Exosomal-miRNas expression and growth factors released by mononuclear cells of CLAD patients in response to extracorporeal photopheresis.

BACKGROUND: CLAD (Chronic Lung Allograft Dysfunction) remains a serious complication following lung transplantation. Some evidence shows that portions of Extracorporeal Photopheresis (ECP)-treated patients improve/stabilize their graft function. In spite of that, data concerning molecular mechanisms are still lacking. Aims of our study were to assess whether ECP effects are mediated by Mononuclear Cells (MNCs) modulation in term of microRNAs (miRNAs) expression and growth factors release. METHODS: Cells from leukapheresis of 16 CLAD patients, at time 0 and 6-months (10 cycles), were cultured for 48h ± PHA (10 ug/ml) or LPS (2 ug/ml). Expression levels of miR-146a-5p, miR-155-5p, miR-31-5p, miR181a-5p, miR-142-3p, miR-16-5p and miR-23b-5p in MNCs-exosomes were evaluated by qRT-PCR, while ELISA assessed different growth factors levels on culture supernatants. RESULTS: Our result showed miR-142-3p down-regulation (p = 0.02) in MNCs of ECP-patients after the 10 cycles and after LPS stimulation (p = 0.005). We also find miR-146a-5p up-regulation in cells after the 10 cycles stimulated with LPS (p = 0.03). Connective tissue growth factor (CTGF) levels significantly decreased in MNCs supernatant (p = 0.04). The effect of ECP is translated into frequency changes of Dendritic Cell (DC) subpopulations and a slight increase in T regulatory cells (Treg) number and a significant decrease in CTGF release. CONCLUSIONS: ECP might affect regulatory T cell functions, since both miR-142 and miR-146a have been shown to be involved in the regulation of suppressor regulatory T cell functions and DCs. On the other side ECP, possibly by regulating macrophage activation, is able to significantly down modulate CTGF release.

Cytological, molecular, cytogenetic, and physiological characterization of a novel immortalized human enteric glial cell line.

Enteric glial cells (EGCs), the major components of the enteric nervous system (ENS), are implicated in the maintenance of gut homeostasis, thereby leading to severe pathological conditions when impaired. However, due to technical difficulties associated with EGCs isolation and cell culture maintenance that results in a lack of valuable in vitro models, their roles in physiological and pathological contexts have been poorly investigated so far. To this aim, we developed for the first time, a human immortalized EGC line (referred as ClK clone) through a validated lentiviral transgene protocol. As a result, ClK phenotypic glial features were confirmed by morphological and molecular evaluations, also providing the consensus karyotype and finely mapping the chromosomal rearrangements as well as HLA-related genotypes. Lastly, we investigated the ATP- and acetylcholine, serotonin and glutamate neurotransmitters mediated intracellular Ca2+ signaling activation and the response of EGCs markers (GFAP, SOX10, S100ß, PLP1, and CCL2) upon inflammatory stimuli, further confirming the glial nature of the analyzed cells. Overall, this contribution provided a novel potential in vitro tool to finely characterize the EGCs behavior under physiological and pathological conditions in humans.

DQA1*0102 DQB1*0602 haplotype distinguishes coeliac disease and its complications from gluten unrelated enteropathies.

BACKGROUND: Duodenal villous atrophy is due not only to coeliac disease and its complications but also to other rare enteropathies unrelated to gluten consumption, defined as noncoeliac enteropathies. The diagnosis of noncoeliac enteropathies remains challenging, and HLA typing has been widely used to exclude coeliac disease if DQ2 and DQ8 alleles are absent. However, the frequency of the various HLA alleles in noncoeliac enteropathies is still unknown. AIMS: To describe the HLA genetic profile of patients affected by noncoeliac enteropathies who have been evaluated at our centres between 2000 and 2021, and to investigate the diagnostic role of HLA typing. METHODS: Genomic DNA was collected from 44 Italian and 19 British adult patients with noncoeliac enteropathies. Patient genotypes were compared with those of healthy Italian and British populations obtained from HLA bone marrow donors' banks. In addition, genotypes were also compared with those of patients with coeliac disease and complicated coeliac disease. RESULTS: Both in the Italian and in the British group, the DQA1*0102 DQB1*0602 haplotype and related alleles occurred significantly more frequently in patients with noncoeliac enteropathies compared to coeliac disease and complicated coeliac disease. CONCLUSIONS: Together with negative HLA-DQ2 and DQ8 haplotypes, the DQA1*0102 DQB1*0602 haplotype can be used to guide the differential diagnosis between coeliac disease and noncoeliac enteropathies.

Mechanisms of Action of Extracorporeal Photopheresis in the Control of Bronchiolitis Obliterans Syndrome (BOS): Involvement of Circulating miRNAs.

