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The role of DNAM-1 family co-receptors on mucosal and circulating T cells in pediatric individuals and Inflammatory Bowel Diseases (IBD) patients
No full textIntroduzione: La mucosa intestinale è sede di un’ampia varietà di cellule T effettrici e della memoria, la cui sopravvivenza e funzioni effettrici sono strettamente regolate. Alterazioni nella regolazione delle cellule T sono fortemente coinvolte nella patogenesi delle malattie infiammatorie croniche dell’intestino (IBD). Il nostro obbiettivo è stato quello di analizzare il pattern di espressione dei co-recettori DNAM-1 e TIGIT sui linfociti T del sangue periferico e mucosali, in soggetti pediatrici sani e pazienti affetti da IBD, oltre allo studiare il loro ruolo nel regolare la proliferazione delle cellule T e le loro funzioni.
Materiali e Metodi: L’espressione di DNAM-1 e TIGIT e la produzione di IFN-γ sono state valutate tramite analisi citofluorimetrica multi-parametrica. La proliferazione delle cellule T è stata misurata tramite il saggio di diluizione del CFSE.
Risultati: La frequenza delle cellule DNAM-1+ è fortemente ridotta nei linfociti T mucosali, rispetto alle loro controparti del sangue periferico, laddove la frequenza delle cellule TIGIT+ è fortemente incrementata.
Entrambi i co-recettori sono down-regolati sui linfociti T CD4+ dei pazienti con IBD attiva. Le cellule T DNAM-1+ e TIGIT+ del sangue periferico mostrano una maggiore capacità di produrre IFN-γ. Nella frazione proliferante dei linfociti T circolanti, stimolati con CD3/CD28, DNAM-1 è arricchito mentre TIGIT è depleto.
La presenza di PVR, ligando comune ad entrambi i co-recettori, aumenta la capacità di proliferazione dei linfociti T, riuscendo a superare il difetto proliferativo delle cellule T DNAM-1+TIGIT+.
Conclusioni: L’espressione differenziale di DNAM-1 e TIGIT nei compartimenti mucosali e circolanti potrebbe essere dovuta ad un differente stato di attivazione/differenziamento e/o a diverse capacità di homing di queste cellule; la down-modulazione di questi recettori sui linfociti T mucosali nei pazienti con IBD attiva potrebbe dipendere dal microambiente infiammatorio degli stessi. Nonostante entrambi i co-recettori identifichino cellule T del sangue periferico funzionalmente competenti, DNAM-1 e TIGIT sono inversamente associati alla capacità proliferativa. Questi dati suggeriscono un ruolo per DNAM-1 e TIGIT nell’immunità mucosale e la patogenesi delle IBD.Introduction: Gut mucosa is home for a variety of effector/memory T cells whose survival and effector functions are tightly regulated. Disturbances in T cells regulation are strongly involved in Inflammatory Bowel Diseases’ pathogenesis. Our aim is to analyze the expression pattern of DNAM-1 and TIGIT co-receptors on mucosal and peripheral blood (PB) T cells in pediatric healthy subjects and IBD patients, and to study their role in regulating T cells proliferation and functions.
Materials and Methods: DNAM-1 and TIGIT expression and IFN-γ production are assessed by multi-parametric cytofluorimetric analysis. T cell proliferation is measured by CFSE dilution assay.
Results: The frequency of DNAM-1+ cells is strongly reduced, whereas that of TIGIT+ cells is increased on mucosal T cells, when compared to PB counterparts. Both co-receptors are down-regulated on mucosal CD4+ T cells from active IBD patients. DNAM-1+ and TIGIT+ PB-T cells show a higher capability to produce IFN-γ. DNAM-1 is enriched and TIGIT is depleted on proliferating (CD3/CD28-stimulated) PB-T cells. The presence of PVR shared ligand increases T cell proliferation, and overcomes the defective proliferative capability of DNAM-1+TIGIT+ T cells.
