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International audience; The present study, conducted in the Galion Bay in Martinique, aims to highlight the temporal and seasonal variations of chlordecone contamination (an organochlorine pollutant) in the ambient environment (seawater) but also in the marine organisms in three main coastal marine habitats (mangroves, seagrass beds and coral reefs). To this end, two methodologies were used to measure and compare the chemical contamination of seawater during 13 months (spot samplings and POCIS technique). In parallel, concentrations of chlordecone and isotopic ratios (C and N) were carried out on marine organisms, collected during two contrasting climatic periods (dry and rainy), to evidence seasonal variations. The results showed that the contamination of seawater displayed significant variations over time and depended on environmental factors such as water flows, which imply dilution and dispersion phenomena. Concerning the marine organisms, the level of contamination varied considerably between the two seasons in seagrass beds with higher levels of contamination during the rainy season. Reef organisms were more moderately affected by this pollution, while mangrove organisms Environnemental Science and Pollution Research 2022 2 showed a high level of chlordecone whatever the season. Finally, isotope analyses highlighted that bioamplification along marine food-webs occurs at each season and each station.

Integrative passive samplers, such as DGT (Diffusive Gradients in Thin-films), are identified in European Technical Guidance Documents as promising tools to improve the quality of the assessment, in the context of the WFD (EU Water Framework Directive). However, DGT results cannot yet be used directly in a regulatory framework to assess the chemical status of water bodies, as DGT labile concentrations cannot be directly compared to the metal AA-EQSmarine water (Annual Average Environmental Quality Standard) established by the WFD, which are defined in the dissolved concentration. Therefore, prior to using DGT results in a regulatory context, for cadmium, nickel and lead, an adaptation of existing AA-EQSmarine water for DGTs should be pursued, ensuring at least the same level of protection. In this sense, in the framework of the MONITOOL project, a robust database of dissolved and labile metal concentrations in transitional and coastal waters, for adapting the existing AA-EQSmarine water for DGT technique, was obtained. Building on these results, this study proposes a methodology and provides values and equations for using DGT results for the chemical status assessment of marine waters, by adapting the EQSmarine water to adapted EQSDGT or predicting dissolved concentrations from DGT results. Based on available dataset, a first simulation of “chemical status” assessment per MONITOOL sampling site using DGT measured labile concentrations was carried out and the results were compared to an assessment based on dissolved concentration to check their compliance. These results demonstrate that the use of DGT passive samplers is appropriate for the metal concentrations level encountered in the marine environment. Further work is recommended to test the effectiveness of the methodology proposed in this study under WFD conditions on more sites and to establish common strategy guidelines for the use of DGT passive samplers in monitoring.

Dans le contexte de la directive cadre sur l’eau (DCE), nous avons mis en place une démonstration à l’échelle nationale afin d’identifier les atouts et les limites des échantillonneurs intégratifs passifs (EIP) pour la surveillance chimique réglementaire des eaux. Ainsi des déploiements de diffusive gradient in thin films (DGT), de polar organic chemical integrative samplers (Pocis) et de membranes silicone (SR) ont été réalisés sur 20 sites (une campagne), mais aussi en continu pendant un an sur trois sites (26 campagnes). En parallèle et pour comparaison, des prélèvements ponctuels d’eau ont été réalisés pour chaque campagne (lors des déploiements et récupérations des EIP). La pertinence mais aussi les limites de ces outils sont discutées au regard des aspects pratiques et des informations générées sur l’état de la contamination chimique des eaux. Les résultats confirment l’intérêt du recours à une surveillance chimique par EIP et démontrent leur applicabilité dans un contexte de réseaux de contrôle réglementaires. Hormis pour certains paramètres pour lesquels il existe des problèmes de contamination des outils, les EIP permettent un abaissement des limites de quantification et, principalement pour les Pocis et les SR, une fréquence de quantification augmentée, qui se traduit par la détection de davantage de molécules, et par des incertitudes diminuées sur les concentrations moyennes annuelles (MA). Enfin, l’évaluation de l’état chimique (comparaison des MA avec les normes de qualité environnementale (NQE)) à partir des MA déterminées par EIP est en cohérence avec celle déterminée à partir d’un échantillonnage ponctuel. Elle permet même dans certains cas de mieux qualifier un état alors que l’échantillonnage ponctuel ne le permet pas.

In order to move forward in the acceptance of a novel contaminant monitoring technique (Diffusive Gradients in Thin-films: DGT) for assessment of marine water bodies, sensu the WFD, an Inter-Laboratories Comparison (ILC) exercise (nine Europeans laboratories) was organized in the framework of the Interreg Atlantic Area MONITOOL project, which focused on the use of the DGT technique for the measurement of WFD priority metals (Cd, Ni and Pb). Reproducible results were obtained for each metal by several laboratories, supporting the assertion that DGT analysis can be performed satisfactorily by laboratories experienced in measuring metals at trace levels in marine environments, even if they have limited practice in DGT analysis. According to the Z-score analysis, among the 9 participating laboratories, 3 had 100 % of satisfactory results for Cd, Ni, and Pb, 3 had80 % satisfactory results and 2 had about 60 % satisfactory results. This work highlights the need to clearly describe the DGT method in order to control sources of contamination during analytical steps, in particular the resin gel retrieval and the elution steps. Such international intercomparison exercise is an important step to develop the laboratory network involved in DGT analysis and contributes to the improvement of data quality.

Molecular profile of Escherichia coli monitored over time by two-dimensional tandem mass spectrometry (2D MS/MS) coupled with a desorption electrospray ionization (DESI) source.

The current study evaluates the effect of seawater physico-chemical characteristics on the relationship between the concentration of metals measured by Diffusive Gradients in Thin films (DGT) passive samplers (i.e., DGT-labile concentration) and the concentrations measured in discrete water samples. Accordingly, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to measure the total dissolved metal concentrations in the discrete water samples and the labile metal concentrations obtained by DGT samplers; additionally, lead and cadmium conditional labile fractions were determined by Anodic Stripping Voltammetry (ASV) and total dissolved nickel was measured by Cathodic Stripping Voltammetry (CSV). It can be concluded that, in general, the median ratios of DGT/ICP and DGT/ASV(CSV) were lower than 1, except for Ni (median ratio close to 1) and Zn (higher than 1). This indicates the importance of speciation and time-integrated concentrations measured using passive sampling techniques, which is in line with the WFD suggestions for improving the chemical assessment of waterbodies. It is the variability in metal content in waters rather than environmental conditions to which the variability of the ratios can be attributed. The ratios were not significantly affected by the temperature, salinity, pH, oxygen, DOC or SPM, giving a great confidence for all the techniques used. Within a regulatory context such as the EU Water Framework Directive this is a great advantage, since the simplicity of not needing to use corrections to minimize the effects of environmental variables could help in implementing DGTs within monitoring networks.

