Peripheral blood CD27+ IgG+ B cells rapidly proliferate and differentiate into immunoglobulin-secreting cells after exposure to low CD154 interaction

Abstract

In vitro CD40 stimulation of human B cells isolated from lymphoid organs is dominated by memory B cells undergoing faster proliferation and higher differentiation than naive B cells. In contrast, we previously reported that blood memory B cells mainly differentiate into immunoglobulin-secreting cells in response to CD40 stimulation. However, variations in CD40-CD154 interaction are now recognized to influence B-cell fate. In this study, we have compared the in vitro response of blood CD27(-) and CD27(-) IgG(-) to CD27(+) and CD27(+) IgG(+) B cells following low-density exposure to CD154 in the presence of a mixture of interleukin-2 (IL-2), IL-4 and IL-10. The evolution of these cell populations was monitored during initiation and following long-term stimulation. Over a 5-day period, CD27(+) B cells underwent differentiation into immunoglobulin-secreting cells more readily than CD27(-) cells, and CD27(+) IgG(+) B cells gave rise to a near homogeneous population of CD19(+) CD27(++) CD38(+) IgG(lo) cells capable of high immunoglobulin G (IgG) secretion. During the same period, CD27(-) IgG(-) B cells partially became CD19(++) CD27(-) CD38(-) IgG(++) cells but showed no IgG secretion. Long-term stimulation revealed that CD27(+) IgG(+) B cells retained a high expansion capacity and could maintain their momentum towards differentiation over naive B cells. In addition, long-term stimulation was driving CD27(-) IgG(-) and total CD19(+) B cells to evolve into similar CD27(+) and CD27(-) subsets, suggesting naive homeostatic proliferation. Overall, these results tend to reconcile memory B cells from blood and lymphoid organs regarding their preferential differentiation capacity compared to naive cells, and further suggest that circulating memory IgG(+) cells may be intrinsically prone to rapid activation upon appropriate stimulation.

Figure 1

CD27⁺ B cells are more efficient than CD27⁻ cells at immunoglobulin secretion. Total CD19⁺ and sorted CD19⁺ CD27⁻ and CD19⁺ CD27⁺ peripheral blood B cells were stimulated with CD154 in the presence of interleukin-2 (IL-2), IL-4 and IL-10. (a) Expansion was determined on days 5, 9 and 14 and (b) immunoglobulin G (IgG) and IgM concentrations were measured at the end of the 5-day culture period. (c) IgG and IgM secretion rates after 14 days of culture. (d) CFSE labelling and CD27 staining were used to track cell division of CD27⁺ (proliferation index 7·9) and CD27⁻ (proliferation index 9·2) populations within total CD19⁺ cells over a 5-day-period. These results are representative of three independent experiments, all performed with different blood samples. Each experiment was performed in triplicate. Error bars can be smaller than symbols and represent variations among triplicates in this experiment.

Figure 2

Heterogeneous populations emerge from blood CD27⁺ and CD27⁻ B cells. CD19, CD27 and surface immunoglobulin G (IgG) expression was evaluated by flow cytometry (> 5000 events) of cultures started with total CD19⁺ and sorted CD19⁺ CD27⁻ (CD27⁻) and CD19⁺ CD27⁺ (CD27⁺) B cells. (a) CD19 and CD27 expression and (b) CD19 and surface IgG expression were measured on days 0, 5 and 14. (c) Phenotypic CD27 and IgG expression on day 14 are shown. These results are representative of two independent experiments and similar results were observed on day 5 in a supplemental short-term assay.

Figure 3

CD27⁻ IgG⁺ cells persist within the CD27⁻ cells following long-term stimulation. (a) Nine IgG transcripts from resting blood CD19⁺ B cells were amplified, sequenced and analysed for the presence of somatic mutations, in parallel with transcripts from (b) unsorted CD19⁺ B cells, (c) sorted CD27⁻ B cells and (d) sorted CD27⁺ B cells stimulated for 14 days with CD154, interleukin-2 (IL-2), IL-4 and IL-10. All characterized IgG transcripts from two donors (1A or 1B) are presented; the number of clones, their origin, and the number of mutated amino acids are indicated. The distribution of R (Replacement: ) and S (Silent; ) mutations is shown only when four or more mutated amino acids were observed. The proportions (%) of R mutations found in FR1, FR2 and FR3 (framework regions; cumulative length: 82 amino acids), and in CDR1 and CDR2 (complementarity determining regions; cumulative length: 22 amino acids) are shown for each sequence. *Clones considered to have arisen independently from antigen selection.

Figure 4

CD27⁺ IgG⁺ cells give rise to CD38⁺ IgG⁺ and CD38⁻ IgG^lo cells. Total CD19⁺ and sorted CD19⁺ CD27⁻ IgG⁻ and CD19⁺ CD27⁺ IgG⁺ B cells, stimulated as described in Table 1, were analysed after 5 days in culture for CD19, CD27 and immunoglobulin G (IgG) expression by flow cytometry (> 500 events). Cell purity before stimulation is shown (d = 0). The proportion of IgG⁺ cells as a function of CD27 expression is presented for each subset. These results are representative of two independent experiments.

Figure 5

Long-term stimulation drives CD27⁺ IgG⁺ cells into plasma cell differentiation. Unsorted blood B cells (CD19⁺) and sorted CD19⁺ CD27⁻ IgG⁻ and CD19⁺ CD27⁺ IgG⁺ populations were stimulated with CD154, interleukin-2 (IL-2), IL-4 and IL-10 for 14 days and monitored for expression of CD19, CD27, CD38 and surface immunoglobulin G (IgG) by flow cytometry (> 10 000 events). All sorted populations were ≥ 94% pure. On day 0, the frequencies of CD27⁺, CD27⁻, CD27⁺ IgG⁺, CD27⁻ IgG⁺ and CD27⁻ IgG⁻ cells within CD19⁺ cells were 21%, 79%, 6%, 4%, and 75%, respectively. The level of CD38 expression (mean fluorescence intensity) on resting B cells varied from 7 to 27. These results are representative of two independent experiments.