α-galactosylceramide-reactive NKT cells increase IgG1 class switch against a C. difficile polysaccharide antigen and enhance immunity against a live pathogen challenge

All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and when conjugated to a protein carrier induces a protective antibody response in animal models. Given that CD1d-restricted Natural Killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG titers were evident in sera from immunized mice. The inclusion of α-GC had a modest influence on isotype switch but increasing the ratio of IgG1/IgG2c. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18 -/- mice were not protected by the PSII/α-GC immunization modality. Absence of NKT cells similarly had a modest effect on isotype switch but ratios of IgG1/IgG2c decreased. These results indicate that α-GC-driven NKT cells move the humoral immune response against C. difficile PSII antigen towards Th2-driven IgG1 and may contribute to augmented protection. This study suggests that NKT activation represents a pathway for additional B cell help that could be used to supplement existing efforts to develop vaccines against polysaccharides derived from C. difficile and other pathogens.

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway

To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered LAMP (lysosome associated membrane protein)-carrier vesicles around multivesicular bodies, as well as the appearance of ‘hourglass’ profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of CHMP6 (charged multi-vesicular body protein 6) and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.

Quantitative consequences of protein carriers in immunopeptidomics and tyrosine phosphorylation MS2 analyses

Utilizing a protein carrier in combination with isobaric labeling to "boost" the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides. Here, we describe two sets of analyses comparing MS2-based quantitation using a 20x protein carrier in pMHC analyses and a high (~100x) and low (~9x) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the control condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward.

In situ synthesis, stabilization and activity of protein-modified gold nanoparticles for biological applications

We demonstrate an in situ synthesis, stabilization and activity of a nanoparticle-based protein carrier platform via the Layer-by-Layer (LbL) technology.