Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-γ-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2Kb-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-γ production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-γ recall responses. Importantly, isolated CD62Lhi KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-γ-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses.

Despite high initial efficacy, targeted therapies eventually fail in advanced cancers, as tumors develop resistance and relapse. In contrast to the substantial body of research on the molecular mechanisms of resistance, understanding of how resistance evolves remains limited. Using an experimental model of ALK positive NSCLC, we explored the evolution of resistance to different clinical ALK inhibitors. We found that resistance can originate from heterogeneous, weakly resistant subpopulations with variable sensitivity to different ALK inhibitors. Instead of the commonly assumed stochastic single hit (epi) mutational transition, or drug-induced reprogramming, we found evidence for a hybrid scenario involving the gradual, multifactorial adaptation to the inhibitors through acquisition of multiple cooperating genetic and epigenetic adaptive changes. Additionally, we found that during this adaptation tumor cells might present unique, temporally restricted collateral sensitivities, absent in therapy naïve or fully resistant cells, suggesting the potential for new therapeutic interventions, directed against evolving resistance.

No abstract

SUMMARY Drug-tolerant “persister” tumor cells underlie emergence of drug-resistant clones and contribute to relapse and disease progression. Here we report that resistance to the BCL-2 targeting drug ABT-199 in models of mantle cell lymphoma and double-hit lymphoma evolves from outgrowth of persister clones displaying loss of 18q21 amplicons that harbor BCL2. Further, persister status is generated via adaptive super-enhancer remodeling that reprograms transcription and offers opportunities for overcoming ABT-199 resistance. Notably, pharmacoproteomic and pharmacogenomic screens revealed that persisters are vulnerable to inhibition of the transcriptional machinery and especially to inhibition of cyclin-dependent kinase 7 (CDK7), which is essential for the transcriptional reprogramming that drives and sustains ABT-199 resistance. Thus, transcription-targeting agents offer new approaches to disable drug resistance in B-cell lymphomas.

Spreading depression severely disrupts ion homeostasis, causes sensory neglect and motor impairment, and is associated with stroke and migraine. Glucose utilization (CMR(glc)) and lactate production rise during spreading depression, but the metabolic changes in different brain cell types are unknown. Uptake of (14)C-labeled compounds known to be preferentially metabolized by the glial tricarboxylic acid cycle was, therefore, examined during unilateral KCl-induced spreading cortical depression in conscious, normoxic rats. [(14)C]Metabolites derived from [(14)C]butyrate in K+ -treated tissue rose 21% compared to that of untreated contralateral control cortex, whereas incorporation of H(14)CO(3) into metabolites in K+ -treated tissue was reduced to 86% of control. Autoradiographic analysis showed that laminar labeling of cerebral cortex by both (14)C-labeled acetate and butyrate was elevated heterogeneously throughout cortex by an average of 23%; the increase was greatest (approximately 40%) in tissue adjacent to the K+ application site. Local uptake of acetate, butyrate, and deoxyglucose showed similar patterns, and monocarboxylic acid uptake was highest in the structures in which apparent loss of labeled metabolites of [6-(14)C]glucose was greatest. Enhancement of net uptake of acetate and butyrate in cerebral cortex during spreading depression is tentatively ascribed to increased astrocyte metabolism.

Stabilization of p53 in erythroid precursors in response to nucleosomal stress underlies the hypoplastic anemia in myelodysplastic syndromes (MDS) with chromosome 5q deletion [del(5q)]. We investigated whether cenersen, a clinically active 20-mer antisense oligonucleotide complementary to TP53 exon10, could suppress p53 expression and restore erythropoiesis in del(5q) MDS. Cenersen treatment of ribosomal protein S-14-deficient erythroblasts significantly reduced cellular p53 and p53-up-regulated modulator of apoptosis expression compared with controls, accompanied by a significant reduction in apoptosis and increased cell proliferation. In a two-stage erythroid differentiation assay, cenersen significantly suppressed nuclear p53 in bone marrow CD34+ cells isolated from patients with del(5q) MDS, whereas erythroid burst recovery increased proportionally to the magnitude of p53 suppression without evidence of del(5q) clonal suppression (r = −0.6; P = 0.005). To explore the effect of p53 suppression on erythropoiesis in vivo, dexamethasone, a glucocorticoid receptor-dependent p53 antagonist, was added to lenalidomide treatment in eight lower-risk, transfusion-dependent, del(5q) MDS patients with acquired drug resistance. Transfusion independence was restored in five patients accompanied by expansion of erythroid precursors and decreased cellular p53 expression. We conclude that targeted suppression of p53 could support effective erythropoiesis in lenalidomide-resistant del(5q) MDS.

Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (≥8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before rejoining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.

No abstract