Expansion of memory Vδ2 T cells following SARS-CoV-2 vaccination revealed by temporal single-cell transcriptomics.

γδ T cells provide rapid cellular immunity against pathogens. Here, we conducted matched single-cell RNA-sequencing and γδ-TCR-sequencing to delineate the molecular changes in γδ T cells during a longitudinal study following mRNA SARS-CoV-2 vaccination. While the first dose of vaccine primes Vδ2 T cells, it is the second administration that significantly boosts their immune response. Specifically, the second vaccination uncovers memory features of Vδ2 T cells, shaped by the induction of AP-1 family transcription factors and characterized by a convergent central memory signature, clonal expansion, and an enhanced effector potential. This temporally distinct effector response of Vδ2 T cells was also confirmed in vitro upon stimulation with SARS-CoV-2 spike-peptides. Indeed, the second challenge triggers a significantly higher production of IFNγ by Vδ2 T cells. Collectively, our findings suggest that mRNA SARS-CoV-2 vaccination might benefit from the establishment of long-lasting central memory Vδ2 T cells to confer protection against SARS-CoV-2 infection.

Transcriptomic profile of TNFhigh MAIT cells is linked to B cell response following SARS-CoV-2 vaccination.

Introduction: Higher frequencies of mucosal-associated invariant T (MAIT) cells were associated with an increased adaptive response to mRNA BNT162b2 SARS-CoV-2 vaccine, however, the mechanistic insights into this relationship are unknown. In the present study, we hypothesized that the TNF response of MAIT cells supports B cell activation following SARS-CoV-2 immunization. Methods: To investigate the effects of repeated SARS-CoV-2 vaccinations on the peripheral blood mononuclear cells (PBMCs), we performed a longitudinal single cell (sc)RNA-seq and scTCR-seq analysis of SARS-CoV-2 vaccinated healthy adults with two doses of the Pfizer-BioNTech BNT162b2 mRNA vaccine. Collection of PBMCs was performed 1 day before, 3 and 17 days after prime vaccination, and 3 days and 3 months following vaccine boost. Based on scRNA/TCR-seq data related to regulatory signals induced by the vaccine, we used computational approaches for the functional pathway enrichment analysis (Reactome), dynamics of the effector cell-polarization (RNA Velocity and CellRank), and cell-cell communication (NicheNet). Results: We identified MAIT cells as an important source of TNF across circulating lymphocytes in response to repeated SARS-CoV-2 BNT162b2 vaccination. The TNFhigh signature of MAIT cells was induced by the second administration of the vaccine. Notably, the increased TNF expression was associated with MAIT cell proliferation and efficient anti-SARS-CoV-2 antibody production. Finally, by decoding the ligand-receptor interactions and incorporating intracellular signaling, we predicted TNFhigh MAIT cell interplay with different B cell subsets. In specific, predicted TNF-mediated activation was selectively directed to conventional switched memory B cells, which are deputed to high-affinity long-term memory. Discussion: Overall, our results indicate that SARS-CoV-2 BNT162b2 vaccination influences MAIT cell frequencies and their transcriptional effector profile with the potential to promote B cell activation. This research also provides a blueprint for the promising use of MAIT cells as cellular adjuvants in mRNA-based vaccines.

What do we mean by long COVID? A scoping review of the cognitive sequelae of SARS-CoV-2 infection.

