Malaria, caused by parasites of the genus Plasmodium, is a leading global driver of infection-related mortality, resulting in 241 million cases of disease and over 600,000 deaths in 2020 (World Malaria Report, 2021). Clinical immunity to malaria (i.e. protection from symptoms) develops only after many years and repeated infections. Consequently, children in endemic areas remain vulnerable to severe illness and death for several years (Black et al., 2010; Schofield and Mueller, 2006; Ryg-Cornejo et al., 2016b), making the slow and incomplete acquisition of immunity a matter of grave concern for human health. The mechanisms underlying this apparently poor development of immunity have been the subject of intense research for decades. Antigenic diversity among Plasmodium parasites is likely one factor; additionally, however, evidence suggests that immune responses generated in hosts with malaria may be defective or sub-optimal, relative to responses against immunizations or other pathogens (Portugal et al., 2013). For example, antibodies against P. falciparum in naturally exposed children are short-lived relative to antibodies against unrelated vaccine antigens (Portugal et al., 2013; Crompton et al., 2010), and P. falciparum infection has been linked to diminished T helper cell responses in children and adults (Ho et al., 1986; Ho et al., 1988; Chizzolini et al., 1990; Rhee et al., 2001; Bejon et al., 2009). These observations raise the question of whether Plasmodium parasites subvert optimal immune responses to facilitate repeated infection of their mammalian hosts. The immune defects associated with Plasmodium infection are not limited to responses against the parasite itself: children with acute malaria exhibit impaired responses to a range of vaccinations (Greenwood et al., 1972; Williamson and Greenwood, 1978), and Plasmodium infection causes declines in pre-existing, unrelated vaccine-induced antibodies and plasma cells in both mice and humans (Banga et al., 2015; Ng et al., 2014). From a public health perspective, it is therefore also important to understand how malaria negatively impacts heterologous immunizations.

One proposed mechanism for the development of sub-optimal or short-lived immune responses in malaria is that Plasmodium parasites may suppress the activity of dendritic cells (DCs), innate immune cells that initiate canonical T cell responses to microbes (evidence for malaria-induced dysfunction reviewed in Wykes and Good, 2008). Some studies have found that P. falciparum-infected RBCs (iRBCs) or parasite products can render human DCs refractory to activation and restrict priming of CD4⁺ T cells in vitro (Urban et al., 1999; Pouniotis et al., 2004; Elliott et al., 2007; Yap et al., 2019; Millington et al., 2006; Pack et al., 2021), and humans with malaria have fewer and less activated circulating DCs than healthy controls (Pinzon-Charry et al., 2013; Woodberry et al., 2012). However, these decreases in circulating DCs during infection could reflect recruitment to infected tissues, and additional in vitro studies have concluded that DCs do become activated and can prime T cells after exposure to iRBCs and parasite products (Coban et al., 2005; Griffith et al., 2009; Götz et al., 2017). Overall, the question of whether malaria suppresses DC activity remains a subject for debate (Wykes and Good, 2008; Struik and Riley, 2004).

Mouse models have provided additional characterizations of DC function during Plasmodium infection, but with similarly conflicting results. Some studies have reported that Plasmodium suppresses DC function under specific circumstances (Wykes et al., 2007a; Sponaas et al., 2012; Wykes et al., 2007b), but others have established that DCs isolated from infected mice can present parasite-derived antigens to activate T cells and generate protective responses (Pouniotis et al., 2004; Wykes et al., 2007a; Sponaas et al., 2012; Sponaas et al., 2006; Voisine et al., 2010; Perry et al., 2004). Whether they actually perform this function in hosts with malaria is less clear. Several papers have sought to test a role for DCs in vivo using a transgenic mouse model that permits deletion of CD11c-expressing cells. These studies concluded that conventional CD11c^hi DCs were important for antigen presentation and T cell responses in malaria (Borges da Silva et al., 2015; Ueffing et al., 2017). However, CD11c is also upregulated on activated B cells, making the results of these studies difficult to interpret. Using more selective genetic tools to ablate antigen presentation specifically in DCs or B cells, we recently demonstrated that B cells, rather than DCs, are the dominant antigen-presenting cells (APCs) that direct activation and polarization of the CD4⁺ T cell response in mice with malaria (Arroyo and Pepper, 2020). In light of this finding, we considered it important to revisit the question of DC dysfunction in mice with malaria.

Historically, the ability of B cells to present antigen to T cells has been studied primarily in the context of cognate interactions with CD4⁺ cells that have already been activated by DCs. However, B cells can also interact with and activate naïve T cells directly. Hong et al. showed recently that whereas DCs were required for activating CD4⁺ T cell responses to a soluble protein antigen, CD4⁺ T cell responses to the same antigen delivered on a nanoparticle required presentation by B cells (Hong et al., 2018). In light of this finding, here we examined whether a differential ability to take up particle-like iRBCs might bias presentation of Plasmodium parasite antigens away from DCs and towards B cells. Further, we tested whether DCs in Plasmodium-infected mice remained competent to activate T cell responses to a heterologous, soluble protein antigen—an approach that allowed us to assess the functional capacity of DCs in hosts with malaria, independent of whether DCs can efficiently obtain iRBC-associated antigen in vivo. We found that DCs are relatively poor phagocytes of iRBCs, which may be one contributing factor in the dominance of B cells as APCs in this disease. We also found that DCs remained competent to activate CD4⁺ T cell responses to soluble antigens in hosts with malaria, suggesting no global dysfunction occurs. However, CD4⁺ T cell polarization, germinal center formation, and antibody production were profoundly altered, a finding that has implications for vaccination schemes in malaria-endemic regions.