Clinical evidence suggests an improvement or stabilization of lung function in a fraction of patients with bronchiolitis obliterans syndrome (BOS) treated by extracorporeal photopheresis (ECP); however, few studies have explored the epigenetic and molecular regulation of this therapy. The aim of present study was to evaluate whether a specific set of miRNAs were significantly regulated by ECP. Total RNA was isolated from serum of patients with established BOS grade 1-2 prior to the start and after 6 months of ECP treatment. We observed a significant downregulation of circulating hsa-miR-155-5p, hsa-miR-146a-5p and hsa-miR-31-5p in BOS patients at the start of ECP when compared to healthy subjects. In responders, increased miR-155-5p and decreased miR-23b-3p expression levels at 6 months were found. SMAD4 mRNA was found to be a common target of these two miRNAs in prediction pathways analysis, and a significant downregulation was found at 6 months in PBMCs of a subgroup of ECP-treated patients. According to previous evidence, the upregulation of miR-155 might be correlated with a pro-tolerogenic modulation of the immune system. Our analysis also suggests that SMAD4 might be a possible target for miR-155-5p. Further longitudinal studies are needed to address the possible role of miR-155 and its downstream targets.

Clonal analysis of immunodominance and cross-reactivity of the CD4 T cell response to SARS-CoV-2.

The identification of CD4+ T cell epitopes is instrumental for the design of subunit vaccines for broad protection against coronaviruses. Here, we demonstrate in COVID-19-recovered individuals a robust CD4+ T cell response to naturally processed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and nucleoprotein (N), including effector, helper, and memory T cells. By characterizing 2943 S-reactive T cell clones from 34 individuals, we found that the receptor-binding domain (RBD) is highly immunogenic and that 33% of RBD-reactive clones and 94% of individuals recognized a conserved immunodominant S346-S365 region comprising nested human leukocyte antigen DR (HLA-DR)- and HLA-DP-restricted epitopes. Using pre- and post-COVID-19 samples and S proteins from endemic coronaviruses, we identified cross-reactive T cells targeting multiple S protein sites. The immunodominant and cross-reactive epitopes identified can inform vaccination strategies to counteract emerging SARS-CoV-2 variants.

Cytokine-Induced Memory-Like NK Cells with High Reactivity against Acute Leukemia Blasts and Solid Tumor Cells Suitable for Adoptive Immunotherapy Approaches.

The limited efficacy of Natural Killer (NK) cell-based immunotherapy results in part from the suboptimal expansion and persistence of the infused cells. Recent reports suggest that the generation of NK cells with memory-like properties upon in vitro activation with defined cytokines might be an effective way of ensuring long-lasting NK cell function in vivo. Here, we demonstrate that activation with IL-12, IL-15 and IL-18 followed by a one-week culture with optimal doses of Interleukin (IL-2) and IL-15 generates substantial numbers of memory-like NK cells able to persist for at least three weeks when injected into NOD scid gamma (NSG) mice. This approach induces haploidentical donor-derived memory-like NK cells that are highly lytic against patients' myeloid or lymphoid leukemia blasts, independent of the presence of alloreactive cell populations in the donor and with negligible reactivity against patients' non-malignant cells. Memory-like NK cells able to lyse autologous tumor cells can also be generated from patients with solid malignancies. The anti-tumor activity of allogenic and autologous memory-like NK cells is significantly greater than that displayed by NK cells stimulated overnight with IL-2, supporting their potential therapeutic value both in patients affected by high-risk acute leukemia after haploidentical hematopoietic stem cell transplantation and in patients with advanced solid malignancies.

Comparative Study of Salivary, Duodenal, and Fecal Microbiota Composition Across Adult Celiac Disease.

BACKGROUND: Growing evidence suggests that an altered microbiota composition contributes to the pathogenesis and clinical features in celiac disease (CD). We performed a comparative analysis of the gut microbiota in adulthood CD to evaluate whether: (i) dysbiosis anticipates mucosal lesions, (ii) gluten-free diet restores eubiosis, (iii) refractory CD has a peculiar microbial signature, and (iv) salivary and fecal communities overlap the mucosal one. METHODS: This is a cross-sectional study where a total of 52 CD patients, including 13 active CD, 29 treated CD, 4 refractory CD, and 6 potential CD, were enrolled in a tertiary center together with 31 controls. A 16S rRNA-based amplicon metagenomics approach was applied to determine the microbiota structure and composition of salivary, duodenal mucosa, and stool samples, followed by appropriate bioinformatic analyses. RESULTS: A reduction of both α- and ß-diversity in CD, already evident in the potential form and achieving nadir in refractory CD, was evident. Taxonomically, mucosa displayed a significant abundance of Proteobacteria and an expansion of Neisseria, especially in active patients, while treated celiacs showed an intermediate profile between active disease and controls. The saliva community mirrored the mucosal one better than stool. CONCLUSION: Expansion of pathobiontic species anticipates villous atrophy and achieves the maximal divergence from controls in refractory CD. Gluten-free diet results in incomplete recovery. The overlapping results between mucosal and salivary samples indicate the use of saliva as a diagnostic fluid.

Reduced Efficacy of Biological Drugs in Psoriatic Patients with HLA-A Bw4-80I KIR Ligands.