BACKGROUND AND PURPOSE: Many COVID-19 patients report persistent symptoms, including cognitive disturbances. We performed a scoping review on this topic, focusing primarily on cognitive manifestations. METHODS: Abstracts and full texts of studies published on PubMed (until May 2023) addressing cognitive involvement persisting after SARS-CoV-2 infection were reviewed, focusing on terms used to name the cognitive syndrome, reported symptoms, their onset time and duration, and testing batteries employed. Reported psychiatric symptoms, their assessment tools, and more general manifestations were also extracted. RESULTS: Among the 947 records identified, 180 studies were included. Only one third of them used a label to define the syndrome. A minority of studies included patients according to stringent temporal criteria of syndrome onset (34%), whereas more studies reported a minimum required symptom duration (77%). The most frequently reported cognitive symptoms were memory and attentional-executive disturbances, and among psychiatric complaints, the most frequent were anxiety symptoms, depression, and sleep disturbances. Most studies reported fatigue among general symptoms. Thirty-six studies employed cognitive measures: screening tests alone (n = 19), full neuropsychological batteries (n = 25), or both (n = 29); 30 studies performed psychiatric testing. Cognitive deficits were demonstrated in 39% of subjects, the most frequently affected domains being attention/executive functions (90%) and memory (67%). CONCLUSIONS: Currently, no agreement exists on a label for post-COVID-19 cognitive syndrome. The time of symptom onset after acute infection and symptom duration are still discussed. Memory and attention-executive complaints and deficits, together with fatigue, anxiety, and depression symptoms, are consistently reported, but the objective evaluation of these symptoms is not standardized.

Selected memory T cells infused post-haploidentical hematopoietic stem cell transplantation persist and hyperexpand.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) with post-transplant cyclophosphamide is a curative treatment for many hematological malignancies, yet a majority of patients still suffers from recurrent infections. Post-transplant infusion of memory T-cells could potentially enhance immunological protection without increasing the risk of eliciting acute graft-versus-host disease, which is mainly induced by naïve T-cells. Here, we performed longitudinal analysis of the lymphocyte compartment in 19 patients who underwent haplo-HSCT previously enrolled in a phase II prospective clinical trial (www.clinicaltrials.gov as #NCT04687982), in which they received post-transplant CD45RA-depleted donor lymphocyte infusions (DLI). T-cell receptor sequencing analysis showed that, surprisingly, CD45RA-depleted DLI do not increase T-cell clonal diversity, but lead to prominent expansion of a selected number of infused memory T-cell clones, suggesting recruitment of these cells in the immune response. Pathogen-specific memory T-cells, including cytomegalovirus (CMV)-specific cells, were engrafted and were able to persist for at least 1 month. Deep immunophenotyping revealed strong polyfunctional effector CMV-specific T-cell responses in the majority of patients, with their expansion correlating with the frequency of CMV-specific cells in the donor. These findings provide a rationale behind the suggested improved protection against viral infections in patients receiving CD45RA-depleted DLI.

Persistence of KIRneg NK cells after haploidentical hematopoietic stem cell transplantation protects from human cytomegalovirus infection/reactivation.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) is a therapeutic option to cure patients affected by hematologic malignancies. The kinetics and the quality of immune-reconstitution (IR) impact the clinical outcome of h-HSCT and limit the onset of life-threatening Human Cytomegalovirus (HCMV) infection/reactivation. Natural Killer (NK) cells are the first lymphocytes that recover after h-HSCT and they can provide rapid innate immune responses against opportunistic pathogens. By performing a longitudinal single-cell analysis of multiparametric flow-cytometry data, we show here that the persistence at high frequencies of CD158b1b2jneg/NKG2Apos/NKG2Cneg/NKp30pos/NKp46pos (KIRneg) NK cells is associated with HCMV infection/reactivation control. These KIRneg NK cells are "unlicensed", and are not terminal-differentiated lymphocytes appearing early during IR and mainly belonging to CD56bright/CD16neg and CD56bright/CD16pos subsets. KIRneg NK cells are enriched in oxidative and glucose metabolism pathways, produce interferon-γ, and are endowed with potent antiviral activity against HCMV ex vivo. Decreased frequencies of KIRneg NK cells early during IR are associated with clinically relevant HCMV replication. Taken together, our findings indicate that the prolonged persistence of KIRneg NK cells after h-HSCT could serve as a biomarker to better predict HCMV infection/reactivation. This phenomenon also paves the way to optimize anti-viral immune responses by enriching post-transplant donor lymphocyte infusions with KIRneg NK cells.

