Author response: Atypical memory B-cells are associated with Plasmodium falciparum anemia through anti-phosphatidylserine antibodies

Field: Medicine (Health Sciences)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Anemia is a common complication of malaria that is characterized by the loss of infected and uninfected erythrocytes. In mouse malaria models, clearance of uninfected erythrocytes is promoted by autoimmune anti-phosphatidylserine (PS) antibodies produced by T-bet+B-cells, which bind to exposed PS in erythrocytes, but the mechanism in patients is still unclear. In Plasmodium falciparum patients with anemia, we show that atypical memory FcRL5+T-bet+ B-cells are expanded and associate both with higher levels of anti-PS antibodies in plasma and with the development of anemia in these patients. No association of anti-PS antibodies or anemia with other B-cell subsets and no association of other antibody specificities with FcRL5+T-bet+ B-cells is observed, revealing high specificity in this response. We also identify FcRL5+T-bet+ B-cells as producers of anti-PS antibodies in ex vivo cultures of naïve human peripheral blood mononuclear cells (PBMC) stimulated with P.-falciparum-infected erythrocyte lysates. These data define a crucial role for atypical memory B-cells and anti-PS autoantibodies in human malarial anemia. https://doi.org/10.7554/eLife.48309.001 Introduction Malaria is still a major global health threat with over 200 million new infections and around 400,000 deaths in 2017 (World Health Organization, 2018). Anemia is a common complication associated with malaria that contributes significantly to the great morbidity and mortality associated with the disease (White, 2018). Despite its high clinical relevance, the mechanisms underlying malarial anemia in patients remain largely unknown. The difficulty in studying this syndrome arises at least in part from its multi-factorial etiology, as malaria causes both the clearance (through complement-mediated lysis or phagocytosis) of infected and uninfected erythrocytes and bone marrow dyserythropoiesis (Lamikanra et al., 2007; White, 2018). The clearance of uninfected erythrocytes is thought to contribute significantly to anemia, because for each erythrocyte lysed directly due to Plasmodium infection, about eight uninfected erythrocytes are killed in P. falciparum infections (Jakeman et al., 1999; Price et al., 2001) and about 34 erythrocytes are killed in P. vivax infections (Collins et al., 2003). The anti-parasite B-cell antibody response that is generated during the malaria blood stage represents an essential component of the protective immune response against this disease (Doolan et al., 2009; Portugal et al., 2013). However, an important autoimmune response is also generated during malaria, in which autoantibodies mediate some of the associated pathologies (Hart et al., 2016; Rivera-Correa and Rodriguez, 2018; Rivera-Correa, 2016). Specifically, autoantibodies that target membrane phosphatidylserine (PS) on uninfected erythrocytes promote anemia during malaria in mouse models, and these autoantibodies correlate with low hemoglobin levels in a cohort of P.-falciparum- and P.-vivax-infected patients with malarial anemia (Barber et al., 2019; Fernandez-Arias et al., 2016), establishing an autoimmune component to malarial anemia. An atypical group of B-cells that express the transcription factor T-bet secrete autoantibodies in different autoimmune mouse models (Rubtsova et al., 2015; Rubtsova et al., 2017) and were found to be major producers of anti-PS antibodies in mouse malaria (Rivera-Correa et al., 2017). The relation of these atypical T-bet+ B-cells to autoantibodies and their role in malarial anemia has not been assessed in malaria patients. B-cells expressing the transcription factor T-bet have been identified in the circulation of individuals from malaria-endemic areas and implicated in the memory response against Plasmodium (Guthmiller et al., 2017), being considered atypical memory B-cells (Obeng-Adjei et al., 2017; Weiss et al., 2009). These cells are characterized as secreting low levels of