Conclusions: The different expression of DNAM-1 and TIGIT on mucosal vs PB-T cells may be related to different activation/differentiation state, and/or homing capability; co-receptor downmodulation on active IBD mucosal T cells may depend on the inflammatory microenvironment. While both co-receptors mark functionally competent PB-T cells, DNAM-1 and TIGIT are inversely associated with proliferative capability. These data suggest a role for DNAM-1 and TIGIT in mucosal immunity and IBD pathogenesis
Regulation of DNAM-1 family receptors and their ligands in T lymphocytes and intestinal epithelial cells
No full textPurpose: DNAM-1 family co-receptors are expressed on T lymphocyte subsets and provide activating (DNAM-1) or inhibitory (TIGIT) signals that regulate T cell functions and proliferation. We previously found that the expression pattern of these co-receptors strikingly differs between circulating and mucosal T cell populations. Moreover, perturbed expression of DNAM-1/ligand system distinctly characterizes infiltrate and epithelial counterpart in the inflamed mucosa microenvironment of active Inflammatory Bowel Disease (IBD) pediatric patients. Here we analyzed the capability of polyclonal TCR-dependent
stimulation or selected cytokines to modulate the expression of DNAM-1 family co-receptors and shared ligands (PVR and Nectin-2) on peripheral blood (PB) T cell subsets and HT-29 colon carcinoma-derived cell line. Results: IL-2 family cytokines or TCR/CD28 stimulation increases the frequency of TIGIT+ T cells, suggesting that such stimuli may partially explain the higher frequency of TIGIT+ mucosal T cells, as compared to PB counterpart. Differently, DNAM-1 levels are increased by TGF-b, and decreased by IL-17A. The dysregulated abundance of these two cytokines in inflamed mucosa microenvironment could underlie the downregulated DNAM-1 expression on mucosal T cells from active IBD patients. Moreover, Nectin-2 expression on HT-29 cells was decreased by TGF-b and IL-10 anti-inflammatory cytokines, suggesting that the reduced amount of these factors may lead to the increased frequency of Nectin-2+ gut epithelial cells recorded in active IBD lesions. Conclusion: Our data suggest that mucosal microenvironment factors shape the physiological expression pattern of DNAM-1 family co-receptor/ligand system and contribute to its alteration in IBD
Natural killer (NK) cells and anti-tumor therapeutic mAb: unexplored interactions
No full textTumor-targeting mAb are widely used in the treatment of a variety of solid and hematopoietic tumors and represent the first immunotherapeutic approach successfully arrived to the clinic. Nevertheless, the role of distinct immune mechanisms in contributing to their therapeutic efficacy is not completely understood and may vary depending on tumor- or antigen/antibody-dependent characteristics. Availability of next-generation, engineered, tumor-targeting mAb, optimized in their capability to recruit selected immune effectors, re-enforces the need for a deeper understanding of the mechanisms underlying anti-tumor mAb functionality. NK cells participate with a major role to innate anti-tumor responses, by exerting cytotoxic activity and producing a vast array of cytokines. As the CD16 (low-affinity FcγRIIIA)-activating receptor is expressed on the majority of NK cells, its effector functions can be ideally recruited against therapeutic mAb-opsonized tumor cells. The exact role of NK cells in determining therapeutic efficacy of tumor-targeting mAb is still unclear and much sought after. This knowledge will be instrumental to design innovative combination schemes with newly validated immunomodulatory agents. We will summarize what is known about the role of NK cells in therapeutic anti-tumor mAb therapy, with particular emphasis on RTX chimeric anti-CD20 mAb, the first one used in clinical practice for treating B cell malignancies
The interplay between anti-CD20 therapeutic antibodies and "memory" Natural Killer cells
No full textPurpose: Our study focuses on the recently described long-lived and highly functional NK cell populations (dubbed memory NK cells), defined by the lack of expression of CD16-associatedFceRIg chain and the ability to produce high amounts of IFNg upon CD16 re-stimulation (1). Particularly relevant are our recent observations demonstrating that the sustained stimulation of NK cells with obinutuzumab (anti-CD20 mAb)-opsonised tumor cells leads to the selective down-regulation of FceRIg chain, along with the priming for enhanced IFNg production (2). Here we want to study the capability of anti-CD20 mAbs to support memory NK cell expansion. Methods: CD56+CD16+CD3-g- (memory) and CD56+CD16+CD3-g+(conventional) NK cells from healthy donors were quantified ex vivo and after 10 day co-culture with anti-CD20 mAb-opsonised CD20+ Raji cells in the presence of IL-2. Two different antiCD20 mAbs, currently employed in the treatment of B cell malignancies were chosen: first generation, reference molecule, rituximab, and next generation, Fc-engineered, obinutuzumab, which shows increased binding affinity to CD16. Results: Almost 55% of healthy donors exhibit a population of memory NK cells, accounting for 5%-70% of total peripheral blood NK cells. We observed that CD56+CD16+CD3- g- (memory) NK cells selectively undergo 2- to 12-fold expansion, upon co-culture with anti-CD20 opsonised targets, with no major differences between different anti-CD20 mAbs; on the opposite, CD56+CD16+CD3-g+ (conventional) NK cell proliferation is not affected by CD16 stimulation. The phenotypic and functional characterization of anti-CD20 mAb-expanded memory NK cells is under investigation. Conclusions: Our data highlight a new aspect of the interplay between therapeutic mAbs and NK cell plasticity, suggesting a potential tool for the clinical exploitment of NK cell effector functions
Expression pattern and functional role of DNAM-1 family co-receptors on mucosal and peripheral blood T cell populations of pediatric subjects and Inflammatory Bowel Diseases (IBD) patients.