Background: Despite extensive research to identify biomarkers of response in patients with non–muscle-invasive bladder cancer (NMIBC), there is no biomarker to date that can serve this purpose. Herein, we report how we leveraged serial urine samples to query a panel of cytokines at varying time points in an attempt to identify predictive biomarkers of response in NMIBC. Methods: Serial urine samples were collected from 50 patients with intermediate- or high-risk NMIBC enrolled in a phase II study, evaluating intravesical BCG ± intradermal HS-410 therapy. Samples were collected at baseline, week 7, week 13, week 28, and at end of treatment. A total of 105 cytokines were analyzed in each sample. To predict outcome of time-to-event (recurrence or progression), univariate and multivariable Cox analyses were performed. Results: Fifteen patients developed recurrence and 4 patients progressed during the follow-up period. Among clinicopathologic variables, ever-smoker versus nonsmoker status was associated with an improved response rate (HR 0.38; 95% confidence interval (CI), 0.14–0.99; P = 0.04). In the most clinically relevant model, the percent change (for 100 units) of IL18-binding protein-a (HR 1.995; 95% CI, 1.16–3.44; P = 0.01), IL23 (HR 1.12; 95% CI, 1.01–1.23; P = 0.03), IL8 (HR 0.27; 95% CI, 0.07–1.08; P = 0.06), and IFNγ-induced protein-10 (HR 0.95; 95% CI, 0.91–0.99; P = 0.04) at week 13 from baseline best predicted time to event. Conclusions: Urinary cytokines provided additional value to clinicopathologic features to predict response to immune-modulating agents in patients with NMIBC. Impact: This study serves as a hypothesis-generating report for future studies to evaluate the role of urine cytokines as a predictive biomarker of response to immune treatments.

This good practice guide is focused on the DGT devices, which are the most widely used passive sampler for metals, covering the following aspects: ■ Principle ■ Handling passive sampling devices for metals ■ Estimation of appropriate field deployment time ■ A Good Practice Guide for the Use of DGTs ■ Passive sampling device preparation and assembly ■ Selection of sampling site and safety precautions ■ Passive sampling device deployment and retrieval ■ Extraction of analytes from passive sampling devices ■ Analysis ■ Calculations

Although increased TIGIT expression on TILs is associated with poor survival in patients with cancer, monotherapy TIGIT antibody blockade has not yet demonstrated significant clinical activity. PD-1 is commonly co-expressed on TILs, and PD-1 mediated SHP-2 signaling inhibits DNAM-1 (CD226), preventing PVR co-stimulation in the setting of TIGIT blockade. The interaction between PD-1 and CD226 explains why TIGIT blockade only translates to clinical benefit in the setting of co-blockade of PD-1/L1, however does not explain why co-blockade of TIGIT and PD-1/L1 does not provide benefit in advanced and PD-1/L1 resistant tumors. In advanced and PD-1/L1 resistant tumors, progressive downregulation of CD226 has been reported. Thus, we hypothesized that the lack of CD226 may underlie the lack of clinical responses to combined TIGIT/PD-1/L1 blockade in PD-1/L1 experienced tumors and we sought to identify alternative costimulatory receptors with high-expression. Analysis of TCGA, as well as single-cell RNASeq from TILs, identified HVEM and LTβR as two costimulatory receptors with higher expression in advanced cancers as compared to CD226. HVEM and LTβR directly activate CD8+ T, natural killer (NK), and myeloid cells when bound by their TNF superfamily ligand, known as LIGHT. LIGHT potentiates effector lymphocyte function through signaling that obviates the co-stimulatory role of DNAM-1. We demonstrated that when the extracellular domain (ECD) of LIGHT is combined with the ECD of TIGIT on a TIGIT-Fc-LIGHT bi-functional fusion protein, the simultaneous blockade of PVR, PVRL2, PVRL3, and Nectin-4 and immune co-stimulation by LIGHT increased CD8+ T and NK infiltration into tumors. This translated into tumor cell killing, regression of established tumors, and improved survival in preclinical models of checkpoint primary and acquired resistance. The translation of TIGIT-Fc-LIGHT activity was evaluated in cynomolgus macaques and was well tolerated at doses up to 40 mg/kg. A series of adaptive immune and proinflammatory cytokines, including IL-2, CCL2, CCL4, IL-10, CXCL10, and CCL17, were induced within two hours of TIGIT-Fc-LIGHT infusion. Additionally, receptor engagement led to the rapid margination of HVEM+CD8+ T cells from the periphery into secondary immune tissues. This network of cytokines and post-dose immune cell margination identified in the monkey, was identical to findings in murine models that ultimately translated into significant anti-tumor responses. Lastly, the combination of TIGIT-Fc-LIGHT with anti-PD(L)1 broadened anti-tumor activity of the checkpoint antibodies in aggressive CPI-resistant tumors. These results suggest that LIGHT could be the differentiator that extends the clinical activity of conventional CPIs into PD-L1 low or CPI acquired resistance tumors. Citation Format: Kyung Jin Yoo, Louis Gonzalez, Kellsey Johannes, Jayalakshmi Miriyala, Karen Lenz, Kristen Campbell, Suresh de Silva, Taylor H. Schreiber, George J. Fromm. LIGHT (TNFSF14) costimulation with TIGIT blockade broadens the activity of checkpoint inhibitors (CPIs) into CPI refractory and resistant tumors through targeted myeloid cell and effector lymphocyte activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4214.

A primary mechanism of cancer immunotherapy resistance involves downregulation of specific antigens or major histocompatibility complex (MHC) based antigen presentation, which renders tumor cells invisible to αβ T cells, but not γδ T cells. Recently, a two-step model of γδ T cell activation has emerged, wherein one butyrophilin (BTN, i.e., BTN2A1) directly binds the γδ TCR but is only activated if certain molecular patterns (e.g., phosphoantigens) facilitate recruitment of a second BTN (i.e., BTN3A1) into a complex to form a BTN2A1/3A1 heterodimer. The BTN2A1/3A1 complex specifically activates the Vγ9Vδ2 T cells found in the peripheral blood in response to phosphoantigen build up in tumor cells. The unique mechanism of action and specificity of γδ TCR/BTN interactions suggests that therapeutic proteins comprising specific BTN heterodimers could be used to target specific γδ T cell populations to tumor cells which lack expression of high affinity neoantigens or MHC class I molecules. In this study, we generated bispecific γδ T cell engagers (GADLEN) containing heterodimeric BTN2A1 and BTN3A1 extracellular domains (ECD) fused via inert Fc linkers to scFv domains targeting a tumor-antigen (CD19 or CD33) to test their ability to modulate Vγ9Vδ2 T cells and to promote tumor cell killing in co-culture assays. GADLEN induced proliferation, degranulation and cytokine production including IFNγ and TNFα in Vγ9Vδ2 T cells but requires co-stimulation of a natural cytotoxicity receptor (via anti-NKG2D) or T-cell co-stimulatory receptor (via anti-CD28), highlighting the parallels of signal 1 and signal 2 requirements of TCR activation in αβ- and γδ- T cells. In Vγ9Vδ2 T cells and tumor co-culture assays, the addition of GADLEN alone enhanced killing of CD19+ lymphoma cells such as Daudi and Raji, expressing known ligands for CD28 (e.g., CD80 and CD86). Similarly, GADLEN enhanced Vγ9Vδ2 T cell-mediated killing of K562 cells engineered to express CD19, which express MICA/B (ligands for NKG2D) on the cell surface. In addition, the CD33-targeted GADLEN alone was able to specifically kill acute myeloid leukemia cells expressing CD33 and costimulatory ligands (e.g., CD80 and CD86) in a mechanism similar to the CD19-targeted GADLEN construct. These results highlight the ability of GADLENs in promoting targeted killing of tumor cells by providing the “active” heterodimeric BTN2A1 and BTN3A1 that is critical to enhance cytotoxic killing by Vγ9Vδ2 T cells. Furthermore, this study provides mechanistic insights into BTN-mediated activation of Vγ9Vδ2 T cells and offers proof of concept for using antigen-targeted butyrophilin heterodimeric fusion proteins for the treatment of cancer. Citation Format: Anne Lai, Arpita Patel, Faraha Brewer, Kinsley Evans, Kellsey Johannes, Louis Gonzalez, Kyung Jin Yoo, George Fromm, Keith Wilson, Taylor H. Schreiber, Suresh de Silva. Bispecific gamma delta T cell engagers containing butyrophilin 2A1/3A1 heterodimeric fusion protein efficiently activate Vg9Vd2 T cells and promote tumor cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3514.

