Vitamin C (VC) or ascorbate is an essential micronutrient for maintaining cell barrier integrity and protecting cells from oxidative damage (Maggini et al., 2007; Webb and Villamor, 2007). Due to a mutation in GULO (L-gulono-gamma-lactone oxidase), humans are unable to synthesize ascorbate and depend on dietary sources to achieve VC adequacy (Nishikimi et al., 1994). Long-term ascorbate deficiency leads to a disease termed scurvy, which has been associated with weakened immune responses and higher susceptibility to pneumonia (Baron, 2009). Although scurvy is rare nowadays, national surveillance data indicate that overt VC deficiency and suboptimal VC status occurs in ~7.1% and ~25% of adults in the US respectively (Schleicher et al., 2009). VC insufficiency is even more prevalent in some subgroups, including the elderly, smokers, those with limited dietary intake, and those with increased oxidative stress due to illnesses (Isola et al., 2019; Cahill and El-Sohemy, 2010; Lima de Araújo et al., 2012; Na et al., 2006). VC deficiency or insufficiency may contribute to the variation of immune responses against infections. Thus, it is critical to understand how VC regulates immune responses at the cellular and molecular level.

In past decades, VC has been examined as a therapeutic treatment for diseases, such as infections and cancers, but the results have been mixed (Nauman et al., 2018; Thomas et al., 2021; Ngo et al., 2019; Magrì et al., 2020; Kuhn et al., 2018; Cerullo et al., 2020; Cheng, 2020; Pecoraro et al., 2019). For instance, the studies of VC oral supplementation related to preventing ‘common cold’ or enhancing immune responses have been highly controversial, often confounded by different administrative routes and quantitative methods (Padayatty et al., 2004; Allan and Arroll, 2014; Douglas and Hemilä, 2005; Lykkesfeldt and Tveden-Nyborg, 2019). The results could potentially be confounded by factors including the pre-existing VC levels in the participants (Prinz et al., 1977; Vallance, 1977; Anderson et al., 1980; Goodwin and Garry, 1983); genetic variants in the specific VC transporters (Cahill and El-Sohemy, 2010); the ill-defined viral pathogens for ‘common cold’; and the evolution of viruses to infect humans regardless of VC status. Notably, excess oral supplementation does not increase the steady-state VC concentration beyond the physiological level maintained by absorption and excretion (Padayatty et al., 2004). Recently, VC has been injected intravenously to achieve a supraphysiological concentration for treating sepsis and cancers (Magrì et al., 2020; Kuhn et al., 2018; Klimant et al., 2018). The outcomes from these studies were ambiguous as to whether or not the ‘mega dose’ VC was effective in treating these diseases (Nauman et al., 2018; Thomas et al., 2021; Ngo et al., 2019; Magrì et al., 2020; Klimant et al., 2018; Fowler et al., 2019; Marik et al., 2017). Nonetheless, oral and intravenous VC supplementation may selectively benefit the population with VC deficiency or insufficiency that may cause inferior outcomes (Consoli et al., 2020; Fisher et al., 2014; Wu et al., 2004).

VC deficiency has been associated with dysregulated immune responses based on historical observation and experimental evidence. For instance, using Gulo-knockout mice that are unable to synthesize VC, studies have shown that VC deficiency impairs the immune response to influenza infection. One study showed that influenza infection in these mice increased the production of pro-inflammatory cytokines (TNF-α, IL-1β) and promoted lung pathology without affecting viral clearance (Li et al., 2006). Another study showed that Gulo-deficient mice are defective in viral clearance due to decreased type I interferon response (Kim et al., 2013). In addition to Gulo-deficient mice, guinea pigs are naturally unable to synthesize VC and have been used as a model to show that VC is important for T-cell-dependent antibody response after immunization (Prinz et al., 1980; Feigen et al., 1982). How VC regulates innate and adaptive immune responses remains unclear. In addition to its antioxidative function, VC is a cofactor for several epigenetic enzymes, including ten-eleven-translocation (TET; Blaschke et al., 2013; Minor et al., 2013; He et al., 2015). TET proteins (TET1, TET2, TET3) are critical for DNA demethylation by oxidizing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and other minor oxidized bases, which are the intermediates for demethylation (Lio and Rao, 2019). Besides enhancing neuronal and hematological differentiations in vitro (Wu and Zhang, 2017), VC facilitates the differentiation of induced regulatory T cells by potentiating TET-mediated demethylation of the Foxp3 intronic enhancer CNS2 (Sasidharan Nair et al., 2016; Yue and Rao, 2020; Someya et al., 2017; Yue et al., 2016). These findings strongly suggested that one of the physiological functions of VC is to ensure the proper regulation of the epigenome that is required for coordinated immune responses.

B cells are responsible for the humoral immunity against pathogens in part by differentiating into plasma cells, the terminally differentiated cells specialized in secreting antibodies. Plasma cells express the key transcription factor (TF) BLIMP1 (encoded by Prdm1) and can be induced by T-cell-derived signals, including CD40 ligand and IL-21. Using a two-step in vitro system that models the T-dependent plasma cell differentiation, we performed a micronutrient analysis and identified VC as a potent enhancer for plasma cell differentiation from mouse and human B cells. This effect on plasma cells is specific to VC as other antioxidants had no significant effect. We identified that early B cell activation (first step) is the critical period requiring the presence of VC, which enables the B cells to become plasma cells after IL-21 stimulation. Intriguingly, VC treatment had only minimal influence on the transcriptome and IL-21 proximal signaling, suggesting the effect of VC is likely via the epigenome. Indeed, VC treatment increases the activity of TET as indicated by the elevated 5hmC level. Genome-wide 5hmC profiling revealed several VC-responsive elements at the Prdm1 locus that the DNA modification statuses were sensitive to VC. Furthermore, we identified that VC augments the association of STAT3 to E27, a critical Prdm1 downstream enhancer, by facilitating TET2/3-dependent DNA demethylation. Our results suggest that an adequate level of VC is essential for proper antibody response and highlight the influence of micronutrients on cell differentiation via epigenetic enzymes.