Natural Killer Cells in SARS-CoV-2 Infection: Pathophysiology and Therapeutic Implications.

Natural Killer (NK) cells are lymphocytes of the innate immunity that play a crucial role in the control of viral infections in the absence of a prior antigen sensitization. Indeed, they display rapid effector functions against target cells with the capability of direct cell killing and antibody-dependent cell-mediated cytotoxicity. Furthermore, NK cells are endowed with immune-modulatory functions innate and adaptive immune responses via the secretion of chemokines/cytokines and by undertaking synergic crosstalks with other innate immune cells, including monocyte/macrophages, dendritic cells and neutrophils. Recently, the Coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally. Although the specific role of NK cells in COVID-19 pathophysiology still need to be explored, mounting evidence indicates that NK cell tissue distribution and effector functions could be affected by SARS-CoV-2 infection and that a prompt NK cell response could determine a good clinical outcome in COVID-19 patients. In this review, we give a comprehensive overview of how SARS-CoV-2 infection interferes with NK cell antiviral effectiveness and their crosstalk with other innate immune cells. We also provide a detailed characterization of the specific NK cell subsets in relation to COVID-19 patient severity generated from publicly available single cell RNA sequencing datasets. Finally, we summarize the possible NK cell-based therapeutic approaches against SARS-CoV-2 infection and the ongoing clinical trials updated at the time of submission of this review. We will also discuss how a deep understanding of NK cell responses could open new possibilities for the treatment and prevention of SARS-CoV-2 infection.

Development of Human ILCs and Impact of Unconventional Cytotoxic Subsets in the Pathophysiology of Inflammatory Diseases and Cancer.

Innate lymphoid cells (ILCs) were firstly described by different independent laboratories in 2008 as tissue-resident innate lymphocytes mirroring the phenotype and function of T helper cells. ILCs have been subdivided into three distinct subgroups, ILC1, ILC2 and ILC3, according to their cytokine and transcriptional profiles. Subsequently, also Natural Killer (NK) cells, that are considered the innate counterpart of cytotoxic CD8 T cells, were attributed to ILC1 subfamily, while lymphoid tissue inducer (LTi) cells were attributed to ILC3 subgroup. Starting from their discovery, significant advances have been made in our understanding of ILC impact in the maintenance of tissue homeostasis, in the protection against pathogens and in tumor immune-surveillance. However, there is still much to learn about ILC ontogenesis especially in humans. In this regard, NK cell developmental intermediates which have been well studied and characterized prior to the discovery of helper ILCs, have been used to shape a model of ILC ontogenesis. Herein, we will provide an overview of the current knowledge about NK cells and helper ILC ontogenesis in humans. We will also focus on the newly disclosed circulating ILC subsets with killing properties, namely unconventional CD56dim NK cells and cytotoxic helper ILCs, by discussing their possible role in ILC ontogenesis and their contribution in both physiological and pathological conditions.

Single-cell profiling identifies impaired adaptive NK cells expanded after HCMV reactivation in haploidentical HSCT.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) represents an efficient curative approach for patients affected by hematologic malignancies in which the reduced intensity conditioning induces a state of immunologic tolerance between donor and recipient. However, opportunistic viral infections greatly affect h-HSCT clinical outcomes. NK cells are the first lymphocytes that recover after transplant and provide a prompt defense against human cytomegalovirus (HCMV) infection/reactivation. By undertaking a longitudinal single-cell computational profiling of multiparametric flow cytometry, we show that HCMV accelerates NK cell immune reconstitution together with the expansion of CD158b1b2jpos/NKG2Aneg/NKG2Cpos/NKp30lo NK cells. The frequency of this subset correlates with HCMV viremia, further increases in recipients experiencing multiple episodes of viral reactivations, and persists for months after the infection. The transcriptional profile of FACS-sorted CD158b1b2jpos NK cells confirmed the ability of HCMV to deregulate NKG2C, NKG2A, and NKp30 gene expression, thus inducing the expansion of NK cells with adaptive traits. These NK cells are characterized by the downmodulation of several gene pathways associated with cell migration, the cell cycle, and effector-functions, as well as by a state of metabolic/cellular exhaustion. This profile reflects the functional impairments of adaptive NK cells to produce IFN-γ, a phenomenon also due to the viral-induced expression of lymphocyte-activation gene 3 (LAG-3) and programmed cell death protein 1 (PD-1) checkpoint inhibitors.

