Molecular characterization of diffuse large B-cell lymphomas associated with hepatitis C virus infection.

Hepatitis C virus (HCV)-associated diffuse large B-cell lymphoma (DLBCL) displays peculiar clinicopathological characteristics, but its molecular landscape is not fully elucidated. In this study, we investigated the clinicopathological and molecular features of 54 patients with HCV-associated DLBCL. The median age was 71 years. An underlying marginal zone lymphoma component was detected in 14.8% of cases. FISH analysis showed rearrangements involving BCL6 in 50.9% of cases, MYC in 11.3% and BCL2 in 3.7%. Lymph2Cx-based assay was successful in 38 cases, recognizing 16 cases (42.1%) as ABC and 16 cases as GCB subtypes, while six resulted unclassified. ABC cases exhibited a higher lymphoma-related mortality (LRM). Next-generation sequencing analysis showed mutations in 158/184 evaluated genes. The most frequently mutated genes were KMT2D (42.6%), SETD1B (33.3%), RERE (29.4%), FAS and PIM1 (27.8%) and TBL1XR1 (25.9%). A mutation in the NOTCH pathway was detected in 25.9% of cases and was associated with worst LRM. Cluster analysis by LymphGen classified 29/54 cases within definite groups, including BN2 in 14 (48.2%), ST2 in seven (24.2%) and MCD and EZB in four each (13.8%). Overall, these results indicate a preferential marginal zone origin for a consistent subgroup of HCV-associated DLBCL cases and suggest potential implications for molecularly targeted therapies.

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.

Phenotypic Variation in Two Siblings Affected with Shwachman-Diamond Syndrome: The Use of Expert Variant Interpreter (eVai) Suggests Clinical Relevance of a Variant in the KMT2A Gene.

Introduction. Shwachman-Diamond Syndrome (SDS) is an autosomal-recessive disorder characterized by neutropenia, pancreatic exocrine insufficiency, skeletal dysplasia, and an increased risk for leukemic transformation. Biallelic mutations in the SBDS gene have been found in about 90% of patients. The clinical spectrum of SDS in patients is wide, and variability has been noticed between different patients, siblings, and even within the same patient over time. Herein, we present two SDS siblings (UPN42 and UPN43) carrying the same SBDS mutations and showing relevant differences in their phenotypic presentation. Study aim. We attempted to understand whether other germline variants, in addition to SBDS, could explain some of the clinical variability noticed between the siblings. Methods. Whole-exome sequencing (WES) was performed. Human Phenotype Ontology (HPO) terms were defined for each patient, and the WES data were analyzed using the eVai and DIVAs platforms. Results. In UPN43, we found and confirmed, using Sanger sequencing, a novel de novo variant (c.10663G > A, p.Gly3555Ser) in the KMT2A gene that is associated with autosomal-dominant Wiedemann−Steiner Syndrome. The variant is classified as pathogenic according to different in silico prediction tools. Interestingly, it was found to be related to some of the HPO terms that describe UPN43. Conclusions. We postulate that the KMT2A variant found in UPN43 has a concomitant and co-occurring clinical effect, in addition to SBDS mutation. This dual molecular effect, supported by in silico prediction, could help to understand some of the clinical variations found among the siblings. In the future, these new data are likely to be useful for personalized medicine and therapy for selected cases.

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.

Feasibility and Efficacy of CD45RA+ Depleted Donor Lymphocytes Infusion After Haploidentical Transplantation With Post-Transplantation Cyclophosphamide in Patients With Hematological Malignancies.

