Healthy and patient type 2 innate lymphoid cells are differently affected by in vitro culture conditions

Background: Type 2 innate lymphoid cells (ILC2s) have emerged as key players in the development of type 2 driven diseases such as allergy and asthma. Due to their low number in the circulation, in vitro expansion is needed to unravel their mechanisms of action. Purpose: The aim of this study is to assess the impact of different culture conditions and address whether the method of expansion may distinctly affect healthy donor or patient-derived ILC2s. Methods: Here, we described the impact of six different culture conditions on the proliferation, phenotype and function of human ILC2s freshly obtained from healthy donors (healthy ILC2s) and allergic patients (patient ILC2s). Results: We showed that the cytokine cocktail or the PHA induced the highest proliferation of healthy ILC2s and patient ILC2s, respectively. We observed that the stromal cells OP9, used as ILC2 feeders, did not boost their proliferation, but impaired the activation marker expression and the function of patient ILC2s. Furthermore, we demonstrated that the culture conditions differently impacted the activation state of c-Kithigh and c-Kitlow ILC2s, in both healthy donors and allergic patients. Last, we also observed that ILC2s expanded only with IL-2 and IL-7 were the most prone to secrete IL-5 and IL-13 upon IL-33 stimulation. In contrast, in patients, the addition of OP9 cells during the expansion restrained their type 2 cytokine secretory functions. Conclusion: This report highlights that culture conditions distinctly impacted on the healthy or patient ILC2 behavior, with important consequences for their study in disease settings

Impact of Immunotherapy on CD4 T Cell Phenotypes and Function in Cancer.

Immunotherapy has become a standard treatment in many cancers and it is based on three main therapeutic axes: immune checkpoint blockade (ICB), vaccination and adoptive cell transfer (ACT). If originally these therapies mainly focused on exploiting CD8 T cells given their role in the direct elimination of tumor cells, increasing evidence highlights the crucial role CD4 T cells play in the antitumor immune response. Indeed, these cells can profoundly modulate the tumor microenvironment (TME) by secreting different types of cytokine or by directly eliminating cancer cells. In this review, we describe how different CD4 T cell subsets can contribute to tumor immune responses during immunotherapy and the novel high-throughput immune monitoring tools that are expected to facilitate the study of CD4 T cells, at antigen-specific and single cell level, thus accelerating bench-to-bed translational research in cancer

Immune system activation by natural products and complex fractions: a network pharmacology approach in cancer treatment.

Natural products and traditional herbal medicine are an important source of alternative bioactive compounds but very few plant-based preparations have been scientifically evaluated and validated for their potential as medical treatments. However, a promising field in the current therapies based on plant-derived compounds is the study of their immunomodulation properties and their capacity to activate the immune system to fight against multifactorial diseases like cancer. In this review we discuss how network pharmacology could help to characterize and validate natural single molecules or more complex preparations as promising cancer therapies based on their multitarget capacities

ILC2s: New Actors in Tumor Immunity

Innate lymphoid cells (ILCs) represent the most recently identified family of innate lymphocytes that act as first responders, maintaining tissue homeostasis and protecting epithelial barriers. In the last few years, group 2 ILCs (ILC2s) have emerged as key regulators in several immunological processes such as asthma and allergy. Whilst ILC2s are currently being evaluated as novel targets for immunotherapy in these diseases, their involvement in tumor immunity has only recently begun to be deciphered. Here, we provide a comprehensive overview of the pleiotropic roles of ILC2s in different tumor settings. Furthermore, we discuss how different therapeutic approaches targeting ILC2s could improve the efficacy of current tumor immunotherapies

Distinct and shared gene expression for human innate versus adaptive helper lymphoid cells

Innate lymphoid cells (ILCs) are the latest identified innate immune cell family. Given their similarity in transcription factor expression and cytokine secretion profiles, ILCs have been considered as the innate phenocopy of CD4 Th cells. Here, we explored the transcriptome of circulating human ILC subsets as opposed to CD4 Th cell subsets. We describe transcriptomic differences between total ILCs and total CD4 Th cells, as well as between paired innate and adaptive cell subsets (ILC1 vs. Th1; ILC2 vs. Th2; and ILC3 vs. Th17 cells). In particular, we observed differences in expression of genes involved in cell trafficking such as CCR1, CCR6 and CXCR3, innate activation and inhibitory functions, including CD119, 2B4, TIGIT, and CTLA-4, and neuropeptide receptors, such as VIPR2. Moreover, we report for the first time on distinct expression of long noncoding RNAs (lncRNAs) in innate vs. adaptive cells, arguing for a potential role of lncRNA in shaping human ILC biology. Altogether, our results point for unique, rather than redundant gene organization in ILCs compared to CD4 Th cells, in regard to kinetics, fine-tuning and spatial organization of the immune response

Inhibition of Membrane-Bound BAFF by the Anti-BAFF Antibody Belimumab.

