ABSTRACT The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G 2 . Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4 + lymphocytes causes G 2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4 + lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4 + lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24 Gag -immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G 2 arrest.
Relatively little is known about features of T cells targeted by HIV in vivo. By applying bioinformatics analysis to mass cytometry (CyTOF)-phenotyped specimens from individuals with viremia and in-vitro-infected cells from uninfected donors, we provide an atlas of the phenotypes of in vivo and in vitro HIV-susceptible cells. T helper 17 (Th17) and α4β1+ cells are preferentially targeted in vivo, whereas T effector memory (Tem), T transitional memory (Ttm), Th1, and Th1/Th17 subsets are targeted in vitro. Multiple proteins—including chemokine and cytokine receptors—are remodeled by HIV in vivo, and these changes are mostly recapitulated in vitro. HIV remodels cells to a T follicular helper (Tfh) phenotype. Using clustering, we uncover a subset of CD29-expressing, Tem-like cells that are highly susceptible to infection in vivo and in vitro and experimentally confirm that susceptibility. These studies provide an in-depth look at features of HIV-susceptible cells in individuals with viremia and demonstrate that some—but not all—HIV-susceptible cells identified in vitro effectively model in vivo susceptibility.
Although T cells are likely players in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity, little is known about the phenotypic features of SARS-CoV-2-specific T cells associated with recovery from severe coronavirus disease 2019 (COVID-19). We analyze T cells from 34 individuals with COVID-19 with severity ranging from mild (outpatient) to critical, culminating in death. Relative to individuals who succumbed, individuals who recovered from severe COVID-19 harbor elevated and increasing numbers of SARS-CoV-2-specific T cells capable of homeostatic proliferation. In contrast, fatal COVID-19 cases display elevated numbers of SARS-CoV-2-specific regulatory T cells and a time-dependent escalation in activated bystander CXCR4+ T cells, as assessed by longitudinal sampling. Together with the demonstration of increased proportions of inflammatory CXCR4+ T cells in the lungs of individuals with severe COVID-19, these results support a model where lung-homing T cells activated through bystander effects contribute to immunopathology, whereas a robust, non-suppressive SARS-CoV-2-specific T cell response limits pathogenesis and promotes recovery from severe COVID-19.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens. https://doi.org/10.7554/eLife.24888.001 eLife digest Seminal plasma, the fluid portion of semen, helps to transport sperm cells to the egg during sexual reproduction. Seminal plasma contains numerous proteins that help the sperm to survive and, in recent years, researchers discovered that it also harbours protein deposits known as amyloid fibrils. Such protein deposits are generally associated with neurodegenerative diseases such as Alzheimer's and Parkinson’s disease, where a build-up of fibrils can damage the nervous system. Semen amyloids, however, are present in the absence of disease, but can boost infection by HIV and other sexually transmitted viruses, by shuttling virus particles to their target cells. Despite these damaging effects, some researchers had suggested that amyloids in semen could be beneficial for humans, though it was unclear what these benefits might be. Roan et al. now set out to assess how semen amyloids affect human sperm activity. The results show that semen amyloids bind to damaged sperm cells and immobilize them, which are then quickly cleared away by immune cells. This could ensure that only the fittest sperm cells reach the egg. These findings suggest that amyloids can potentially serve beneficial roles for reproduction. A next step will be to investigate how semen amyloids trap unwanted sperm and how immune cells know when to remove it. More research is needed to investigate if problems in these processes could lead to infertility in men. https://doi.org/10.7554/eLife.24888.002 Introduction Seminal plasma (SP) is a unique biological fluid, harboring unusually high concentrations of proteases and protease inhibitors (Laflamme and Wolfner, 2013), immunomodulatory cytokines such as TGF-β (Robertson et al., 2002), and metals such as zinc, all of which serve important functions in promoting reproductive success. Thus far, SP