Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

Characterization of the Intraclonal Complexity of Chronic Lymphocytic Leukemia B Cells: Potential Influences of B-Cell Receptor Crosstalk with Other Stimuli.

Chronic lymphocytic leukemia (CLL) clones contain subpopulations differing in time since the last cell division ("age"): recently born, proliferative (PF; CXCR4DimCD5Bright), intermediate (IF; CXCR4IntCD5Int), and resting (RF; CXCR4BrightCD5Dim) fractions. Herein, we used deuterium (2H) incorporation into newly synthesized DNA in patients to refine the kinetics of CLL subpopulations by characterizing two additional CXCR4/CD5 fractions, i.e., double dim (DDF; CXCR4DimCD5Dim) and double bright (DBF; CXCR4BrightCD5Bright); and intraclonal fractions differing in surface membrane (sm) IgM and IgD densities. Although DDF was enriched in recently divided cells and DBF in older cells, PF and RF remained the most enriched in youngest and oldest cells, respectively. Similarly, smIgMHigh and smIgDHigh cells were the youngest, and smIgMLow and smIgDLow were the oldest, when using smIG levels as discriminator. Surprisingly, the cells closest to the last stimulatory event bore high levels of smIG, and stimulating via TLR9 and smIG yielded a phenotype more consistent with the in vivo setting. Finally, older cells were less sensitive to in vivo inhibition by ibrutinib. Collectively, these data define additional intraclonal subpopulations with divergent ages and phenotypes and suggest that BCR engagement alone is not responsible for the smIG levels found in vivo, and the differential sensitivity of distinct fractions to ibrutinib might account, in part, for therapeutic relapse.

Effects of Deacetylase Inhibition on the Activation of the Antioxidant Response and Aerobic Metabolism in Cellular Models of Fanconi Anemia.

Fanconi anemia (FA) is a rare genetic disease characterized by a dysfunctional DNA repair and an oxidative stress accumulation due to defective mitochondrial energy metabolism, not counteracted by endogenous antioxidant defenses, which appear down-expressed compared to the control. Since the antioxidant response lack could depend on the hypoacetylation of genes coding for detoxifying enzymes, we treated lymphoblasts and fibroblasts mutated for the FANC-A gene with some histone deacetylase inhibitors (HDACi), namely, valproic acid (VPA), beta-hydroxybutyrate (OHB), and EX527 (a Sirt1 inhibitor), under basal conditions and after hydrogen peroxide addition. The results show that VPA increased catalase and glutathione reductase expression and activity, corrected the metabolic defect, lowered lipid peroxidation, restored the mitochondrial fusion and fission balance, and improved mitomycin survival. In contrast, OHB, despite a slight increase in antioxidant enzyme expressions, exacerbated the metabolic defect, increasing oxidative stress production, probably because it also acts as an oxidative phosphorylation metabolite, while EX527 showed no effect. In conclusion, the data suggest that VPA could be a promising drug to modulate the gene expression in FA cells, confirming that the antioxidant response modulation plays a pivotal in FA pathogenesis as it acts on both oxidative stress levels and the mitochondrial metabolism and dynamics quality.

Altered Mitochondrial Dynamic in Lymphoblasts and Fibroblasts Mutated for FANCA-A Gene: The Central Role of DRP1.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and aplastic anemia. So far, 23 genes are involved in this pathology, and their mutations lead to a defect in DNA repair. In recent years, it has been observed that FA cells also display mitochondrial metabolism defects, causing an accumulation of intracellular lipids and oxidative damage. However, the molecular mechanisms involved in the metabolic alterations have not yet been elucidated. In this work, by using lymphoblasts and fibroblasts mutated for the FANC-A gene, oxidative phosphorylation (OxPhos) and mitochondria dynamics markers expression was analyzed. Results show that the metabolic defect does not depend on an altered expression of the proteins involved in OxPhos. However, FA cells are characterized by increased uncoupling protein UCP2 expression. FANC-A mutation is also associated with DRP1 overexpression that causes an imbalance in the mitochondrial dynamic toward fission and lower expression of Parkin and Beclin1. Treatment with P110, a specific inhibitor of DRP1, shows a partial mitochondrial function recovery and the decrement of DRP1 and UCP2 expression, suggesting a pivotal role of the mitochondrial dynamics in the etiopathology of Fanconi anemia.

CLL stereotyped B-cell receptor immunoglobulin sequences are recurrent in the B-cell repertoire of healthy individuals: Apparent lack of central and early peripheral tolerance censoring.

