Peer review of: Cholesterol metabolism drives regulatory B cell IL-10 through provision of geranylgeranyl pyropho (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this manuscript, Bibby et al propose that the induction of a regulatory program in human B cells 
is dependent on cholesterol metabolism. They show that synthesis of the metabolic intermediate 
geranylgeranyl pyrophosphate (GGPP) was required to specifically drive IL-10 production. 
Furthermore, GGPP-dependent protein modifications controlled signaling through PI3K, which in 
turn promoted BLIMP1-dependent IL-10 production. 

This is fundamentally an interesting topic and this reviewer is prepared to accept that the 
pathways proposed by the authors impact IL-10 production from B lineage cells. However, in its 
current form the study falls short of providing clarity but rather confuses the issue. Both IL-10 and 
Blimp1 expression are tightly linked to discrete differentiation stages of B lineage cells (ie plasma 
blasts and plasma cells). Thus, it is not correct to explore these pathways without taking this into 
account. 

Major points 

Il-10 production from B lineage cells has been shown to be largely restricted to plasma blasts (eg 
Matsumoto et al Immunity 2014). Similarly, Blimp1 expression is limited to plasma cells as widely 
documented. Firstly, this needs to be acknowledged and discussed in the manuscript. The authors 
state that “Currently there is no defined transcription factor that regulates IL-10 in human B cells” 
While this maybe true for human B cells, the role of Blimp1 in IL-10 production from many 
different cell types is well established for mouse. 

Secondly, it is essential to examine how the proposed pathways (inhibition of HMG-CoA reductase 
or GGTase) impact on plasma cell differentiation and other basic processes such as proliferation 
and survival. Antibody production should be part of the parameters measured. The authors look at 
proliferation and survival, but only after 40h. This is too early to expect to see major differences. 
What happens at later time points? 

Overall, I am concerned about the main readout in this manuscript. In most experiments the 
population of IL10+ B cells is very small (2-3%) and a substantial fraction of this small population 
sits at the very low end of the gate and may actually constitute background (eg Fig. 3G or Fig 5I). 
This makes me wonder how relevant the population is in vivo and whether or not the authors 
measure the right population/time point? 

In this context: Fig. S4A shows a very distinct population of IL-10+ B lineage cells that sits 
separate from all other cells. What are these cells? The interpretation of the authors that ‘all B cell 
populations contribute to IL-10 production’ is hard to follow given this result. If the authors were 
able to pinpoint this population and show how it is specifically regulated, this would make the 
study much more attractive. It just does not make sense to claim that naïve and transitional B 
cells produce Il-10 in a Blimp1 dependent manner. These cells do not express Blimp1. 

Finally, MKD patient B cells need to be fully characterized with respect to the above listed 
parameters and pathways. It is clear that these patients have a completely altered B cell 
compartment, both in terms of subset composition and activation status. Hence, with clear 
dissection of the role of differentiation, it is impossible to draw firm conclusions. 




Reviewer #2 (Remarks to the Author): 

This study provides evidence that geranylgeranylation is important for TLR9-mediated induction of 
IL10-producing human B cells. This is shown using inhibitors of HMGCoA reductase, GGTase and 
FTase, and analysis by FACS, qPCR and ELISA. Induction of another cytokine, TNF, is unaffected 
by the inhibitor treatments. TLR9-mediated activation of Akt and to a lesser extent Erk is 
antagonized by the GGTi and GSK is identified as a likely enzyme that needs to be repressed by 
Akt during IL10 induction. Inhibitor and genetic experiments show that PI3Kd is required for TLR9-
induced IL10. Gene expression analysis of TLR9 activated control and GGTi treated B cells leads to 
identification of several TFs as being induced in a geranylgeranyl-dependent manner. Blimp1 is 
focused on as IL10+ B cells have previously been shown to express Blimp1, and shRNA mediated 
knockdown of Blimp1 leads to reduced IL10 but not TNF. Finally, B cells from human patients with 
hypomorphic mutations in mevalonate kinase (MKD patients) are shown to make less IL10 upon 
CpG stimulation. Overall, this study contains a set of interesting insights about requirements for 
TLR9-induced IL10 production in B cells. However, there are also a number of weaknesses that 
currently limit enthusiasm. Since the study is focused on human B cells, it is performed entirely in 
vitro, which is understandable. However, the extensive reliance on chemical inhibitors raises 
concerns about off target effects. More generally, while geranylgeranlylation is implicated as being 
important in regulatory B cell IL10 production, the key question of what protein(s) needs to be 
geranylgeranylated is not answered. 

