DOI: 10.5281/zenodo.17084542
title: antiCD19_CART_NTX_Dandelion_BCR_TCR

description: Single-cell 5′ multiomic profiling of human bone marrow and blood lymphocytes before and after anti CD19 CAR T therapy therapy for HLA desensitization

We performed five single-cell captures (NTX1–NTX5) on primary human bone marrow and blood using the 10x Genomics Chromium Single Cell 5′ workflow. Library types included 5′ gene expression (GEX), cell-surface protein profiling via CITE-seq (TotalSeq C), and paired V(D)J repertoires (TCR and BCR). Cell inputs per capture were: NTX1 (20,000 plasma cells; BM before therapy), NTX2 (14,000 memory B cells + 4,000 T cells + 2,000 NK; BM before therapy), NTX3 (14,000 plasma cells & memory B cells + 4,000 T + 2,000 NK; blood before therapy), NTX4 (14,000 plasma cells + 4,000 T + 2,000 NK; BM after therapy), and NTX5 (14,000 T + 6,000 NK; blood after therapy). Libraries were quantified and sequenced on an Illumina NextSeq 500 (GEX: High Output v2 150 cycles; VDJ: Mid Output v2 300 cycles). Processed outputs include CellBender-filtered matrices and integrated AnnData (h5ad) objects for joint analyses.Five 10x Genomics Chromium Single Cell 5′ captures (NTX1–NTX5) with matched GEX, CITE-seq (TotalSeq C), and V(D)J libraries (TCR and BCR as applicable). BM vs blood; before vs after therapy design: NTX1 (BM pre), NTX2 (BM pre), NTX3 (blood pre), NTX4 (BM post), NTX5 (blood post). CITE-seq prepared with Feature Barcode Library Kit and TotalSeq C panel; V(D)J enrichment using Human T-cell and B-cell kits; sequenced on Illumina NextSeq 500

brief overview of library generation#Samples represent before-therapy and after-therapy time points; no ex vivo stimulation prior to capture.Cell isolation by density gradient; FACS enrichment of targeted lymphocyte subsets; immediate encapsulation using 10x Genomics Chromium Controller (Single Cell 5′).10x Genomics Single Cell 5′ Library & Gel Bead Kit and Feature Barcode Library Kit. CITE-seq libraries prepared with TotalSeq C antibodies and Single Index Kit N Set A. V(D)J target enrichment using Chromium Single Cell V(D)J Enrichment Kits (Human T cells and B cells). Final libraries quantified by Qubit HS DNA; size distribution assessed with Fragment Analyzer (Agilent) HS NGS Fragment Kit (1–6000 bp). Sequencing performed on Illumina NextSeq 500 (GEX: High Output v2, 150 cycles; VDJ: Mid Output v2, 300 cycles).Demultiplexing with cellranger mkfastq.Gene expression quantified with cellranger count (5′ + Feature Barcoding).V(D)J reconstructed with cellranger vdj for TCR/BCR libraries.Background removal on GEX with CellBender; integration and analysis using Scanpy/AnnData (h5ad outputs).CITE-seq features mapped using TotalSeq C antibody reference; UMI counting performed as per 10x Feature Barcode pipeline.Human GRCh38 (hg38) for GEX; 10x vdj_GRCh38 human reference for V(D)J.CellBender-filtered 10x HDF5 matrices per capture (e.g., cellbender_output_NTX_1_filtered.h5).Integrated single-cell objects in AnnData format (.h5ad) with per-cell metadata (e.g., processed_adata_NTX_all_samples.h5ad), and per-cell V(D)J tables (per_cell_TCR_table.csv; my_merged_contigs.tsv).

The uploaded files contain the environments, and base files for the analyses described in the M&M section of our Manuscript

B cell receptor V(D)J single cell processingRaw 10x Genomics BCR FASTQ files from four samples (NTX 1–4) were processed with Dandelion 1.5.0 (Singularity image scdandelion_latest.sif) (62)—identical to the TCR pipeline. Command line execution inside the container was: dandelion preprocess --all-contigs --filter_to_high_confidence yielding highconfidence‑ all_contig_dandelion.tsv files. Each table was (i) annotated with the sample prefix, (ii) barcode harmonization (iii) filtered to retain productive contigs only. All BCR contigs were concatenated and cells harbouring > 1 productive IGH or IGK/IGL chain were removed. For remaining singlepair cells, the contig with the highest UMI count per chain was selected and pivoted into a ‑percell matrix containing‑ IGH_cdr3, IGH_v_call, IGH_j_call, IGL/IGK_cdr3, IGL/IGK_v_call, IGL/IGK_j_call (Supplementary Table S5). The table was joined to the integrated TotalVI AnnData object; non‑overlapping barcodes were discarded. A flag has_BCR indicates cells with at least one productive chain. All downstream Python and R scripts were executed inside the Immcantation suite container (immcantation/suite:4.5.0, (63)), which embeds a minimal Miniconda 23.5 installation supplying required dependencies.

T cell receptor V(D)J single cell processingRaw 10x Genomics TCR FASTQ files from four samples (NTX 2–5) were processed with Dandelion 1.5.0 (Singularity image scdandelion_latest.sif, (62). Inside the container we executed dandelion preprocess --all-contigs --filter_to_high_confidence to generate highconfidence‑ all_contig_dandelion.tsv files. For each table we (i) extracted TRA/TRB rows, (ii) normalised barcodes (iii) kept only contigs flagged productive. Cells harbouring > 1 productive TRA or TRB chain (multiplets) were discarded. Productive singlepair cells were saved to a ‑percell matrix containing‑ TRA_cdr3, TRA_v_call, TRA_j_call, TRB_cdr3, TRB_v_call, TRB_j_call and exported. The table was joined to a pre‑processed TotalVI AnnData object (Scanpy 1.10.1) using exact barcodes. All productive singlepair‑ contigs were concatenated into a single Dandelion object. UMAP overlays and CAR‑T annotationAnalyses were executed in Python 3.11 inside the Immcantation suite container (immcantation/suite:4.5.0, (63)) which bundles Anndata 0.10.6, Scanpy 1.10.1, Dandelion 1.5.0, Matplotlib 3.9, NumPy 1.26 and related dependencies.

 

Citations

62.    Suo C, Polanski K, Dann E, Lindeboom RGH, Vilarrasa-Blasi R, Vento-Tormo R, Haniffa M, Meyer KB, Dratva LM, Tuong ZK, Clatworthy MR, Teichmann SA. Dandelion uses the single-cell adaptive immune receptor repertoire to explore lymphocyte developmental origins. Nat Biotechnol. 2024;42(1):40-51.63.    Gabernet G, Marquez S, Bjornson R, Peltzer A, Meng H, Aron E, Lee NY, Jensen CG, Ladd D, Polster M, Hanssen F, Heumos S, nf-core c, Yaari G, Kowarik MC, Nahnsen S, Kleinstein SH. nf-core/airrflow: An adaptive immune receptor repertoire analysis workflow employing the Immcantation framework. PLoS Comput Biol. 2024;20(7):e1012265.