Profound differences in T cell receptor (TCR) repertoire and functional profiles between human and murine γδ T cells pose significant challenges for translational γδ T cell research. Therefore, we generated humanized immune system (HIS) NBSGW (NOD,B6.PrkdcscidIl2rγ-/-KitW41/W41) mice reconstituted with human fetal liver CD34+ hematopoietic stem and progenitor cells (HSPCs) enabling evaluation of human γδ T cells in vivo. The HIS mice accurately recapitulate the TCR-associated thymic programming of human γδ T cells-alongside αβ T cell development-and their peripheral effector functions, including the generation of phosphoantigen-reactive Vγ9Vδ2 T cells uniquely found in humans. Moreover, terminal deoxynucleotidyl transferase (TdT) is identified as a key regulator of type 3 Vδ2 T cell development. These findings demonstrate that HIS mice are a powerful model to screen human γδ T cell-targeting immunotherapies and to obtain mechanistic insights into human γδ T cell biology.

The co-inhibitory programmed death (PD)-1 signaling pathway plays a major role in the context of tumor -spe-cific T cell responses. Conversely, it also contributes to the maintenance of peripheral tolerance, as patients receiving anti-PD-1 treatment are prone to developing immune-related adverse events. Yet, the physiological role of the PD-1/PDL-1 axis in T cell homeostasis is still poorly understood. Herein, we show that under steady-state conditions, the absence of PD-1 signaling led to a preferential expansion of CD8+ T cells in the liver. These cells exhibit an oligoclonal T cell receptor (TCR) repertoire and a terminally differentiated exhaustion profile. The transcription factor EOMES is required for the clonal expansion and acquisition of this differentiation program. Finally, single-cell transcriptomics coupled with TCR repertoire analysis support the notion that these cells arise locally from liver-resident memory CD8+ T cells. Overall, we show a role for PD-1 signaling in liver memory T cell homeostasis.

Severe COVID-19 disease is associated with dysregulation of the myeloid compartment during acute infection. Survivors frequently experience long-lasting sequelae, but little is known about the eventual persistence of this immune alteration. Herein, we evaluated TLR-induced cytokine responses in a cohort of mild to critical patients during acute or convalescent phases (n = 97). In the acute phase, we observed impaired cytokine production by monocytes in the patients with the most severe COVID-19. This capacity was globally restored in convalescent patients. However, we observed increased responsiveness to TLR1/2 ligation in patients who recovered from severe disease, indicating that these cells display distinct functional properties at the different stages of the disease. In patients with acute severe COVID-19, we identified a specific transcriptomic and epigenomic state in monocytes that can account for their functional refractoriness. The molecular profile of monocytes from recovering patients was distinct and characterized by increased chromatin accessibility at activating protein 1 (AP1) and MAF loci. These results demonstrate that severe COVID-19 infection has a profound impact on the differentiation status and function of circulating monocytes, during both the acute and the convalescent phases, in a completely distinct manner. This could have important implications for our understanding of short-and long-term COVID-19???related morbidity.

Cancer is caused primarily by genomic alterations resulting in deregulation of gene regulatory circuits in key growth, apoptosis, or DNA repair pathways. Multiple genes associated with the initiation and development of tumors are also regulated at the level of mRNA decay, through the recruitment of RNA-binding proteins to AU-rich elements (AREs) located in their 3′-untranslated regions. One of these ARE-binding proteins, tristetraprolin (TTP; encoded by Zfp36), is consistently dysregulated in many human malignancies. Herein, using regulated overexpression or conditional ablation in the context of cutaneous chemical carcinogenesis, we show that TTP represents a critical regulator of skin tumorigenesis. We provide evidence that TTP controlled both tumor-associated inflammation and key oncogenic pathways in neoplastic epidermal cells. We identify Areg as a direct target of TTP in keratinocytes and show that EGFR signaling potentially contributed to exacerbated tumor formation. Finally, single-cell RNA-Seq analysis indicated that ZFP36 was downregulated in human malignant keratinocytes. We conclude that TTP expression by epidermal cells played a major role in the control of skin tumorigenesis.

c-Jun N-terminal protein kinase 1 (JNK1) is involved in multiple biological processes but its implication in inflammatory skin diseases is still poorly defined. Herein, we studied the role of JNK1 in the context of Aldara ® -induced skin inflammation. We observed that constitutive ablation of JNK1 reduced Aldara ® -induced acanthosis and expression of inflammatory markers. Conditional deletion of JNK1 in myeloid cells led to reduced skin inflammation, a finding that was associated with impaired Aldara ® -induced inflammasome activation in vitro . Next, we evaluated the specific role of JNK1 in epidermal cells. We observed reduced Aldara ® -induced acanthosis despite similar levels of inflammatory markers. Transcriptomic and epigenomic analysis of keratinocytes revealed the potential involvement of JNK1 in the EGFR signaling pathway. Finally, we show that inhibition of the EGFR pathway reduced Aldara ® -induced acanthosis. Taken together, these data indicate that JNK1 plays a dual role in the context of psoriasis by regulating the production of inflammatory cytokines by myeloid cells and the sensitivity of keratinocytes to EGFR ligands. These results suggest that JNK1 could represent a valuable therapeutic target in the context of psoriasis.

