Author response:
eLife Assessment
This study provides useful insights into the ways in which germinal center B cell metabolism, particularly lipid metabolism, affects cellular responses. The authors use sophisticated mouse models to demonstrate that ether lipids are relevant for B cell homeostasis and efficient humoral responses. Although the data were collected from in vitro and in vivo experiments and analyzed using solid and validated methodology, more careful experiments and extensive revision of the manuscript will be required to strengthen the authors' conclusions.
In addition to praise for the eLife system and transparency (public posting of the reviews; along with an opportunity to address them), we are grateful for the decision of the Editors to select this submission for in-depth peer review and to the referees for the thoughtful and constructive comments.
In overview, we mostly agree with the specific comments and evaluation of strengths of what the work adds as well as with indications of limitations and caveats that apply to the breadth of conclusions. One can view these as a combination of weaknesses, of instances of reading more into the work than what it says, and of important future directions opened up by the findings we report. Regarding the positives, we appreciate the reviewers' appraisal that our work unveils a novel mechanism in which the peroxisomal enzyme PexRAP mediates B cell intrinsic ether lipid synthesis and promotes a humoral immune response. We are gratified by a recognition that a main contribution of the work is to show that a spatial lipidomic analysis can set the stage for discovery of new molecular processes in biology that are supported by using 2-dimensional imaging mass spectrometry techniques and cell type specific conditional knockout mouse models.
By and large, the technical issues are items we will strive to improve. Ultimately, an over-arching issue in research publications in this epoch are the questions "when is enough enough?" and "what, or how much, advance will be broadly important in moving biological and biomedical research forward?" It appears that one limitation troubling the reviews centers on whether the mechanism of increased ROS and multi-modal death - supported most by the in vitro evidence - applies to germinal center B cells in situ, versus either a mechanism for decreased GC that mostly applies to the pre-GC clonal amplification (or recruitment into GC). Overall, we agree that this leap could benefit from additional evidence - but as resources ended we instead leave that question for the future other than the findings with S1pr2-CreERT2-driven deletion leading to less GC B cells. While we strove to be very careful in framing such a connection as an inference in the posted manuscript, we will revisit the matter via rechecking the wording when revising the text after trying to get some specific evidence.
In the more granular part of this provisional response (below), we will outline our plan prompted by the reviewers but also comment on a few points of disagreement or refinement (longer and more detailed explanation). The plan includes more detailed analysis of B cell compartments, surface level of immunoglobulin, Tfh cell population, a refinement of GC B cell markers, and the ex vivo GC B cell analysis for ROS, proliferation, and cell death. We will also edit the text to provide more detailed information and clarify our interpretation to prevent the confusion of our results. At a practical level, some evidence likely is technologically impractical, and an unfortunate determinant is the lack of further sponsored funding for further work. The detailed point-by-point response to the reviewer’s comments is below.
Public Reviews:
Reviewer #1 (Public review):
In this manuscript, Sung Hoon Cho et al. presents a novel investigation into the role of PexRAP, an intermediary in ether lipid biosynthesis, in B cell function, particularly during the Germinal Center (GC) reaction. The authors profile lipid composition in activated B cells both in vitro and in vivo, revealing the significance of PexRAP. Using a combination of animal models and imaging mass spectrometry, they demonstrate that PexRAP is specifically required in B cells. They further establish that its activity is critical upon antigen encounter, shaping B cell survival during the GC reaction.
Mechanistically, they show that ether lipid synthesis is necessary to modulate reactive oxygen species (ROS) levels and prevent membrane peroxidation.
Highlights of the Manuscript:
The authors perform exhaustive imaging mass spectrometry (IMS) analyses of B cells, including GC B cells, to explore ether lipid metabolism during the humoral response. This approach is particularly noteworthy given the challenge of limited cell availability in GC reactions, which often hampers metabolomic studies. IMS proves to be a valuable tool in overcoming this limitation, allowing detailed exploration of GC metabolism.
