Busulfan-fludarabine versus busulfan-cyclophosphamide for allogeneic transplant in acute myeloid leukemia: long term analysis of GITMO AML-R2 trial.

We report the long-term results of a randomized trial (GITMO, AML-R2), comparing 1:1 the combination of busulfan and cyclophosphamide (BuCy2, n = 125) and the combination of busulfan and fludarabine (BuFlu, n = 127) as conditioning regimen in acute myeloid leukemia patients (median age 51 years, range 40-65) undergoing allogeneic hematopoietic stem cell transplantation. With a median follow-up of 6 years, significantly better non-relapse mortality (NRM) was confirmed in BuFlu recipients, which is sustained up to 4 years after transplant (10% vs. 20%, p = 0.0388). This difference was higher in patients older than 51 years (11% in BuFlu vs. 27% in BuCy2, p = 0.0262). The cumulative incidence of relapse, which was the first cause of death in the entire study population, did not differ between the two randomized arms. Similarly, the leukemia-free survival (LFS) and overall survival (OS) were not different in the two cohorts, even when stratifying patients per median age. Graft-and relapse-free survival (GRFS) in BuFlu arm vs. the BuCy2 arm was 25% vs. 20% at 4 years and 20% vs. 17% at 10 years. Hence, the benefit gained by NRM reduction is not offsets by an increased relapse. Leukemia relapse remains a major concern, urging the development of new therapeutic approaches.

Spike-Specific Memory B Cell Response in Hematopoietic Cell Transplantation Recipients following Multiple mRNA-1273 Vaccinations: A Longitudinal Observational Study.

Preventing SARS-CoV-2 infection is of utmost importance in allogeneic hematopoietic cell transplantation patients (allo-HCT), given their heightened susceptibility to adverse outcomes associated with SARS-CoV-2 infection. However, limited data are available regarding the immune response to COVID-19 vaccines in these subjects, particularly concerning the generation and persistence of spike-specific memory response. Here, we analyzed the spike-specific memory B cells in a cohort of allo-HCT recipients vaccinated with multiple doses of the mRNA-1273 vaccine and monitored the spike-specific antibody response from baseline up to one month after the fourth dose. After the primary vaccine series, the frequency of spike-specific B cells, detected within the pool of Ig-switched CD19+ cells, significantly increased. The booster dose further induced a significant expansion, reaching up to 0.28% of spike-specific B cells. The kinetics of this expansion were slower in the allo-HCT recipients compared to healthy controls. Spike-specific IgG and ACE2/RBD binding inhibition activity were observed in 80% of the allo-HCT recipients after the first two doses, with a significant increase after the third and fourth booster doses, including in the subjects who did not respond to the primary vaccine series. Additionally, 87% of the allo-HCT recipients exhibited positive cross-inhibition activity against the BA.1 variant. Our findings provide evidence that allo-HCT recipients need repeated doses of the mRNA-1273 vaccine to induceSARS-CoV-2 specific immune response similar to that observed in healthy individuals. This is particularly crucial for vulnerable individuals who may exhibit a limited response to the primary series of SARS-CoV-2 vaccination.

A novel potent class I HDAC inhibitor reverses the STAT4/p66Shc apoptotic defect in B cells from chronic lymphocytic leukemia patients.

