Activation of human CD4+ T cells by targeting MHC class II epitopes to endosomal compartments using human CD1 tail sequences.

Distinct CD4(+) T-cell epitopes within the same protein can be optimally processed and loaded into major histocompatibility complex (MHC) class II molecules in disparate endosomal compartments. The CD1 protein isoforms traffic to these same endosomal compartments as directed by unique cytoplasmic tail sequences, therefore we reasoned that antigen/CD1 chimeras containing the different CD1 cytoplasmic tail sequences could optimally target antigens to the MHC class II antigen presentation pathway. Evaluation of trafficking patterns revealed that all four human CD1-derived targeting sequences delivered antigen to the MHC class II antigen presentation pathway, to early/recycling, early/sorting and late endosomes/lysosomes. There was a preferential requirement for different CD1 targeting sequences for the optimal presentation of an MHC class II epitope in the following hierarchy: CD1b > CD1d = CD1c > > > CD1a or untargeted antigen. Therefore, the substitution of the CD1 ectodomain with heterologous proteins results in their traffic to distinct intracellular locations that intersect with MHC class II and this differential distribution leads to specific functional outcomes with respect to MHC class II antigen presentation. These findings may have implications in designing DNA vaccines, providing a greater variety of tools to generate T-cell responses against microbial pathogens or tumours.

Natural killer cell subsets differentially reject embryonic stem cells based on licensing.

BACKGROUND: Embryonic stem cells (ESC) and induced pluripotent stem cells provide great promise to the future of medicine. Because immune rejection represents a major obstacle to the success of all stem cell-based therapies, many recent studies have sought to determine the key immune mediators involved in ESC rejection. The role of natural killer (NK) cells and specifically the role of NK cell licensing is not well understood in ESC rejection. METHODS: Mouse or human ESCs were subjected to cytotoxicity assays involving their respective species-matched activated NK cells. Mouse ESCs were then transplanted to allogeneic recipients after depletion of NK cell subsets in the host. ESC engraftment was analyzed by bioluminescent imaging. RESULTS: Depletion of all NK cells in vivo resulted in the greatest amount of ESC engraftment, confirming a role for NK cells in ESC rejection. Importantly, depletion of the Ly49C/I or Ly49G2 NK cell subsets resulted in differential ESC engraftment and rejection. This indicates that NK cell rejection of allogeneic ESC is highly differential based on the presence of licensed NK cells. Blocking NKG2D in vitro resulted in less killing of mESC by allogeneic NK cells, indicating NKG2D is a likely mechanism for NK-mediated killing of mESC. CONCLUSIONS: In this study, we show that expression of inhibitory Ly49s correlates with the ability of NK cells to kill murine ESC in an NKG2D-dependent manner. This further suggests that the rejection of similar stem cell transplants in humans will be dependent upon the presence of licensed NK cells.

Valosin-containing protein gene mutations: cellular phenotypes relevant to neurodegeneration.

Previously, we identified valosin-containing protein (VCP) as a mediator of ER stress-induced cell death. Mutations in the VCP gene including R93, R155, and R191 have been described that manifest clinically as hereditary inclusion body myopathy with Paget's disease of bone and frontotemporal dementia. In addition, other studies have demonstrated that as a consequence of a mutation generated in the second ATP binding domain of VCP (K524A), cells accumulated large cytoplasmic vacuoles and underwent programmed cell death. In order to better understand the biochemical and molecular consequences of the clinically relevant VCP mutations as well as the genetically engineered ATPase-inactive mutant K524A and any relationship these may have to ER stress-induced cell death, we introduced analogous mutations separately and together into the human VCP gene and evaluated their effect on proteasome activity, Huntingtin protein aggregation and ER stress-induced cell death. Our results indicate that the VCP K524A mutant and the triple mutant VCP R93C-R155C-K524A block protein degradation, trigger Huntingtin aggregate formation, and render cells highly susceptible to ER stress-induced cell death as compared to VCPWT or other VCP mutants.