Development and optimization of the human allogeneic mixed lymphocyte islet (MLIC) and acinar (MLAC) coculture system

Abstract

Although human islet allotransplantation has been shown to be effective in restoring normoglycaemia to diabetic recipients, the long-term success rate is relatively low. The poor results of human islet transplantation are surprising given the relative ease of establishing functioning islet allografts in rodent models with minimal immunosuppression and have been attributed to a number of factors of which rejection may play a major part. The impracticality of studying the immunogenicity of human islet in vivo has led us to develop a model specifically to study the allogenic immune response to the major components of isolated human islet preparations. To try and overcome the variability associated with this methodology, this study was designed to develop and optimize the mixed lymphocyte coculture as an in vitro model for the initial alloresponse to both freshly isolated intact human islets (MLIC) and acinar tissue pieces (MLAC). For this, titration of the islets and acinar tissue, as well as kinetic studies of the response, were used to select optimum conditions for the MLIC which were shown to be ten islets and ten acinar tissue pieces with a coculture duration of 7–9 days. The optimized studies included the use of non-tissue culture microwells to deter fibroblast growth, RPMI + 10% human AB serum to reduce the background lymphocyte response and a low concentration of dithizone to stain the islets prior to handpicking. Freshly isolated human islets were found to stimulate allogeneic lymphocytic proliferation; however, differences in the relative response to islets and acinar tissue led us to test the effect of soluble products from the acinar cells on lymphocyte proliferation in the mixed lymphocyte reaction. An inhibitory effect of the soluble products of acinar cells on the allogeneic lymphocyte proliferative response was found to contribute to the reduced response of the MLAC compared to the MLIC. Human islets were shown to stimulate an allogeneic immune response in vitro, and this MLIC model provides a method for assessing the specific immunogenicity of human isolated islet preparations and associated acinar tissue in transplantation.