Abstract
Human B-cell differentiation has been extensively investigated on genomic and transcriptomic grounds; however, no studies have accomplished so far detailed analysis of antigen-dependent maturation-associated human B-cell populations from a proteomic perspective. Here, we investigate for the first time the quantitative proteomic profiles of B-cells undergoing antigen-dependent maturation using a label-free LC-MS/MS approach applied on 5 purified B-cell subpopulations (naive, centroblasts, centrocytes, memory and plasma B-cells) from human tonsils (data are available via ProteomeXchange with identifier PXD006191). Our results revealed that the actual differences among these B-cell subpopulations are a combination of expression of a few maturation stage-specific proteins within each B-cell subset and maturation-associated changes in relative protein expression levels, which are related with metabolic regulation. The considerable overlap of the proteome of the 5 studied B-cell subsets strengthens the key role of the regulation of the stoichiometry of molecules associated with metabolic regulation and programming, among other signaling cascades (such as antigen recognition and presentation and cell survival) crucial for the transition between each B-cell maturation stage.
Highlights
The final stages of antigen-dependent human B-cell differentiation leading toclonal B-cell expansion and affinity maturation are associated with activation of naive Bcells to their terminal differentiation into antibody-secreting plasma cells (PC) and memory B-cells, in secondary lymphoid tissues (SLT) such as tonsils [1]
A minimum of 150 x 106 tonsil cells were stained in parallel in several different tubes (15 min at room temperature (RT) in the darkness) with the following 8-color combination panel of monoclonal antibodies: CD45 conjugated with fluorescein isothiocyanate (CD45-FITC), CD184 conjugated with phycoerythrin (CD184-protein existence (PE)), CD38 conjugated with peridinin chlorophyll protein-Cy5.5 (CD38-PerCPCy5.5), CD10 conjugated with PE- Cy7 (CD10-PECy7), CD20 and CD19 conjugated with APC (CD20-APC, CD19-APC), CD3 conjugated with allophycocyanine-H7 (CD3-APCH7), and CD27 conjugated with Brilliant VioletTM 421 (CD27-BV421) [22]
Two main groups were observed; group 1 included only the PC while group 2 included naive B-cells, CB, CC, and memory B-cells. Since such clustering was mainly due to the limited amount of sample processed for PC as discussed above, we further investigated the differences in protein expression profiles of the cell populations included in the second group
Summary
The final stages of antigen-dependent human B-cell differentiation leading to (oligo)clonal B-cell expansion and affinity maturation are associated with activation of naive Bcells to their terminal differentiation into antibody-secreting plasma cells (PC) and memory B-cells, in secondary lymphoid tissues (SLT) such as tonsils [1]. Within the GC, the dark zone—where proliferation and somatic hypermutation occur—hosts the centroblasts (CB), which further differentiate into smaller centrocytes (CC) that enter the light zone of the GC Both GC B-cell populations display a considerable overlap in their gene expression profiles, despite showing a clearly distinct morphological appearance [4, 5]
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