The data presented is highly relevant, especially in light of recent studies suggesting a pivotal role for lipid metabolism in GC B cells. While these studies primarily focus on mitochondrial function, this manuscript uniquely investigates peroxisomes, which are linked to mitochondria and contribute to fatty acid oxidation (FAO). By extending the study of lipid metabolism beyond mitochondria to include peroxisomes, the authors add a critical dimension to our understanding of B cell biology.
Additionally, the metabolic plasticity of B cells poses challenges for studying metabolism, as genetic deletions from the beginning of B cell development often result in compensatory adaptations. To address this, the authors employ an acute loss-of-function approach using two conditional, cell-type-specific gene inactivation mouse models: one targeting B cells after the establishment of a pre-immune B cell population (Dhrs7b^f/f, huCD20-CreERT2) and the other during the GC reaction (Dhrs7b^f/f; S1pr2-CreERT2). This strategy is elegant and well-suited to studying the role of metabolism in B cell activation.
Overall, this manuscript is a significant contribution to the field, providing robust evidence for the fundamental role of lipid metabolism during the GC reaction and unveiling a novel function for peroxisomes in B cells.
We appreciate these positive reactions and response, and agree with the overview and summary of the paper's approaches and strengths.
However, several major points need to be addressed:
Major Comments:
Figures 1 and 2
The authors conclude, based on the results from these two figures, that PexRAP promotes the homeostatic maintenance and proliferation of B cells. In this section, the authors first use a tamoxifen-inducible full Dhrs7b knockout (KO) and afterwards Dhrs7bΔ/Δ-B model to specifically characterize the role of this molecule in B cells. They characterize the B and T cell compartments using flow cytometry (FACS) and examine the establishment of the GC reaction using FACS and immunofluorescence. They conclude that B cell numbers are reduced, and the GC reaction is defective upon stimulation, showing a reduction in the total percentage of GC cells, particularly in the light zone (LZ).
The analysis of the steady-state B cell compartment should also be improved. This includes a more detailed characterization of MZ and B1 populations, given the role of lipid metabolism and lipid peroxidation in these subtypes.
Suggestions for Improvement:
B Cell compartment characterization: A deeper characterization of the B cell compartment in non-immunized mice is needed, including analysis of Marginal Zone (MZ) maturation and a more detailed examination of the B1 compartment. This is especially important given the role of specific lipid metabolism in these cell types. The phenotyping of the B cell compartment should also include an analysis of immunoglobulin levels on the membrane, considering the impact of lipids on membrane composition.
Although the manuscript is focused on post-ontogenic B cell regulation in Ab responses, we believe we will be able to polish a revised manuscript through addition of results of analyses suggested by this point in the review: measurement of surface IgM on and phenotyping of various B cell subsets, including MZB and B1 B cells, to extend the data in Supplemental Fig 1H and I. Depending on the level of support, new immunization experiments to score Tfh and analyze a few of their functional molecules as part of a B cell paper may be feasible.
- GC Response Analysis Upon Immunization: The GC response characterization should include additional data on the T cell compartment, specifically the presence and function of Tfh cells. In Fig. 1H, the distribution of the LZ appears strikingly different. However, the authors have not addressed this in the text. A more thorough characterization of centroblasts and centrocytes using CXCR4 and CD86 markers is needed.
The gating strategy used to characterize GC cells (GL7+CD95+ in IgD− cells) is suboptimal. A more robust analysis of GC cells should be performed in total B220+CD138− cells.
We first want to apologize the mislabeling of LZ and DZ in Fig 1H. The greenish-yellow colored region (GL7+ CD35+) indicate the DZ and the cyan-colored region (GL7+ CD35+) indicates the LZ.
As a technical note, we experienced high background noise with GL7 staining uniquely with PexRAP deficient (Dhrs7bf/f; Rosa26-CreERT2) mice (i.e., not WT control mice). The high background noise of GL7 staining was not observed in B cell specific KO of PexRAP (Dhrs7bf/f; huCD20-CreERT2). Two formal possibilities to account for this staining issue would be if either the expression of the GL7 epitope were repressed by PexRAP or the proper positioning of GL7+ cells in germinal center region were defective in PexRAP-deficient mice (e.g., due to an effect on positioning cues from cell types other than B cells). In a revised manuscript, we will fix the labeling error and further discuss the GL7 issue, while taking care not to be thought to conclude that there is a positioning problem or derepression of GL7 (an activation antigen on T cells as well as B cells).
