Quantitative characterization of CTLA4 trafficking and turnover using a combinedin vitroandin silicoapproach

Abstract

12CTLA4 is an essential negative regulator of T cell immune responses and is a key checkpoint regulating 13 autoimmunity and anti-tumour immunity. Genetic mutations resulting in a quantitative defect in CTLA4 are 14 associated with the development of an immune dysregulation syndrome. Endocytosis of CTLA4 is rapid and 15 continuous with subsequent degradation or recycling. CTLA4 has two natural ligands, the surface transmembrane 16 proteins CD80 and CD86 that are shared with the T cell co-stimulatory receptor CD28. Upon ligation with 17 CD80/CD86, CTLA4 can remove these ligands from the opposing cells by transendocytosis. The efficiency of 18 ligand removal is thought to be highly dependent on the processes involved in CTLA4 trafficking. With a combined 19 in vitro-in silico study, we quantify the rates of CTLA4 internalization, recycling and degradation. We incorporate 20 experimental data from cell lines and primary human T cells. Our model provides a framework for exploring the 21 impact of altered affinity of natural ligands or therapeutic anti-CTLA4 antibodies and for predicting the effect of 22 clinically relevant CTLA4 pathway mutations. The presented methodology for extracting trafficking rates can be 23 transferred to the study of other transmembrane proteins. 24 1 In a healthy organism, the immune system has to maintain a balance between immune activation and inhibition. 26 The decision between these two outcomes is influenced by receptors which are not specific for any antigenic stimulus. 27CD28 and CTLA4 are two such transmembrane receptors expressed by lymphocytes with opposing regulatory functions. 28 These receptors bind to the same co-stimulatory ligands, CD80 and CD86, which are expressed by antigen presenting 29 cells [1, 2]. While ligation of CD28 with co-stimulatory ligands is essential for full T cell activation and effector 30 functions [3][4][5], CTLA4 inhibits excess or aberrant co-stimulation of T cells by competing for CD28 ligands and 31 thereby preventing uncontrolled T cell activation and clonal expansion of cells specific for healthy tissues [6][7][8]. 32CTLA4 molecules are mostly observed in cytoplasmic vesicles [9][10][11][12] by the virtue of their interaction with the µ2 33 subunit of the clathrin adaptor protein complex AP2 [13][14][15][16]. In contrast, CD28 is present on the plasma membrane 34 with a slow turnover rate [17]. The unusual localization of CTLA4 raises questions about the mode of CTLA4 action. 35 For example, it is not clear how the particular distribution of CTLA4 molecules results from dynamic trafficking between 36 cytosol and plasma membrane. The impact of various parameters affecting internalization, degradation and recycling 37 rates all have the potential to influence the cellular distribution and function of CTLA4. Accordingly, mathematical 38 models are useful in understanding and predicting the impact of changes in these parameters on CTLA4 localization 39 and capacity to elicit suppressive function. 40 Early data suggested that CT...

A local regulatory T cell feedback circuit maintains immune homeostasis by pruning self-activated T cells

“…Costimulatory signaling and inhibition by CTLA4 (DC:TC2 and TR:DC1) This component describes binding kinetics of CD80jCD86, CD28, and CTLA4 and the trans-endocytosis of CD80jCD86 by CTLA4 (Collins et al, 2002;Jansson et al, 2005;Khailaie et al, 2017;van der Merwe et al, 1997;Qureshi et al, 2011;Suga ´r et al, 2017).…”