is also the only human biological fluid known to contain endogenous amyloid fibrils in a non-disease state (Usmani et al., 2014). Two classes of semen amyloids have been identified: those derived from proteolytic fragments of prostatic acid phosphatase (PAP) which polymerize to form amyloids named semen-derived enhancer of viral infection (SEVI), and those derived from PSA-generated fragments of semenogelins which polymerize to form amyloids named SEM fibrils. Both sets of amyloids markedly enhance HIV infection by electrostatically binding HIV virions and increasing their propensity to bind to and infect cellular targets (Münch et al., 2007; Kim et al., 2010; Roan et al., 2011; Arnold et al., 2012; Roan et al., 2014). Because the ability of semen and SP to enhance HIV infection directly correlates with endogenous levels of these fibrils (Kim et al., 2010; Roan et al., 2014), inhibiting the activity of semen amyloids may decrease HIV transmission rates. Indeed, semen fibrils are currently being pursued as targets for HIV microbicide development (Olsen et al., 2010; Roan et al., 2010; Hartjen et al., 2012; Lump et al., 2015). While the effects of SP amyloids on HIV infection have been extensively studied, the normal physiological function of these fibrils is unclear. SEM proteins have undergone extensive positive selection over evolutionary time (Hurle et al., 2007; Ferreira et al., 2013), suggesting an important role for these proteins in evolutionary fitness. Studies analyzing orthologs of the human amyloidogenic SEM peptide from 12 non-human primate species revealed that the amyloidogenic potential of these orthologous peptides and their virus-enhancing properties are conserved amongst great apes (Roan et al., 2014). Whether this selective pressure occurs at the level of the fibrils or the parent protein is not known, but the existence of amyloidogenic semen peptides from multiple primate species suggest that these structures may serve a physiological function. Furthermore, the observation that SP amyloids promote infection by multiple sexually transmitted viruses (Tang et al., 2013; Torres et al., 2015) suggests that they should be selected against during primate evolution unless they serve a significant physiological purpose. Here, we examined the effects of semen amyloid fibrils on sperm function, and show that they participate in sperm selection and disposal. Results Semen fibrils inhibit fusion of spermatozoa to oocytes Because parallels exist between the fusion of HIV to cells and the fusion of sperm to oocyte (Doncel, 2006), we first examined whether semen fibrils promote fertilization. Because endogenous semen fibrils behave similarly to synthetic versions of the fibrils and are difficult to isolate as purified materials (Roan et al., 2014; Usmani et al., 2014), we used fibrils derived from synthetic peptides for the majority of our studies. Synthetic SEVI and SEM peptides were confirmed to form fibrils by thioflavin T (ThT) staining and electron microscopy (Figure 1—figure supplement 1). These synthetic fibrils, like their endogenous counterparts, include both fibrils and fibrillar oligomers as well as prefibrillar oligomers, as determined by their reactivity with amyloid conformer-specific antibodies OC and A11 (Figure 1—figure supplement 2). In the remainder of this manuscript, we use the term ‘fibrils’ to refer to the synthetic form of the amyloids, and ‘endogenous amyloids’ when fibrils were purified from semen. For ethical reasons, we conducted in vitro fertilization (IVF) using mouse instead of human gametes. Given that the infection-promoting effects of the fibrils are driven by electrostatic forces and is not receptor-specific (Roan et al., 2009, 2011), if fertilization is enhanced by the fibrils, then the effect should be species-independent. Contrary to our hypothesis, both SEVI and SEM fibrils decreased IVF rates in a dose-dependent manner (Figure 1). Significant inhibition of IVF was observed at fibril concentrations of 50 µg/ml, and near complete inhibition was achieved with 250 µg/ml. Because concentrations of amyloidogenic peptides in semen range from 28 to 267 µg/ml (Münch et al., 2007; Roan et al., 2011, 2014), these results suggest that physiologically relevant concentrations of semen fibrils suppress IVF. This inhibition was not due to fibril-induced cytotoxicity to the spermatozoa, oocytes, or embryos, as evidenced by propidium iodide (PI) staining experiments (data not shown). Figure 1 with 3 supplements see all Download asset Open asset SEVI and SEM fibrils inhibit IVF in a dose-dependent manner. The indicated concentrations