Allogeneic stem cell transplantation from haploidentical donor using post-transplantation cyclophosphamide has been used to cure hematological diseases. Because of slow immunological reconstitution, there is an increased incidence of viral infection. The aim of our study was to prospectively evaluate the efficacy and the feasibility of a CD45RA+ depleted donor lymphocytes infusion (DLI) in terms of reduction of viral infection early after haploidentical transplantation. This a prospective single-center study. We enrolled 23 patients, of whom 19 were evaluable. Graft-versus-host disease (GVHD) prophylaxis was the same for all patients. The primary endpoint was 100-day cumulative incidence of viral infections. The primary endpoint was met, because the 100-day cumulative incidence of viral infection was 32%. The median time from transplantation to first CD45RA+ depleted DLI was 55 days (range, 46-63). 28% of patients had cytomegalovirus reactivation, no patients reactivated human herpesvirus-6; 1 patient developed BK virus related hemorrhagic cystitis. Most of the patients received the planned 3 infusions. Only 1 patient had development of grade 2 acute GVHD, and 2 patients had moderate chronic GVHD. All evaluable patients were off immunosuppressive therapy at last follow-up. The median follow-up was 12 months (range, 3-23), the 1-year overall survival and progression-free survival were 79% and 75%, respectively; the 100-day and 1-year non-relapse mortality were 5% and 12%, respectively. CD45RA+ depleted DLI are feasible in patients treated with haploidentical transplantation. The toxic profile is good with a low risk for development of both acute and chronic GVHD.

Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity.

Adoptive T cell transfer (ACT) immunotherapy benefits from early differentiated stem cell memory T (Tscm) cells capable of persisting in the long term and generating potent antitumor effectors. Due to their paucity ex vivo, Tscm cells can be derived from naive precursors, but the molecular signals at the basis of Tscm cell generation are ill-defined. We found that less differentiated human circulating CD8+ T cells display substantial antioxidant capacity ex vivo compared with more differentiated central and effector memory T cells. Limiting ROS metabolism with antioxidants during naive T cell activation hindered terminal differentiation, while allowing expansion and generation of Tscm cells. N-acetylcysteine (NAC), the most effective molecule in this regard, induced transcriptional and metabolic programs characteristic of self-renewing memory T cells. Upon ACT, NAC-generated Tscm cells established long-term memory in vivo and exerted more potent antitumor immunity in a xenogeneic model when redirected with CD19-specific CAR, highlighting the translational relevance of NAC as a simple and inexpensive method to improve ACT.

Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

Human T memory stem (TSCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8+ T-cell differentiation and allows the generation of CD45RO- CD45RA+ CCR7+ CD27+ CD95+ -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human TSCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy.

Transducin-like enhancer of split-1 is expressed and functional in human macrophages.

Macrophages display heterogeneous phenotypes, including the classical M1 proinflammatory and the alternative M2 anti-inflammatory polarization states. The transducin-like enhancer of split-1 (TLE1) is a transcriptional corepressor whose functions in macrophages have not been studied yet. We report that TLE1 is highly expressed in human alternative macrophages in vitro and in atherosclerotic plaques as well as in adipose tissue M1/M2 mixed macrophages. TLE1 silencing in alternative macrophages decreases the expression of the M2 markers IL-1Ra and IL-10, while it exacerbates TNFα and CCL3 induction by lipopolysaccharide. Hence, TLE1 is expressed in human macrophages where it has potential anti-inflammatory and alternative phenotype promoting properties.

Macrophage phenotypes and their modulation in atherosclerosis.

Atherosclerosis is the result of a chronic inflammatory response in the arterial wall related to uptake of low-density lipoprotein by macrophages and their subsequent transformation in foam cells. Monocyte-derived macrophages are the principal mediators of tissue homeostasis and repair, response to pathogens and inflammation. However, macrophages are a homogeneous cell population presenting a continuum phenotypic spectrum with, at the extremes, the classically Th-1 polarized M1 and alternatively Th-2 polarized M2 macrophage phenotypes, which have been well described. Moreover, M2 macrophages also present several subtypes often termed M2a, b, c and d, each of them expressing specific markers and exhibiting specialized properties. Macrophage plasticity is mirrored also in the atherosclerotic lesions, where different stimuli can influence the phenotype giving rise to a complex system of subpopulations, such as Mox, Mhem, M(Hb) and M4 macrophages. An abundant literature has described the potential modulators of the reciprocal skewing between pro-inflammatory M1 and anti-inflammatory M2 macrophages including lesion stage and localization, miRNA, transcription factors such as PPARγ, KLF4 and NR4A family members, high-density lipoproteins and plaque lipid content, pathways such as the rapamycin-mTOR1 pathway, molecules such as thioredoxin-1, infection by helminths and irradiation. We hope to provide an overview of the macrophage phenotype complexity in cardiovascular diseases, particularly atherosclerosis.

miR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages.

Liver X receptors (LXRα and LXRß) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRß. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity.