory T (Treg) cells were phenotypically characterized. Results: 90% of children with AD were sensitized to food antigens before introducing them into the diet, and 100% developed FA. Phenotypic analysis showed a significantly higher percentage of CTLA-4 and PD-1 expressing Treg cells in AD patients than in healthy controls. Basophils from patients exhibited a marked reduction in the expression of CD300a, higher expression of Fc+RI and CXCR4, and to some extent higher expression of CD63 and CD300c. Conclusions: Infants with moderate-to-severe AD are at high risk of being sensitized to food allergens. Therefore, to avoid allergic reactions, broad-spectrum sensitization studies are necessary before introducing complementary diet. Increased expression of CTLA-4 and PD-1 suggests greater suppressive potential of Treg cells in infants with AD than healthy controls. Furthermore, our results suggest a role for CD300 molecules on circulating basophils as possible biomarkers for FA susceptibility.This study has been funded by the Department of Health, Basque Government through the Project 2019111031 and by a grant from the Agencia Estatal de Investigación Project PID2019- 109583RB-I00/AEI/10.13039/501100011033. OZ is recipient of a postdoctoral contract funded by Instituto de Salud Carlos IIIContratos Sara Borrell 2017 (CD17/0128) and the European Social Fund (ESF)-The ESF invests in your future. IT is recipient of a predoctoral contract funded by the Department of Education, Basque Government (PRE_2020_2_0007). AO is recipient of a fellowship from the Jesús de Gangoiti Barrera Foundation (FJGB20/007). GA-P is the recipient of a fellowship from the BBK Fundazioa (BBK/PRACT/20/001). FB is an Ikerbasque Research Professor, Ikerbasque, Basque Foundation for Science

Monocytes Phenotype and cytokine Production in human immunodeficiency Virus-1 infected Patients receiving a Modified Vaccinia ankara-Based hiV-1 Vaccine: relationship to cD300 Molecules expression

A modified vaccinia Ankara-based HIV-1 vaccine clade B (MVA-B) has been tested for safety and immunogenicity in low-risk human immunodeficiency virus (HIV)-uninfected individuals and as a therapeutic vaccine in HIV-1-infected individuals on combined antiretroviral therapy (cART). As a therapeutic vaccine, MVA-B was safe and broadly immunogenic; however, patients still showed a viral rebound upon treatment interruption. Monocytes are an important part of the viral reservoir and several studies suggest that they are partly responsible for the chronic inflammation observed in cART-treated HIV-infected people. The CD300 family of receptors has an important role in several diseases, including viral infections. Monocytes express CD300a, c, e, and f molecules and lipopolysaccharide (LPS) and other stimuli regulate their expression. However, the expression and function of CD300 receptors on monocytes in HIV infection is still unknown. In this work, we investigated for the first time the expression of CD300 molecules and the cytokine production in response to LPS on monocytes from HIV-1-infected patients before and after vaccination with MVA-B. Our results showed that CD300 receptors expression on monocytes from HIV-1-infected patients correlates with markers of HIV infection progression and immune inflammation. Specifically, we observed a positive correlation between the expression of CD300e and CD300f receptors on monocytes with the number of CD4+ T cells of HIV-1-infected patients before vaccination. We also saw a positive correlation between the expression of the inhibitory receptor CD300f and the expression of CD163 on monocytes from HIV-1-infected individuals before and after vaccination. In addition, monocytes exhibited a higher cytokine production in response to LPS after vaccination, almost at the same levels of monocytes from healthy donors. Furthermore, we also described a correlation in the expression of CD300e and CD300f receptors with TNF-α production in response to LPS, only in monocytes of HIV-1-infected patients before vaccination. Altogether, our results describe the impact of HIV-1 and of the MVA-B vaccine in cytokine production and monocytes phenotype.This study was supported by grants from “Plan Estatal de I+ D+ I 2013–2016, ISCIII-Subdirección de Evaluación y Fomento de la Investigación-Fondo Europeo de Desarrollo Regional (FEDER) (Grants PI13/00889, PI15/00480), and Marie Curie Actions, Career Integration Grant, European Commission (Grant CIG 631674).” The study was also partially supported by grants: EC10-153, TRA-094, SAF2015-66193-R,RIS [Red Temática Cooperativa de Grupos de Investigación en Sida del Fondo de Investigación Sanitaria (FIS)], HIVACAT (Catalan Program for the development of HIV-1 vaccines). JV is recipient of a predoctoral contract from the Department of Education, Language Policy, and Culture, Basque Government and a fellowship from the Jesús de Gangoiti Barrera Foundation