Our results paint a nuanced picture of DC function during Plasmodium infection. DCs do not take up iRBCs efficiently in vivo, compared to their uptake of soluble antigen. This relatively poor ability to capture RBC-associated antigen may contribute to the importance of B cells as APCs in this infection setting, since the majority of iRBCs were found to associate with B cells, along with RPMs. However, differential uptake is likely not the only factor biasing antigen presentation toward B cells, since DCs do acquire sufficient antigen in infected mice to activate CD4⁺ T cells ex vivo (Pouniotis et al., 2004; Sponaas et al., 2012; Sponaas et al., 2006; Voisine et al., 2010; Perry et al., 2004). We speculate that some of this antigen may be acquired by DCs in soluble form after being shed from iRBCs (Beeson et al., 2016).

Although they did not capture iRBCs efficiently, DCs in both uninfected and infected mice readily took up soluble antigen and expressed the Class II and costimulatory molecules necessary to activate CD4⁺ T cells. Further, although the composition of the DC subset changed during infection, the magnitude of the T cell response to soluble protein immunization was similar in both uninfected and infected mice, indicating that DCs were competent to activate CD4⁺ T cells in a Plasmodium-infected spleen. The use of different markers to define DC subsets complicates direct comparisons between our findings and previous studies, but Sponaas et al. identified CD8^- CD11b⁺ DCs from infected mice as the most robust activators of CD4⁺ T cells ex vivo Sponaas et al., 2006; we believe that at the timepoints examined, this subset largely overlaps with the CD11b⁺ CD64⁺ moDC population that we identified here as the main DC subset capturing soluble antigen in infected mice. In other contexts, moDCs have been found fully capable of antigen presentation and T cell activation (Plantinga et al., 2013; Gieseler et al., 1998).

Altogether, our findings are consistent with previous studies showing that DCs isolated from infected mice are capable of activating CD4⁺ T cells in vitro. In addition, though, our work provides a readout of interactions between DCs and CD4⁺ T cells within malarial hosts rather than ex vivo. This is important because the splenic architecture undergoes profound alteration in mice and humans with malaria, with dissolution of marginal zones and blurring of borders between the T cell zones and B cell follicles; it has been suggested that these architectural disruptions contribute to the observed delay in establishment of germinal centers, and subsequent failure to make optimal antibody responses (Achtman et al., 2003; Stevenson and Kraal, 1989; Beattie et al., 2006; Urban et al., 2005; Cadman et al., 2008; Keitany et al., 2016). These tissue changes have already begun to occur 5 days after infection, when we immunized mice with GP, and reach their peak during the window in which the GP-specific response is being initiated (Achtman et al., 2003; Stevenson and Kraal, 1989; Cadman et al., 2008; Ryg-Cornejo et al., 2016a). Our data suggest that even within this dramatically altered tissue environment, DCs are able to interact with both antigen and CD4⁺ T cells in a way that permits robust expansion of the latter, ruling out the existence of global DC dysfunction in the malaria-inflamed spleen.

At the same time, we did observe a near-complete defect in differentiation of GP-specific Tfh and GC Tfh cells in mice that were immunized with GP 5 days after Plasmodium infection. We and others previously showed that ICOS-mediated interactions between B and T cells support maintenance of Tfh cells, while cognate B-T interactions are necessary for the GC Tfh subset (Pepper et al., 2011). In light of this, we infer that the loss of GP-specific Tfh and GC Tfh cells in Plasmodium-infected mice is due to disruption of both cognate and non-cognate interactions between GP-specific T cells and B cells. Here, alteration of the splenic architecture may play an important role, with the blurring of the T-B border potentially making it more difficult for activated T cells to locate B cells. The inflammatory milieu of the infected spleen may also negatively impact Tfh development; we and others have found that blocking inflammatory cytokines enhances the differentiation of Tfh cells and GC B cells during infections with other strains of Plasmodium (Keitany et al., 2016; Ryg-Cornejo et al., 2016a). Finally, it is interesting to speculate whether the BCR-independent binding of iRBCs that we observed may dilute or misdirect the antigen-specific B cell response in hosts with malaria. Indeed, Plasmodium infection is known to induce polyclonal, nonspecific B cell activation as well as activation of B cells autoreactive for RBC antigens (Rivera-Correa et al., 2020; Rivera-Correa et al., 2019; Fernandez-Arias et al., 2016; Sardinha et al., 2002). This could also contribute to the failure of GP-specific B cells to expand and support the differentiation of cognate Tfh cells.

An important question is whether the disruption to humoral responses that we observe in Plasmodium-infected spleens is a malaria-specific phenomenon. Daugan et al. previously found that mice infected with LCMV simultaneous with or 4 days before heterologous immunization developed fewer immunogen-specific antibody-secreting cells and lower specific antibody titers, suggesting that other pathogens can induce similar immune dysfunction (Daugan et al., 2016). However, the same study found intact responses to heterologous immunization in mice infected with vesicular stomatitis virus, indicating that not all infections disrupt heterologous immunity. A separate study found that mice infected with Plasmodium two days after heterologous immunization exhibited defective antibody responses to the heterologous antigen, but immunized mice infected with LCMV or Listeria monocytogenes did not (Banga et al., 2015). Taken together with the contrasting results from Daugan et al., 2016, which differed in the timing of immunization relative to infection, these data may indicate that LCMV disrupts humoral responses within a narrower window of time than Plasmodium. Nevertheless, we favor the hypothesis that multiple pathogenic infections can interfere with the robust development of heterologous responses. The precise mechanisms of disruption appear to differ in different infections, but likely involve host-derived cytokines and chemokines, with type 1 interferons, lymphotoxin, and interferon gamma being implicated in studies using LCMV, Toxoplasma gondii, and Plasmodium respectively (Keitany et al., 2016; Daugan et al., 2016; Glatman Zaretsky et al., 2012). Typically in these studies, defects in the humoral response correlate with elevated inflammation and disruption of the splenic architecture; as inflammation resolves, the infection is cleared, and the tissue reorganizes, germinal centers and B cell responses also recover.