Chronic Lymphocytic Leukemia (CLL) patients have a defective expression of the proapoptotic protein p66Shc and of its transcriptional factor STAT4, which evoke molecular abnormalities, impairing apoptosis and worsening disease prognosis and severity. p66Shc expression is epigenetically controlled and transcriptionally modulated by STAT4; epigenetic modifiers are deregulated in CLL cells and specific histone deacetylases (HDACs) like HDAC1, are overexpressed. Reactivation of STAT4/p66Shc expression may represent an attractive and challenging strategy to reverse CLL apoptosis defects. New selective class I HDAC inhibitors (HDACis, 6a-g) were developed with increased potency over existing agents and preferentially interfering with the CLL-relevant isoform HDAC1, to unveil the role of class I HDACs in the upregulation of STAT4 expression, which upregulates p66Shc expression and hence normalizes CLL cell apoptosis. 6c (chlopynostat) was identified as a potent HDAC1i with a superior profile over entinostat. 6c induces marked apoptosis of CLL cells compared with SAHA, which was associated with an upregulation of STAT4/p66Shc protein expression. The role of HDAC1, but not HDAC3, in the epigenetic upregulation of STAT4/p66Shc was demonstrated for the first time in CLL cells and was validated in siRNA-induced HDAC1/HDAC3 knock-down EBV-B cells. To sum up, HDAC1 inhibition is necessary to reactivate STAT4/p66Shc expression in patients with CLL. 6c is one of the most potent HDAC1is known to date and represents a novel pharmacological tool for reversing the impairment of the STAT4/p66Shc apoptotic machinery.

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia.

The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eµ-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adapter whose deficiency has been implicated in disease aggressiveness both in CLL and in the Eµ-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from Eµ-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL.

p66Shc deficiency in CLL cells enhances PD-L1 expression and suppresses immune synapse formation.

Introduction: Escape from immunosurveillance is a hallmark of chronic lymphocytic leukemia (CLL) cells. In the protective niche of lymphoid organs, leukemic cells suppress the ability of T lymphocytes to form the immune synapse (IS), thereby hampering T-cell mediated anti-tumoral activities. By binding its cognate receptor PD-1 at the surface of T lymphocytes, the inhibitory ligand PD-L1, which is overexpressed in CLL cells, mediates the T-cell suppressive activities of CLL cells. However, the molecular mechanism underlying PD-L1 overexpression in CLL cells remains unknown. We have previously reported a defective expression of the pro-apoptotic and pro-oxidant adaptor p66Shc in CLL cells, which is causally related to an impairment in intracellular reactive oxygen species (ROS) production and to the activation of the ROS-sensitive transcription factor NF-κB. The fact that PD-L1 expression is regulated by NF-κB suggests a mechanistic relationship between p66Shc deficiency and PD-L1 overexpression in CLL cells. Methods: 62 treatment-naive CLL patients and 43 healthy donors were included in this study. PD-L1 and p66Shc expression was quantified in B cells by flow cytometry and qRT-PCR. IS architecture and local signaling was assessed by flow cytometry and confocal microscopy. CD8+ cell killing activity was assessed by flow cytometry. Results: Here we show that residual p66Shc expression in leukemic cells isolated both from CLL patients and from the CLL mouse model Eµ-TCL1 inversely correlated with PD-L1 expression. We also show that the PD-L1 increase prevented leukemic cells from forming ISs with T lymphocytes. Reconstitution of p66Shc, but not of a ROS-defective mutant, in both CLL cells and the CLL-derived cell line MEC-1, enhanced intracellular ROS and decreased PD-L1 expression. Similar results were obtained following treatment of CLL cells with H2O2 as exogenous source of ROS, that normalized PD-L1 expression and recovered IS formation. Discussion: Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to enhance PD-L1 expression and provides a mechanistic basis for the suppression of T cell-mediated anti-tumoral functions in the immunosuppressive lymphoid niche.

HLA class I expression on human platelets is highly variable and correlates with distinct allele group frequencies.

