CD4 regulatory T cells (Tregs) can be subdivided into two subsets according to Ly-6C expression in the periphery. Phenotypic analysis, imaging, and adoptive-transfer experiments of peripheral Ly-6C(-) and Ly-6C(+) Tregs reveal that the nonexpression of Ly-6C by ∼70% of peripheral Tregs depends on TCR signaling events. Interestingly, Ly-6C(-) Tregs express higher surface amounts of key immunosuppressive molecules than do Ly-6C(+) Tregs and produce constitutively anti-inflammatory cytokines. In line with their phenotype, Ly-6C(+) Tregs exhibit poor suppressive capacities in vitro and in vivo. Finally, although Ly-6C(-) Tregs maintain their numbers with age, Ly-6C(+) Tregs gradually disappear. Altogether, our data strongly suggest that both the survival and suppressive functions of peripheral CD4 Tregs rely on their ability to receive strong TCR signals.

Despite progress, our understanding of psychiatric and neurological illnesses remains poor, at least in part due to the inability to access neurons directly from patients. Currently, there are in vitro models available but significant work remains, including the search for a less invasive, inexpensive and rapid method to obtain neuronal-like cells with the capacity to deliver reproducible results. Here, we present a new protocol to transdifferentiate human circulating monocytes into neuronal-like cells in 20 days and without the need for viral insertion or reprograming. We have thoroughly characterized these monocyte-derived-neuronal-like cells (MDNCs) through various approaches including immunofluorescence (IF), flow cytometry, qRT-PCR, single cell mRNA sequencing, electrophysiology and pharmacological techniques. These MDNCs resembled human neurons early in development, expressed a variety of neuroprogenitor and neuronal genes as well as several neuroprogenitor and neuronal proteins and also presented electrical activity. In addition, when these neuronal-like cells were exposed to either dopamine or colchicine, they responded similarly to neurons by retracting their neuronal arborizations. More importantly, MDNCs exhibited reproducible differentiation rates, arborizations and expression of dopamine 1 receptors (DR1) on separate sequential samples from the same individual. Differentiation efficiency measured by cell morphology was on average 11.9 ± 1.4% (mean, SEM, n = 38,819 cells from 15 donors). To provide context and help researchers decide which in vitro model of neuronal development is best suited to address their scientific question,we compared our results with those of other in vitro models currently available and exposed advantages and disadvantages of each paradigm.

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN-I). What other functions pDCs exert in vivo during viral infections is controversial and more studies are needed to understand their orchestration. Here, we characterize indepth and link pDC activation states in animals infected by mouse cytomegalovirus, by combining Ifnb1 reporter mice with flow cytometry, single-cell RNA sequencing, confocal microscopy and a cognate CD4 T cell activation assay. We show that IFN-I production and T cell activation were performed by the same pDC, but sequentially in time and in different micro-anatomical locations. In addition, we show that pDC commitment to IFN-I production was marked early on by their downregulation of LIFR and promoted by cell-intrinsic TNF signaling. We propose a novel model of how individual pDCs are endowed to exert different functions in vivo during a viral infection in a manner tightly orchestrated in time and space.

The activation of tumor-specific effector immune cells is key for successful immunotherapy and vaccination is a powerful strategy to induce such adaptive immune responses. However, the generation of effective anticancer vaccines is challenging. To overcome these challenges, a novel straight-forward strategy of adjuvant-induced tumor antigen assembly to generate nanovaccines with superior antigen/adjuvant loading efficiency is developed. To protect nanovaccines in circulation and to introduce additional functionalities, a biocompatible polyphenol coating is installed. The resulting functionalizable nanovaccines are equipped with a pH (low) insertion peptide (pHLIP) to facilitate endolysosomal escape and to promote cytoplasmic localization, with the aim to enhance cross-presentation of the antigen by dendritic cells to effectively activate CD8 T cell. The results demonstrate that pHLIP-functionalized model nanovaccine can induce endolysosomal escape and enhance CD8 T cell activation both in vitro and in vivo. Furthermore, based on the adjuvant-induced antigen assembly, nanovaccines of the clinically relevant tumor-associated antigen NY-ESO-1 are generated and show excellent capacity to elicit NY-ESO-1-specific CD8 T cell activation, demonstrating a high potential of this functionalizable nanovaccine formulation strategy for clinical applications.