antibodies and by inhibitory phenotypic markers such as FcRL5 (Sullivan et al., 2015). The secreted-antibody specificity of these atypical memory B-cells has not been characterized because of their reduced effector function and minimal antibody secretion in-vitro (Portugal et al., 2015). Hence studying these FcRL5+T-bet+ B-cells during acute stage malaria in a population from a non-endemic area, such as European travelers, represents a unique opportunity to study the antibody specificity that results from recent primary activation. In this study, we focused on measuring the levels of atypical FcRL5+T-bet+ B-cells in the circulation of P.-falciparum-infected returned travelers and its relation to autoantibodies and anemia development. Our results show that atypical FcRL5+T-bet+ B-cells are greatly expanded in acute malaria in P.-falciparum-infected patients and correlate with both anemia development and plasma anti-PS antibody levels in these patients. In vitro studies confirmed that the activation of FcRL5+T-bet+ B-cells by P.-falciparum-infected erythrocytes induces the secretion of anti-PS autoantibodies. All together, these findings attribute a role to atypical FcRL5+T-bet+ B-cells and anti-PS antibodies in the pathogenesis of human malarial anemia. Targeting these cells could have a therapeutic benefit in treating anemia during malaria. Results Specific autoantibodies correlate with malarial anemia and erythrocyte lysis capacity in P.-falciparum-infected patients In this study, we focused on samples (24 patients, 31 unique samples) from a cohort of P.-falciparum-infected returned travelers from Germany, who acquired malaria while visiting Africa. This cohort suffered from mild anemia with average hemoglobin levels of 12.4 g/dL (males) and 10.2 g/dL (females) (normal range is 13.8 to 17.2 and 12.1 to 15.1 g/dL, respectively) (Table 1). Table 1 Clinical information from P.-falciparum-infected returned German travelers. https://doi.org/10.7554/eLife.48309.002 Subject IDDay of sampling&Hemoglobin (g/dl)*Hemoglobin (g/dl)**Thrombocyte count (1000/µl)*Parasite count/µl **, #Red blood cell (RBC) count (million/µl)**SexAgeType of patient$Country of infection100310.313.4135132,0004.6m51VFRNigeria31ND13.4ND132,0004.6m51VFRNigeria10187.312.12701,860,0003.89f56TGambia248.312.15891,860,0003.89f56TGambia102613.516.2140<52,8005.28m63TUganda27ND16.2ND<52,8005.28m63TUganda103213.814.45446,9004.69m38VFRGuinea104312.613.6130470,0004.7m56TMadagascar713.713.6321470,0004.7m56TMadagascar10557.88.11351,050,0003.5f53VFRKenya106210.811.9963,9004.26m43VFRGhana107311.612.448>430,0004.3m52VFRGhana1211.412.4475>430,0004.3m52VFRGhana108010.310.31561763.83m50VFRBenin109210.510.898163.73m62VFRGhana110312.313.5128366,8005.24f20VFRTanzania111113.514.423144,6004.82m35TNigeria112313.113.770160,2005.34m26VFRBenin113410.713.774340,0004.25m39VFRUnknown114316.618.75348405.96m26TGhana115012.612.65314,7624.84m62VFRGhana116212.512.98026,9174.58f43VFRCameroon410.412.910626,9174.58f43VFRCameroon11741519.532492,8006.16m46TNigeria11811112.120296,1004.23m39TUganda119111.911.912329,8004.02f25VFRIvory Coast311.611.911629,8004.02f25VFRIvory Coast120016.116.110278965.36m38VFRGuinea Bissau121214.415.71194965.48m34VFRNigeria1222ND13.5ND745.15m18VFRTogo123212.814.26049,8004.98m52VFRGhana &Days since treatment start to sampling, *Measurement at day of sampling, **Measurement at day of presentation, #Parasitemia expressed in infected erythrocytes per µl of blood, $Tourist (T), Visiting Friend or Relative (VFR). ND, not determined. As described before in other cohorts with mild anemia (Fernandes et al., 2008; Sumbele et al., 2016), hemoglobin levels in this cohort do not significantly correlate with parasitemia (Figure 1A), confirming that direct erythrocyte infection by Plasmodium is not a major cause of anemia and indicating that other mechanisms must contribute to this pathology. This is in agreement with previous findings reporting major losses of uninfected erythrocytes and dyserythropoiesis during malaria (White, 2018). Figure 1 Download asset Open asset Specific autoantibodies correlate with malarial anemia in P.