Extracellular Vesicles as Biomarkers of Acute Graft-vs.-Host Disease After Haploidentical Stem Cell Transplantation and Post-Transplant Cyclophosphamide.

Even with high-dose post-transplant cyclophosphamide (PT-Cy) which was initially introduced for graft-versus-host disease (GvHD) prevention in the setting of HLA-haploidentical transplantation, both acute and chronic GvHDs remain a major clinical challenge. Despite improvements in the understanding of the pathogenesis of both acute and chronic GvHDs, reliable biomarkers that predict their onset have yet to be identified. We recently studied the potential correlation between extracellular vesicles (EVs) and the onset of acute (a)GvHD in transplant recipients from related and unrelated donors. In the present study, we further investigated the role of the expression profile of membrane proteins and their microRNA (miRNA) cargo (miRNA100, miRNA155, and miRNA194) in predicting the onset of aGvHD in haploidentical transplant recipients with PT-Cy. Thirty-two consecutive patients were included. We evaluated the expression profile of EVs, by flow cytometry, and their miRNA cargo, by real-time PCR, at baseline, prior, and at different time points following transplant. Using logistic regression and Cox proportional hazard models, a significant association between expression profiles of antigens such as CD146, CD31, CD140a, CD120a, CD26, CD144, and CD30 on EVs, and their miRNA cargo with the onset of aGvHD was observed. Moreover, we also investigated a potential correlation between EV expression profile and cargo with plasma biomarkers (e.g., ST2, sTNFR1, and REG3a) that had been associated with aGVHD previously. This analysis showed that the combination of CD146, sTNFR1, and miR100 or miR194 strongly correlated with the onset of aGvHD (AUROC >0.975). A large prospective multicenter study is currently in progress to validate our findings.

Biomarkers for acute and chronic graft versus host disease: state of the art.

INTRODUCTION: Despite significant advances in treatment and prevention, graft-versus-host disease (GVHD) still represents the main cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Thus, considerable research efforts have been made to find and validate reliable biomarkers for diagnosis, prognosis, and risk stratification of GVHD. AREAS COVERED: In this review the most recent evidences on different types of biomarkers studied for GVHD, such as genetic, plasmatic, cellular markers, and those associated with microbiome, were summarized. A comprehensive search of peer-review literature was performed in PubMed including meta-analysis, preclinical and clinical trials, using the terms: cellular and plasma biomarkers, graft-versus-host disease, cytokines, and allogeneic hematopoietic stem cell transplantation. EXPERT OPINION: In the near future, several validated biomarkers will be available to help clinicians in the diagnosis of GVHD, the identification of patients at high risk of GVHD development and in patients' stratification according to its severity. Then, immunosuppressive treatment could be tailored to each patient's real needs. However, more efforts are needed to achieve this goal. Although most of the proposed biomarkers currently lack validation with large-scale clinical data, their study led to improved knowledge of the biological basis of GVHD, and ultimately to implementation of GHVD treatment.

Cancer Immunotherapy by Blocking Immune Checkpoints on Innate Lymphocytes.