Polyfunctional HIV-1 specific response by CD8+ T lymphocytes expressing high levels of CD300a

CD300a receptor is found on different CD8+ T cell subsets and its expression has been associated to a more cytotoxic molecular signature. CD300a has an important role in some viral infections and its expression levels are known to be modulated by human immunodeficiency virus (HIV)−1 infection on several cell types. The main objective of this work was to investigate CD300a expression and its regulation during HIV-1 specific CD8+ T cell responses. CD300a receptor expression was analysed by multiparametric flow cytometry on CD8+ T lymphocytes from HIV negative donors, naive HIV-1+ individuals and HIV-1+ subjects under suppressive combined antiretroviral therapy (cART). HIV-1 specific CD8+ T cell response was studied by stimulating cells with HIV-1 derived peptides or with a Gag HIV-1 peptide. Our results showed that HIV-1 specific CD8+ T cells expressing higher levels of CD300a were more polyfunctional showing an increased degranulation and cytokine production. Moreover, we observed an up-regulation of CD300a expression after Gag HIV-1 peptide stimulation. Finally, our results demonstrated an inverse correlation between CD300a expression on CD8+ T lymphocytes and HIV disease progression markers. In conclusion, CD300a expression is associated to a better and more polyfunctional HIV-1 specific CD8+ T cell responseThis study was supported by a grant from “Plan Estatal de I+ D+ I 2013–2016, ISCIII-Subdirección de Evaluación y Fomento de la Investigación- Fondo Europeo de Desarrollo Regional (FEDER) (Grant PI13/00889)” and Marie Curie Actions, Career Integration Grant, European Commission (Grant CIG 631674)

IL-12/15/18-induced cell death and mitochondrial dynamics of human NK cells

Natural killer (NK) cells are lymphocytes with potent antitumor functions and, consequently, several NK cell-based strategies have been developed for cancer immunotherapy. A remarkable therapeutic approach is the adoptive transfer of NK cells stimulated with IL-12, IL-15 and IL-18. This cytokine stimulation endows NK cells with properties that resemble immunological memory and, for this reason, they are known as cytokine-induced memorylike (CIML) NK cells. Very promising results have been reported in clinical trials and yet, there are still unknown aspects of CIML NK cells. Here, we have conducted a preliminary study of their mitochondrial dynamics. Our results show that upon IL-12/15/18 stimulation the viability of NK cells decreased and an increment in mitochondrial superoxide levels was observed. In addition, we found that mitochondria appeared slightly elongated and their cristae density decreased following IL-12/15/18 stimulation, possibly in a process mediated by the low levels of optic atrophy type 1 (OPA1) protein. Interestingly, although mitophagy was slightly impaired, an increase in autophagic flux was observed, which might explain the reduced viability and the accumulation of unfit mitochondria. Our findings could be of relevance in order to design new strategies intended to improve the mitochondrial fitness of IL-12/15/18-stimulated NK cells with the aim of improving their therapeutic efficacy.The research in FB lab was supported by Fundacioı́n AECCSpanish Association Against Cancer Foundation (PROYE16074BORR) and Health Department, Basque Government (2022333018) and BBK Fundazioa (BBK22/3196). The research in LS lab was supported by Italian Cancer Research Association (AIRC) grant IG19991. IT and AA-I are recipient of a predoctoral contract funded by the Department of Education, Basque Government (PRE_2021_2_0215 and PRE_2022_1_0063). AL-P is recipient of a predoctoral contract funded by La Caixa Foundation (100010434; LCF/BQ/DI22/ 11940012). GA-P is recipient of a predoctoral contract funded by Fundacioı́n AECC-Spanish Association Against Cancer Foundation (PRDVZ21440ASTA). GA-P and AA-I are recipient of grants from Jesuı́s de Gangoiti Barrera Foundation (FJGB21/001 and FJBG21/005). AS is recipient of a grant from Margarita Salas program, for the requalification of the Spanish university system 2021-2023, financed by European Union - Next Generation EU. LA is an Ikerbasque Research Fellow and FB is an Ikerbasque Research Professor, Ikerbasque, Basque Foundation for Science

