Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion

Abstract

The contribution of proliferation to B lymphocyte homeostasis and antigen responses is largely unknown. We quantified the replication history of mouse and human B lymphocyte subsets by calculating the ratio between genomic coding joints and signal joints on kappa-deleting recombination excision circles (KREC) of the IGK-deleting rearrangement. This approach was validated with in vitro proliferation studies. We demonstrate that naive mature B lymphocytes, but not transitional B lymphocytes, undergo in vivo homeostatic proliferation in the absence of somatic mutations in the periphery. T cell-dependent B cell proliferation was substantially higher and showed higher frequencies of somatic hypermutation than T cell-independent responses, fitting with the robustness and high affinity of T cell-dependent antibody responses. More extensive proliferation and somatic hypermutation in antigen-experienced B lymphocytes from human adults compared to children indicated consecutive responses upon additional antigen exposures. Our combined observations unravel the contribution of proliferation to both B lymphocyte homeostasis and antigen-induced B cell expansion. We propose an important role for both processes in humoral immunity. These new insights will support the understanding of peripheral B cell regeneration after hematopoietic stem cell transplantation or B cell-directed antibody therapy, and the identification of defects in homeostatic or antigen-induced B cell proliferation in patients with common variable immunodeficiency or another antibody deficiency.

Figure 1.

Detection of coding joints and signal joints of kappa-deleting rearrangements in mice (A) and man (B). V(D)J recombination on the IGK locus results in a Vκ-Jκ coding joint. Subsequent rearrangement between the IRS1 (mouse) or intronRSS (human) and the RS (mouse) and Kde (human) elements can make the IGK allele nonfunctional by deleting the Cκ exons and the enhancers. Consequently, the coding joint precludes any further rearrangements in the IGK locus and therefore remains present in the genome. The KREC with the corresponding signal joint is a stable double-stranded, circular DNA structure. The coding joint in the genome and the signal joint on the episomal excision circle can be quantified via RQ-PCR using the indicated primers and TaqMan probes. The murine IGK locus contains two intronRSS sequences, IRS1 and IRS2, of which IRS1 has the most conserved RSS sequence and is >10-fold more frequently found in rearrangements to RS (reference and unpublished data).

Figure 2.

Quantification of the replication history of B cells using KRECs. (A) When a B lymphocyte with an intronRSS–Kde rearrangement divides, both daughter cells inherit the intronRSS–Kde coding joint in the genome. However, the signal joint, which is on the episomal KREC, will be inherited by only one of the two daughter cells. Crucially, the ΔC_T of the PCRs detecting the coding joint and the signal joint exactly represent the number of cell divisions a B lymphocyte has undergone because both processes have an exponential increase with base number 2. (B) The ΔC_T between the coding joint and the signal joint is shown for mouse splenic B cells that were cultured in vitro with polyclonal stimulation and were sorted based on the CFSE staining intensity as measure for 0, 1, 2, 3, and 4 cell divisions.

Figure 3.

Generation of a human control B cell line with one intronRSS–Kde coding joint and one artificially introduced signal joint per genome. (A) Schematic representation of the Southern blot probes that recognize the IGK alleles of the U698-M cell line and the KREC signal joint construct that was introduced into the cell line. The Kde probe recognizes both IGK alleles, whereas the Kde-RSS probe recognizes allele 1, which does not harbor an intronRSS–Kde rearrangement, and the KREC construct. (B) Southern blot of the original U698-M cell line and three single clones that contain a unique insertion of the signal joint construct.

Figure 4.

Replication history of mouse B cell subsets. (A) Scheme of the differentiation stages of mature B cell development in mice. B cell subsets were isolated based on the indicated marker expression from spleen and Peyer's patches. (B) The replication history of each B cell subset. The ΔC_T between the coding joint and the signal joint PCRs represents the number of cell divisions, which are given for each subset. The data are the means of three independent sorts and are presented as mean ± SD.

Figure 5.

Replication history of human B lymphocyte subsets from childhood tonsil. (A) Scheme of the differentiation stages of mature B cell development. The B cell subsets that contain the intronRSS–Kde rearrangement were isolated from bone marrow and tonsil samples of young children using the indicated markers. (B) The replication history of each B cell subset. The ΔC_T between the coding joint and the signal joint PCRs, which represents the number of cell divisions is given for each subset. The data are the means of independently sorted subsets from three children and are presented as mean ± SD.

Figure 6.

Replication history of the four main B lymphocyte subsets in adult peripheral blood. (A) Schematic overview of the four main blood B lymphocyte subsets and the markers that were used to isolate them from adult peripheral blood. (B) The replication history of these subsets was determined as the ΔC_T of the coding joint and the signal joint PCR of subsets isolated from four or five donor samples (represented as mean ± SD).

Figure 7.

Quantification of SHM in human B cell subsets. The IgκREHMA assay was modified from Andersen et al. (37) for use on genomice DNA. (A) Schematic representation of a Vκ3-20 to Jκ rearrangement on genomic DNA. The Vκ3-20 gene segment contains two Fnu4HI restriction sites in a SHM hot-spot in the CDR1 region. In addition, a KpnI site is located downstream of the Fnu4HI sites in the FR2 region. Vκ3-20–Jκ rearrangements were PCR amplified with one HEX-labeled Vκ3-20–intron forward and two Jκ reverse primers that recognize all five Jκ gene segments (61). Digestion of these PCR products with both Fnu4HI and KpnI resulted in unmutated fragments of 244 and 247 bp and mutated fragments of 262 bp. (B and C) Spectratyping of Vκ3-20–Jκ rearrangements of CD5⁺ B lymphocytes and memory B lymphocytes after double digest with Fnu4HI and KpnI. CD5⁺ B lymphocytes did not have a replication history and showed no SHM, whereas memory B lymphocytes in adult blood had undergone 11 cell cycles in the periphery and showed 30% mutated Vκ3-20–Jκ rearrangements. (D and E) The percentage of mutated Vκ3-20–Jκ rearrangements was measured in the same DNA samples that were previously used for determining the replication history (data represent means ± SD).