High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations

Abstract

B-cell receptor (BCR) diversity is achieved centrally by rearrangement of Variable, Diversity, and Joining genes, and peripherally by somatic hypermutation and class-switching of the rearranged genes. Peripheral B-cell populations are subject to both negative and positive selection events in the course of their development that have the potential to shape the BCR repertoire. The origin of IgM(+)IgD(+)CD27(+) (IgM memory) cells is controversial. It has been suggested that they may be a prediversified, antigen-independent, population of cells or that they are a population of cells that develop in response to T-independent antigens. Most recently, it was suggested that the majority of IgM memory cells are directly related to switched memory cells and are early emigrants from the germinal center reaction. Advances in sequencing technology have enabled us to undertake large scale IGH repertoire analysis of transitional, naive, IgM memory and switched memory B-cell populations. We find that the memory B-cell repertoires differ from the transitional and naive repertoires, and that the IgM memory repertoire is distinct from that of class-switched memory. Thus we conclude that a large proportion of IgM memory cells develop in response to different stimuli than for class-switched memory cell development.

Figure 1

Sorting human B-cell subsets. (A) Human peripheral B cells (CD19⁺) were stained and sorted into 4 populations, according to their expression of CD27 and IgD: IgD⁺CD27^– (naive and transitional), IgD⁺CD27⁺ (IgM memory), IgD^–CD27⁺, and IgD^–CD27^– B cells. Numbers indicate the percentage of subsets in boxes. (B) From the IgD⁺CD27^– gate, naive and transitional cells were further divided into: CD10⁺ transitional and CD10^– naive cells. Numbers indicate the percentage within the IgD⁺CD27^– gate. Data are representative of 3 separate experiments.

Figure 2

Frequency of clonal expansion in different B-cell populations. Large clonal expansions were observed in antigen-experienced memory populations. Sequencing of Ig gene rearrangements showed that some occurred more than once, and were considered to belong to a clonal family. Members of the same clone (with the same junctional CDR3 sequence) were identified. The size of each family was noted but only 1 member of each family was used in subsequent analyses. We plotted the frequency distribution of sequences between clones of different size for transitional (IgD⁺CD27⁻CD10⁺, n = 433), naive (IgD⁺CD27⁻CD10⁻, n = 662), switched memory (IgD⁻ and either IgG⁺ or IgA⁺, n = 1293) and IgM memory (IgD⁺CD27⁺, n = 1209) populations. Clone size of 1 indicates no related sequences were found.

Figure 3

IGHJ gene and IGHV family usage. IGHV family and IGHJ genes were identified from the Ig gene sequences using V-QUEST. (A) The relative frequency of 6 IGHJ genes, and (B) 7 IGHV gene families in transitional (IgD⁺CD27⁻CD10⁺, n = 433), naive (IgD⁺CD27⁻CD10⁻, n = 662), switched memory (IgD⁻ and either IgG⁺ or IgA⁺, n = 1293) and IgM memory (IgD⁺CD27⁺, n = 1209) populations. “m,“ “s,” “n” and “t” indicate significant differences compared with IgM memory, switched memory, naive, and transitional cells, respectively. *P < .05; **P < .005; ***P < .0005.

Figure 4

Three-dimensional landscapes of IGHV gene usage in conjunction with IGHJ usage show variation between antigen-inexperienced and memory B-cell populations. Landscapes illustrate the pairing of individual IGHV and IGHJ gene combinations in the Ig gene sequences, as identified by V-Quest. The relative frequency (y-axis) of individual IGHV genes (x-axis) using IGHJ genes (z-axis) in (A) Transitional (IgD⁺CD27⁻CD10⁺, n = 433), (B) Naive (IgD⁺CD27⁻CD10⁻, n = 662), (C) Switched memory (IgD⁻ and either IgG⁺ or IgA⁺, n = 1293), and (D) IgM memory (IgD⁺CD27⁺, n = 1209) populations is shown. Allelic variations of each IGHV gene are not shown. P values for χ² comparisons of individual IGHV gene usage between the different groups are indicated in Table 2.

Figure 5

Four subsets of B cells are distinguished from one another in their Ig CDR3 properties. The amino acid sequence of the Ig CDR3 region was determined from V-Quest for each sequence and subjected to ProtParam analysis to determine the peptide characteristics for transitional (T, n = 433), naive (N, n = 662), switched memory (S, n = 1293), and IgM memory (M, n = 1209) populations. Error bars are SEM. *P < .05; **P < .005; ***P < .0005.