Partitioning stable and unstable expression level variation in cell populations: A theoretical framework and its application to the T cell receptor.

Phenotypic variation in the copy number of gene products expressed by cells or tissues has been the focus of intense investigation. To what extent the observed differences in cellular expression levels are persistent or transient is an intriguing question. Here, we develop a quantitative framework that resolves the expression variation into stable and unstable components. The difference between the expression means in two cohorts isolated from any cell population is shown to converge to an asymptotic value, with a characteristic time, τT, that measures the timescale of the unstable dynamics. The asymptotic difference in the means, relative to the initial value, measures the stable proportion of the original population variance [Formula: see text]. Empowered by this insight, we analysed the T-cell receptor (TCR) expression variation in CD4 T cells. About 70% of TCR expression variance is stable in a diverse polyclonal population, while over 80% of the variance in an isogenic TCR transgenic population is volatile. In both populations the TCR levels fluctuate with a characteristic time of 32 hours. This systematic characterisation of the expression variation dynamics, relying on time series of cohorts' means, can be combined with technologies that measure gene or protein expression in single cells or in bulk.

Activation-induced cytidine deaminase and active DNA demethylation.

The regulation of demethylation in vertebrates has begun to be elucidated in the past decade. However, a possible involvement of activation-induced cytidine deaminase (AID) in this process remains uncertain. We survey the data supporting or casting doubt on such a role, and propose that there is no strong evidence for an involvement of AID in genome-wide active demethylation processes. Conversely, we present evidence that favors AID involvement in gene-specific demethylation events underlying cell differentiation.

In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria.

Dendritic cells (DCs) are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP) of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin)-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip), with Plasmodium chabaudi AS (Pc) parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs) by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection.

A novel activation-induced cytidine deaminase (AID) mutation in Brazilian patients with hyper-IgM type 2 syndrome.

Activation-induced cytidine deaminase (AID) is a DNA editing protein that plays an essential role in three major events of immunoglobulin (Ig) diversification: somatic hypermutation, class switch recombination and Ig gene conversion. Mutations in the AID gene (AICDA) have been found in patients with autosomal recessive Hyper-IgM (HIGM) syndrome type 2. Here, two 9- and 14-year-old Brazilian sisters, from a consanguineous family, were diagnosed with HIGM2 syndrome. Sequencing analysis of the exons from AICDA revealed that both patients are homozygous for a single C to G transversion in the third position of codon 15, which replaces a conserved Phenylalanine with a Leucine. To our knowledge, this is a new AICDA mutation found in HIGM2 patients. Functional studies confirm that the homologous murine mutation leads to a dysfunctional protein with diminished intrinsic cytidine deaminase activity and is unable to rescue CSR when introduced in Aicda(-/-)stimulated murine B cells. We briefly discuss the relevance of AICDA mutations found in patients for the biology of this molecule.

In Vivo Clonal Analysis Reveals Random Monoallelic Expression in Lymphocytes That Traces Back to Hematopoietic Stem Cells.

Evaluating the epigenetic landscape in the stem cell compartment at the single-cell level is essential to assess the cells' heterogeneity and predict their fate. Here, using a genome-wide transcriptomics approach in vivo, we evaluated the allelic expression imbalance in the progeny of single hematopoietic cells (HSCs) as a read-out of epigenetic marking. After 4 months of extensive proliferation and differentiation, we found that X-chromosome inactivation (XCI) is tightly maintained in all single-HSC derived hematopoietic cells. In contrast, the vast majority of the autosomal genes did not show clonal patterns of random monoallelic expression (RME). However, a persistent allele-specific autosomal transcription in HSCs and their progeny was found in a rare number of cases, none of which has been previously reported. These data show that: 1) XCI and RME in the autosomal chromosomes are driven by different mechanisms; 2) the previously reported high frequency of genes under RME in clones expanded in vitro (up to 15%) is not found in clones undergoing multiple differentiation steps in vivo; 3) prior to differentiation, HSCs have stable patterns of autosomal RME. We propose that most RME patterns in autosomal chromosomes are erased and established de novo during cell lineage differentiation.