The initial motivation for this study was to investigate mechanisms underlying our previous discovery that B cells, rather than DCs, served as the dominant APC population shaping the CD4⁺ T cell response during Plasmodium infection. By expanding previously established assays of iRBC uptake to include examination of B cells, we found that the majority of iRBCs associate with splenic B cells rather than with DCs, and that B cells preferentially bind iRBCs over nRBCs. This finding is consistent with a previous study showing that B cells were the primary APCs that activated CD4⁺ T cell responses when antigen was delivered in nanoparticle form, whereas DCs were key for initiating responses to the same antigen in soluble form (Hong et al., 2018). In that study, B cell uptake of nanoparticle-associated antigen was BCR-dependent and performed by antigen-specific B cells. Here, we found that B cells can preferentially bind iRBCs independent of the BCR, although this does not preclude additional BCR-dependent binding by B cells specific for parasite antigens, such as those we have characterized extensively in previous studies (Krishnamurty et al., 2016; Kim et al., 2019; Keitany et al., 2016; Thouvenel et al., 2021). This selective but BCR-independent binding is intriguing; it suggests the existence of antigen-nonspecific interactions between iRBCs and B cells that might explain the reported activation of nonspecific polyclonal or autoreactive B cells during malaria. The literature suggests a number of possible such interaction pathways: receptors expressed by B cells might bind opsonizing antibody, complement, exposed phosphatidylserines or parasite proteins on the iRBC surface (Vijay et al., 2021; Boyle et al., 2015; Donati et al., 2004). Once taken up, iRBCs contain immunostimulatory ligands, such as parasite RNA and DNA, that could activate B cells through Toll-like receptor signaling, alone or in conjunction with signaling from any of the above pathways (Baccarella et al., 2013; Sharma et al., 2011; Wu et al., 2010; Rivera-Correa et al., 2017). The iRBCs used in this study were isolated from mice 6–7 days post-infection, a time when some anti-parasite antibody may be present on the iRBC surface; however, we did not observe any defect in B cell binding of iRBCs in vitro when we isolated iRBCs from μMT mice (which lack antibodies) or added an Fc receptor-blocking antibody (data not shown). Further dissection of interactions between iRBCs and B cells therefore remains an important avenue for future research.

One key aspect of our study with relevance for human health is its insight into the detrimental effects of ongoing Plasmodium infection on antibody responses to immunization. Such effects have been appreciated for decades, most notably through measurement of poor responses to childhood vaccinations in children from endemic regions (Greenwood et al., 1972; Williamson and Greenwood, 1978; Banga et al., 2015). Malaria also may affect antibody avidity (Cadman et al., 2008). But the mechanisms underlying these malaria-associated decreases in antibody titers have mostly remained unknown, although one paper did identify increased turnover of serum antibodies as a contributing factor (Sardinha et al., 2002). We recently found that blood-stage-induced inflammation disrupts the development of sporozoite-specific B cells and antibodies in mice (Keitany et al., 2016), perhaps providing an explanation for the relative scarcity of CSP-specific memory B cells in naturally exposed humans (Wipasa et al., 2010; Jahnmatz et al., 2022). In the present study, we have further elucidated the cellular basis for defective antibody production during malaria: we showed that immunization with adjuvanted protein during acute Plasmodium infection activates immunogen-specific CD4⁺ T cells, but fails to elicit the Tfh cells necessary to support a robust immunogen-specific B cell response, correlating with diminished B cell expansion and decreased serum antibody titers. The detrimental effect on antibody production is transient, suggesting that interactions between B and T cells eventually recover as the infection is cleared to sub-patent levels. However, in endemic areas, many clinically immune humans carry patent levels of parasites in their blood during much of the year (Ryg-Cornejo et al., 2016b). It remains to be seen whether asymptomatic parasitemia affects vaccine efficacy similarly to acute malaria. Even so, a strategy in which vaccine is administered together with anti-Plasmodium chemoprophylaxis has recently shown great promise (Chandramohan et al., 2021), raising our hope that a greater understanding of malaria’s effects on the immune system will enable continued improvement of human protection from this and other diseases.

All data generated or analyzed during this study are included in the manuscript, figures, and provided source data files for Figures 1–5.

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Decision letter after peer review:

Thank you for submitting your article "Plasmodium infection disrupts the T follicular helper cell response to heterologous immunization" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Tadatsugu Taniguchi as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Michelle J Boyle (Reviewer #2).

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:

1) In regard to reviewer #2 conceptual concerns (from 1) to 3)), please discuss these points carefully.

2) Authors should clarify whether the impairment of B-T cell interaction is due to direct BCR interaction with iRBC, or indirect response to extrinsic factors induced by malaria infection, by using MD4 mice

3) Does P. chabaudi infection have any effects on B cell uptake of subsequent antigens, such as soluble antigen PE or particulate antigen CFSE-labeled P. yoelii iRBC?

Reviewer #1 (Recommendations for the authors):

This manuscript clearly demonstrates that murine malaria infection with Plasmodium chabaudi impairs B cells' interaction with T cells, rather than DCs interaction with T cells. The authors elegantly showed that DCs were activated, capable of acquiring antigens and priming T cells during P. chabaudi infection. B cells are the main APC to capture particulate antigens such as infected RBC (iRBC), while DCs preferentially take up soluble antigens. This study is important to understand how ongoing infections such as malaria may negatively affect heterologous immunizations.

