Igh and Igk loci use different folding principles for V gene recombination due to distinct chromosomal architectures of pro-B and pre-B cells.

Extended loop extrusion across the immunoglobulin heavy-chain (Igh) locus facilitates VH-DJH recombination following downregulation of the cohesin-release factor Wapl by Pax5, resulting in global changes in the chromosomal architecture of pro-B cells. Here, we demonstrate that chromatin looping and VK-JK recombination at the Igk locus were insensitive to Wapl upregulation in pre-B cells. Notably, the Wapl protein was expressed at a 2.2-fold higher level in pre-B cells compared with pro-B cells, which resulted in a distinct chromosomal architecture with normal loop sizes in pre-B cells. High-resolution chromosomal contact analysis of the Igk locus identified multiple internal loops, which likely juxtapose VK and JK elements to facilitate VK-JK recombination. The higher Wapl expression in Igµ-transgenic pre-B cells prevented extended loop extrusion at the Igh locus, leading to recombination of only the 6 most 3' proximal VH genes and likely to allelic exclusion of all other VH genes in pre-B cells. These results suggest that pro-B and pre-B cells with their distinct chromosomal architectures use different chromatin folding principles for V gene recombination, thereby enabling allelic exclusion at the Igh locus, when the Igk locus is recombined.

Developmental dynamics of two bipotent thymic epithelial progenitor types.

T cell development in the thymus is essential for cellular immunity and depends on the organotypic thymic epithelial microenvironment. In comparison with other organs, the size and cellular composition of the thymus are unusually dynamic, as exemplified by rapid growth and high T cell output during early stages of development, followed by a gradual loss of functional thymic epithelial cells and diminished naive T cell production with age1-10. Single-cell RNA sequencing (scRNA-seq) has uncovered an unexpected heterogeneity of cell types in the thymic epithelium of young and aged adult mice11-18; however, the identities and developmental dynamics of putative pre- and postnatal epithelial progenitors have remained unresolved1,12,16,17,19-27. Here we combine scRNA-seq and a new CRISPR-Cas9-based cellular barcoding system in mice to determine qualitative and quantitative changes in the thymic epithelium over time. This dual approach enabled us to identify two principal progenitor populations: an early bipotent progenitor type biased towards cortical epithelium and a postnatal bipotent progenitor population biased towards medullary epithelium. We further demonstrate that continuous autocrine provision of Fgf7 leads to sustained expansion of thymic microenvironments without exhausting the epithelial progenitor pools, suggesting a strategy to modulate the extent of thymopoietic activity.

Cohesin-dependence of neuronal gene expression relates to chromatin loop length.

Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here, we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in mouse primary cortical neurons. Immediate early genes (IEGs) remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the IEGs Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterized by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend on cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts.

Pax5 regulates B cell immunity by promoting PI3K signaling via PTEN down-regulation.

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

Neuronal genes deregulated in Cornelia de Lange Syndrome respond to removal and re-expression of cohesin.

Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterize the transcriptional profile of cortical neurons from CdLS patients and find deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupts 3D genome organization and transcriptional control in post-mitotic cortical mouse neurons, demonstrating that cohesin is continuously required for neuronal gene expression. The genes affected by acute depletion of cohesin belong to similar gene ontology classes and show significant numerical overlap with genes deregulated in CdLS. Interestingly, reconstitution of cohesin function largely rescues altered gene expression, including the expression of genes deregulated in CdLS.

Synergistic, context-dependent, and hierarchical functions of epithelial components in thymic microenvironments.

Specialized niche environments specify and maintain stem and progenitor cells, but little is known about the identities and functional interactions of niche components in vivo. Here, we describe a modular system for the generation of artificial thymopoietic environments in the mouse embryo. Thymic epithelium that lacks hematopoietic function but is physiologically accessible for hematopoietic progenitor cells is functionalized by individual and combinatorial expression of four factors, the chemokines Ccl25 and Cxcl12, the cytokine Scf, and the Notch ligand DLL4. The distinct phenotypes and variable numbers of hematopoietic cells in the resulting epithelial environments reveal synergistic, context-dependent, and hierarchical interactions among effector molecules. The surprisingly simple rules determining hematopoietic properties enable the in vivo engineering of artificial environments conducive to the presence of distinct myeloid or T or B lymphoid lineage precursors; moreover, synthetic environments facilitate the procurement of physiological progenitor cell types for analytical purposes and future therapeutic applications.

Three chemokine receptors cooperatively regulate homing of hematopoietic progenitors to the embryonic mouse thymus.

The thymus lacks self-renewing hematopoietic cells, and thymopoiesis fails rapidly when the migration of progenitor cells to the thymus ceases. Hence, the process of thymus homing is an essential step for T-cell development and cellular immunity. Despite decades of research, the molecular details of thymus homing have not been elucidated fully. Here, we show that chemotaxis is the key mechanism regulating thymus homing in the mouse embryo. We determined the number of early thymic progenitors in the thymic rudiments of mice deficient for one, two, or three of the chemokine receptor genes, chemokine (C-C motif) receptor 9 (Ccr9), chemokine (C-C motif) receptor 7 (Ccr7), and chemokine (C-X-C motif) receptor 4 (Cxcr4). In the absence of all three chemokine receptors, thymus homing was reduced about 100-fold both before and after vascularization of the thymic rudiment. In the absence of only two of these three chemokine receptor genes, thymus homing was much less affected (only two- to 10-fold), indicating that the chemotactic regulation of thymus homing is remarkably robust. Our results reveal the redundant roles of Ccr9, Ccr7, and Cxcr4 for thymic homing and provide a framework to examine the regulation of progenitor homing in the postnatal thymus.

Modulation of the specific allergic response by mite allergens encapsulated into liposomes.

Liposomes are non toxic and biodegradable lipid vesicles, which are safe and effective adjuvants to induce Th1-skewed immune response. Therefore, the encapsulation of allergens into liposomes could be an attractive alternative for specific allergy immunotherapy. Previously, we obtained DPPC iposomes encapsulating purified allergens from Dermatophagoides siboney, with suitable stability and extremely reduced allergenicity. In this study, Balb/c mice were immunized with allergens ncapsulated into liposomes (LP) and the induced immune response was evaluated in comparison with allergens dissolved in PBS (PBSA) or adsorbed in Alum (AL). The use of Alum or Liposomes induced a strong allergen specific IgG response. However, total IgE serum levels in the AL group were very high, while levels found in LP group were not significantly different from the control group receiving only PBS. The IgG2a/IgG1 subclass ratio was raised in the LP group. Allergen specific IgE, as measured by PCA assay, was similar for LP and PBSA groups, and approximately the half of the reaction size found in AL group. After allergen challenge by inhalation route, peripheral blood and airway eosinophil counts increased significantly in AL, but not in LP group. Additionally, histopathological analysis of lung tissue sections obtained from challenged mice indicated a reduced cellular infiltration in mice immunized with liposomes. These results support the potential use of liposomal formulations for allergen vaccines.