Mutant IL7R collaborates with MYC to induce T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric cancer. Amongst the wide array of driver mutations, 10% of T-ALL patients display gain-of-function mutations in the IL-7 receptor α chain (IL-7Rα, encoded by IL7R), which occur in different molecular subtypes of this disease. However, it is still unclear whether IL-7R mutational activation is sufficient to transform T-cell precursors. Also, which genes cooperate with IL7R to drive leukemogenesis remain poorly defined. Here, we demonstrate that mutant IL7R alone is capable of inducing T-ALL with long-latency in stable transgenic zebrafish and transformation is associated with MYC transcriptional activation. Additionally, we find that mutant IL7R collaborates with Myc to induce early onset T-ALL in transgenic zebrafish, supporting a model where these pathways collaborate to drive leukemogenesis. T-ALLs co-expressing mutant IL7R and Myc activate STAT5 and AKT pathways, harbor reduced numbers of apoptotic cells and remake tumors in transplanted zebrafish faster than T-ALLs expressing Myc alone. Moreover, limiting-dilution cell transplantation experiments reveal that activated IL-7R signaling increases the overall frequency of leukemia propagating cells. Our work highlights a synergy between mutant IL7R and Myc in inducing T-ALL and demonstrates that mutant IL7R enriches for leukemia propagating potential.

Saliva molecular testing bypassing RNA extraction is suitable for monitoring and diagnosing SARS-CoV-2 infection in children.

BACKGROUND: Adults are being vaccinated against SARS-CoV-2 worldwide, but the longitudinal protection of these vaccines is uncertain, given the ongoing appearance of SARS-CoV-2 variants. Children remain largely unvaccinated and are susceptible to infection, with studies reporting that they actively transmit the virus even when asymptomatic, thus affecting the community. METHODS: We investigated if saliva is an effective sample for detecting SARS-CoV-2 RNA and antibodies in children, and associated viral RNA levels to infectivity. For that, we used a saliva-based SARS-CoV-2 RT-qPCR test, preceded or not by RNA extraction, in 85 children aged 10 years and under, admitted to the hospital regardless of COVID-19 symptomatology. Amongst these, 29 (63.0%) presented at least one COVID-19 symptom, 46 (54.1%) were positive for SARS-CoV-2 infection, 28 (32.9%) were under the age of 1, and the mean (SD) age was 3.8 (3.4) years. Saliva samples were collected up to 48 h after a nasopharyngeal swab-RT-qPCR test. RESULTS: In children aged 10 years and under, the sensitivity, specificity, and accuracy of saliva-RT-qPCR tests compared to NP swab-RT-qPCR were, respectively, 84.8% (71.8%-92.4%), 100% (91.0%-100%), and 91.8% (84.0%-96.6%) with RNA extraction, and 81.8% (68.0%-90.5%), 100% (91.0%-100%), and 90.4% (82.1%-95.0%) without RNA extraction. Rescue of infectious particles from saliva was limited to CT values below 26. In addition, we found significant IgM positive responses to SARS-CoV-2 in children positive for SARS-CoV-2 by NP swab and negative by saliva compared to other groups, indicating late infection onset (>7-10 days). CONCLUSIONS: Saliva is a suitable sample type for diagnosing children aged 10 years and under, including infants aged <1 year, even bypassing RNA extraction methods. Importantly, the detected viral RNA levels were significantly above the infectivity threshold in several samples. Further investigation is required to correlate SARS-CoV-2 RNA levels to viral transmission.

AID from bony fish catalyzes class switch recombination.

Class switch recombination was the last of the lymphocyte-specific DNA modification reactions to appear in the evolution of the adaptive immune system. It is absent in cartilaginous and bony fish, and it is common to all tetrapods. Class switching is initiated by activation-induced cytidine deaminase (AID), an enzyme expressed in cartilaginous and bony fish that is also required for somatic hypermutation. Fish AID differs from orthologs found in tetrapods in several respects, including its catalytic domain and carboxy-terminal region, both of which are essential for the switching reaction. To determine whether evolution of class switch recombination required alterations in AID, we assayed AID from Japanese puffer and zebra fish for class-switching activity in mouse B cells. We find that fish AID catalyzes class switch recombination in mammalian B cells. Thus, AID had the potential to catalyze this reaction before the teleost and tetrapod lineages diverged, suggesting that the later appearance of a class-switching reaction was dependent on the evolution of switch regions and multiple constant regions in the IgH locus.