VC has long been known for its broad effect on immune responses. Long-term VC deficiency results in scurvy, a disease historically associated with increased susceptibility to infections (Baron, 2009; Kuhn et al., 2018; Hemilä, 2017). While recent studies have focused on VC supplements for disease treatments or prevention (Nauman et al., 2018; Ngo et al., 2019; Magrì et al., 2020; Kuhn et al., 2018; Douglas and Hemilä, 2005; Marik et al., 2017; Fisher et al., 2014; Wu et al., 2004; Hemilä, 2017), relatively little is known about the mechanism about how VC deficiency may affect the immune response. Here, we provide an additional evidence that VC controls the differentiation of plasma cells that are essential for humoral immunity.

Using an in vitro two-step model of plasma cell differentiation, we performed a limited micronutrient analysis and identified VC as a strong potentiator of plasma cell differentiation in mouse and human B cells (Figure 1C, D, F, and G). The effect of VC on plasma cells is independent of its general antioxidant function (Figure 1C–E). We further identified that the main effect of VC occurred during the initial activation step (first step; Figure 3A), prior to IL-21 stimulation. Intriguingly, after 4 days of first-step culture, the percentage of plasma cells (Figure 3—figure supplement 1, Figure 3B), capacity of proximal IL-21/STAT3 signaling (Figure 4), and transcriptomes (Figure 5C) were remarkably similar between control and VC-treated cells. As recent studies suggested that VC is a cofactor for α-ketoglutarate (αKG)-dependent epigenetic enzymes, we speculated that VC might support plasma cell differentiation by remodeling the epigenome via TET. Indeed, the positive effect of VC on plasma cells required TET2 and TET3 (Figure 6), and the analysis of the genome-wide 5hmC distribution revealed a significant difference between control and VC-treated cells (Figure 7). Our data demonstrated that VC facilitates plasma cell differentiation via TET.

The minimal difference in transcriptomes between control and VC groups on day 4 (Figure 5C) was surprising in light of the substantial effect of VC on plasma cell differentiation (Figure 1). The lack of difference in the transcriptome was likely not due to the sensitivity as we were able to detect a significant difference in RNA expression between activated and naïve B cells (Figure 5A and B). A previous study reported the coupled changes in DNA methylation and RNA expression at the later stage of plasma cell differentiation (Barwick et al., 2016). Given 5hmC modification precedes DNA demethylation, we would expect 5hmC modification to appear prior to transcriptional changes. Indeed, our results and another study on plasma cell differentiation (Scharer et al., 2018) support the notion that epigenome remodeling (e.g., 5hmC and chromatin accessibility) appears to precede transcriptional changes; and the changes in the epigenome may not necessarily result in immediate transcriptional responses.

Since Prdm1/BLIMP1 is the TF essential for plasma cell differentiation, we speculate that VC may affect the 5hmC modification at the Prdm1 locus and render it permissive to STAT3-mediated upregulation. Upon examining the 5hmC at the Prdm1 locus, we identified at least 14 VC-induced DhmRs in the B cells cultured with VC (VC^Day4) that we termed ‘ascorbate-responsive elements’ (E_AR; Figure 8B), including the regions with gained 5hmC or DNA demethylation after VC treatment. One noticeable E_AR is E58, an undescribed cis-element located at +58 kb (Kwon et al., 2009). While E58 contains a STAT3 motif and is permissive to STAT3 binding in T cells (Kwon et al., 2009), STAT3 does not associate with E58 in B cells in our system. Instead, we showed that STAT3 binds to the 3′ enhancer E27 and promoter. The binding was significantly increased in the presence of VC and correlated with DNA demethylation at E27, thus qualifying E27 as an E_AR that undergoes VC-induced, TET-mediated DNA demethylation (Figure 9). Sequence analysis of E27 in mice and the corresponding region in humans showed that STAT3 and two of the CpG motifs are largely conserved (Figure 9—figure supplement 2). How the DNA methylation status of these neighboring CpGs affects STAT3 binding remains unclear. One possibility is that DNA methylation may decrease nucleosome accessibility (Lio et al., 2016; Choy et al., 2010), thus decreasing the STAT3 binding to the motif. The detailed mechanism remains to be addressed.

Our data is in accordance with a recent report showing that VC can promote plasma cell differentiation via TET enzymes (Qi et al., 2020). Similar to our results, Qi et al. showed that the effect of VC on plasma cells is dependent on TET, and TET-deficient affects VC-assisted DNA demethylation at the Prdm1 intronic enhancers. While the previous study provided in vivo data, we have identified detailed molecular mechanisms of how VC enhances plasma cell differentiation. For instance, we dissected the two-step culture system and have shown that VC is critical during the initial B cell activation (first step). The effect of VC is unique and cannot be substituted by other antioxidants. Moreover, we demonstrated that VC has no immediate effect on the transcriptome and IL-21/STAT3 signaling after the first-step culture. Our data demonstrated that the main effect of VC is to remodel the epigenome. Furthermore, our genome-wide profiling of 5hmC revealed several E_AR at the Prdm1 locus, including a potential novel element E58 and the 3′ enhancer E27. Together, the studies here and Qi et al. have presented a clear picture of the importance of VC in regulating B cell differentiation.

