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Phosphatidylinositol 3-kinase delta (PI3KCD) is a critical signaling enzyme for B cell development, activation, function and immune regulation. Gain-of-function mutations in PI3KCD result in the congenital immunodeficiency known as Activated PI3KCD Syndrome (APDS). APDS patients are prone to repeated infections and other serious clinical manifestations. Here, we determine how B cell-intrinsic expression of the APDS-associated PI3KCDE1021K mutation impacts immune responses?to the protozoan parasite Trypanosoma congolense. PI3KCDE1021K/B mice exhibit a significant expansion of IL10-expressing?B cells within the spleen and peritoneal cavity, which was associated with impaired control of T. congolense infection. Despite the generation of robust germinal center, plasma cell and antibody responses, PI3KCDE1021K/B mice show elevation in the first wave of parasitemia and increased mortality. We further characterize the phenotype of the expanded IL10-producing B cell population in PI3KCDE1021K/B mice, which show hallmarks of innate-like regulatory B cells (Breg) and expression of multiple inhibitory molecules. This Breg expansion is associated with reduced IFN¦Ã/IL10 ratio, reduced TNF¦Á production and impaired activation of myeloid cells, likely compromising the innate response to infection. These findings highlight the profound impact of dysregulated PI3KCD activity on regulatory B cells that can functionally impair innate immune responses controlling a systemic parasite protozoan disease. Author summaryB cells and antibodies play a critical role in the immune response to Trypanosome parasites. Molecular signaling networks within B cells can control the type of response generated during infection. Here, we studied how a genetic variant in the signaling enzyme PI3KCD, previously linked to human immune deficiencies, impacts B cell responses to Trypanosome infection. We find that mice expressing the PI3KCDE1021K mutation in their B cells show impaired control of Trypanosome infection, and alterations in several aspects of the immune response. Specifically, we noted these mice poorly control parasite growth within the first week of infection, a timeframe where specific antibody responses have not yet been generated. We noted an altered balance between pro-inflammatory and anti-inflammatory cytokine mediators produced within the first week of infection. This was associated with high numbers of regulatory B cells expressing multiple molecules capable of inhibiting other cells of the immune system. We further found that these mice show functional alterations in other critical immune cell types, such as macrophages and T cells. These findings highlight the impact of dysregulated PI3KCD activity on regulatory B cells that can impair immune responses controlling a systemic parasite protozoan disease.

The levels of transcription factor Ets1 are high in resting B and T cells, but are downregulated by signaling through antigen receptors and Toll-like receptors (TLRs). Loss of Ets1 in mice leads to excessive immune cell activation and development of an autoimmune syndrome and reduced Ets1 expression has been observed in human PBMCs in the context of autoimmune diseases. In B cells, Ets1 serves to prevent premature activation and differentiation to antibody-secreting cells. Given these important roles for Ets1 in the immune response, stringent control of Ets1 gene expression levels is required for homeostasis. However, the genetic regulatory elements that control expression of the Ets1 gene remain relatively unknown. Here we identify a topologically-associating domain (TAD) in the chromatin of B cells that includes the mouse Ets1 gene locus and describe an interaction hub that extends over 100?kb upstream and into the gene body. Additionally, we compile epigenetic datasets to find several putative regulatory elements within the interaction hub by identifying regions of high DNA accessibility and enrichment of active enhancer histone marks. Using reporter constructs, we determine that DNA sequences within this interaction hub are sufficient to direct reporter gene expression in lymphoid tissues of transgenic mice. Further analysis indicates that the reporter construct drives faithful expression of the reporter gene in mouse B cells, but variegated expression in T cells, suggesting the existence of T cell regulatory elements outside this region. To investigate how the downregulation of Ets1 transcription is associated with alterations in the epigenetic landscape of stimulated B cells, we performed ATAC-seq in resting and BCR-stimulated primary B cells and identified four regions within and upstream of the Ets1 locus that undergo changes in chromatin accessibility that correlate to Ets1 gene expression. Interestingly, functional analysis of several putative Ets1 regulatory elements using luciferase constructs suggested a high level of functional redundancy. Taken together our studies reveal a complex network of regulatory elements and transcription factors that coordinate the B cell-specific expression of Ets1.

BackgroundCNS stromal cells, especially fibroblasts and endothelial cells, support leukocyte accumulation through upregulation of adhesion molecules and lymphoid chemokines. While chronically activated fibroblast networks can drive pathogenic immune cell aggregates known as tertiary lymphoid structures (TLS), early stromal cell activation during CNS infection can support anti-viral T cells. However, the cell types and factors driving early stromal cell activation is poorly explored.AimsA neurotropic murine coronavirus (mCoV) infection model was used to better characterize signals that promote fibroblast networks supporting accumulation of antiviral lymphocytes. Based on the early appearance of IgD+ B cells with unknown functions during several CNS infections, we probed their potential to activate stromal cells through lymphotoxin ¦Â (LT¦Â), a molecule critical in maintaining fibroblast-networks in lymphoid tissues as well as promoting TLS in autoimmunity and cancers.ResultsKinetic analysis of stromal cell activation in olfactory bulbs and brains revealed that upregulation of adhesion molecules and lymphoid chemokines Ccl19, Ccl21 and Cxcl13 closely tracked viral replication. Immunohistochemistry revealed that upregulation of the fibroblast marker podoplanin (PDPN) at meningeal and perivascular sites mirrored kinetics of RNA expression. Moreover, both B cells and T cells colocalized to areas of PDPN reactivity, supporting a potential role in regulating stromal cell activation. However, specific depletion of LT¦Â from B cells using Mb1-creERT2 x Lt¦Âfl/fl mice had no effect on T or B cell recruitment or viral replication. B cell depletion by anti-CD20 antibody also had no adverse effects. Surprisingly, LT¦ÂR agonism reduced viral control and parenchymal T cell localization despite increasing stromal cell lymphoid chemokines and PDPN. Additional assessment of direct stromal cell activation by the viral RNA mimic poly I:C showed induction of Pdpn and Ccl19 preceding Ltb.ConclusionsNeither B cell-derived LT¦Â or B cells are primary drivers of stromal cell activation networks in the CNS following mCoV infection. Although supplementary agonist mediated LT¦ÂR engagement confirmed a role for LT¦Â in enhancing PDPN and lymphoid chemokine expression, it impeded T cell migration to the CNS parenchyma and viral control. Our data overall indicate that stromal cells can integrate LT¦ÂR signals to tune their activation, but that LT¦Â is not necessarily essential and can even dysregulate protective antiviral T cell functions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03491-7.