Indirect CD4+ T cell protection against mouse gamma-herpesvirus infection via interferon gamma.

CD4+ T cells play a key role in γ-herpesvirus infection control. However, the mechanisms involved are unclear. Murine herpesvirus type 4 (MuHV-4) allows relevant immune pathways to be dissected experimentally in mice. In the lungs, it colonizes myeloid cells, which can express MHC class II (MHCII), and type 1 alveolar epithelial cells (AEC1), which lack it. Nevertheless, CD4+ T cells can control AEC1 infection, and this control depends on MHCII expression in myeloid cells. Interferon-gamma (IFNγ) is a major component of CD4+ T cell-dependent MuHV-4 control. Here, we show that the action of IFNγ is also indirect, as CD4+ T cell-mediated control of AEC1 infection depended on IFNγ receptor (IFNγR1) expression in CD11c+ cells. Indirect control also depended on natural killer (NK) cells. Together, the data suggest that the activation of MHCII+ CD11c+ antigen-presenting cells is key to the CD4+ T cell/NK cell protection axis. By contrast, CD8+ T cell control of AEC1 infection appeared to operate independently. IMPORTANCE: CD4+ T cells are critical for the control of gamma-herpesvirus infection; they act indirectly, by recruiting natural killer (NK) cells to attack infected target cells. Here, we report that the CD4+ T cell/NK cell axis of gamma-herpesvirus control requires interferon-γ engagement of CD11c+ dendritic cells. This mechanism of CD4+ T cell control releases the need for the direct engagement of CD4+ T cells with virus-infected cells and may be a common strategy for host control of immune-evasive pathogens.

Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8(+) Cytolytic T Cell Responses.

Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and ß) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8(+) T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8(+) cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8(+) CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8(+) OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy.

Indirect CD4+ T cell protection against persistent MCMV infection by NK cells requires IFNγ.

Host control of mouse cytomegalovirus (MCMV) infection of MHCII- salivary gland acinar cells is mediated by CD4+ T cells, but how they protect is unclear. Here, we show CD4+ T cells control MCMV indirectly in the salivary gland, via IFNγ engagement with uninfected, but antigen+ MHCII+ APC and recruitment of NK cells to infected cell foci. This immune mechanism renders direct contact of CD4+ T cells with infected cells unnecessary and may represent a host strategy to overcome viral immune evasion.

Floral scent changes in response to pollen removal are rare in buzz-pollinated Solanum.

MAIN CONCLUSION: One of seven Solanum taxa studied displayed associations between pollen presence and floral scent composition and volume, suggesting buzz-pollinated plants rarely use scent as an honest cue for foraging pollinators. Floral scent influences the recruitment, learning, and behaviour of floral visitors. Variation in floral scent can provide information on the amount of reward available or whether a flower has been visited recently and may be particularly important in species with visually concealed rewards. In many buzz-pollinated flowers, tubular anthers opening via small apical pores (poricidal anthers) visually conceal pollen and appear similar regardless of pollen quantity within the anther. We investigated whether pollen removal changes floral scent composition and emission rate in seven taxa of buzz-pollinated Solanum (Solanaceae). We found that pollen removal reduced both the overall emission of floral scent and the emission of specific compounds (linalool and farnesol) in S. lumholtzianum. Our findings suggest that in six out of seven buzz-pollinated taxa studied here, floral scent could not be used as a signal by visitors as it does not contain information on pollen availability.

Nectar cardenolides and floral volatiles mediate a specialized wasp pollination system.

Specialization in plant pollination systems can arise from traits that function as filters of flower visitors. This may involve chemical traits such as floral volatiles that selectively attract favoured visitors and non-volatile nectar constituents that selectively deter disfavoured visitors through taste or longer-term toxic effects or both. We explored the functions of floral chemical traits in the African milkweed Gomphocarpus physocarpus, which is pollinated almost exclusively by vespid wasps, despite having nectar that is highly accessible to other insects such as honeybees. We demonstrated that the nectar of wasp-pollinated G. physocarpus contains cardenolides that had greater toxic effects on Apis mellifera honeybees than on Vespula germanica wasps, and also reduced feeding rates by honeybees. Behavioural experiments using natural compositions of nectar compounds showed that these interactions are mediated by non-volatile nectar chemistry. We also identified volatile compounds with acetic acid as a main component in the floral scent of G. physocarpus that elicited electrophysiological responses in wasp antennae. Mixtures of these compounds were behaviourally effective for attraction of V. germanica wasps. The results show the importance of both volatile and non-volatile chemical traits as filters that lead to specialization in plant pollination systems.