DNA methylation is a dynamic process and undergoes substantial remodeling during differentiation. Studies have shown that differentiation from naïve B cells to plasma cells and memory B cells is accompanied by remodeling of the methylome, with more regions being hypomethylated that is consistent with the role of TET enzymes in cell differentiations (Kulis et al., 2015; Oakes et al., 2016; Lai et al., 2013). Therefore, these TET-dependent differentiations may be influenced by the levels of VC. We speculated that methylome remodeling may partially contribute to the increased effectiveness of memory B cells to differentiate into germinal center B cells and plasma cells. Interestingly, a previous report showed that human memory B cells, compared to naïve B cells, require a lower level of STAT3 to differentiate into plasma cells (Deenick et al., 2013). As DNA hypomethylation may enhance STAT3 binding, DNA demethylation may contribute to the lower requirement for STAT3 in memory B cells. In addition, given the lower methylation levels in memory compared to naïve B cells, we hypothesize that the differentiation of plasma cells from naïve B cells may be preferentially affected by VC deficiency.

VC has long been associated with immune function but the notion continues to be controversial. While VC supplementation does not prevent ‘common cold,’ a comprehensive meta-analysis of 29 clinical trials showed that VC supplementation is associated with shortened duration of symptoms (Hemilä and Chalker, 2013). However, how VC may enhance antiviral immunity remains unclear. Giving the importance of early IgM produced by antibody-secreting cells in controlling infection (Nguyen et al., 2017), it is possible that VC deficiency may delay the plasma cell differentiation to result in inferior control of the viruses at the early phase. Here, our in vitro findings provided a mechanistic explanation of how VC may affect humoral immune response. It is currently unclear how VC deficiency affects B cell function in vivo. Future experiments using genetic VC-deficient animal models (hamsters or Gulo-deficient mice) will provide critical insight into the physiological function of VC in immune response.

Increasing evidence has revealed the link between the metabolome and epigenome in the immune system (Lio and Huang, 2020a). For instance, VC stabilizes the in vitro-induced regulatory T cells by TET-mediated DNA demethylation at the intronic enhancer of the key TF Foxp3 (Sasidharan Nair et al., 2016; Someya et al., 2017; Yue et al., 2016). Another example is αKG, an intermediate in the Kreb cycle and a substrate for many epigenetic enzymes. Studies have shown that αKG modulates macrophage function by facilitating histone demethylation (Liu et al., 2017) and promoting IL-2-sensitive gene expression in T cells by TET-mediated DNA demethylation (Yue et al., 2021). Our results and others have shown that VC is a vital micronutrient required for efficient plasma cell differentiation (Qi et al., 2020). Based on these observations, we hypothesize that epigenetic enzymes may act as rheostats to control appropriate gene transcription by integrating the availability of micronutrients or metabolites. Future studies will be needed to address how micronutrients, including VC, contribute to immune response via epigenetic remodeling.

The data have been deposited to National Center for Biotechnology Information Gene Expression Omnibus (GEO GSE183681).

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Decision letter after peer review:

Thank you for submitting your article "Epigenetic Remodeling by Vitamin C Potentiates the Differentiation of Mouse and Human Plasma Cells" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Betty Diamond as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Paolo Casali (Reviewer #2); Frances Lee (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

For revision, we would like the authors to focus on the following:

(1) Provide ChIP-seq data on Stat3 binding in the Prdm1 gene,

(2) Address the applicability of the Vitamin C enhancement of plasma cell differentiation by including data from additional culture conditions,

(3) Improve Discussion section of the manuscript.

Reviewer #1 (Recommendations for the authors):

Overall this is an interesting paper that describes a role for VC in augmenting B cell differentiation to PC. The authors have impressive data showing the effects in an in vitro system that works well in the mice and less well in human systems where there is more variation and small fold changes.

1) Deletion of the TET enzymes shows that the effects of VC are dependent in part on these enzymes. What other pathways are involved for the rest of the increase in PC formation?

2) As mentioned above, showing by ChIP that STAT3 binding is enhanced would close the circle of the premise. Without it the paper has a hole that needs filling.

3) I am surprised that the treatment with VC changes results in only 5 differentially expressed transcripts and no protein encoding genes, yet the DNA methylation pattern is disrupted during that time. Other studies have implied that there are changes in DNA methylation during B cell differentiation that are coupled to some extent with transcriptional changes. This should at least be discussed in more depth.

4) I am concerned as mentioned above about the interpretation of the changes in 5hmC following VC treatment. Naïve cells have the most 5hmC by dot dot blot and the heat map in all of the experiments. Most of the naïve sites are lost by Day 4 under either condition (SupFigure 4). To me it looks like the TET enzymes are inhibited and fail to continue to convert 5hmC to the downstream nucleosides either actively by TET or passive through replication (Figure 7A, D). A smaller number of sites accumulate 5hmC in the VC samples (SupFig4B), presumably by demethylating a 5mC->5hmC. Do the cells in your culture divide? If so, how could 5hmC be maintained? Is E58 in that upper grouping of new 5hmC sites (SupFig4B)? Please provide a clearer explanation of the interpretation/logic.

Reviewer #2 (Recommendations for the authors):

This paper is of interest to cell biologists and immunologists who study the molecular mechanisms underpinning the maturation of the antibody response. The work provides new insight into the overall activity of Vit C on B lymphocytes by showing that in these cells Vit C promotes differentiation to plasma cells by activation of Tet2/Te3 epigenetic elements with no contribution of its antioxidant activity. Additional experiments using Vit C oxidant inhibitors would strengthen the overall trust of the paper on the solely epigenetic impact of Vit C on B cell plasma cell differentiation through Tet2/Tet3 activation.

