Abstract Objective As a negative regulator of T cells, Programmed Death Ligand 1 (PD-L1) is both an indicator and inhibitor of anti -tumor immune responses, which has led to confusion about its ...prognostic significance. We investigated the primary source of PD-L1 expression in epithelial ovarian cancer and its relationship to tumor-infiltrating lymphocytes (TIL) and associated gene products. Methods Tissue microarrays containing high-grade serous carcinomas (HGSC) and endometrioid, clear cell and mucinous ovarian cancers from optimally debulked patients were assessed by immunohistochemistry for expression of PD-L1 and other markers (CD68, CD3, CD8, PD-1, CD103, FoxP3 and CD25). The Cancer Genome Atlas was interrogated for associations between PD-L1 expression and immune-related transcriptional and genomic features of HGSC. Results PD-L1 was primarily expressed by tumor-associated CD68+ macrophages rather than tumor cells. PD-L1+ cells frequently co-localized with CD8, CD4 and PD-1+ TIL, CD25+ FoxP3+ Tregs, and other TIL subsets. PD-L1+ cells were prognostically favorable in HGSC. Moreover, the presence of both PD-L1+ cells and CD8 TIL was associated with better prognosis than CD8 TIL alone. PD-L1 gene expression was independent of BRCA status. At the transcriptional level, PD-L1 was associated with both cytolytic (granzyme B, T-bet and IFN-γ) and suppressive (PD-1, CTLA-4, LAG3 and IDO-1) gene products. Conclusions PD-L1 is primarily expressed by macrophages in ovarian cancer and is strongly associated with both cytolytic and regulatory TIL subsets, resulting in a net positive association with survival. Tumors containing PD-L1+ macrophages appear caught in an immunological stalemate that may require multi-pronged immunotherapy to alleviate.
Resident memory T cells (T
) are a recently identified subset of long-lived memory T cells that are characterized in terms of their unique surface phenotype combined with a non-recirculating pattern ...of localization to non-lymphoid, peripheral tissues. T
have quickly become a key area of focus in understanding immune responses to microbial infection in so-called "barrier" tissues, and appear to be particularly critical for protection against repeat exposure at the same site. More recently, tumor-infiltrating T cells with canonical T
features are being identified in human cancers, in particular cancers of epithelial origin, and their presence is broadly found to be associated with favorable long-term prognosis. Moreover, recent studies have shown that these "resident memory-like" tumor-infiltrating lymphocytes (referred to herein as TIL
) are uniquely activated in melanoma patients undergoing PD-1 directed checkpoint blockade therapy. Accordingly, there is much interest at present regarding the biology of these cells and their precise role in anti-cancer immunity. Herein, we review the current state of the literature regarding TIL
with a specific emphasis on their specificity, origins, and relationship to conventional pathogen-specific T
and speculate upon the way(s) in which they might contribute to improved prognosis for cancer patients. We discuss the growing body of evidence that suggests TIL
may represent a population of bona-fide tumor-reactive T cells and the attractive possibility of leveraging this cell population for future immunotherapy.
Extracellular bacteria that spread via the vasculature employ invasive mechanisms that mirror those of metastatic tumor cells, including intravasation into the bloodstream and survival during ...hematogenous dissemination, arrestation despite blood flow, and extravasation into distant tissue sites. Several invasive bacteria have been shown to exploit normal platelet function during infection. Due to their inherent ability to interact with and influence other cell types, platelets play a critical role in alteration of endothelial barrier permeability, and their role in cancer metastasis has been well established. The highly invasive bacterium and causative agent of syphilis, Treponema pallidum subspecies pallidum, readily crosses the endothelial, blood-brain and placental barriers. However, the mechanisms underlying this unusual and important aspect of T. pallidum pathogenesis are incompletely understood. In this study we use darkfield microscopy in combination with flow cytometry to establish that T. pallidum interacts with platelets. We also investigate the dynamics of this interaction and show T. pallidum is able to activate platelets and preferentially interacts with activated platelets. Platelet-interacting treponemes consistently exhibit altered kinematic (movement) parameters compared to free treponemes, and T. pallidum-platelet interactions are reversible. This study provides insight into host cell interactions at play during T. pallidum infection and suggests that T. pallidum may exploit platelet function to aid in establishment of disseminated infection.
The presence of CD8(+) tumor-infiltrating lymphocytes (TIL) is associated with prolonged survival in high-grade serous ovarian cancer (HGSC) and other epithelial cancers. Survival is most strongly ...associated with intraepithelial versus intrastromal CD8(+) TILs; however, the mechanisms that promote the intraepithelial localization of TILs remain poorly understood. We hypothesized that intraepithelial CD8(+) TILs, like normal mucosal intraepithelial lymphocytes, might express CD103, a subunit of αE/β7 integrin, which binds E-cadherin on epithelial cells.
A large collection of primary ovarian tumors (HGSC, endometrioid, mucinous, and clear cell) was analyzed by immunohistochemistry for the presence of TIL-expressing CD103. The activation and differentiation status of CD103(+) TILs were assessed by flow cytometry. The prognostic significance of TIL subsets was evaluated by Kaplan-Meier analysis.