AU-rich element (ARE)-mediated mRNA decay represents a key mechanism to avoid excessive production of inflammatory cytokines. Tristetraprolin (TTP, encoded byZfp36) is a major ARE-binding protein, sinceZfp36(-/-)mice develop a complex multiorgan inflammatory syndrome that shares many features with spondyloarthritis. The role of TTP in intestinal homeostasis is not known. Herein, we show thatZfp36(-/-)mice do not develop any histological signs of gut pathology. However, they display a clear increase in intestinal inflammatory markers and discrete alterations in microbiota composition. Importantly, oral antibiotic treatment reduced both local and systemic joint and skin inflammation. We further show that absence of overt intestinal pathology is associated with local expansion of regulatory T cells. We demonstrate that this is related to increased vitamin A metabolism by gut dendritic cells, and identify RALDH2 as a direct target of TTP. In conclusion, these data bring insights into the interplay between microbiota-dependent gut and systemic inflammation during immune-mediated disorders, such as spondyloarthritis.

Memory CD8+ T cells have the ability to provide lifelong immunity against pathogens. Although memory features generally arise after challenge with a foreign antigen, naïve CD8 single positive (SP) thymocytes may acquire phenotypic and functional characteristics of memory cells in response to cytokines such as interleukin-4. This process is associated with the induction of the T-box transcription factor Eomesodermin (EOMES). However, the underlying molecular mechanisms remain ill-defined. Using epigenomic profiling, we show that these innate memory CD8SP cells acquire only a portion of the active enhancer repertoire of conventional memory cells. This reprograming is secondary to EOMES recruitment, mostly to RUNX3-bound enhancers. Furthermore, EOMES is found within chromatin-associated complexes containing BRG1 and promotes the recruitment of this chromatin remodelling factor. Also, the in vivo acquisition of EOMES-dependent program is BRG1-dependent. In conclusion, our results support a strong epigenetic basis for the EOMES-driven establishment of CD8+ T cell innate memory program.

Monocytes play a major role in the defense against pathogens. They are rapidly mobilized to inflamed sites where they exert both proinflammatory and regulatory effector functions. It is still poorly understood how this dynamic and exceptionally plastic system is controlled at the molecular level. Herein, we evaluated the differentiation process that occurs in Ly6C hi monocytes during oral infection by Toxoplasma gondii . Flow cytometry and single-cell analysis revealed distinct activation status and gene expression profiles in the bone marrow, the spleen and the lamina propria of infected mice. We provide further evidence that acquisition of effector functions, such as the capacity to produce interleukin-27, is accompanied by distinct waves of epigenetic programming, highlighting a role for STAT1/IRF1 in the bone marrow and AP-1/NF-κB in the periphery. This work broadens our understanding of the molecular events that occur in vivo during monocyte differentiation in response to inflammatory cues.

The oil-in-water emulsion Adjuvant System 03 (AS03) is one of the few adjuvants used in licensed vaccines. Previous work indicates that AS03 induces a local and transient inflammatory response that contributes to its adjuvant effect. However, the molecular mechanisms involved in its immunostimulatory properties are ill-defined. Upon intramuscular injection in mice, AS03 elicited a rapid and transient downregulation of lipid metabolism-related genes in the draining lymph node. In vitro, these modifications were associated with profound changes in lipid composition, alteration of endoplasmic reticulum (ER) morphology and activation of the unfolded protein response pathway. In vivo, treatment with a chemical chaperone or deletion of the ER stress sensor kinase IRE1α in myeloid cells decreased AS03-induced cytokine production and its capacity to elicit high affinity antigen-specific antibodies. In summary, our results indicate that IRE1α is a sensor for the metabolic changes induced by AS03 in monocytic cells and may constitute a canonical pathway that could be exploited for the design of novel vaccine adjuvants.

