In this review, we first consider the inherent structural constraints for binding of a peptide to MHC class II molecules. Such parameters at the site of TCR recognition are dependent upon the efficient generation of the antigenic determinant during natural processing of the whole protein antigen. Strikingly, only a minor fraction of such potential determinants on an antigen are presented in an immunodominant manner, while the remaining peptides are silent (cryptic). Why one determinant is selected while the majority are neglected is still unresolved, but we review the experimental evidence pertaining to this choice. Thus, features of the antigen remote from the actual determinant can either steer processing toward disclosure or revelation of a determinant, or interfere with the binding of peptides to MHC (hinderotopy). The evidence is reviewed for "MHC-guided processing," where the unfolding antigen binds at an early stage to an MHC molecule through its most available and affine agretope and then is trimmed down to final size, while the rest of the molecule, including cryptic determinants, is discarded. Different MHC molecules can compete for determinants at an early stage of processing when the antigen is close to its original length. There are shifts in the hierarchy of display of dominant and cryptic determinants, and these shifts relate to local inflammatory states, to changes in the state or composition of the APC population, and to aspects of exogenous vs endogenous processing. The impact of this differential display of determinants on tolerance and autoimmunity is discussed.

Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host’s lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation.

BackgroundSecondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials.ResultsThe new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse.ConclusionMIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.