Análise transcriptómica de Single-cell de linfócitos efectores do recetor Fc-like 6 em coelhos: um novo modelo translacional para estudos de imunidade humana.

For this project, our goals are: 1) Establishing the first atlas for rabbit immune cells. Most of the knowledge on the functional and structural characteristics and expression of antibodies was developed through the research of rabbit immunoglobulins. Recent studies have provided stronger evidence of the translational value between rabbit and human models given closer genetic identity than that found between mice and humans. A relevant example is the marked interspecies disparity found for mouse FCRL6, which has a disparate primary structure and cellular expression pattern to its human counterpart. These major differences have posed a major challenge for functionally modelling FCRL6 and establishing physiologic understanding of this clinically significant receptor. Given that, rabbits have fundamentally shaped our understanding of the adaptive immune system, exhibit closer genetic similarity with humans than mice, and share a conserved FCRL6 receptor-gene, this project proposes to advance the rabbit as a model for the study of immunoreceptors in B, T and NK cell lymphocytes. This will be the first known single-cell analysis of these immune subsets in rabbits and will provide a highly significant resource for immunologists worldwide. We will perform a comparative analysis of transcriptomic features and developmental pathways with those already established for human and mouse lymphocytes. We will use this to establish the first atlas for rabbit lymphocytes in the spleen and thymus. 2) Single-cell transcriptomic analysis of rabbit immune effector lymphocytes- Through the atlas, we will analyze the expression pattern of immunoreceptors with a particular focus on FCRL6. This will allow us to determine if the pattern of expression and inherent functional regulation of this receptor is maintained in both species. This will be crucial to uncover rFCRL6 ligands and establish novel transcriptomic understanding of rabbit lymphocytes. This will allow innovation in the fields of immunology, advance veterinary development of vaccines for rabbit farms and provide a unique view into rabbit immune effector lymphocytes. 3) Uncover new aspects of FCRL6 evolution - We have recently described a mass duplication event for the FCRL6 gene in Armadillo. Through this work, we obtained preliminary results that indicate a putative FCRL6-like gene at a neighbouring locus, present in rabbit. Here we will advance the analysis of this newly identified receptor-gene. We will also investigate the evolution of the domain(s) responsible for FCRL6 ligand binding and characterize the selection forces at play. 4) Determine structure/function relationships for FCRL6 - HLA-DR/MHCII, one of the most polymorphic genes in adaptive immunity, is an FCRL6 ligand. Importantly, FCRL6 is linked to tumour immunity and is predictive of overall survival in human malignancies. We will perform binding assays for rFCRL6 and a putative FCRL6-like receptor to determine if they share the same ligand as the human counterpart. Additionally, we will test if this receptor displays the same inhibitory features observed in the human immunoreceptor. We will probe molecular and functional aspects of these FCRL6 ligand relationships, define features of the FCRL6/MHCII contact interface, and determine how it may be manipulated to modulate immune responses to cancer and host defence.