Astrocytic trans-Differentiation Completes a Multicellular Paracrine Feedback Loop Required for Medulloblastoma Tumor Growth.

The tumor microenvironment (TME) is critical for tumor progression. However, the establishment and function of the TME remain obscure because of its complex cellular composition. Using a mouse genetic system called mosaic analysis with double markers (MADMs), we delineated TME evolution at single-cell resolution in sonic hedgehog (SHH)-activated medulloblastomas that originate from unipotent granule neuron progenitors in the brain. First, we found that astrocytes within the TME (TuAstrocytes) were trans-differentiated from tumor granule neuron precursors (GNPs), which normally never differentiate into astrocytes. Second, we identified that TME-derived IGF1 promotes tumor progression. Third, we uncovered that insulin-like growth factor 1 (IGF1) is produced by tumor-associated microglia in response to interleukin-4 (IL-4) stimulation. Finally, we found that IL-4 is secreted by TuAstrocytes. Collectively, our studies reveal an evolutionary process that produces a multi-lateral network within the TME of medulloblastoma: a fraction of tumor cells trans-differentiate into TuAstrocytes, which, in turn, produce IL-4 that stimulates microglia to produce IGF1 to promote tumor progression.

Multiple Immune and Genetic Mechanisms Contribute to Cmv5s-Driven Susceptibility and Tissue Damage during Acute Murine Cytomegalovirus Infection.

The MHC class I molecule H-2Dk conveys resistance to acute murine CMV infection in both C57L (H-2Dk transgenic) and MA/My mice. M.H2k/b mice are on an MA/My background aside from a C57L-derived region spanning the MHC (Cmv5s), which diminishes this resistance and causes significant spleen histopathology. To hone in on the effector elements within the Cmv5s interval, we generated several Cmv5-recombinant congenic mouse strains and screened them in vivo, allowing us to narrow the phenotype-associated interval >6-fold and segment the genetic mechanism to at least two independent loci within the MHC region. In addition, we sought to further characterize the Cmv5s-associated phenotypes in their temporal appearance and potential direct relationship to viral load. To this end, we found that Cmv5s histopathology and NK cell activation could not be fully mirrored in the MA/My mice with increased viral dose, and that marginal zone destruction was the first apparent Cmv5s phenotype, being reliably quantified as early as 2 d postinfection in the M.H2k/b mice, prior to divergence in viral load, weight loss, or NK cell phenotype. Finally, we further dissect NK cell involvement, finding no intrinsic differences in NK cell function, despite increased upregulation of activation markers and checkpoint receptors. In conclusion, these data dissect the genetic and immunologic underpinnings of Cmv5 and reveal a model in which polymorphism within the MHC region of the genome leads to the development of tissue damage and corrupts protective NK cell immunity during acute viral infection.

Altered-Self MHC Class I Sensing via Functionally Disparate Paired NK Cell Receptors Counters Murine Cytomegalovirus gp34-Mediated Immune Evasion.

The murine CMV (MCMV) immunoevasin m04/gp34 escorts MHC class I (MHC I) molecules to the surface of infected cells where these complexes bind Ly49 inhibitory receptors (IRs) and prevent NK cell attack. Nonetheless, certain self-MHC I-binding Ly49 activating and inhibitory receptors are able to promote robust NK cell expansion and antiviral immunity during MCMV infection. A basis for MHC I-dependent NK cell sensing of MCMV-infected targets and control of MCMV infection however remains unclear. In this study, we discovered that the Ly49R activation receptor is selectively triggered during MCMV infection on antiviral NK cells licensed by the Ly49G2 IR. Ly49R activating receptor recognition of MCMV-infected targets is dependent on MHC I Dk and MCMV gp34 expression. Remarkably, although Ly49R is critical for Ly49G2-dependent antiviral immunity, blockade of the activation receptor in Ly49G2-deficient mice has no impact on virus control, suggesting that paired Ly49G2 MCMV sensing might enable Ly49R+ NK cells to better engage viral targets. Indeed, MCMV gp34 facilitates Ly49G2 binding to infected cells, and the IR is required to counter gp34-mediated immune evasion. A specific requirement for Ly49G2 in antiviral immunity is further explained by its capacity to license cytokine receptor signaling pathways and enhance Ly49R+ NK cell proliferation during infection. These findings advance our understanding of the molecular basis for functionally disparate self-receptor enhancement of antiviral NK cell immunity.

c-Myb Coordinates Survival and the Expression of Genes That Are Critical for the Pre-BCR Checkpoint.