Nanovaccines, co-delivering antigen and invariant natural killer T (iNKT) cell agonists, proved to be very effective in inducing anti-tumor T cell responses due to their exceptional helper function. However, it is known that iNKT cells are not equally present in all lymphoid organs and nanoparticles do not get evenly distributed to all immune compartments. In this study, we evaluated the effect of the vaccination route on iNKT cell help to T and B cell responses for the first time in an antigen and agonist co-delivery setting. Intravenous administration of PLGA nanoparticles was mainly targeting liver and spleen where iNKT1 cells are abundant and induced the highest serum IFN-y levels, T cell cytotoxicity, and Th-1 type antibody responses. In comparison, after subcutaneous or intranodal injections, nanoparticles mostly drained or remained in regional lymph nodes where iNKT17 cells were abundant. After subcutaneous and intranodal injections, antigen-specific IgG2 c production was hampered and IFN-y production, as well as cytotoxic T cell responses, depended on sporadic systemic drainage. Therapeutic anti-tumor experiments also demonstrated a clear advantage of intravenous injection over intranodal or subcutaneous vaccinations. Moreover, tumor control could be further improved by PD-1 immune checkpoint blockade after intravenous vaccination, but not by intranodal vaccination. Anti PD-1 antibody combination mainly exerts its effect by prolonging the cytotoxicity of T cells. Nanovaccines also demonstrated synergism with anti-4-1BB agonistic antibody treatment in controlling tumor growth. We conclude that nanovaccines containing iNKT cell agonists shall be preferentially administered intravenously, to optimally reach cellular partners for inducing effective anti-tumor immune responses.

Mounting an effective immune response relies critically on the coordinated interactions between adaptive and innate compartments. How and where immune cells from these different compartments interact is still poorly understood. Here, we demonstrate that the cross-talk between invariant natural killer T cells (iNKT) and CD8+T cells in the spleen, essential for initiating productive immune responses, is biphasic and occurs at 2 distinct sites. Codelivery of antigen and adjuvant to antigen-presenting cells results in: 1) initial short-lived interactions (0 to 6 h), between CD8+T cells, dendritic cells (DCs), and iNKT cells recruited outside the white pulp; 2) followed by long-lasting contacts (12 to 24 h) between iNKT cells, DCs, and CD8+T cells occurring in a 3-way interaction profile within the white pulp. Both CXCR3 and CCR4 are essential to orchestrate this highly dynamic process and play nonredundant in T cell memory generation. While CXCR3 promotes memory T cells, CCR4 supports short-lived effector cell generation. We believe our work provides insights into the initiation of T cell responses in the spleen and their consequences for T cell differentiation.

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab′ fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

Dendritic cells (DC) are able to elicit anti-tumoral CD8+ T cell responses by cross-presenting exogenous antigens in association with major histocompatibility complex (MHC) class I molecules. Therefore they are crucial actors in cell-based cancer immunotherapy. Although apoptotic cells are usually considered to be the best source of antigens, live cells are also able to provide antigens for cross-presentation by DC. We have recently shown that prophylactic immunotherapy by DC after capture of antigens from live B16 melanoma cells induced strong CD8+ T-cell responses and protection against a lethal tumor challenge in vivo in C57Bl/6 mice. Here, we showed that DC cross-presenting antigens from live B16 cells can also inhibit melanoma lung dissemination in a therapeutic protocol in mice. DC were first incubated with live tumor cells for antigen uptake and processing, then purified and irradiated for safety prior to injection. This treatment induced stronger tumor-specific CD8+ T-cell responses than treatment by DC cross-presenting antigens from apoptotic cells. Apoptotic B16 cells induced more IL-10 secretion by DC than live B16 cells. They underwent strong native antigen degradation and led to the expression of fewer MHC class I/epitope complexes on the surface of DC than live cells. Therefore, the possibility to use live cells as sources of tumor antigens must be taken into account to improve the efficiency of cancer immunotherapy.