This study investigates the relationships among Ni, Cd and Pb's different chemical forms determined by different methodologies in coastal and transitional waters across a broad geographical scale. Concentrations were measured in spot samples and through passive sampling (DGT). High variability of metal concentrations was found among sampling sites and methodologies due to natural water fluctuations rather than to a given metal or method. Total dissolved metal concentrations in spot samples were lower than the EQS-WFD values. The labile fractions of Cd and Pb, measured in spot samples by Anodic Stripping Voltammetry and by DGT-ICPMS, were highly correlated. Similar labilities were found for Cd, while for Pb, the ASV labile fraction was ≈50% lower. These results reflect the pool of mobile and labile species available towards each technique kinetic window, and they seem not to be affected by discrete sampling flaws.

Background A primary mechanism of cancer immunotherapy resistance involves downregulation of specific antigens or major histocompatibility complex based antigen presentation, which renders tumor cells invisible to alpha-beta T cells, but not gamma-delta T cells. Recently, a two-step model of gamma-delta T cell activation has emerged, wherein one butyrophilin (BTN, ie. BTN2A1) directly binds the gamma-delta TCR but is only activated if certain molecular patterns (eg. phosphoantigens) facilitate recruitment of a second BTN (ie. BTN3A1) into a complex to form a BTN2A1/3A1 heterodimer. The BTN2A1/3A1 complex specifically activates the predominant gamma-delta T cell population in the peripheral blood, comprising the Vg9d2 T cell receptor (TCR), but does not activate the primary gamma-delta T cell population in mucosal tissues, comprising the Vg4 TCR. The unique mechanism of action and specificity of gamma-delta TCR/BTN interactions suggests that therapeutic proteins comprising specific BTN heterodimers could be used to target specific gamma-delta T cell populations, with a lower risk of off-target activation common with CD3-directed T cell engagers. Methods Human BTN2A1/3A1-Fc-CD19scFv and mouse BTNL1/6-Fc-CD19scFv heterodimeric fusion proteins were purified and binding to CD19 or the respective gamma-delta TCRs was assessed by ELISA, Octet and flow cytometry using gd T-cells isolated from human peripheral blood and mouse intestinal tissue. The functionality of the constructs to activate gamma-delta T cells and mediate killing of tumor cells was assessed using live cell imaging in vitro as well as a murine B-cell lymphoma model in vivo. Results The CD19-targeting scFv domains of the BTN heterodimer fusion proteins bound to human and mouse CD19 with low nanomolar affinity. The BTN2A1/3A1-Fc-CD19scFv compound specifically bound to the Vg9d2 TCR on human gd T cells while the mouse BTNL1/6-Fc-CD19scFv bound to Vg7d4 TCR on mouse gd T cells. Both compounds were able to activate gd T cells in a co-culture assay resulting in degranulation and increased surface expression of CD107a and also increased apoptosis of CD19+ tumor cells. Intraperitoneal administration of the mouse BTNL1/6-Fc-CD19scFv led to anti-tumor effects in A20 tumor bearing BALB/c mice. Phenotyping from BTNL1/6-Fc-CD19scFv treated mice revealed profound and rapid expansion of the endogenous gamma-delta T cells in the circulation and tumor, with concomitant depletion of peripheral CD19+ B-cells, confirming the mechanism of action of the heterodimer as a gamma-delta T cell specific engager. Conclusions These results provide proof of mechanism for in vivo manipulation of gamma-delta T cells using antigen-targeted butyrophilin heterodimeric fusion proteins for the treatment of cancer.

We detected OH 18 cm main line emission from 5 of 7 positions in the intermediate-velocity molecular cloud (IVMC) G211+63. These are the first detections of hydroxyl in IVMCs. Our OH detections at AV levels ranging from 0.24 to 0.27 mag reinforce the notion that these lines can efficiently trace diffuse, low-extinction molecular gas. N(OH) is derived for a range of excitation temperatures (5–30 K) and likely falls between 1 and 16 × 1013cm−2⁠. The corresponding N(H2) values are consistent both with estimates derived from E(B − V) and CO data. Estimates of the cloud mass from the OH data range from 26 to 820 M⊙ for distances from 100 to 400 pc and Tex from 5 to 10 K. These values are 3–20 times less than the virial mass of the cloud. It is likely IVMCs resemble high-latitude clouds in this respect. If so, they are transient structures breaking up on time-scales of ∼106yr⁠.

The presence of ten metals (Cd, Ni, Pb, Cr, Cu, Zn, Al, Fe, Mn, and Co) was investigated in the final discharge of six facilities, including four wastewater treatment plants, which were continuously discharging treated wastewater to the coastal environment in Gran Canaria Island. A four-day sampling campaign was carried out at each facility in July 2020, in which both the spot samplings technique and the diffusive gradient in thin-film technique (DGT) were carried out to measure total dissolved metals and the in situ labile metal fraction, respectively. After the necessary sample preparation steps, measurements were carried out by ICP-MS for both samplings. Raw data referred to the spot total dissolved and DGT-labile metal concentrations were reported. In general, the average metal concentrations were dispersed in a broad range. As expected, the highest metal contents were found in those facilities with larger industrial contributions. The values of annual average environmental quality standards (AA-EQS) were used to assess the total dissolved metal concentrations for every metal in every final discharge. In only one of the studied facilities, some metals (Ni and Zn) exceeded these EQS within the receiving waterbody, highlighting the need for more efficient treatment targeted towards a specific discharging-water quality. In addition, the total dissolved and labile metal daily fluxes of discharge were calculated to estimate the contribution of every effluent to the receiving water bodies.

