RNA isolation

Study Design This study aimed to understand mechanisms that underlie decreased vaccine efficacy during maternal schistosomiasis, specifically focusing on the B cell response. Using our previously established maternal schistosomiasis murine model (13), we generated pups from Schistosoma mansoni infected and sham infected mothers. Sample size for each experiment was limited by liter size. The number of independent experiments is indicated in the figure legend. Pups were excluded if the dam stopped breastfeeding the pups before 14 days of age, determined by weanling weight. Outliers were excluded by the ROUT method with Q = 0.1% to ensure only definitive outliers were removed.


Study Design
This study aimed to understand mechanisms that underlie decreased vaccine efficacy during maternal schistosomiasis, specifically focusing on the B cell response. Using our previously established maternal schistosomiasis murine model (13), we generated pups from Schistosoma mansoni infected and sham infected mothers. Sample size for each experiment was limited by liter size. The number of independent experiments is indicated in the figure legend. Pups were excluded if the dam stopped breastfeeding the pups before 14 days of age, determined by weanling weight. Outliers were excluded by the ROUT method with Q = 0.1% to ensure only definitive outliers were removed.
To determine developmental B cell defects in pups from S. mansoni infected mothers that could lead to a diminished vaccine response, flow cytometry and single cell RNA sequencing were coupled in the bone marrow and peripheral lymph nodes. V(D)J sequencing was performed to establish if maternal infection also altered the homeostatic and germinal center B cell repertoires.
Due to the sex-specific nature of the results, and an increase of Xist expression in males from infected mothers, we performed a pulldown using biotinylated probes for Xist and extracted the DNA for a methylation array. Silencing of Xist in whole bone marrow in vitro determined the relationship between dysfunctional Xist expression and the BCR development. Finally, adoptive transfers were conducted to test the origin, longevity, and persistence of these defects.
Experiments were not blinded.
Mice 4get homozygous (Il4 tm1Lk )(strain# 004190) mice and Rag1 -/-(C.129S7(B6)-Rag1 tm1Mom /J)(strain# 003145) mice were obtained from The Jackson Laboratory and bred at the University of Utah animal facilities. Experiments were performed in strict accordance with the NIH guide for the Care and Use of Laboratory animals and institutional guidelines for animal care at the University of Utah under approved protocols (18-09001 and 21-07009). At six weeks of age, 4get homozygous females were infected with infected with ~35 Schistosoma mansoni cercaria, as previously described (13), or sham infected. Six weeks post S. mansoni or sham infection, females were paired to KN2 homozygous (Il4 tm1(CD2)Mmrs ) (16)) males and remained paired until they reached humane endpoints. To generate 4getKN2 and KN2 homozygous littermates, 4getKN2 females were infected and mated to KN2 homozygous males as described above. Pups from mixed genotype litters were genotyped by flow cytometry before use.

In vivo treatments
Pups were weaned and genotyped (as necessary) at 28 days old. Steady state experiments were performed between 28-35 days of age. For immunization experiments, pups were immunized with 1/10 th the human dose of Tetanus/Diphtheria commercial vaccine (Grifols Therapeutics Inc (TDVAX) #13533013101) subcutaneous in the rear footpad. Mice were sacrificed at 14-days post-primary immunization or administered a secondary immunization at over 60 days postprimary immunization and sacrificed 3 days post-secondary immunization. For all experiments, littermates were used, and groups were split by sex and infection status of the mother.

Cell isolation for single cell suspension
For bone marrow harvesting, femurs were collected, and the tip connected to the knee was clipped on a bias to expose the bone marrow. Bones were placed cut side down into a 200µL microcentrifuge tube with a hole in the bottom and ~50µL of Dulbecco's Modified Eagle Medium (DMEM)(Corning #10-013-CV). This tube was then placed into a 1.5mL microcentrifuge tube and then pulsed in a microcentrifuge at ≥ 10,000 RPM for 15 seconds at 4°C. The 200µL tube with the flushed bones were then discarded. Erythrocytes were lysed using 1X lysis buffer (10X BD Pharm Lyse™, BD Biosciences #555899)(diluted to 1X) for 1 minute.
The lysis was quenched using 1% fetal bovine serum (FBS)(Gibco #26140-079) in DMEM and centrifuged at 200xg for 5 minutes at 4° C. The supernatant was discarded and washed with 1X phosphate-buffered saline (PBS) before cell surface staining.
Popliteal lymph nodes (PLNs) and blood were collected and processed as previously described (88). In brief, lymph nodes were collected, smashed through a 100µm cell strainer, and washed with 15mL of DMEM.

