Biology subjects

Attaf, N.

Publications and source records attributed to Attaf, N..

4 recordsLinked to original sources

bioRxiv2024

FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cells

Parallel analysis of phenotype, transcriptome and antigen receptor sequence in single B cells is a useful method for tracking B cell activation and maturation during immune responses. However, in most cases, the specificity and affinity of the B cell antigen receptor cannot be inferred from its sequence. Antibody cloning and expression from single B cells is then required for functional assays. Here we propose a method that integrates FACS-based 5-end single-cell RNA sequencing (FB5P-seq) and monoclonal antibody cloning for integrative analysis of single B cells. Starting from a cell suspension, single B cells are FACS-sorted into 96-well plates for reverse transcription, cDNA barcoding and amplification. A fraction of the single-cell cDNA is used for preparing 5-end RNA-seq libraries that are sequenced for retrieving transcriptome-wide gene expression and paired BCR sequences. The archived cDNA of selected cells of interest is used as input for cloning heavy and light chain variable regions into antibody expression plasmid vectors. The corresponding monoclonal antibodies are produced by transient transfection of a eukaryotic producing cell line and purified for functional assays. We provide detailed step-by-step instructions and describe results obtained on ovalbumin-specific murine germinal center B cells after immunization. Our method is robust, flexible, cost-effective, and applicable to different B cell types and species. We anticipate it will be useful for mapping antigen specificity and affinity of rare B cell subsets characterized by defined gene expression and/or antigen receptor sequence.

immunology↗

bioRxiv2024

Specific pre-plasma cell states and local proliferation at the dark zone - medulla interface characterize germinal center-derived plasma cell differentiation in lymph node

High affinity antibody-producing plasma cells (PC) generated in germinal centers (GC) are crucial for durable immunity after vaccination or infection. The selection of high affinity B cells in the GC light zone instructs PC differentiation in a subset of cells, but the cellular transitions and spatial organization of GC to PC differentiation remain poorly understood. Here, we have used a mouse model to track GC-derived B cells with integrative single-cell and spatial analyses in draining lymph node after immunization or infection. We first identified putative PrePC cells in scRNA-seq datasets, then enriched those cells through their specific surface phenotype for further analysis of their gene expression trajectories and BCR repertoire. We found a continuum of actively proliferating transitional states bridging selected LZ GC B cells and recently exported PCs, with gradually increasing levels of endoplasmic reticulum stress-associated genes and Ig transcripts. Spatial analyses revealed that recently differentiated PC continued their maturation and affinity-restricted proliferation at a previously uncharacterized interface between the DZ and extensions of the lymph node medulla. Our findings provide insights into the intermediate stages and microenvironmental factors involved in the differentiation of GC B cells into PC, with implications for vaccine development and understanding antibody responses.

immunology↗

bioRxiv2022

Functional plasticity and recurrent cell states of malignant B cells in follicular lymphoma

Follicular lymphoma (FL) derives from malignant transformation of germinal center (GC) B cells. FL malignant B cells are heterogeneous and diverge from their GC B cell-of-origin, but the diversity, function, and location of malignant B cell states remain to be addressed. Based on integrative single-cell RNA-seq, we identified and studied recurrent FL malignant B cell states and dynamics. Most FL B cells spanned a continuum of states from proliferating GC-like to quiescent memory (Mem)-like cell states. That GC-to-Mem axis was the main source of intra-tumor transcriptional heterogeneity. While FL B cell states were independent from subclonal B cell receptor genetics divergence, T follicular helper (TFH) cell-derived signals controlled the transition from Mem-like to GC-like states. GC-like, TFH-activated and Mem-like FL B cells tended to occupy distinct niches within and around tumor follicles. Our study characterizes novel malignant cell states recurrent in B cell lymphomas, and highlights the functional plasticity of malignant B cells.

cancer biology↗

bioRxiv2020

An NF-κB/IRF1 axis programs cDC1s to drive anti-tumor immunity

Conventional type 1 dendritic cells (cDC1s) are critical for anti-tumor immunity. They acquire antigens from dying tumor cells and cross-present them to CD8+ T cells, promoting the expansion of tumor-specific cytotoxic T cells. However, the signaling pathways that govern the anti-tumor functions of cDC1s are poorly understood. We mapped the molecular pathways regulating intra-tumoral cDC1 maturation using single cell RNA sequencing. We identified NF-{kappa}B and IFN pathways as being highly enriched in a subset of functionally mature cDC1s. The specific targeting of NF-{kappa}B or IFN pathways in cDC1s prevented the recruitment and activation of CD8+ T cells and the control of tumor growth. We identified an NF-{kappa}B-dependent IFN{gamma}-regulated gene network in cDC1s, including cytokines and chemokines specialized in the recruitment and activation of cytotoxic T cells. We used single cell transcriptomes to map the trajectory of intra-tumoral cDC1 maturation which revealed the dynamic reprogramming of tumor-infiltrating cDC1s by NF-{kappa}B and IFN signaling pathways. This maturation process was perturbed by specific inactivation of either NF-{kappa}B or IRF1 in cDC1s, resulting in impaired expression of IFN-{gamma}-responsive genes and consequently a failure to efficiently recruit and activate anti-tumoral CD8+ T cells. Finally, we demonstrate the relevance of these findings to cancer patients, showing that activation of the NF-{kappa}B/IRF1 axis in association with cDC1s is linked with improved clinical outcome. The NF-{kappa}B/IRF1 axis in cDC1s may therefore represent an important focal point for the development new diagnostic and therapeutic approaches to improve cancer immunotherapy. One Sentence SummaryNF-{kappa}B and IRF1 coordinate intra-tumoral cDC1 maturation and control of immunogenic tumor growth.

immunology↗