Immune checkpoints refer to a plethora of inhibitory pathways of the immune system that play a crucial role in maintaining self-tolerance and in tuning the duration and amplitude of physiological immune responses to minimize collateral tissue damages. The breakdown of this delicate balance leads to pathological conditions, including cancer. Indeed, tumor cells can develop multiple mechanisms to escape from immune system defense, including the activation of immune checkpoint pathways. The development of monoclonal antibodies, targeting inhibitory immune checkpoints, has provided an immense breakthrough in cancer therapy. Immune checkpoint inhibitors (ICI), initially developed to reverse functional exhaustion in T cells, recently emerged as important actors in natural killer (NK)-cell-based immunotherapy. Moreover, the discovery that also helper innate lymphoid cells (ILCs) express inhibitory immune checkpoints, suggests that these molecules might be targeted on ILCs, to modulate their functions in the tumor microenvironment. Recently, other strategies to achieve immune checkpoint blockade have been developed, including miRNA exploiting systems. Herein, we provide an overview of the current knowledge on inhibitory immune checkpoints on NK cells and ILCs and we discuss how to target these innate lymphocytes by ICI in both solid tumors and hematological malignancies.

Extracellular Vesicles After Allogeneic Hematopoietic Cell Transplantation: Emerging Role in Post-Transplant Complications.

Extracellular vesicles (EVs) play an important role in the cellular crosstalk by transferring bioactive molecules through biological barriers from a cell to another, thus influencing recipient cell functions and phenotype. Therefore, EVs are increasingly being explored as biomarkers of disease progression or response to therapy and as potential therapeutic agents in different contexts including in hematological malignancies. Recently, an EV role has emerged in allogeneic hematopoietic cell transplantation (allo-HCT) as well. Allogeneic hematopoietic cell transplantation often represents the only curative option in several hematological disorders, but it is associated with potentially life-threatening complications that can have a significant impact on clinical outcomes. The most common complications have been well-established and include graft-versus-host disease and infections. Furthermore, relapse remains an important cause of treatment failure. The aim of this review is to summarize the current knowledge, the potential applications, and clinical relevance of EVs in allo-HCT. Herein, we will mainly focus on the immune-modulating properties of EVs, in particular those derived from mesenchymal stromal cells, as potential therapeutic strategy to improve allo-HCT outcome. Moreover, we will briefly describe the main findings on EVs as biomarkers to monitor graft-versus-host disease onset and tumor relapse.

Increased VEGF levels in a case of papillary tumor of the pineal region with intracranial hemorrhage: a potential surrogate indicator of tumor angiogenesis and aggressiveness?

Pineal tumors are rare, about 1% of all intracranial tumors. At variance with pineocytomas, usually characterized by a good prognosis, papillary tumors behave more aggressively. Owing to their rarity, little is known about their biology and clinical behavior, moreover conflicting data on prognosis have been reported. Here we present an unusual case of papillary neuroepithelial tumor of the pineal region in a 40-year-old man who was admitted in a state of unconsciousness due to the presence of intracranial hemorrhage. After 21 days from admission, he underwent third ventriculostomy for hydrocephalus and biopsy of the lesion. Since bleeding manifestations are uncommonly associated with this kind of tumors, we performed some additional non routine laboratory tests in order to identify biological indicators of disease course and abnormal angiogenesis. Coagulation screening tests were performed to rule out the presence of coagulopathy and vascular endothelial growth factor (VEGF ) levels were measured in plasma as marker of tumor angiogenic potential. Histologic evaluation confirmed the diagnosis of a papillary tumor of the pineal region with the presence of tiny vessel lumens that may account for increased angiogenesis Coagulation screening was normal and VEGF levels were extremely high if compared to healthy individuals. After 20 months of follow-up the tumor mass, radiotherapy treated, appeared dramatically reduced at MRI evaluation, and, interestingly, VEGF levels, although still higher than in healthy individuals, resulted significantly decreased as compared to those measured at time of first hospital admission suggesting a role for VEGF as indicator of tumor aggressiveness. In conclusion, measurement of angiogenesis circulating soluble markers could have an additional feedback in the diagnosis, therapy and monitoring the disease in patients with very rare CNS tumors as papillary tumors of pineal region that have non univocal clinical behavior and prognosis.

Pretransplant active disease status and HLA class II mismatching are associated with increased incidence and severity of cytokine release syndrome after haploidentical transplantation with posttransplant cyclophosphamide.