BackgroundOne of the pharmacodynamic measures of biologic compounds is an assessment of target receptor/ligand occupancy (RO). If the primary mechanism for drug clearance is through target binding, drug exposure may increase once RO is saturated. SL-172154 (SIRPα-Fc-CD40L) and SL-279252 (PD1-Fc-OX40L) are two bi-functional fusion proteins in phase I clinical trials (NCT04406623 and NCT03894618). The binding interaction between a biologic compound and its targets (CD47 or PD-L1) typically does not stimulate migration from the blood, allowing direct measurement of drug and target. Evaluation of RO for immune agonists (CD40 and OX40) is challenging because agonists can stimulate lymphocytes to rapidly extravasate from the peripheral blood following infusion, thus precluding direct RO measurement as target cells are no longer present in the blood (figure 1a).MethodsTo assess full receptor engagement of CD40 or OX40, the formula shown in figure 1b was derived. The formula captures cells that rapidly migrated from the blood, combined with those that remained in the blood within 2 hours post infusion using multiparameter FACS analysis. SAS JMP was used to calculate and visualize all parameters.ResultsWithin 2 hours of SL-172154 infusion at 1 mg/kg (n=3), CD40+ B cell counts decreased from a pre-dose average by 87%. Of the CD40+ B cells remaining in the blood, ~95% were bound with SL-172154. CD40+ B cell counts recovered in the blood by the next dose, and as counts increased so did the proportion of cells that were bound with SL-172154. Similarly, within 2 hours of SL-279252 infusion at 1 mg/kg (n=10), CD4+OX40+ T cell counts decreased from a pre-dose average by 41.5% (range 0 – 70%). Of the CD4+OX40+ cells remaining in the blood, ~32% were bound with SL-279252. CD4+OX40+ cells returned to pre-treatment numbers over a 7-day interval and nearly all cells remained bound with SL-279252. Taken together, these data demonstrate that a large proportion of CD40+ (SL-172154) or OX40+ (SL-279252) cells bind drug immediately post infusion, migrate from the blood, and slowly return to the blood with drug still bound to the cell surface.Abstract 3 Figure 1CD4+ OX40+ T cells and SL-279252ConclusionsAdministration of SL-172154 (CD40) and SL-279252 (OX40) stimulated rapid egress of target cells from the blood. An integrated assessment, termed ‘receptor engagement’, was developed to derive RO both on circulating cells and those that rapidly marginated. When CD40+ or OX40+ cells returned to the blood, they remained drug-bound, indicating that the compounds may piggy-back on target cells into tissues.AcknowledgementsThanks are extended to study participants; Takeda Pharmaceutical Company, Boston, MA, United States; Cathrine Leonowens, PhD, Nuventra Pharma Sciences, Durham, NC, United States and Cadence Communications and Research, Thousand Oaks, CA, United States. This study is funded by Shattuck Labs, Inc. Austin, TX and Durham, NC, USAEthics ApprovalThis study is being conducted in full conformity with the Declaration of Helsinki and was approved by all IRBs/ethics committees from each clinical site participating in the study. Specific approval numbers can be provided upon request.

BackgroundSIRPα-Fc-CD40L is a hexameric, bi-functional fusion protein consisting of SIRPα (binding affinity to CD47 is 0.628 nM) linked to CD40L (binding affinity to CD40 is 4.74 nM) through an Fc linker protein.1 By augmenting antigen processing and promoting antigen presenting cell (APC) maturation, this molecule is designed to bridge innate and adaptive immunity, enhancing tumor cell phagocytosis and antigen cross-presentation to CD8 T cells.MethodsThe first-in-human, Phase 1 dose escalation study is evaluating SL-172154 as monotherapy in patients (pts) with platinum resistant ovarian, fallopian tube and primary peritoneal cancers. Objectives include evaluation of safety, dose-limiting toxicity (DLT) and recommended phase 2 dose (RP2D), pharmacokinetic (PK) parameters, pharmacodynamic (PD) effects and antitumor activity based on RECIST.ResultsAs of 6 July 2021, 14 heavily pretreated pts (median age, 67 years) were enrolled and treated with intravenous (IV) administration of SL-172154 across 4 dose levels on 2 schedules: schedule 1 (day 1, 8, 15, 29, Q2 weeks) at 0.1, 0.3 mg/kg and schedule 2 (weekly) at 0.3, 1.0, 3.0 mg/kg. The most common treatment-related (>20%) adverse events (AEs) of any grade (G) were fatigue (n=7, 50%), infusion-related reactions (IRR) (n=6, 43%), nausea (n=4, 29%), and decreased appetite (n=3, 21%). Treatment-related IRRs (G1/G2) generally occurred near the end of infusion or immediately post-infusion; the full dose was able to be delivered in each IRR event, and subsequent infusions in patients having IRRs were managed with pre-medications. No treatment related ≥G3 AEs or DLTs have occurred. CD47 receptor occupancy (RO) on leukocytes approached 90% at 1.0 and 3.0 mg/kg. Minimal binding to CD47+ red blood cells was observed at all dose levels. CD40 RO on B cells was >60% at doses ≥0.1 mg/kg and 75%–100% at 1.0 and 3.0 mg/kg. Rapid, transient B cell and monocyte margination was observed following infusion of SL-172154 and was consistent with dose-dependent increases in IL-12, MCP-1, MIP-1β, MIP-1α, and MDC. No appreciable increases in IL-6 or TNFα were noted and there was no correlation between IRRs and cytokine increases. Among 12 evaluable pts, the best response was stable disease in 3 pts.ConclusionsSL-172154 has been well tolerated with no evidence of anemia, thrombocytopenia, liver dysfunction or cytokine release syndrome. A unique serum cytokine signature consistent with CD40 RO and activation has been observed and this signature is maintained following repeat dosing. Dose escalation is ongoing.AcknowledgementsThanks are extended to study participants; Cathrine Leonowens, PhD, Nuventra Pharma Sciences, Durham, NC, United States and Cadence Communications and Research, Thousand Oaks, CA, United States. This study is funded by Shattuck Labs, Inc. Austin, TX and Durham, NC, United States.Trial RegistrationNCT04406623Referencesde Silva S, Fromm G, Shuptrine CW, Johannes K, Patel A, Yoo KJ, et al. CD40 enhances type I interferon responses downstream of CD47 blockade, bridging innate and adaptive immunity. Cancer Immunol Res 2020; 8: 230–245.Ethics ApprovalThis study is being conducted in full conformity with the Declaration of Helsinki and was approved by all IRBs/ethics committees from each clinical site participating in the study. Specific approval numbers can be provided upon request.

International audience; High abundances of juvenile fish in certain ports suggest they might provide alternative nursery habitats for several species. To further investigate this possibility, post-settlement growth, metal uptake and body condition were estimated in 127 juveniles of two seabream species, collected in 2014-15, inside and outside the highly polluted ports of the Bay of Toulon. This showed that differences in local pollution levels (here in Hg, Cu, Pb and Zn) are not consistently mirrored within fish flesh. Muscle metal concentrations, below sanitary thresholds for both species, were higher in ports for Cu, Pb and V only. Otherwise, fish muscle composition principally differed by species or by year. Juvenile growth and condition were equivalent at all sites. Higher prey abundance in certain ports might therefore compensate the deleterious effects of pollution, resulting in similar sizes and body conditions for departing juvenile fish than in nearby natural habitats.