B cell activating factor of the TNF family (BAFF, also known as BLyS), a cytokine that regulates homeostasis of peripheral B cells, is elevated in the circulation of patients with autoimmune diseases such as systemic lupus erythematosus (SLE). BAFF is synthetized as a membrane-bound protein that can be processed to a soluble form after cleavage at a furin consensus sequence, a site that in principle can be recognized by any of the several proteases of the pro-protein convertase family. Belimumab is a human antibody approved for the treatment of SLE, often cited as specific for the soluble form of BAFF. Here we show in different experimental systems, including in a monocytic cell line (U937) that naturally expresses BAFF, that belimumab binds to membrane-bound BAFF with similar EC50 as the positive control atacicept, which is a decoy receptor for both BAFF and the related cytokine APRIL (a proliferation inducing ligand). In U937 cells, binding of both reagents was only detectable in furin-deficient U937 cells, showing that furin is the main BAFF processing protease in these cells. In CHO cells expressing membrane-bound BAFF lacking the stalk region, belimumab inhibited the activity of membrane-bound BAFF less efficiently than atacicept, while in furin-deficient U937 cells, belimumab inhibited membrane-bound BAFF and residual soluble BAFF as efficiently as atacicept. These reagents did not activate complement or antibody-dependent cell cytotoxicity upon binding to membrane-bound BAFF in vitro. In conclusion, our data show that belimumab can inhibit membrane-bound BAFF, and that BAFF in U937 cells is processed by furin

Human innate lymphoid cells (ILCs): Toward a uniform immune-phenotyping.

Helper innate lymphoid cells (ILCs), the most recently identified population of the ILC family, play a fundamental role in the restoration of tissue integrity, in the protection against infiltrating pathogens as well as in tumor immune-surveillance. ILCs have been divided into three main subsets, ILC1, ILC2, and ILC3, that can be specifically activated by different signals coming either indirectly from pathogens or from other cell populations, including cancer cells. Following activation, ILCs are in turn able to promptly secrete a wide range of soluble mediators that modulate effector cell functions. The discovery and the study of these immune cells is now offering important opportunities for innovative therapies of allergic airway diseases, inflammatory disorders and might be crucial for the discovery of new targets for the therapy of cancer. It is therefore fundamental that the scientific community establishes harmonized guidelines to obtain a consensus in the identification and phenotypical and functional characterization of ILCs. © 2018 International Clinical Cytometry Society

Severe COVID-19 patients exhibit an ILC2 NKG2D⁺ population in their impaired ILC compartment.

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The SOCS3-independent expression of IDO2 supports the homeostatic generation of T regulatory cells by human dendritic cells.

Dendritic cells (DCs) are professional APCs that have a role in the initiation of adaptive immune responses and tolerance. Among the tolerogenic mechanisms, the expression of the enzyme IDO1 represents an effective tool to generate T regulatory cells. In humans, different DC subsets express IDO1, but less is known about the IDO1-related enzyme IDO2. In this study, we found a different pattern of expression and regulation between IDO1 and IDO2 in human circulating DCs. At the protein level, IDO1 is expressed only in circulating myeloid DCs (mDCs) and is modulated by PGE2, whereas IDO2 is expressed in both mDCs and plasmacytoid DCs and is not modulated by PGE2. In healthy subjects, IDO1 expression requires the presence of PGE2 and needs continuous transcription and translation, whereas IDO2 expression is constitutive, independent from suppressor of cytokine signaling 3 activity. Conversely, in patients suffering from inflammatory arthritis, circulating DCs express both IDO1 and IDO2. At the functional level, both mDCs and plasmacytoid DCs generate T regulatory cells through an IDO1/IDO2-dependent mechanism. We conclude that, in humans, whereas IDO1 provides an additional mechanism of tolerance induced by proinflammatory mediators, IDO2 is stably expressed in steady-state conditions and may contribute to the homeostatic tolerogenic capacity of DCs