Continuous acquisition of MHC:peptide complexes by recipient cells contributes to the generation of anti-graft CD8+T cell immunity

Understanding the evolution of the direct and indirect pathways of allorecognition following tissue transplantation is essential in the design of tolerance-promoting protocols. On the basis that donor bone marrow-derived antigen presenting cells are eliminated within days of transplantation, it has been argued that the indirect response represents the major threat to long term transplant survival, and is consequently the key target for regulation. However, the detection of MHC transfer between cells, and particularly the capture of MHC:peptide complexes by dendritic cells, led us to propose a third, semi-direct, pathway of MHC allorecognition. Persistence of this pathway would lead to sustained activation of direct pathway T cells, arguably persisting for the life of the transplant. In this study, we focused on the contribution of acquired MHC class I, on recipient DCs, during the life span of a skin graft. We observed that MHC class I acquisition by recipient DCs occurs for at least one month following transplantation and may be the main source of alloantigen that drives CD8+ cytotoxic T cell responses. In addition, acquired MHC class I-peptide complexes stimulate T cell responses in vivo further emphasizing the need to regulate both pathways to induce indefinite survival of the graft.

In vitro and in vivo insulinotropic properties of the multifunctional frog skin peptide hymenochirin-1B: a structure–activity study

Hymenochirin-1b (Hym-1B; IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is a cationic, α-helical amphibian host-defense peptide with antimicrobial, anticancer, and immunomodulatory properties. This study investigates the abilities of the peptide and nine analogues containing substitutions of Pro5, Glu6, and Asp9 by either l-lysine or d-lysine to stimulate insulin release in vitro using BRIN-BD11 clonal β cells or isolated mouse islets and in vivo using mice fed a high-fat diet to produce obesity and insulin resistance. Hym-1B produced a significant and concentration-dependent increase in the rate of insulin release from BRIN-BD11 cells without cytotoxicity at concentrations up to 1 µM with a threshold concentration of 1 nM. The threshold concentrations for the analogues were: [P5K], [E6K], [D9K], [P5K, E6K] and [E6K, D9k] 0.003 nM, [E6K, D9K] and [D9k] 0.01 nM, [P5K, D9K] 0.1 nM and [E6k] 0.3 nM. All peptides displayed cytotoxicity at concentrations ≥1 µM except the [P5K] and [D9k] analogues which were non-toxic at 3 µM. The potency and maximum rate of insulin release from mouse islets produced by the [P5K] peptide were significantly greater than produced by Hym-1B. Neither Hym-1B nor the [P5K] analogue at 1 µM concentration had an effect on membrane depolarization or intracellular Ca2+. The [P5K] analogue (1 µM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and stimulated GLP-1 secretion from GLUTag cells. Down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of [P5K]hym-1B. Intraperitoneal administration of the [P5K] and [D9k] analogues (75 nmol/kg body weight) to high-fat-fed mice with insulin resistance significantly enhanced glucose tolerance with a concomitant increase in insulin secretion. We conclude that [P5K]hym-1B and [D9k]hym-1B show potential for development into anti-diabetic agents.