While the gating strategy for an overall population of GC B cells is fairly standard even in the current literature, the question about using CD138 staining to exclude early plasmablasts (i.e., analyze B220+ CD138neg vs B220+ CD138+) is interesting. In addition, some papers like to use GL7+ CD38neg for GC B cells instead of GL7+ Fas (CD95)+, and we thank the reviewer for suggesting the analysis of centroblasts and centrocytes. For the revision, we will try to secure resources to revisit the immunizations and analyze them for these other facets of GC B cells (including CXCR4/CD86) and for their GL7+ CD38neg. B220+ CD138- and B220+ CD138+ cell populations.
We agree that comparison of the Rosa26-CreERT2 results to those with B cell-specific loss-of-function raise a tantalizing possibility that Tfh cells also are influenced by PexRAP. Although the manuscript is focused on post-ontogenic B cell regulation in Ab responses, we hope to add a new immunization experiments that scores Tfh and analyzes a few of their functional molecules could be added to this B cell paper, depending on the ability to wheedle enough support / fiscal resources.
- The authors claim that Dhrs7b supports the homeostatic maintenance of quiescent B cells in vivo and promotes effective proliferation. This conclusion is primarily based on experiments where CTV-labeled PexRAP-deficient B cells were adoptively transferred into μMT mice (Fig. 2D-F). However, we recommend reviewing the flow plots of CTV in Fig. 2E, as they appear out of scale. More importantly, the low recovery of PexRAP-deficient B cells post-adoptive transfer weakens the robustness of the results and is insufficient to conclusively support the role of PexRAP in B cell proliferation in vivo.
In the revision, we will edit the text and try to adjust the digitized cytometry data to allow more dynamic range to the right side of the upper panels in Fig. 2E, and otherwise to improve the presentation of the in vivo CTV result. However, we feel impelled to push back respectfully on some of the concern raised here. First, it seems to gloss over the presentation of multiple facets of evidence. The conclusion about maintenance derives primarily from Fig. 2C, which shows a rapid, statistically significant decrease in B cell numbers (extending the finding of Fig. 1D, a more substantial decrease after a bit longer a period). As noted in the text, the rate of de novo B cell production does not suffice to explain the magnitude of the decrease.
In terms of proliferation, we will improve presentation of the Methods but the bottom line is that the recovery efficiency is not bad (comparing to prior published work) inasmuch as transferred B cells do not uniformly home to spleen. In a setting where BAFF is in ample supply in vivo, we transferred equal numbers of cells that were equally labeled with CTV and counted B cells. The CTV result might be affected by lower recovered B cell with PexRAP deficiency, generally, the frequencies of CTVlow divided population are not changed very much. However, it is precisely because of the pitfalls of in vivo analyses that we included complementary data with survival and proliferation in vitro. The proliferation was attenuated in PexRAP-deficient B cells in vitro; this evidence supports the conclusion that proliferation of PexRAP knockout B cells is reduced. It is likely that PexRAP deficient B cells also have defect in viability in vivo as we observed the reduced B cell number in PexRAP-deficient mice. As the reviewer noticed, the presence of a defect in cycling does, in the transfer experiments, limit the ability to interpret a lower yield of B cell population after adoptive transfer into µMT recipient mice as evidence pertaining to death rates. We will edit the text of the revision with these points in mind.
- In vitro stimulation experiments: These experiments need improvement. The authors have used anti-CD40 and BAFF for B cell stimulation; however, it would be beneficial to also include anti-IgM in the stimulation cocktail. In Fig. 2G, CTV plots do not show clear defects in proliferation, yet the authors quantify the percentage of cells with more than three divisions. These plots should clearly display the gating strategy. Additionally, details about histogram normalization and potential defects in cell numbers are missing. A more in-depth analysis of apoptosis is also required to determine whether the observed defects are due to impaired proliferation or reduced survival.