The Mouse Cytomegalovirus G Protein-Coupled Receptor Homolog, M33, Coordinates Key Features of In Vivo Infection via Distinct Components of Its Signaling Repertoire.

Common to all cytomegalovirus (CMV) genomes analyzed to date is the presence of G protein-coupled receptors (GPCR). Animal models of CMV provide insights into their role in viral fitness. The mouse cytomegalovirus (MCMV) GPCR, M33, facilitates dendritic cell (DC)-dependent viremia, the extravasation of blood-borne infected DCs to the salivary gland, and the frequency of reactivation events from latently infected tissue explants. Constitutive G protein-coupled M33 signaling is required for these phenotypes, although the contribution of distinct biochemical pathways activated by M33 is unknown. M33 engages Gq/11 to constitutively activate phospholipase C ß (PLCß) and downstream cyclic AMP response-element binding protein (CREB) in vitro. Identification of a MCMV M33 mutant (M33ΔC38) for which CREB signaling was disabled but PLCß activation was preserved provided the opportunity to investigate their relevance in vivo. Following intranasal infection with MCMV M33ΔC38, the absence of M33 CREB Gq/11-dependent signaling correlated with reduced mobilization of lytically-infected DCs to the draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33ΔC38-infected DCs within the vascular compartment extravasated to the salivary glands via a pertussis toxin-sensitive, Gi/o-dependent, and CREB-independent mechanism. In the context of MCMV latency, spleen explants from M33ΔC38-infected mice were markedly attenuated for reactivation. Taken together, these data demonstrate that key features of the MCMV life cycle are coordinated in diverse tissues by distinct pathways of the M33 signaling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular "switches" that regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analyzed to date possess GPCR homologs with phylogenetic evidence for independent gene capture events, signifying important in vivo roles. The mouse CMV (MCMV) GPCR homolog, designated M33, is important for cell-associated virus spread and the establishment and/or reactivation of latent MCMV infection. The signaling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signaling pathways for in vivo M33 function. In this report, we showed that temporal and tissue-specific M33 signaling was required to facilitate in vivo infection. Understanding the relevance of the viral GPCR signaling profiles for in vivo function will provide opportunities for future targeted interventions.

CD4+ T Cells Control Murine Cytomegalovirus Infection Indirectly.

CD4+ T cells are key to controlling cytomegalovirus infections. Salivary gland infection by murine cytomegalovirus (MCMV) provides a way to identify mechanisms. CD11c+ dendritic cells (DC) disseminate MCMV to the salivary glands, where they transfer infection to acinar cells. Antiviral CD4+ T cells are often considered to be directly cytotoxic for cells expressing major histocompatibility complex class II (MHCII). However, persistently infected salivary gland acinar cells are MHCII- and are presumably inaccessible to direct CD4 T cell recognition. Here, we show that CD4+ T cell depletion amplified infection of MHCII- acinar cells but not MHCII+ cells. MCMV-infected mice with disrupted MHCII on CD11c+ cells showed increased MHCII- acinar infection; antiviral CD4+ T cells were still primed, but their recruitment to the salivary glands was reduced, suggesting that engagement with local MHCII+ DC is important for antiviral protection. As MCMV downregulates MHCII on infected DC, the DC participating in CD4 protection may thus be uninfected. NK cells and gamma interferon (IFN-γ) may also contribute to CD4+ T cell-dependent virus control: CD4 T cell depletion reduced NK cell recruitment to the salivary glands, and both NK cell and IFN-γ depletion equalized infection between MHCII-disrupted and control mice. Taken together, these results suggest that CD4+ T cells protect indirectly against infected acinar cells in the salivary gland via DC engagement, requiring the recruitment of NK cells and the action of IFN-γ. Congruence of these results with an established CD4+ T cell/NK cell axis of gammaherpesvirus infection control suggests a common mode of defense against evasive viruses. IMPORTANCE Cytomegalovirus infections commonly cause problems in immunocompromised patients and in pregnancy. We lack effective vaccines. CD4+ T cells play an important role in normal infection control, yet how they act has been unknown. Using murine cytomegalovirus as an accessible model, we show that CD4+ T cells are unlikely to recognize infected cells directly. We propose that CD4+ T cells interact with uninfected cells that present viral antigens and recruit other immune cells to attack infected targets. These data present a new outlook on understanding how CD4+ T cell-directed control protects against persistent cytomegalovirus infection.