Abstract Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. However, a limitation of the current autologous approach is that CAR T-cells must be manufactured on a "per patient basis". To overcome this limitation, we have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" engineered CAR+ T-cell-based frozen products. Our allogenic platform utilizes the Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, significantly reducing the potential for T-cells bearing alloreactive TCR’s to mediate Graft-versus-Host Disease (GvHD). We have previously demonstrated the precise and efficient disruption of the the TRAC gene by gene editing, yielding up to 85% of TCRαβ-negative cells. This allows the production of TCRαβ-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. In the clinic, the proof of concept of the applicability of our allogeneic platform was achieved with early compassionate use for patients treated with UCART19, an allogeneic engineered CAR T-cell product directed against CD19. UCART19 clinical trials are currently ongoing. Here, we have developed T-cells targeting CD22 which is expressed on tumor cells from the majority of patients with B-cells leukemia. In a first step, we have screened multiple antigen recognition domains in the context of different CAR architectures to identify effective CAR candidates displaying activity against cells expressing variable levels of the CD22 antigen. As a safety feature, T-cells are engineered to co-express a depletion gene, rendering them sensitive to the monoclonal antibody rituximab. Several constructs of depletion genes have been evaluated in the context of the CD22 CAR. In addition, experiments in an orthotopic ALL mouse model using UCART22 cells demonstrated important anti-tumor activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non-alloreactive CD22 specific T-cells from a single healthy donor can offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of leukemic patients. UCART22 could also offer an alternative to patients who may relapse with CD19-negative tumors after CD19 CAR T-cell treatment. Citation Format: Agnès Gouble, Cécile Schiffer-Mannioui, Severine Thomas, Anne-Sophie Gautron, Laurent Poirot, Julianne Smith. UCART22: allogenic adoptive immunotherapy of leukemia by targeting CD22 with CAR T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3763. doi:10.1158/1538-7445.AM2017-3763

The adjuvant properties of the saponin QS-21 have been known for decades. It is a component of the Adjuvant System AS01 that is used in several vaccine candidates. QS-21 strongly potentiates both cellular and humoral immune responses to purified antigens, yet how it activates immune cells is largely unknown. Here, we report that QS-21 directly activated human monocyte-derived dendritic cells (moDCs) and promoted a pro-inflammatory transcriptional program. Cholesterol-dependent QS-21 endocytosis followed by lysosomal destabilization and Syk kinase activation were prerequisites for this response. Cathepsin B, a lysosomal cysteine protease, was essential for moDC activation in vitro and contributed to the adjuvant effects of QS-21 in vivo. Collectively, these findings provide new insights into the pathways involved in the direct activation of antigen-presenting cells by a clinically relevant QS-21 formulation.

Saponins represent a promising class of vaccine adjuvant. Together with the TLR4-ligand MPL, QS-21 is part of the Adjuvant System AS01, a key component of the malaria and zoster candidate vaccines that display demonstrated clinical efficacy. However, the mechanism of action of QS-21 in this liposomal formulation is poorly understood. Upon intra-muscular immunisation, we observed that QS-21 rapidly accumulated in CD169+ resident macrophages of the draining lymph node where it elicited a local innate immune response. Depletion of these cells abrogated QS-21-mediated innate cell recruitment to the lymph node, dendritic cell (DC) phenotypic maturation as well as the adjuvant effect on T-cell and antibody responses to co-administered antigens. DCs rather than lymph node-resident macrophages were directly involved in T-cell priming by QS-21, as revealed by the decrease in antigen-specific T-cell response in Batf3-/- mice. Further analysis showed that the adjuvant effect of QS-21 depended on the integration of Caspase-1 and MyD88 pathways, at least in part through the local release of HMGB1. Taken together, this work unravels the key role of lymph node sentinel macrophage in controlling the adjuvant effect of a molecule proven to improve vaccine response in humans.

The last few years have seen the increasing development of new DNA targeting molecular tools and strategies for precise genome editing. However, opportunities subsist to either improve or expand the current toolbox and further broaden the scope of possible biotechnological applications. Here we report the discovery and the characterization of BurrH, a new modular DNA binding protein from Burkholderia rhizoxinica that is composed of highly polymorphic DNA targeting modules. We also engineered this scaffold to create a new class of designer nucleases that can be used to efficiently induce in vivo targeted mutagenesis and targeted gene insertion at a desired locus.

TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.

Background: The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases.Results: In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE:: FokI and FokI:: TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines.Conclusion: Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases.

A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of > 15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.

Transcription activator-like effector nucleases are readily targetable 'molecular scissors' for genome engineering applications. These artificial nucleases offer high specificity coupled with simplicity in design that results from the ability to serially chain transcription activator-like effector repeat arrays to target individual DNA bases. However, these benefits come at the cost of an appreciably large multimeric protein complex, in which DNA cleavage is governed by the nonspecific FokI nuclease domain. Here we report a significant improvement to the standard transcription activator-like effector nuclease architecture by leveraging the partially specific I-TevI catalytic domain to create a new class of monomeric, DNA-cleaving enzymes. In vivo yeast, plant and mammalian cell assays demonstrate that the half-size, single-polypeptide compact transcription activator-like effector nucleases exhibit overall activity and specificity comparable to currently available designer nucleases. In addition, we harness the catalytic mechanism of I-TevI to generate novel compact transcription activator-like effector nuclease-based nicking enzymes that display a greater than 25-fold increase in relative targeted gene correction efficacy.

Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it.