Flow cytometry and antibodies
To stain cells for flow cytometry, cells were harvested as described above and resuspended in the appropriate volume of FACS buffer (2%FBS, 5mM EDTA in 1X PBS) as previously described  Bone marrow B cells gating was done after first gating out Dump + cells , and transitional B cells (B220 + CD19 + IgM -IgD + ) gating was adopted previously published studies (21-23). PLN germinal center (GC) B cells are described as Viability -CD19 + GL-7 + FAS + .
All flow cytometry experiments were run on an Attune NxT (Invitrogen), and data were analyzed with FlowJo.

Tetanus neutralization assays
Modified tetanus neutralization assays (89) were performed by first coating an Immulon 4 HBX 96 well plate (Fisher Scientific #14-245-153) with 10µg/mL of trisialoganglioside-GT1b from bovine brain (Sigma-Aldrich # G3767-1MG) in ethanol overnight. The next day, the plate was blocked with 1% BSA for two hours at room temperature. Meanwhile, serum from Td immunized pups were incubated with tetanus toxin for two hours at 37°C. After flicking off the blocking solution, the serum incubated with tetanus was added to the plate and let to sit at room temperature for two hours. After washing, anti-tetanus toxoid rabbit serum (VWR # BOSSBS-11772R) was added to the plate for two hours at room temperature. After washing, anti-rabbit IgG HRP (Abcam # ab6721) was added to the plate and left to incubate for two hours at 37°C.
Finally, SuperAqua Blue ELISA substrate (Thermo Fisher Scientific #00-4203-56) was added to each well before reading plate at 405nm with spectrophotometer.

Adoptive cell transfers
Steady state experiments were performed by harvesting whole bone marrow from male 4getKN2 pups from infected and control mothers as described above. Cell suspensions were pooled before counting using a hemocytometer. Three million cells were then injected intravenously into 4-6- week-old Rag1 -/recipients. Recipients were bled weekly to check lymphocyte reconstitution. At 5 weeks post-transfer, recipients were euthanized, and cells were collected for flow cytometric analysis.
Immunization experiments began by partially irradiating Rag1 -/with 300 grays. The following day, donor cells were collected from bone marrow of males from infected and control mothers.
Non-labelled cells were then counted using a hemocytometer and 5x10 5 cells were transferred intravenously into each Rag1 -/male recipient mouse. Recipients were allowed 5 weeks to reconstitute before transferring 5x10 5 T cells from wildtype 4getKN2 females. The following day, mice we immunized in the footpad with 1/10 th the human dose of the commercial Tetanus/Diphtheria vaccine listed above. Fourteen days after immunization, mice were euthanized, and cells were harvested for analysis.

Tetanus toxin fluorophore conjugation
Tetanus toxin (List Labs #190B) was conjugated to using the Lightning Link ® FITC conjugation kit (Abcam #ab102884) per manufacturer instruction. Ovalbumin conjugated to Biotin (Abcam #ab201795) was used in conjunction with the conjugated Tetanus toxin to control for nonspecific B cell binding. The decoy was incubated with streptavidin-BV786 (BD Biosciences #563858) for 10 minutes at RT before mixing with the conjugated Tetanus toxin antibody. Cells were stained with 0.01µL of conjugated Tetanus toxin for 20 minutes 4° C and washed twice with 1X PBS before surface antibody staining.