Reviewer #3 (Recommendations for the authors):

This paper titled "Epigenetic remodeling by Vit C potentiates the differentiation of mouse and human plasma cells" is a very interesting paper with novel findings and mechanisms of Vit C in plasma cell differentiation. They use a 2-step IL-4, CD40L, then IL-21 culture system of total B cells with and without Vit C to generate plasma cell secreting IgM, IgG1, and IgE in mice. They elegantly show that Vit C enhances plasma cell differentiation and that the mechanisms involve TET activity and DNA demethylation. These data may help to support Linus Pauling's claims that Vit C prevents and alleviates the common cold if the model can be applied broadly. Thus, the paper is interesting and a novel study. Overall, it is important and exciting.

Some questions should be addressed.

1. The mouse model of proliferation and differentiation is for IgM, IgG1 (Mouse) and IgE thus, is this also important for Type 1 immune responses such as viral infection? Is this a model of IgE plasma cell generation and thus Vit C exacerbates allergic disease or is this a model for all plasma cells IgE and not for IgG2a or IgG2b in mice and (IgG1 in human)?

2. Most of the cultures used total B cells from mice (which would have included memory and naive B cells). Interestingly, the cultures were taken for only 7 days. Later timepoints may have also been informative. Were later time points taken to provide ample time for naive B cell to plasma cell differentiation?

3. From the mouse data, there appeared to be a lot of variability in the results with Vit C. Some experiments showed 50% CD138+ plasma cell responses while others were as high as 80% CD138+ fractions. Could the authors explain?

4. The human experiments uses only naive B cells cultured with IL-21, CpG and antihuman CD40 or IL21 and pansorbin. Very few CD38 high cells were identified. Do they also have CD27 staining since CD19loCD38hiCD27hi have been important markers of early plasma cells? Is there more information about the isotypes by in the supernatants or elispots? Were there later time points for the plasma cell cultures to provide enough time for naive B cell to plasma cell differentiation?

5. The human responses appeared more modest and quite variable (with some subject's naive B cells with anti-CD40, CpG, and IL-21 did not increase at all). Pansorbin and IL-21 showed more consistent increases which were more modest. BCR stimulation is quite important for naive B cell activation. Is there a reason to use the stimulation described above? Do the authors have an explanation for this?

6. The assessment at the different phases of the cultures were interesting and important.

7. The TET studies are interesting and identifying the BLIMP-1 locus together with an enhancer site is interesting. With his expertise in the conditional CD19 cre TET mice, these experiments were clever and quite revealing. Are there other possible sites such as IRF4 or Xbp1 notable in these mice?

8. The paper reads well in the introduction and results. However, the discussion could be better written. The summary is helpful but additional thoughts of the implications of this study, limitations of interpretation due to the current in vitro stimulation models, and considerations of Vit C in memory versus naive B cell subsets, would have provided some context of this work.

Essential revisions:

For revision, we would like the authors to focus on the following:

(1) Provide ChIP-seq data on Stat3 binding in the Prdm1 gene,

We have performed STAT3 ChIP-seq using B cells stimulated with IL-21. The result showed that the STAT3 association with Prdm1 3’ enhancer E27 and its binding is increased in the presence of VC.

(2) Address the applicability of the Vitamin C enhancement of plasma cell differentiation by including data from additional culture conditions,

We have included additional culture conditions for both mouse and human B cells. For instance, we have shown that:

1. VC stereoisomer erythobic acid could also facilitate plasma cell differentiation (Figure 9—figure supplement 1-2);

2. Enzymatic oxidation of VC abolished the plasma cell enhancing activity (Figure 1—figure supplement 3);

3. VC could enhance plasma cell differentiation and antibody secretion in B cells stimulated with LPS and cytokines (Figure 1—figure supplement 4);

4. VC had a similar positive effect on plasma cell generation from polyclonal and monoclonal (BCR-knockin) naïve B cells (Figure 2—figure supplement 1);

5. In an extended 3-step culture, VC further increased the percentage of plasma cells that bear characteristics of mature plasma cells (Figure 2—figure supplement 3);

6. VC positively affects the differentiation of antibody secreting cells (or plasmablasts) from naïve human B cells that were stimulated with an addition condition (Figure 1—figure supplement 5).

(3) Improve Discussion section of the manuscript.

We have included additional discussions regarding the implication of our findings and the limitations of our study.

Reviewer #1 (Recommendations for the authors):

Overall this is an interesting paper that describes a role for VC in augmenting B cell differentiation to PC. The authors have impressive data showing the effects in an in vitro system that works well in the mice and less well in human systems where there is more variation and small fold changes.

1) Deletion of the TET enzymes shows that the effects of VC are dependent in part on these enzymes. What other pathways are involved for the rest of the increase in PC formation?

VC is an antioxidant and a co-factor for other 2-oxoglutarate-dependent dioxygenases (2OGDDs), including most histone demethylases. For instance, previous reports have demonstrated that VC can promote JHDM1a/1b (2OGDDs) to demethylate H3K36me2/3 and enhance the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells (PMID 22100412). Another study showed that VC enhances KDM3a/b-dependent H3K9me2 demethylation in mouse embryonic stem cells (PMID 28706564). While our data showed that TET enzymes are necessary, it remains possible that other histone demethylases may be required for the effect of VC on PC formation.

Besides histone methylation, VC is implicated in the hypoxic response by enhancing the activity of prolyl hydroxylase domain (PHD) proteins that are also 2OGDDs (reviewed by PMID 25540771). In the presence of oxygen, PHD proteins hydroxylate the prolines on hypoxia-inducible factor-1/2 (HIF-1/2) and the proteasomemediated degradation of HIFs. Presumably, in the absence of VC, an increased HIF1/2 level may result in a hypoxic response despite the presence of oxygen. One study showed that hypoxia favors the in vitro differentiation of plasma cells from humans (28463531) and mice (27798169), suggesting that the absence of VC (increased HIF-1/2 levels) may promote PB/PC formation. How hypoxia may influence the positive effect of VC on PC in our culture system remains to be determined.

