Final Report Summary - DERMACRO (Probing the function of dermal macrophages by lectin targeting for potential application to autoimmune diseases.) Targeting of dermal macrophages for the control of resident memory T cells residing in the skin.State of the art Autoimmune diseases originate from a breakdown in immune tolerance leading to responses against self-antigens and chronic inflammation. This results in progressive destruction of the patient’s organs, such as skin, kidney, or joints. Available treatments rely on anti-inflammatory drugs, which compromise the ability of the immune system to fight infections and cancer. Abnormal differentiation or activation of T cells is a critical turning point in most autoimmune diseases. Cutaneous antigen-presenting cells (APCs) that control the fate of T cells therefore represent an attractive and easily accessible option as a specific therapeutic target. Several subpopulations of APCs can be distinguished in human skin, based on their function, localization (dermis or epidermis) and capacity to traffic towards skin-draining lymph nodes upon stimulation. Epidermal Langerhans cells (LCs) and CD1a+ dermal dendritic cells (dDCs) traffic towards skin-draining lymph nodes upon stimulation. Conversely, dermal macrophages are permanently resident APCs. All skin APCs capture glycosylated proteins via lectins, and anti-lectin antibodies have been used to specifically load antigens onto APC subsets. We propose here a novel approach to target skin APCs, based on purified viral envelope proteins. Indeed, arboviruses are naturally inoculated into the skin by insect bites and have a specific glycosylation pattern which is recognized by lectins. Scientific progress and results Targeting dermal macrophagesThe soluble envelope protein 2 of Chikungunya virus (CHIK-sE2) showed a high affinity in vitro for lectins DC-SIGN/CD209 and Macrophage Mannose Receptor/CD206, which are expressed by dermal macrophages. To evaluate its targeting capacity, CHIK-sE2 was coupled to a fluorescent dye and incubated with a mixture of all skin APCs isolated from healthy human explants. CHIK-sE2 specifically bound to dermal macrophages, but not to Langerhans cells, CD1a+ dDCs, or non-immune cells. We have traced fluorescent CHIK-sE2 into intracellular compartments of targeted dermal macrophages by confocal microscopy, demonstrating its efficient uptake. Based on our promising results, we proceeded to functional assays to further validate CHIK-sE2 as an efficient agent for the delivery of antigens to dermal macrophages.Presentation of CHIK-sE2-carried antigens to human TRMResident memory T cells (TRM) are derived from effector T cells that colonize the skin to mediate adaptive immune responses. Reactivation of TRM upon secondary exposure to their cognate antigen is a critical step to ensure a complete and rapid response. Nevertheless, the activation state of TRM must be tightly regulated to avoid autoimmune reactions. The mechanisms that ensure this control are unknown yet, but might involve cutaneous antigen-presenting cells such as skin-resident dermal macrophages. Thus, CHIK-sE2 represents an ideal tool to specifically load dermal macrophages with a given antigen and to study its presentation to TRM. First, we determined which immunodominant antigenic peptides alone, derived from skin-tropic microorganisms (Varizella Zoster Virus) or vaccines inoculated into the skin (Clostridium tetanii, Mycobacterium tuberculosis), could be most efficiently presented to CD4+ TRM from a majority of skin donors. We found that a peptide from the Tetanus Toxin (TT) yielded the most frequent responses. Thus, we coupled CHIK-sE2 to this TT peptide and exposed dermal macrophages to the resulting conjugate. Interestingly, antigen presentation by targeted macrophages to TT-specific TRM isolated from the same donor appeared to decrease TRM activation by other APCs. Upon confirmation of this negative influence in experiments with CD4+ T cell clones of defined specificity, we intend to propose our results for publication by the end of the year.Presentation of CHIK-sE2-carried antigens to ovalbumin-specific T cells in vivoIn parallel to these human ex vivo assays, we would like to establish a mouse model of cutaneous TRM reactivation, in order to dissect the molecular mechanisms that control TRM interactions with dermal resident APCs. To this aim, we will perform cutaneous immunization by the model antigen ovalbumin (OVA), leading to the recruitment into the skin and TRM differentiation of OVA-specific transgenic T cells. This will allow us to study in vivo the reactivation of OVA-specific TRM by dermal APCs targeted by OVA-coupled CHIK-sE2. In a preliminary functional assay, we coupled CHIK-sE2 to OVA to evaluate antigen presentation to TCR-transgenic CD4+ T cells in vivo. When inoculated intradermally into C57BL/6 mice, CHIK-sE2/OVA promoted the proliferation of OVA-specific CD4+ T cells in skin-draining lymph nodes in the absence of adjuvant, suggesting the involvement of lymph node-resident APCs rather than skin-emigrating DCs. This project will be proposed in a collaborative grant application to be submitted in 2017.Prospects of the research career development and integration of the fellow.Thanks to the Career Integration Grant, I have been able to demonstrate financial independence shortly after being hired by the Centre National de la Recherche Scientifique (CNRS). This allowed me to initiate other research projects and new, exciting collaborations. I am now established at the laboratory CNRS UPR3572 “Immunopathology and Therapeutic Chemistry” in Strasbourg as a permanent researcher (Chargé de Recherche) in the team “Immune-microenvironment interactions in health and disease” of Dr. C. G. Mueller. Because I am employed in this dynamic research team of a renowned institute, I am now able to foster my independence level by developing my managerial skills and scientific network locally and internationally. In this perspective, I have acquired in 2016 the university degree “Habilitation à diriger des recherches” which, in France, is required to recruit and officially supervise Ph.D. students. This was a critically important step to supply the necessary workforce for the expansion of the team that I belong to. Indeed, in addition to the CIG-funded study of dermal macrophages, I have been directly responsible for implementing new research programs, including the development of an innervated and vascularized immunocompetent tissue-engineered human skin model, as well as investigations on the role of autophagy in the homeostasis and function of Langerhans cells.