No full textPURPOSE: To investigate the role of DNAM-1 (activating) and Tigit (inhibitory) co-receptors on circulating and mucosal “innate-like” (CD56+ and CD4-CD8-) and “classical” (CD4+ and CD8+) T cell populations, and their potential role in Inflammatory Bowel Disease (IBD) pathogenesis.
METHODS: We analysed the distribution of DNAM-1 family members on peripheral blood and mucosal (colon biopsy) T cell populations cells from pediatric IBD patients and age-matched controls, with a multiparameter citofluorimetric approach.
RESULTS: Peripheral blood “classical” and “innate-like” T cell populations differently express DNAM-1 family co-receptors; within each subset, mucosa addressin (CCR6+ or CD103+)-expressing cells display a distinct profile, as compared to negative ones.
DNAM-1 expression positively correlates with an enhanced capability to produce IFN γ, within both “classical” and “innate-like” peripheral blood T cells; a comparable enrichment in the functional capability is observed in Tigit+ “classical”, but not “innate-like” T cells.
The expression pattern of DNAM-1 and Tigit strikingly differs between tissue and peripheral T cell compartments: DNAM-1+ cells are strongly reduced on all analyzed mucosal subsets, whereas the frequency of Tigit+ cells is significatively augmented on mucosal CD4+ and DN, but not on CD8+ T cells; moreover, Tigit surface levels are significatively reduced on mucosal T cells of active IBD lesions.
DISCUSSION & CONCLUSIONS: Our results show that DNAM-family co-receptors are differently expressed on distinct T cell subsets; moreover, coreceptor expression strikingly differs between a relatively ligand-free environment (peripheral blood), and a ligand-rich tissue (intestinal mucosa). Ligand presence in the environment may also influence coreceptor levels in inflamed vs non-inflamed mucosa populations. These results may underline a primary role for DNAM-1 family coreceptors in the fine-tuning of mucosal immunity and in IBD pathogenesis.PURPOSE: To investigate the role of DNAM-1 (activating) and Tigit (inhibitory) co-receptors on circulating and mucosal “innate-like” (CD56+ and CD4-CD8-) and “classical” (CD4+ and CD8+) T cell populations, and their potential role in Inflammatory Bowel Disease (IBD) pathogenesis.
METHODS: We analysed the distribution of DNAM-1 family members on peripheral blood and mucosal (colon biopsy) T cell populations cells from pediatric IBD patients and age-matched controls, with a multiparameter citofluorimetric approach.
RESULTS: Peripheral blood “classical” and “innate-like” T cell populations differently express DNAM-1 family co-receptors; within each subset, mucosa addressin (CCR6+ or CD103+)-expressing cells display a distinct profile, as compared to negative ones.