2) As mentioned above, showing by ChIP that STAT3 binding is enhanced would close the circle of the premise. Without it the paper has a hole that needs filling.

We really appreciate this comment that has led to our new discovery of another ascorbate-responsive element E27. While the previously proposed element E58 may regulate Prdm1 expression, it is clear from our new ChIPseq data that STAT3 does not associate with E58 in B cells cultured in our system. Instead, STAT3 binds to E27 and the binding was enhanced by VC. Additional response to the comment was started above and is shown below again.

“We were unable to completely reproduce the previous T cell STAT3 ChIP-seq by Kwon et al., 2009 (Dr. Warren Leonard’s lab) due to the discontinuation of the antibody (Santa Cruz). Therefore, we have tested four antiSTAT3 antibodies (Cell Signaling Technology rabbit monoclonal #4904, #12640, #9145; Millipore rabbit polyclonal #06-596), and found that only the rabbit mAb #9145 anti-phospho-Stat3 (Tyr705) from CST generated specific signals for ChIP, which was quantified by qPCR using an IL-21-induced positive control Mcl1.

Surprisingly, the ChIP-seq result showed that the STAT3 binding patterns differ between B cells and the previously published data from T cells (New Figure 8D and S8-1B). The difference may likely be due to cell types, culture conditions, and the source of antibodies. Nonetheless, our STAT3 ChIP-seq data showed that VC could enhance STAT3 binding at the Prdm1 promoter and E27, a previously identified enhancer with a functional STAT3 motif at +27kb (New Figure 8D)(Kwon et al., 2009). In order to prove that the DNA modification of this element was indeed regulated by VC, we analyzed the DNA methylation using bisulfite nanopore sequencing and showed that VC induced DNA demethylation at E27 (NEW Figure 9A). Importantly, both VC-enhanced DNA methylation (NEW Figure 9B) and STAT3 binding (NEW Figure 9C) at E27 were abolished in the Tet2/3deficient B cells. Our data strongly corroborate with the previously identified STAT3 binding site at E27 by Kwon et al., where they showed that the STAT3 motif is responsible for IL-21-induced Prdm1 expression. Our results further demonstrated that E27 is regulated by DNA methylation and is thus sensitive to the status of VC.”

3) I am surprised that the treatment with VC changes results in only 5 differentially expressed transcripts and no protein encoding genes, yet the DNA methylation pattern is disrupted during that time. Other studies have implied that there are changes in DNA methylation during B cell differentiation that are coupled to some extent with transcriptional changes. This should at least be discussed in more depth.

We agree with the Reviewer that the similar transcriptomes between B cells cultured with or without VC (Mock and VC) is surprising.

As mentioned by the Reviewer, most previous studies of plasma cell differentiation induced stimulation at the beginning of experiments (e.g., Shi et al., PMID 25894659) and observed substantial changes in the transcriptome. In another study (Barwick et al., PMID 27500631), the transcriptome and methylome also differ between naïve B cells and in vitro PB and PC after LPS stimulation. In fact, our results are consistent with the previous reports: we detected substantial changes in RNA expression after B cell activation (Figure 5A-B), accompanied by changes in the genome-wide distribution of 5hmC (Figure 7C-D).

As a DNA methylation intermediate, 5hmC often precedes the changes in DNA hypomethylation. The in vivo plasma cell differentiation experiments from Barwick et al., showed that the major changes in RNA expression and DNA methylation occur later in B cells that have undergone at least 8 cell divisions. Therefore, it is possible that the changes in 5hmC precede RNA expression and DNA methylation in our system.

We have included the following paragraph to the Discussion:

Line 399

The minimal difference in transcriptomes between control and VC groups on day 4 (Figure 5C) was surprising in light of the substantial effect of VC on plasma cell differentiation (Figure 1). The lack of difference in the transcriptome was likely not due to the sensitivity, as we were able to detect a significant difference in RNA expression between activated and naïve B cells (Figure 5A-B). A previous study reported the coupled changes in DNA methylation and RNA expression at the later stage of plasma cell differentiation⁶⁹. Given 5hmC modification precedes DNA demethylation, we would expect 5hmC modification appear prior to transcriptional changes. Indeed, our results and another study on plasma cell differentiation⁷⁰ support the notion that epigenome remodeling (e.g., 5hmC and chromatin accessibility) appears to precede transcriptional changes; and the changes in the epigenome may not necessarily result in immediate transcriptional responses.

4) I am concerned as mentioned above about the interpretation of the changes in 5hmC following VC treatment. Naïve cells have the most 5hmC by dot dot blot and the heat map in all of the experiments. Most of the naïve sites are lost by Day 4 under either condition (SupFigure 4). To me it looks like the TET enzymes are inhibited and fail to continue to convert 5hmC to the downstream nucleosides either actively by TET or passive through replication (Figure 7A, D). A smaller number of sites accumulate 5hmC in the VC samples (SupFig4B), presumably by demethylating a 5mC->5hmC. Do the cells in your culture divide? If so, how could 5hmC be maintained? Is E58 in that upper grouping of new 5hmC sites (SupFig4B)? Please provide a clearer explanation of the interpretation/logic.

As the Reviewer suggested above, we have included a better explanation of how TET functions and the fate of 5hmC after DNA replication (Figure 7—figure supplement 1). During DNA replication, the 5mC at CpG motif will pair with the unmodified cytosine on the newly synthesized complementary strand, creating a “hemi-methylated CpG”. Hemi-methylated CpG is recognized by the maintenance DNA methyltransferase complex DNMT1 and UHRF1, which is required for methylating the unmodified cytosine at the hemi-methylated CpG. However, the 5hmC-containing CpG motif is not recognized by the DNMT1 complex, and thus the CpG on the newly synthesized strand is not methylated. As a result, after rounds of cell division, the percentage of 5hmC on the original DNA will be diluted by the newly synthesized DNA. This explains why naïve B cells have more 5hmC than the activated B cells and lose most of the 5hmC peaks after divisions. In fact, in the 40LB co-culture, naïve B cells expand ~40 times after four days of culture, equating to around five divisions.