BACKGROUND: Biological drugs (biologics) are a highly effective therapy for the moderate to severe form of psoriasis, an immune-mediated dermatosis with a strong immunogenetic component. The interaction between human leukocyte antigen (HLA) class I ligands and killer immunoglobulin-like receptors (KIR) has a functional significance in the education of natural killer (NK) cells, and can thus influence the response to biologics. OBJECTIVE: In this study, we investigated the impact of HLA-A and -B KIR ligands in the response to biologics in a cohort of psoriatic patients. METHODS: Eighty-five patients with moderate to severe psoriasis treated with biologics (adalimumab, etanercept, infliximab, ustekinumab and secukinumab) were enrolled in the study. Clinical response was evaluated as patients attaining 50%, 75% or 90% reduction in the Psoriasis Area and Severity Index (PASI) (PASI 50, 75 or 90, respectively) over 6 months' follow-up. Poor response was defined as PASI 50, and in this case patients shifted to treatment with a different biologic. Fifty-two patients (61.2%) showed excellent response (PASI 90) to the first biologic, while 33 patients (38.8%), needed two or more biologics before reaching an excellent response (PASI 90) and were considered difficult to treat. RESULTS: Only HLA-A Bw4-80I ligands were associated with the response to biologics; in particular, they were linked with reduced response both at univariable analysis (odds ratio [OR] 3.11, 95% confidence interval [CI] 1.19-8.07; p = 0.019) and multivariable analysis (OR 5.02, 95% CI 1.40-17.97; p = 0.013). CONCLUSION: We suggest that the HLA-A Bw4-80I epitope could be a marker of reduced responsiveness to biologics. The possible reason for this is an increase of tumour necrosis factor (TNF)-α and the silencing of NK cells through the predominant interaction with the KIR3DL/S pair. HLA-KIR affinities might lead to a more efficient way to prescribe biologics.

Decreased interferon-γ production by NK cells from KIR haplotype B carriers in hepatitis C virus infection.

BACKGROUND AND AIMS: Different population genetics studies showed that interactions between killer-cell immunoglobulin-like receptors (KIR) and HLA play a role in viral disease outcome, but functional correlates are missing. Building upon our previous work pointing to a regulatory role for KIR3DL1/DS1 in hepatitis C virus (HCV) infection, we analysed whether its expression may affect natural killer (NK) cell function in the presence or absence of its principal ligand HLA-Bw4 in KIR haplotype A and B carriers, which are characterized by a different representation of activating and inhibitory KIRs. METHODS: We performed KIR and HLA class I genotypic analysis in 54 healthy donors (HD) and 50 HCV+ subjects and examined NK cell cytokine secretion and degranulation in the context of KIR3DL1-HLA-Bw4 match stratified by KIR haplotype. RESULTS: KIR3DL1-HLA-Bw4 match induced functional NK cell modulation, reflected by reduced interferon (IFN)γ production in haplotype B HCV+ patients compared to HD. This functional impairment could be ascribed to the KIR3DS1 negative HCV-infected patient population, whose NK cells also showed a significantly decreased proportion of KIR3DL1. Haplotype A HCV-infected patients showed increased NK cell degranulation compared with HD in the absence of KIR-HLA-Bw4 match and this activity was associated with increased phosphorylation of signal transducer and activator of transcription (STAT) 1. CONCLUSIONS: Our data show that NK cells from HCV+ patients have an unbalanced ability to produce IFNγ and to kill target cells in haplotype A and B carriers, suggesting the existence of complex functional differences governed by KIR-HLA interaction, particularly on KIR3DL1 expressing NK cells.

Luminex® xMAP® technology is an effective strategy for high-definition human leukocyte antigen typing of cord blood units prior to listing.

INTRODUCTION: Allele-level donor-recipient match at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci impacts the outcome after cord blood transplantation for hematologic malignancies and modifies the strategy of donor selection. High definition of both class I and II HLA loci at time of listing is a way to improve the attractiveness of cord blood bank inventories, reducing the time for donor search and procurement and simplifying donor choice, in particular, for patients of non-European heritage. METHODS: In 2014, Luminex® xMAP® technology was introduced in our laboratory practice and was applied to cord blood units typing. In this study, we evaluated the impact of this strategy in comparison with the platform in use until 2013, relying on LiPA reverse polymerase chain reaction-sequence-specific oligonucleotide (revPCR-SSO) plus polymerase chain reaction-sequence-specific primer (PCR-SSP). RESULTS: In 2014, the time for testing was shorter (141 vs 181 days on average), the number of test repetitions was lower (in particular for HLA-A locus, p = 0.026), and the cost reduced (240.7 vs 395.6 euros per unit on average) compared to 2013, demonstrating that Luminex xMAP technology is superior to the previous approach. CONCLUSION: Luminex xMAP platform has useful application in cord blood banking programs, to achieve high-definition HLA typing of cord blood units at the time of banking in a quick, accurate, and cost-effective manner.