Memory NK cell features exploitable in anticancer immunotherapy

Besides 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

Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses. Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcεRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcεRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli. These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional progra

Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming

Natural Killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human Cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C+Fc∝RI≥- long-lived “memory” NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterised yet. Here we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells, and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansion. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting, and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy

Tumor-associated and immunochemotherapy-dependent long-term alterations of the peripheral blood NK cell compartment in DLBCL patients

Natural Killer (NK) cells are a key component of tumor immunosurveillance and thus play an important role in rituximab-dependent killing of lymphoma cells via an antibody-dependent cellular cytotoxicity (ADCC) mechanism. We evaluated the phenotypic and functional assets of peripheral blood NK cell subsets in 32 newly-diagnosed diffuse large B-cell lymphoma (DLBCL) patients and in 27 healthy controls. We further monitored long-term modifications of patient NK cells for up to 12 months after rituximab-based immunochemotherapy. At diagnosis, patients showed a higher percentage of CD56dim and CD16C NK cells, and a higher frequency of GrzBC cells in CD56dim, CD56bright, and CD16C NK cell subsets than healthy controls. Conversely, DLBCL NK cell killing and interferon g (IFNg) production capability were comparable to those derived from healthy subjects. Notably, NK cells from refractory/relapsed patients exhibited a lower “natural” cytotoxicity. A marked and prolonged therapy-induced reduction of both “natural” and CD16- dependent NK cytotoxic activities was accompanied by the down-modulation of CD16 and NKG2D activating receptors, particularly in the CD56dim subset. However, reduced NK cell killing was not associated with defective lytic granule content or IFNg production capability. This study firstly describes tumor-associated and therapy-induced alterations of the systemic NK cell compartment in DLBCL patients. As these alterations may negatively impact rituximab-based therapy efficacy, our work may provide useful information for improving immunochemotherapeutic strategies

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

The role of DNAM-1 family co-receptors on mucosal and circulating T cells in pediatric individuals and Inflammatory Bowel Diseases (IBD) patients

Introduzione: 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

Purpose: 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

Tumor-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

Purpose: 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.

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 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