Since 5hmC is not replicated, all newly gained 5hmC are generated by TET-mediated oxidation of 5mC. Therefore, in Figure 7—figure supplement 2, those VC^up regions (Figure 7-figure supplement 2, top panel) represent de novo 5mC oxidation into 5hmC by TET enzymes that are presumably recruited by TFs. Meanwhile, the common regions (Figure 7—figure supplement 2, middle panel) are likely caused by the continuous recruitment of TET at the same regions that are enriched in 5hmC in naïve B cells. A recent study has demonstrated that the de novo DNA methyltransferase DNMT3A/B and TET can be simultaneously recruited to certain loci, where the cytosines undergo constant methylation turnover (32471981). We speculate that these common regions may represent these cytosines that recruit both DNMT3A/B and TET. Therefore, after the CpG on newly synthesized DNA is not methylated by DNMT1, the CpG is later methylated by DNMT3A/B, which are capable of methylating unmodified cytosines, and then oxidized by TET. It is still currently unknown how 5hmC is the preferential oxidative product of TET, which can oxidize 5hmC further into other minor oxidized cytosines (31965999).

Reviewer #2 (Recommendations for the authors):

This paper is of interest to cell biologists and immunologists who study the molecular mechanisms underpinning the maturation of the antibody response. The work provides new insight into the overall activity of Vit C on B lymphocytes by showing that in these cells Vit C promotes differentiation to plasma cells by activation of Tet2/Te3 epigenetic elements with no contribution of its antioxidant activity. Additional experiments using Vit C oxidant inhibitors would strengthen the overall trust of the paper on the solely epigenetic impact of Vit C on B cell plasma cell differentiation through Tet2/Tet3 activation.

We would like to thank you for the suggestion. We have performed additional experiments to explore the mechanism of how VC may affect plasma cell (PC) differentiation using our system. Note that in most experiments, the redox-stable version of VC 2-phospho-ascorbic acid (P-AA) was used. In the experiments below, both P-AA and L-ascorbic acid (L-AA) were used and they were collectively represented by VC.

First, we have explored whether erythobic acid (EA), a stereoisomer of L-AA, may have the same activity on PC (NEW Figure 1—figure supplement 2). EA differs from L-AA by the orientation of the hydroxyl group on the 5^th carbon. Our results showed that EA can promote PC differentiation with a lower activity compared to L-AA. This data suggest that the conformation of VC is not absolutely required for the PC enhancing activity.

Second, we tested whether oxidized L-AA is able to promote PC differentiation. To generate dehydroascorbic acid (DHA) from L-AA, we oxidized L-AA with ascorbate oxidase (AAO) in vitro prior to adding the reactions to B cell cultures. We found that AAO oxidation diminished the effect of L-AA on PC differentiation (NEW Figure 1figure supplement 3). As a control, the AAO-resistant P-AA was not affected by AAO (NEW Figure 1—figure supplement 3). These results demonstrated that the oxidation of VC inhibits its ability to facilitate the differentiation of PC.

Reviewer #3 (Recommendations for the authors):

This paper titled "Epigenetic remodeling by Vit C potentiates the differentiation of mouse and human plasma cells" is a very interesting paper with novel findings and mechanisms of Vit C in plasma cell differentiation. They use a 2-step IL-4, CD40L, then IL-21 culture system of total B cells with and without Vit C to generate plasma cell secreting IgM, IgG1, and IgE in mice. They elegantly show that Vit C enhances plasma cell differentiation and that the mechanisms involve TET activity and DNA demethylation. These data may help to support Linus Pauling's claims that Vit C prevents and alleviates the common cold if the model can be applied broadly. Thus, the paper is interesting and a novel study. Overall, it is important and exciting.

Thank you very much for the comments and suggestions. We have performed additional experiments and have strengthened the discussion. Please see below for the point-to-point response.

Some questions should be addressed.

1. The mouse model of proliferation and differentiation is for IgM, IgG1 (Mouse) and IgE thus, is this also important for Type 1 immune responses such as viral infection?

As noted by the Reviewer, the 40LB co-culture system generated Type 2 associated isotypes (IgG1, IgE). We have attempted to induce other isotypes using the 40LB system (with TGF-b or with IFN-g) but were not successful. Therefore, to induce the switching to other isotypes, we stimulated mouse B cells with LPS and a combination of cytokines following a previous study (PMID 11007474), including LPS alone (for IgM and IgG3), LPS+IFN-g (IgG2c), and LPS+IL-4 (IgG1). Similar to the observation using 40LB co-culture, the addition of VC increased the percentage of plasma cells and the secretion of antibodies with multiple isotypes (NEW Figure 1figure supplement 4). The lower VC-dependency in the LPS+/-cytokine culture compared to 40LB could potentially be attributed to multiple factors, including different signaling pathways (TLR vs CD40/BAFFR) and the lower proliferation rate (thus lower passive DNA demethylation). Nonetheless, our results showed that VC is able to promote plasma cell differentiation regardless of the switched isotypes.

Is this a model of IgE plasma cell generation and thus Vit C exacerbates allergic disease or is this a model for all plasma cells IgE and not for IgG2a or IgG2b in mice and (IgG1 in human)?

