Zhoujie Ding

How antibodies use complement to regulate antibody responses.

Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed.

IgE-mediated enhancement of CD4+ T cell responses in mice requires antigen presentation by CD11c+ cells and not by B cells.

IgE antibodies, administered to mice together with their specific antigen, enhance antibody and CD4+ T cell responses to this antigen. The effect is dependent on the low affinity receptor for IgE, CD23, and the receptor must be expressed on B cells. In vitro, IgE-antigen complexes are endocytosed via CD23 on B cells, which subsequently present the antigen to CD4+ T cells. This mechanism has been suggested to explain also IgE-mediated enhancement of immune responses in vivo. We recently found that CD23+ B cells capture IgE-antigen complexes in peripheral blood and rapidly transport them to B cell follicles in the spleen. This provides an alternative explanation for the requirement for CD23+ B cells. The aim of the present study was to determine whether B-cell mediated antigen presentation of IgE-antigen complexes explains the enhancing effect of IgE on immune responses in vivo. The ability of spleen cells, taken from mice 1–4 h after immunization with IgE-antigen, to present antigen to specific CD4+ T cells was analyzed. Antigen presentation was intact when spleens were depleted of CD19+ cells (i.e., primarily B cells) but was severely impaired after depletion of CD11c+ cells (i.e., primarily dendritic cells). In agreement with this, the ability of IgE to enhance proliferation of CD4+ T cells was abolished in CD11c-DTR mice conditionally depleted of CD11c+ cells. Finally, the lack of IgE-mediated enhancemen of CD4+ T cell responses in CD23-/- mice could be rescued by transfer of MHC-II-compatible as well as by MHC-II-incompatible CD23+ B cells. These findings argue against the idea that IgE-mediated enhancement of specific CD4+ T cell responses in vivo is caused by increased antigen presentation by B cells. A model where CD23+ B cells act as antigen transporting cells, delivering antigen to CD11c+ cells for presentation to T cells is consistent with available experimental data.

Complement-Activating IgM Enhances the Humoral but Not the T Cell Immune Response in Mice

IgM antibodies specific for a certain antigen can enhance antibody responses when administered together with this antigen, a process believed to require complement activation by IgM. However, recent data show that a knock-in mouse strain, Cμ13, which only produces IgM unable to activate complement, has normal antibody responses. Moreover, the recently discovered murine IgM Fc receptor (FcµR or TOSO/FAIM3) was shown to affect antibody responses. This prompted the re-investigation of whether complement activation by specific IgM is indeed required for enhancement of antibody responses and whether the mutation in Cµ13 IgM also caused impaired binding to FcµR. The results show that IgM from Cµ13 and wildtype mice bound equally well to the murine FcµR. In spite of this, specific Cμ13 IgM administered together with sheep red blood cells or keyhole limpet hemocyanine was a very poor enhancer of the antibody and germinal center responses as compared with wildtype IgM. Within seconds after immunization, wildtype IgM induced deposition of C3 on sheep red blood cells in the blood. IgM which efficiently enhanced the T-dependent humoral immune response had no effect on activation of specific CD4+ T cells as measured by cell numbers, cell division, blast transformation, or expression of the activation markers LFA-1 and CD44 in vivo. These observations confirm the importance of complement for the ability of specific IgM to enhance antibody responses and suggest that there is a divergence between the regulation of T- and B-cell responses by IgM.

Marginal Zone B Cells Transport IgG3-Immune Complexes to Splenic Follicles

Ag administered together with specific IgG3 induces a higher Ab response than Ag administered alone, an effect requiring the presence of complement receptors 1 and 2 (CR1/2). In this study, we have investigated the fate of Ag, the development of germinal centers (GCs), and the Ab response after i.v. administration of IgG3 anti-trinitrophenyl (TNP) in complex with OVA-TNP. After 2 h, OVA-TNP was detected on marginal zone (MZ) B cells, and a substantial amount of Ag was detected in splenic follicles and colocalized with follicular dendritic cells (FDCs). After 10 d, the percentage of GCs and the IgG responses were markedly higher than in mice immunized with uncomplexed OVA-TNP. The effects of IgG3 were dependent on CR1/2 known to be expressed on B cells and FDCs. Using bone marrow chimeric mice, we demonstrate that an optimal response to IgG3-Ag complexes requires that CR1/2 is expressed on both cell types. These data suggest that CR1/2+ MZ B cells transport IgG3-Ag-C complexes from the MZ to the follicles, where they are captured by FDCs and induce GCs and IgG production. This pathway for initiating the transport of Ags into splenic follicles complements previously known B-cell dependent pathways where Ag is transported by 1) MZ B cells, binding large Ags-IgM-C complexes via CR1/2; 2) recirculating B cells, binding Ag via BCR; or 3) recirculating B cells, binding IgE-Ag complexes via the low-affinity receptor for IgE, CD23.

