We have previously developed functional in vitro germinal centers (GCs) using naïve murine B cells. The model was studied in two dimensions (2-D) in culture plates. In these murine in vitro GCs, immunoglobulin (Ig) class switching, somatic hypermutation, selection of the high affinity B cells, and affinity maturation were demonstrated. These activities are important to the goal of studying vaccines in vitro. In the in vitro GC, follicular dendritic cells (FDCs) serve two main functions: to facilitate T cell-B cell interaction and to potentiate B cell viability. Both of these functions enable and facilitate activation of specific B cells, antibody production, and differentiation into plasma cells.
In 1968, Szakal and Hanna (J. Immunol. 101, 949-962; Exp. Mol. Pathol. 8, 75-89) and Nossal et al. (J. Exp. Med. 127, 277-290) published the first descriptions and electron micrographs of what are now known as follicular dendritic cells (FDCs). Both groups used 125I-labeled antigens and examined autoradiographs of the follicles in rodent spleens or lymph nodes using electron microscopy. Both groups found that radiolabel persisted on or near the surface of highly convoluted fine cell processes of dendritic-type cells with peculiar, irregularly shaped, euchromatic nuclei. The fine cell processes formed an elaborate meshwork around passing lymphocytes, allowing extensive cell-cell contact. Several names have been used for these cells but a nomenclature committee recommended the name “follicular dendritic cell” and the abbreviation “FDC” and these have been generally adopted (Tew et al. (1982) J. Reticuloendothelial Soc. 31, 371-380).
The ability of FDCs to trap and retain antigen-antibody complexes, together with their follicular location, distinguishes them from other cells, including other dendritic cells (DCs). FDCs bearing specific antigens are required for full development of GCs (Kosco et al. (1992) J. Immunol. 148, 2331-2339; Tew et al. (1990) Immunol. Rev. 117, 185-211) and are believed to be involved in Ig class switching, production of B memory cells, selection of somatically mutated B cells with high affinity receptors, affinity maturation, induction of secondary antibody responses, and regulation of serum IgG with high affinity antibodies (Tew et al. (1990) Immunol. Rev. 117, 185-211; Berek & Ziegner (1993) Immunol. Today 14, 400-404; MacLennan & Gray (1986) Immunol. Rev. 91, 61-85; Kraal et al. (1982) Nature 298, 377-379; Liu et al. (1996) Immunity 4, 241-250; Tsiagbe et al. (1992) Immunol. Rev. 126, 113-141). Many researchers have worked with FDCs in culture in 2D with the general idea of mimicking an in vivo GC. An appreciation of the accessory functions of FDCs and regulation of these functions is important to an understanding of fully functional and mature antibody responses.
FDC development is B cell-dependent; FDCs are not detectable in, for example, SCID mice, mice treated with anti-mu (to remove B cells), or mice lacking the mu chain (where B cells do not develop) (MacLennan & Gray (1986) Immunol. Rev. 91, 61-85; Kapasi et al. (1993) J. Immunol. 150, 2648-2658). In T cell-deficient mice (e.g., nude mice), FDCs do develop, although the development is retarded and the FDCs do not appear to express many FDC markers (Tew et al. (1979) Aust. J. Exp. Biol. Med. Sci. 57, 401-414).
Reconstitution of FDCs in SCID mice occurs best when both B cells and T cells are adoptively transplanted, suggesting that T cells are also involved in FDC development (Kapasi et al. (1993) J. Immunol. 150, 2648-2658). Disruption of LT/TNF or the cognate receptors disrupts lymph node organogenesis and interferes with the development of FDC networks (De Togni et al. (1994) Science 264, 703-707; Rennert et al. (1996) J. Exp. Med. 184, 1999-2006; Chaplin & Fu (1998) Curr. Opin. Immunol. 10, 289-297; Endres et al. (1999) J. Exp. Med. 189, 159-168; Ansel et al. (2000) Nature 406, 309-314). As summarized by Debard et al. (1999), it is known that a lack of LTα, LTβ, TNFαR1, and LTIβR interferes with the development of FDC networks (Semin. Immunol. 11, 183-191). B cells are an important source of LTα/β heterotrimers, consistent with data indicating that FDC development is B cell-dependent (Endres et al. (1999) J. Exp. Med. 189, 159-168; Ansel et al. (2000) Nature 406, 309-314; Fu et al. (1998) J. Exp. Med. 187, 1009-1018).
The functional element of a mammalian lymph node is the follicle, which develops a GC when stimulated by an antigen. The GC is an active area in a lymph node, where important interactions occur in the development of an effective humoral immune response. Upon antigen stimulation, follicles are replicated and an active human lymph node may have dozens of active follicles, with functioning GCs. Interactions between B cells, T cells, and FDCs take place in GCs. Various studies of GCs in vivo indicate that the following events occur there:                immunoglobulin (Ig) class switching,        rapid B cell proliferation (GC dark zone),        production of B memory cells,        accumulation of select populations of antigen specific T cells and B cells,        hypermutation,        selection of somatically mutated B cells with high affinity receptors,        apoptosis of low affinity B cells,        affinity maturation,        induction of secondary antibody responses, and        regulation of serum immunoglobulin G (IgG) with high affinity antibodies.        
Similarly, data from in vitro GC models indicate that FDCs are involved in:                stimulating B cell proliferation with mitogens and it can also be demonstrated with antigen (Ag),        promoting production of antibodies including recall antibody responses,        producing chemokines that attract B cells and certain populations of T cells, and        blocking apoptosis of B cells.        
While T cells are necessary for B cell responses to T cell-dependent antigens, they are not sufficient for the development of fully functional and mature antibody responses that are required with most vaccines. FDCs provide important assistance needed for the B cells to achieve their full potential (Tew et al. (2001) Trends Immunol. 22, 361-367).
Humoral responses in vaccine assessment can be examined using an artificial immune system (AIS). Accessory functions of follicular dendritic cells and regulation of these functions are important to an understanding of fully functional and mature antibody responses.
Important molecules have been characterized by blocking ligands and receptors on FDCs or B cells. FDCs trap antigen-antibody complexes and provide intact antigen for interaction with B cell receptors (BCRs) on GC B cells; this antigen-BCR interaction provides a positive signal for B cell activation and differentiation. Engagement of CD21 in the B cell co-receptor complex by complement derived FDC-CD21L delivers an important co-signal. Coligation of BCR and CD21 facilitates association of the two receptors and the cytoplasmic tail of CD19 is phosphorylated by a tyrosine kinase associated with the B cell receptor complex (Carter et al. (1997) J. Immunol. 158, 3062-3069). This co-signal dramatically augments stimulation delivered by engagement of BCR by antigen and blockade of FDC-CD21L reduces the immune responses ˜10- to ˜1.000-fold.
USPTO Backgrounds
USPTO Backgrounds