Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity

CTLA-4: a moving target in immunotherapy

AbstractCD28 and CTLA-4 are members of a family of immunoglobulin-related receptors that are responsible for various aspects of T-cell immune regulation. The family includes CD28, CTLA-4, and ICOS as well as other proteins, including PD-1, BTLA, and TIGIT. These receptors have both stimulatory (CD28, ICOS) and inhibitory roles (CTLA-4, PD-1, BTLA, and TIGIT) in T-cell function. Increasingly, these pathways are targeted as part of immune modulatory strategies to treat cancers, referred to generically as immune checkpoint blockade, and conversely to treat autoimmunity and CTLA-4 deficiency. Here, we focus on the biology of the CD28/CTLA-4 pathway as a framework for understanding the impacts of therapeutic manipulation of this pathway.</jats:p

Quantitative characterization of CTLA4 trafficking and turnover using a combined <i>in vitro</i> and <i>in silico</i> approach

AbstractCTLA4 is an essential negative regulator of T cell immune responses and is a key checkpoint regulating autoimmunity and anti-tumour immunity. Genetic mutations resulting in a quantitative defect in CTLA4 are associated with the development of an immune dysregulation syndrome. Endocytosis of CTLA4 is rapid and continuous with subsequent degradation or recycling. CTLA4 has two natural ligands, the surface transmembrane proteins CD80 and CD86 that are shared with the T cell co-stimulatory receptor CD28. Upon ligation with CD80/CD86, CTLA4 can remove these ligands from the opposing cells by transendocytosis. The efficiency of ligand removal is thought to be highly dependent on the processes involved in CTLA4 trafficking. With a combined in vitro-in silico study, we quantify the rates of CTLA4 internalization, recycling and degradation. We incorporate experimental data from cell lines and primary human T cells. Our model provides a framework for exploring the impact of altered affinity of natural ligands or therapeutic anti-CTLA4 antibodies and for predicting the effect of clinically relevant CTLA4 pathway mutations. The presented methodology for extracting trafficking rates can be transferred to the study of other transmembrane proteins.</jats:p

CD86 Is a Selective CD28 Ligand Supporting FoxP3+ Regulatory T Cell Homeostasis in the Presence of High Levels of CTLA-4

CD80 and CD86 are expressed on antigen presenting cells and are required to engage their shared receptor, CD28, for the costimulation of CD4 T cells. It is unclear why two stimulatory ligands with overlapping roles have evolved. CD80 and CD86 also bind the regulatory molecule CTLA-4. We explored the role of CD80 and CD86 in the homeostasis and proliferation of CD4+FoxP3+ regulatory T cells (Treg), which constitutively express high levels of CTLA-4 yet are critically dependent upon CD28 signals. We observed that CD86 was the dominant ligand for Treg proliferation, survival, and maintenance of a regulatory phenotype, with higher expression of CTLA-4, ICOS, and OX40. We also explored whether CD80-CD28 interactions were specifically compromised by CTLA-4 and found that antibody blockade, clinical deficiency of CTLA-4 and CRISPR-Cas9 deletion of CTLA-4 all improved Treg survival following CD80 stimulation. Taken together, our data suggest that CD86 is the dominant costimulatory ligand for Treg homeostasis, despite its lower affinity for CD28, because CD80-CD28 interactions are selectively impaired by the high levels of CTLA-4. These data suggest a cell intrinsic role for CTLA-4 in regulating CD28 costimulation by direct competition for CD80, and indicate that that CD80 and CD86 have discrete roles in CD28 costimulation of CD4 T cells in the presence of high levels of CTLA-4.</jats:p

Characterization of CTLA4 Trafficking and Implications for Its Function

CTLA4 is an essential negative regulator of T-cell immune responses and a key checkpoint regulating autoimmunity and antitumor responses. Genetic mutations resulting in quantitative defects in the CTLA4 pathway are also associated with the development of immune dysregulation syndromes in humans. It has been proposed that CTLA4 functions to remove its ligands CD80 and CD86 from opposing cells by a process known as transendocytosis. A quantitative characterization of CTLA4 synthesis, endocytosis, degradation, and recycling and how these affect its function is currently lacking. In a combined in vitro and in silico study, we developed a mathematical model and identified these trafficking parameters. Our model predicts optimal ligand removal in an intermediate affinity range. The intracellular CTLA4 pool as well as fast internalization, recovery of free CTLA4 from internalized complexes, and recycling is critical for sustained functionality. CD80-CTLA4 interactions are predicted to dominate over CD86-CTLA4. Implications of these findings in the context of control of antigen-presenting cells by regulatory T cells and of pathologic genetic deficiencies are discussed. The presented mathematical model can be reused in the community beyond these questions to better understand other trafficking receptors and study the impact of CTLA4 targeting drugs

The in vitro characterization of a gelatin scaffold, prepared by cryogelation and assessed in vivo as a dermal replacement in wound repair

Abstract A sheet gelatin scaffold with attached silicone pseudoepidermal layer for wound repair purposes was produced by a cryogelation technique. The resulting scaffold possessed an interconnected macroporous structure with a pore size distribution of 131±17μm at one surface decreasing to 30±8μm at the attached silicone surface. The dynamic storage modulus (G′) and mechanical stability were comparable to the clinical gold standard dermal regeneration template, Integra®. The scaffolds were seeded in vitro with human primary dermal fibroblasts. The gelatin based material was not only non-cytotoxic, but over a 28day culture period also demonstrated advantages in cell migration, proliferation and distribution within the matrix when compared with Integra®. When seeded with human keratinocytes, the neoepidermal layer that formed over the cryogel scaffold appeared to be more advanced and mature when compared with that formed over Integra®. The in vivo application of the gelatin scaffold in a porcine wound healing model showed that the material supports wound healing by allowing host cellular infiltration, biointegration and remodelling. The results of our in vitro and in vivo studies suggest that the gelatin based scaffold produced by a cryogelation technique is a promising material for dermal substitution, wound healing and other potential biomedical applications