Sterol and lipid metabolism in bees.

BACKGROUND: Bees provide essential pollination services for many food crops and are critical in supporting wild plant diversity. However, the dietary landscape of pollen food sources for social and solitary bees has changed because of agricultural intensification and habitat loss. For this reason, understanding the basic nutrient metabolism and meeting the nutritional needs of bees is becoming an urgent requirement for agriculture and conservation. We know that pollen is the principal source of dietary fat and sterols for pollinators, but a precise understanding of what the essential nutrients are and how much is needed is not yet clear. Sterols are key for producing the hormones that control development and may be present in cell membranes, where fatty-acid-containing species are important structural and signalling molecules (phospholipids) or to supply, store and distribute energy (glycerides). AIM OF THE REVIEW: In this critical review, we examine the current general understanding of sterol and lipid metabolism of social and solitary bees from a variety of literature sources and discuss implications for bee health. KEY SCIENTIFIC CONCEPTS OF REVIEW: We found that while eusocial bees are resilient to some dietary variation in sterol supply the scope for this is limited. The evidence of both de novo lipogenesis and a dietary need for particular fatty acids (FAs) shows that FA metabolism in insects is analogous to mammals but with distinct features. Bees rely on their dietary intake for essential sterols and lipids in a way that is dependent upon pollen availability.

Hyper-CVAD versus dose-adjusted EPOCH as initial treatment for adults with acute lymphoblastic leukemia.

OBJECTIVES: We recently performed a single-arm phase II trial of DA-EPOCH in adults with acute lymphoblastic leukemia (ALL). We sought to compare these results to those with standard Hyper-CVAD. METHODS: We created a retrospective matched cohort of patients who received Hyper-CVAD (n = 69) at our center and otherwise met eligibility criteria for the DA-EPOCH trial (n = 53). RESULTS: Our outcomes support the use of Hyper-CVAD over DA-EPOCH in Ph- disease for both overall survival (OS; HR 0.18, p = .004) and event-free survival (EFS; HR 0.51, p = .06). In contrast, outcomes were similar in Ph+ disease (OS HR 0.97, p = .96; EFS HR 0.65, p = .21). Rates of morphologic remission and measurable residual-disease negativity were similar between the regimens. Hyper-CVAD was associated with significantly more febrile neutropenia (OR 1.9, p = .03) and a greater incidence of Grade 4 or 5 adverse events (20% vs. 6%). Average transfusions per cycle of both red blood cells (p < .001) and platelets (p < .001) were five-fold higher with Hyper-CVAD. CONCLUSIONS: Our findings support continued use of Hyper-CVAD for Ph- ALL but suggest that DA-EPOCH may be a reasonable alternative for Ph+ ALL. These data also highlight a potential role for DA-EPOCH in resource-limited settings or when more intense therapy is not feasible.

Cerebrospinal fluid flow cytometry and risk of central nervous system relapse after hyperCVAD in adults with acute lymphoblastic leukemia.

