Imprinting the fate of antigen-reactive B cells through the affinity of the B cell receptor

Abstract

Long-lived plasma cells (PCs) and memory B cells (B(mem)) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short- or long-lived PCs and B(mem). Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither B(mem) nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but B(mem) remain extinct. In contrast, lower affinity interactions show tempered GCs, producing B(mem) and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity.

FIGURE 1

Tg λ and κ mice show disparate BCR affinities. A, Generation strategy and B cell phenotype of Tg λ and κ mice. Flow cytometric analysis of donor splenic B cells revealed that Tg mice have an equivalent yet higher frequency of marginal zone (MZ; B220⁺CD23^lowCD21^highIgM^high) compared with follicular (FO; B220⁺CD23^highCD21^intIgM^int) subsets than non-Tg mice. Newly formed (NF; B220⁺CD23⁻CD21⁻IgM^int) immigrants were unchanged. Dot plots shown are gated on the B220⁺ population. B and C, Chromatographically purified V_H17.2.25 Id⁺ Igs from the sera of naive Tg λ and κ mice were standardized and serial dilutions were tested for their ability to bind a high or low hapten ratio (NP₂₅ vs NP₄) by ELISA. D, A competitive ELISA was performed using the purified serum Tg Ig to measure average affinities of λ and κ Abs for NP and NIP as described in Materials and Methods. Hapten inhibition curves are shown that represent the percentage of maximal Ab binding to a constant amount of Ag when serial dilutions of competing soluble NP or NIP monomeric hapten (μM) is present. Measurements of the IC₅₀ values are depicted by the dotted horizontal line (NP, Tg κ = 1.86 × 10⁻³ M, Tg λ = 2.21 × 10⁻⁴ M; NIP, Tg κ = 4.69 × 10⁻⁴ M, Tg λ = 2.07 × 10⁻⁵ M). Results are from three independent experiments.

FIGURE 2

Adoptively transferred Tg λ B cells show accelerated proliferation and a failure to acquire GC phenotype and exhibit altered levels of B cell maturation-related gene expression in vivo. A, Donor Tg B cells were quantified by flow cytometry from recipient spleens on days 5, 7, and 10 after immunization with NP-KLH (NP), NIP-KLH (NIP), or CFA alone (naive). B, Ex vivo flow cytometric analysis on spleen cells from NP or NIP immune recipients. Fluorescence intensity of the GC marker PNA on Tg cells (B220⁺Id⁺) is shown by open histograms. Naive controls are shown in shaded histograms. C, Relative levels of mRNA expression of the indicated genes was measured by real time RT-PCR on purified donor Tg B cells from day 7 immune recipient spleens. The data are given as mean ± SD (n = 3) and are representative of three experiments.

FIGURE 3

Serum IgG1 and IgG2a titers as well as the frequency of splenic PCs produced by transferred Tg λ B cells are reduced early on in the humoral immune response. Tg κ or λ cells were adoptively transferred and recipients immunized with NP, NIP, or CFA alone (naive). Serum and spleens from recipients were collected at days 5, 7, and 10 following challenge. Left panels show total levels of Tg NP-specific serum IgG1, IgG2a, and IgM as measured by ELISA using allotype-specific detection mAbs. Right panels show splenic Tg anti-NP IgG1, IgG2a, and IgM PCs quantified by ELISPOT from the same recipients. The data shown are mean ± SD (n = 3) and are representative of three experiments.

FIGURE 4

BCR affinity determines the generation of long-lived BM PCs in noncompeting and competing environments. A and B, Recipient mice received either Tg λ (A) or Tg κ (B) donor B cells and were immunized with NP or NIP. Three weeks later, BM cells were analyzed by flow cytometry for fluorescence intensity of CD138 on PC_pre (Ly5.1⁺sIgG⁺Ly6C⁺). C and D, PC_pre, shown in A and B, were sort purified and transferred into naive 2° recipients. Two weeks later, the capacity of transferred Tg PC_pre to produce anti-NP IgG1/2a^a BM PCs was quantified by ELISPOT. E, Equal numbers of Tg λ and κ B cells were transferred into 1° recipients singly or combined. Recipients were immunized with NP, NIP, or CFA alone (naive). Confocal image analysis of recipient spleens was performed 7 days later using fluorescently labeled mAbs to Ly5.1, B220, κ, and PNA. Insets in upper two rows show PNA staining only, whereas insets in lower row show κ staining only. F, Tg λ and κ B cells were cotransferred into 1° recipients and immunized with NP, NIP, or CFA (naive). After 30 days, the frequency of BM PCs produced from transferred Tg B cells was quantified by ELISPOT to dually detect anti-NP IgG1/2a^a+κ⁺- or IgG1/2a^a+λ⁺-secreting cells. Data shown are representative of four experiments.

FIGURE 5

Innate BCR affinity determines affinity maturation. A, Tg κ or Tg λ cells were adoptively transferred into 1° recipients and immunized with NP or CFA (naive). Spleens were removed at day 7 postimmunization, and transferred Tg B cells were stained with mAbs to B220, V_H17.2.25 Id, IgG, and graded dilutions of fluorescently labeled NP or NIP. Histograms shown from flow cytometric analyses are gated on B220⁺Id⁺ Tg κ or λ cells. Surface IgG expression was equivalent between Tg λ and κ B cells (gray histogram, naive Tg B cells; black histogram, immune Tg B cells). B and C, SHM analysis of individual V_H17.2.25 transcripts from Tg λ and κ BM PCs. Tg κ or λ B cells were adoptively transferred into 1° recipients and immunized with NP. After 4 wk, CD138⁺ BM PCs were purified by magnetic bead selection. cDNA from purified RNA was amplified using V_H17.2.25-specific primers and transcripts were sequenced as described in Materials and Methods. Silent and replacement mutations are denoted by lowercase and uppercase nucleotides, respectively. Sequences shown are a composite of two independent experiments.