X-Chromosome Inactivation and Autosomal Random Monoallelic Expression as "Faux Amis".

X-chromosome inactivation (XCI) and random monoallelic expression of autosomal genes (RMAE) are two paradigms of gene expression regulation where, at the single cell level, genes can be expressed from either the maternal or paternal alleles. X-chromosome inactivation takes place in female marsupial and placental mammals, while RMAE has been described in mammals and also other species. Although the outcome of both processes results in random monoallelic expression and mosaicism at the cellular level, there are many important differences. We provide here a brief sketch of the history behind the discovery of XCI and RMAE. Moreover, we review some of the distinctive features of these two phenomena, with respect to when in development they are established, their roles in dosage compensation and cellular phenotypic diversity, and the molecular mechanisms underlying their initiation and stability.

The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing.

The repurposing of the CRISPR/Cas bacterial defense system against bacteriophages as simple and flexible molecular tools has revolutionized the field of gene editing. These tools are now widely used in basic research and clinical trials involving human somatic cells. However, a global moratorium on all clinical uses of human germline editing has been proposed because the technology still lacks the required efficacy and safety. Here we focus on the approaches developed since 2013 to decrease the frequency of unwanted mutations (the off-targets) during CRISPR-based gene editing.

Interleukin-7 receptor α mutational activation can initiate precursor B-cell acute lymphoblastic leukemia.

Interleukin-7 receptor α (encoded by IL7R) is essential for lymphoid development. Whether acute lymphoblastic leukemia (ALL)-related IL7R gain-of-function mutations can trigger leukemogenesis remains unclear. Here, we demonstrate that lymphoid-restricted mutant IL7R, expressed at physiological levels in conditional knock-in mice, establishes a pre-leukemic stage in which B-cell precursors display self-renewal ability, initiating leukemia resembling PAX5 P80R or Ph-like human B-ALL. Full transformation associates with transcriptional upregulation of oncogenes such as Myc or Bcl2, downregulation of tumor suppressors such as Ikzf1 or Arid2, and major IL-7R signaling upregulation (involving JAK/STAT5 and PI3K/mTOR), required for leukemia cell viability. Accordingly, maximal signaling drives full penetrance and early leukemia onset in homozygous IL7R mutant animals. Notably, we identify 2 transcriptional subgroups in mouse and human Ph-like ALL, and show that dactolisib and sphingosine-kinase inhibitors are potential treatment avenues for IL-7R-related cases. Our model, a resource to explore the pathophysiology and therapeutic vulnerabilities of B-ALL, demonstrates that IL7R can initiate this malignancy.

Independent recruitment of Igh alleles in V(D)J recombination.

How the vast majority of B cells express only one of the two alleles at their immunoglobulin loci remains a biological puzzle. Here, in mice reconstituted with a single haematopoietic stem cell, we demonstrate that each of the two immunoglobulin heavy chain (Igh) alleles has a similar probability to be the first to undergo V(H) to DJ(H) rearrangement. We also observe this similar probability in clones from multipotent and common lymphoid precursors. The extreme biases in the expression of the alleles that we find in more differentiated subsets are mostly due to constraints imposed by early rearrangements. Our data demonstrate that each of the two Igh alleles in a B cell behaves independently of the other, up to the moment when a successful rearrangement in one allele triggers a feedback mechanism that prevents further recombination.

Activation-Induced Cytidine Deaminase Does Not Impact Murine Meiotic Recombination.