of SEVI and SEM fibrils were added to mouse spermatozoa and oocytes, and monitored for IVF rates as detailed in the Materials and methods section. *p<0.05 (two-tailed Student’s t test). n.s. = non-significant. Error bars reflect variation between different experiments conducted using gametes from different mice, and correspond to data averaged from 3 to 5 experiments. In experiments with SEVI, the number of oocytes fertilized were 197/272 (0 µg/ml SEVI), 112/179 (10 µg/ml SEVI), 67/219 (50 µg/ml SEVI), and 0/77 (250 µg/ml SEVI). In experiments with SEM fibrils, the number of oocytes fertilized were 78/116 (0 µg/ml SEM), 91/162 (10 µg/ml SEM), 39/128 (50 µg/ml SEM), and 6/35 (250 µg/ml SEM). The 250 µg/ml condition lacks error bars as it was only tested in two experiments due to limited cell numbers; in both of these experiments treatment with 250 µg/ml SEVI led to complete abrogation of IVF (0% fertilized oocytes). https://doi.org/10.7554/eLife.24888.003 Semen fibrils immobilize spermatozoa To clarify the mechanism underlying reduced IVF rates in the presence of the fibrils, we performed live cell imaging. We found that the fibrils seem to inhibit fusion of sperm to oocyte by entrapping mouse sperm cells (Figure 1—figure supplement 3; Video 1). Fibrils similarly entrapped human spermatozoa in a dose-dependent manner, as assessed by both manual quantitation as well as computer-assisted sperm analysis (CASA) (Figure 2A, Figure 2—figure supplement 1). Close examination of fibril-exposed human spermatozoa by cryosection electron microscopy revealed that the fibrils directly interacted with the plasma membranes of both sperm heads and tails, and that points of contact tended to extend the membrane away from the base of the sperm head and tail (Figure 2B). To confirm that endogenous amyloids also associate with sperm, we fractionated human SP pooled from 20 donors and obtained a fraction containing endogenous amyloids as demonstrated by ThT binding (Figure 2—figure supplement 2). Microscopic analysis showed that these purified endogenous amyloids associated with spermatozoa, as did endogenous amyloids present in fresh liquefied ejaculates (Figure 2—figure supplement 3). Furthermore, purified endogenous amyloids, like synthetic fibrils, efficiently entrapped spermatozoa (Figure 2C). Figure 2 with 4 supplements see all Download asset Open asset Semen fibrils directly bind and immobilize human spermatozoa. (A) Human spermatozoa incubated with semen fibrils were imaged at 37°C for 5–10 min and then assessed for % entrapped spermatozoa as described in the Materials and methods section. Native peptide corresponds to monomeric, non-fibrillized peptide. (B) Spermatozoa were incubated in the absence (i, ii) or presence (iii, iv) of SEM fibrils and then imaged by sectioning electron microscopy. Image in panel (iii) shows two sperm heads and image in panel (iv) shows two sperm tails, with arrows highlighting examples of interactions between the fibrils and the tail. (C) Spermatozoa treated with fractions containing (Positive Fraction) or lacking (Negative Fraction) endogenous semen amyloids were imaged for 5–10 min at 37°C and then assessed for % entrapped spermatozoa as described in the Materials and methods section. Treatment of spermatozoa with synthetic SEVI fibrils was used as a positive control for entrapment. The buffer only and negative fraction controls exhibited 0% entrapment. (D) Sperm motility was assessed before (i) or after (ii–vii) perfusion with 50 µg/ml of SEM fibrils (ii), SEM1(68–85) (iii), SEM1(108–159) (iv), SEVI (v), the O. garnettii (Galago) SEM2 repeat amyloid fibrils (vi), or Aβ(1–42) (vii). Within each pair of images, the first corresponds to t = 0, whereas the second corresponds to t = 0.6 s. Numbers correspond to the number of beats that occurred within the length of each movie (total time = 2 s). In instances where two numbers are shown, the first corresponds to the spermatozoon on the left and the second to the spermatozoon on the right. Red text highlights samples where spermatozoa were immobilized. Yellow arrows highlight tail regions within the second frame that moved relative to the first frame. Scale bars = 5 µm. Data for each treatment are representative of at least two independent experiments examining >5 individual spermatozoa per treatment. https://doi.org/10.7554/eLife.24888.007 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Semen fibrils entrap mouse spermatozoa. 