Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typified by multiple relapses after therapy. These tumors are genetically characterized by B-cell leukemia/lymphoma 2 (BCL2) translocation and mutation of genes involved in chromatin modification. By analyzing purified tumor cells, we identified additional novel recurrently mutated genes and confirmed mutations of one or more chromatin modifier genes within 96% of FL tumors and two or more in 76% of tumors. We defined the hierarchy of somatic mutations arising during tumor evolution by analyzing the phylogenetic relationship of somatic mutations across the coding genomes of 59 sequentially acquired biopsies from 22 patients. Among all somatically mutated genes, CREBBP mutations were most significantly enriched within the earliest inferable progenitor. These mutations were associated with a signature of decreased antigen presentation characterized by reduced transcript and protein abundance of MHC class II on tumor B cells, in line with the role of CREBBP in promoting class II transactivator (CIITA)-dependent transcriptional activation of these genes. CREBBP mutant B cells stimulated less proliferation of T cells in vitro compared with wild-type B cells from the same tumor. Transcriptional signatures of tumor-infiltrating T cells were indicative of reduced proliferation, and this corresponded to decreased frequencies of tumor-infiltrating CD4 helper T cells and CD8 memory cytotoxic T cells. These observations therefore implicate CREBBP mutation as an early event in FL evolution that contributes to immune evasion via decreased antigen presentation. lymphoma | exome | hierarchy | antigen presentation | CREBBP

An aged circulatory environment can activate microglia, reduce neural precursor cell activity, and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of Vascular Cell Adhesion Molecule 1 (VCAM1), a protein that facilitates vascular-immune cell interactions. Concomitantly, the shed, soluble form of VCAM1 is prominently increased in plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and young mouse hippocampi. Systemic anti-VCAM1 antibody or genetic ablation of VCAM1 in BECs counteracts the detrimental effects of aged plasma on young brains and reverses aging aspects including microglial reactivity and cognitive deficits in old mouse brains. Together, these findings establish brain endothelial VCAM1 at the blood-brain barrier (BBB) as a possible target to treat age-related neurodegeneration.

Key Points• Analysis of coding genomes of FL tumor subpopulations reveals striking clonal diversity at diagnosis and progression.• Within a hierarchy of somatic evolution of FL coding genomes, many recurrent mutations are subclonal at diagnosis.Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies. (Blood. 2013;121(9):1604-1611) IntroductionFollicular lymphoma (FL) is a common form of non-Hodgkin lymphoma (NHL) arising from mature B cells. FL tumor cells share identical immunoglobulin (Ig) gene rearrangements, indicating that the transforming founder mutation(s) occurs subsequent to VDJ recombination. These cells express markers of mature B-lineage including surface-Ig and CD19, and germinal center B-cell markers such as LMO2, CD10, and BCL6. 1 The B-cell marker CD20, which is also the target of the anti-lymphoma therapy Rituximab, 2,3 is expressed to a variable degree on FL tumors. 4,5 CD20 levels can predict primary responsiveness of diverse lymphomas to Rituximab,6 and changes in CD20 expression contribute to Rituximab resistance and relapse. 7,8 For instance, CD20 expression levels are associated with survival in patients with aggressive lymphomas treated with or without Rituximab. 9 These studies each found CD20 to be variably expressed on cells within the same tumor, suggesting that this may be a marker of underlying clonal diversity.The genetic hallmark of FL is the t(14;18)(q32;q21) translocation that places the antiapoptotic BCL2 oncogene under control of the Ig heavy-chain enhancer. 10 This lesion is present in Ͼ 90% of FL cases, 11 but is also detectable in the majority of older healthy adults suggesting that it is not sufficient to induce clinical disease. 12 Recently, mutation of a histone methyltransferase gene, MLL2, was identified in 89% of FL cases, indicating that this may also be a founder mutation. 13 Genes encodin...

