Are human V\u3b42pos T cells really resistant to aging and Human Cytomegalovirus infection?

In their recent paper, Weili Xu et al. [1] described the different behaviors of V\u3b41pos and V\u3b42pos T cell subsets in response to lifelong stress and claimed that V\u3b42pos T cells are not affected by aging and Human Cytomegalovirus (HCMV) infection. While we agree that these two \u3b3\u3b4 T cell subsets diverge both in phenotype/function and in tissue distribution, we are somewhat surprised that authors did not take into account the several previously published and contradictory experimental evidence in regards to senescence of V\u3b42pos T cells [2,3]. These latter studies reported that HCMV infection not only induces a clonal expansion of a distinct V\u3b39neg/V\u3b42pos T cell subset, but also determines a concomitant adaptive differentiation from CD27high na\uefve cells to CD27low/neg terminal-effectors. However, Weili Xu et al. argued that the expression and kinetics of both CD27 and CD45RA surface markers do not change and follow the homeostatic changes of V\u3b42pos T cells. This statement goes in the opposite direction to previously reported findings as the CD27/CD45RA phenotype has been shown to mark the maturation and differentiation (TNa\uefve, TCentral-Memory, Teffector-Memory and TEffectory-Memory RA) of V\u3b42pos T cells. Indeed, the different surface expression of both CD27 and CD45 parallel the progressive decrease of telomere length, the proliferative capacity as well as the different effector-functions and resistance to death of V\u3b42+ T cells in response to antigens and homeostatic cytokines [4,5]. Hence, we believe that these controversial issues require further discussion beyond the unilateral conclusion given by the study of Weili Xu et al

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

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), \u3b3\u3b4 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

Chemotherapy accelerates immune-senescence and functional impairments of Vδ2pos T cells in elderly patients affected by liver metastatic colorectal cancer.

Human (gamma delta) γδ T cells are unconventional innate-like lymphocytes displaying a broad array of anti-tumor activities with promising perspectives in cancer immunotherapy. In this context, Vδ2pos T cells represent the preferential target of several immunotherapy protocols against solid tumors. However, the impact of both aging and chemotherapy (CHT) on Vδ2pos T cells is still unknown. The present study evaluates with multi-parametric flow cytometry the frequencies, terminal differentiation, senescence and effector-functions of peripheral blood and tumor infiltrating Vδ2pos T cells purified from liver metastases (CLM) of patients affected by colorectal cancer (CRC) compared to those of sex- and age-matched healthy donors. The peripheral blood of CLM patients underwent CHT is characterized by decreased amounts of Vδ2pos T cells showing a relative increase of terminally-differentiated CD27neg/CD45RApos (TEMRA) cells. The enrichment of this latter subset is associated with an increased expression of the senescent marker CD57. The acquisition of CD57 on TEMRA Vδ2pos T cells is also coupled with impairments in cytotoxicity and production of TNF-α and IFN-γ. These features resemble the acquisition of an immune-senescent profile by Vδ2pos T cells from CLM patients that received CHT, a phenomenon that is also associated with the loss of the co-stimulatory marker CD28 and with the induced expression of CD16. The group of CLM patients underwent CHT and older than 60 years old showed higher frequencies of CD57pos and TEMRA Vδ2pos T cells. Similar results were found for tumor infiltrating Vδ2pos T cell subset purified from CLM specimens of patients treated with CHT. The toxicity of CHT regimens also affects the homeostasis of Vδ2pos T cells by inducing higher frequencies of circulating CD57pos TEMRA subset in CLM underwent CHT and younger than 60 years old. Taken together, our data demonstrate that the enrichment of senescent Vδ2pos T cells in CLM patients is not only induced by patients' aging but also by the toxicity of CHT that further accelerates the accumulation of CD57pos TEMRA cells highly dysfunctional in their anti-tumor activities. These results are important to both predict the clinical outcome of CLM and to optimize those protocols of cell cancer immunotherapy employing unconventional Vδ2pos T cells

Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors

Uncontrolled insertion of gene transfer vectors into the human genome is raising significant safety concerns for their clinical use. The wild-type adeno-associated virus (AAV) can insert its genome at a specific site in human chromosome 19 (AAVS1) through the activity of a specific replicase/integrase protein (Rep) binding both the AAVS1 and the viral inverted terminal repeats (ITRs). AAV-derived vectors, however, do not carry the rep gene and cannot maintain site-specific integration properties. We describe a novel hybrid vector carrying an integration cassette flanked by AAV ITRs and a tightly regulated, drug-inducible Rep expression cassette in the framework of a high-capacity, helper-dependent adenoviral (Ad) vector. Rep-dependent integration of ITR-flanked cassettes of intact size and function was obtained in human primary cells and cell lines in the absence of selection. The majority of integrations were site specific and occurred within a 1000-bp region of the AAVS1. Genome-wide sequencing of integration junctions indicates that nonspecific integrations occurred predominantly in intergenic regions. Site-specific integration was obtained also in vivo, in an AAVS1 transgenic mouse model: upon a single tail vein administration of a nontoxic dose of Ad/AAV vectors, AAVS1-specific integrations were detected and sequenced in DNA obtained from the liver of all animals in which Rep expression was induced by drug treatment. Nonrandom integration of double-stranded DNA can therefore be obtained ex vivo and in vivo by the use of hybrid Ad/AAV vectors, in the absence of toxicity and with efficiency compatible with gene therapy applications

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

Bright expression of CD91 identifies highly activated human dendritic cells that can be expanded by defensins

CD91 is a scavenger receptor expressed by different immune cells and its ligands defensins have been demonstrated to contribute to immune responses against infections and tumors. We previously demonstrated that CD91 is expressed on human monocyte-derived dendritic cells (moDCs) and that human defensins stimulate in vitro the activation of these cells. In this study, we observed that CD91 is expressed at different levels on two distinct moDC subsets: CD91dim and CD91bright moDCs. Although CD91bright moDCs represented a small proportion of total moDCs, this subset showed higher levels of activation and maturation markers compared to CD91dim moDCs. The frequency of CD91bright moDCs increased by ~50% after in vitro stimulation with recombinant Human Neutrophil Peptide-1 (rHNP-1) and recombinant Human Beta Defensin-1 (rHBD-1), while lipopolysaccharide (LPS) stimulation decreased it by ~35%. Both defensins up-regulated moDC expression of CD80, CD40, CD83 and HLA-DR, although to a lower extent compared with LPS. Notably, upon culture with rHNP-1 and rHBD-1, CD91bright moDCs maintained their higher activation/maturation status, while this was lost upon culture with LPS. Our findings suggest that defensins promote the differentiation into activated CD91bright DCs and may encourage the exploitation of the CD91/defensins axis as a novel therapeutic strategy to potentiate antimicrobial and antitumor immune response

Transcription factor binding sites are genetic determinants of retroviral integration in the human genome.

Gamma-retroviruses and lentiviruses integrate non-randomly in mammalian genomes, with specific preferences for active chromatin, promoters and regulatory regions. Gene transfer vectors derived from gamma-retroviruses target at high frequency genes involved in the control of growth, development and differentiation of the target cell, and may induce insertional tumors or pre-neoplastic clonal expansions in patients treated by gene therapy. The gene expression program of the target cell is apparently instrumental in directing gamma-retroviral integration, although the molecular basis of this phenomenon is poorly understood. We report a bioinformatic analysis of the distribution of transcription factor binding sites (TFBSs) flanking >4,000 integrated proviruses in human hematopoietic and non-hematopoietic cells. We show that gamma-retroviral, but not lentiviral vectors, integrate in genomic regions enriched in cell-type specific subsets of TFBSs, independently from their relative position with respect to genes and transcription start sites. Analysis of sequences flanking the integration sites of Moloney leukemia virus (MLV)- and human immunodeficiency virus (HIV)-derived vectors carrying mutations in their long terminal repeats (LTRs), and of HIV vectors packaged with an MLV integrase, indicates that the MLV integrase and LTR enhancer are the viral determinants of the selection of TFBS-rich regions in the genome. This study identifies TFBSs as differential genomic determinants of retroviral target site selection in the human genome, and suggests that transcription factors binding the LTR enhancer may synergize with the integrase in tethering retroviral pre-integration complexes to transcriptionally active regulatory regions. Our data indicate that gamma-retroviruses and lentiviruses have evolved dramatically different strategies to interact with the host cell chromatin, and predict a higher risk in using gamma-retroviral vs. lentiviral vectors for human gene therapy applications