Overall, the experimental designs are straightforward, and the manuscript is well-written. However, there were several limitations in this study.

Specific comments:

1. The mechanism of how the prior capture of iRBC by B cells lead to the impairment of B-T interaction was not understood. It is unclear whether the impairment of B-T cell interaction is due to direct BCR interaction with iRBC, or an indirect response to extrinsic factors induced by malaria infection.

2. Would malaria infection in MD4 mouse that carries transgenic BCR that does not recognize malaria parasite impair subsequent B cell response to HEL immunization? This may clarify whether the impairment of subsequent B cell response is BCR-specific. If malaria impairs subsequent B cell response to HEL in MD4 mouse, it might suggest that other cell types and B cell-extrinsic factors might be involved in causing the impaired B cell responses, instead of malaria affecting B cells directly.

3. MD4 mice were mentioned in the Methods in vitro RBC binding, although none of the figures described the usage of MD4 mice. This experiment data might be important to show whether RBC binding to B cells is mediated through BCR.

4. Does P. chabaudi infection have any effects on B cell uptake of subsequent antigens, such as soluble antigen PE or particulate antigen CFSE-labeled P. yoelii iRBC?

5. Is this phenomenon specific to malaria infection? Does malaria-irrelevant particulate immunization affect T-B interaction of subsequent heterologous immunization?

6. Despite the impaired Tfh and GC 8 days after immunization following malaria infection, Figure 5F showed GP-specific IgG eventually increased to the same level as the uninfected immunized mice on day 23. Did the authors check whether these mice had a delayed Tfh and GC response that eventually increase on day 23? Are these antibody responses derived from GC, or GC-independent response?

7. Does recovery from malaria infection by antimalarial treatment rescue the B cell response to subsequent heterologous immunization?

8. Figure 1C shows more nRBC was taken up than iRBC in B cells, but Line 142 states that "B cells bound significantly more iRBC than nRBC. Is there a mistake in the figure arrangement? Why do B cells take up for naïve RBC than iRBC?

9. Figure S1 C and D are confusing. CD45.1+ CD45.2+ mouse did not receive labeled iRBC, but why iRBC was detected as much as 40% in the spleen of this naïve mouse?

Reviewer #2 (Recommendations for the authors):

The data presented support the conclusions of the paper, and my concerns are largely conceptional in how we understand this data in the context of malaria infection in vaccination in endemic areas

1) The data is presented based on the idea that antigen uptake and presentation differ between particle and soluble antigens, and that during malaria infection particle uptake is more important due to circulating iRBCs. However, during parasite invasion of RBCs, the parasite sheds large amounts of antigen into the circulation, at least some of which would then be found in a soluble form in the circulation. Can the authors comment on this aspect of infection and if/how this may impact the interpretation of results? Do authors assume that any soluble antigen taken up and presented (via DCs?) during infection would be impacted as for GP66 soluble antigen? Or could an interaction on immune responses where the antigen is presented via both particle and soluble pathways?

2) Impact of particle antigen opsonisation on antigen uptake and presentation. The authors use parasites isolated from mice who have been infected for 6-7 days to investigate the ability of different subsets to update particle antigens. At this time point, have mice developed antibody responses that opsonise these parasites, or are antibody levels low and parasites opsonised? Would opsonised parasites, such as those coated with sera from children in a setting of chronic infection, have a different pattern/ability to be opsonised by different immune cell subsets? And/or would opsonisation change how the DC and other cell types are processing/presenting antigens? While these issues could be addressed experimentally either now or in the future, the manuscript should at least consider this issue because, during a human infection in areas of high exposure, individuals are likely to have reasonable levels of antibodies with opsonised parasites circulating.

3) While authors show that malaria infection disrupts the response to soluble antigens, the relevance directly to vaccination should be considered carefully, specifically because vaccines of soluble antigens are largely given alongside adjuvants which also will modulate DC function. Again, this could be addressed experimentally now or in the future, but definitely should be mentioned and considered when interpreting the results.

Prior to publication, I suggest the following edits/queries for clarity.

Is the legend in Figure 1 correct? Line 142 states "B cells bound significantly more iRBCs than nRBCs …" but the figure is the opposite as labelled. I assumed that the labels have been switched, but best to check and match back to the text as needed.

In Figure 1 – suggestion to also analyse the data as RBC+ (% of total cell subset) – ie RBC+ DCs/% of total DCs, to understand the relative capacity of each subset to uptake antigen as a % of all those cells in that subset.

In Figure 2 – it looks like that are hardly any double positive cells – ie those which have taken up both PE and iRBCs at the same time – is this expected and consistent or just in the gating example? Does it suggest the specialisation of both B cells and DCs to be better able to uptake soluble or particle antigens in some way or suggestive of different subsets?

I don't understand Figure 3C – why is the PE+ DCs in the naive mice 0? Additionally, the text states in line 226 "we observed equivalent or slightly higher frequencies" but Figure 3C has a clear significantly increased uptake in infected mice.

At day 23, when antibody levels to soluble antigen (Figure 5F) have recovered to levels of uninfected mice – are robust Tfh/GC B cells detected? Ie, is there evidence of germinal centre recovery? Is this due to long-term soluble antigen in DCs that is able to then activate naïve T/B cells down the track?

Figure 5 – shows reduced IgG, but would be beneficial to also look at the specific subclasses of these antibodies, particularly given the importance of cytophilic subclasses in protection.

Essential revisions:

1) In regard to reviewer #2 conceptual concerns (from 1) to 3)), please discuss these points carefully.