Introduction: The leukemic cells of patients with chronic lymphocytic leukemia (CLL) are often unique, expressing remarkably similar IGHV-IGHD-IGHJ gene rearrangements, "stereotyped BCRs". The B-cell receptors (BCRs) on CLL cells are also distinctive in often deriving from autoreactive B lymphocytes, leading to the assumption of a defect in immune tolerance. Results: Using bulk and single-cell immunoglobulin heavy and light chain variable domain sequencing, we enumerated CLL stereotype-like IGHV-IGHD-IGHJ sequences (CLL-SLS) in B cells from cord blood (CB) and adult peripheral blood (PBMC) and bone marrow (BM of healthy donors. CLL-SLS were found at similar frequencies among CB, BM, and PBMC, suggesting that age does not influence CLL-SLS levels. Moreover, the frequencies of CLL-SLS did not differ among B lymphocytes in the BM at early stages of development, and only re-circulating marginal zone B cells contained significantly higher CLL-SLS frequencies than other mature B-cell subpopulations. Although we identified CLL-SLS corresponding to most of the CLL major stereotyped subsets, CLL-SLS frequencies did not correlate with those found in patients. Interestingly, in CB samples, half of the CLL-SLS identified were attributed to two IGHV-mutated subsets. We also found satellite CLL-SLS among the same normal samples, and they were also enriched in naïve B cells but unexpectedly, these were ~10-fold higher than standard CLL-SLS. In general, IGHV-mutated CLL-SLS subsets were enriched among antigen-experienced B-cell subpopulations, and IGHV-unmutated CLL-SLS were found mostly in antigen-inexperienced B cells. Nevertheless, CLL-SLS with an IGHV-mutation status matching that of CLL clones varied among the normal B-cell subpopulations, suggesting that specific CLL-SLS could originate from distinct subpopulations of normal B cells. Lastly, using single-cell DNA sequencing, we identified paired IGH and IGL rearrangements in normal B lymphocytes resembling those of stereotyped BCRs in CLL, although some differed from those in patients based on IG isotype or somatic mutation. Discussion: CLL-SLS are present in normal B-lymphocyte populations at all stages of development. Thus, despite their autoreactive profile they are not deleted by central tolerance mechanisms, possibly because the level of autoreactivity is not registered as dangerous by deletion mechanisms or because editing of L-chain variable genes occurred which our experimental approach could not identify.

Unexpected CD5+ B Cell Lymphocytosis during SARS-CoV-2 Infection: Relevance for the Pathophysiology of Chronic Lymphocytic Leukemia.

Recently, cases of fortuitous discovery of Chronic Lymphocytic Leukemia (CLL) during hospitalization for Coronavirus disease (COVID-19) have been reported. These patients did not show a monoclonal B cell expansion before COVID-19 but were diagnosed with CLL upon a sudden lymphocytosis that occurred during hospitalization. The (hyper)lymphocytosis during COVID-19 was also described in patients with overt CLL disease. Contextually, lymphocytosis is an unexpected phenomenon since it is an uncommon feature in the COVID-19 patient population, who rather tend to experience lymphopenia. Thus, lymphocytosis that arises during COVID-19 infection is a thought-provoking behavior, strikingly in contrast with that observed in non-CLL individuals. Herein, we speculate about the possible mechanisms involved with the observed phenomenon. Many of the plausible explanations might have an adverse impact on these CLL patients and further clinical and laboratory investigations might be desirable.

Old and New Facts and Speculations on the Role of the B Cell Receptor in the Origin of Chronic Lymphocytic Leukemia.

The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli.

Characterizing Features of Human Circulating B Cells Carrying CLL-Like Stereotyped Immunoglobulin Rearrangements.

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5+ B cells, nor in specific B-cell subpopulations or the CD5+ cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin.

B cell receptor isotypes differentially associate with cell signaling, kinetics, and outcome in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the B cell receptor (BCR) plays a critical role in disease development and progression, as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlying BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each coexpressed by more than 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (Ig) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization associated with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B cell fate decisions, possibly opening new avenues for CLL therapy.

SH3BGRL3 binds to myosin 1c in a calcium dependent manner and modulates migration in the MDA-MB-231 cell line.

