Graphical AbstractHighlights d Mouse and human tumors harbor relatively undifferentiated Tcf1 + PD-1 + CD8 + T cells d These intratumoral cells have expansion, regeneration, and differentiation capacity d They produce differentiated Tcf1 À PD-1 + CD8 + T cells in response to immunotherapy d These stem-like cells are critical for tumor control in response to immunotherapy In BriefSince chronic activation promotes terminal T cell differentiation (exhaustion), it has remained unclear how checkpoint blockade mediates a proliferative response of tumorinfiltrating T cells. Siddiqui et al. identify intratumoral, tumor-reactive Tcf1 + PD-1 + CD8 + T cells that display stem-like properties and that promote tumor control in response to vaccination and checkpoint blockade immunotherapy. SUMMARYCheckpoint blockade mediates a proliferative response of tumor-infiltrating CD8 + T lymphocytes (TILs). The origin of this response has remained elusive because chronic activation promotes terminal differentiation or exhaustion of tumor-specific T cells. Here we identified a subset of tumor-reactive TILs bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcf1. Tcf1 + PD-1 + TILs mediated the proliferative response to immunotherapy, generating both Tcf1 + PD-1 + and differentiated Tcf1 À PD-1 + cells. Ablation of Tcf1 + PD-1 + TILs restricted responses to immunotherapy. Tcf1 was not required for the generation of Tcf1 + PD-1 + TILs but was essential for the stem-like functions of these cells. Human TCF1 + PD-1 + cells were detected among tumor-reactive CD8 + T cells in the blood of melanoma patients and among TILs of primary melanomas. Thus, immune checkpoint blockade relies not on reversal of T cell exhaustion programs, but on the proliferation of a stem-like TIL subset.

Aging is an inherently stochastic process, and its hallmark is heterogeneity between organisms, cell types, and clonal populations, even in identical environments. The replicative lifespan of primary human cells is telomere dependent; however, its heterogeneity is not understood. We show that mitochondrial superoxide production increases with replicative age in human fibroblasts despite an adaptive UCP-2–dependent mitochondrial uncoupling. This mitochondrial dysfunction is accompanied by compromised [Ca2+]i homeostasis and other indicators of a retrograde response in senescent cells. Replicative senescence of human fibroblasts is delayed by mild mitochondrial uncoupling. Uncoupling reduces mitochondrial superoxide generation, slows down telomere shortening, and delays formation of telomeric γ-H2A.X foci. This indicates mitochondrial production of reactive oxygen species (ROS) as one of the causes of replicative senescence. By sorting early senescent (SES) cells from young proliferating fibroblast cultures, we show that SES cells have higher ROS levels, dysfunctional mitochondria, shorter telomeres, and telomeric γ-H2A.X foci. We propose that mitochondrial ROS is a major determinant of telomere-dependent senescence at the single-cell level that is responsible for cell-to-cell variation in replicative lifespan.

CCL21Ser is one of the ligands for chemokine receptor CCR7. Kozai et al. report that CCL21Ser is essential for the accumulation of developing thymocytes in the thymic medulla and the establishment of self-tolerance in T cells, indicating a functional inequality among CCR7 ligands in vivo.

Chronic therapy with nitroglycerin results in a rapid development of nitrate tolerance, which is associated with an increased production of reactive oxygen species. We have recently shown that mitochondria are an important source of nitroglycerin-induced oxidants and that the nitroglycerin-bioactivating mitochondrial aldehyde dehydrogenase is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on aldehyde dehydrogenase activity and its restoration by dihydrolipoic acid. In vivo tolerance in Wistar rats was induced by infusion of nitroglycerin (6.6 g/kg/min, 4 days). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to nitroglycerin. Chronic nitroglycerin infusion lead to impaired vascular responses to nitroglycerin and decreased dehydrogenase activity, which was corrected by dihydrolipoic acid co-incubation. Superoxide, peroxynitrite, and nitroglycerin itself were highly efficient in inhibiting mitochondrial and yeast aldehyde dehydrogenase activity, which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate aldehyde dehydrogenase thereby leading to nitrate tolerance. Glutathionylation obviously amplifies oxidative inactivation of the enzyme providing another regulatory pathway. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores mitochondrial aldehyde dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance.Organic nitrates such as nitroglycerin (glyceryl trinitrate, GTN) 3 have been used for over a century in the therapy of cardiovascular diseases like myocardial infarction, unstable angina, and arterial hypertension (1). However, the usefulness of organic nitrates is limited by tolerance, which develops shortly after onset of treatment. The mechanisms underlying nitrate tolerance remain only in part defined and are most likely multifactorial (2). Previously, we found that 3 days of nitrate treatment doubled vascular superoxide (O 2 . ) production (3), which was also found in human bypass material from GTNtreated patients (4). Chen et al. (5) identified the mitochondrial aldehyde dehydrogenase (ALDH-2) as a GTN-metabolizing enzyme and a possible important component in the processes leading to tolerance. This concept was supported by recent studies in ALDH-2-deficient mice (ALDH-2 Ϫ/Ϫ ) (6). Our laboratory further substantiated this concept in an animal model of in vivo tolerance and extended previous observations by demonstrating that mitochondria are a major source of reactive oxygen species formation in response to acute and chronic GTN challenges (7,8). The importance of the ALDH-2 concept for clinical nitrate tolerance was proven by two in...

Host defense depends on orchestrated cell migration guided by chemokines that elicit selective but biased signaling pathways to control chemotaxis. Here, we showed that different inflammatory stimuli provoked oligomerization of the chemokine receptor CCR7, enabling human dendritic cells and T cell subpopulations to process guidance cues not only through classical G protein-dependent signaling but also by integrating an oligomer-dependent Src kinase signaling pathway. Efficient CCR7-driven migration depends on a hydrophobic oligomerization interface near the conserved NPXXY motif of G protein-coupled receptors as shown by mutagenesis screen and a CCR7-SNP demonstrating super-oligomer characteristics leading to enhanced Src activity and superior chemotaxis. Furthermore, Src phosphorylates oligomeric CCR7, thereby creating a docking site for SH2-domain-bearing signaling molecules. Finally, we identified CCL21-biased signaling that involved the phosphatase SHP2 to control efficient cell migration. Collectively, our data showed that CCR7 oligomers serve as molecular hubs regulating distinct signaling pathways.