DNAM-1 expression positively correlates with an enhanced capability to produce IFN γ, within both “classical” and “innate-like” peripheral blood T cells; a comparable enrichment in the functional capability is observed in Tigi
Memory NK cell features exploitable in anticancer immunotherapy
Full text linkBesides their innate ability to rapidly produce effector cytokines and kill virus-infected or transformed cells, natural killer (NK) cells display a strong capability to adapt to environmental modifications and to differentiate into long-lived, hyperfunctional populations, dubbed memory or memory-like NK cells. Despite significant progress in the field of NK cell-based immunotherapies, some factors including their short life span and the occurrence of a tumor-dependent functional exhaustion have limited their clinical efficacy so that strategies aimed at overcoming these limitations represent one of the main current challenges in the field. In this scenario, the exploitation of NK cell memory may have a considerable potential. This article summarizes recent evidence in the literature on the peculiar features that render memory NK cells an attractive tool for antitumor immunotherapy, including their long-term survival and in vivo persistence, the resistance to tumor-dependent immunosuppressive microenvironment, the amplified functional responses to IgG-opsonized tumor cells, and in vitro expansion capability. Along with highlighting these issues, we speculate that memory NK cell-based adoptive immunotherapy settings would greatly take advantage from the combination with tumor-targeting therapeutic antibodies (mAbs), as a strategy to fully unleash their clinical efficacy
Regulation and trafficking of the HLA-E molecules during monocyte-macrophage differentiation
No full textHLA-E is a nonclassical HLA-class I molecule whose best known role is to protect from the natural killer cells. More recently, an additional function more similar to that of classical HLA-class I molecules, i.e., antigen presentation to T cells, is emerging. However, much remains to be explored about the intracellular trafficking of the HLA-E molecules. With the use of 3 different cellular contexts, 2 monocytic cell lines, U937 and THP1, and peripheral blood monocytes, we show here a remarkable increase of HLA-E during monocyte-macrophage differentiation. This goes independently from the classical HLA-class I, the main source of HLA-E-specific peptides, which is found strongly up-regulated upon differentiation of peripheral blood monocytes but not at all in the case of U937 and THP1 cell lines. Although in all cases, there was a moderate increase of HLA-E expressed in the cell surface, lysis by natural killer cells is comparably restored by an anti-NKG2A antibody in untreated as well as in PMA-differentiated U937 cells. Instead, the great majority of the HLA-E is retained in the vesicles of the autophagy-lysosome network, where they colocalize with the microtubule-associated protein light chain 3, as well as with the lysosomal-associated membrane protein 1. We conclude that differently from the classical HLA-class I molecules, the primary destination of the newly synthesized HLA-E molecules in macrophages is, rather than the cell membrane, the intracellular autophagy-lysosomal vesicles where they are stored and where they can encounter the exogenous antigens
CD16 pre-ligation by defucosylated tumor-targeting mAb sensitizes human NK cells to γc cytokine stimulation via PI3K/mTOR axis
No full textObinutuzumab is a glycoengineered tumor-targeting anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for the FcγRIIIA/CD16 receptor, which was recently approved for clinical use in CLL and follicular lymphoma. Here we extend our previous observation that, in human NK cells, the sustained CD16 ligation by obinutuzumab-opsonized targets leads to a markedly enhanced IFN-γ production upon a subsequent cytokine re-stimulation. The increased IFN-γ competence in response to IL-2 or IL-15 is attributable to post-transcriptional regulation, as it does not correlate with the upregulation of IFN-γ mRNA levels. Different from the reference molecule rituximab, we observe that the stimulation with obinutuzumab promotes the upregulation of microRNA (miR)-155 expression. A similar trend was also observed in NK cells from untreated CLL patients stimulated with obinutuzumab-opsonized autologous leukemia. miR-155 upregulation associates with reduced levels of SHIP-1 inositol phosphatase, which acts in constraining PI3K-dependent signals, by virtue of its ability to mediate phosphatidylinositol 3,4,5-trisphosphate (PIP3) de-phosphorylation. Downstream of PI3K, the phosphorylation status of mammalian target of rapamycin (mTOR) effector molecule, S6, results in amplified response to IL-2 or IL-15 stimulation in obinutuzumab-experienced cells. Importantly, NK cell treatment with the PI3K or mTOR inhibitors, idelalisib and rapamycin, respectively, prevents the enhanced cytokine responsiveness, thus, highlighting the relevance of the PI3K/mTOR axis in CD16-dependent priming. The enhanced IFN-γ competence may be envisaged to potentiate the immunoregulatory role of NK cells in a therapeutic setting
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