1. Immunology and Inflammation
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Atypical memory B-cells are associated with Plasmodium falciparum anemia through anti-phosphatidylserine antibodies

  1. Juan Rivera-Correa
  2. Maria Sophia Mackroth
  3. Thomas Jacobs
  4. Julian Schulze zur Wiesch
  5. Thierry Rolling
  6. Ana Rodriguez  Is a corresponding author
  1. New York University School of Medicine, United States
  2. University Medical Center Hamburg-Eppendorf, Germany
  3. Bernhard Nocht Institute for Tropical Medicine, Germany
  4. German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems, Germany
Research Article
Cite this article as: eLife 2019;8:e48309 doi: 10.7554/eLife.48309
7 figures, 2 tables and 1 additional file

Figures

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
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 3 with 3 supplements
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—figure supplement 1
Atypical MBCs do not correlate significantly with patient background.

Comparison of the percentage of atypical MBCs in the circulation of P. falciparum patients by background (visiting friends or relatives (VFR) and tourists). Significant assessed by unpaired Student's t test.

https://doi.org/10.7554/eLife.48309.008
Figure 3—figure supplement 2
Atypical MBCs do not correlate significantly with patient gender.

Comparison of the percentage of atypical MBCs in the circulation of P. falciparum patients by gender. Significant assessed by unpaired Student's t test.

https://doi.org/10.7554/eLife.48309.010
Figure 3—figure supplement 3
The time after treatment at which samples were collected correlates significantly with atypical MBCs but not with hemoglobin levels.

Non-parametric Spearman Correlation analysis comparing the days after treatment when samples were collected and levels of (A) atypical MBCs and (B) hemoglobin.

https://doi.org/10.7554/eLife.48309.012
Figure 4 with 6 supplements
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—figure supplement 1
Gating strategy for relevant B-cell sub-populations.

B-cell subpopulations of human PBMC (Weiss et al., 2009).

https://doi.org/10.7554/eLife.48309.016
Figure 4—figure supplement 2
Expression of T-bet in FcRL5+ cells compared to classical MBCs.

Significance assessed by unpaired Student's t test. **p≤0.01.

https://doi.org/10.7554/eLife.48309.017
Figure 4—figure supplement 3
Atypical MBCs do not correlate significantly with parasitemia.

Non-parametric Spearman correlation analysis comparing the percentage of atypical MBCs and parasite levels.

https://doi.org/10.7554/eLife.48309.019
Figure 4—figure supplement 4
Atypical MBCs correlate significantly with patient's age.

Non-parametric Spearman correlation analysis comparing the percentage of atypical MBCs and patient's age.

https://doi.org/10.7554/eLife.48309.021
Figure 4—figure supplement 5
Atypical MBCs do not correlate significantly with thrombocyte levels.

Non-parametric Spearman correlation analysis comparing the percentage of atypical MBCs and thrombocyte levels in the circulation.

https://doi.org/10.7554/eLife.48309.023
Figure 4—figure supplement 6
Correlations of other B-cell subsets with hemoglobin levels in P. falciparum patients.

Non-parametric Spearman correlation analysis of relevant B-cells subsets from the PBMC of P. falciparum patients: (A) naïve B-cells (CD27CD21+CD10), (B) immature B-cells (CD10+), and (C) plasma cells (CD27+CD21CD20) compared with hemoglobin levels.

https://doi.org/10.7554/eLife.48309.025
Figure 5 with 3 supplements
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—figure supplement 1
Anti-PS IgG antibodies do not correlate with other B-cell subsets in P. falciparum patients.

Non-parametric Spearman Correlation analysis of (A) naïve B-cells (CD27CD21+CD10), (B) immature B-cells (CD10+), and (C) plasma cells (CD27+CD21CD20) with anti-PS IgG antibody levels from the plasma of P. falciparum patients.

https://doi.org/10.7554/eLife.48309.029
Figure 5—figure supplement 2
Correlations of anti-RBC IgG antibodies with B-cell subsets in P. falciparum patients.

Non-parametric Spearman Correlation analysis of (A) atypical MBCs (CD27CD21FcRL5+), (B) classical MBCs (CD27+CD21+), (C) naïve B-cells (CD27CD21+CD10), (D) immature B-cells (CD10+), and (E) plasma cells or plasmablasts (CD27+CD21CD20) with anti-erythrocyte lysate IgG antibody levels from the plasma of P. falciparum patients.

https://doi.org/10.7554/eLife.48309.031
Figure 5—figure supplement 3
Anti-DNA IgG antibodies do not correlate with the B-cell subsets analyzed in P. falciparum patients.

Non-parametric Spearman correlation of (A) atypical MBCs (CD27CD21FcRL5+), (B) classical MBCs (CD27+CD21+), (C) naïve B-cells (CD27CD21+CD10), (D) immature B-cells (CD10+), and (E) plasma cells or plasmablasts (CD27+CD21CD20) with anti-DNA lysate IgG antibody levels from the plasma of P. falciparum patients.

https://doi.org/10.7554/eLife.48309.033
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 7 with 2 supplements
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—figure supplement 1
Total antibody-secreting cells among the CD27+- and FcLR5+-enriched PBMC.