-falciparum-infected returned travelers. Non-parametric Spearman correlation analysis comparing hemoglobin with (A) parasitemia, (B) anti-PS IgG antibodies, (C) anti-PfEBA IgG antibodies, (D) anti-erythrocyte IgG antibodies and (E) anti-DNA IgG antibodies. https://doi.org/10.7554/eLife.48309.003 Figure 1—source data 1 Source data for Figure 1 . https://doi.org/10.7554/eLife.48309.004 Download elife-48309-fig1-data1-v2.zip As we had previously observed that autoimmune anti-PS antibodies induce anemia during malaria in a mouse model (Fernandez-Arias et al., 2016; Rivera-Correa et al., 2017), we determined whether hemoglobin levels correlated with autoimmune anti-PS IgG antibodies in our cohort. We observed an inverse correlation between anti-PS antibodies and hemoglobin levels (Figure 1B), which was not found for IgG antibodies against the P. falciparum erythrocyte binding antigen (PfEBA) (Figure 1C), suggesting that an autoimmune response contributes to the development of anemia in malaria. To further dissect the role that autoantibodies could be playing during malarial anemia in P.-falciparum-infected patients, we analyzed other relevant autoantibodies: anti-erythrocyte and anti-DNA IgG antibodies. As expected, our results show that plasma anti-erythrocyte IgG antibodies also correlate with the development of malarial anemia, showing a significant negative correlation with hemoglobin from the samples of P.-falciparum-infected returned travelers (Figure 1D). These antibodies recognized all kinds of antigens in human erythrocyte lysates (Mourão et al., 2018; Mourão et al., 2016), including PS. The similarities in the correlations between anti-PS and anti-erythrocyte IgG antibodies and anemia may indicate that anti-PS antibodies are the major antibody specificity driving anemia. In addition, our results show that anti-DNA IgG antibodies, which are typical in autoimmune diseases (Tsokos, 2011), do not correlate with anemia (Figure 1E), suggesting that the specificity of the autoimmune antibody response is important for the development of anemia. Collectively, these initial correlations point to an autoimmune origin of malarial anemia, and confirm that the cohort of P.-falciparum-infected returned travelers is an adequate study group for testing the contribution of atypical B-cells in malarial anemia. As malarial anemia is characterized by the lysis of uninfected erythrocytes and by an increase in lactate dehydrogenase (LDH) in the plasma (Fendel et al., 2010), we determined whether anti-PS IgG antibodies correlate with the levels of LDH in the patient's plasma (Figure 2A). We observed a significant positive correlation, which suggests that autoimmune anti-PS IgG antibodies may induce the lysis of erythrocytes during malaria. We next determined whether erythrocyte lysis capacity was increased in the plasma of P.-falciparum-infected patients. Using an in vitro complement lysis assay of human erythrocytes exposing PS, we observed that plasma from patients was significantly more effective than healthy control plasma in lysing human erythrocytes (Figure 2B). Figure 2 Download asset Open asset Plasma from P. falciparum patients mediates erythrocyte lysis, which can be partially inhibited by Annexin V. (A,B) Correlation of plasma anti-PS IgG antibodies with the LDH levels (A) or with the erythrocyte lysis capacity (B) of the plasma of P. falciparum patients. (C) Complement-mediated lysis of erythrocytes exposing PS by P. falciparum patient's plasma compared to plasma from uninfected controls, expressed as percentage of maximal lysis. (D) Complement-mediated lysis of erythrocytes exposing PS, pre-incubated or not with Annexin V, before incubation with the plasma of P. falciparum patients (n = 6). Results show the means and standard deviations of triplicated determinations. Significance was assessed by nonparametric Spearman correlation analysis (A,B) or unpaired Student's t-test (C,D). *p≤0.05, **p≤0.01. https://doi.org/10.7554/eLife.48309.005 Figure 2—source data 1 Source data for Figure 2. https://doi.org/10.7554/eLife.48309.006 Download elife-48309-fig2-data1-v2.zip We then