Abstract
In response to T-cell-dependent antigens, mature B cells in the secondary lymphoid organs are stimulated to form germinal centers (GCs), which are histological structures deputed to antibody affinity maturation, a process associated with immunoglobulin gene editing by somatic hypermutation (SHM) and class switch recombination (CSR). GC B cells are heterogeneous and transition across multiple stages before being eliminated by apoptosis or committing to post-GC differentiation as memory B cells or plasma cells. In order to explore the dynamics of SHM and CSR during the GC reaction, we identified GC subpopulations by single-cell (sc) transcriptomics and analyzed the load of immunoglobulin variable (V) region mutations as well as the isotype class distribution in each subpopulation. The results showed that the large majority of GC B cells display a quantitatively similar mutational load in the V regions and analogous IGH isotype class distribution, except for the precursors of memory B cells (PreM) and plasma cells (PBL). PreM showed a bimodal pattern with about half of the cells displaying high V region germline identity and enrichment for unswitched IGH, while the rest of the cells carried a mutational load similar to the bulk of GC B cells and showed a switched isotype. PBL displayed a bias toward expression of IGHG and higher V region germline identity compared to the bulk of GC B cells. Genes implicated in SHM and CSR were significantly induced in specific GC subpopulations, consistent with the occurrence of SHM in dark zone cells and suggesting that CSR can occur within the GC.
Highlights
Germinal centers (GCs) are histological structures, which form in the secondary lymphoid organs in response to antigenic challenge
Confirming our previous observations [7], 99% of the cells were assigned to clusters that displayed features of dark zone (DZ), light zone (LZ) or Intermediate germinal centers (GCs) B cells as well as clusters representing the precursors of plasma cells and memory B cells (Figure 1A)
Among the genes expressed at higher level in IGHM cells with high IGV mutational load we detected FCRL2, which representing a hallmark of a specific GC subpopulation (FCRL2/3), appeared to be expressed in a small fraction of other populations (Figure S5). These results indicate that the large majority of GC B cells show a similar pattern of isotype distribution dominated by switched cells; cells committing to memory B cell or plasma cell differentiation display unique repertoires characterized by enrichment for IGHM and IGHG, respectively
Summary
Germinal centers (GCs) are histological structures, which form in the secondary lymphoid organs in response to antigenic challenge. They represent the site of antibody affinity maturation, based on the acquisition of mutations in the immunoglobulin variable regions followed by selection based on the affinity for the antigen [1]. A small subset of cells in the GC are primed to become memory B cells or plasma cells These precursors of post-GC effector B cells are clearly distinct and recent sc-RNAseq studies have further expanded their transcriptome characterization [7, 10,11,12,13]
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