TLR4 Promotes B Cell Maturation: Independence and Cooperation with B Lymphocyte-Activating Factor

Abstract

We have previously shown that TLR4 triggering promotes the generation of CD23+CD93+ transitional T2-like cells in vitro from mouse B cell precursors, suggesting a possible role for this receptor in B cell maturation. In this study, we perform an extensive study of cell surface markers and functional properties of B cells matured in vitro with LPS, comparatively with the well-known B cell maturation factor B lymphocyte-activating factor (BAFF). LPS increased generation of CD23+ transitional B cells in a TLR4-dependent way, upregulating IgD and CD21 and downregulating CD93, without inducing cell proliferation, in a manner essentially equivalent to BAFF. For both BAFF and LPS, functional maturation of the IgM+CD23+CD93+ cells was confirmed by their higher proliferative response to anti-CD40 plus IL-4 compared with IgM+CD23negCD93+ cells. BAFF-R-Fc–mediated neutralization experiments showed that TLR4-induced B cell maturation was independent of BAFF. Distinct from BAFF, maturation by LPS relied on the activation of canonical NF-κB pathway, and the two factors together had complementary effects, leading to higher numbers of IgM+CD23+CD93+ cells with their simultaneous addition. Importantly, BCR cross-linking abrogated the generation of CD23+ B cells by LPS or BAFF, indicating that signals mimicking central tolerance act on both systems. Addition of cyclosporin A reverted BCR-mediated inhibition, both for BAFF and LPS, suggesting similar regulation of signaling pathways by calcineurin. Finally, LPS-injected mice showed a rapid increase of mature B cells in the bone marrow, suggesting that TLR4 signaling may effectively stimulate B cell maturation in vivo, acting as an accessory stimulus in B cell development, complementary to the BAFF physiological pathway.

IL-4 Regulates Bim Expression and Promotes B Cell Maturation in Synergy with BAFF Conferring Resistance to Cell Death at Negative Selection Checkpoints

“…Incubation of cells in the presence of BAFF for the last 18 h of culture in this system induces a 2-to 4-fold increase in the percentage and number of the CD23 + B cells compared with unstimulated control (Fig. 1A, 1C), in accordance with the well-known role of BAFF in B cell development at a transitional stage (13)(14)(15)28). Using BAFF as our comparative standard, we tested the effect of the incubation of B cells with IL-4 for the last 18 h of culture on developing B lymphocytes, analyzing changes in the expression of surface maturation markers by flow cytometry.…”

“…In contrast to the relatively well-resolved roles of IL-4 in mature B cell activation, less clear is its role in B cell maturation and whether it can interfere on the pivotal roles of BCR/BAFF-R with a potential cooperation with BAFF in the control of autoreactivity at negative selection checkpoints. We have previously described that TLR4 agonists can affect B cell maturation in vitro by stimulating generation of T2-like B cells from BM developing B cells and cooperating with BAFF to enhance this process (27,28). These results suggested that TLR4 agonists can act as alternate/complementary factors for BAFF.…”

“…As previously described (27,28), even without addition of any supplemental growth factors, this system allows spontaneous differentiation of purified B cell precursors with generation of a heterogeneous population of IgM + B cells ( Fig. 1 + fraction E (11).…”

Toll-like receptor 4-mediated signaling regulates IL-7-driven proliferation and differentiation of B-cell precursors

“…24 Recent studies by Hayashi et al indicate that TLR4 signaling also plays a role during late stages of B-cell development by facilitating the generation of immature or transitional B cells. 14 The present study further demonstrates that early B-cell development is subject to regulation by TLR4 signaling. The pro-B-and pre-B-cell population was found to be enlarged in the bone marrow of TLR4-mutant, C3H/HeJ mice, indicating a physiological role of TLR4 in early B lymphopoiesis.…”

“…13 A more recent study, however, indicates that LPS may facilitate B-cell maturation by acting as an accessory stimulus to complement the BAFF physiological pathway of B-cell development. 14 Therefore, the precise role of TLR4 signaling in the early stages of B-cell development requires more in-depth analysis. In the present study, we used in vitro (cell culture) and in vivo (adoptive transfer) approaches to systemically analyze the impact of TLR4 signaling on the proliferation, survival and differentiation of B-cell precursors.…”