Deconstructing the germinal center, one cell at a time

Abstract

Successful vaccination relies on driving the immune response towards high specificity, affinity and longevity. Germinal centers facilitate the evolution of antigen-specific B cells by iterative rounds of diversification, selection, and differentiation to memory and plasma cells. Experimental evidence points to B cell receptor affinity and amount of antigen presented to follicular helper T cells as main drivers of clonal evolution. Concurrent studies suggest that modifiers of cognate contact, temporal mechanisms, and stochastic factors can also shape diversity and influence differentiation to memory and plasma cells, but molecular pathways driving these selection decisions are unresolved. Due to rapid cycles of transcriptional change in the germinal center, single-cell resolution is imperative to dissect mechanisms dictating the mature antigen-specific repertoire. Future studies linking high-resolution analysis of this diverse evolving population with cellular outcome are needed to fully understand the complex mechanisms of selection driving antigen-specific humoral immunity.

Figure 1. Immunization-driven antigen-specific immunity

Immunization with protein antigen primes naïve antigen-specific B cells and T cells separately. Activated B cells uptake bound antigen, processing and presenting antigenic peptide on MHCII to T_FH cells and a germinal center is formed. The population of germinal center (GC) B cells undergoes evolution toward higher antigenic affinity and specificity, marked by continual antigenic binding, processing, and presentation to cognate T_FH cells, which deliver selection signals resulting in further diversification or exit to join the memory compartment (Mem) or differentiate to plasma cells (PC), which secrete specific, high-affinity antibodies (Abs).

Figure 2. Stage-specific regulation of genetic programs in the GC

Gene expression of single cells in the GC assorted into four distinct patterns of gene expression (Stages 1–4). Gene clusters were determined according to patterns of similar expression using the machine learning algorithm t-distributed stochastic neighbor embedding (t-SNE), and order of cyclic progression computationally inferred by the trajectory detection algorithm Wanderlust. Genes in colored boxes change significantly between the stages marked in the colored semicircles in the direction indicated by the arrow. Stages 1–2 represent cognate control in the light zone (LZ), where germinal center (GC) B cells pick up antigen trapped on the follicular dendritic cell network, processing and presenting them to limiting numbers of cognate follicular helper T cells (Tfh), which select B cells based on levels of cognate antigenic peptide. Between Stages 2–3, selected B cells travel to the dark zone (DZ), upregulating genes associated with somatic hypermutation and proliferation, as well as the chemokine receptor Cxcr4, which enables spatial migration. From Stages 3–4, GC B cells diversify the B cell receptor and proliferate, maintaining high levels of Aicda and Mki67, among others. Finally, from Stages 4–1, GC B cells re-enter the LZ, downregulating genes necessary for affinity maturation and prepare to re-express the new B cell receptor to pick up and present antigen once again.