Distinguishing Mast Cell Progenitors from Mature Mast Cells in Mice

Mast cells originate from the bone marrow and develop into c-kit+ FcɛRI+ cells. Both mast cell progenitors (MCp) and mature mast cells express these cell surface markers, and ways validated to distinguish between the two maturation forms with flow cytometry have been lacking. Here, we show that primary peritoneal MCp from naïve mice expressed high levels of integrin β7 and had a low side scatter (SSC) light profile; whereas mature mast cells expressed lower levels of integrin β7 and had a high SSC light profile. The maturation statuses of the cells were confirmed using three main strategies: (1) MCp, but not mature mast cells, were shown to be depleted by sublethal whole-body γ-irradiation. (2) The MCp were small and immature in terms of granule formation, whereas the mature mast cells were larger and had fully developed metachromatic granules. (3) The MCp had fewer transcripts of mast cell-specific proteases and the enzyme responsible for sulfation of heparin than mature mast cells. Moreover, isolated peritoneal MCp gave rise to mast cells when cultured in vitro. To summarize, we have defined MCp and mature mast cells in naïve mice by flow cytometry. Using this strategy, mast cell maturation can be studied in vivo.

IgE-mediated enhancement of CD4(+) T cell responses requires antigen presentation by CD8α(-) conventional dendritic cells.

IgE, forming an immune complex with small proteins, can enhance the specific antibody and CD4+ T cell responses in vivo. The effects require the presence of CD23 (Fcε-receptor II)+ B cells, which capture IgE-complexed antigens (Ag) in the circulation and transport them to splenic B cell follicles. In addition, also CD11c+ cells, which do not express CD23, are required for IgE-mediated enhancement of T cell responses. This suggests that some type of dendritic cell obtains IgE-Ag complexes from B cells and presents antigenic peptides to T cells. To elucidate the nature of this dendritic cell, mice were immunized with ovalbumin (OVA)-specific IgE and OVA, and different populations of CD11c+ cells, obtained from the spleens four hours after immunization, were tested for their ability to present OVA. CD8α− conventional dendritic cells (cDCs) were much more efficient in inducing specific CD4+ T cell proliferation ex vivo than were CD8α+ cDCs or plasmacytoid dendritic cells. Thus, IgE-Ag complexes administered intravenously are rapidly transported to the spleen by recirculating B cells where they are delivered to CD8α− cDCs which induce proliferation of CD4+ T cells.

B Cell-mediated Antigen Transport to Splenic Follicles

To the Editor An effective antibody response requires that antibodies go through affinity maturation and class switch recombination. These reactions largely take place in germinal centres which develop in B cell follicles of secondary lymphoid organs. An important step in this process is antigen deposition on follicular dendritic cells (FDC). Antigen can be transported from the periphery to follicles either via small channels (conduits) or bound to various cell types [1, 2]. Generally, antigen transport in lymph nodes is better understood than in the spleen, mostly because lymph nodes are more accessible to visualization. Here, we highlight recent observations which demonstrate that antigens can enter splenic follicles with the help of B cells in three different ways: via the low-affinity receptor for IgE (CD23) [3], via the B cell receptor (BCR) [4] and via the complement receptors CD21/CD35 [5, 6] (Fig. 1). Marginal zone (MZ) B cells, only residing in spleen and not in lymph nodes, continuously shuttle between the MZ and the follicles with as much as a 20% exchange of cells between the two compartments per hour [7, 8]. MZ B cells express high levels of CD21/CD35. The shuttling takes place independently of whether these receptors are ligated. Once complement-opsonized immune complexes are present in the MZ, they will bind to MZ B cells and be delivered to FDC [5, 6]. Whereas MZ B cells require that antigen first be transported to the MZ, recirculating B cells survey the periphery and migrate to secondary lymphoid organs where they reside for approximately one day before returning to the circulation. Hence, it would seem beneficial for the immune system to utilize them for antigen transport, and indeed, two such situations have been described. First, B cells in the blood can capture IgE–antigen complexes via CD23 and rapidly transport them to splenic follicles [3]. Subsequently, a potent germinal centre response, massive proliferation of specific CD4 T cells and a specific IgG response developed [3, 9, 10]. As CD23 is a C-type lectin and other receptors of this family are pattern recognition receptors, hypothetically CD23 could bind directly to certain pathogens without using IgE antibodies as a bridge. Second, antigens from the respiratory tract can reach the spleen bound to cognate B cells [4]. Twenty-four hours after intranasal administration, viruslike particles were

IgG3-antigen complexes are deposited on follicular dendritic cells in the presence of C1q and C3

IgG3, passively administered together with small proteins, induces enhanced primary humoral responses against these proteins. We previously found that, within 2 h of immunization, marginal zone (MZ) B cells capture IgG3-antigen complexes and transport them into splenic follicles and that this requires the presence of complement receptors 1 and 2. We have here investigated the localization of IgG3 anti-2, 4, 6-trinitrophenyl (TNP)/biotin-ovalbumin-TNP immune complexes in the follicles and the involvement of classical versus total complement activation in this process. The majority (50–90%) of antigen inside the follicles of mice immunized with IgG3-antigen complexes co-localized with the follicular dendritic cell (FDC) network. Capture of antigen by MZ B cells as well as antigen deposition on FDC was severely impaired in mice lacking C1q or C3, and lack of either C1q or C3 also impaired the ability of IgG3 to enhance antibody responses. Finally, IgG3 efficiently primed for a memory response against small proteins as well as against the large protein keyhole limpet hemocyanine.