BACKGROUND: Human leukocyte antigen (HLA) class I molecules are expressed on platelets and can represent a source of alloimmunization in recipients of platelet transfusions. HLA mismatch between donors and recipients may be associated with the induction of anti-HLA antibodies, which can culminate in refractoriness to platelet transfusions. In the present study we analyzed HLA allele group frequencies and HLA expression levels on human platelets from blood donors. MATERIALS AND METHODS: Platelet-rich plasma was collected from 139 donors to monitor platelet HLA class I expression by flow cytometry. DNA from donors with high and low platelet HLA expression was used in the genotype studies. Frequencies of large and normal-sized platelet subpopulations were determined and HLA class I expression was studied. Mean platelet volume (MPV) and platelet large-cell ratio (P-LCR) were analyzed in both groups of donors. RESULTS: The analysis showed variable platelet HLA class I expression with significant differences among donors. HLA class I allele group frequencies in donors with high and low platelet HLA expression showed distinctive genotypic features strictly related to expression level. The main allele groups found in samples with high platelet HLA class I expression were HLA-A*02, -A*68, -B*15, -B*49, and -C*03. Platelet HLA class I expression did not change over time or during freezing-thawing cycles. The analysis of platelet subpopulations showed a statistically significant higher expression of HLA class I molecules on large platelets than on normal-sized platelets. Moreover, donors with high HLA class I expression showed a higher frequency of large platelets (p<0.0001). The analysis of P-LCR in both groups of donors showed a statistically significant difference (p<0.05) within high HLA-expressing donors. DISCUSSION: Our data suggest an allele-dependent expression of HLA class I molecules on human platelets with distinct HLA allele group frequencies and different platelet subpopulation frequencies among blood donors.

Human colorectal cancer: upregulation of the adaptor protein Rai in TILs leads to cell dysfunction by sustaining GSK-3 activation and PD-1 expression.

BACKGROUND: The immunosuppressive tumor microenvironment (TME) of colorectal cancer (CRC) is a major hurdle for immune checkpoint inhibitor-based therapies. Hence characterization of the signaling pathways driving T cell exhaustion within TME is a critical need for the discovery of novel therapeutic targets and the development of effective therapies. We previously showed that (i) the adaptor protein Rai is a negative regulator of T cell receptor signaling and T helper 1 (Th1)/Th17 cell differentiation; and (ii) Rai deficiency is implicated in the hyperactive phenotype of T cells in autoimmune diseases. METHODS: The expression level of Rai was measured by qRT-PCR in paired peripheral blood T cells and T cells infiltrating tumor tissue and the normal adjacent tissue in CRC patients. The impact of hypoxia-inducible factor (HIF)-1α on Rai expression was evaluated in T cells exposed to hypoxia and by performing chromatin immunoprecipitation assays and RNA interference assays. The mechanism by which upregulation of Rai in T cells promotes T cell exhaustion were evaluated by flow cytometric, qRT-PCR and western blot analyses. RESULTS: We show that Rai is a novel HIF-1α-responsive gene that is upregulated in tumor infiltrating lymphocytes of CRC patients compared to patient-matched circulating T cells. Rai upregulation in T cells promoted Programmed cell Death protein (PD)-1 expression and impaired antigen-dependent degranulation of CD8+ T cells by inhibiting phospho-inactivation of glycogen synthase kinase (GSK)-3, a central regulator of PD-1 expression and T cell-mediated anti-tumor immunity. CONCLUSIONS: Our data identify Rai as a hitherto unknown regulator of the TME-induced exhausted phenotype of human T cells.

Polo-like kinase 1 regulates immune synapse assembly and cytotoxic T cell function by driving microtubule dynamics.

Elimination of virally infected or tumoral cells is mediated by cytotoxic T cells (CTL). Upon antigen recognition, CTLs assemble a specialized signaling and secretory domain at the interface with their target, the immune synapse (IS). During IS formation, CTLs acquire a transient polarity, marked by re-orientation of the centrosome and microtubule cytoskeleton toward the IS, thus directing the transport and delivery of the lytic granules to the target cell. Based on the implication that the kinase Aurora A has a role in CTL function, we hypothesized that its substrate, the mitotic regulator Polo-like kinase 1 (PLK1), might participate in CTL IS assembly. We demonstrate that PLK1 is phosphorylated upon TCR triggering and polarizes to the IS. PLK1 silencing or inhibition results in impaired IS assembly and function, as witnessed by defective synaptic accumulation of T cell receptors (TCRs), as well as compromised centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing. This function is achieved by coupling early signaling to microtubule dynamics, a function pivotal for CTL-mediated cytotoxicity. These results identify PLK1 as a new player in CTL IS assembly and function.

