Quantitative determination of absolute and relative protein amounts is an essential requirement for most current bottom-up proteomics applications, but protein quantitation estimates are affected by several sources of variability such as sample preparation, mass spectrometric acquisition, and data analysis. Among them, sample digestion has attracted much attention from the proteomics community, as protein quantitation by bottom-up proteomics relies on the efficiency and reproducibility of protein enzymatic digestion, with the presence of missed cleavages, nonspecific cleavages, or even the use of different proteases having been postulated as important sources of variation in protein quantitation. Here we evaluated both in-solution and filter-aided digestion protocols and assessed their influence in the estimation of protein abundances using five E. coli mixtures with known amounts of spiked proteins. We observed that replicates of trypsin specificity digestion protocols are highly reproducible in terms of peptide quantitation, with digestion technique and the chosen proteolytic enzyme being the major sources of variability in peptide quantitation. Finally, we also evaluated the result of including peptides with missed cleavages in protein quantitation and observed no significant differences in precision, accuracy, specificity, and sensitivity compared with the use of fully tryptic peptides.

Chronic antigenic stimulation can trigger the differentiation of antigen-experienced CD4+ T cells into T regulatory type 1 (TR1) cells, a subset of interleukin-10-producing Treg cells that do not express FOXP3. The identities of the progenitor(s) and transcriptional regulators of this T-cell subset remain unclear. Here, we show that the peptide-major histocompatibility complex class II (pMHCII) monospecific immunoregulatory T-cell pools that arise in vivo in different genetic backgrounds in response to pMHCII-coated nanoparticles (pMHCII-NPs) are invariably comprised of oligoclonal subpools of T follicular helper (TFH) and TR1 cells with a nearly identical clonotypic composition but different functional properties and transcription factor expression profiles. Pseudotime analyses of scRNAseq data and multidimensional mass cytometry revealed progressive downregulation and upregulation of TFH and TR1 markers, respectively. Furthermore, pMHCII-NPs trigger cognate TR1 cell formation in TFH cell-transfused immunodeficient hosts, and T-cell-specific deletion of Bcl6 or Irf4 blunts both the TFH expansion and TR1 formation induced by pMHCII-NPs. In contrast, deletion of Prdm1 selectively abrogates the TFH-to-TR1 conversion. Bcl6 and Prdm1 are also necessary for anti-CD3 mAb-induced TR1 formation. Thus, TFH cells can differentiate into TR1 cells in vivo, and BLIMP1 is a gatekeeper of this cellular reprogramming event.

Down syndrome (DS), caused by trisomy of chromosome 21, is the most common genetic cause of intellectual disability. We recently discovered that green tea extracts containing epigallocatechin-3-gallate (EGCG) improve cognition in mice transgenic for Dyrk1a (TgDyrk1A) and in a trisomic DS mouse model (Ts65Dn). Interestingly, paired with cognitive stimulation, green tea has beneficial pro-cognitive effects in DS individuals. Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase 1A (DYRK1A) is a major candidate to explain the cognitive phenotypes of DS, and inhibiting its activity is a promising pro-cognitive therapy. DYRK1A kinase activity can be normalized in the hippocampus of transgenic DYRK1A mice administering green tea extracts, but also submitting the animals to environmental enrichment (EE). However, many other mechanisms could also explain the pro-cognitive effects of green tea extracts and EE. To underpin the overall alterations arising upon DYRK1A overexpression and the molecular processes underneath the pro-cognitive effects, we used quantitative proteomics. We investigated the hippocampal (phospho)proteome in basal conditions and after treatment with a green tea extract containing EGCG and/or EE in TgDyrk1A and control mice. We found that Dyrk1A overexpression alters protein and phosphoprotein levels of key postsynaptic and plasticity-related pathways and that these alterations were rescued upon the cognitive enhancer treatments.

Assembly of soluble peptide-major histocompatibility complex class II (pMHCII) monomers into multimeric structures enables the detection of antigen-specific CD4+ T cells in biological samples and, in some configurations, their reprogramming in vivo. Unfortunately, current MHCII-αβ chain heterodimerization strategies are typically associated with low production yields and require the use of foreign affinity tags for purification, precluding therapeutic applications in humans. Here, we show that fusion of peptide-tethered or empty MHCII-αβ chains to the IgG1-Fc mutated to form knob-into-hole structures results in the assembly of highly stable pMHCII monomers. This design enables the expression and rapid purification of challenging pMHCII types at high yields without the need for leucine zippers and purification affinity tags. Importantly, this design increases the antigen-receptor signaling potency of multimerized derivatives useful for therapeutic applications and facilitates the detection and amplification of low-avidity T cell specificities in biological samples using flow cytometry.