We have addressed these points with additional discussion and references to the relevant literature; please see our responses to Reviewer #2, Public Review Points 1-3, below.

2) Authors should clarify whether the impairment of B-T cell interaction is due to direct BCR interaction with iRBC, or indirect response to extrinsic factors induced by malaria infection, by using MD4 mice

We have addressed this point extensively in our responses to Reviewer #1, Specific Comments 1 and 2, below.

3) Does P. chabaudi infection have any effects on B cell uptake of subsequent antigens, such as soluble antigen PE or particulate antigen CFSE-labeled P. yoelii iRBC?

We now provide data showing the effect of P. chabaudi infection on B cell uptake of soluble PE; Please see our response to Reviewer #1, Specific Comment #4.

Reviewer #1 (Recommendations for the authors):

This manuscript clearly demonstrates that murine malaria infection with Plasmodium chabaudi impairs B cells' interaction with T cells, rather than DCs interaction with T cells. The authors elegantly showed that DCs were activated, capable of acquiring antigens and priming T cells during P. chabaudi infection. B cells are the main APC to capture particulate antigens such as infected RBC (iRBC), while DCs preferentially take up soluble antigens. This study is important to understand how ongoing infections such as malaria may negatively affect heterologous immunizations.

Overall, the experimental designs are straightforward, and the manuscript is well-written. However, there were several limitations in this study.

Specific comments:

1. The mechanism of how the prior capture of iRBC by B cells lead to the impairment of B-T interaction was not understood. It is unclear whether the impairment of B-T cell interaction is due to direct BCR interaction with iRBC, or an indirect response to extrinsic factors induced by malaria infection.

We believe we have carefully demonstrated that impairment of B-T interactions does not require specific BCR-antigen interactions between B cells and iRBCs (for a complete explanation of this point, please see the response to the next comment). However, the question remains whether direct, antigen-nonspecific iRBC-B cell interactions (i.e., not mediated by the BCR) or additional extrinsic factors, or a combination, are responsible for the observed defects in Tfh and GC B cell populations.

Existing studies from other infection models are informative in answering this question. Daugan et al. (Front Immunol 2016; PMID 27994594) previously published experiments similar to ours, but used LCMV instead of Plasmodium. That is, they immunized uninfected or LCMV-infected mice with the well-studied immunogen NPP-CGG and measured NP-specific antibody production and other parameters. They found that LCMV infection concurrent with immunization (or 4-8 days before) significantly decreased the numbers of NP-specific splenic antibody-secreting cells and IgG1 titers, and caused major disruptions to splenic architecture. These defects were shown to require type I interferon (T1IFN) signaling in B cells. However, T1IFN is unlikely to be solely responsible for the observed phenotypes, because simultaneous infection with VSV, another virus that also induces T1IFN, did not cause any defects in NP-specific antibody production. Contrasting with the work of Daugan et al., Banga et al. (PloS One 2015; PMID 25919588) found that infecting with LCMV (or with Listeria monocytogenes) two days after heterologous immunization did not disrupt immunogen-specific responses, whereas P. yoelii did. Examining both these studies, we hypothesize that both LCMV and Plasmodium infections can disrupt humoral responses, but that LCMV does so within a narrower time frame, thereby yielding different results depending on whether infection comes a few days before or a few days after immunization.

Complementing these studies of heterologous immunization, additional publications have reported that cytokines induced by several different pathogenic infections drive disruption of germinal centers and decreases in antibody titers specific for the pathogen itself, often correlated with disordered splenic architecture. Glatman Zaretsky et al. (Infect Immun 2012; PMID 22851754) showed that Toxoplasma gondii infection causes transient disruption of splenic architecture and loss of defined GCs by microscopy. These defects were partially due to decreased lymphotoxin expression by B cells, and were rescued by a lymphotoxin receptor agonist. Similarly, we previously reported that blood-stage Plasmodium infection disrupted germinal center responses to a Plasmodium liver-stage antigen (Keitany et al. Cell Rep 2016; PMID 28009289). In this context, however, the same lymphotoxin receptor agonist had no effect on GCs; instead, blockade of the pro-inflammatory cytokine interferon γ partially restored antibody responses to the liver-stage antigen. Overall, we favor the hypothesis that several different pathogens can disrupt GCs and antibody responses indirectly by inducing inflammation and a disordered splenic environment; however, the precise mechanisms of disruption likely differ from infection to infection, with different cytokines or other effectors playing key roles in some but not other settings. Importantly, not all pathogens disrupt antibody production, since again, infection with VSV or L. monocytogenes did not affect immunogen-specific titers in immunized mice (Daugan Front Immunol 2016; Banga et al. 2015). We have now addressed this topic at length in the Discussion (lines 399-418).

The existence of indirect, inflammation- or cytokine-related mechanisms that may interfere with germinal center formation and antibody production does not preclude additional direct interactions between B cells and iRBCs that might also affect B cell function. We address this possibility more fully in the response to the next comment.

2. Would malaria infection in MD4 mouse that carries transgenic BCR that does not recognize malaria parasite impair subsequent B cell response to HEL immunization? This may clarify whether the impairment of subsequent B cell response is BCR-specific. If malaria impairs subsequent B cell response to HEL in MD4 mouse, it might suggest that other cell types and B cell-extrinsic factors might be involved in causing the impaired B cell responses, instead of malaria affecting B cells directly.

The question of whether the impairments we observe require BCR-specific interactions with iRBCs is an important one. However, we believe that the experiment the reviewer proposes to address this question has technical limitations; further, we assert that we have already provided data to address a requirement for BCR specificity.