studied the relation of anti-PS IgG levels and the erythrocyte lysis capacity in the patient's plasma, as determined using the in vitro complement lysis assay. We observed a direct correlation between anti-PS and erythrocyte lysis capacity (Figure 2C), which suggests that anti-PS IgG antibodies may contribute to anemia in malaria by inducing the complement-mediated lysis of uninfected erythrocytes. To determine the anti-PS specificity of the erythrocyte lysis, we pre-incubated the erythrocytes with annexin V, a protein that specifically binds to PS and inhibits the binding of anti-PS antibodies (Fernandez-Arias et al., 2016; van Engeland et al., 1998), finding a partial reduction of the erythrocyte lysis capacity in the plasma samples (Figure 2D). It is likely that other antibody specificities (Mourão et al., 2018; Mourão et al., 2016) in addition to anti-PS also contribute to erythrocyte lysis in malaria patients. Taken together, these results suggest that anti-PS antibodies mediate the lysis of uninfected erythrocytes that expose PS during malaria. Atypical memory FcRL5+T-bet+ B-cells are greatly expanded in P.-falciparum-infected patients Because our previous studies in mice and previous reports in human malaria patients had shown a large increase in atypical memory B-cell (MBC) population upon infection with Plasmodium (Patgaonkar et al., 2018; Pérez-Mazliah et al., 2018; Portugal et al., 2015; Rivera-Correa et al., 2017; Sullivan et al., 2016; Weiss et al., 2009), we next analyzed the total levels of atypical MBCs in peripheral blood mononuclear cells (PBMC) in the cohort of P.-falciparum-infected returned travelers. We characterized atypical MBCs by the double expression of FcRL5 and T-bet, as both markers are highly expressed and characteristic of this population (Figure 3A). This population is known to be elevated in malaria patients from endemic areas (Obeng-Adjei et al., 2017; Sullivan et al., 2016). After gating out non-B-cells (CD19–), we observed that FcRL5+T-bet+ B-cells are indeed expanded in the PBMC samples from P.-falciparum-infected German returned travelers when compared to samples from uninfected German controls (Figure 3B). Figure 3 with 3 supplements see all Download asset Open asset Atypical MBCs expand in P.-falciparum-infected patients and decline after treatment. (A) Gating strategy for the characterization of FcRL5+ T-bet+ B-cells (CD19+) with representative plots of one uninfected control and one P. falciparum patient. (B) Percentage of CD19+ FcRL5+ T-bet+ B-cells in samples from uninfected controls and P. falciparum patients. Significance assessed by unpaired Student's t test. ****p≤0.0001. https://doi.org/10.7554/eLife.48309.007 Figure 3—source data 1 Source data for Figure 3. https://doi.org/10.7554/eLife.48309.014 Download elife-48309-fig3-data1-v2.zip We further analyzed the cohort of P.-falciparum-infected returned travelers by considering two different groups: 1) tourists, who reported to be naïve to malaria, and 2) those visiting friends or relatives (VFR), who reported having at least one previous episode of malaria (Table 1). We did not observe any significant difference in the levels of atypical MBCs in the PBMC between these two groups (Figure 3—figure supplement 1). We also observed no significant gender difference between the levels of FcRL5+T-bet+ B-cells in PBMC (Figure 3—figure supplement 2). Furthermore, we found no significant correlation between the time after treatment at which samples were obtained (ranging from 0 to 31 days, Table 1) and the level of hemoglobin, which indicates that the variations in hemoglobin levels are not just a consequence of time after parasite clearance. We also observed a significant direct correlation between the levels of atypical MBCs and the days after treatment, which suggests that the levels of these cells continue to increase after treatment. This increase is compatible with the activation of atypical MBCs during infection and their continuing proliferation after parasite clearance (Figure 3—figure supplement 3). Altogether, these initial results suggest an expansion of atypical FcRL5+T-bet+ B-cells in P.