Journal: Cell Reports Methods

Article Title: TRAPnSeq allows high-throughput profiling of antigen-specific antibody-secreting cells

doi: 10.1016/j.crmeth.2023.100522

Figure Lengend Snippet: Antigen-specific IgG plasma cells isolated from the bone marrow using TRAPnSeq show higher affinity compared with splenic/draining lymph node (dLN) B cells (A) Schematic of the experimental workflow for Igk secretion trap technology. Mice were immunized with hIL-4Rα and single cells prepared from bone marrow (BM), dLN, and spleen. B cells and plasma cells (PCs) were enriched by negative selection. Only BMPCs were cell surface biotinylated and incubated with αIgk secretion trap. All cells were stained with αIgG along with B cell and PC markers and barcoded antigens for detection of antigen-specific B cells and PCs. Antigen + /IgG + B cells and PCs were sorted, and single-cell sequencing was performed to determine gene expression, VH:VL sequences, and antigen specificity. Selected clones were expressed by transient transfection of 293F cells and antigen binding tested by ELISA and Biacore. (B) Flow cytometry of antigen-specific IgG that is captured on the surface of PCs in hIL-4Rα-immunized mice. (C) Visualization of antigen specificity based on barcode signal of hIL-6Rα (non-specific) and hIL-4Rα (specific) in BMPCs (n = 1,134) (top) and spleen/dLN B cells and PCs (n = 3,586) (bottom). (D) UMAP of B cells and PCs isolated from spleen/dLN and BM of challenged mice shows localization of spleen/dLN B cell cluster (n = 3,197) in blue, BM-PC cluster (n = 1,063) in red, and spleen/dLN-PC cluster (n = 242) in violet (top). UMAP of B cells and PCs shows hIL-4Rα-specific cells in 3 clusters based on antigen specificity score (bottom). (E) Graph shows binding affinity of antibodies cloned from BMPCs (n = 19) and spleen (Sp)-B cells (n = 16). In (E), each symbol indicates individual antibodies. Asterisks indicate statistical significance as calculated with Mann-Whitney test. ∗∗∗p value ≤ 0.001. See also Table S1 .

Article Snippet: To enrich the V(D)J aliquot for BCR sequences, the Chromium Automated Single Cell Mouse and Human BCR Amplification & Library Construction Kit (10x Genomics) was used.

Techniques: Clinical Proteomics, Isolation, Selection, Incubation, Staining, Sequencing, Gene Expression, Clone Assay, Transfection, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: TRAPnSeq allows high-throughput profiling of antigen-specific antibody-secreting cells

doi: 10.1016/j.crmeth.2023.100522

Figure Lengend Snippet: IgG secretion trap can be coupled with single-cell gene expression and BCR profiling to characterize antigen-specific PCs (A) Graphs show percentage of similarity to germline V region between hIL-4Rα-specific and non-specific cells isolated from BMPCs and Sp/dLN B cells from challenged mice. (B) Venn diagram shows clonal overlap based on CDR3 nucleotide sequence from heavy and light chains of clones isolated from BMPCs and Sp/dLN B cells. (C) Volcano plot comparison of differential gene expression between hIL-4Rα-specific and non-antigen specific BMPCs. (D) Heatmap of scaled expression shows transcriptional profile of the most differentially expressed genes expressed in hIL-4Rα-specific and non-antigen-specific BMPCs. (E) UMAP shows scaled gene expression of Cd24a , Ppob , Fkbp11 , Ssr2 , Tmem176a , Tmem176b , Ly6d , and Cd74 in BMPCs. In (A), dots represent individual antibodies. Asterisks indicate statistical significance as calculated with Mann-Whitney test. ∗∗∗p value ≤ 0.001. See also Table S2 .

Article Snippet: To enrich the V(D)J aliquot for BCR sequences, the Chromium Automated Single Cell Mouse and Human BCR Amplification & Library Construction Kit (10x Genomics) was used.

Techniques: Gene Expression, Isolation, Sequencing, Clone Assay, Comparison, Expressing, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: TRAPnSeq allows high-throughput profiling of antigen-specific antibody-secreting cells

doi: 10.1016/j.crmeth.2023.100522

Figure Lengend Snippet: Identification and profiling of allergen-specific IgE PCs in HDM-exposed mice using TRAPnSeq (A) Schematic of the IgE secretion trap experimental workflow. IgE Venus Blimp-1 mCherry mice were exposed to HDM 3× a week for 15 weeks. Single-cell preparations from lung draining lymph nodes (dLN) and BM cells were enriched for B cells and PCs. Cell surface was biotinylated and incubated with FceRIα secretion trap to allow capture of secreted IgE antibody and stained with an antibody mix containing fluorochrome-labeled anti-IgE along with barcoded antigens (Der p1, Der p2, Der f1, and Ole e1) for detection of antigen-specific IgE PCs. Labeled IgE PCs were sorted and single-cell sequencing performed to determine gene expression, VH:VL sequences, and antigen specificity. Monoclonal IgE antibodies were generated from selected clones and tested for antigen binding by ELISA and for functionality in vivo using passive cutaneous anaphylaxis assay. (B) Flow plots of BM and dLN from mice exposed to HDM. PCs were gated as single cells, live, DUMP − , and CD138 + / − Blimp-1 + cells (DUMP gate included CD3, CD11b, IgD, IgM, CD49b, and Ly6g cells). IgE-secreting PCs were sorted as IgE − Venus + cells. (C) UMAP of IgE + PCs based on VDJ-BCR expression in red over non-IgE PCs in gray. (D) IgE, IgG, IgA, and IgM PC average scaled gene expression of the top 4 differentially expressed genes from both dLN and BM. (E) Visualization of antigen specificity based on barcode expression of Der p1, Der p2, and Der f1 vs. the negative control Ole e1. (F) Antigen specificity validated by ELISA for a subset of IgE antibodies generated from IgE-secreting cells. Representative binding curves of IgE antibodies to Der p1 (top left), Der p2 (top right), Der f1 (bottom left) and to the negative control antigen Ole e1 (bottom right). (G) PCA (mast cell degranulation) was assayed by intradermal injection of IgE mAb 3_1 and IgE mAb 12_2 for Der p1 and IgE mAb 8_2 and IgE mAb 21_2 for Der p2. After 24 h, the mice were challenged intravenously with Der p1 or Der p2 diluted in 0.5% Evans blue dye. Evans blue dye was extracted from ear tissue and measured spectrophotometrically. Plot shows Evans blue dye extravasation in the tissue quantified as nanograms of Evans blue per milligram of tissue as a measure of local mast cell degranulation. Each mouse is indicated in the plot with a square symbol. See also Table S3 .

Article Snippet: To enrich the V(D)J aliquot for BCR sequences, the Chromium Automated Single Cell Mouse and Human BCR Amplification & Library Construction Kit (10x Genomics) was used.

Techniques: Incubation, Staining, Labeling, Sequencing, Gene Expression, Generated, Clone Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Expressing, Negative Control, Injection

Journal: Cell Reports Methods

Article Title: TRAPnSeq allows high-throughput profiling of antigen-specific antibody-secreting cells

doi: 10.1016/j.crmeth.2023.100522

Figure Lengend Snippet:

Article Snippet: To enrich the V(D)J aliquot for BCR sequences, the Chromium Automated Single Cell Mouse and Human BCR Amplification & Library Construction Kit (10x Genomics) was used.

Techniques: Blocking Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Isolation, Conjugation Assay, Expressing, Plasmid Preparation, Software, Saline