As suggested by reviewer, testing additional forms of B cell activation can help explore the generality (or lack thereof) of findings. We plan to test anti-IgM stimulation together with anti-CD40 + BAFF as well as anti-IgM + TLR7/8, and add the data to a revised and final manuscript.
With regards to Fig. 2G (and 2H), in the revised manuscript we will refine the presentation (add a demonstration of the gating, and explicate histogram normalization of FlowJo).
It is an interesting issue in bioscience, but in our presentation 'representative data' really are pretty representative, so a senior author is reminded of a comment Tak Mak made about a reduction (of proliferation, if memory serves) to 0.7 x control. [His point in a comment to referees at a symposium related that to a salary reduction by 30% :) A mathematical alternative is to point out that across four rounds of division for WT cells, a reduction to 0.7x efficiency at each cycle means about 1/4 as many progeny.]
We will try to edit the revision (Methods, Legends, Results, Discussion] to address better the points of the last two sentences of the comment, and improve the details that could assist in replication or comparisons (e.g., if someone develops a PexRAP inhibitor as potential therapeutic).
For the present, please note that the cell numbers at the end of the cultures are currently shown in Fig 2, panel I. Analogous culture results are shown in Fig 8, panels I, J, albeit with harvesting at day 5 instead of day 4. So, a difference of ≥ 3x needs to be explained. As noted above, a division efficiency reduced to 0.7x normal might account for such a decrease, but in practice the data of Fig. 2I show that the number of PexRAP-deficient B cells at day 4 is similar to the number plated before activation, and yet there has been a reasonable amount of divisions. So cell numbers in the culture of mutant B cells are constant because cycling is active but decreased and insufficient to allow increased numbers ("proliferation" in the true sense) as programmed death is increased. In line with this evidence, Fig 8G-H document higher death rates [i.e., frequencies of cleaved caspase3+ cell and Annexin V+ cells] of PexRAP-deficient B cells compared to controls. Thus, the in vitro data lead to the conclusion that both decreased division rates and increased death operate after this form of stimulation.
An inference is that this is the case in vivo as well - note that recoveries differed by ~3x (Fig. 2D), and the decrease in divisions (presentation of which will be improved) was meaningful but of lesser magnitude (Fig. 2E, F).
Reviewer #2 (Public review):
Summary:
In this study, Cho et al. investigate the role of ether lipid biosynthesis in B cell biology, particularly focusing on GC B cell, by inducible deletion of PexRAP, an enzyme responsible for the synthesis of ether lipids.
Strengths:
Overall, the data are well-presented, the paper is well-written and provides valuable mechanistic insights into the importance of PexRAP enzyme in GC B cell proliferation.
We appreciate this positive response and agree with the overview and summary of the paper's approaches and strengths.
Weaknesses:
More detailed mechanisms of the impaired GC B cell proliferation by PexRAP deficiency remain to be further investigated. In the minor part, there are issues with the interpretation of the data which might cause confusion for the readers.
Issues about contributions of cell cycling and divisions on the one hand, and susceptibility to death on the other, were discussed above, amplifying on the current manuscript text. The aggregate data support a model in which both processes are impacted for mature B cells in general, and mechanistically the evidence and work focus on the increased ROS and modes of death. Although the data in Fig. 7 do provide evidence that GC B cells themselves are affected, we agree that resource limitations had militated against developing further evidence about cycling specifically for GC B cells. We will hope to be able to obtain sufficient data from some specific analysis of proliferation in vivo (e.g., Ki67 or BrdU) as well as ROS and death ex vivo when harvesting new samples from mice immunized to analyze GC B cells for CXCR4/CD86, CD38, CD138 as indicated by Reviewer 1. As suggested by Reviewer 2, we will further discuss the possible mechanism(s) by which proliferation of PexRAP-deficient B cells is impaired. We also will edit the text of a revision where to enhance clarity of data interpretation - at a minimum, to be very clear that caution is warranted in assuming that GC B cells will exhibit the same mechanisms as cultures in vitro-stimulated B cells.
