miR-15/16 Restrain Memory T Cell Differentiation, Cell Cycle, and Survival.

Coordinate control of T cell proliferation, survival, and differentiation are essential for host protection from pathogens and cancer. Long-lived memory cells, whose precursors are formed during the initial immunological insult, provide protection from future encounters, and their generation is the goal of many vaccination strategies. microRNAs (miRNAs) are key nodes in regulatory networks that shape effective T cell responses through the fine-tuning of thousands of genes. Here, using compound conditional mutant mice to eliminate miR-15/16 family miRNAs in T cells, we show that miR-15/16 restrict T cell cycle, survival, and memory T cell differentiation. High throughput sequencing of RNA isolated by cross-linking immunoprecipitation of AGO2 combined with gene expression analysis in miR-15/16-deficient T cells indicates that these effects are mediated through the direct inhibition of an extensive network of target genes within pathways critical to cell cycle, survival, and memory.

Antigen Complexed with a TLR9 Agonist Bolsters c-Myc and mTORC1 Activity in Germinal Center B Lymphocytes.

The germinal center (GC) is the anatomical site where humoral immunity evolves. B cells undergo cycles of proliferation and selection to produce high-affinity Abs against Ag. Direct linkage of a TLR9 agonist (CpG) to a T-dependent Ag increases the number of GC B cells. We used a T-dependent Ag complexed with CpG and a genetic model for ablating the TLR9 signaling adaptor molecule MyD88 specifically in B cells (B-MyD88- mice) together with transcriptomics to determine how this innate pathway positively regulates the GC. GC B cells from complex Ag-immunized B-MyD88- mice were defective in inducing gene expression signatures downstream of c-Myc and mTORC1. In agreement with the latter gene signature, ribosomal protein S6 phosphorylation was increased in GC B cells from wild-type mice compared with B-MyD88- mice. However, GC B cell expression of a c-Myc protein reporter was enhanced by CpG attached to Ag in both wild-type and B-MyD88- mice, indicating a B cell-extrinsic effect on c-Myc protein expression combined with a B cell-intrinsic enhancement of gene expression downstream of c-Myc. Both mTORC1 activity and c-Myc are directly induced by T cell help, indicating that TLR9 signaling in GC B cells either enhances their access to T cell help or directly influences these pathways to further enhance the effect of T cell help. Taken together, these findings indicate that TLR9 signaling in the GC could provide a surrogate prosurvival stimulus, "TLR help," thus lowering the threshold for selection and increasing the magnitude of the GC response.

The Formin mDia1 Regulates Acute Lymphoblastic Leukemia Engraftment, Migration, and Progression in vivo.

Leukemias typically arise in the bone marrow and then spread to the blood and into other tissues. To disseminate into tissues, leukemia cells migrate into the blood stream and then exit the circulation by migrating across vascular endothelial barriers. Formin proteins regulate cytoskeletal remodeling and cell migration of normal and malignant cells. The Formin mDia1 is highly expressed in transformed lymphocytes and regulates lymphocyte migration. However, the role of mDia1 in regulating leukemia progression in vivo is unknown. Here, we investigated how mDia1 mediates the ability of leukemia cells to migrate and disseminate in vivo. For these studies, we used a mouse model of Bcr-Abl pre-B cell acute lymphoblastic leukemia. Our data showed that mDia1-deficient leukemia cells have reduced chemotaxis and ability to complete transendothelial migration in vitro. In vivo, mDia1 deficiency reduced the ability of leukemia cells to engraft in recipient mice. Furthermore, leukemia dissemination to various tissues and leukemia progression were inhibited by mDia1 depletion. Finally, mDia1 depletion in leukemia cells resulted in prolonged survival of recipient mice in a leukemia transfer model. Overall, our data show that the Formin mDia1 mediates leukemia cell migration, and drives leukemia engraftment and progression in vivo, suggesting that targeting mDia1 could provide a new method for treatment of leukemia.

Thymic tuft cells promote an IL-4-enriched medulla and shape thymocyte development.

The thymus is responsible for generating a diverse yet self-tolerant pool of T cells1. Although the thymic medulla consists mostly of developing and mature AIRE+ epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought2. Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers3. Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function.

Myosin-IIA regulates leukemia engraftment and brain infiltration in a mouse model of acute lymphoblastic leukemia.

Leukemia dissemination (the spread of leukemia cells from the bone marrow) and relapse are associated with poor prognosis. Often, relapse occurs in peripheral organs, such as the CNS, which acts as a sanctuary site for leukemia cells to escape anti-cancer treatments. Similar to normal leukocyte migration, leukemia dissemination entails migration of cells from the blood circulation into tissues by extravasation. To extravasate, leukemia cells cross through vascular endothelial walls via a process called transendothelial migration, which requires cytoskeletal remodeling. However, the specific molecular players in leukemia extravasation are not fully known. We examined the role of myosin-IIA a cytoskeletal class II myosin motor protein, in leukemia progression and dissemination into the CNS by use of a mouse model of Bcr-Abl-driven B cell acute lymphoblastic leukemia. Small hairpin RNA-mediated depletion of myosin-IIA did not affect apoptosis or the growth rate of B cell acute lymphoblastic leukemia cells. However, in an in vivo leukemia transfer model, myosin-IIA depletion slowed leukemia progression and prolonged survival, in part, by reducing the ability of B cell acute lymphoblastic leukemia cells to engraft efficiently. Finally, myosin-IIA inhibition, either by small hairpin RNA depletion or chemical inhibition by blebbistatin, drastically reduced CNS infiltration of leukemia cells. The effects on leukemia cell entry into tissues were mostly a result of the requirement for myosin-IIA to enable leukemia cells to complete the transendothelial migration process during extravasation. Overall, our data implicate myosin-IIA as a key mediator of leukemia cell migration, making it a promising target to inhibit leukemia dissemination in vivo and potentially reduce leukemia relapses.