Unfortunately, we have not examined the role of VC using an allergy model. However, we speculate that in vivo transfer of IgE-secreting antigen-specific plasma cells generated with the 40LB system will likely exacerbate allergy or asthma pathology.

2. Most of the cultures used total B cells from mice (which would have included memory and naive B cells). Interestingly, the cultures were taken for only 7 days. Later timepoints may have also been informative. Were later time points taken to provide ample time for naive B cell to plasma cell differentiation?

We appreciate the insightful comments.

While the mice we used are between 6-12 weeks old, it is possible to have memory B cells in the starting population. To mitigate this concern about memory B cells, we isolated B cells from IgHCGG mice, a strain expressing BCR-specific for chicken γ globulin (CGG) generated by Dr. Gabriel Victora (PMID 30181412). In IgHCGG, the IgH and Igk from a CGG-specific antibody were joint and knocked into the IgH locus. Around 80% of CD19⁺ B cells express the CGG-specific receptor, the expression of which restricts the recombination of endogenous IgH and Igk/l. Therefore, in the absence of cognate antigen CGG, most follicular B cells express IgM and presumably to have a naïve phenotype. We isolated the B cells from B6 and IgHCGG and cultured them using the 40LB system as in Figure 1. The result showed no significant difference between the two groups (NEW Figure 2—figure supplement 1). This result suggests that VC can enhance plasma cell generation from naïve mouse B cells. Whether VC has a differential effect on memory cells remains to be determined.

For the human B cells experiment, we also provided data to verify the purity of naïve B cells (NEW Figure 1figure supplement 5). The typical purity is >95%. Whether human memory B cells may have different requirements for VC remained to be studied but were discussed (in Discussion).

To address the comment about the later timepoint, we have cultured the B cells for an extended period (11 days) with a 3-step culture (NEW Figure 2—figure supplement 3A). Consistent with the day 7 results (Figure 1), a significantly higher percentage of plasma cells were generated in the presence of VC (NEW Figure 2—figure supplement 3B), suggesting VC may not simply accelerate the kinetics of plasma cell generation. Interestingly, the majority of the CD138⁺ plasma cells from the VC group downregulated CD19 (NEW Figure 2—figure supplement 3C), suggesting these CD138⁺ cells may have a higher resemblance to in vivo mature plasma cells.

3. From the mouse data, there appeared to be a lot of variability in the results with Vit C. Some experiments showed 50% CD138+ plasma cell responses while others were as high as 80% CD138+ fractions. Could the authors explain?

Thank you for pointing out the inherent variability. While the intraexperiment variability is very low, the variability is occasionally higher between experiments. We attempted to identify the source of the variability and found that the condition of 40LB (>10 passages) may affect the outcome. It is possible that the 40LB cells decrease the expression of CD40L and BAFF after extended culture and thus resulting in lower overall plasma cell generation.

Another source of variability potentially comes from the small variation in the B cell seeding density. We have tested and found that higher starting cell numbers during 1^st culture usually resulted in an overall lower plasma cell generation after 2^nd culture. One possibility is the competition between B cells for CD40L and BAFF. Another possibility is that B cells may produce factor(s) that decrease the differentiation of plasma cells.

4. The human experiments uses only naive B cells cultured with IL-21, CpG and antihuman CD40 or IL21 and pansorbin. Very few CD38 high cells were identified. Do they also have CD27 staining since CD19loCD38hiCD27hi have been important markers of early plasma cells? Is there more information about the isotypes by in the supernatants or elispots? Were there later time points for the plasma cell cultures to provide enough time for naive B cell to plasma cell differentiation?

Unfortunately, CD27 was not included in the initial staining panel as we followed the protocol and gating scheme from an earlier publication that included IL-21 in the plasma cell culture (PMID 16339522). However, we have performed an additional stimulation condition and have incorporated CD27 in the analysis (NEW Figure 2—figure supplement 5B-C). Consistent with the earlier results (Figure 1F-G), VC could enhance plasma cell differentiation in the 3^rd culture condition. A similar result was observed when using CD38^hiCD27⁺ as the marker for early plasma cells. These early plasma cells cultured with VC had a lower surface expression of CD19. Longer team culture may be needed to observe the complete CD19 downregulation and the expression of additional plasma cell features.

We have not performed ELISA or ELISPOT with the human samples. As noted above, we have performed a later time point culture with mouse cells (NEW Figure 2—figure supplement 3) but have not been successful in longer-term human B cell culture.

5. The human responses appeared more modest and quite variable (with some subject's naive B cells with anti-CD40, CpG, and IL-21 did not increase at all). Pansorbin and IL-21 showed more consistent increases which were more modest. BCR stimulation is quite important for naive B cell activation. Is there a reason to use the stimulation described above? Do the authors have an explanation for this?

The main reasons for using these conditions were based on previous publications (PMID 16339522) where they have used IL-21 in their culture system. Initially, we tested multiple conditions from the study and attempted to include conditions that involved both CD40 or BCR stimulations. However, we were not able to reliably generate antigen secreting cells using anti-IgM-F(ab')2 due to abnormally high cell death. Since pansorbin (S. aureus Cowan) has a high level of Protein A on the bacteria surface, we reasoned that it would trigger BCR signals via Protein A-Ig crosslinking (PMID 6974188) in addition to activating multiple TLRs. Therefore, we chose the two conditions shown in Figure 1 where CD40+CpG+IL-21 mimics the T-dependent stimulation, while pansorbin+IL21 (triggering BCR and TLRs) mimics T-independent stimulation.

We have investigated the reason behind the lack of increase after VC culture for some subjects. See Author response image 1 for the breakdown of the results by experiment. Each experiment was performed with PBMC from non-overlapping donors. We found that the responsiveness to VC varied among individual donors. Experiments 3 and 4 have low overall ASC differentiation and responsiveness to VC (Author response image 1). Further investigations are needed to identify the factors that have contributed to the variability.