scRNASeq experiments
Single cell suspensions were isolated from tissue as described above then sorted using either a FACSAria or a Sony MA800 or MA900 cell sorter. Cells were processed as previously described (13). In brief, sorted cells were pelleted by centrifugation and resuspended in 1% BSA (bovine serum albumin) in 1X PBS and counted by hemocytometer and resuspended to a concentration of 1,200 cells/µL to run on the 10x platform. Paired-end RNASeq (125 cycles) was performed via an Agilent HiSeq next-generation sequencer.

scRNASeq analysis
Raw data from scRNASeq experiments in this manuscript can be found in the NCBI's Gene Expression Omnibus database. Sequencing reads were processed by using a 10x Genomics CellRanger pipeline and further analyzed using R Studio. Prior to analysis, low quality cells (greater than 15% mitochondrial gene representation and/or fewer than 200 and/or more than 6000 genes per cell) were filtered out before clustering and differential expression testing (DEGs) by using the Seurat package (90)(91)(92). The biological identities of cell clusters were annotated by referencing a published scRNASeq data set (93) and by surveying known transcriptional cell markers in the scRNASeq data set.

V(D)J scRNASeq and analysis
Cell isolation, cell sorting, single cells clustering, and analysis were performed as described above. Data sets were analyzed using both scRepertoire (94) and VDJView (95).

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were isolated and sorted using the strategies outline above. After sorting, cells were cytospun at 400xg for 5 minutes onto microscope slides (VWR #48311-703). Slides were prepared and stained using established protocols (45). Two Cy3-labeled 20-nucleotide oligo probes were designed to recognize regions within exon 1 (a gift from Montserrat Anguera). Slides were imaged at 63x using a Leica SP8 DIVE multiphoton microscope.

Xist chromatin isolation by RNA purification (ChIRP)
Whole bone marrow was isolated from femurs as described above. Cells from 3-4 mice were pooled before further processing. The RNA pulldown was done using a previously established protocol (98, 99) with the following adaptations; 1) cells were crosslinked with 1% formaldehyde for 20 minutes; 2) after hybridization and washing, DNA was isolated using PhOH:Chloroform:Isoamyl (Invitrogen #15593-031).

Illumina® Infinium HD Mouse Methylation Assay
Bisulfite conversion of DNA was performed using the EZ DNA Methylation-Gold kit (Zymo, catalog #D5006) as per manufacture's instruction. Infinium HD methylation microbead array (#20041558) was performed by the DNA sequencing core at the University of Utah following manufacturer's protocols. Data was processed and analyzed using the Illumina GenomeStudio Software.

Enzyme-linked immunosorbent assay (ELISA)
ELISA data used was published in (13), but split by sex of the pups and reanalyzed. Avidity ELISA were performed by following the above ELISA protocol with an additional 15-minute agitated incubation with 0.5M ammonium thiocyanate for treated wells or 15-minute incubation with 1X PBS for control wells before secondary staining as described in (100).

RNA isolation and Quantitative polymerase chain reaction
RNA isolation and q-RT-PCR were performed as previously described (88). In short, 30,000-50,000 cells were sorted into 500µL of TRIzol™ LS (Invitrogen # 10296010) and stored at -80° C until RNA extraction as previously described (Fairfax, 2013 #271). RNA was used for cDNA synthesis using Superscript IV VILO (Invitrogen # 11756050) for qPCR analysis. qPCR was performed using TaqMan™  MFI was analyzed using the ProcartaPlex Analyst 1.0 Software.

Flow-FISH
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of control and S. mansoni chronically infected 4get females. Whole blood was lysed using three times using 1X diluted lysis buffer (BD Biosciences #349202). Xist probes used were the same as used for as described by (Arrigucci et al., 2017).

Statistical Analysis
For scRNASeq experiments, p-values and adjusted p-values were calculated while doing differential gene expression analysis, which uses a non-parametric Wilcoxon rank sum test. All  UMAPs generated after sorting CD45 lo B220 + CD19 + IgM + IgD +/bone marrow cells from pups from control and S. mansoni infected mothers. Each group represents a pool of >4 mice. Transcriptional markers shown as FeaturePlots used to identify different clusters.