The rootstock shape microbial diversity and functionality in the rhizosphere of Vitis vinifera L. cultivar Falanghina.

The rhizosphere effect occurring at the root-soil interface has increasingly been shown to play a key role in plant fitness and soil functionality, influencing plants resilience. Here, for the first time, we investigated whether the rootstock genotype on which Vitis vinifera L. cultivar Falanghina is grafted can influence the rhizosphere microbiome. Specifically, we evaluated to which extent the 5BB and 1103P rootstocks are able to shape microbial diversity of rhizosphere environment. Moreover, we explored the potential function of microbial community and its shift under plant genotype influence. We investigated seven vineyards subjected to the same pedo-climatic conditions, similar age, training system and management and collected twelve rhizosphere soil samples for metagenomic analyses and composite soil samples for physical-chemical properties. In this study, we used 16S rRNA gene-based metagenomic analysis to investigate the rhizosphere bacterial diversity and composition. Liner discriminant analysis effect size (LEFSe) was conducted for metagenomic biomarker discovery. The functional composition of sampled communities was determined using PICRUSt, which is based on marker gene sequencing profiles. Soil analyses involved the determination of texture, pH, Cation Exchange Capacity (CSC), Organic Carbon (OC), electrical conductivity (EC), calcium (Ca), magnesium (Mg), potassium (K) content, Phosphorous (P), nitrogen (N). The latter revealed that soil features were quite homogenous. The metagenomic data showed that the bacterial alpha-diversity (Observed OTUs) significantly increased in 1103P rhizosphere microbiota. Irrespective of cultivar, Pseudomonadota was the dominant phylum, followed by Actinomycetota > Bacteroidota > Thermoproteota. However, Actinomycetota was the major marker phyla differentiating the rhizosphere microbial communities associated with the different rootstock types. At the genus level, several taxa belonging to Actinomycetota and Alphaproteobacteria classes were enriched in 1103P genotype rhizosphere. Investigating the potential functional profile, we found that most key enzyme-encoding genes involved in N cycling were significantly more abundant in 5BB rootstock rhizosphere soil. However, we found that 1103P rhizosphere was enriched in genes involved in C cycle and Plant Growth Promotion (PGP) functionality. Our results suggest that the different rootstocks not only recruit specific bacterial communities, but also specific functional traits within the same environment.

Rationale for the evaluation of renal functional reserve in allogeneic stem cell transplantation candidates: a pilot study.

Background: The main purpose of our study was to evaluate the ability of renal functional reserve (RFR) to stratify the risk of acute kidney injury (AKI) occurrence within 100 days of hematopoietic stem cell transplantation (HSCT) and to predict any functional recovery or the onset of chronic kidney disease. A secondary aim was to identify the clinical/laboratory risk factors for the occurrence of AKI. Methods: The study design is prospective observational. We enrolled 48 patients with normal basal glomerular filtration rate (bGFR) who underwent allogenic HSCT. A multiparameter assessment and the Renal Functional Reserve Test (RFR-T) using an oral protein load stress test were performed 15 days before the HSCT. Results: Different RFRs corresponded to the same bGFR values. Of 48 patients, 29 (60%) developed AKI. Comparing the AKI group with the group that did not develop AKI, no statistically significant difference emerged in any characteristic related to demographic, clinical or multiparameter assessment variables except for the estimated GFR (eGFR). eGFR ≤100 mL/min/1.73 m2 was significantly related to the risk of developing AKI (Fisher's exact test, P = .001). Moreover, RFR-T was lower in AKI+ patients vs AKI- patients, but did not allow statistical significance (28% vs 40%). In AKI patients, RFR >20% was associated with complete functional recovery (one-sided Fisher's exact test, P = .041). The risk of failure to recover increases significantly when RFR ≤20% (odds ratio = 5.50, 95% confidence interval = 1.06-28.4). Conclusion: RFR identifies subclinical functional deterioration conditions essential for post-AKI recovery. In our cohort of patients with no kidney disease (NKD), the degree of pre-HSCT eGFR is associated with AKI risk, and a reduction in pre-HSCT RFR above a threshold of 20% is related to complete renal functional recovery post-AKI. Identifying eGFR first and RFR second could help select patients who might benefit from changes in transplant management or early nephrological assessment.

SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly.

CTL-mediated killing of virally infected or malignant cells is orchestrated at the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent elimination of infected cells.

Ultrasound Detection of Intracranial Hypertension in Brain Injuries.

In recent years, the measurement of optic nerve sheath diameter with ultrasound to detect the presence of increased intracranial pressure has widely spread. It can be qualitatively and effectively used to identify intracranial hypertension. Intracranial pressure can rise due to acute injury, cerebral bleeding, hydrocephalus, brain tumors and other space-occupying abnormalities, and it is linked to a high death rate. The purpose of this review is to give a general overview of the most relevant scientific publications on ultrasonographic evaluation of the optic nerve in case of brain injuries published in the last 30 years, as well as to analyze the limits of the most extensively used B-scan approach. Fifty-two papers chosen from the PubMed medical database were analyzed in this review. Our findings revealed that ocular ultrasound is an useful diagnostic tool in the management of intracranial hypertension when it exceeds a certain value or after head trauma. As a result, an ultrasound of the optic nerve can be extremely helpful in guiding diagnosis and treatment. The blooming effect is one of the most critical restrictions to consider when using B-scan ultrasonography. Since amplitude-scan ultrasound, also known as A-scan, does not have this limit, these two diagnostic techniques should always be used together for a more full, accurate, and trustworthy ultrasound examination, ensuring more data objectivity.

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19.

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage.

Assessing gene function in human B cells: CRISPR/Cas9-based gene editing and mRNA-based gene expression in healthy and tumor cells.

Robust methods for manipulation of human B cells, isolated from healthy donors and patients with B cell disorders, has the potential to significantly accelerate B cell research. Our work describes a step-by-step protocol to perform electroporation-based screening of gene function in B cells through the use of Cas9 ribonuclecomplexes and in vitro produced mRNA.

Hypoxia Induces Autophagy in Human Dendritic Cells: Involvement of Class III PI3K/Vps34.

Hypoxia is a component of both physiological and pathological conditions, including inflammation, solid tumors, and lymphoid tissues, where O2 demand is not balanced by O2 supply. During their lifespan, dendritic cells (DCs) are exposed to different pO2 and activate different adaptive responses, including autophagy, to preserve their viability and functions. Autophagy plays multiple roles in DC physiology. Very recently, we demonstrated that hypoxia shapes autophagy in DCs upon their differentiation state. Here, we proposed a role for PI3Ks, and especially class III PI3K/Vps34, that could be relevant in hypoxia-induced autophagy, in either immature or mature DCs. Hypoxia inhibited mTOR phosphorylation and activated a pro-autophagic program. By using different pharmacological inhibitors, we demonstrated that hypoxia-induced autophagy was mediated by PI3Ks, especially by Vps34. Furthermore, Vps34 expression was enhanced by LPS, a TLR4 ligand, along with the promotion of autophagy under hypoxia. The Vps34 inhibitor, SAR405, abolished hypoxia-induced autophagy, inhibited pro-survival signaling and viability, and increased the expression of proinflammatory cytokines. Our results underlined the impact of autophagy in the maintenance of DC homeostasis at both cell survival and inflammatory response levels, therefore, contributing to a better understanding of the significance of autophagy in DC physiology and pathology.