Fig 1.
Maternal S. mansoni infection leads to a humoral anti-SEA response in offspring and a reduction in follicular dendritic cells.
(A) anti- Schistosoma mansoni egg antigen (SEA) specific IgG1 antibody titers in serum of naïve 4get/KN2 mice born to infected or uninfected mothers. Each point represents an individual mouse. (B) Analysis of the correlation between maternal and offspring anti-SEA specific IgG1. (C) Anti-SEA IgG1 titers in naïve KN2 homozygous and 4get/KN2 mice born to infected and uninfected mothers at 35, 60, and 90 days of age. (D-E) Flow cytometry plots of plasma cells (IgD-CD19+/-CD138+) in popliteal and hepatic lymph nodes from naïve 4get/KN2 pups born to infected and uninfected mothers. (F) Tile confocal imaging of naïve popliteal lymph nodes of 4get/KN2 mice at 28–35 days of age, scale bar: 200 μm. Sections were stained for CD21/35 (red) FDC-M2 (gray) and CD31 (blue) with the respective CD21/35 area quantification. (G) Flow cytometry analysis of FDCs (CD21/35+FDC-M2+) gated from CD45- with the total number of FDC in popliteal lymph node from pups 28–35 days of age. (H) Frequency of CD4+ GFP+ in the blood of naïve 4get/KN2 mice. (I) Flow cytometry analysis of the expression of BaffR in CD19+ B cells, memory B cells and plasma cells. Flow plots were concatenated samples, with 3–6 mice per group and representative of six independent experiments. Confocal microscopy data is representative of n >3 mice per group from two biologically independent experiments. Statistical significance was calculated by unpaired Student's t-test. Error bar denotes mean ± SEM. Correlation analysis was calculated by Pearson correlation coefficient.
Fig 2.
iNKT cells are the main cellular source of IL-4 in peripheral lymph nodes at steady state, and maternal S. mansoni infection reduces their secretion of IL-4.
Gating strategy in popliteal lymph nodes for invariant NKT cell (αGalCer+TCRαβ+ DX-5+), αβ T cells (TCRβ+CD3+), γδT cells (TCRβ-CD3+), NK T cells (TCRβ+DX-5+), NK cells (TCRβ-DX-5+) with respective staining control (unloaded tetramer) analyzed by flow cytometry in (A) pups from uninfected mother and (B) pups from infected mother at 28–35 days of age. Flow plots are concatenated from >4 mice per group with two biologically independent experiments.
Fig 3.
Maternal schistosomiasis leads to reduced TFH cells and IL-4 secretion in response to Tetanus/Diphtheria immunization.
4get/KN2 offspring born to S. mansoni infected mothers or uninfected control mothers were immunized in the rear footpad with 1/10th of the human dose of an aluminum adjuvanted Tetanus/Diphtheria vaccine. (A) Tile confocal imaging of 10-micron cryo-sections of popliteal lymph nodes at day-8 post-immunization, stained for HuCD2 (red), GL-7 (gray), and CD4 (green). Scale bar: 200 μm and respective quantitation of germinal center area. Flow cytometry analysis from popliteal lymph nodes of (B) T follicular helper cells (PD-1+CXCR5+, gated from CD4+), (C) intracellular IL-21 and INF-gamma co-production by TFH (D) IL-4 production and secretion by TFH (E) Th2 effector (GFP+IL-7R-) and Th2 memory (GFP+IL-7R+). (F) Frequencies of effector Th2, memory Th2 and TFH cells at 8 days post immunization. (G) Flow cytometry of germinal center B cells (CD19+GL-7+FAS+) (H) Memory IgG1+CD27+ B cells (I) Bulk memory B cells (CD27+CD19+) (J) Cell frequencies of GC B cells and plasma cells (K) Frequency of bulk memory B cell in popliteal lymph node. Confocal microscopy images are representative of four independent experiments. Flow plots are concatenated from n >3 mice from at least two biologically independent experiments. Statistical significance was calculated using Student’s t-test.
Fig 4.
Pups from infected mothers exhibit reduced GC B cells, TFH, and FDC 14 days post immunization with Tetanus/Diphtheria.