Combination antiretroviral therapies (cART) can lead to normal life expectancy in HIV-infected persons, and people aged >50 yrs represent the fastest growing HIV group. Although HIV and aging are independently associated with impaired humoral immunity, immune status in people aging with HIV is relatively unexplored. In this study influenza vaccination was used to probe age associated perturbations in the B cell compartment of HIV-negative “healthy controls” (HC) and virologically controlled HIV-infected participants on cART (HIV) (n=124), grouped by age as young (

HIV-infected patients of all ages frequently underperform in response to seasonal influenza vaccination, despite virologic control of HIV. The molecular mechanisms governing this impairment, as well as predictive biomarkers for responsiveness, remain unknown. This study was performed in samples obtained prevaccination (T0) from HIV-infected children who received the 2012–2013 seasonal influenza vaccine. Response status was determined based on established criterion for hemagglutination inhibition titer; participants with a hemagglutination titer ≥1:40 plus a ≥4-fold increase over T0 at 3 wk postvaccination were designated as responders. All children had a history of prior influenza vaccinations. At T0, the frequencies of CD4 T cell subsets, including peripheral T follicular helper (pTfh) cells, which provide help to B cells for developing into Ab-secreting cells, were similar between responders and nonresponders. However, in response to in vitro stimulation with influenza A/California/7/2009 (H1N1) Ag, differential gene expression related to pTfh cell function was observed by Fluidigm high-density RT-PCR between responders and nonresponders. In responders, H1N1 stimulation at T0 also resulted in CXCR5 induction (mRNA and protein) in CD4 T cells and IL21 gene induction in pTfh cells that were strongly associated with H1N1-specific B cell responses postvaccination. In contrast, CD4 T cells of nonresponders exhibited increased expression of IL2 and STAT5 genes, which are known to antagonize peripheral Tfh cell function. These results suggest that the quality of pTfh cells at the time of immunization is important for influenza vaccine responses and provide a rationale for targeted, ex vivo Ag-driven molecular profiling of purified immune cells to detect predictive biomarkers of the vaccine response.

In highly dynamic systems, such as transitional and coastal waters, establishing their chemical status is challenging. MONITOOL is an exciting European project consisting of 16 Partners covering the Atlantic region from the Canary Islands to the Scottish Highlands and Islands, which aims to address this complex analytical challenge, responding to European Directive demands for the assessment of the chemical status of transitional and coastal waters. Diffusive Gradient in Thin Films (DGT), and passive samplers (PS), in general, are already widely used in investigative monitoring and there is an increasing interest in their use for the environmental assessment of water bodies, within European policies requirements. The main barrier hindering the regulatory acceptance of PS for compliance checking is the lack of appropriate Environmental Quality Standards (EQS). EQSs for metals are defined in the dissolved fraction, preventing the use of DGT-labile concentrations for the establishment of the chemical status of water bodies. The first sampling campaigns were performed during winter 2017/2018 in 4 selected sites (transitional and coastal sites) in each consortium region (8 regions). All partners followed the same protocol for sampling and analysis to minimize the operational variability. Priority metals (Cd, Ni, Pb) and other specific metals (Al, Ag, Cu, Cr, Co, Fe, Mn, Zn) were analysed in waters and in the DGT resins. Statistical analysis is being applied to study relationships between metal concentrations in DGT and in grab water samples. Suitable EQS for DGTs will be calculated on basis the statistical relations obtained previously. This will permit a better implementation of the Water Framework Directive in variable systems like transitional and coastal waters. The work presented here shows initial DGT results from the Irish sampling sites for selected target metals.

Approximately 20-40% of cancer patients who initially benefit from PD-1/L1 checkpoint blockade later develop progressive disease, which is associated with genetic and/or phenotypic changes in tumor cells that enable acquired resistance. Tumors from patients who have developed acquired resistance to checkpoint blockade (CPI-AR) typically demonstrate downregulation of specific antigens or proteins involved in antigen presentation on MHC class I, and dysregulation of interferon response pathways. With increasing CPI use across a large number of tumor types, the proportion of cancer patients with CPI-AR is increasing. Pre-clinical tumor models which mimic CPI-AR in humans are needed. We generated in vivo CPI-AR tumor models by serially passaging common CPI responsive murine syngeneic tumor cell lines (i.e. CT26) in vivo, followed by the excision and ex vivo expansion of the tumors that failed to respond to anti-PD1. This was repeated until passaged tumors no longer responded to anti-PD1 therapy. Transcriptomes of CT26 parental, CT26/CPI-AR, and B16.F10 melanoma (considered CPI primary resistant, CPI-PR) tumors were sequenced either under normal culture conditions or following 24 hour exposure to IFNg to assess interferon responsiveness. Here we present the genomic characterization of the CPI -PR and -AR models. Paradoxically, CPI-AR tumors maintain a state of type I/II IFN gene hyperactivation and increased expression of genes involved in MHC class I mediated antigen presentation/processing. Despite this transcriptional hyperactivity, CPI-AR tumor cells have a decreased capacity to translate and traffic these associated proteins to the cell membrane. Interestingly, upon challenge with IFNg, CPI-AR tumors down-regulate gene expression for PD-L1, TAP1, TAP2, β2M and other key pathways typically up-regulated by IFN stimulation. Collectively, the CPI-AR and -PR tumor models reflect many of the genomic and phenotypic changes reported in CPI-AR cancer patients. The CT26/CPI-AR tumors reproduce a state of IFN hypo-responsiveness and genetic/protein dysregulation. These clinically relevant models can be used to screen therapeutics that may translate to meaningful benefit for CPI-AR cancer patients. Citation Format: George J. Fromm, Danish Memon, Suresh de Silva, Kyung Jin Yoo, Kellsey Johannes, Casey Shuptrine, Jaya Miriyala, Arpita Patel, Fatima Rangwala, Zachary Opheim, Thuy-Ai Nguyen, Louis Gonzalez, Fatima Rangwala, Taylor H. Schreiber, Matthew D. Hellmann, Martin L. Miller, Taylor H. Schreiber. The development of an in vivo model of checkpoint acquired resistance, reveals a program of interferon hyperstimulation, resulting in dysregulation of MHC class I, protein translation/trafficking, and other unique pathways, that may be useful for guiding clinical strategy in patients with phenotypic similarities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1697.

A major mechanism of acquired resistance to immune checkpoint inhibition involves downregulation of antigen presentation, including the MHC I complex itself. Downregulation of antigen presentation on MHC I can render tumor cells invisible to any α/β T cell directed therapy. γδ T cells (γδT) are a small subset of the overall T cell compartment, but are characterized by increased cytolytic capacity relative to α/β T cells. Rather than MHC I, γδT recognize target cells via a complex of heterodimerized butyrophilin (BTN) proteins. Thus, display of BTN heterodimers on the surface of tumor cells may enhance immunity to tumors that have downregulated MHC I, or which express low abundance or low affinity antigens. We have previously reported the generation of a heterodimeric BTN protein targeting the CD19 antigen, referred to as BTN2A1/3A1-Fc-CD19scFv. While the BTN2A1/3A1 complex is a potent activator of γδT expressing the Vγ9δ2 T cell receptor (TCR), which is the major γδT population in human peripheral blood, Vγ9δ2 T cells are not the predominant γδT in many tissues. The murine equivalent of BTN2A1/3A1-Fc-CD19scFv, BTNL1/6-Fc-CD19scFv, stimulated specific proliferation and increased the cytolytic capacity of peripheral blood γδT in mice, but not other tissue-restricted GDT populations. These data suggested that distinct BTN heterodimers may preferentially activate tissue-restricted subsets of GDT. To characterize potential differences between peripheral blood and tissue-restricted γδT , we performed a multi-layered analysis, including single-cell sequencing of γδ TCR from paired peripheral blood and tumor tissues from human cancer patients such as melanoma, prostate and colon cancer. These data identified tissue-specific preferences for individual γδ TCRs, with corresponding tissue-specific preferences for individual BTN proteins. Based on this information, we generated a panel of distinct heterodimeric BTN proteins, and show that specific γδT populations are preferentially activated by specific BTN heterodimers in a lock-and-key fashion. These data are a necessary pre-requisite for designing γδT specific therapeutics that may target both immune neglected and acquired resistant tumors that have limited visibility to α/β T cell directed approaches. Citation Format: Suresh de Silva, George Fromm, Anne Lai, Louis Gonzalez, Arpita Patel, Kyung Jin Yoo, Kellsey Johannes, Kinsley Evans, Keith Wilson, Taylor H. Schreiber. Antigen-specific targeting of tissue-resident gamma delta T cells with recombinant butyrophilin heterodimeric fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1736.

