In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functional properties of ex vivo effector-memory (EM) CD45RA−CCR7− T lymphocytes present within the circulating CD8+ T cell pool of healthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28 expression into four subsets. EM1 (CD27+CD28+) and EM4 (CD27−CD28+) T cells express low levels of effector mediators such as granzyme B and perforin and high levels of CD127/IL-7Rα. EM1 cells also have a relatively short replicative history and display strong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA−CCR7+) cells. In contrast, EM2 (CD27+CD28−) and EM3 (CD27−CD28−) cells express mediators characteristic of effector cells, whereby EM3 cells display stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiated effector (CD45RA+CCR7−) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss of CCR7, CD28, and CD27 both characterize CD8+ T cell differentiation. Finally, memory CD8+ T cells not only include central-memory cells but also EM1 cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid and peripheral tissues, respectively.

Therapeutic harnessing of adaptive immunity via checkpoint inhibition has transformed the treatment of many cancers. Despite unprecedented long-term responses, most patients do not respond to these therapies. Immunotherapy non-responders often harbor high levels of circulating myeloid-derived suppressor cells (MDSCs)-an immunosuppressive innate cell population. Through genetic and pharmacological approaches, we uncovered a pathway governing MDSC abundance in multiple cancer types. Therapeutic liver-X nuclear receptor (LXR) agonism reduced MDSC abundance in murine models and in patients treated in a first-in-human dose escalation phase 1 trial. MDSC depletion was associated with activation of cytotoxic T lymphocyte (CTL) responses in mice and patients. The LXR transcriptional target ApoE mediated these effects in mice, where LXR/ApoE activation therapy elicited robust anti-tumor responses and also enhanced T cell activation during various immune-based therapies. We implicate the LXR/ApoE axis in the regulation of innate immune suppression and as a target for enhancing the efficacy of cancer immunotherapy in patients.

Although numerous chemokines act on monocytes, none of them is specific for these cells. Here, we show that breast and kidney–expressed chemokine (BRAK) is a highly selective monocyte chemoattractant. Migration efficacy and Bordetella pertussis toxin–sensitive Ca2+ mobilization responses to BRAK were strongly enhanced after treatment of monocytes with the cyclic AMP–elevating agents prostaglandin E2 and forskolin. BRAK is the first monocyte-selective chemokine, as other types of blood leukocytes or monocyte-derived dendritic cells and macrophages did not respond. Expression in normal skin keratinocytes and dermal fibroblasts as well as lamina propria cells in normal intestinal tissues suggests a homeostatic rather than an inflammatory function for this chemokine. In addition, macrophages were frequently found to colocalize with BRAK-producing fibroblasts. We propose that BRAK is involved in the generation of tissue macrophages by recruiting extravasated precursors to fibroblasts, which are known to secrete essential cytokines for macrophage development.

After antigenic challenge, naive T lymphocytes enter a program of proliferation and differentiation during the course of which they acquire effector functions and may ultimately become memory cells. In humans, the pathways of effector and memory T-cell differentiation remain poorly defined. Here we describe the properties of 2 CD8 ؉ T-lymphocyte subsets, RA ؉ CCR7 ؊ 27 ؉ 28 ؉ and RA ؉ CCR7 ؊ 27 ؉ 28 ؊ , in human peripheral blood. These cells display phenotypic and functional features that are intermediate between naive and effector T cells. Like naive T lymphocytes, both subsets show relatively long telomeres. However, unlike the naive population, these T cells exhibit reduced levels of T-cell receptor excision circles (TRECs), indicating they have undergone additional rounds of in vivo cell division. Furthermore, we show that they also share effector-type properties. At equivalent in vivo replicative history, the 2 subsets express high levels of Fas/CD95 and CD11a, as well as increasing levels of effector mediators such as granzyme B, perforin, interferon ␥, and tumor necrosis factor ␣. Both display partial ex vivo cytolytic activity and can be found among cytomegalovirus-specific cytolytic T cells. Taken together, our data point to the presence of T cells with intermediate effector-like functions and suggest that these subsets consist of T lymphocytes that are evolving toward a more differentiated effector or effector-memory stage.