CD103(+) TILs were present in all major ovarian cancer subtypes and were most abundant in HGSC. CD103(+) TILs were preferentially localized to epithelial regions of tumors and were comprised predominantly of CD8(+) T cells expressing activation (HLA-DR, Ki-67, PD-1) and cytolytic (TIA-1) markers, as well as CD56(+) NK cells. Tumor infiltration by CD103(+) TILs was strongly associated with patient survival in HGSC. Tumors containing CD8(+) TILs that were CD103(-) showed poor prognosis equivalent to tumors lacking CD8(+) TILs altogether.
CD103(+) TILs comprise intraepithelial, activated CD8(+) T cells, and NK cells and are strongly associated with patient survival in HGSC. CD103 may serve as a useful marker for enriching the most beneficial subsets of TILs for immunotherapy.
Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned ...by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.
Analytical methods that enable visualization of nanomaterials derived from solution self‐assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated ...emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization‐driven block copolymer (BCP) self‐assembly in organic media at the sub‐diffraction scale. Four different dyes were successfully used for single‐colour super‐resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual‐colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well‐established for aqueous systems, the results highlight the potential of super‐resolution microscopy techniques for the interrogation of self‐assembly processes in organic media.
Super‐resolution: Stimulated emission depletion microscopy (STED) and single‐molecule localization microscopy (SMLM) have been employed to map living crystallization‐driven block copolymer (BCP) self‐assembly in organic media and determine micelle length distributions in situ.
αE(CD103)β7 is a TGFβ-regulated integrin that mediates retention of lymphocytes in peripheral tissues by binding to E-cadherin expressed on epithelial cells. We recently reported that αE(CD103)β7 ...specifically demarcates intraepithelial CD8(+) tumor-infiltrating lymphocytes (CD8 TIL) in ovarian cancer and that CD103(+) TIL have a surface profile consistent with an active effector phenotype (HLA-DR(+), Ki67(+), and CD127(lo)). These findings led us to hypothesize that, over time, CD103-mediated retention of CD8 TIL within the tumor epithelium might result in chronic stimulation by tumor antigen, which in turn might lead to an exhausted phenotype. To investigate this possibility, we evaluated PD-1 expression in a large cohort of ovarian tumors (N = 489) with known CD103(+) TIL content. PD-1(+) cells were present in 38.5% of high-grade serous carcinomas (HGSC), but were less prevalent in other histologic subtypes. PD-1(+) TIL were strongly associated with increased disease-specific survival in HGSC (HR, 0.4864; P = 0.0007). Multicolor immunohistochemistry and flow cytometry revealed a high degree of PD-1 and CD103 coexpression, specifically within the CD8 TIL compartment. PD-1(+)CD103(+) CD8 TIL were quiescent when assessed directly ex vivo yet were capable of robust cytokine production after pharmacologic stimulation. Moreover, they showed negligible expression of additional exhaustion-associated markers, including TIM-3, CTLA-4, and LAG-3. Thus, as hypothesized, CD103(+) CD8 TIL express PD-1 and appear quiescent in the tumor microenvironment. However, these cells retain functional competence and demonstrate strong prognostic significance. We speculate that, after standard treatment, PD-1(+)CD103(+) CD8 TIL might regain functional antitumor activity, an effect that potentially could be augmented by immune modulation.
Two major challenges in proteomics are the large number of proteins and their broad dynamic range in the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to ...selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances, and we observed greater than threefold improvement in low-abundance human-protein identification and quantitation metrics. This methodology should be broadly applicable to many organisms, proteases and proteomic pipelines.
Redox transitions of uranium from U(VI) to U(IV) in low-temperature sediments govern the mobility of uranium in the environment and the accumulation of uranium in ore bodies, and inform our ...understanding of Earth’s geochemical history. The molecular-scale mechanistic pathways of these transitions determine the U(IV) products formed, thus influencing uranium isotope fractionation, reoxidation, and transport in sediments. Studies that improve our understanding of these pathways have the potential to substantially advance process understanding across a number of earth sciences disciplines. Detailed mechanistic information regarding uranium redox transitions in field sediments is largely nonexistent, owing to the difficulty of directly observing molecular-scale processes in the subsurface and the compositional/physical complexity of subsurface systems. Here, we present results from an in situ study of uranium redox transitions occurring in aquifer sediments under sulfate-reducing conditions. Based on molecular-scale spectroscopic, pore-scale geochemical, and macroscale aqueous evidence, we propose a biotic–abiotic transition pathway in which biomass-hosted mackinawite (FeS) is an electron source to reduce U(VI) to U(IV), which subsequently reacts with biomass to produce monomeric U(IV) species. A species resembling nanoscale uraninite is also present, implying the operation of at least two redox transition pathways. The presence of multiple pathways in low-temperature sediments unifies apparently contrasting prior observations and helps to explain sustained uranium reduction under disparate biogeochemical conditions. These findings have direct implications for our understanding of uranium bioremediation, ore formation, and global geochemical processes.
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