(A) TFH (PD-1+CXCR5+, gated from CD4+) flow plots, frequency, and cell number from draining PLN. (B) Th2 effector (GFP+IL-7R-) and Th2 memory (GFP+IL-7R+) from reactive lymph node (C) Plasma cells (IgD-CD19+/-CD138+) (D) FDC (CD21/35+FDC-M2+CD45-) frequencies (E) Median fluorescence intensity of CD21/35 on follicular dendritic cells. (F) Cryosections of reactive popliteal lymph node stained with CD21/35 (red), GL-7 (green), and FDC-M2 (blue). Scale bar: 200um. (G) Bar graph of the area of follicular dendritic cells the B cell follicles of draining lymph nodes. (H) Correlation analysis of offspring anti SEA titers to titers of anti-diphtheria and anti-tetanus 14 days post-immunization. Flow plots and confocal microscopy data are representative of three independent experiments with 3–6 mice per group. Statistical analysis was calculated with Student’s t-test. Correlation was expressed as Pearson correlation coefficient. Error bar denotes mean ± SEM.
Fig 5.
Cellular T and B responses are reduced in 4get/KN2 offspring from infected mothers >60 days post-primary immunization.
4get/KN2 28–35 days old were immunized in the hind footpad as described in the Materials and Methods. Draining popliteal lymph nodes were collected >60 days post primary immunization (A) GC B cells (CD19+GL-7+FAS+) frequencies (B) TFH(PD-1+CXCR5+, gated from CD4+) frequencies, (C) Th2 responses (GFP+IL-7R +/-) (D) Flow cytometry analysis of CD21/35 cell count (E) FDC (CD21/35+FDC-M2+CD45-) frequency. Flow data is concatenated representative of n n>4 mice per group and two independent experiments. Statistical analysis was calculated with Student’s t-test.
Fig 6.
Memory B cell response is impaired during secondary challenge in offspring that were prenatally exposed to SEA antigens.
(A) Schematic of experimental timeline (B) Flow cytometry of GC B cells (CD19+GL-7+FAS+), (C) TFH (PD-1+CXCR5+, gated from CD4+) (D) Follicular dendritic cells (CD21/35+FDCM2+ CD45-) (E) Tile confocal imaging of draining lymph node in response to secondary immunization with alum adjuvanted Tetanus/Diphtheria, cryosections were stained for CD21/35 (red), FDC-M2 (gray) and CD31 (blue). (F) Bulk memory B cells (CD19+CD27+) and (G) IgG1 memory response (IgG1+CD27+). Flow data are representative of n>4 mice per group and three independent experiments (except panel F and G, one independent experiment). Tile confocal imaging is representative of two independent experiments with n>3 mice per group per experiment.
Fig 7.
Maternal schistosomiasis induced modulation of B cell cell-cycle and proliferation is dependent on egg antigen exposure.
(A, B) 4get homozygous females were infected with a low dose of S. mansoni cercariae to produce either single-sex infection (verified by anti-SEA ELISA and perfusion of the mother), or mixed-sex patent infection. Offspring born to mothers with either patent (egg exposure), single-sex (adult antigen exposure only), or control uninfected mothers were immunized at 28–35 days of age with commercial alum adjuvanted Tetanus/Diphtheria. At 8 days post-immunization, CD45+live cells were sorted and processed for 10x Genomics single-cell sequencing. Expression levels of genes significantly altered in follicular B cluster 2 and germinal B cell clusters. In these plots, each dot represents a single cell. Normalized expression values were used, and random noise was added to show the distribution of data points. The box plots show interquartile range and the median value (bold horizontal bar). The average expression value per sample is indicated by the dot. Wilcoxon’s test was used for statistical comparisons. (C) Flow cytometry analysis of Ebf-1 and Ki-67 at steady state in CD19+ and plasma B cells. (D) Flow cytometry analysis of Ebf-1 and Ki-67 at day 14 post tetanus/diphtheria immunization. (E) Total number of plasma cells in the popliteal lymph node at steady-state and Day 14 post-tetanus/diphtheria immunization. Statistical analysis for C-E was calculated with Student’s t-test with Welch’s correction.