Cytokine release syndrome (CRS) represents a life-threatening side effect after haploidentical stem cell transplantation (Haplo-SCT) with posttransplant cyclophosphamide (PT-Cy). Factors predictive of CRS development is still a matter of debate. We retrospectively analyzed 102 consecutive patients receiving a bone marrow (BM) (n = 42) or peripheral blood stem cells (PBSC) (n = 60) Haplo-SCT with PT-Cy. The two cohorts were similar in main patients' characteristics besides disease type (P = .02). Cumulative incidence of grades 1, 2, and ≥3 CRS was 80%, 52%, and 15% at a median of 2, 4, and 7 days, respectively. Moderate/High-grade fever (39°-41°), grade 1 and grade ≥3 CRS occurred more frequently after PBSC relative to BM grafts (68% vs 33%, P = .0005; 87% vs 71%, P = .009; 20% vs 7%, P = .07). Only patients experiencing grade ≥3 CRS had a worse outcome in terms of 1-year overall survival (OS) and nonrelapse mortality (NRM): 39% vs 80% (P = .002) and 40% vs 8% (P = .005), respectively. By univariate analysis the only factors associated with the increased risk of ≥3 CRS were pretransplant disease status (8% for complete remission, 11% for partial remission, and 38% for active disease, P = .002), HLA-DRB1 mismatching (57% vs 14%, P = .007), and PBSC graft (P = .07). By multivariable analysis, only pretransplant disease status (hazard ratio, HR: 6.84, P = .005) and HLA-DRB1 mismatching (HR: 17.19, P = .003) remained independent predictors of grade ≥3 CRS. Only grade ≥3 CRS is clinically relevant for the final outcome of patients receiving Haplo-SCT with PT-Cy, is more frequent after a PBSC graft and is associated with pretransplant active disease and HLA-DRB1 mismatching.

Innate Immune Responses in the Outcome of Haploidentical Hematopoietic Stem Cell Transplantation to Cure Hematologic Malignancies.

In the context of allogeneic transplant platforms, human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) represents one of the latest and most promising curative strategies for patients affected by high-risk hematologic malignancies. Indeed, this platform ensures a suitable stem cell source immediately available for virtually any patents in need. Moreover, the establishment in recipients of a state of immunologic tolerance toward grafted hematopoietic stem cells (HSCs) remarkably improves the clinical outcome of this transplant procedure in terms of overall and disease free survival. However, the HLA-mismatch between donors and recipients has not been yet fully exploited in order to optimize the Graft vs. Leukemia effect. Furthermore, the efficacy of haplo-HSCT is currently hampered by several life-threatening side effects including the onset of Graft vs. Host Disease (GvHD) and the occurrence of opportunistic viral infections. In this context, the quality and the kinetic of the immune cell reconstitution (IR) certainly play a major role and several experimental efforts have been greatly endorsed to better understand and accelerate the post-transplant recovery of a fully competent immune system in haplo-HSCT. In particular, the IR of innate immune system is receiving a growing interest, as it recovers much earlier than T and B cells and it is able to rapidly exert protective effects against both tumor relapses, GvHD and the onset of life-threatening opportunistic infections. Herein, we review our current knowledge in regard to the kinetic and clinical impact of Natural Killer (NK), γδ and Innate lymphoid cells (ILCs) IRs in both allogeneic and haplo-HSCT. The present paper also provides an overview of those new therapeutic strategies currently being implemented to boost the alloreactivity of the above-mentioned innate immune effectors in order to ameliorate the prognosis of patients affected by hematologic malignancies and undergone transplant procedures.

On the Way to Become a Natural Killer Cell.