With regard to the proposed experiment of immunizing MD4 mice with HEL in the presence or absence of malaria infection: MD4 mice, in which B cells express a transgenic receptor specific for HEL, can be expected to mount a massive, monoclonal response to direct immunization with HEL that would be very different from the physiological context of a polyclonal B cell population. We are doubtful that this experimental setup would be informative for the question at hand, especially because we are studying the effects of B-Tfh interactions, which are already limiting in the physiological setting of a polyclonal B cell response, but would be massively unbalanced in an MD4 mouse where all B cells express the receptor for HEL.

Usually, investigators studying MD4 B cell responses generate a more physiological setting by adoptively transferring a small but detectable number of MD4 transgenic B cells into a mouse with a normal polyclonal B cell population, and immunizing that mouse. We maintain that this approach is essentially what we have done in our study, except that instead of using transferred. transgenic cells to identify a B cell population of known specificity, we have used tetramers to detect a specific population of endogenous B cells in a polyclonal setting. By examining GP-specific B cells in our immunization experiments, we restricted our analysis to B cells that could not have had any BCR-mediated, antigen-specific interactions with iRBCs (because the GP antigen is not present in the iRBCs; it is delivered as a soluble protein antigen, 5 days after initiation of infection). Because we see dysfunction in the GP-specific T and B cell populations despite the absence of this antigen within iRBCs, we can conclude that the disruptions to these populations are not due to antigen-specific iRBC-BCR interactions.

We do also show (using MD4 B cells in Figure S1B) that selective interactions between iRBCs and B cells do not require an antigen-specific BCR. Thus, it is still possible that direct interactions between iRBCs and B cells (that are independent of antigen binding to the BCR) are responsible for disrupting subsequent adaptive responses, perhaps in addition to the more indirect factors that we discuss in the response to Comment #1 above. We are very interested in this possibility, which is discussed in lines 428-436 of the manuscript. But the use of MD4 B cells would not address this specific question. Instead, we would need to identify an alternative pathway or receptor that mediates the iRBC-B cell interaction, and study the effects of blocking that pathway on downstream adaptive responses. We have spent considerable time and energy on this question, but have not yet been able to identify such a pathway; this remains a matter for further study.

3. MD4 mice were mentioned in the Methods in vitro RBC binding, although none of the figures described the usage of MD4 mice. This experiment data might be important to show whether RBC binding to B cells is mediated through BCR.

Cells from MD4 mice were used in Figure S1B to show that in vitro binding of iRBCs to B cells did not require interaction with an antigen-specific BCR. We agree that this is an important point and have revised the text (lines 152-156) to outline it more clearly.

4. Does P. chabaudi infection have any effects on B cell uptake of subsequent antigens, such as soluble antigen PE or particulate antigen CFSE-labeled P. yoelii iRBC?

We examined uptake of PE by B cells in P. chabaudi-infected mice (5 days post-infection) compared to naïve mice. There was a trend towards increased uptake in the infected mice, but this difference was not significant. These data are taken from the same samples that did reveal a significant increase in PE uptake by DCs in infected mice (Figure 3C). We have now included the B cell data in the paper as Figure 3D, and discussed them in lines 231-232.

5. Is this phenomenon specific to malaria infection? Does malaria-irrelevant particulate immunization affect T-B interaction of subsequent heterologous immunization?

We do not believe this phenomenon is specific to malaria infection; please see the extensive discussion of this point in the response to Comment #1 above. We would hypothesize that malaria-irrelevant particle immunization (as with nanoparticles) would not affect T-B interactions for subsequent heterologous immunizations, however, since the disruption seems to be associated with the massive inflammation and splenic disorganization that occurs following certain infections.

6. Despite the impaired Tfh and GC 8 days after immunization following malaria infection, Figure 5F showed GP-specific IgG eventually increased to the same level as the uninfected immunized mice on day 23. Did the authors check whether these mice had a delayed Tfh and GC response that eventually increase on day 23? Are these antibody responses derived from GC, or GC-independent response?

We have now examined GP-specific T cell numbers and polarization between days 23 and 35 post-immunization. We found that although a defect persists in the percentage of GP66-specific T cells that exhibit a GC Tfh phenotype at later timepoints, the absolute number of GC Tfh cells is not significantly defective in infected mice at these times. Concurrently there is a slight (though nonsignificant) increase in the total numbers of GP66+ T cells in the infected mice; we believe that this modest overall expansion permits recovery of the GC Tfh population numbers despite the continued defect in their frequency. These findings are consistent with our observation that antibody levels recover in infected mice by 3 weeks post-infection. We have added these data to Figure 4 (E-G) and discuss them in lines 283-293.

7. Does recovery from malaria infection by antimalarial treatment rescue the B cell response to subsequent heterologous immunization?

We have shown previously that drug-mediated clearance of blood-stage Plasmodium infection restores GC and antibody responses to a liver-stage-specific antigen, which normally are disrupted by emergence of the blood-stage (Keitany et al. Cell Rep 2016). We have also shown that antimalarial drug treatment restores GC responses in mice lacking the innate immune sensor CGAS, which have higher parasitemia, exacerbated splenic disruption, and diminished GC responses following P. yoelii infection (Hahn et al., JCI Insight 2018). Based on these results we hypothesize that drug-mediated clearance of blood-stage infection would also rescue B cell responses to heterologous immunization.

8. Figure 1C shows more nRBC was taken up than iRBC in B cells, but Line 142 states that "B cells bound significantly more iRBC than nRBC. Is there a mistake in the figure arrangement? Why do B cells take up for naïve RBC than iRBC?

The symbols in the figure legend were switched in error; the filled circles are actually iRBC+ and the outlined circles are nRBC+. We regret the error and appreciate the reviewer bringing it to our attention. We have corrected the figure.

