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Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15Rβ (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.

Background T cell-based immunotherapies including immune checkpoint blockade and chimeric antigen receptor T cells can induce durable responses in patients with cancer. However, clinical efficacy is limited due to the ability of cancer cells to evade immune surveillance. While T cells have been the primary focus of immunotherapy, recent research has highlighted the importance of natural killer (NK) cells in directly recognizing and eliminating tumor cells and playing a key role in the set-up of an effective adaptive immune response. The remarkable potential of NK cells for cancer immunotherapy is demonstrated by their ability to broadly identify stressed cells, irrespective of the presence of neoantigens, and their ability to fight tumors that have lost their major histocompatibility complex class I (MHC I) expression due to acquired resistance mechanisms.However, like T cells, NK cells can become dysfunctional within the tumor microenvironment. Strategies to enhance and reinvigorate NK cell activity hold potential for bolstering cancer immunotherapy.Methods In this study, we conducted a high-throughput screen to identify molecules that could enhance primary human NK cell function. After compound validation, we investigated the effect of the top performing compounds on dysfunctional NK cells that were generated by a newly developed in vitro platform. Functional activity of NK cells was investigated using compounds alone and in combination with checkpoint inhibitor blockade. The findings were validated on patient-derived intratumoral dysfunctional NK cells from different cancer types.Results The screening approach led to the identification of a Casitas B-lineage lymphoma (Cbl-b) inhibitor enhancing the activity of primary human NK cells. Furthermore, the Cbl-b inhibitor was able to reinvigorate the activity of in vitro generated and patient-derived dysfunctional NK cells. Finally, Cbl-b inhibition combined with T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade further increased the cytotoxic potential and reinvigoration of both in vitro generated and patient-derived intratumoral dysfunctional NK cells.Conclusions These findings underscore the relevance of Cbl-b inhibition in overcoming NK cell dysfunctionality with the potential to complement existing immunotherapies and improve outcomes for patients with cancer.

Background: Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood., Methods: Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec-6 and Siglec-8. For functional characterization, each receptor was evaluated biochemically and in ex vivo and in vivo inhibition models of IgE and non-IgE-mediated MC activation in Siglec-6- or Siglec-8-expressing transgenic mice., Results: Siglec-6 and Siglec-8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec-6 and Siglec-8 interacted with a large cluster of proteins involved in IgE and non-IgE-mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL-4 and IL-33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec-6 and Siglec-8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec-6. Indeed, Siglec-6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti-Siglec-6 antibody significantly inhibited SCF-mediated MC activation more in comparison to targeting Siglec-8., Conclusion: These data demonstrate a central role for Siglec-6 and Siglec-8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec-6 has a role distinct from that of Siglec-8 in regulating MC function and represents a distinct potential therapeutic target in mast cell-driven diseases., (© 2024 Allakos Inc. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Mast cells (MC) are key drivers of allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 is an immunoregulatory receptor found on MCs. While it is recognized that engaging Siglecs with antibodies mediates inhibition across immune cells, the mechanisms that govern this agonism are not understood. Here we generated Siglec-6 mAb clones (AK01 to AK18) to better understand Siglec-6-mediated agonism. Siglec-6 mAbs displayed epitope-dependent receptor internalization and inhibitory activity. We identified a Siglec-6 mAb (AK04) that required Fc-mediated interaction for receptor internalization and induced inhibition and antibody-dependent cellular phagocytosis against MCs. AK04-mediated MC inhibition required Siglec-6 immunoreceptor tyrosine-based inhibitory motif (ITIM) and ITIM-like domains and was associated with receptor cluster formation containing inhibitory phosphatases. Treatment of humanized mice with AK04 inhibited systemic anaphylaxis with a single dose and reduced MCs with chronic dosing. Our findings suggest Siglec-6 activity is epitope dependent and highlight an agonistic Siglec-6 mAb as a potential therapeutic approach in allergic disease., (© 2022. The Author(s).)

Mast cells are tissue-resident cells that contribute to allergic diseases, among others, due to excessive or inappropriate cellular activation and degranulation. Therapeutic approaches to modulate mast cell activation are urgently needed. Siglec-6 is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptor selectively expressed by mast cells, making it a promising target for therapeutic intervention. However, the effects of its engagement on mast cells are poorly defined. Siglec-6 expression and endocytosis on primary human mast cells and mast cell lines were assessed by flow cytometry. SIGLEC6 mRNA expression was examined by single-cell RNAseq in esophageal tissue biopsy samples. The ability of Siglec-6 engagement or co-engagement to prevent primary mast cell activation was determined based on assessments of mediator and cytokine secretion and degranulation markers. Siglec-6 was highly expressed by all mast cells examined, and the SIGLEC6 transcript was restricted to mast cells in esophageal biopsy samples. Siglec-6 endocytosis occurred with delayed kinetics relative to the related receptor Siglec-8. Co-crosslinking of Siglec-6 with FcεRIα enhanced the inhibition of mast cell activation and diminished downstream ERK1/2 and p38 phosphorylation. The selective, stable expression and potent inhibitory capacity of Siglec-6 on human mast cells are favorable for its use as a therapeutic target in mast cell-driven diseases.