-falciparum-infected patients following acute infection. Atypical memory FcRL5+T-bet+ B-cells correlate with hemoglobin levels in P.-falciparum-infected returned travelers We next sought to determine whether atypical MBCs correlate with hemoglobin levels in P. falciparum patients. For this purpose, we performed a B-cell sub-population gating analysis in PBMC samples from our cohort. Following classical gating strategies for all relevant B-cell (CD19+) sub-populations from human PBMC (Weiss et al., 2009), we analyzed: (i) naïve B-cells (CD27–CD21+CD10–), (ii) immature B-cells (CD10+), (iii) plasma cells (CD27+CD21–CD20–), (iv) classical MBCs (CD27+CD21+) and (v) atypical MBCs (FcRL5+T-bet+) (Figure 4—figure supplement 1). To define the atypical and classical MBC populations better, we analyzed the expression of T-bet in FcRL5+ cells compared to classical MBCs, finding that the expression of T-bet is significantly higher in FcRL5+ cells (Figure 4—figure supplement 2). We found a significant inverse correlation between hemoglobin levels and atypical MBCs levels in the PBMC samples from P. falciparum patients (Figure 4A). Atypical MBCs did not significantly correlate with other relevant parameters such as parasitemia (Figure 4—figure supplement 3), but did correlate positively with the age of the patient (Figure 4—figure supplement 4), supporting previous studies that initially denominated these cells as Age-associated B-cells (Phalke and Marrack, 2018). We also analyzed a possible relation of atypical MBCs with thrombocytopenia, another complication that frequently accompanies malaria and has an autoimmune component to its pathology (Lacerda et al., 2011). We did not observe any significant correlation between the levels of atypical MBCs and thrombocyte counts in our cohort of patients (Figure 4—figure supplement 5), suggesting that atypical MBCs specifically correlate with anemia and not with other malaria-associated complications. Figure 4 with 6 supplements see all Download asset Open asset The atypical MBC subset correlates with the development of anemia in P. falciparum patients. Correlation analysis of atypical (A) and classical (B) MBC subsets from the PBMC of P. falciparum patients compared with hemoglobin levels. Significance was assessed by non-parametric Spearman correlation analysis. https://doi.org/10.7554/eLife.48309.015 Figure 4—source data 1 Source data for Figure 4. https://doi.org/10.7554/eLife.48309.027 Download elife-48309-fig4-data1-v2.zip Interestingly, we observed that classical memory B-cells (CD27+CD21+) had a significant positive correlation with hemoglobin levels (Figure 4B). We did not find any significant correlation between hemoglobin level and percentage of naïve B-cells or immature B-cells, but observed a significant positive correlation with percentage of plasma cells (Figure 4—figure supplement 6). Taken together, these results suggest that atypical MBCs, but not other B-cell subtypes, are specifically implicated in malaria-induced anemia in patients. Atypical memory B-cells correlate with anti-PS IgG antibodies in P.-falciparum-infected patients During Plasmodium infections in mice, T-bet+ B-cells secrete anti-PS IgG antibodies that induce premature clearance of uninfected erythrocytes, promoting malarial anemia (Fernandez-Arias et al., 2016; Rivera-Correa et al., 2017). The role of anti-PS IgG antibodies and the B-cells that secrete them during malarial anemia in P.-falciparum-infected patients has not been studied before. In this cohort of P.