9. Figure S1 C and D are confusing. CD45.1+ CD45.2+ mouse did not receive labeled iRBC, but why iRBC was detected as much as 40% in the spleen of this naïve mouse?

The experiment depicted in Figures S1 C and D was designed to test whether B cells actually bound injected iRBCs in vivo, or whether the binding occurred during processing of the tissue. With this experimental setup (injecting labeled iRBCs into CD45.2+ mice, then excising and disrupting the spleen together with an untreated CD45.1+ CD45.2+ spleen), iRBC signal from in vivo uptake should be observed only in CD45.2+ splenocytes, whereas iRBC binding that occurs during tissue processing will be distributed between the two genotypes. Thus, the ~40% of iRBC signal observed in CD45.1+ CD45.2+ B cells leads us to conclude that much of the observed B cell binding from our in vivo experiments occurs during processing, as we state in the text (lines 151-152). Even so, in vitro experiments clearly show that B cells selectively bind iRBCs over naïve RBCs in a setting where processing is not a confounder (Figure S1B). To clear up any confusion, we have expanded the description of the experiment and its interpretation in the Supplemental Figure Legend.

Reviewer #2 (Recommendations for the authors):

The data presented support the conclusions of the paper, and my concerns are largely conceptional in how we understand this data in the context of malaria infection in vaccination in endemic areas

1) The data is presented based on the idea that antigen uptake and presentation differ between particle and soluble antigens, and that during malaria infection particle uptake is more important due to circulating iRBCs. However, during parasite invasion of RBCs, the parasite sheds large amounts of antigen into the circulation, at least some of which would then be found in a soluble form in the circulation. Can the authors comment on this aspect of infection and if/how this may impact the interpretation of results? Do authors assume that any soluble antigen taken up and presented (via DCs?) during infection would be impacted as for GP66 soluble antigen? Or could an interaction on immune responses where the antigen is presented via both particle and soluble pathways?

This is an important point and we have now discussed it further in the text (lines 111-115, 204-210, and 356-357). In our previously published study, where we extensively characterized CD4 T cell responses to the GP66 epitope expressed by P. yoelii, the epitope was fused to a parasite protein (Hep17) that localizes to the parasitophorous vacuole membrane, and so we do assume that the majority of this antigen is encountered by APCs in the context of an iRBC, rather than shed in soluble form. In contrast, some merozoite surface antigens such as cleaved MSP1 are shed copiously from the parasite coat upon RBC invasion, and therefore would be expected to exist in soluble as well as parasite-associated form.

Unfortunately, our laboratory does not currently have tetramer reagents or access to transgenic mice that would allow us to assess T cell responses specific for shed or soluble parasite antigens. But a previous study from Stephens et al. (Blood 2005; PMID 15890689) reported that T cells with a transgenic TCR specific for an epitope in the shed portion of MSP1 could boost antibody production when transferred into T cell-deficient mice infected with P. chabaudi, suggesting that at least some fraction of the MSP1-specific T cells differentiate into T helper cells capable of supporting B cell activity. However, antibody production was significantly delayed in this setting compared to its usual kinetics in wild-type mice. Further side-by-side characterization would be needed to assess differentiation of these MSP1-specific transgenic T cells during infection, and determine whether they are primed from B cells or from DCs (or a combination).

We will note that we have extensively characterized B cell responses to MSP1 during both infection and immunization. While robust and T-dependent, MSP1-specific B cell responses in infected mice are delayed relative to their kinetics in mice immunized with recombinant MSP1 or other protein antigens. This may indicate that MSP1-specific T cell activation or cognate B-T interactions are defective in infected mice relative to immunized mice, despite the presumed presence of soluble (shed) MSP1 during infection. If this is the case, it suggests that the defects we describe in the current manuscript exist for both particle-associated and soluble parasite antigens. However, as we mentioned above, a careful characterization of MSP-1-specific T cell differentiation is needed to really understand this, and that requires additional tools that we can’t easily access at this time.

2) Impact of particle antigen opsonisation on antigen uptake and presentation. The authors use parasites isolated from mice who have been infected for 6-7 days to investigate the ability of different subsets to update particle antigens. At this time point, have mice developed antibody responses that opsonise these parasites, or are antibody levels low and parasites opsonised? Would opsonised parasites, such as those coated with sera from children in a setting of chronic infection, have a different pattern/ability to be opsonised by different immune cell subsets? And/or would opsonisation change how the DC and other cell types are processing/presenting antigens? While these issues could be addressed experimentally either now or in the future, the manuscript should at least consider this issue because, during a human infection in areas of high exposure, individuals are likely to have reasonable levels of antibodies with opsonised parasites circulating.

We ourselves have been very interested in the question of whether host antibodies (or other host factors such as complement) might affect uptake of iRBCs. As the reviewer notes, the iRBCs we use in our experiments are taken from mice 6-7 days post-infection, at which time some amount of anti-parasite antibody has developed. We spent a considerable amount of time trying to measure effects of opsonizing antibody, or even deposited complement, on uptake of iRBCs. However, we did not see any change in B cell binding of iRBCs in vitro when we blocked complement receptor with anti-CD21; blocked antibody receptors (Fc receptors) with anti-CD16/CD32 or excess normal mouse serum; or used iRBCs taken from complement-depleted mice (treated with cobra venom factor) or from uMT mice (which entirely lack B cells and antibody). Thus, we have not been able to find any role for opsonizing antibody (or complement) in iRBC uptake. We have not included these experiments in the manuscript because they yielded only negative data, and we were not able to design positive controls robust enough to give us confidence that the experiments were technically sound (and therefore that the negative results were due to the underlying biology and not to experiment failure). We have added a discussion point about this issue (lines 438-442).

