CD73 expression identifies a subset of IgM+ antigen-experienced cells with memory attributes that is T cell and CD40 signalling dependent

Abstract

B-cell memory was long characterized as isotype-switched, somatically mutated and germinal centre (GC)-derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B-cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype-switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T-dependent versus T-independent responses. We report that CD73 expression identifies a subset of antigen-experienced IgM⁺ cells that share attributes of functional B-cell memory. This subset is reduced in the spleens of T-cell-deficient and CD40-deficient mice and in mixed marrow chimeras made with mutant and wild-type marrow, the proportion of CD73⁺ IgM memory is restored in the T-cell-deficient donor compartment but not in the CD40-deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age-associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B-1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC-associated memory generation during B-cell differentiation, CD40-signalling can influence the composition of the unswitched memory B-cell pool. They also raise the possibility that a fraction of ABCs may represent T-cell-dependent IgM memory.

Keywords: CD40; CD73; IgM; T cells; age-associated B cells; memory.

Figure 1

Gating strategy for identification of antigen‐experienced B cells in unimmunized mice. B220⁺ cells in the spleen were sequentially gated as IgD^– CD21⁻ CD93⁻ CD23⁺ to identify total antigen‐experienced cells and then as IgM⁺ and IgM^– for identification of the unswitched and switched components, respectively (a). Gating for identification of CD73⁺, CD80⁺ and CD273⁺ cells on IgM⁺ (b) and IgM^– (c) antigen‐experienced cells.

Figure 2

IgM⁺ antigen‐experienced cells share features of memory. Expression of transcripts (as labelLed) in sorted IgM⁺ and IgM^– antigen‐experienced cells from unimmunized mice relative to 24‐hr lipopolysaccharide (LPS) blasts (a). Immunoglobulin in supernatants of sorted cells stimulated with 10 μg/ml LPS in the absence (without) or presence (with) of 1 μg/ml aphidicolin for 84 hr. Activated B cells are spleen cells pre‐stimulated with LPS for 72 hr and re‐stimulated with or without aphidicolin for 48 hr (b). (ASCs)/10⁵ cells that were stimulated with LPS ± aphidicolin for 72 hr. Activated B cells are spleen cells pre‐stimulated with LPS for 72 hr and re‐stimulated with or without aphidicolin for 48 hr (c). Data are shown as mean ± SD of replicates (a), mean ± SEM of triplicate cultures (b), and mean ± SEM of triplicate cultures, with cells from each culture loaded onto six wells each for ASC assay (c).

Figure 3

Switched memory subsets are reduced in mice lacking T cells and CD40. Frequencies of switched memory cells, and the proportion of CD73⁺, CD80⁺ and CD273⁺ subsets in the spleens of B6 and TCR‐β ^−/− (TCRb^–) mice (a, b) and of BALB/c and CD40^−/− (CD40^–) mice (c, d). Data are shown as mean ± SEM of 8–12 mice from three or four experiments. *P ≤ 8·8 × 10⁻⁴.

Figure 4

IgM memory subsets are reduced in TCR‐β ^−/− and CD40^−/− mice. Frequencies of IgM memory cells and the proportion of CD73⁺, CD80⁺ and CD273⁺ subsets in the spleens of B6 and TCR‐β ^−/− (TCRb^–) mice (a, b), BALB/c and CD40^−/− (CD40^–) mice (c, d) and B6 and TCR‐δ ^−/− (TCRd^–) mice. Data are shown as mean ± SEM of 8–12 mice from three or four experiments. *P = 0·03, **P ≤ 0·002.

Figure 5

The CD73⁺ subset of IgM memory requires CD40 ligation. Frequencies of CD73⁺, CD80⁺ and CD273⁺ subsets of IgM memory in the spleens of bone marrow (BM) chimeras made with a 1 : 1 mixture of BM from wild‐type (WT; B6.SJL) and TCR‐β ^– δ ^‐– (TCRb^–d^–) mice (a–c) and from WT (BALB/c) and CD40^−/− (CD40^−/−) mice (d–f). The donors were identified as B220⁺ CD45.1⁺ and B220⁺ CD45.2⁺ (a–c) and as B220⁺ CD40⁺ and B220⁺ CD40⁻ (d–f). Data are shown as mean ± SEM of 10 chimeras (WT/TCRb^–d^–) and six chimeras (WT/CD40^−/−) from two experiments. *P ≤ 0·03, **P ≤ 1·2 × 10⁻⁵.

Figure 6

Transfer of peripheral T cells rescues the CD73⁺ subset of IgM memory in T‐cell‐deficient mice. Frequencies of IgM memory subsets (a–c) and switched memory subsets (d–f) on gated CD45.2 cells in the spleen of TCR‐β ^– δ ^– mice 6 weeks after adoptive transfer of congenic wild‐type (WT) CD45.1 spleen cells. Data are shown as mean ± SEM of 10 recipients from two experiments. *P ≤ 0·04, **P ≤ 0·01, ***P ≤ 0.009.

Figure 7

CD40 deficiency affects CD73 expression on age‐associated B cells (ABCs) and peripheral T cells. Frequency of CD73⁺ cells on gated blood neutrophils (a, n = 10), peritoneal cavity B‐1 B cells (b, n = 10), splenic regulatory T (Treg) cells (c, n = 10), marginal zone (MZ) B cells (d, n = 14), ABCs (e, n = 15), B regulatory (Breg) cells (f, n = 12), Peyer's patch follicular helper T (Tfh) (g, n = 8) and splenic CD4⁺ T (h, n = 9) and CD8⁺ T cells (i, n = 9) in BALB/c and CD40^−/− mice. (j, k) Frequency of CD73⁺ cells on gated CD4⁺ and CD8⁺ (n = 8) T cells in B6 and μMT mice. Data are shown as mean ± SEM of 8–15 mice, as indicated, from two to five experiments. *P ≤ 2·4 × 10⁻⁹, **P = 0·01.