-falciparum-infected German returned travelers, we observed an inverse correlation between anti-PS IgG antibodies and hemoglobin, suggesting a role of these autoantibodies in promoting anemia in this cohort (Figure 1B). As both FcRL5+T-bet+ atypical B-cells and anti-PS IgG antibodies correlate with hemoglobin levels in our cohort, we assessed the relationship between the levels of FcRL5+T-bet+ atypical B-cells and anti-PS IgG antibodies. Our results show a significant positive correlation between FcRL5+T-bet+ atypical MBCs and anti-PS IgG antibodies (Figure 5A), possibly implicating these cells as the major producers of the antibodies. In accordance with the hemoglobin results (Figure 4B), we also found a significant inverse relationship between anti-PS IgG antibodies and classical MBCs (CD27+CD21+) (Figure 5B). As shown before for hemoglobin (Figure 4—figure supplement 6), neither naïve nor immature B-cells presented a significant correlation with the levels of plasma anti-PS IgG antibodies (Figure 5—figure supplement 1). Plasma cells, which previously correlated with hemoglobin levels, did not correlate with anti-PS IgG antibodies, suggesting that their role in anemia may be mediated through the secretion of antibodies that have different specificities. These results establish a relationship between atypical MBCs and anti-PS IgG antibodies. Figure 5 with 3 supplements see all Download asset Open asset Anti-PS IgG antibodies show distinct correlations with classical and atypical MBC subsets in P. falciparum patients. Correlation analysis of levels of (A) atypical and classical (B) MBCs with anti-PS IgG antibody levels from the plasma of P. falciparum patients. (C) Correlation analysis of atypical and classical MBC levels. Significance was assessed by non-parametric Spearman correlation analysis. https://doi.org/10.7554/eLife.48309.028 Figure 5—source data 1 Source data for Figure 5. https://doi.org/10.7554/eLife.48309.035 Download elife-48309-fig5-data1-v2.zip As an inverse relationship between classical and FcRL5+T-bet+ atypical MBCs was found with hemoglobin and anti-PS antibodies, we analyzed whether there was any correlation between the levels of these two populations. Accordingly, we found a significant negative correlation between the levels of classical and FcRL5+T-bet+ atypical MBCs (Figure 5C). These results suggest a possible relationship between classical and atypical MBC populations, but may also be interpreted as the result of a robust proliferation of atypical MBCs, which would decrease the proportion of classical MBCs among the CD19+ population even if their actual numbers had not decreased. In addition, we assessed the levels of two other autoantibodies (anti-erythrocyte and anti-DNA) to further characterize the autoantibody repertoire that correlates with malaria anemia in P.-falciparum-infected patients. We first analyzed the relationship between anti-erythrocyte IgG antibodies and all of the B-cell sub populations. The analysis of memory B-cell subsets and anti-erythrocyte IgG antibodies resembles the anti-PS IgG antibodies correlations, showing a positive significant correlation between FcRL5+T-bet+ atypical MBCs. No significant correlation was found with classical memory, naïve or immature B-cell subsets. A positive correlation was observed with plasma cells, suggesting that other autoantibodies besides anti-PS are produced by these cells, possibly explaining the previous correlation of plasma cells with hemoglobin levels (Figure 5—figure supplement 2). We also analyzed possible correlations of the levels of plasma anti-DNA antibodies with all of the B-cell subpopulations (Figure 5—figure supplement 3). We did not observe any significant correlation with atypical MBCs, suggesting that anti-DNA antibodies are not predominantly produced by this subpopulation of B-cells. The lack of correlation with other B-cell sub-populations (naïve, immature, plasma cell, and classical memory) does not provide any indications on the B-cell subtype that produces these antibodies. Anti-DNA antibodies do not correlate with hemoglobin levels in our cohort (Figure 1E), so it is likely that they do not play a role in this pathology and therefore are not expected to correlate with relevant B-cells subsets that contribute to anemia. Atypical memory B-cells do not correlate with anti-PfEBA antibodies in P.-falciparum-infected patients We also assessed the relationship between plasma anti-parasite antibodies (anti-PfEBA) and the different B-cell subsets in our cohort of P.