Author response image 1

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6. The assessment at the different phases of the cultures were interesting and important.

Thank you very much for the compliment.

7. The TET studies are interesting and identifying the BLIMP-1 locus together with an enhancer site is interesting. With his expertise in the conditional CD19 cre TET mice, these experiments were clever and quite revealing. Are there other possible sites such as IRF4 or Xbp1 notable in these mice?

We really appreciate the positive comments. While the expression of Irf4 and Xbp1 did not change significantly in B cells activated for four days in the presence of VC, we did detect significant 5hmC modification changes at the loci (Author response image 2). For instance, at Xbp1 locus (left), VC induced an additional intronic 5hmC modification. The common 5hmC peaks between naïve and Mock^Day4 likely undergo demethylation in VCDay4, a scenario similar to E27 at Prdm1. Similarly, VC is required for 5hmC modification at Irf4 locus (right). Most importantly, VC similarly facilitates STAT3 association at Xbp1 promoter and Irf4 gene. The role of VC on genome-wide STAT3 binding will be the subject of further study.

Author response image 2

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8. The paper reads well in the introduction and results. However, the discussion could be better written. The summary is helpful but additional thoughts of the implications of this study, limitations of interpretation due to the current in vitro stimulation models, and considerations of Vit C in memory versus naive B cell subsets, would have provided some context of this work.

Thank you for the constructive comments. We have added included additional discussion (listed below).

“DNA methylation is a dynamic process and undergoes substantial remodeling during differentiation. Studies have shown that the differentiation from naïve B cells to plasma cells and memory B cells is accompanied by the remodeling of methylome, with more regions being hypomethylated that is consistent with the role of TET enzymes in cell differentiations^74-76. Therefore, these TET-dependent differentiations may be influenced by the levels of VC. We speculated that methylome remodeling may partially contribute to the increased effectiveness of memory B cells to differentiate into germinal center B cells and plasma cells. Interestingly, a previous report showed that human memory B cells, compared to naïve B cells, require a lower level of STAT3 to differentiate into plasma cells⁷⁷. As DNA hypomethylation may enhance STAT3 binding, DNA demethylation may contribute to the lower requirement for STAT3 in memory B cells. In addition, given the lower methylation levels in memory compared to naïve B cells, we hypothesize that the differentiation of plasma cells from naïve B cells may be preferentially affected by VC deficiency.

VC has long been associated with immune function but the notion continues to be controversial. While VC supplementation does not prevent “common cold”, a comprehensive meta-analysis of 29 clinical trials showed that VC supplementation is associated with shortened duration of symptoms⁷⁸. However, how VC may enhance anti-viral immunity remained unclear. Giving the importance of early IgM produced by antibody secreting cells in controlling infection⁷⁹, it is possible that VC deficiency may delay the plasma cell differentiation to result in inferior control of the viruses at the early phase. Here, our in vitro findings provided a mechanistic explanation of how VC may affect humoral immune response. It is currently unclear how VC-deficiency affects B cell function in vivo. Future experiments using genetic VC-deficient animal models (hamsters or Gulo-deficient mice) will provide critical insight into the physiological function of VC in immune response.”

Author details

1. Heng-Yi Chen

   Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review and editing, Performed most experiments

   Contributed equally with

   Ana Almonte-Loya

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9283-6144

2. Ana Almonte-Loya

   1. Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States
   2. Division of Gene Expression and Signaling, La Jolla Institute for Immunology, San Diego, CA, United States

   Contribution

   Data curation, Software, Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review and editing, Performed bioinformatics analyses

   Contributed equally with

   Heng-Yi Chen

   Competing interests

   No competing interests declared

3. Fang-Yun Lay

   Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States

   Contribution

   Data curation, Formal analysis, Investigation, Performed human cell experiments

   Competing interests

   No competing interests declared

4. Michael Hsu

   Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States

   Contribution

   Data curation, Formal analysis, Writing – review and editing, Performed Nanopore bisulfite sequencing and related bioinformatics analyses

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3852-1461

5. Eric Johnson

   Division of Gene Expression and Signaling, La Jolla Institute for Immunology, San Diego, CA, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Edahí González-Avalos

   Division of Gene Expression and Signaling, La Jolla Institute for Immunology, San Diego, CA, United States

   Contribution

   Software, Advice on bioinformatics analyses

   Competing interests

   No competing interests declared

7. Jieyun Yin

   Division of Gene Expression and Signaling, La Jolla Institute for Immunology, San Diego, CA, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Richard S Bruno

   Human Nutrition Program, The Ohio State University, Columbus, OH, United States

   Contribution

   Validation, Writing – review and editing

   Competing interests

   No competing interests declared

9. Qin Ma

   1. Biomedical Informatics, The Ohio State University, Columbus, OH, United States
   2. Pelotonia Institute for Immuno-Oncology, The James Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, United States

   Contribution

   Software, Advice on bioinformatics analyses

   Competing interests

   No competing interests declared

10. Hazem E Ghoneim

    1. Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States
    2. Pelotonia Institute for Immuno-Oncology, The James Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, United States

    Contribution

    Resources, Validation

    Competing interests

    No competing interests declared

11. Daniel J Wozniak

    Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States

    Contribution

    Resources

    Competing interests

    No competing interests declared

12. Fiona E Harrison

    Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, United States

    Contribution

    Resources, Writing – review and editing

    Competing interests

    No competing interests declared

13. Chan-Wang Jerry Lio

    1. Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States
    2. Division of Gene Expression and Signaling, La Jolla Institute for Immunology, San Diego, CA, United States
    3. Pelotonia Institute for Immuno-Oncology, The James Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, United States

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    lio.4@osu.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3876-6741

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