105 mouse spermatozoa stained with Hoechst 33342 (green) were incubated in the absence (A) or presence (B) of 50 µg/ml of semen fibrils at 37°C in a final volume of 100 µl for 15–20 min. Images were acquired for 20 s with an interval of 1 s on an LSM710 confocal microscope (Zeiss) using a 20X air objective. https://doi.org/10.7554/eLife.24888.012 To examine the effects of the fibrils on sperm motility at the single cell level, we assayed the motility of spermatozoa from freshly ejaculated human semen by video microscopy. Under HEPES-buffered conditions, spermatozoa attached by their heads to coverslips exhibited continuous beating motions of the tail (Video 2A). Remarkably, within 10 min after perfusion of the SEM fibrils, some spermatozoa became completely immotile, while others exhibited twitching movements (Video 2B, Figure 2D). In the presence of the fibrils, 89.8 ± 9.3% of spermatozoa (average data from three donors) were fully or partly immobilized as defined by at least 50% of the tail being surface-immobilized. Of note, this immobilization is distinct from the previously described ability of the SEM1 holoprotein to limit sperm motility by binding to the sperm-associated EPPIN complex (Mitra et al., 2010), since that activity, mapped to cysteine 239 of SEM1, is not part of the amyloidogenic SEM1 fragment (Roan et al., 2014). SEM fibril-induced immobilization did not cause changes in PI uptake, mitochondrial activity, or ability to capacitate relative to control peptide (Figure 2—figure supplement 4), showing that immobilized spermatozoa were not compromised in viability due to exposure to amyloid structures. In addition, perfusion of seminal proteases onto fibril-immobilized spermatozoa partially restored sperm motility (Video 3) further confirming lack of cytotoxicity. Because proteolytic degradation of amyloidogenic peptides in semen occurs gradually over the course of liquefaction (Roan et al., 2014), immobilization of sperm is likely most efficient during the earliest stages post-ejaculation. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Spermatozoa from fresh ejaculates are immobilized by SEM fibrils. (A) Spermatozoa were isolated as detailed in the supplemental experimental procedures, attached onto coverslips, and examined for motility by live microscopy. (B) Cells were then perfused with 50 µg/ml SEM1(86–107) fibrils and motility was assessed by video microscopy after 10 min. Of note, the same four spermatozoa are shown in the two panels. https://doi.org/10.7554/eLife.24888.013 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Immobilization of surface-associated sperm cells by SEM1(86–107) fibrils is reversible. Motile spermatozoa were examined before (A) and 10 min after (B) perfusion with SEM1(86–107) amyloid fibrils. 0.45 µm-filtered seminal plasma was then perfused in at a concentration of 20% as a source of seminal proteases, and 20 min later sperm cells were assessed for motility (C). Data are representative of n = 6 experiments from two sperm donors. https://doi.org/10.7554/eLife.24888.014 We further demonstrated that the ability of SEM fibrils to immobilize spermatozoa was linked to its fibrillar structure by showing that SEM1(68–85), a naturally-occurring SEM-derived peptide in semen that does not form fibrils (Roan et al., 2011), did not inhibit motility (Video 4A, Figure 2D). Motility was also not inhibited by SEM1(108–159), a SEM-derived peptide that is non-fibrillar but highly cationic (pI = 10.12) (Roan et al., 2011) (Video 4B, Figure 2D). In contrast, human SEVI (Münch et al., 2007) and the previously described SEM-derived fibril from the non-human primate Otolemur garnettii (referred to as Galago) (Roan et al., 2014) both immobilized spermatozoa (Video 5A,B, Figure 2D), while Aβ(1–42) fibrils, not naturally present in semen, did not cause immobilization (Video 5C, Figure 2D). All together, these data suggest that semen amyloid fibrils, and not native semen peptides or pathological amyloids, are distinct in their ability to immobilize sperm cells. The differential effects of semen fibrils vs. Aβ(1–42) fibrils on sperm motility could be due to differences in fibril charge and/or distribution of amyloid conformers. Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg PSA-generated non-fibrillar fragments SEM1(68–85) and SEM1(108–159) do not inhibit sperm motility. Motile spermatozoa were examined for motility before and 10 min after perfusion with 50 µg/ml SEM1(68–85) (A) or SEM1(108–159) (B). https://doi.org/10.7554/eLife.24888.015 Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg SEVI and O. garnettii SEM2 repeat fibrils inhibit sperm motility, whereas Aβ(1–42) fibrils do not. Motile spermatozoa were examined for motility before and 10 min after perfusion with 50 µg/ml SEVI fibrils (A), O. garnettii (Galago) SEM2 repeat fibrils (B), or Aβ(1–42) fibrils (C). An Aβ(1–42) fibril concentration of 100 µg/ml, corresponding to an equimolar amount of 50 µg/ml SEM1(86–107), was also tested and did not inhibit sperm motility (data not shown). https://doi.org/10.7554/eLife.24888.016 Semen fibrils promote phagocytosis of damaged spermatozoa Having established that semen fibrils do not promote fusion of sperm to egg, but that they do uniquely immobilize sperm cells, we next sought to address what effect this may have on reproduction. Sexual intercourse elicits a massive infiltration of neutrophils and macrophages into the female reproductive tract (FRT) (Pandya and Cohen, 1985; Sharkey et al., 2012), presumably to mediate clearance of microorganisms and remnant sperm cells, and perhaps to filter out morphologically abnormal and/or non-functional sperm cells (Tomlinson et al., 1992; Oren-Benaroya et al., 2007). Thus, we next tested whether spermatozoa entrapped by the fibrils are preferentially phagocytosed. Macrophages were differentiated from human monocytes obtained from female donors, confirmed for phagocytic activity (Figure 3—figure supplement 1A), and then imaged by confocal microscopy following incubation with fluorescently-labeled spermatozoa. Macrophages that had taken up multiple sperm cells could be readily observed (Figure 3—figure supplement 1B, Videos 6 and 7). Higher throughput imaging with the Amnis Imagestream revealed that some macrophages had taken up a single spermatozoon while others had taken up multiple ones (Figure 3—figure supplement 1C). Having demonstrated the ability to assess phagocytosis of spermatozoa in vitro, we next used a biochemical method to assess whether fibrils increase this process. Spermatozoa were added to cultured macrophages, and at various timepoints the macrophages were washed extensively to remove surface-associated spermatozoa and cell lysates were prepared for Western blotting. Acetylated tubulin, a ciliary protein expressed at high levels in sperm but not in somatic cells, was used as a marker for phagocytosed sperm cells. As shown in Figure 3A, phagocytosis of spermatozoa was apparent by 3 hr, and markedly increased by semen fibrils. Acetylated tubulin was not detected when the assay was performed at 4°C to prevent phagocytic activity (Figure 3A), verifying detection of actual phagocytosis as opposed to surface binding of sperm cells. Figure 3 with 5 supplements see all Download asset Open asset Semen fibrils promote phagocytosis of sperm cells. (A) Spermatozoa were added to monocyte-derived macrophages for the indicated number of hours in the presence or absence of 100 µg/ml SEM fibrils, washed, and then blotted for acetylated tubulin (to detect spermatozoa) or β-actin (to detect macrophages). Negative controls include macrophages in the absence of spermatozoa, and incubation of macrophages with spermatozoa at 4°C to prevent phagocytosis. (B) Motile sperm cells purified by the swim-up method were labeled with eFluor 670 and then left at room temperature or damaged by five sequential rounds of freeze-thaw with liquid nitrogen. Spermatozoa were then added to monocyte-derived macrophages for 0.5 hr at 37°C in the presence or absence of 100 µg/ml SEM fibrils, washed, and then assessed by flow cytometry. Macrophages were identified by gating on CD14+CD33+ cells, and phagocytosis was assessed by determining the percentages of macrophages that were eFluor 670+. Results are representative of data from five different donors. (C) Comparison of eFluor 670+ macrophages after incubation with labeled spermatozoa at 37°C vs. 4°C (temperature at which phagocytosis is inhibited). These data suggest that in the presence of SEM fibrils, a small number of macrophages have surface-associated spermatozoa. (D) Macrophage-mediated phagocytosis of healthy vs. damaged spermatozoa in the presence of SEM fibrils was compared in triplicates. Shown values are those where the levels of eFluor 670+ macrophages in the presence of the SEM fibrils under 4°C conditions were subtracted out. This normalization was performed to discount surface-associated spermatozoa (which is present to some extent as demonstrated in panel C) from the analysis. *p<0.05 (by 2-tailed t test). https://doi.org/10.7554/eLife.24888.017 Video 6 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Internalization of spermatozoa by macrophage (rotational view). Monocyte-derived macrophages labeled with a membrane dye (green) were incubated with sperm cells (red) for 3 hr and then imaged by confocal microscopy. https://doi.org/10.7554/eLife.24888.023 