CD300a inhibits CD16-mediated NK cell effector functions in HIV-1-infected patients

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) through CD16 has a critical role in anti-human immunodeficiency virus (HIV) responses. CD300a, an inhibitory receptor highly expressed on NK cells, has the capacity to diminish NK cell killing of pseudorabies-infected cells. CD300a expression is altered during HIV-1 infection on B cells and CD4+ T lymphocytes. In this work, we have investigated in a cross-sectional study the expression and function of CD300a on NK cells from healthy donors, untreated HIV-1 infected subjects and patients on combined antiretroviral treatment (cART). First, we detected an expansion of a CD300a-expressing CD56neg NK cell subset in untreated HIV-1 infected patients. In addition, an association between CD300a expression and other NK cell surface receptors was observed in both healthy and HIV-1 infected people. Notably, we also described that CD300a exerted an inhibitory effect in CD16-mediated effector functions, including degranulation and cytokine production, in all donors and NK cell subsets and, more importantly, this inhibitory effect was higher in HIV-1 infected patients. Therefore, the CD300a inhibitory receptor could be proposed as a new target for therapies aimed to improve NK cells effector functions in HIV-1 infected patients

CD300a identifies a CD4R memory T cell subset with a higher susceptibility to HIV-1 infection

Human CD300a is known to promote the infection by dengue and other enveloped viruses and is overexpressed on CD4R T cells from HIV-1-infected patients.We found that infected CD4RRAS T cells from untreated HIV-1-infected patients were mostly CD300aR. Furthermore, CD300a expressing CD4RRAS T cells from healthy donors were significantly more infected by HIV-1 in vitro than CD300aS cells. CD300a might represent a biomarker of susceptibility to HIV-1 infection on memory CD4R T lymphocytes.The study was supported by a grant from ‘Plan Estatal de IþDþI 2013–2016, ISCIII-Subdirección de Evaluación y Fomento de la Investigación-Fondo Europeo de Desarrollo Regional (FEDER) (Grant PI13/00889)’ and Marie Curie Actions, Career Integration Grant, European Commission (Grant CIG 631674)

T Cell Activation, Highly Armed Cytotoxic Cells and a Shift in Monocytes CD300 Receptors Expression Is Characteristic of Patients With Severe COVID-19

COVID-19 manifests with a wide diversity of clinical phenotypes characterized by dysfunctional and exaggerated host immune responses. Many results have been described on the status of the immune system of patients infected with SARS-CoV-2, but there are still aspects that have not been fully characterized or understood. In this study, we have analyzed a cohort of patients with mild, moderate and severe disease.We performed flow cytometric studies and correlated the data with the clinical characteristics and clinical laboratory values of the patients. Both conventional and unsupervised data analyses concluded that patients with severe disease are characterized, among others, by a higher state of activation in all T cell subsets (CD4, CD8, double negative and T follicular helper cells), higher expression of perforin and granzyme B in cytotoxic cells, expansion of adaptive NK cells and the accumulation of activated and immature dysfunctional monocytes which are identified by a low expression of HLA-DR and an intriguing shift in the expression pattern of CD300 receptors.More importantly, correlation analysis showed a strong association between the alterations in the immune cells and the clinical signs of severity. These results indicate that patients with severe COVID-19 have a broad perturbation of their immune system, and they will help to understand the immunopathogenesis of COVID-19.This work is supported by a grant from the Agencia Estatal de Investigación Project PID2019-109583RBI00/ AEI/10.13039/501100011033 and a grant from the Department of Health, Basque Government (2020111045). OZ is recipient of a postdoctoral contract funded by Instituto de Salud Carlos III-Contratos Sara Borrell 2017 (CD17/0128) and the European Social Fund (ESF)-The ESF invests in your future. GA-P is recipient of a fellowship from the BBK Fundazioa (1543/2006_0001) and from the Jesús de Gangoiti Barrera Foundation (FJGB20/002). IT is recipient of a predoctoral contract funded by the Department of Education, Basque Government (PRE_2020_2_007). AO is recipient of a fellowship from the Jesús de Gangoiti Barrera Foundation (FJGB20/007). FB is an Ikerbasque Research Professor, Ikerbasque, Basque Foundation for Science