International audience; The study presents the efforts on including the polarimetric data to the routine inversion of the radiometric ground-based measurements for characterization of the atmospheric aerosols and analysis of the obtained advantages in retrieval results. First, to operationally process the large amount of polarimetric data the data preparation tool was developed. The AERONET inversion code adapted for inversion of both intensity and polarization measurements was used for processing. Second, in order to estimate the effect from utilization of polarimetric information on aerosol retrieval results, both synthetic data and the real measurements were processed using developed routine and analyzed. The sensitivity study has been carried out using simulated data based on three main aerosol models: desert dust, urban industrial and urban clean aerosols. The test investigated the effects of utilization of polarization data in the presence of random noise, bias in measurements of optical thickness and angular pointing shift. The results demonstrate the advantage of polarization data utilization in the cases of aerosols with pronounced concentration of fine particles. Further, the extended set of AERONET observations was processed. The data for three sites have been used: GSFC, USA (clean urban aerosol dominated by fine particles), Beijing, China (polluted industrial aerosol characterized by pronounced mixture of both fine and coarse modes) and Dakar, Senegal (desert dust dominated by coarse particles). The results revealed considerable advantage of polarimetric data applying for characterizing fine mode dominated aerosols including industrial pollution (Beijing). The use of polarization corrects particle size distribution by decreasing overestimated fine mode and increasing the coarse mode. It also increases underestimated real part of the refractive index and improves the retrieval of the fraction of spherical particles due to high sensitivity of polarization to particle shape. Overall, the study demonstrates a substantial value of polarimetric data for improving aerosol characterization.

The authors wish to make the following corrections to this paper [...]

International audience; The ADAM (A Surface Reflectance Database for ESA's Earth Observation Missions) product (a climatological database coupled to its companion calculation toolkit) enables users to simulate realistic hyperspectral and directional global Earth surface reflectances (i.e., top-of-canopy/bottom-of-atmosphere) over the 240-4000 nm spectral range (at 1-nm resolution) and in any illumination/observation geometry, at 0.1° x 0.1° spatial resolution for a typical year. ADAM aims to support the preparation of optical Earth observation missions as well as the design of operational processing chains for the retrieval of atmospheric parameters by characterizing the expected surface reflectance, accounting for its anisotropy. Firstly, we describe (1) the methods used in the development of the gridded monthly ADAM climatologies (over land surfaces: monthly means of normalized reflectances derived from MODIS observations in seven spectral bands for the year 2005; over oceans: monthly means over the 1999-2009 period of chlorophyll content from SeaWiFS and of wind speed from SeaWinds), and (2) the underlying modeling approaches of ADAM toolkit to simulate the spectro-directional variations of the reflectance depending on the assigned surface type. Secondly, we evaluate ADAM simulation performances over land surfaces. A comparison against POLDER multi-spectral/multi-directional measurements for year 2008 shows reliable simulation results with root mean square differences below 0.027 and R2 values above 0.9 for most of the 14 land cover IGBP classes investigated, with no significant bias identified. Only for the "Snow and ice" class is the performance lower pointing to a limitation of climatological data to represent actual snow properties. An evaluation of the modeled reflectance in the specific backscatter direction against CALIPSO data reveals that ADAM tends to overestimate (underestimate) the so-called "hot-spot" by a factor of about 1.5 (1.5 to 2) for barren (vegetated) surfaces.

International audience; The construction of ports has caused substantial habitat destruction in coastal areas previously used as nursery grounds by many fish species, with consequences to fish stocks. These artificial coastal areas might provide alternative nursery habitats for several species for juvenile fish abundances and growth in ports, although their contribution to adult stocks had never been estimated. The variability of otolith composition in the juveniles of two Diplodus species was investigated in three contrasting port areas and two adjacent coastal juvenile habitats of the Bay of Toulon (northwestern Mediterranean) in order to determine the possible use of otolith fingerprints as natural tags for the identification of juvenile fishes in ports. The global accuracy of discrimination between ports and coastal areas was very high (94%) irrespective of species, suggesting that otolith fingerprints can be used with confidence to retrospectively identify past residency in the ports of this bay. However, Ba was systematically the most discriminating element, since its concentrations in otoliths were generally higher outside ports than in inside them, probably due to river runoff. Moreover, otolith signatures varied greatly by species and between sampling sites. Furthermore, although Cu and Pb concentrations in water were at least 2.3–34-fold higher inside ports than outside, this was not consistently reflected in fish otoliths, confirming that spatial differences in otolith concentrations depend on the species and do not directly reflect differences in environmental contamination levels. Therefore, it seems unlikely that otolith microchemistry could provide a universal fingerprint capable of discriminating ports from other coastal areas. Nevertheless, the contribution of ports to adult fish populations can be determined well by establishing a library of otolith fingerprints for all juvenile habitats.

HIV infection changes the lymph node (LN) tissue architecture, potentially impairing the immunologic response to antigenic challenge. The tissue-resident immune cell dynamics in virologically suppressed HIV+ patients on combination antiretroviral therapy (cART) are not clear. We obtained LN biopsies before and 10 to 14 days after trivalent seasonal influenza immunization from healthy controls (HCs) and HIV+ volunteers on cART to investigate CD4+ T follicular helper (Tfh) and B cell dynamics by flow cytometry and quantitative imaging analysis. Prior to vaccination, compared with those in HCs, HIV+ LNs exhibited an altered follicular architecture, but harbored higher numbers of Tfh cells and increased IgG+ follicular memory B cells. Moreover, Tfh cell numbers were dependent upon preservation of the follicular dendritic cell (FDC) network and were predictive of the magnitude of the vaccine-induced IgG responses. Interestingly, postvaccination LN samples in HIV+ participants had significantly (P = 0.0179) reduced Tfh cell numbers compared with prevaccination samples, without evidence for peripheral Tfh (pTfh) cell reduction. We conclude that influenza vaccination alters the cellularity of draining LNs of HIV+ persons in conjunction with development of antigen-specific humoral responses. The underlying mechanism of Tfh cell decline warrants further investigation, as it could bear implications for the rational design of HIV vaccines.