BACKGROUND: Potential involvement of the central nervous system (CNS) by acute lymphoblastic leukemia is typically evaluated by a conventional cytospin (CC) of cerebrospinal fluid (CSF). Multiparameter flow cytometry (MFC) is generally more sensitive and specific than morphology, but data to guide its use versus CC are limited. METHODS: This study identified 92 patients who had MFC performed on their initial CSF specimen and received at least 4 cycles of hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with methotrexate and cytarabine (hyperCVAD) as their initial treatment. RESULTS: Eighteen (20%) were CSF+ by MFC at the baseline, and only 6 of these patients were positive by CC. In contrast, 0 of 51 patients who were negative by MFC and had CC available were positive by CC. Despite the receipt of significantly more intra-CSF chemotherapy (P < .001), the cumulative incidence of CNS relapse by MFC was 22% among CSF+ patients versus 5% among those who were CSF- (P = .044). No such association was observed between CNS relapse and CC results (P = .42). None of the 74 CSF- patients became CSF+ during their initial treatment despite being tested a median of 5 times (range, 2-10). CSF positivity by MFC was the factor most strongly associated with CNS relapse in a series of univariate Cox models (hazard ratio, 3.7; P = .067). The initial CSF status by MFC had no significant impact on overall or event-free survival. CONCLUSIONS: MFC of CSF is superior to CC of CSF in identifying adults at high risk for CNS relapse after treatment with hyperCVAD. Surveillance of CSF by MFC has limited utility.

The impact of B-cell-directed therapy on SARS-CoV-2 vaccine efficacy in chronic lymphocytic leukaemia.

Prior reports evaluating SARS-CoV-2 vaccine efficacy in chronic lymphocytic leukaemia (CLL) used semiquantitative measurements of anti-S to evaluate immunity; however, neutralization assays were used to assess functional immunity in the trials leading to vaccine approval. Here, we identified decreased rates of seroconversion in vaccinated CLL patients and lower anti-S levels compared to healthy controls. Notably, we demonstrated similar results with the Roche anti-S assay and neutralization activity. Durable responses were seen at six months; augmentation with boosters was possible in responding patients. Absence of normal B cells, frequently seen in patients receiving Bruton tyrosine kinase and B-cell lymphoma 2 inhibitors, was a strong predictor of lack of seroconversion.

A Live Olfactory Mouse Cytomegalovirus Vaccine, Attenuated for Systemic Spread, Protects against Superinfection.

Vaccination against the betaherpesvirus, human cytomegalovirus (HCMV) is a public health goal. However, HCMV has proved difficult to vaccinate against. Vaccination against single HCMV determinants has not worked, suggesting that immunity to a wider antigenic profile may be required. Live attenuated vaccines provide the best prospects for protection, but the question remains as to how to balance vaccine virulence with safety. Animal models of HCMV infection provide insights into identifying targets for virus attenuation and understanding how host immunity blocks natural, mucosal infection. Here, we evaluated the vaccine potential of a mouse cytomegalovirus (MCMV) vaccine deleted of a viral G protein-coupled receptor (GPCR), designated M33, that renders it attenuated for systemic spread. A single noninvasive olfactory ΔM33 MCMV vaccine replicated locally, but as a result of the loss of the M33 GPCR, it failed to spread systemically and was attenuated for latent infection. Vaccination did not prevent host entry of a superinfecting MCMV but spread from the mucosa was blocked. This approach to vaccine design may provide a viable alternative for a safe and effective betaherpesvirus vaccine. IMPORTANCE Human cytomegalovirus (HCMV) is the most common cause of congenital infection for which a vaccine is not yet available. Subunit vaccine candidates have failed to achieve licensure. A live HCMV vaccine may prove more efficacious, but it faces safety hurdles which include its propensity to persist and to establish latency. Understanding how pathogens infect guide rational vaccine design. However, HCMV infections are asymptomatic and thus difficult to capture. Animal models of experimental infection provide insight. Here, we investigated the vaccine potential of a mouse cytomegalovirus (MCMV) attenuated for systemic spread and latency. We used olfactory vaccination and virus challenge to mimic its natural acquisition. We provide proof of concept that a single olfactory MCMV that is deficient in systemic spread can protect against wild-type MCMV superinfection and dissemination. This approach of deleting functional counterpart genes in HCMV may provide safe and effective vaccination against congenital HCMV disease.

Murine Cytomegalovirus MCK-2 Facilitates In Vivo Infection Transfer from Dendritic Cells to Salivary Gland Acinar Cells.

