PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, ...incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC′ sheet, which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC′ sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1·ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3–4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.
Background: The inhibitory leukocyte receptor PD-1 binds two ligands, PD-L1 and PD-L2.
Results: Nuclear magnetic resonance analysis and rigorous binding and thermodynamic measurements reveal the structure of, and the mode of ligand recognition by, PD-1.
Conclusion: PD-L1 and PD-L2 bind differently to PD-1 and much more weakly than expected.
Significance: Potent inhibitory signaling can be initiated by weakly interacting receptors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large ...receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.
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•Local, steric phosphatase exclusion by antibodies drives immune receptor signaling•Agonists targeting activating and inhibitory receptors can be rationally designed•A bespoke PD-1 agonist exemplifies a new class of treatments for autoimmunity•Antibody engineering that limits agonism improves immune checkpoint receptor blockade
Antibodies can initiate immune receptor signaling. Lippert et al. provide mechanistic insight into this effect, showing that antibody agonists trigger receptor signaling by locally excluding receptor-type protein tyrosine phosphatases from sites of antibody-receptor engagement. This finding provides a framework for the engineering of therapeutic antibodies.
People with HIV (PWH) may be particularly vulnerable to the psychological impacts of COVID-19. To assess this, participants were recruited from two established cohorts of PWH and HIV- adults with the ...available pre-pandemic baseline data and completed the Beck Depression Inventory-II (BDI-II), Beck Anxiety Inventory (BAI), Alcohol Use Identification Test (AUDIT), National Institute on Drug Abuse Quick Screen (NIDA-QS), and Pittsburgh Sleep Quality Index (PSQI) at two distinct intra-pandemic time periods. All outcomes were evaluated using generalized linear mixed models. In total, 87 participants completed all the questionnaires; 45 were PWH and 42 were HIV-. The pre-pandemic mean BDI-II, BAI, AUDIT and PSQI scores were higher in the PWH cohort. After the onset of the pandemic, the mean BDI-II, AUDIT and PSQI scores increased within the sample as a whole (
< 0.001,
= 0.029 and
= 0.046, respectively). The intra-pandemic mean BDI-II scores fell slightly for both groups and the AUDIT scores increased slightly for the PWH group and fell slightly for the HIV- group, but not significantly. The intra-pandemic PSQI scores increased sharply for both groups. The percentage of PWH and HIV- participants who moved into a more severe category of depression was identical (18%), but more PWH met the criteria for clinical evaluation. The BAI and NIDA-QS scores did not increase significantly. In conclusion, the measures of mental health symptoms and alcohol use increased in both groups after the onset of the pandemic. Although there were no significant differences in the changes between the groups, the PWH had higher baseline scores and the changes in this group had more clinical impacts.
Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic ...similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.
Purpose
Pediatric breast malignancies are rare, and descriptions in the literature are limited. The purpose of our study was to compare pediatric and adult breast malignancy.
Methods
We performed a ...retrospective cohort study using the National Cancer Data Base comparing patients ≤21 years to those >21 years at diagnosis (1998–2012). Generalized linear models estimated differences in demographic, tumor, and treatment characteristics. Cox regression was used to compare overall survival.
Results
Of 1,999,181 cases of invasive breast malignancies, 477 (0.02%) occurred in patients ≤21 years. Ninety-nine percent of adult patients had invasive carcinoma compared with 64.8% of pediatric patients with the remaining patients having sarcoma, malignant phyllodes, or malignancy not otherwise specified (
p
< 0.001). Pediatric patients were twice as likely to have an undifferentiated malignancy relative risk (RR) 2.19; 95% confidence interval (CI) 1.72–3.79. Half of adults presented with Stage I disease compared with only 22.7% of pediatric patients (
p
< 0.001). Pediatric patients were 40% more likely to have positive axillary nodes (RR 1.42; 95% CI 1.10–1.84). Among patients with invasive carcinoma, pediatric patients were more than four times as likely to receive a bilateral than a unilateral mastectomy compared with adults (RR 4.56; 95% CI 3.19–6.53). There was no difference in overall survival between children and adults.