Video 7 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Internalization of spermatozoa by macrophage (z-stacks view). Monocyte-derived macrophages labeled with a membrane dye (green) were incubated with sperm cells (red) for 3 hr and then imaged by confocal microscopy. https://doi.org/10.7554/eLife.24888.024 To quantify spermatozoa uptake by macrophages at the single-cell level, we developed a FACS-based phagocytosis assay. Spermatozoa were fluorescently labeled with eFluor 670 and then co-cultured with macrophages. Cell surface expression of CD14 and CD33 was used to differentiate the macrophages from the sperm cells. Macrophages that had taken up spermatozoa were identified by eFluor 670 fluorescence. As demonstrated in Figure 3—figure supplement 2A, a distinct population of macrophages that had phagocytosed spermatozoa was readily apparent after 0.5 hr of co-culture. This population was abrogated when the assay was conducted in the presence of the phagocytosis inhibitor cytochalasin D or when the assay was conducted at 4°C instead of 37°C (Figure 3—figure supplement 2A). Thus, detection of sperm-harboring macrophages was not due to leakage of dye from spermatozoa, or from cell-surface binding of the spermatozoa to the macrophages. Furthermore, >99% of the macrophages at the time of harvest were viable (Figure 3—figure supplement 2B), excluding non-specific effects due to autofluorescence of dying cells. To assess whether semen fibrils promote phagocytosis of spermatozoa, we conducted the FACS-based phagocytosis assay in the absence and presence of semen fibrils. Consistent with the Western blot data, addition of fibrils increased phagocytosis of spermatozoa, from 4.61% to 22.6% (Figure 3B). Because removal of damaged or defective sperm cells from the reproductive tract may be important to rapidly clear the lower FRT of potentially immunogenic male antigens, we also assessed phagocytosis of damaged spermatozoa. Sperm cells damaged by multiple freeze/thaw cycles in liquid nitrogen or electromagnetic radiation were taken up at higher rates than healthy sperm cells (Figure 3—figure supplement 3A). Because liquid nitrogen exerted a more potent effect (up to 3.4-fold increased uptake, as opposed to up to 2.3-fold increased uptake for radiation-damaged spermatozoa), we selected this method of damage induction for subsequent experiments. In the absence of the fibrils, the rate of phagocytosis of liquid nitrogen-damaged spermatozoa was higher than that of fresh spermatozoa (31.3% versus 4.61%, respectively); this was also observed in the presence of the fibrils but at higher percentages (49.9% versus 22.6%, respectively) (Figure 3B). Importantly, the absolute numbers of spermatozoa added to each co-culture condition were the same to allow direct comparisons between conditions. Interestingly, with longer incubation times high levels of phagocytosis of both healthy and damaged sperm cells were achieved in the presence of fibrils, suggesting that the fibrils promote the kinetics of sperm phagocytosis (Figure 3—figure supplement 3B). Confocal microscopy revealed that fibrils also increased the number of spermatozoa engulfed by individual macrophages, frequently resulting in a single macrophage endocytosing more than a dozen sperm heads (Figure 3—figure supplement 4, Videos 8 and 9). Video 8 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg High number of spermatozoa internalized by a single macrophage in the presence of semen fibrils (rotational view). Monocyte-derived macrophages labeled with a membrane dye (green) were incubated with sperm cells (red) for 3 hr in the presence of 100 µg/ml SEM fibrils and then imaged by confocal microscopy. https://doi.org/10.7554/eLife.24888.025 Video 9 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg High number of spermatozoa internalized by a single macrophage in the presence of semen fibrils (z-stacks view). Monocyte-derived macrophages labeled with a membrane dye (green) were incubated with sperm cells (red) for 3 hr in the presence of 100 µg/ml SEM fibrils and then imaged by confocal microscopy. https://doi.org/10.7554/eLife.24888.026 We consistently observed in multiple donors that in the presence of the fibrils damaged spermatozoa are preferentially phagocytosed over healthy ones (Figure 3B, Figure 3—figure supplement 3A). To confirm this phenomenon, healthy and damaged spermatozoa were co-cultured with macrophages in the absence or presence of SEM fibrils, both at 37°C as well as 4°C to block phagocytic activity. As shown in Figure 3C, the highest level of phagocytosis was observed