3) While authors show that malaria infection disrupts the response to soluble antigens, the relevance directly to vaccination should be considered carefully, specifically because vaccines of soluble antigens are largely given alongside adjuvants which also will modulate DC function. Again, this could be addressed experimentally now or in the future, but definitely should be mentioned and considered when interpreting the results.

Whenever we performed soluble protein immunizations to examine adaptive immune responses in this study, the immunogen was delivered in adjuvant, specifically Σ Adjuvant System (SAS), as described in the Methods. This adjuvant contains the Monophosphoryl Lipid A component from Salmonella in an oil-water emulsion, and as such, its formulation is at least roughly similar to the AS01 adjuvant used in Mosquirix (RTS,S), the only licensed anti-malaria vaccine, as well as other vaccines currently used in humans. SAS has been shown to elicit very high titers of neutralizing antibodies in mice (Sastry et al., PloS One 2017, PMID 29073183). Therefore our results should have relevance for vaccination in humans. We have modified the manuscript text (lines 454-455) to highlight that in this study, protein immunogens were administered with adjuvant.

Prior to publication, I suggest the following edits/queries for clarity.

Is the legend in Figure 1 correct? Line 142 states "B cells bound significantly more iRBCs than nRBCs …" but the figure is the opposite as labelled. I assumed that the labels have been switched, but best to check and match back to the text as needed.

The symbols in the figure legend were switched in error; the filled circles are actually iRBC+ and the open circles are nRBC+. We regret the error and appreciate the reviewer bringing it to our attention. We have corrected the figure.

In Figure 1 – suggestion to also analyse the data as RBC+ (% of total cell subset) – ie RBC+ DCs/% of total DCs, to understand the relative capacity of each subset to uptake antigen as a % of all those cells in that subset.

The data are presented this way (as RBC+ % of total cell subset) in Figure 2C.

In Figure 2 – it looks like that are hardly any double positive cells – ie those which have taken up both PE and iRBCs at the same time – is this expected and consistent or just in the gating example? Does it suggest the specialisation of both B cells and DCs to be better able to uptake soluble or particle antigens in some way or suggestive of different subsets?

We consistently observed this phenomenon across all our experiments—i.e. that very few cells took up both PE and iRBCs. We can suggest at least three factors that may underlie this: (1) an intrinsic relative specialization of B cells and DCs at picking up soluble versus particulate antigen, which we describe and characterize in Figure 2 and lines 186-192; (2) spatial organization within the spleen that may affect movement of, or access to, soluble versus particulate antigen; and (3) the overall very low frequency of splenocytes that pick up either label, which statistically would also result in an even lower frequency of cells picking up both labels. These factors are not mutually exclusive. We now discuss this point explicitly (lines 172-175).

I don't understand Figure 3C – why is the PE+ DCs in the naive mice 0? Additionally, the text states in line 226 "we observed equivalent or slightly higher frequencies" but Figure 3C has a clear significantly increased uptake in infected mice.

The absolute number of DCs that picked up PE in naïve mice in these experiments was between 500-5000 cells per mouse. We had graphed these numbers on a linear scale with the y- axis tick marks representing the indicated number multiplied by 10^-4 (which we indicated in the y-axis label). In this format, the values did appear very close to zero, especially next to the data points from infected mice. For better resolution of the data, we have changed the y-axis of this graph to a log scale in the revision.

Regarding the reviewer’s point about line 226:

“we had not wanted to overstate the increase in PE uptake in infected mice, since four of the six mice in this group were only slightly higher than in the naïve group while two additional mice had much higher uptake. However, the reviewer is correct that the statistics show a significant increase in the affected mice, so we have revised our text to state this clearly (line 229).”

At day 23, when antibody levels to soluble antigen (Figure 5F) have recovered to levels of uninfected mice – are robust Tfh/GC B cells detected? Ie, is there evidence of germinal centre recovery? Is this due to long-term soluble antigen in DCs that is able to then activate naïve T/B cells down the track?

Please see our response to Reviewer 1, Specific Comment #6, with regard to recovery of Tfh cells by Day 23 and beyond. We have not examined GC B cell numbers at this later timepoint, but the recovered production of IgG in infected mice (Figure 5F) by this time, combined with the presence of near normal numbers of Tfh cells (Figure 4G), strongly suggests that germinal centers have recovered as well. Since total antigen-specific T cell numbers are intact in these mice even at early timepoints, we favor the hypothesis that resolving inflammation permits resumption of normal cognate and non-cognate interactions between B and T cells that allows recovery of germinal centers.

Figure 5 – shows reduced IgG, but would be beneficial to also look at the specific subclasses of these antibodies, particularly given the importance of cytophilic subclasses in protection.

We agree that it would be interesting to examine specific subclasses of IgG in this setting. Unfortunately, we do not have any more stored serum samples that would facilitate the rapid assessment of this question. However, we have performed extensive analyses of antigen-specific IgG+ B cells in mice with malaria and found that the great majority of the class-switched population is dependent on T cell help (Harms Pritchard and Pepper, manuscript under revision). Thus, we would hypothesize that all IgG subclasses elicited by our immunization would be affected in hosts with malaria, since our model is that B-T interactions are severely disrupted in these mice.

Author details

1. Mary F Fontana

   Department of Immunology, University of Washington School of Medicine, Seattle, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   maryffontana@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3630-181X

2. Erica Ollmann Saphire

   Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology, La Jolla, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

3. Marion Pepper

   Department of Immunology, University of Washington School of Medicine, Seattle, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing – review and editing

   For correspondence

   mpepper@uw.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7278-0147

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