The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans. Human POT1 appears to interact with telomeres both through direct binding to the 3 overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex. The influence of POT1 on telomerase activity has not been studied at the molecular level. We show here that POT1 negatively effects telomerase activity in vitro. We find that the DNA binding activity of POT1 is required for telomerase inhibition. Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase.Telomeres, the nucleoprotein complexes at the ends of eukaryotic linear chromosomes, serve a number of vital cellular functions. Telomere capping protects the chromosome ends from nucleolytic degradation and provides a mechanism for cells to distinguish natural from broken ends, which signal DNA damage and are substrates for DNA repair processes (reviewed in references 7 and 13). The DNA component of telomeres is composed of tandem, simple repeats (TTAGGG in mammals), terminating with a 3Ј-protruding single strand of the guanosine-rich strand. Due to lack of a template to replicate the 3Ј overhang, most human somatic cells lose terminal DNA with each division; thus, telomeres also provide a buffer between the ends and the more internally located coding region of the genome (12,19). In germ line cells and some cells in highly proliferative tissues, telomere loss is counterbalanced through the activities of the ribonucleoprotein telomerase. In vitro assays reveal that catalytically active telomerase is minimally composed of an RNA (TER) and a protein (TERT) subunit (5,38,39). Telomerase adds telomeric repeats by iteratively reverse transcribing the template portion of its RNA subunit, using the 3Ј single-strand telomeric overhang as a primer (18, 42; reviewed in references 20 and 22).Proteins that specifically bind the telomere single-strand overhang have been identified in numerous organisms. The 3Ј telomeric overhang of the ciliate Oxytricha nova is bound by OnTEBP, a heterodimeric end-binding protein composed of an ␣ subunit and a ␤ subunit (14, 17). Crystal structures reveal that the ␣ subunit contains three oligonucleotide/oligosaccharide binding (OB) folds: the first two OB folds bind telomeric DNA with sequence specificity, and the third participates in protein-protein interactions with the ␤ subunit (21). A search for homologs of the OnTEBP ␣ subunit identified the Pot1 protein in Schizosaccharomyces pombe and human based on a weak sequence similarity with the first OB fold in the ␣ subunit (3). Genetic studies with S. pombe demonstrate a role for S. pombe POT1 in telomere end protection, since deletion of the pot1 gene results in the rapid loss of ...

The Escherichia coli replisome contains three polymerases, one more than necessary to duplicate the two parental strands. Using single-molecule studies, we reveal two advantages conferred by the third polymerase. First, dipolymerase replisomes are inefficient at synthesizing lagging strands, leaving single-strand gaps, whereas tripolymerase replisomes fill strands almost to completion. Second, tripolymerase replisomes are much more processive than dipolymerase replisomes. These features account for the unexpected three-polymerase-structure of bacterial replisomes.

Replicative polymerases are tethered to DNA by sliding clamps for processive DNA synthesis. Despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off DNA for each Okazaki fragment. In the 'collision release' model, the lagging strand polymerase collides with the 5 0 terminus of an earlier completed fragment, which triggers it to release from DNA and from the clamp. This report examines the mechanism of collision release by the Escherichia coli Pol III polymerase. We find that collision with a 5 0 terminus does not trigger polymerase release. Instead, the loss of ssDNA on filling in a fragment triggers polymerase to release from the clamp and DNA. Two ssDNA-binding elements are involved, the s subunit of the clamp loader complex and an OB domain within the DNA polymerase itself. The s subunit acts as a switch to enhance polymerase binding at a primed site but not at a nick. The OB domain acts as a sensor that regulates the affinity of Pol III to the clamp in the presence of ssDNA.

SummaryAlthough homologous recombination (HR) is considered an accurate form of DNA repair, genetics suggest that Escherichia coli (E. coli) translesion DNA polymerase (pol) IV (DinB) promotes error-prone recombination during stress which allows cells to overcome adverse conditions. How pol IV functions and is regulated during recombination under stress, however, is unknown. We show that pol IV is highly proficient in error-prone recombination, and is preferentially recruited to D-loops at stress-induced concentrations in vitro. Unexpectedly, we find that high-fidelity pol II switches to exonuclease mode at D-loops which is stimulated by topological stress and reduced deoxy-ribonucleotide pools observed during stationary-phase. The exonuclease activity of pol II enables it to compete with pol IV which likely suppresses error-prone recombination. These findings indicate that preferential D-loop extension by pol IV facilitates error-prone recombination and explain how pol II reduces such errors in vivo.