CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

Characterization of two in vivo challenge models to measure functional activity of monoclonal antibodies to Plasmodium falciparum circumsporozoite protein.

BACKGROUND: New strategies are needed to reduce the incidence of malaria, and promising approaches include the development of vaccines and monoclonal antibodies (mAbs) that target the circumsporozoite protein (CSP). To select the best candidates and speed development, it is essential to standardize preclinical assays to measure the potency of such interventions in animal models. METHODS: Two assay configurations were studied using transgenic Plasmodium berghei expressing Plasmodium falciparum full-length circumsporozoite protein. The assays measured (1) reduction in parasite infection of the liver (liver burden) following an intravenous (i.v) administration of sporozoites and (2) protection from parasitaemia following mosquito bite challenge. Two human CSP mAbs, AB311 and AB317, were compared for their ability to inhibit infection. Multiple independent experiments were conducted to define assay variability and resultant impact on the ability to discriminate differences in mAb functional activity. RESULTS: Overall, the assays produced highly consistent results in that all individual experiments showed greater functional activity for AB317 compared to AB311 as calculated by the dose required for 50% inhibition (ID50) as well as the serum concentration required for 50% inhibition (IC50). The data were then used to model experimental designs with adequate statistical power to rigorously screen, compare, and rank order novel anti-CSP mAbs. CONCLUSION: The results indicate that in vivo assays described here can provide reliable information for comparing the functional activity of mAbs. The results also provide guidance regarding selection of the appropriate experimental design, dose selection, and group sizes.

Optimization of an in vivo model to study immunity to Plasmodium falciparum pre-erythrocytic stages.

BACKGROUND: The circumsporozoite protein (CSP) of Plasmodium is a key surface antigen that induces antibodies and T-cells, conferring immune protection in animal models and humans. However, much of the work on CSP and immunity has been developed based on studies using rodent or non-human primate CSP antigens, which may not be entirely translatable to CSP expressed by human malaria parasites, especially considering the host specificity of the different species. METHODS: Using a genetically engineered strain of Plasmodium berghei that expresses luciferase, GFP and the Plasmodium falciparum orthologue of CSP, the effect of laboratory preparation, mosquito treatment and mouse factors on sporozoite infectivity was assessed using an in vivo bioluminescence assay on mice. This assay was compared with a PCR-based protection assay using an already described monoclonal antibody that can provide sterile protection against sporozoite challenge. RESULTS: Bioluminescence assay demonstrated similar detection levels of the quantity and kinetics of liver-stage infection, compared to PCR-based detection. This assay was used to evaluate treatment of sporozoite and delivery method on mouse infectivity, as well as the effects of age, sex and strain of mice. Finally, this assay was used to test the protective capacity of monoclonal antibody AB317; results strongly recapitulate the findings of previous work on this antibody. CONCLUSIONS: The PbGFP-Luc line and in vivo bioluminescence imaging provide highly sensitive read-outs of liver-stage infection in mice, and this method can be useful to reliably evaluate potency of pre-erythrocytic interventions.

Native Wolbachia infection and larval competition stress shape fitness and West Nile virus infection in Culex quinquefasciatus mosquitoes.

Introduction: Wolbachia transinfections established in key mosquito vectors, including Aedes aegypti are typically associated with pathogen blocking-reduced susceptibility to infection with key pathogens and reduced likelihood those pathogens are transmitted to new hosts. Host-symbiont-virus interactions are less well understood in mosquitoes like Culex quinquefasciatus, which naturally harbor Wolbachia, with pathogen blocking observed in some populations but not others, potentially due to innate differences in their Wolbachia load. In nature, mosquito larvae are often subject to developmental stresses associated with larval competition, which can lead to reduced body size and differential susceptibility to arbovirus infection. Methods: In this study, we sought to understand whether competition stress and Wolbachia infection in Cx. quinquefasciatus combine to impact host fitness and susceptibility to infection with West Nile virus. We reared Wolbachia-infected and uninfected Cx. quinquefasciatus larvae under three competition stress levels, increasing larval density without increasing the amount of food supplied. We then monitored larval development and survival, measured wing length and quantified Wolbachia density in adults, and then challenged mosquitoes from each treatment group orally with West Nile virus. Results and Discussion: We observed that high competition stress extended development time, decreased the likelihood of eclosion, decreased body size, and increased susceptibility to West Nile virus (WNV) infection. We also observed that Wolbachia infection reduced WNV load under low competition stress, and significantly improved the rate of survival for larval reared under higher competition stress. Consequently, our data suggest that native Wolbachia infection in Cx. quinquefasciatus has differential consequences for host fitness and susceptibility to WNV infection depending on competition stress.

Mosquito Communities Vary across Landscape and Vertical Strata in Indian River County, Florida.

Mosquito and arbovirus surveillance is essential to the protection of public health. A majority of surveys are undertaken at ground level. However, mosquitoes shelter, breed, and quest for hosts across vertical strata, thus limiting our ability to fully describe mosquito and arboviral communities. To elucidate patterns of mosquito vertical stratification, canopy traps were constructed to sample mosquitoes at heights of 1.5, 5.0, and 8.7 m across three different landscape types in a Florida coastal conservation area. We assessed trapping efforts using individual-based rarefaction and extrapolation. The effects of height, landscape, site location, and sampling date on mosquito community composition were parsed out using permutational ANOVA on a Hellinger-transformed Bray-Curtis dissimilarity abundance matrix. Lastly, a generalized linear mixed effects model (GLMM) was used to explore species-specific vertical patterns. We observed differences in sampling effort and community composition structure across various heights and landscapes. Our GLMM revealed significant effects of trap height for Aedes taeniorhynchus, Anopheles crucians, Anopheles quadrimaculatus, and Culex coronator, but not for Culex nigripalpus, the ultra-dominant species present in this area. Together these data provide evidence that height and landscape significantly affect mosquito community structures and highlight a need to develop sampling regimes to target specific vector and nuisance species at their preferred height and across different landscape types.