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FR104 is a monovalent pegylated Fab' Ab, antagonist of CD28, under development for treatment of transplant rejection and autoimmune diseases. In contrast to CD80/86 antagonists (CTLA4-Ig), FR104 selectively blunts CD28 costimulation while sparing CTLA-4 and PD-L1 coinhibitory signals. In the present work, FR104 has been evaluated in a first-in-human study to evaluate the safety, pharmacokinetics, pharmacodynamics, and potency of i.v. administrations in healthy subjects. Sixty-four subjects were randomly assigned to four single ascending dose groups, two double dose groups and four single ascending dose groups challenged with keyhole limpet hemocyanin. Subjects were followed up over a maximum of 113 d. Overall, the pharmacokinetics of FR104 after a single and double infusions was approximately linear at doses ≥0.200 mg/kg. CD28 receptor occupancy by FR104 was saturated at the first sampling time point (0.5 h) at doses above 0.02 mg/kg and returned to 50% in a dose-dependent manner, by day 15 (0.020 mg/kg) to 85 (1.500 mg/kg). FR104 was well tolerated, with no evidence of cytokine-release syndrome and no impact on blood lymphocyte subsets. Inhibition of anti-keyhole limpet hemocyanin Ab response was dose-dependent in FR104 recipients and was already apparent at a dose of 0.02 mg/kg. Abs to FR104 were detected in 22/46 (48%) of FR104 recipients and only 1/46 (2.2%) was detected during drug exposure. In conclusion, selective blockade of CD28 with FR104 was safe and well tolerated at the doses tested. The observed immunosuppressive activity indicated that FR104 has potential to show clinical activity in the treatment of immune-mediated diseases.

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Dendritic cells (DCs) are major antigen presenting cells that can efficiently prime immune responses. However, the roles of skin resident Langerhans cells (LCs) in eliciting immune responses have not been fully understood. We here demonstrate for the first time that LCs in cynomolgus macaque skin are capable of inducing antiviral-specific immune responses in vivo. Targeting HIV-Gag or influenza hemagglutinin antigens to skin LCs using recombinant fusion proteins of anti-Langerin antibody and antigens resulted in the induction of the viral antigen-specific responses. We further demonstrated that such antigen-specific immune responses elicited by skin LCs were greatly enhanced by TLR ligands (TLR-Ls), polyriboinosinic polyribocytidylic acid (poly(I:C)) and R848. These enhancements were not due to the direct actions of TLR-Ls on LCs, but mainly dependent on TNF-α secreted from macrophages and neutrophils recruited to local tissues. Skin LC activation and migration out of the epidermis are associated with macrophage and neutrophil infiltration into the tissues. More importantly, blocking TNF-α abrogated the activation and migration of skin LCs. This study highlights that the cross-talk between innate immune cells in local tissues is an important component for the establishment of adaptive immunity. Understanding the importance of local immune networks will help us to design new and effective vaccines against microbial pathogens.

FIG 2. Basophils from allergic patients do not respond to TSLP and do not express IL-7Ra. Whole blood samples from HDs (empty bars, n 5 5) and allergic patients (black bars, n 5 8) were stimulated for 6 hours with IL-3, TSLP, anti-Fc 3RI antibody, or medium and analyzed by FCM. Gating strategy on whole blood (A). Intracellular P-STAT5 expression in basophils (left) and mDCs (right) (B). CD203c (left) and CCR3 (right) expression on basophils (C). % of IL-7Ra-and TSLPR-expressing basophils (left) or mDCs (right) (D). Data are shown as mean 6 SEM.

The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine-targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti-Langerin-HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti-HIV immune response without requirement of an adjuvant. Although the co-injection of the TLR-7/8 synthetic ligand, R-848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA-DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine-targeted epidermal LCs and R-848. Keywords:Fluorescence imaging r Langerhans cell r Nonhuman primate r Skin r Vaccination Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Dr. Catherine Chapon e-mail: catherine.chapon@cea.fr IntroductionFluorescence imaging is a powerful and minimally invasive technology for real-time visualization in vivo of the dynamics of the behavior of immune cells in their natural microenvironment [1][2][3][4][5][6].C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 690Nina Salabert et al. Eur. J. Immunol. 2016. 46: 689-700 It also facilitates the characterization of the fate of vaccine antigens in the organism allowing unprecedented progress in the understanding of vaccine-induced immune response mechanisms [7,8]. Up to now, most in vivo studies have used transgenic mouse models expressing fluorescent molecules under specific promoters. However, relevance of mouse models to the study of human vaccines is questionable. Major differences between the two species have been reported, when comparing mature immune system [9][10][11][12][13][14]. Mice models turned out to be little predictive of vaccine immunogenicity and efficacy in humans, even when highly advanced transgenic and humanized animals were used [15]. Currently, due to their close genetic relatedness with humans, and immune compartment similarities in particular, nonhuman primates (NHP) are considered to be the most appropriate model species for predicting vaccine immunogenicity and efficacy in humans [16,17]. The development of new strategies for immunization [17,18] requires a better understanding of molecular and cellular change...

Background: Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Besides, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent by which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods. Methods:In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor ® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC.Results: Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly,we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa). Conclusions:In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.