Specific comments: 
1. The GGTi used in this study (GGTi-298) inhibits geranylgeranyl transferase I. The authors didn't 
test the inhibition of GGTaseII (Rab geranylgeranyl transferase). Although GGTaseII inhibitors are 
less available, it is notable that the authors used one such inhibitor, psoromic acid, in a study 
earlier this year (Nat Commun 10, 498). Moreover, given the concerns about inhibitor off-target 
effects, it seems important to show that key findings made with single inhibitors can be confirmed 
where possible with a second inhibitor for that enzyme (or by a second approach as in 2). 
2. The data in Figure 5F show the ability of the authors to achieve knockdown of gene expression 
in human B cells. Given the central conclusion of the study regarding the role of GGTase and 
geranylgeranylation in TLR9-mediated IL10 induction, it would seem appropriate to show that this 
result is obtained in siRNA-mediated knockdown cells (and not just with chemical inhibitors). 
3. Since the mevalonate pathway has two main outcomes (cholesterol and isoprenoids), it would 
seem appropriate to demonstrate whether or not addition of a cholesterol-committed metabolite 
such as squalene to the statin treated cells has any effect on IL-10. 
4. Given the crucial role of small G-proteins in many B cell activation events, one wonders if the 
selective effect observed in this study is the dependence of IL10 on such isoprenylated proteins or, 
rather, the lack of TNF dependence on them. The effect of GGTi treatment on various other B cell 
activation outputs (other cytokines, activation marker expression, proliferation, viability) needs to 
be shown. 
5. The authors state: "Isoprenyl modifications almost exclusively regulate the localization of Ras 
superfamily proteins", and so investigated selected Ras family signaling pathways downstream of 
TLR9. However, the cited review does not make this statement and the quantitatively dominant 
targets of geranylgeranylation are still poorly defined. 
6. It is stated: "Gene set enrichment analysis identified defective Ras (KRAS) and PI3K-Akt-mTOR 
signaling pathways in MKD patients when compared to healthy donors, in line with our own 
observations (Figure 6F)". However, KRAS is farnesylated not geranylgeranylated according to the 
cited review (it can be geranylgeranylated, but usually when FTase is first inhibited or GGTaseI is 
overexpressed (cross-prenylation)). Also, when interrogating these data, the ERK signaling shown 
earlier to have an effect on IL-10 is not mentioned. 
7. It is stated that addition of GGPP rescues the defective IL10 production in CpG stimulated MKD 
B cells. However, the rescue does not appear to be statistically significant (Fig. 6C). While it is 
recognized that the patients are rare, it would have been more compelling if additional data (such 
as technical repeats) were available. Comparison to the effect of squalene addition would also 
have been informative. 

Minor 
1. "Cholesterol metabolism" as referenced in the title seems to vague given that the paper focuses 
on GGPP (and protein prenylation) rather than cholesterol. A more appropriate title might be: 
"GGPP is required for IL-10 production by regulatory B-cells" 
2. The name of the FTi inhibitor and the ERK inhibitor needs to be provided. The names of the 
inhibitors should be provided in the main text or figure legends and not just in the methods. 
3. The focus on PI3K-AKT as one of the downstream pathways is reasonable but this is quite 
possibly just one of many downstream pathways involved. It is important not to suggest that there 
is a selective role of geranylgeranylation in the activation of this pathway. For example, the data 
show that there is a reduction in pERK as well as pAKT. 
4. The differences between these findings for B cell IL10 expression and the earlier findings of 
cholesterol biosynthesis requirements in T cell IL10 production (Nat Commun 10, 498) is not 
adequately discussed.