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Bispecific T cell-engagers (BTEs) are engineered antibodies that redirect T cells to target antigen-expressing tumors. BTEs targeting tumor-specific antigens such as interleukin 13 receptor alpha 2 (IL13RA2) and epidermal growth factor receptor variant III (EGFRvIII) have been developed for glioblastoma (GBM). However, there is limited mechanistic understanding of the action of BTE since prior studies were mostly conducted in immunocompromised animal models. To close this gap, the function of BTEs was assessed in the immunosuppressive tumor microenvironment (TME) of orthotopic and genetically engineered mouse models (GEMM) with intact immune systems. Method: sA BTE that bridges CD3 epsilon on murine T cells to IL13RA2-positive GBM cells was developed, and the therapeutic mechanism was investigated in immunocompetent mouse models of GBM. Multicolor flow cytometry, single-cell RNA sequencing (scRNA-seq), multiplex immunofluorescence, and multiparametric MRI across multiple preclinical models of GBM were used to evaluate the mechanism of action and response. Results: BTE-mediated interactions between murine T cells and GBM cells triggered T cell activation and antigen-dependent killing of GBM cells. BTE treatment significantly extended the survival of mice bearing IL13RA2-expressing orthotopic glioma and de novo forming GBM in the GEMM. Quantified parametric MRI validated the survival data, showing a reduction in glioma volume and decreased glioma viability. Flow cytometric and scRNA-seq analyses of the TME revealed robust increases in activated and memory T cells and decreases in immunosuppressive myeloid cells within the brains of mice following BTE treatment. Conclusions: Our data demonstrate that the survival benefits of BTEs in preclinical models of glioma are due to the ability to engage the host immune system in direct killing, induction of immunological memory, and modulation of the TME. These findings provide a deeper insight into the mechanism of BTE actions in GBM.

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.