The cytomegaloviruses (CMVs) spread systemically via myeloid cells and demonstrate broad tissue tropism. Human CMV (HCMV) UL128 encodes a component of the virion pentameric complex (PC) that is important for entry into epithelial cells and cell-cell spread in vitro. It possesses N-terminal amino acid sequences similar to those of CC chemokines. While the species specificity of HCMV precludes confirmation of UL128 function in vivo, UL128-like counterparts in experimental animals have demonstrated a role in salivary gland infection. How they achieve this has not been defined, although effects on monocyte tropism and immune evasion have been proposed. By tracking infected cells following lung infection, we show that although the UL128-like protein in mouse CMV (MCMV) (designated MCK-2) facilitated entry into lung macrophages, it was dispensable for subsequent viremia mediated by CD11c+ dendritic cells (DCs) and extravasation to the salivary glands. Notably, MCK-2 was important for the transfer of MCMV infection from DCs to salivary gland acinar epithelial cells. Acinar cell infection of MCMVs deleted of MCK-2 was not rescued by T-cell depletion, arguing against an immune evasion mechanism for MCK-2 in the salivary glands. In contrast to lung infection, peritoneal MCMV inoculation yields mixed monocyte/DC viremia. In this setting, MCK-2 again promoted DC-dependent infection of salivary gland acinar cells, but it was not required for monocyte-dependent spread to the lung. Thus, the action of MCK-2 in MCMV spread was specific to DC-acinar cell interactions. IMPORTANCE Cytomegaloviruses (CMVs) establish myeloid cell-associated viremias and persistent shedding from the salivary glands. In vitro studies with human CMV (HCMV) have implicated HCMV UL128 in epithelial tropism, but its role in vivo is unknown. Here, we analyzed how a murine CMV (MCMV) protein with similar physical properties, designated MCK-2, contributes to host colonization. We demonstrate that MCK-2 is dispensable for initial systemic spread from primary infection sites but within the salivary gland facilitates the transfer of infection from dendritic cells (DCs) to epithelial acinar cells. Virus transfer from extravasated monocytes to the lungs did not require MCK-2, indicating a tissue-specific effect. These results provide new information about how persistent viral tropism determinants operate in vivo.

Olfactory Entry Promotes Herpesvirus Recombination.

Herpesvirus genomes show abundant evidence of past recombination. Its functional importance is unknown. A key question is whether recombinant viruses can outpace the immunity induced by their parents to reach higher loads. We tested this by coinfecting mice with attenuated mutants of murid herpesvirus 4 (MuHV-4). Infection by the natural olfactory route routinely allowed mutant viruses to reconstitute wild-type genotypes and reach normal viral loads. Lung coinfections rescued much less well. Attenuated murine cytomegalovirus mutants similarly showed recombinational rescue via the nose but not the lungs. These infections spread similarly, so route-specific rescue implied that recombination occurred close to the olfactory entry site. Rescue of replication-deficient MuHV-4 confirmed this, showing that coinfection occurred in the first encountered olfactory cells. This worked even with asynchronous inoculation, implying that a defective virus can wait here for later rescue. Virions entering the nose get caught on respiratory mucus, which the respiratory epithelial cilia push back toward the olfactory surface. Early infection was correspondingly focused on the anterior olfactory edge. Thus, by concentrating incoming infection into a small area, olfactory entry seems to promote functionally significant recombination. IMPORTANCE All organisms depend on genetic diversity to cope with environmental change. Small viruses rely on frequent point mutations. This is harder for herpesviruses because they have larger genomes. Recombination provides another means of genetic optimization. Human herpesviruses often coinfect, and they show evidence of past recombination, but whether this is rare and incidental or functionally important is unknown. We showed that herpesviruses entering mice via the natural olfactory route meet reliably enough for recombination routinely to repair crippling mutations and restore normal viral loads. It appeared to occur in the first encountered olfactory cells and reflected a concentration of infection at the anterior olfactory edge. Thus, natural host entry incorporates a significant capacity for herpesvirus recombination.

HLA-B leader and survivorship after HLA-mismatched unrelated donor transplantation.

