Effects of tumor grade and dexamethasone on myeloid cells in patients with glioma.

Efforts to reduce immunosuppression in the solid tumor microenvironment by blocking the recruitment or polarization of tumor associated macrophages (TAM), or myeloid derived suppressor cells (MDSCs), have gained momentum in recent years. Expanding our knowledge of the immune cell types, cytokines, or recruitment factors that are associated with high-grade disease, both within the tumor and in circulation, is critical to identifying novel targets for immunotherapy. Furthermore, a better understanding of how therapeutic regimens, such as Dexamethasone (Dex), chemotherapy, and radiation, impact these factors will facilitate the design of therapies that can be targeted to the appropriate populations and retain efficacy when administered in combination with standard of care regimens. Here we perform quantitative analysis of tissue microarrays made of samples taken from grades I-III astrocytoma and glioblastoma (GBM, grade IV astrocytoma) to evaluate infiltration of myeloid markers CD163, CD68, CD33, and S100A9. Serum, flow cytometric, and Nanostring analysis allowed us to further elucidate the impact of Dex treatment on systemic biomarkers, circulating cells, and functional markers within tumor tissue. We found that common myeloid markers were elevated in Dex-treated grade I astrocytoma and GBM compared to non-neoplastic brain tissue and grade II-III astrocytomas. Cell frequencies in these samples differed significantly from those in Dex-naïve patients in a pattern that depended on tumor grade. In contrast, observed changes in serum chemokines or circulating monocytes were independent of disease state and were due to Dex treatment alone. Furthermore, these changes seen in blood were often not reflected within the tumor tissue. Conclusions: Our findings highlight the importance of considering perioperative treatment as well as disease grade when assessing novel therapeutic targets or biomarkers of disease.

Developmental arrest of T cells in Rpl22-deficient mice is dependent upon multiple p53 effectors.

αß and γδ lineage T cells are thought to arise from a common CD4(-)CD8(-) progenitor in the thymus. However, the molecular pathways controlling fate selection and maturation of these two lineages remain poorly understood. We demonstrated recently that a ubiquitously expressed ribosomal protein, Rpl22, is selectively required for the development of αß lineage T cells. Germline ablation of Rpl22 impairs development of αß lineage, but not γδ lineage, T cells through activation of a p53-dependent checkpoint. In this study, we investigate the downstream effectors used by p53 to impair T cell development. We found that many p53 targets were induced in Rpl22(-/-) thymocytes, including miR-34a, PUMA, p21(waf), Bax, and Noxa. Notably, the proapoptotic factor Bim, while not a direct p53 target, was also strongly induced in Rpl22(-/-) T cells. Gain-of-function analysis indicated that overexpression of miR-34a caused a developmental arrest reminiscent of that induced by p53 in Rpl22-deficient T cells; however, only a few p53 targets alleviated developmental arrest when individually ablated by gene targeting or knockdown. Co-elimination of PUMA and Bim resulted in a nearly complete restoration of development of Rpl22(-/-) thymocytes, indicating that p53-mediated arrest is enforced principally through effects on cell survival. Surprisingly, co-elimination of the primary p53 regulators of cell cycle arrest (p21(waf)) and apoptosis (PUMA) actually abrogated the partial rescue caused by loss of PUMA alone, suggesting that the G1 checkpoint protein p21(waf) facilitates thymocyte development in some contexts.

Coordinated regulation of cell cycle transcripts by p53-Inducible microRNAs, miR-192 and miR-215.

Cell cycle arrest in response to DNA damage is an important antitumorigenic mechanism. MicroRNAs (miRNAs) were recently shown to play key regulatory roles in cell cycle progression. For example, miR-34a is induced in response to p53 activation and mediates G(1) arrest by down-regulating multiple cell cycle-related transcripts. Here we show that genotoxic stress promotes the p53-dependent up-regulation of the homologous miRNAs miR-192 and miR-215. Like miR-34a, activation of miR-192/215 induces cell cycle arrest, suggesting that multiple miRNA families operate in the p53 network. Furthermore, we define a downstream gene expression signature for miR-192/215 expression, which includes a number of transcripts that regulate G(1) and G(2) checkpoints. Of these transcripts, 18 transcripts are direct targets of miR-192/215, and the observed cell cycle arrest likely results from a cooperative effect among the modulations of these genes by the miRNAs. Our results showing a role for miR-192/215 in cell proliferation combined with recent observations that these miRNAs are underexpressed in primary cancers support the idea that miR-192 and miR-215 function as tumor suppressors.

Enforced expression of Spi-B reverses T lineage commitment and blocks beta-selection.

The molecular changes that restrict multipotent murine thymocytes to the T cell lineage and render them responsive to Ag receptor signals remain poorly understood. In this study, we report our analysis of the role of the Ets transcription factor, Spi-B, in this process. Spi-B expression is acutely induced coincident with T cell lineage commitment at the CD4(-)CD8(-)CD44(-)CD25(+) (DN3) stage of thymocyte development and is then down-regulated as thymocytes respond to pre-TCR signals and develop beyond the beta-selection checkpoint to the CD4(-)CD8(-)CD44(-)CD25(-) (DN4) stage. We found that dysregulation of Spi-B expression in DN3 thymocytes resulted in a dose-dependent perturbation of thymocyte development. Indeed, DN3 thymocytes expressing approximately five times the endogenous level of Spi-B were arrested at the beta-selection checkpoint, due to impaired induction of Egr proteins, which are important molecular effectors of the beta-selection checkpoint. T lineage-committed DN3 thymocytes expressing even higher levels of Spi-B were diverted to the dendritic cell lineage. Thus, we demonstrate that the prescribed modulation of Spi-B expression is important for T lineage commitment and differentiation beyond the beta-selection checkpoint; and we provide insight into the mechanism underlying perturbation of development when that expression pattern is disrupted.