Memory B cells are biased towards terminal differentiation: a strategy that may prevent repertoire freezing

Abstract

Isolation of large numbers of surface IgD+CD38- naive and surface IgD-CD38- memory B cells allowed us to study the intrinsic differences between these two populations. Upon in vitro culture with IL-2 and IL-10, human CD40-activated memory B cells undergo terminal differentiation into plasma cells more readily than do naive B cells, as they give rise to five- to eightfold more plasma cells and three- to fourfold more secreted immunoglobulins. By contrast, naive B cells give rise to a larger number of nondifferentiated B blasts. Saturating concentrations of CD40 ligand, which fully inhibit naive B cell differentiation, only partially affect that of memory B cells. The propensity of memory B cells to undergo terminal plasma cell differentiation may explain the extensive extra follicular plasma cell reaction and the limited germinal center reaction observed in vivo after secondary immunizations, which contrast with primary responses in carrier-primed animals. This unique feature of memory B cells may confer two important capacities to the immune system: (a) the rapid generation of a large number of effector cells to efficiently eliminate the pathogens; and (b) the prevention of the overexpansion and chronic accumulation of one particular memory B cell clone that would freeze the available peripheral repertoire.

Figure 1

Naive and memory cells differentiation at the end of secondary cultures, followed by CD20 and CD38 expression. PCs are CD20^−/lowCD38^high and nondifferentiated B blasts (BLASTS) are CD20⁺CD38^low (37). (A) Naive B cells cultured with IL-2 and IL-10 over CD40L-transfected L cells. (B) Naive B cells cultured with IL-2 and IL-10 over parental nontransfected L cells together with an anti-CD40L– blocking antibody at 2 μg/ml. (C) Memory B cells cultured with IL-2 and IL-10 over CD40L-transfected L cells. (D) Memory B cells cultured IL-2 and IL-10 over nontransfected L cells together with an anti-CD40L– blocking antibody at 2 μg/ml.

Figure 2

Morphology and intracellular Ig content. Giemsa staining of sorted CD20^−/lowCD38^high PC (A, original magnification: 1,000) and CD20⁺CD38^low B blasts (B, original magnification: 1,000). Red anti-Igκ + λ light chain staining of sorted CD20^−/lowCD38^high PCs (C, original magnification: 1,000) and CD20⁺CD38^low B blasts (D, original magnification: 1,000). Red anti-IgM staining of sorted CD20^−/lowCD38^high PCs derived from naive B cells (E, original magnification: 400) and from memory B cells (F, original magnification: 400).

Figure 3

CD40L inhibits B cell differentiation during secondary cultures in a dose-dependent fashion. (A) Percentages of CD20^−/lowCD38^high PCs derived from naive (closed circles) and memory (open circles) B cells. (B) Percentages of CD20⁺CD38^low nondifferentiated B blasts derived from naive (closed circles) and memory (open circles) B cells. (C) Total numbers of CD20^−/lowCD38^high PCs derived from naive (closed circles) and memory (open circles) B cells. The number of CD40L molecules per B cell in secondary cultures was measured and used as x-axis (a negative value artificially represents cultures with parental fibroblasts in the presence of a blocking antibody to CD40L at 2 μg/ml). Cell input was 1.5 × 10⁵ at the beginning of secondary cultures. Each circle represents an individual value. Mean values are linked (plain and dotted lines represent naive and memory cell cultures, respectively). Standard deviations are vertical bars.

Figure 4

. CD40L inhibits Ig production during secondary cultures in a dose-dependent fashion. The culture conditions are the same as described in Fig. 3. (A) Total IgG + IgA + IgM production from 10⁶ cells of naive (closed circles) and of memory B cell cultures (open circles). (B) IgA production. (C) IgG production. (D) IgM production. Each circle (closed and open correspond to naive and memory cell cultures, respectively) represents an individual value. Mean values are linked (plain and dotted lines represent naive and memory cell cultures, respectively). Standard deviations are vertical bars.

Figure 5

Naive and memory B cells undergo comparable proliferation and expansion during 12 d of cultures with IL-2 and IL-10 over CD40L-transfected L cells. (A) [³H]thymidine uptake by cultured naive (closed circles) and memory (open circles) B cells. (B) Cell numbers of cultured naive (closed circles) and memory (open circles) B cells. Initial cell inputs were 2.5 × 10⁴ for [³H]thymidine uptake and 1.5 × 10⁵ for the viable cell numbers.

Figure 6

Memory, but not naive, B cells preferentially undergo plasma cell differentiation even after BCR triggering. Naive and memory B cells were cultured for 3 d with anti-Igκ and anti-Igλ antibodies, together with IL-2, IL-10, and CD40L. After washing, cells were recultured with IL-2, IL-10, and different concentrations of CD40L for 4 d. (A) CD38⁺CD20⁻ plasma cells and CD38^lowCD20⁺ undifferentiated B cells generated from naive B cells. (B) CD38⁺CD20⁻ plasma cells and CD38^lowCD20⁺ undifferentiated B cells generated from memory B cells. This is one representative of two experiments.

Figure 7

Memory B cells are biased towards terminal plasma cell differentiation in vivo and in vitro. Naive B cells predominantly give rise to germinal center reaction within a rat spleen after primary immunization with DNP-KLH in KLH-primed animals (A original magnification: 40; B original magnification: 200). The rats were given BrdU in their drinking water for 48 h before they were killed. Red stains BrdU, blue stains DNP-binding cells, brown stains total B cells. MZ, marginal zone; PALS, periarteriolar lymphoid sheath. Consistent with this in vivo finding, human naive B cells predominantly give rise to proliferating B blasts upon activation in vitro (C). Memory B cells predominantly give rise to plasma cell reaction along the outer edges of the periarteriolar lymphoid sheath and within the red pulp of a rat spleen after 2 d of secondary immunization with DNP-KLH (D and E). The rats have received BrdU in their drinking water for 48 h before killing. DNP-specific plasma blasts are cells with strong blue cytoplasmic staining. Consistent with this in vivo finding, human memory B cells predominantly give rise to plasma cells upon activation in vitro (F). The figures on immunohistology are derived from Y.-J. Liu and I.C.M. MacLennan (40).