Activation-induced cytidine deaminase (AID) was first described as the triggering enzyme of the B-cell-specific reactions that edit the immunoglobulin genes, namely somatic hypermutation, gene conversion, and class switch recombination. Over the years, AID was also detected in cells other than lymphocytes, and it has been assigned additional roles in the innate defense against transforming retroviruses, in retrotransposition restriction and in DNA demethylation. Notably, AID expression was found in germline tissues, and in heterologous systems it can induce the double-strand breaks required for the initiation of meiotic recombination and proper gamete formation. However, because AID-deficient mice are fully fertile, the molecule is not essential for meiosis. Thus, the remaining question that we addressed here is whether AID influences the frequency of meiotic recombination in mice. We measured the recombination events in the meiosis of male and female mice F1 hybrids of C57BL/6J and BALB/c, in Aicda+/+ and Aicda-/- background by using a panel of single-nucleotide polymorphisms that distinguishes C57BL/6J from BALB/c genome across the 19 autosomes. In agreement with the literature, we found that the frequency of recombination in the female germline was greater than in male germline, both in the Aicda+/+ and Aicda-/- backgrounds. No statistical difference was found in the average recombination events between Aicda+/+ and Aidca-/- animals, either in females or males. In addition, the recombination frequencies between single-nucleotide polymorphisms flanking the immunoglobulin heavy and immunoglobulin kappa loci was also not different. We conclude that AID has a minor impact, if any, on the overall frequency of meiotic recombination.

A Novel Quantitative Fluorescent Reporter Assay for RAG Targets and RAG Activity.

Recombination-Activating Genes (RAG) 1 and 2 form the site specific recombinase that mediates V(D)J recombination, a process of DNA editing required for lymphocyte development and responsible for their diverse repertoire of antigen receptors. Mistargeted RAG activity associates with genome alteration and is responsible for various lymphoid tumors. Moreover several non-lymphoid tumors express RAG ectopically. A practical and powerful tool to perform quantitative assessment of RAG activity and to score putative RAG-Recognition signal sequences (RSS) is required in the fields of immunology, oncology, gene therapy, and development. Here we report the detailed characterization of a novel fluorescence-based reporter of RAG activity, named GFPi, a tool that allows measuring recombination efficiency (RE) by simple flow cytometry analysis. GFPi can be produced both as a plasmid for transient transfection experiments in cell lines or as a retrovirus for stable integration in the genome, thus supporting ex vivo and in vivo studies. The GFPi assay faithfully quantified endogenous and ectopic RAG activity as tested in genetically modified fibroblasts, tumor derived cell lines, developing pre-B cells, and hematopoietic cells. The GFPi assay also successfully ranked the RE of various RSS pairs, including bona fide RSS associated with V(D)J segments, artificial consensus sequences modified or not at specific nucleotides known to affect their efficiencies, or cryptic RSS involved in RAG-dependent activation of oncogenes. Our work validates the GFPi reporter as a practical quantitative tool for the study of RAG activity and RSS efficiencies. It should turn useful for the study of RAG-mediated V(D)J and aberrant rearrangements, lineage commitment, and vertebrate evolution.

Activation-induced cytidine deaminase structure and functions: a species comparative view.

In the ten years since the discovery of activation-induced cytidine deaminase (AID) there has been considerable effort to understand the mechanisms behind this enzyme's ability to target and modify immunoglobulin genes leading to somatic hypermutation and class switch recombination. While the majority of research has focused on mouse and human models of AID function, work on other species, from lamprey to rabbit and sheep, has taught us much about the scope of functions of the AID mutator. This review takes a species-comparative approach to what has been learned about the AID mutator enzyme and its role in humoral immunity.

Regulation of hypermutation by activation-induced cytidine deaminase phosphorylation.

Activation-induced cytidine deaminase (AID) initiates Ig class switch recombination and somatic hypermutation by producing U:G mismatches in DNA. These mismatches also have the potential to induce DNA damage including double-stranded breaks and chromosome translocations; therefore, strict regulation of AID is important for maintaining genomic stability. In addition to transcriptional regulation, it has been proposed that phosphorylation can also modulate AID activity. Using a combination of MS and immunochemical approaches we found that 5-15% of the AID expressed in activated B cells was phosphorylated at serine-38 (p38AID). This form of AID was enriched in the chromatin fraction in activated B cells, suggesting a role for phosphorylation in targeting AID to DNA. Consistent with this idea, serine-38 to alanine mutant AID (AID(S38A)) showed diminished somatic hypermutation activity on artificial and physiological DNA targets. We conclude that a small fraction of AID is phosphorylated in activated B cells and that the modified form contributes disproportionately to hypermutation.