Total number of anti-IgM spots of antibody-secreting cells (ASCs) from either CD27+- or FcRL5+-enriched PBMC that were stimulated with P.-falciparum-infected erythrocyte lysate. Significant assessed by unpaired Student's t test, ****p<0.0001.

https://doi.org/10.7554/eLife.48309.039
Figure 7—figure supplement 2
Stimulation with P. falciparum Histidine Rich Protein II (HRPII) does not stimulate the expansion of atypical MBCs in vitro.

Stimulation in vitro of naïve PBMC from healthy US donors (n = 3) with medium, P. falciparum HRPII, uninfected erythrocyte lysate (uLysate) or P.-falciparum-infected erythrocyte lysate (iLysate). Significance was assessed by one-way Anova. *p<0.05, **p<0.01.

https://doi.org/10.7554/eLife.48309.040

Tables

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 infection
100310.313.4135132,0004.6m51VFRNigeria
31ND13.4ND132,0004.6m51VFRNigeria
10187.312.12701,860,0003.89f56TGambia
248.312.15891,860,0003.89f56TGambia
102613.516.2140<52,8005.28m63TUganda
27ND16.2ND<52,8005.28m63TUganda
103213.814.45446,9004.69m38VFRGuinea
104312.613.6130470,0004.7m56TMadagascar
713.713.6321470,0004.7m56TMadagascar
10557.88.11351,050,0003.5f53VFRKenya
106210.811.9963,9004.26m43VFRGhana
107311.612.448>430,0004.3m52VFRGhana
1211.412.4475>430,0004.3m52VFRGhana
108010.310.31561763.83m50VFRBenin
109210.510.898163.73m62VFRGhana
110312.313.5128366,8005.24f20VFRTanzania
111113.514.423144,6004.82m35TNigeria
112313.113.770160,2005.34m26VFRBenin
113410.713.774340,0004.25m39VFRUnknown
114316.618.75348405.96m26TGhana
115012.612.65314,7624.84m62VFRGhana
116212.512.98026,9174.58f43VFRCameroon
410.412.910626,9174.58f43VFRCameroon
11741519.532492,8006.16m46TNigeria
11811112.120296,1004.23m39TUganda
119111.911.912329,8004.02f25VFRIvory Coast
311.611.911629,8004.02f25VFRIvory Coast
120016.116.110278965.36m38VFRGuinea Bissau
121214.415.71194965.48m34VFRNigeria
1222ND13.5ND745.15m18VFRTogo
123212.814.26049,8004.98m52VFRGhana
  1. &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.

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional information
Biological sample (Homo sapiens)CPD backed cellsInterstate Blood bank
AntibodyAnti-human CD20 (mouse monoclonal)Biolegend3023041:100
AntibodyAnti- T-bet (mouse monoclonal)Biolegend6448101:100
AntibodyAnti-human CD11c (mouse monoclonal)Biolegend3016041:100
AntibodyAnti-human CD27 (mouse monoclonal)Biolegend3028061:100
AntibodyAnti-human CD21 (mouse monoclonal)Biolegend354910 (FITC)
354906 (APC)
1:100
AntibodyAnti-human FcRL5 (mouse monoclonal)Biolegend3403061:100
AntibodyAnti-human CD10 (mouse monoclonal)Biolegend3122101:100
AntibodyAnti-human CD19 (mouse monoclonal)Biolegend302281:100
AntibodyAnti-human IgM- HRP (goat polyclonal)MilliporeAP114P1:2000
AntibodyAnti-human IgG-HRP (goat polyclonal)GE HealthcareNA9331:2000
AntibodyAnti-human FcRL5-biotin (mouse monoclonal)Miltenyi Biotec130-105-9931:100
AntibodyAnti-human IgM unlabeled (mouse monoclonal)Biolegend314–50215 μg/ml
AntibodyAnti-human IgM-biotin(mouse monoclonal)EMD Millipore4115431 μg/ml
Peptide, recombinant proteinP. falciparum Erythrocyte Binding AntigenBEI Resources
MR-4
#MRA-116215 μg/ml
Commercial assay or kitTrue-Nuclear Transcription Factor Buffer SetBiolegend424401
Commercial assay or kitMycoAlert Mycoplasma Detection KitLonzaLT07-118
Commercial assay or kitTMB substrateBD Biosciences555214
Commercial assay or kitCD27 Microbeads humanMiltenyi Biotec130-051-601
Chemical compound, drugIonomycinLife technologiesI242222.5 µM
Chemical compound, drugFicoll-Paquee PlusGE Life Sciences17144002
Software, algorithmGraphPad PRISMGraphPad PRISM
OtherPhosphatidylserineSigma-AldrichP776920 μg/ml
OtherCalf Thymus DNASigma-AldrichD452210 μg/ml
OtherStop bufferBiolegend423001
OtherAnnexin VBiolegend6409020.5 µM
OtherX- VIVO 15 mediaLonza04-418Q
OtherHuman AB healthy plasmaSigma-AldrichH4522

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures.

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