Immunomodulation of mast cell (MC) activity is warranted in allergic and inflammatory diseases where MCs have a central role in pathogenesis. Targeting Siglec-8, an inhibitory receptor on MCs and eosinophils, has shown promising activity in preclinical and clinical studies. While the intracellular pathways that regulate Siglec-8 activity in eosinophils have been well studied, the signaling mechanisms that lead to MC inhibition have not been fully elucidated. Here, we evaluate the intracellular signaling pathways of Siglec-8-mediated inhibition in primary MCs using an anti-Siglec-8 monoclonal antibody (mAb). Phospho-proteomic profiling of FcεRI-activated MCs revealed Siglec-8 mAb-treatment globally inhibited proximal and downstream kinases, leading to attenuated MC activation and degranulation. In fact, Siglec-8 was found to directly interact with FcεRI signaling molecules. Siglec-8 inhibition was dependent on both cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that interact with the SH2 containing protein phosphatase Shp-2 upon Siglec-8 phosphorylation. Taken together, these data support a model in which Siglec-8 regulates proximal FcεRI-induced phosphorylation events through phosphatase recruitment and interaction with FcεRIγ, resulting in global inhibition of MCs upon Siglec-8 mAb engagement., Competing Interests: WK, AW, SG, JS, KC, JL, ZB, TL, EB, KL, AX and BY were employed by Allakos Inc. GN was employed by LM Biostat Consulting Inc. The authors declare that this study received funding from Allakos Inc. The funder had the following involvement with the study: data collection, analysis, interpretation, study design, and writing of this article., (Copyright © 2022 Korver, Wong, Gebremeskel, Negri, Schanin, Chang, Leung, Benet, Luu, Brock, Luehrsen, Xu and Youngblood.)

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection represents a global health crisis. Immune cell activation via pattern recognition receptors has been implicated as a driver of the hyperinflammatory response seen in COVID-19. However, our understanding of the specific immune responses to SARS-CoV-2 remains limited. Mast cells (MCs) and eosinophils are innate immune cells that play pathogenic roles in many inflammatory responses. Here we report MC-derived proteases and eosinophil-associated mediators are elevated in COVID-19 patient sera and lung tissues. Stimulation of viral-sensing toll-like receptors in vitro and administration of synthetic viral RNA in vivo induced features of hyperinflammation, including cytokine elevation, immune cell airway infiltration, and MC-protease production-effects suppressed by an anti-Siglec-8 monoclonal antibody which selectively inhibits MCs and depletes eosinophils. Similarly, anti-Siglec-8 treatment reduced disease severity and airway inflammation in a respiratory viral infection model. These results suggest that MC and eosinophil activation are associated with COVID-19 inflammation and anti-Siglec-8 antibodies are a potential therapeutic approach for attenuating excessive inflammation during viral infections., Competing Interests: JS, SG, MB, TL, EB, AX, AW, JL,WK, and BY are employees of Allakos and/or own stock options in Allakos. BB and RS did not perform any of the experiments but are paid consultants on the scientific advisory board of Allakos, Inc., and own stock in Allakos. BB and RS are coinventors on existing Siglec-8–related patents and thus may be entitled to a share of royalties received by Johns Hopkins University from Allakos, Inc. on the potential sales of such products. BB and RS are also cofounders of Allakos, which makes him subject to certain restrictions under university policy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gebremeskel, Schanin, Coyle, Butuci, Luu, Brock, Xu, Wong, Leung, Korver, Morin, Schleimer, Bochner and Youngblood.)

Introduction: Opioid overdose is a growing problem in the US. Often, residents are first responders to community and in-hospital opioid overdoses, and so, hands-on naloxone administration education is necessary. While residents get a brief algorithm on suspected opioid overdose during their mandatory American Heart Association basic life support training, there is a lack of hands-on standardized curricula on how to administer this lifesaving medication., Methods: To fill this gap, we developed a hands-on workshop for medical trainees on how to respond to an opioid overdose. Trainees who completed our workshop left with a first-responder naloxone kit using the Massachusetts statewide open prescription. All attendees were asked to take a voluntary pre- and posttraining survey., Results: A total of 80 trainees from a variety of specialties and training levels participated in this workshop. We were able to successfully link the pre- and postdata of 29 participants. Trainees were assessed on comfort in administering naloxone as a first responder and in teaching patients how to administer naloxone (via a 5-point Likert scale) and on percentage of time they prescribed naloxone to high-risk patient populations. We saw statistically significant increases in comfort in using naloxone and comfort in teaching patients to administer naloxone., Discussion: This innovative curriculum provides an adaptable, short, and effective workshop with hands-on practice for medical trainees at a variety of training levels. The workshop can efficiently train future health care professionals how to approach an opioid overdose., Competing Interests: Thuy Luu is now an employee of Biogen., (Copyright © 2020 Jawa et al.)

Although HIV (human immunodeficiency virus) testing for all women has been promoted by Vietnam's Ministry of Health since 2000, test acceptance rates in this country were reported to be less than 30% in the community. This country has been facing the barriers to approach the national services towards transmission prevention from mother to child including HIV testing during antenatal care (ANC) towards mothers. Here, we aim to assess the socioeconomic inequalities in HIV testing during ANC among Vietnamese women. This study used available data from the Vietnam Multiple Indicator Cluster Survey 2014. Overall, the prevalence of HIV testing during antenatal care was 30% and the concentrate index (CCI) was 0.1926. There was significant inequality between women classified as poor and rich, and when stratified by social characteristics, inequality was found in women aged 15-49 years (CCI: 0.4), living in rural areas (CCI: 0.3), belonging to ethnic minorities (CCI: 0.5) and having primary or less education (CCI: 0.4). In the multivariate logistic regression analysis, ethnicity and socioeconomic status were significant factors associated with HIV testing during ANC. We found the prevalence of HIV testing during ANC was low, and its inequalities were associated with age, living area, ethnicity, education, and economic status.