The c-Myb transcription factor is required for adult hematopoiesis, yet little is known about c-Myb function during lineage-specific differentiation due to the embryonic lethality of Myb-null mutations. We previously used tissue-specific inactivation of the murine Myb locus to demonstrate that c-Myb is required for differentiation to the pro-B cell stage, survival during the pro-B cell stage, and the pro-B to pre-B cell transition during B lymphopoiesis. However, few downstream mediators of c-Myb-regulated function have been identified. We demonstrate that c-Myb regulates the intrinsic survival of CD19+ pro-B cells in the absence of IL-7 by repressing expression of the proapoptotic proteins Bmf and Bim and that levels of Bmf and Bim mRNA are further repressed by IL-7 signaling in pro-B cells. c-Myb regulates two crucial components of the IL-7 signaling pathway: the IL-7Rα-chain and the negative regulator SOCS3 in CD19+ pro-B cells. Bypassing IL-7R signaling through constitutive activation of Stat5b largely rescues survival of c-Myb-deficient pro-B cells, whereas constitutively active Akt is much less effective. However, rescue of pro-B cell survival is not sufficient to rescue proliferation of pro-B cells or the pro-B to small pre-B cell transition, and we further demonstrate that c-Myb-deficient large pre-B cells are hypoproliferative. Analysis of genes crucial for the pre-BCR checkpoint demonstrates that, in addition to IL-7Rα, the genes encoding λ5, cyclin D3, and CXCR4 are downregulated in the absence of c-Myb, and λ5 is a direct c-Myb target. Thus, c-Myb coordinates survival with the expression of genes that are required during the pre-BCR checkpoint.

c-Myb promotes the survival of CD4+CD8+ double-positive thymocytes through upregulation of Bcl-xL.

Mechanisms that regulate the lifespan of CD4(+)CD8(+) double-positive (DP) thymocytes help shape the peripheral T cell repertoire. However, the molecular mechanisms controlling DP thymocyte survival remain poorly understood. The Myb proto-oncogene encodes a transcription factor required during multiple stages of T cell development. We demonstrate that Myb mRNA expression is upregulated as thymocytes differentiate from the double-negative into the metabolically quiescent, small, preselection DP stage during T cell development. Using a conditional deletion mouse model, we demonstrate that Myb-deficient DP thymocytes undergo premature apoptosis, resulting in a limited Tcralpha repertoire biased toward 5' Jalpha segment usage. Premature apoptosis occurs specifically in the small preselection DP compartment in an alphabetaTCR-independent manner and is a consequence of decreased Bcl-xL expression. Forced Bcl-xL expression is able to rescue survival, and reintroduction of c-Myb restores both Bcl-xL expression and the small preselection DP compartment. We further demonstrate that c-Myb promotes transcription at the Bcl2l1 locus via a genetic pathway that is independent of the expression of T cell-specific factor-1 or RORgammat, two transcription factors that induce Bcl-xL expression in T cell development. Thus, Bcl-xL is a novel mediator of c-Myb activity during normal T cell development.

c-Myb is required for pro-B cell differentiation.

The c-Myb transcription factor is required for normal adult hematopoiesis. However, the embryonic lethality of Myb-null mutations has been an impediment to identifying roles for c-Myb during lymphocyte development. We have used tissue-specific inactivation of the Myb locus in early progenitor cells to demonstrate that c-Myb is absolutely required for the differentiation of CD19(+) B-lineage cells and B cell differentiation is profoundly blocked beyond the pre-pro-B cell stage in Myb(f/f) Mb1-cre mice. We demonstrate that c-Myb is required for the intrinsic survival of CD19(+) pro-B cells as well as the proper expression of the alpha-chain of the IL-7 receptor (CD127) and Ebf1. However, survival of c-Myb-deficient CD19(+) pro-B cells cannot be rescued by transduction with CD127-producing retrovirus, suggesting that c-Myb controls a survival pathway independent of CD127. Furthermore, c-Myb-deficient progenitor cells inefficiently generate CD19(+) B-lineage cells during stromal cell culture but this process can be partially rescued with exogenous Ebf1. Thus, c-Myb does not appear to be required for commitment to B cell differentiation but is crucial for B cell differentiation to the CD19(+) pro-B cell stage as well as survival of CD19(+) pro-B cells. Surprisingly, forced c-Myb expression in lymphoid-primed multipotent progenitors favors differentiation toward the myeloid lineage, suggesting that proper c-Myb expression is crucial for B-lineage development.

Deletion of the SAPS1 subunit of protein phosphatase 6 in mice increases radiosensitivity and impairs the cellular DNA damage response.

Cellular responses to DNA damage include activation of DNA-dependent protein kinase (DNA-PK) through, among others, the serine/threonine protein phosphatase 6 (PP6). We previously showed that recognition of DNA-PKcs is mediated by the SAPS1 PP6 regulatory subunit. Here, we report and characterize a SAPS1 null mouse and investigate the effects of deletion on DNA damage signaling and repair. Strikingly, neither SAPS1-null animals nor cells derived from them show gross defects, unless subjected to DNA damage by radiation or chemical agents. The overall survival of SAPS1-null animals following whole body irradiation is significantly shortened as compared to wild-type mice, and the clonogenic survival of null cells subjected to ionizing radiation is reduced. The dephosphorylation of DNA damage/repair markers, such as γH2AX, p53 and Kap1, is diminished in SAPS1-null cells as compared to wild-type controls. Our results demonstrate that loss of SAPS1 confers sensitivity to DNA damage and confirms previously reported cellular phenotypes of SAPS1 knock-down in human glioma cells. The results support a role for PP6 regulatory subunit SAPS1 in DNA damage responses, and offer a novel target for sensitization to enhance current tumor therapies, with a potential for limited deleterious side effects.