-falciparum-infected patients. As specific plasma autoantibodies (anti-PS and anti-erythrocyte, but not anti-DNA) correlate distinctly with the atypical MBC subset and with anemia development, we questioned whether there is any correlation between any of the different B-cell subsets and anti-parasite antibodies (anti-PfEBA). This analysis showed no significant correlation between anti-PfEBA IgG antibodies and any of the B-cells subsets assessed (Figure 6). Anti-PfEBA IgG antibodies presented no significant correlation with anemia in patients (Figure 1C), so the lack of correlation with FcRL5+T-bet+ atypical MBCs, which tightly correlate with anemia, is expected and further supports the lack of involvement of anti-parasite antibodies in malarial anemia. Figure 6 Download asset Open asset There is no significant correlation of anti-parasite PfEBA antibodies with relevant B-cell subsets from P. falciparum patients. Correlation analysis of (A) atypical MBCs, (B) classical MBCs, (C) plasma cells, (D) naïve B-cells, and (E) immature B-cells with anti-P. falciparum (PfEBA) IgG antibody levels from the plasma of P. falciparum patients. Significance was assessed by non-parametric Spearman correlation analysis. https://doi.org/10.7554/eLife.48309.036 Figure 6—source data 1 Source data for Figure 6. https://doi.org/10.7554/eLife.48309.037 Download elife-48309-fig6-data1-v2.zip FcRL5+T-bet+ atypical B-cells secrete anti-PS antibodies upon stimulation with P.-falciparum-infected erythrocytes in vitro We have observed a strong significant correlation between FcRL5+T-bet+ B-cells, anti-PS antibodies and malarial anemia in a cohort of P.-falciparum-infected patients (Figures 3 and 4). As these data from patient samples are limited to the analysis of correlations between different parameters, we aimed to determine directly whether activation of FcRL5+T-bet+ atypical B-cells can induce the secretion of anti-PS antibodies. Expansion of T-bet+ B-cells and secretion of anti-PS antibodies into the culture medium was observed in vitro after incubation of PBMC from healthy donors with lysates of P.-falciparum-infected erythrocytes (Rivera-Correa et al., 2017). To determine whether the T-bet+ B-cells that are activated in these experiments are also FcRL5+ and, more importantly, whether they specifically secrete anti-PS antibodies, we incubated PBMC from healthy US individuals with P.-falciparum-infected erythrocyte lysates. We observed a robust expansion of FcRL5+T-bet+ B-cells compared to cells incubated with uninfected erythrocyte lysate or with no stimulation (Figure 7A). To investigate specifically whether these in vitro P.-falciparum-induced FcRL5+ T-bet+ atypical B-cells secrete anti-PS antibodies, we first enriched this population by selecting FcRL5+ cells from PBMC stimulated with P.-falciparum-infected erythrocyte lysate. Remarkably, ELISPOT analysis of the antibody specificity of enriched FcRL5+ cells showed significantly increased numbers of anti-PS-specific B-cells in the FcRL5+ population when compared to enriched CD27+ cells, which would represent predominantly classical memory B-cells and plasmablast/plasma cells (Figure 7B). No significant difference is observed between the number of FcRL5+antibody-secreting cells (ASCs) producing anti-PS and the number of anti-PfEBA cells, suggesting that the FcLR5+ subset can efficiently produce both autoimmune and anti-parasite antibodies. Figure 7 with 2 supplements see all Download asset Open asset P. falciparum drives the expansion of human FcRL5+ T-bet+ B-cells that secrete anti-PS antibodies in vitro. (A) Percentage of T-bet+FcRL5+ B-cells that expanded from the PBMCs of a healthy naïve donor after in-vitro exposure to either uninfected erythrocyte lysate (uLysate) or P. -falciparum-infected erythrocyte lysate (iLysate). (B) ELISPOT of enriched populations for either FcRL5 (gray bars) or CD27 (black bars) from PBMCs of healthy naïve US donors after in-vitro exposure to P.-falciparum-infected erythrocyte lysate (iLysate) (N = 3). ASC, antibody-secreting cells. Significance assessed by unpaired Student's t test. **p≤0.01, ***p≤0.001. https://doi.org/10.7554/eLife.48309.038 Figure 7—source data 1 Source data for Figure 7. https://doi.org/10.7554/eLife.48309.041 Download elife-48309-fig7-data1-v2.zip As FcRL5 i