with damaged spermatozoa in the presence of the fibrils, in line with our prior experiments. However, macrophage/sperm samples incubated at 4°C with SEM fibrils also exhibited positive events within the phagocytosis gate, suggesting that SEM caused some level of sperm sticking to the macrophage surface (Figure 3C). To normalize for the contribution of cell-surface sticking, we subtracted out the contribution of the positive events at 4°C for each sample. Levels of phagocytosis of damaged spermatozoa in the presence of fibrils remained significantly higher than that of healthy spermatozoa under these conditions (Figure 3D). All together, these results suggest that: (1) macrophages preferentially phagocytose damaged spermatozoa both in the absence and presence of semen fibrils, consistent with prior reports that increased levels of macrophages are associated with decreased levels of abnormal spermatozoa in semen (Tomlinson et al., 1992), and (2) within the first hour, the highest levels of damaged sperm phagocytosis are observed in the presence of semen fibrils. To directly assess the preference for phagocytosis of damaged spermatozoa, we established a competition assay enabling visualization of normal and damaged spermatozoa within the same well by labeling the two populations with different fluorescent dyes. Notably,
SUMMARY Although T cells are likely players in SARS-CoV-2 immunity, little is known about the phenotypic features of SARS-CoV-2-specific T cells associated with recovery from severe COVID-19. We analyzed T cells from longitudinal specimens of 34 COVID-19 patients with severities ranging from mild (outpatient) to critical culminating in death. Relative to patients that succumbed, individuals that recovered from severe COVID-19 harbored elevated and increasing numbers of SARS-CoV-2-specific T cells capable of homeostatic proliferation. In contrast, fatal COVID-19 displayed elevated numbers of SARS-CoV-2-specific regulatory T cells and a time-dependent escalation in activated bystander CXCR4+ T cells. Together with the demonstration of increased proportions of inflammatory CXCR4+ T cells in the lungs of severe COVID-19 patients, these results support a model whereby lung-homing T cells activated through bystander effects contribute to immunopathology, while a robust, non-suppressive SARS-CoV-2-specific T cell response limits pathogenesis and promotes recovery from severe COVID-19. Graphical Abstract HIGHLIGHTS Dysfunctional spike-specific T cells are characteristic of severe COVID-19 Spike-specific CD127+ Th1 cells are increased in survivors of severe COVID-19 Spike-specific Tregs and IL6+ CD8+ T cells are increased in fatal COVID-19 Escalation of activated lung-homing CXCR4+ T cells associates with fatal COVID-19 BRIEF SUMMARY By conducting CyTOF on total and SARS-CoV-2-specific T cells from longitudinal specimens spanning the entire spectrum of COVID-19 diseases, Neidleman et al. demonstrate that spike-specific Th1 cells capable of IL7-dependent homeostatic proliferation predict survival from severe COVID-19, while Tregs and IL6+ CD8+ T cells recognizing spike predict fatal outcome. Fatal COVID-19 is characterized by escalating activation of bystander CXCR4+ T cells in the lungs. Boosting SARS-CoV-2-specific CD4+ T effector responses while diminishing CXCR4-mediated homing may help recovery from severe disease.
Summary There is an urgent need for prophylactic and therapeutic vaccines against human immunodeficiency virus (HIV). Mucosal immunization strategies have great potential to elicit both mucosal and systemic cellular immunity required to protect against HIV‐induced aquired immune deficiency syndrome (AIDS). However, mucosal immunizations with soluble protein antigens generally require adjuvants. In this study, we tested two mutants of the heat‐labile enterotoxin (LT) from Escherichia coli , LTK63: with no measurable ADP‐ribosyltransferase activity, and LTR72: with residual ADP‐ribosyltransferase activity, as mucosal adjuvants for induction of cytotoxic T lymphocyte (CTL) responses to coadministered HIV gag p55 protein. We found that intranasal (i.n.) immunizations with HIV gag p55 protein coadministered with LTK63 or LTR72 induced systemic CTL responses comparable to that obtained following intramuscular (i.m.) immunizations with the same adjuvants. Moreover, oral coadministration of LTR72, but not LTK63, resulted in local as well as systemic p55‐specific CTL responses in mesenteric lymph nodes (MLN) and spleens (SP) of the immunized mice. These data have important implications for current efforts to develop a safe vaccine against HIV.
