Metabolic insufficiency underlies intratumoral cytotoxic T cell dysfunction

T cells have the remarkable ability to recognize and remove abnormal cells with precision, a feature that is very desirable for the treatment of cancer. However, while T cells specific for tumor antigens are primed and can infiltrate tumors, they are quickly rendered dysfunctional, through both cell intrinsic and cell extrinsic mechanisms. One way that tumors cripple T cell function is through the generation of an immunosuppressive microenvironment that is chronically inflamed, hypoxic, and nutrient poor. T cell activation and subsequent generation of effector function is bioenergetically demanding, requiring large amounts of metabolic intermediates to effectively proliferate, produce cytokines, and lyse target cells. We hypothesized that T cell dysfunction in cancer is due, in part, to metabolic insufficiency caused by chronic activation in metabolically dearth conditions. Using single-cell metabolic assays and extracellular flux analysis, we show that CD8+ cytotoxic T cells that infiltrate tumors demonstrate a progressive loss of mitochondrial function and mass, concomitant with upregulation of markers that correlate with T cell exhaustion. This mitochondrial dysfunction occurs independently of coinhibitory molecule signaling and specifically in the tumor microenvironment. This results in a failure to generate an adequate pool of ATP and in inability to effectively translate effector gene transcripts. This in stark contrast to T cells responding to an acute viral infection, where activated effector T cells demonstrate increased mitochondrial mass and ATP reserve. Further, artificial induction of mitochondrial dysfunction in T cells results in upregulation of coinhibitory molecules and an ‘exhausted-like’ phenotype, suggesting that metabolic insufficiency underlies the dysfunctional phenotype in cancer. Taken together, our data support a model in which tumor-infiltrating T cells have metabolic needs that cannot be met, resulting in failed effector function and tumor growth. Our studies also suggest that modulation or reprogramming of the altered metabolism of intratumoral T cells represents a potential strategy to reinvigorate dysfunctional T cells for the immunotherapeutic treatment of cancer

Hirschsprung disease in twins: A case series

Introduction: Hirschsprung's disease (HD) occurs secondary to failed migration of neural crest cells in embryonic life and results in a dysfunctional, mechanically obstructed colon. Despite an increased occurrence amongst family members, predominantly amongst siblings, HD in twins is extremely rare with approximately 21 total cases documented in the literature. Case presentations: This case series includes 2 cases of concordant expression of HD in twins: one dizygotic set and the other monozygotic. This is contrary to prior studies which suggest that monozygotic twins more frequently exhibit disease discordance, where one twin is affected while the other is not, over disease concordance. In each case, the twins demonstrated either variability in presentation or location of pathology, which ultimately delayed diagnosis and further management. The complex etiology makes it difficult to predict the likelihood of disease inheritance. A comprehensive literature review is also included to consider the rarity of this occurrence and evaluate the variation of concordant disease expression. Conclusion: Further understanding of the inheritance pattern will help guide management and provide us with more information for genetic counseling

Early TCR Signaling Induces Rapid Aerobic Glycolysis Enabling Distinct Acute T Cell Effector Functions

To fulfill bioenergetic demands of activation, T cells perform aerobic glycolysis, a process common to highly proliferative cells in which glucose is fermented into lactate rather than oxidized in mitochondria. However, the signaling events that initiate aerobic glycolysis in T cells remain unclear. We show T cell activation rapidly induces glycolysis independent of transcription, translation, CD28, and Akt and not involving increased glucose uptake or activity of glycolytic enzymes. Rather, TCR signaling promotes activation of pyruvate dehydrogenase kinase 1 (PDHK1), inhibiting mitochondrial import of pyruvate and facilitating breakdown into lactate. Inhibition of PDHK1 reveals this switch is required acutely for cytokine synthesis but dispensable for cytotoxicity. Functionally, cytokine synthesis is modulated via lactate dehydrogenase, which represses cytokine mRNA translation when aerobic glycolysis is disengaged. Our data provide mechanistic insight to metabolic contribution to effector T cell function and suggest that T cell function may be finely tuned through modulation of glycolytic activity. Menk et al. show rapid induction of aerobic glycolysis after activation of effector T cells that is required for acute cytokine production. These data provide mechanistic insight into the regulation of T cell function through nutrient availability

Expression of the AR-EGFP transgene in founder mice.

<p>A) Testis whole cell extracts from 6 founder mice having germ line transmission of the AR-GFP transgene and extracts from Cos7 cells infected with an adenovirus expressing EGFP (AdEGFP) by western blot. B) Whole cell extracts from the 2460 founder strain containing the AR-EGFP transgene were assayed for EGFP expression by western blot.</p

Expression of the AR-EGFP transgene rescues full spermatogenesis in a SCARKO background.

<p>Testes cross sections stained with PAS-Hematoxylin are shown for postnatal day 105 mouse littermates (founder 2464) that express only Cre recombinase (Control), Cre recombinase in the presence of a floxed AR gene (SCARKO), and a SCARKO mouse expressing the AR-EGFP transgene (Rescue). Bars = 100 μm, magnification: 40x objective.</p

Summary of spermatogenesis rescue by specific founder strains.

<p>No spermatids present</p><p>+ A few elongated spermatids present</p><p>++ Complete spermatogenesis in >10% of tubule cross sections</p><p>+++ Complete spermatogenesis in >50% of tubule cross sections</p><p>++++ Complete spermatogenesis in >90% of tubule cross sections</p><p>Summary of spermatogenesis rescue by specific founder strains.</p

AR is expressed stage-specifically in Sertoli cells of rescue mice.

<p>Testis cross sections from SCARKO, wild type and rescue mice were probed with antisera against AR. Long green arrows denote AR immunostaining (red immunofluorescence) in peritubular cell nuclei. Short yellow arrows show AR expression in Leydig cells. White arrowheads denote AR immunostaining in Sertoli cell nuclei. Stages of the seminiferous epithelium for tubule cross sections are denoted by Roman numerals (I-XII). Bar = 100 μm, magnification: 20x objective.</p

Characterization of three BACs containing parts of the AR gene.

<p>(A) Top: a map of the AR gene and flanking region is shown. Below: maps of the BACs mAR BAC-196 (RP23-102011), mAR BAC-192 (RP24-352D1) and mAR BAC-185 (RP23-316P7) are shown. AR exons are denoted by rectangles on the lines. <i>SalI</i> restiction sites are denoted with an S. Boxes above exons 1 and 8 denote the probes used for Southern blotting. The BACs contained either extended 5’ sequence, the complete AR gene or extended 3’ sequence, respectively. The BACs were end sequenced to determine their insertion sites. (B) Restriction digests followed by PFGE and Southern blotting were performed to ensure that preperations of the BACs were correct. The <i>SalI</i> digested BACs were fractionated by PFGE and subjected to Southern blotting using ³²P random primed labelled AR exon 1 and AR exon 8 probes. mAR BAC-192 was positive for both probes while mAR BAC-196 only probed positive for exon 8 and mAR BAC-185 probed positive only for exon1.</p