Hematopoietic cell transplantation (HCT) from HLA-mismatched unrelated donors can cure life-threatening blood disorders, but its success is limited by graft-versus-host disease (GVHD). HLA-B leaders encode methionine (M) or threonine (T) at position 2 and give rise to TT, MT, or MM genotypes. The dimorphic HLA-B leader informs GVHD risk in HLA-B-mismatched HCT. If the leader influences outcome in other HLA-mismatched transplant settings, the success of HCT could be improved for future patients. We determined leader genotypes for 10 415 patients receiving a transplant between 1988 and 2016 from unrelated donors with one HLA-A, HLA-B, HLA-C, HLA-DRB1, or HLA-DQB1 mismatch. Multivariate regression methods were used to evaluate risks associated with patient leader genotype according to the mismatched HLA locus and with HLA-A, HLA-B, HLA-C, HLA-DRB1, or HLA-DQB1 mismatching according to patient leader genotype. The impact of the patient leader genotype on acute GVHD and mortality varied across different mismatched HLA loci. Nonrelapse mortality was higher among HLA-DQB1-mismatched MM patients compared with HLA-DQB1-mismatched TT patients (hazard ratio, 1.35; P = .01). Grades III to IV GVHD risk was higher among HLA-DRB1-mismatched MM or MT patients compared with HLA-DRB1-mismatched TT patients (odds ratio, 2.52 and 1.51, respectively). Patients tolerated a single HLA-DQB1 mismatch better than mismatches at other loci. Outcome after HLA-mismatched transplantation depends on the HLA-B leader dimorphism and the mismatched HLA locus. The patient's leader variant provides new information on the limits of HLA mismatching. The success of HLA-mismatched unrelated transplantation might be enhanced through the judicious selection of mismatched donors for a patient's leader genotype.

Sexual harassment from patient to medical student: a cross-sectional survey.

BACKGROUND: There is little existing research investigating SH/SA specifically from patients to students. This study aims to assess the prevalence and impact of SH and SA from patient to medical student. METHODS: A cross-sectional survey study was administered via electronic email list to all current medical students at the University of Washington School of Medicine (n = 1183) over a two-week period in 2019. The survey questions addressed respondents' experiences with SH/SA from patients, frequency of reporting, and impact on feelings of burnout. RESULTS: Three hundred eleven responses were received for a response rate of 26%; 268 complete responses were included in the final analysis. Overall, 56% of respondents reported ever experiencing SH from a patient. SH from a patient was reported by significantly more of those who identify as female compared to male (66% vs 31%; p < .001). Similar frequency of experiencing SH within the last year were reported by females and males (90% vs 88%; p = .96). Clinical students were more likely to have ever experienced SH compared to preclinical students (61% vs 39%; p < .001). The majority (86%) of respondents who experienced SH/SA did not report it in an official capacity. Those who identify as female were more likely to report that SH from a patient contributed to feelings of burnout (21% vs 5% for male; p = .02). Behaviors consistent with SA were experienced by 16% of respondents, with similar frequency between females and males. CONCLUSIONS: This study demonstrates that patient to medical student SH/SA is a common occurrence, particularly among students identifying as female. It also highlights the significant impact of SH/SA incidents on feelings of burnout.

Agri-environment scheme nectar chemistry can suppress the social epidemiology of parasites in an important pollinator.

Emergent infectious diseases are one of the main drivers of species loss. Emergent infection with the microsporidian Nosema bombi has been implicated in the population and range declines of a suite of North American bumblebees, a group of important pollinators. Previous work has shown that phytochemicals found in pollen and nectar can negatively impact parasites in individuals, but how this relates to social epidemiology and by extension whether plants can be effectively used as pollinator disease management strategies remains unexplored. Here, we undertook a comprehensive screen of UK agri-environment scheme (AES) plants, a programme designed to benefit pollinators and wider biodiversity in agricultural settings, for phytochemicals in pollen and nectar using liquid chromatography and mass spectrometry. Caffeine, which occurs across a range of plant families, was identified in the nectar of sainfoin (Onobrychis viciifolia), a component of UK AES and a major global crop. We showed that caffeine significantly reduces N. bombi infection intensity, both prophylactically and therapeutically, in individual bumblebees (Bombus terrestris), and, for the first time, that such effects impact social epidemiology, with colonies reared from wild-caught queens having both lower prevalence and intensity of infection. Furthermore, infection prevalence was lower in foraging bumblebees from caffeine-treated colonies, suggesting a likely reduction in population-level transmission. Combined, these results show that N. bombi is less likely to be transmitted intracolonially when bumblebees consume naturally available caffeine, and that this may in turn reduce environmental prevalence. Consequently, our results demonstrate that floral phytochemicals at ecologically relevant concentrations can impact pollinator disease epidemiology and that planting strategies that increase floral abundance to support biodiversity could be co-opted as disease management tools.