Heat-labile enterotoxin (LT) from Escherichia coli and Cholera toxin (CT) from Vibro cholerae are known to be potent mucosal immunogens. These toxins have 80% sequence homology and a similar tertiary structure (,), and both elicit potent serum IgG and mucosal IgA responses (,). Moreover, both also serve as excellent adjuvants for coadministered antigens. However, they are toxic in their native state and both produce accumulation of intestinal fluid and watery diarrhea (). LT is the cause of traveler's diarrhea, whereas CT causes cholera. In order to make use of the adjuvanticity of these molecules but reduce their toxicity, several mutants have been generated by site-directed mutagenesis. Of these, there are two mutants of the enzymatic A subunit, LTK63 and LTR72, that maintain a high level of immunogenicity and have significant potential as adjuvants. This chapter will focus on the use of LTK63 and LTR72 as intramuscular and intranasal adjuvants for the induction of cytotoxic T lymphocytes (CTL) activity against coadministered protein immunogens.
CD4 T lymphocytes belong to diverse cellular subsets whose sensitivity or resistance to HIV-associated killing remains to be defined. Working with lymphoid cells from human tonsils, we characterized the HIV-associated depletion of various CD4 T cell subsets using mass cytometry and single-cell RNA-seq. CD4 T cell subsets preferentially killed by HIV are phenotypically distinct from those resistant to HIV-associated cell death, in a manner not fully accounted for by their susceptibility to productive infection. Preferentially-killed subsets express CXCR5 and CXCR4 while preferentially-infected subsets exhibit an activated and exhausted effector memory cell phenotype. Single-cell RNA-seq analysis reveals that the subsets of preferentially-killed cells express genes favoring abortive infection and pyroptosis. These studies emphasize a complex interplay between HIV and distinct tissue-based CD4 T cell subsets, and the important contribution of abortive infection and inflammatory programmed cell death to the overall depletion of CD4 T cells that accompanies untreated HIV infection.
In 1758, Rousseau announced that he had adopted "vitam impendere vero" (dedicate life to truth) as a personal pledge. Despite the dramatic nature of this declaration, no scholar has yet approached Rousseau's work through the lens of truth or truthseeking. What did it mean for Rousseau to lead a life dedicated to truth? This book presents Rousseau's normative account of truthseeking, his account of what human beings must do if they hope to discover the truths essential to human happiness. Rousseau's writings constitute a practical guide to these truths; they describe how he arrived at them and how others might as well. In reading Rousseau through the lens of truth, Neidleman traverses the entirety of Rousseau's corpus, and, in the process, reveals a series of symmetries among the disparate themes treated in those texts. The first section of the book lays out Rousseau's general philosophy of truth and truthseeking. The second section follows Rousseau down four distinct pathways to truth: reverie, republicanism, religion, and reason. With a strong grounding in both the Anglophone and Francophone scholarship on Rousseau, this book will appeal to scholars across a broad range of disciplines.
In infected people, the HIV-1 envelope glycoprotein (Env) constantly evolves to escape the immune response while retaining the essential elements needed to mediate viral entry into target cells. The extensive genetic variation of Env is particularly striking in the V1/V2 hypervariable domains. In this study, we investigated the trade-off, in terms of fusion efficiency, for encoding V1/V2 domains of different lengths. We found that natural variations in V1/V2 length exert a profound impact on HIV-1 entry. Variants encoding compact V1/V2 domains mediated fusion with higher efficiencies than related Envs encoding longer V1/V2 domains. By exchanging the V1/V2 domains between Envs of the same infected person or between two persons linked by a transmission event, we further demonstrated that V1/V2 domains critically influence both Env incorporation into viral particles and fusion to primary CD4 T cells and monocyte-derived dendritic cells. Shortening the V1/V2 domains consistently increased Env incorporation and fusion, whereas lengthening the V1/V2 domains decreased Env incorporation and fusion. Given that in a new host transmitted founder viruses are distinguished by compact Envs with fewer glycosylation sites, our study points to fusion and possibly Env incorporation into virions as limiting steps for transmission of HIV-1 to a new host and suggests that the length and/or the N-glycosylation profile of the V1/V2 domain influences these early steps in the HIV life cycle.