Natural Killer (NK) cells are innate lymphocytes playing pivotal roles in host defense and immune-surveillance. The homeostatic modulation of germ-line encoded/non-rearranged activating and inhibitory NK cell receptors (NKRs) determines the capability of these innate lymphocytes to either spare "self" cells or to kill viral-infected, tumor-transformed and heterologous cell targets. However, despite being discovered more than 40 years ago, several aspects of NK cell biology remain unknown or are still being debated. In particular, our knowledge of human NK cell ontogenesis and differentiation is still in its infancy as the majority of our experimental evidence on this topic mainly comes from findings obtained in vitro or with animal models in vivo. Although both the generation and the maintenance of human NK cells are sustained by hematopoietic stem cells (HSCs), the precise site(s) of NK cell development are still poorly defined. Indeed, HSCs and hematopoietic precursors are localized in different anatomical compartments that also change their ontogenic commitments before and after birth as well as in aging. Currently, the main site of NK cell generation and maturation in adulthood is considered the bone marrow, where their interactions with stromal cells, cytokines, growth factors, and other soluble molecules support and drive maturation. Different sequential stages of NK cell development have been identified on the basis of the differential expression of specific markers and NKRs as well as on the acquisition of specific effector-functions. All these phenotypic and functional features are key in inducing and regulating homing, activation and tissue-residency of NK cells in different human anatomic sites, where different homeostatic mechanisms ensure a perfect balance between immune tolerance and immune-surveillance. The present review summarizes our current knowledge on human NK cell ontogenesis and on the related pathways orchestrating a proper maturation, functions, and distributions.

Costimulatory Molecules and Immune Checkpoints Are Differentially Expressed on Different Subsets of Dendritic Cells.

Dendritic cells (DCs) play a crucial role in initiating and shaping immune responses. The effects of DCs on adaptive immune responses depend partly on functional specialization of distinct DC subsets, and partly on the activation state of DCs, which is largely dictated by environmental signals. Fully activated immunostimulatory DCs express high levels of costimulatory molecules, produce pro-inflammatory cytokines, and stimulate T cell proliferation, whereas tolerogenic DCs express low levels of costimulatory molecules, produce immunomodulatory cytokines and impair T cell proliferation. Relevant to the increasing use of immune checkpoint blockade in cancer treatment, signals generated from inhibitory checkpoint molecules on DC surface may also contribute to the inhibitory properties of tolerogenic DCs. Yet, our knowledge on the expression of inhibitory molecules on human DC subsets is fragmentary. Therefore, in this study, we investigated the expression of three immune checkpoints on peripheral blood DC subsets, in basal conditions and upon exposure to pro-inflammatory and anti-inflammatory stimuli, by using a flow cytometric panel that allows a direct comparison of the activatory/inhibitory phenotype of DC-lineage and inflammatory DC subsets. We demonstrated that functionally distinct DC subsets are characterized by differential expression of activatory and inhibitory molecules, and that cDC1s in particular are endowed with a unique immune checkpoint repertoire characterized by high TIM-3 expression, scarce PD-L1 expression and lack of ILT2. Notably, this unique cDC1 repertoire was subverted in a group of patients with myelodysplastic syndromes included in the study. Applied to the characterization of DCs in the tumor microenvironment, this panel has the potential to provide valuable information to be used for investigating the role of DC subsets in cancer, guiding DC-targeting treatments, and possibly identifying predictive biomarkers for clinical response to cancer immunotherapy.

Hepatic Natural Killer Cells: Organ-Specific Sentinels of Liver Immune Homeostasis and Physiopathology.

The liver is considered a preferential tissue for NK cells residency. In humans, almost 50% of all intrahepatic lymphocytes are NK cells that are strongly imprinted in a liver-specific manner and show a broad spectrum of cellular heterogeneity. Hepatic NK (he-NK) cells play key roles in tuning liver immune response in both physiological and pathological conditions. Therefore, there is a pressing need to comprehensively characterize human he-NK cells to better understand the related mechanisms regulating their effector-functions within the dynamic balance between immune-tolerance and immune-surveillance. This is of particular relevance in the liver that is the only solid organ whose parenchyma is constantly challenged on daily basis by millions of foreign antigens drained from the gut. Therefore, the present review summarizes our current knowledge on he-NK cells in the light of the latest discoveries in the field of NK cell biology and clinical relevance.