Conclusions
Pediatric breast malignancies are more advanced at presentation, and there is variability in treatment practices. Adult and pediatric patients with invasive carcinoma have similar overall survival.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Multiple aspects of Black young men who have sex with men's (YMSM) identities cause them to be differentially targeted for arrest and incarceration. However, limited research has explored structural ...drivers of Black YMSM' criminal justice involvement, particularly co-occurring forms of discrimination. This article examines the temporal relationship between perceived racial discrimination, perceived sexual orientation discrimination, and community-level HIV discrimination and criminal justice involvement among Black YMSM in North Carolina. The study followed 465 Black YMSM from November 2013 to October 2016 who were recruited for a randomized controlled trial to test an internet-based intervention for Black YMSM living with, and at risk for HIV; participants completed online surveys at baseline, 3, 6, and 12 months. Logistic regression was used to explore the relationship between the three predictors at baseline (i.e., perceived racism and sexual orientation discrimination and community-level HIV discrimination) and criminal justice involvement at follow-up. All three predictor variables were significantly associated with subsequent criminal justice involvement in separate regression models that adjusted for other covariates: HIV discrimination (aOR = 1.06 1.01-1.11), perceived sexual orientation discrimination (aOR = 1.12 1.00-1.27), and perceived racism (aOR = 1.26 1.12-1.42). Perceived racism remained significant in the model with all three predictors (aOR = 1.29 1.07-1.55). Racism did not modify the relationship between HIV discrimination and perceived sexual orientation discrimination and criminal justice involvement. This study expands existing research by exploring racism as a structural driver of criminal justice involvement; we subsequently examined whether racism modified the effect of the two other predictors. It also contributes to research on co-occurring discrimination by examining their impact on an underrepresented population.
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CEKLJ, FFLJ, NUK, ODKLJ, PEFLJ, UPUK
Programmed death-1 (PD-1) is a critical mediator of virus-specific CD8+ T-cell exhaustion. Here, we examined the expression of PD-1 on simian immunodeficiency virus (SIV)-specific CD8+ T cells and ...its possible involvement in regulation of cytokine production, proliferation, and survival of these cells. The majority of SIV-specific CD8+ T cells expressed a PD-1high phenotype, independent of their differentiation status, in all tissues tested. PD-1 expression gradually declined on CD8+ T cells specific for SIV-derived epitopes that had undergone mutational escape, indicating that antigen-specific TCR stimulation is the primary determinant of PD-1 expression. SIV-specific PD-1highCD8+ T cells produced IFN-γ, TNF-α, and IL-2 under cognate peptide stimulation. While CD8+ T cells that proliferated in response to antigen had a PD-1high phenotype, it was determined that there was a reduced proliferative capacity of PD-1high compared with PD-1low SIV-specific CD8+ T cells. PD-1high SIV-specific CD8+ T cells were highly susceptible to cell death leading to loss of such cells after in vitro stimulation. Thus, PD-1 is a negative regulator of SIV-specific CD8+ T cells, operating predominantly through the induction of cell death. Manipulation of the interaction of PD-1 with its ligands could thus potentially restore the CD8+ T-cell responses in SIV infection.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the ...TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Meleagris gallopavo silvestris (Eastern Wild Turkey) habitat was altered in the Southeast by the introduction of Pinus spp. plantations to agricultural areas through the Conservation Reserve Program. ...However, the preponderance of M. gallopavo silvestris research has focused on extensive Pinus spp. plantations that lack the cover-type diversity that typifies the Southeast. From May–July 1998 and 1999, we monitored 36 radio-tagged M. gallopavo silvestris in Burke County, GA to investigate habitat use in landscapes intensively managed for agriculture and silviculture. We used compositional analysis to ide.gify habitats selected by male and female M. gallopavo silvestris during summers. Proportions of habitat types within the home range were different from habitats at radio-locations of males and females. Hardwood stands and fields were the most-selected habitat types by M. gallopavo silvestris in the summer. However, within home ranges, males and females also selected closed-canopy Pinus spp. habitats. Hens with broods did not preferentially select planted Pinus spp. habitats, but their use of Pinus spp. stands was greater than use of agricultural fields. The replacement of agricultural fields by closed-canopy Pinus spp. plantations may have improved habitat quality for M. gallopavo silvestris in some areas of the Southeast by diversifying the landscape. Our results suggest that closed-canopy planted Pinus spp. cover types are not detrimental to M. gallopavo silvestris when well distributed with fields and mature hardwood drains.
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BFBNIB, DOBA, IZUM, KILJ, NMLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
We observed two instances of Eastern Wild Turkey (Meleagris gallopavo silvestris) hens renesting after hatching their first clutches. These observations constitute for this species the third report ...of renesting after successfully hatching a clutch and, in one instance, the first record of two clutches hatching during the same breeding season. After the successful hatching of the second clutch, the hen raised the poults for at least 16 d. We observed the same female with two poults 19 days after hatching her first clutch.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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