International audience; The Meteosat Second Generation (MSG) geostationary platform equipped with the Spinning Enhanced Visible and Infrared Imager (SEVIRI) instrument provides observations of the Earth every 15 min since 2004. Based on those measurements, we present a new method called North African Sandstorm Survey (NASCube) to: (i) generate day/night remote sensing images in order to detect sandstorms over the Sahara and Saudi Arabia; and (ii) estimate day and night aerosol optical depth (AOD). This paper presents a method to create true color day and night images from the SEVIRI instrument level 1.5 products and the complete operational data processing system to detect sandstorms and quantify the AOD over the desert areas of North Africa and Saudi Arabia. The designed retrieval algorithms are essentially based on the use of artificial neural networks (ANN), which seems to be well suited to this issue. Our methods are validated against two different datasets, namely the Deep Blue NASA moderate-resolution imaging spectroradiometer (MODIS) product and AErosol RObotic NETwork (AERONET) acquisitions located in desert areas. It is shown that NASCube products deliver better estimations for high AOD (>0.2) over land areas than Deep Blue products. The open-public web platform will help researchers to identify, quantify and retrieve the impact of sandstorms over desert regions.

Vaccine-induced protection against infection by HIV or highly pathogenic and virulent SIV strains has been limited. In a proof-of-concept study, we show that a novel vaccine approach significantly protects rhesus macaques from mucosal infection by the highly pathogenic strain SIVmac251. We vaccinated three cohorts of 12 macaques each with live, irradiated vaccine cells secreting the modified endoplasmic reticulum chaperone gp96-Ig. Cohort 1 was vaccinated with cells secreting gp96SIVIg carrying SIV peptides. In addition, Cohort 2 received recombinant envelope protein SIV-gp120. Cohort 3 was injected with cells secreting gp96-Ig (no SIV Ags) vaccines. Cohort 2 was protected from infection. After seven rectal challenges with highly pathogenic SIVmac251, the hazard ratio was 0.27, corresponding to a highly significant, 73% reduced risk for viral acquisition. The apparent success of the novel vaccine modality recommends further study.

The implementation of the Water Framework Directive (WFD) requires the monitoring of micropollutants in the aquatic environments to prevent any damages to both human health and ecosystems. In this context, a number of organic and inorganic compounds have been selected as priority pollutants and their measurement is necessary to ensure that water-quality standards are maintained (EC, 2008; EC, 2013; EC, 2015). For two decades, passive sampling methods have been developed for the monitoring of organic and inorganic compounds. Passive samplers allow measuring these compounds at trace levels by accumulation and concentration over long-term exposure. Moreover, the use of integrative passive samplers (IPS) allows a better representativeness of measurements because it takes into account the episodic pollution (Miège et al., 2015). Such passive sampling techniques have been recommended in the European Commission Guidance Document on surface water chemical monitoring, as complementary methods to improve the level of confidence in water monitoring data in comparison with conventional spot sampling (EC, 2009). In the context of the WFD monitoring programs, French government has mobilized the expertise of AQUAREF for two main objectives: - Demonstrate, in-situ, the interest to deploy IPS for monitoring of priority substances in aquatic environments; - Diffuse protocols and guidelines on the use and analyses of IPS, and initiate the formation of future stakeholders. We present the in situ campaigns to demonstrate theIPS relevance. Conventional IPS will be deployed: POCIS (Polar Organic Chemical Sampler), silicone membrane and DGT (Diffusive Gradients in Thin films). In this poster, we will focus on micropollutants needed to be controlled in the water matrix (the more hydrophilic ones), not in biota1. In a first round, the contamination by around 50 micropollutants on 3 sites will be measured every 2weeks, continuously during one year. In a second round, the contamination by around 100 micropollutants on 20 sites spread throughout France will be studied during a 2 weeks long campaign. Results obtained with IPS will be compared with spot water samples. This large scale study should highlight the interest to use IPS for WFD monitoring program by increasing the frequency of quantification and obtaining a better temporal representativeness of the contamination. In this context, the formation of stakeholders is also a strategic challenge; we present the current gaps and our actions on this aspect at a national scale.

International audience; The MODIS instruments have been flying onboard the Terra and Aqua platforms and have acquired Earth observation data since early 2000 and mid 2002, respectively. After atmospheric correction, the collected data allows the monitoring of the land cover dynamics. Here, we describe a data processing scheme to generate Earth reflectance and emissivity time series at a sub-kilometer spatial resolution and with a period of 8 days. The data processing scheme removes residual cloud and aerosol contamination in the MODIS products, applies directional correction, and fills the gaps resulting from persistent cloud cover. The resulting database, referred to FondsDeSol, offers a significant improvement with respect to the first version proposed in (Gonzalez et al., 2010), and covers a period of ten years against only one year for the first version. The first motivation of the database is to improve the estimation of at sensor radiances for the design of future sensor in the optical domain. Nevertheless, such database opens the way to new research topics like land surface dynamics, land cover changes, and inter-annual variations due to climate perturbations.; Les instruments MODIS volent sur les plateformes TERRA et AQUA et acquièrent des données d'observation de la Terre depuis début 2000 et mi-2002 respectivement. Après correction atmosphérique, les données collectées permettent le suivi de la dynamique de l'occupation des sols. Dans ce papier, nous décrivons le traitement des données permettant la génération de séries temporelles de réflectances et d'émissivités de la surface à une résolution spatiale sub-kilométrique et avec une période de 8 jours. Au cours du traitement des données, les nuages et la contamination par les aérosols résiduels sont éliminés des produits MODIS, une correction directionnelle est appliquée et les trous résultants d'une couverture nuageuse persistante sont comblés. La base de données FondsDeSol ainsi obtenue offre une amélioration significative au regard de la première version proposée dans (Gonzalez et al. 2010), et couvre une période de 10 ans au lieu de 1 ans pour la première version. La première motivation de cette nouvelle base est d'améliorer l'estimation des luminances pour aider au développement de nouveaux capteurs optiques. Néanmoins, une telle base de données ouvre la voie à de nouveaux sujets de recherche en dynamique de la surface, pour l'étude du changement dans l'occupation des sols ou des variations inter-annuelles dues aux perturbations climatiques.

HIV-infected patients of all ages frequently underperform in responsiveness to seasonal influenza vaccination despite virologic control of HIV. Molecular mechanisms governing this impairment as well as predictive biomarkers for responsiveness remain unknown. This study was performed in pre-vaccination samples (T0) of HIV-infected children who received the 2012–2013 seasonal influenza vaccine. Response status was determined based on established criteria of hemagglutination inhibition (HAI) titer; participants with HAI ≥ 1:40 plus ≥ 4-fold increase over T0 at three weeks post-vaccination (T1) were designated as responders. All children had a history of prior influenza vaccinations. At T0, frequencies of CD4 T cell subsets, including peripheral T follicular helper (pTfh) cells which provide help to B cells for developing into Ab secreting cells were similar between responders and non-responders. However, in response to in vitro stimulation with H1N1 antigen, differential gene expression related to pTfh function was observed by Fluidigm high density RT-PCR between responders and non-responders. In responders, H1N1 stimulation at pre-vaccination also resulted in CXCR5 induction (mRNA and protein) in CD4 T cells and IL21 gene induction in pTfh cells that strongly associated with H1N1-specific B cell responses post-vaccination. In contrast, CD4 T cells of non-responders exhibited increased expression of IL2 and STAT5 genes which are known to antagonize pTfh function. These results suggest that the quality of pTfh at the time of immunization are important for influenza vaccine responses and provide a rationale for targeted, ex vivo antigen-driven molecular profiling of purified immune cells to detect predictive biomarkers of vaccine response.