BACKGROUND: The human SH3 domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues. However, its function is largely undetermined. The protein was found to be overexpressed in several tumors, and recent work suggested a possible relationship with EGFR family members. We aimed at further highlighting on these issues and investigated SH3BGRL3 molecular interactions and its role in cellular migration ability. RESULTS: We first engineered the ErbB2-overexpressing SKBR3 cells to express exogenous SH3BGRL3, as well as wild type Myo1c or different deletion mutants. Confocal microscopy analysis indicated that SH3BGRL3 co-localized with Myo1c and ErbB2 at plasma membranes. However, co-immunoprecipitation assays and mass spectrometry demonstrated that SH3BGRL3 did not directly bind ErbB2, but specifically recognized Myo1c, on its IQ-bearing neck region. Importantly, the interaction with Myo1c was Ca2+-dependent. A role for SH3BGRL3 in cell migration was also assessed, as RNA interference of SH3BGRL3 in MDA-MB-231 cells, used as a classical migration model, remarkably impaired the migration ability of these cells. On the other side, its over-expression increased cell motility. CONCLUSION: The results of this study provide insights for the formulation of novel hypotheses on the putative role of SH3BGRL3 protein in the regulation of myosin-cytoskeleton dialog and in cell migration. It could be envisaged the SH3BGRL3-Myo1c interaction as a regulation mechanism for cytoskeleton dynamics. It is well known that, at low Ca2+ concentrations, the IQ domains of Myo1c are bound by calmodulin. Here we found that binding of Myo1c to SH3BGRL3 requires instead the presence of Ca2+. Thus, it could be hypothesized that Myo1c conformation may be modulated by Ca2+-driven mechanisms that involve alternative binding by calmodulin or SH3BGRL3, for the regulation of cytoskeletal activity.

Post-Transformation IGHV-IGHD-IGHJ Mutations in Chronic Lymphocytic Leukemia B Cells: Implications for Mutational Mechanisms and Impact on Clinical Course.

Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.

Myeloid-derived suppressor cell subtypes differentially influence T-cell function, T-helper subset differentiation, and clinical course in CLL.

Cancer pathogenesis involves the interplay of tumor- and microenvironment-derived stimuli. Here we focused on the influence of an immunomodulatory cell type, myeloid-derived suppressor cells (MDSCs), and their lineage-related subtypes on autologous T lymphocytes. Although MDSCs as a group correlated with an immunosuppressive Th repertoire and worse clinical course, MDSC subtypes (polymorphonuclear, PMN-MDSC, and monocytic, M-MDSCs) were often functionally discordant. In vivo, PMN-MDSCs existed in higher numbers, correlated with different Th-subsets, and more strongly associated with poor clinical course than M-MDSCs. In vitro, PMN-MDSCs were more efficient at blocking T-cell growth and promoted Th17 differentiation. Conversely, in vitro M-MDSCs varied in their ability to suppress T-cell proliferation, due to the action of TNFα, and promoted a more immunostimulatory Th compartment. Ibrutinib therapy impacted MDSCs differentially as well, since after initiating therapy, PMN-MDSC numbers progressively declined, whereas M-MDSC numbers were unaffected, leading to a set of less immunosuppressive Th cells. Consistent with this, clinical improvement based on decreasing CLL-cell numbers correlated with the decrease in PMN-MDSCs. Collectively, the data support a balance between PMN-MDSC and M-MDSC numbers and function influencing CLL disease course.

Berberine affects mitochondrial activity and cell growth of leukemic cells from chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (CLL) results from accumulation of leukemic cells that are subject to iterative re-activation cycles and clonal expansion in lymphoid tissues. The effects of the well-tolerated alkaloid Berberine (BRB), used for treating metabolic disorders, were studied on ex-vivo leukemic cells activated in vitro by microenvironment stimuli. BRB decreased expression of survival/proliferation-associated molecules (e.g. Mcl-1/Bcl-xL) and inhibited stimulation-induced cell cycle entry, irrespective of TP53 alterations or chromosomal abnormalities. CLL cells rely on oxidative phosphorylation for their bioenergetics, particularly during the activation process. In this context, BRB triggered mitochondrial dysfunction and aberrant cellular energetic metabolism. Decreased ATP production and NADH recycling, associated with mitochondrial uncoupling, were not compensated by increased lactic fermentation. Antioxidant defenses were affected and could not correct the altered intracellular redox homeostasis. The data thus indicated that the cytotoxic/cytostatic action of BRB at 10-30 µM might be mediated, at least in part, by BRB-induced impairment of oxidative phosphorylation and the associated increment of oxidative damage, with consequent inhibition of cell activation and eventual cell death. Bioenergetics and cell survival were instead unaffected in normal B lymphocytes at the same BRB concentrations. Interestingly, BRB lowered the apoptotic threshold of ABT-199/Venetoclax, a promising BH3-mimetic whose cytotoxic activity is counteracted by high Mcl-1/Bcl-xL expression and increased mitochondrial oxidative phosphorylation. Our results indicate that, while CLL cells are in the process of building their survival and cycling armamentarium, the presence of BRB affects this process.

