Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

The ability to identify and characterise individual cells of the immune system under label-free conditions would be a significant advantage in biomedical and clinical studies where untouched and unmodified cells are required. We present a multi-modal system capable of simultaneously acquiring both single point Raman spectra and digital holographic images of single cells. We use this combined approach to identify and discriminate between immune cell populations CD4+ T cells, B cells and monocytes. We investigate several approaches to interpret the phase images including signal intensity histograms and texture analysis. Both modalities are independently able to discriminate between cell subsets and dual-modality may therefore be used a means for validation. We demonstrate here sensitivities achieved in the range of 86.8% to 100%, and specificities in the range of 85.4% to 100%. Additionally each modality provides information not available from the other providing both a molecular and a morphological signature of each cell.

Objective HLA–B27 is associated with the inflammatory spondyloarthritides (SpA), although subtypes HLA–B*27:06 and HLA–B*27:09 are not. These subtypes differ from the HLA–B*27:05 disease‐associated allele primarily at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen‐binding groove. Dimerization of HLA–B27 during assembly has been implicated in disease onset. The purpose of this study was to investigate the factors that influence differences in dimerization between disease‐associated and non–disease‐associated HLA–B27 alleles. Methods HLA–B*27:05 and mutants resembling the HLA–B*27:06 and 09 subtypes were expressed in the rat C58 T cell line, the human CEM T cell line and its calnexin‐deficient variant CEM.NKR. Immunoprecipitation, pulse–chase experiments, flow cytometry, and immunoblotting were performed to study the assembly kinetics, heavy‐chain dimerization, and chaperone associations. Results By expressing HLA–B*27:05, 06‐like, and 09 alleles on a restrictive rat transporter associated with antigen processing background, we demonstrate that a tyrosine expressed at p116, either alone or together with an aspartic acid residue at p114, inhibited HLA–B27 dimerization and increased the assembly rate. F‐pocket residues altered the associations with chaperones of the early major histocompatibility complex class I folding pathway. Calnexin was demonstrated to participate in endoplasmic reticulum (ER) stress–mediated degradation of dimers, whereas the oxidoreductase ERp57 does not appear to influence dimerization. Conclusion Residues within the F pocket of the peptide‐binding groove, which differ between disease‐associated and non–disease‐associated HLA–B27 subtypes, can influence the assembly process and heavy‐chain dimerization, events which have been linked to the initiation of disease pathogenesis.

Extracellular vesicles (EVs) are released by most cell types and have been associated with multiple immunomodulatory functions. MHC class I molecules have crucial roles in antigen presentation and in eliciting immune responses and are known to be incorporated into EVs. However, the MHC class I immunopeptidome of EVs has not been established. Here, using a small-scale immunoisolation of the antigen serotypes HLA-A*02:01 and HLA-B*27:05 expressed on the Epstein-Barr virus-transformed B cell line Jesthom and MS of the eluted peptides from both cells and EVs, we identified 516 peptides that bind either HLA-A*02:01 or HLA-B*27:05. Of importance, the predicted serotype-binding affinities and peptide-anchor motifs did not significantly differ between the peptide pools isolated from cells or EVs, indicating that during EV biogenesis, no obvious editing of the MHC class I immunopeptidome occurs. These results, for the first time, establish EVs as a source of MHC class I peptides that can be used for the study of the immunopeptidome and in the discovery of potential neoantigens for immunotherapies.

No abstract