A CD4+ T Cell-NK Cell Axis of Gammaherpesvirus Control.

CD4+ T cells are essential to control herpesviruses. Murid herpesvirus 4 (MuHV-4)-driven lung disease in CD4+ T-cell-deficient mice provides a well-studied example. Protective CD4+ T cells have been hypothesized to kill infected cells directly. However, removing major histocompatibility complex class II (MHCII) from LysM+ or CD11c+ cells increased MuHV-4 replication not in those cells but in type 1 alveolar epithelial cells, which lack MHCII, LysM, or CD11c. Disruption of MHCII in infected cells had no effect. Therefore, CD4+ T cells engaged uninfected presenting cells and protected indirectly. Mice lacking MHCII in LysM+ or CD11c+ cells maintained systemic antiviral CD4+ T cell responses, but recruited fewer CD4+ T cells into infected lungs. NK cell infiltration was also reduced, and NK cell depletion normalized infection between MHCII-deficient and control mice. Therefore, NK cell recruitment seemed to be an important component of CD4+ T-cell-dependent protection. Disruption of viral CD8+ T cell evasion made this defense redundant, suggesting that it is important mainly to control CD8-evasive pathogens.IMPORTANCE Gammaherpesviruses are widespread and cause cancers. CD4+ T cells are a key defense. We found that they defend indirectly, engaging uninfected presenting cells and recruiting innate immune cells to attack infected targets. This segregation of CD4+ T cells from immediate contact with infection helps the immune system to cope with viral evasion. Priming this defense by vaccination offers a way to protect against gammaherpesvirus-induced cancers.

Pollen sterols are associated with phylogeny and environment but not with pollinator guilds.

Phytosterols are primary plant metabolites that have fundamental structural and regulatory functions. They are also essential nutrients for phytophagous insects, including pollinators, that cannot synthesize sterols. Despite the well-described composition and diversity in vegetative plant tissues, few studies have examined phytosterol diversity in pollen. We quantified 25 pollen phytosterols in 122 plant species (105 genera, 51 families) to determine their composition and diversity across plant taxa. We searched literature and databases for plant phylogeny, environmental conditions, and pollinator guilds of the species to examine the relationships with pollen sterols. 24-methylenecholesterol, sitosterol and isofucosterol were the most common and abundant pollen sterols. We found phylogenetic clustering of twelve individual sterols, total sterol content and sterol diversity, and of sterol groupings that reflect their underlying biosynthesis pathway (C-24 alkylation, ring B desaturation). Plants originating in tropical-like climates (higher mean annual temperature, lower temperature seasonality, higher precipitation in wettest quarter) were more likely to record higher pollen sterol content. However, pollen sterol composition and content showed no clear relationship with pollinator guilds. Our study is the first to show that pollen sterol diversity is phylogenetically clustered and that pollen sterol content may adapt to environmental conditions.

Limited protection against γ-herpesvirus infection by replication-deficient virus particles.

The γ-herpesviruses have proved hard to vaccination against, with no convincing protection against long-term latent infection by recombinant viral subunits. In experimental settings, whole-virus vaccines have proved more effective, even when the vaccine virus itself establishes latent infection poorly. The main alternative is replication-deficient virus particles. Here high-dose, replication-deficient murid herpesvirus-4 only protected mice partially against wild-type infection. By contrast, latency-deficient but replication-competent vaccine protected mice strongly, even when delivered non-invasively to the olfactory epithelium. Thus, this approach seems to provide the best chance of a safe and effective γ-herpesvirus vaccine.