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Degeneration is the process whereby Clostridium acetobutylicum ATCC 824 loses the capacity to produce acetone and butanol after repeated vegetative transfers or in continuous culture. Two degenerate mutants (M5 and DG1) of C. acetobutylicum ATCC 824 do not contain the four genes (ctfA, ctfB, adc, and aad) for acetone and butanol formation. Strain ATCC 824 contains a 210-kb plasmid (pSOL1) which is absent in M5 and DG1. pSOL1 carries the four acetone and butanol formation genes. A restriction map of pSOL1 was constructed by using ApaI, SmaI, SstII, and NarI digestions. M5 and DG1 could be complemented for acetone and butanol formation by expressing the corresponding genes (ctfA, ctfB, and adc for acetone; aad for butanol) on the plasmid. Degeneration of this strain thus appears to be the result of pSOL1 loss.The American Heritage Dictionary defines the term "degenerate" as "having fallen or descended to a state below what is considered normal or desirable." Within the solventogenic clostridial community, the term "degenerate" is used to describe mutants which have one common trait, namely, that they produce more acids (butyrate and acetate) and little or no solvents (butanol, acetone, and ethanol). Degeneracy as applied to solventogenic clostridia appears to have no implications about the nature or origin of the mutation(s).It has long been observed that upon serial subculturing or in continuous culture, solventogenic clostridia undergo a degenerative process which affects both morphological and physiological features and leads to mutant strains with impaired solvent formation capabilities (14). Weizmann's original strain of Clostridium acetobutylicum was found (20) to degenerate after 10 to 20 transfers when the transfers were made during the acidogenic phase of the fermentation. Solvent formation before the 50th transfer dropped to 0.5 to 2.0% of the starch fermented, and the spore formation capability was almost completely abolished. Attempts to recover "normal" solvent-producing strains from degenerate strains by culture with growth factors and various salts and by inducing sporulation were only partially successful. However, when transfers were made following sporulation and heat shocking, a solvent yield of 24.7% was retained after 150 transfers (more than 2 years).Jones et al. observed that certain morphological and cytological changes, which could be correlated with growth and physiological changes, occurred in C. acetobutylicum P262 during solvent production (13). The number of swollen, cigarshaped clostridial forms could be directly correlated with the production of solvents. Initiation of solvent production and clostridial-stage formation are essential for sporulation in different Clostridium species (12, 18). Thus, degenerate mutants would be expected to be asporogenous. However, the existence of an asporogenous strain which is a good solvent producer (17) should be noted, because it demonstrates that degeneracy (assumed here to imply impaired solvent formation ability) is not necessarily equivalent t...

A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was identified immediately upstream of the previously cloned ctfA (J. W. Cary, D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett, Appl. Environ. Microbiol. 56:1576-1583

Despite evidence that γδ T cells play an important role during malaria, their precise role remains unclear. During murine malaria induced by Plasmodium chabaudi infection and in human P. falciparum infection, we found that γδ T cells expanded rapidly after resolution of acute parasitemia, in contrast to αβ T cells that expanded at the acute stage and then declined. Single-cell sequencing showed that TRAV15N-1 (Vδ6.3) γδ T cells were clonally expanded in mice and had convergent complementarity-determining region 3 sequences. These γδ T cells expressed specific cytokines, M-CSF, CCL5, CCL3, which are known to act on myeloid cells, indicating that this γδ T cell subset might have distinct functions. Both γδ T cells and M-CSF were necessary for preventing parasitemic recurrence. These findings point to an M-CSF-producing γδ T cell subset that fulfills a specialized protective role in the later stage of malaria infection when αβ T cells have declined.

Normal stem cells from a variety of tissues display unique metabolic properties compared to their more differentiated progeny. However, relatively little is known about heterogeneity of metabolic properties cancer stem cells, also called tumor initiating cells (TICs). In this study we show that, analogous to some normal stem cells, breast TICs have distinct metabolic properties compared to non-tumorigenic cancer cells (NTCs). Transcriptome profiling using RNA-Seq revealed TICs under-express genes involved in mitochondrial biology and mitochondrial oxidative phosphorylation and metabolic analyses revealed TICs preferentially perform glycolysis over oxidative phosphorylation compared to NTCs. Mechanistic analyses demonstrated that decreased expression and activity of pyruvate dehydrogenase (Pdh), a key regulator of oxidative phosphorylation, play a critical role in promoting the pro-glycolytic phenotype of TICs. Metabolic reprogramming via forced activation of Pdh preferentially eliminates TICs both in vitro and in vivo. Our findings reveal unique metabolic properties of TICs and demonstrate that metabolic reprogramming represents a promising strategy for targeting these cells.