Peer review process
Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, public reviews, and a provisional response from the authors.
Read more about eLife’s peer review process.Editors
- Reviewing EditorMia SmithUniversity of Colorado Anschutz Medical Campus, Aurora, United States of America
- Senior EditorTadatsugu TaniguchiThe University of Tokyo, Tokyo, Japan
Reviewer #1 (Public review):
The work presented by Cheung et al. used a quantitative proteomics method to capture molecular changes in B cells exposed to LPS and IL-4, a combination of stimuli activating naive B cells. Amino acid transporters, cholesterol biosynthetic enzymes, ribosomal components, and other proteins involved in cell proliferation were found to increase in stimulated B cells. Experiments involving genetic loss-of-function (SLC7A5), pharmacological inhibition (HMGCR, SQLE, prenylation), and functional rescue by metabolites (mevalonate, GGPP) validated the proteomics data and revealed that amino acid uptake, cholesterol/mevalonate biosynthesis, and cholesterol uptake played a crucial role in B cell proliferation, survival, biogenesis, and immunoglobulin class switching. Experiments involving cholesterol-free medium showed that both biosynthesis and LDLR-mediated uptake catered to the cholesterol demand of LPS/IL-4-stimulated B cells. A role for protein prenylation in LDLR-mediated cholesterol uptake was postulated and backed by divergent effects of GGPP rescue in the presence and absence of cholesterol in culture medium.
Strengths:
The discovery was made by proteome-wide profiling and unbiased computational analysis. The discovered proteins were functionally validated using appropriate tools and approaches. The metabolic processes identified and prioritized from this comprehensive survey and systematic validation are highly likely to represent mechanisms of high importance and influence. Analysis of immune cell metabolism at the protein level is relatively compared to transcriptomic and metabolomic analysis.
The conclusions from functional validation experiments were supported by clear data and based on rational interpretations. This was enabled by well-established readouts/analytical methods used to analyze cell proliferation, viability, size, cholesterol content, and transporter/enzyme function. The data generated from these experiments strongly support the conclusions.
This work reveals a complex, yet intriguing, relationship between cholesterol metabolism and protein prenylation as they serve to promote B cell activation. The effects of pharmacological inhibition and metabolite replenishment on the cholesterol content and activation of B cells were precisely determined and logically interpreted.
Weaknesses:
The findings of this study were obtained almost exclusively from ex vivo B cell stimulation experiments. Their contribution to B cell state and B-cell-mediated immune responses in vivo was not explored. Without in vivo data, the study still provides valuable mechanistic information and insights, but it remains unknown, and there is no discussion about how the identified mechanisms may play out in B cell immunity.
The role of HMGCR, SQLE, and prenylation in B cell activation was assessed using pharmacological inhibitors. Evidence from other loss-of-function approaches, which could strengthen the conclusions, does not exist. This is a moderate weakness.
Reviewer #2 (Public review):
This study uses mass spectrometry to quantify how LPS and IL-4 modify the mouse B cell proteome as naïve cells undergo blastogenesis and enter the cell cycle. This analysis revealed changes in key proteins involved in amino acid transport and cholesterol biosynthesis. Genetic and pharmacological experiments indicated important roles for these metabolic processes in B cell proliferation.
This work provides new information about the regulation of TI B cell responses by changes in cell metabolism and also a comprehensive mass spectrometry dataset, which will be an important general resource for future studies. The experiments are thorough and carefully carried out. The majority of conclusions are backed up by data that is shown to be highly significant statistically.
The study would be strengthened by additional experiments to determine whether the detected changes are unique to stimulation with LPS + IL-4 or more generic responses of resting B cells to mitogenic agonists.