Tracing CLL-biased stereotyped immunoglobulin gene rearrangements in normal B cell subsets using a high-throughput immunogenetic approach.

BACKGROUND: B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5+ and CD5- B cells from the spleen and peripheral blood (PB). METHODS: Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5+ and CD5- cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution. RESULTS: CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5+ B (0.57%) cells compared to CD5- B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family. CONCLUSIONS: CBS-IG receptors appear to represent a part of the "public" BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.

IGLV3-21*01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling.

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

Endogenous retroviruses are associated with hippocampus-based memory impairment.

Retrotransposons compose a staggering 40% of the mammalian genome. Among them, endogenous retroviruses (ERV) represent sequences that closely resemble the proviruses created from exogenous retroviral infection. ERVs make up 8 to 10% of human and mouse genomes and range from evolutionarily ancient sequences to recent acquisitions. Studies in Drosophila have provided a causal link between genomic retroviral elements and cognitive decline; however, in mammals, the role of ERVs in learning and memory remains unclear. Here we studied 2 independent murine models for ERV activation: muMT strain (lacking B cells and antibody production) and intracerebroventricular injection of streptozotocin (ICVI-STZ). We conducted behavioral assessments (contextual fear memory and spatial learning), as well as gene and protein analysis (RNA sequencing, PCR, immunohistochemistry, and western blot assays). Mice lacking mitochondrial antiviral-signaling protein (MAVS) and mice lacking stimulator of IFN genes protein (STING), 2 downstream sensors of ERV activation, provided confirmation of ERV impact. We found that muMT mice and ICVI-STZ mice induced hippocampal ERV activation, as shown by increased gene and protein expression of the Gag sequence of the transposable element intracisternal A-particle. ERV activation was accompanied by significant hippocampus-related memory impairment in both models. Notably, the deficiency of the MAVS pathway was protective against ICVI-STZ-induced cognitive pathology. Overall, our results demonstrate that ERV activation is associated with cognitive impairment in mice. Moreover, they provide a molecular target for strategies aimed at attenuating retroviral element sensing, via MAVS, to treat dementia and neuropsychiatric disorders.

Measurement of Leukemic B-Cell Growth Kinetics in Patients with Chronic Lymphocytic Leukemia.

Cell proliferation plays a central role in the pathogenesis of every neoplastic disease as well as many other types of illness. Labeling of newly replicated DNA with deuterium (2H), a nonradioactive isotope of hydrogen, administered to the patients in drinking water (2H2O) is a safe and reliable method to measure the in vivo birth rates of cells. Here, we describe a protocol to measure chronic lymphocytic leukemia B-cell birth/proliferation and death rates over time using this approach.

Novel Method for High-Throughput Full-Length IGHV-D-J Sequencing of the Immune Repertoire from Bulk B-Cells with Single-Cell Resolution.

Efficient and accurate high-throughput DNA sequencing of the adaptive immune receptor repertoire (AIRR) is necessary to study immune diversity in healthy subjects and disease-related conditions. The high complexity and diversity of the AIRR coupled with the limited amount of starting material, which can compromise identification of the full biological diversity makes such sequencing particularly challenging. AIRR sequencing protocols often fail to fully capture the sampled AIRR diversity, especially for samples containing restricted numbers of B lymphocytes. Here, we describe a library preparation method for immunoglobulin sequencing that results in an exhaustive full-length repertoire where virtually every sampled B-cell is sequenced. This maximizes the likelihood of identifying and quantifying the entire IGHV-D-J repertoire of a sample, including the detection of rearrangements present in only one cell in the starting population. The methodology establishes the importance of circumventing genetic material dilution in the preamplification phases and incorporates the use of certain described concepts: (1) balancing the starting material amount and depth of sequencing, (2) avoiding IGHV gene-specific amplification, and (3) using Unique Molecular Identifier. Together, this methodology is highly efficient, in particular for detecting rare rearrangements in the sampled population and when only a limited amount of starting material is available.

Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.