TNFR superfamily (TNFRSF)4 (OX40, CD134) and TNFRSF25 are costimulatory receptors that influence CD4+ and CD8+ T cell responses to cognate Ag. Independently, these receptors have been described to stimulate overlapping functions, including enhanced proliferation and activation for both regulatory T cells (CD4+Foxp3+; Tregs) and conventional T cells (CD4+Foxp3− or CD8+Foxp3−; Tconvs). To determine the relative functionality of TNFRSF4 and TNFRSF25 in T cell immunity, the activity of TNFRSF4 and TNFRS25 agonistic Abs was compared in the context of both traditional protein/adjuvant (OVA/aluminum hydroxide) and CD8+-specific heat shock protein-based (gp96-Ig) vaccine approaches. These studies demonstrate that both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine-induced CD8+ T cell proliferation following both primary and secondary Ag challenge. In contrast, the activities of TNFRSF4 and TNFRSF25 were observed to be divergent in the costimulation of CD4+ T cell immunity. TNFRSF4 agonists were potent costimulators of OVA/aluminum hydroxide-induced CD4+ Tconv proliferation, but they only weakly costimulated Treg proliferation and IgG2a production, whereas TNFRSF25 agonists were strong costimulators of Treg proliferation, producers of IgG1, IgG2a, and IgG2b, and weak costimulators of CD4+ Tconv proliferation. Interestingly, Ag-specific cellular and humoral responses were uncoupled upon secondary immunization, which was dramatically affected by the presence of TNFRSF4 or TNFRSF25 costimulation. These studies highlight the overlapping but nonredundant activities of TNFRSF4 and TNFRSF25 in T cell immunity, which may guide the application of receptor agonistic agents as vaccine adjuvants for infectious disease and tumor immunity.

In this study, we report that addition of checkpoint inhibition and T cell costimulators into the Heat Biologics gp96-Ig-based vaccine platform elicits stronger antigen-specific CD8+ T cell activation and expansion, stimulates memory precursor cell activation, and enhances rejection of murine tumors. Despite the dramatic successes of checkpoint inhibitors in limited populations of cancer patients, 60-90% of patients are still failing to respond to these therapies. It is widely believed that approaches such as ours that target multiple facets of the immune system, including combinations with checkpoint inhibition and costimulator agonism, will be required to improve patient outcomes. Heat Biologics' first-generation ImPACT vaccine is a cell-based therapy that utilizes representative tumor cell lines expressing the antigen chaperone, gp96-Ig, to efficiently prime antigen-specific CD8+ T cells. Heat recently developed a next generation vaccine called ComPACT (COMbination Pan-Antigen Cytotoxic Therapy) that incorporates expression of a T cell costimulator (OX40L-Ig) and gp96-Ig within a single vaccine cell line. In multiple murine tumor and tumor-naive models, ComPACT elicits more potent activation of antigen-specific T cells as well as expansion of CD127+KLRG1- memory precursor cells, leading to a detectable memory response. Furthermore, ComPACT lacks the toxic systemic inflammatory cytokine production and proliferation of non-specific CD4+ T cells and Tregs observed with systemic administration of OX40 agonist antibodies. ComPACT also leads to high frequencies of IFNγ+, TNFα+, granzyme B+ and IL-2+ antigen-specific CD8+ T cells in mice at both priming and boosting, which increases rejection of established murine melanoma (B16.F10) and colon cancer (CT26) tumors and increased overall survival. Here, we have further assessed ImPACT and ComPACT-OX40L in combination with the anti-PD1 checkpoint inhibitor and with expression of an additional cell-secreted T cell costimulator, TL1A-Ig. In various murine models we show that these treatments synergize effectively to amplify antigen-specific T cells, program a functional memory response, and eliminate tumors. The combination of ComPACT-OX40L/TL1A with αPD1 or αPD-L1 may therefore translate into an efficacious approach to treat human cancers. Citation Format: Louise Giffin, Louis Gonzalez, Jason Rose, Jeff Hutchins. Gp96-Ig/costimulator combination platform improves T cell priming and enhances immunity, memory, and tumor elimination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1732.

Induction of mucosal immunity is critical for protection from enteric pathogens. Heat shock protein gp96 is one of the primary peptide and protein chaperones located in the endoplasmic reticulum. We reported previously that a cell-secreted gp96-Ig fusion protein (gp96-Ig) mediated strong systemic, antigen-specific CD8-CTL expansion in vivo. We now evaluate the mucosal immune response to stimulation by secreted gp96 using allogeneic NIH-3T3 transfected with ovalbumin (OVA) and gp96-Ig. A single intraperitoneal NIH-3T3-OVA-gp96-Ig immunization caused significant homing of OVA-specific TCR transgenic CD8 cells (OT-I) to Peyer's patches, to the intraepithelial compartment and to the lamina propria. Intraperitoneal immunization with cells secreting gp96-Ig provided stronger mucosal immunity than the same dose instilled vaginally or rectally or injected subcutaneously or intradermally. Our results provide the first evidence that cell-based gp96-Ig-secreting vaccines may serve as a potent modality to induce mucosal immunity.

The MODIS instruments have been flying onboard the Terra and Aqua platforms and acquire Earth observation data since early 2000 and mid 2002. Data processing allows the monitoring of the land cover dynamic. Here, a data processing scheme is described to generate Earth reflectance and emissivity time series at a sub-kilometer spatial resolution and with a period of 8 days. The data processing allows the identification of artifacts generated by clouds, aerosols or other unwanted effects that corrupt the time series. In addition, a bidirectional reflectance model, depending on the surface cover type, is applied to i) normalize the reflectance to a constant viewing geometry and ii) compute a land surface albedo. A web-service tool has also been developed for an easy analysis of the reflectance images and their time-evolution. It is expected that the database and the tool will help researchers to identify and quantify the anthropogenic impact on the Earth surface cover, and the impact of naturallyinduced disturbances such as fires, floods or droughts.

TNF-alpha is a highly pleiotropic cytokine and plays an important role in regulating HIV-1 replication. It may compromise the integrity of the blood-brain-barrier and, thus, may contribute to the neurotoxicity of HIV-1-infection. Both intravenous drug abuse (IDU) and HIV infection can increase TNF-alpha activity, but little information is available on the effects of a combination of these factors on TNF-alpha. We investigated plasma TNF-alpha levels and mRNA in the peripheral monocytes of 166 men and women in three groups: HIV-1-positive IDUs, HIV-1-negative IDUs, and HIV-negative non-IDU control participants. HIV-1-positive IDUs had higher TNF-alpha levels than HIV-1-negative IDUs who, in turn, had higher levels than controls. TNF-alpha mRNA expression in peripheral monocytes was significantly increased in both HIV-1-positive and negative IDUs compared to controls. These findings show that the effects of HIV infection and intravenous drug use may be additive in increasing TNF-alpha levels. Given the multiple effects of TNF-alpha in HIV infection, additional investigation of its role is needed.