Marie H. Kosco

Localization of antibody-forming cells in draining lymphoid organs during long-term maintenance of the antibody response

Previous studies on the “spontaneous antibody response” have included in vitro steps and it is possible that the response is an in vitro artifact. The objective of the present study was to induce a spontaneous antibody response entirely in vivo and determine if the response is localized and if the magnitude of the response is related to the location of persisting antigen. Antigen was injected into the right hind footpads of mice, and lymph nodes on the right side were draining and lymph nodes on the left side were controls. Antibody‐forming cells (AFCs) were enumerated in both draining and nondraining nodes 2 weeks, 2 months, and 1 year after secondary immunization. Four days prior to determining AFC number, the mice were severely bled to stimulate AFC production. Thousands of AFCs were found in the draining lymph nodes and the numbers were dramatic in nodes closest to the injection site that retain the most antigen. In contrast, the vast majority of nondraining nodes lacked any AFCs. One year after immunization, the response was almost exclusively in the popliteal node, draining the foot where antigen was administered a year earlier. These results are consistent with previous data on the spontaneous response and support the hypothesis that antigen retained on FDCs is essential in the maintenance of serum antibody levels.

FDC-Iccosome Mediated Antigen Delivery to Germinal Center B Cells, Antigen Processing and Presentation to T Cells

Antigen trapped by follicular dendritic cells (FDC) is retained for long periods as intact molecules (1). An antigen must be internalized, partially degraded and reexpressed on the surface of an antigen presenting cell to be recognized by T cells (2). Consequently, it was difficult to explain how antigen retained by FDC could be presented to T cells, which are required for germinal center (GC) development (3) and can be demonstrated in GCs (4,5). Recently, antigen specific B cells were shown to process and present antigen in a highly efficient manner in vitro (6-8). Germinal center B cells have the requisite features of good antigen presenting cells, i.e., they are metabolically active, rich in la, and include cells specific for the FDC-trapped antigen. Thus, GC B cells could present antigen obtained from FDC to GC T cells.

Germinal center B cells and mixed leukocyte reactions

The present study was undertaken to determine if germinal center (GC) B cells are sufficiently activated to stimulate mixed leukocyte reactions (MLR). Percoll density fractionation and a panning technique with peanut agglutinin (PNA) were used to isolate GC B cells from the lymph nodes of immune mice. The GC B cells were treated with mitomycin C or irradiation and used to stimulate allogeneic or syngeneic splenic T cells in the MLR. Controls included high‐density (HD) B cells prepared from spleens of the same mice and HD B cells activated with lipopolysaccharide (LPS) and dextran sulfate. GC B cells bound high amounts of PNA (i.e., PNAhi). Similarly, the LPS‐dextran sulfate‐activated B cells were PNAhl. Treatment with neuraminidase rendered the PNA HD B cells PNAhl. GC B cells and the LPS‐dextran sulfate‐activated HD B cells stimulated a potent MLR, while the untreated HD B cells did not. However, following neuraminidase treatment, the resulting PNAhl HD B cell population was able to induce an MLR. The PNA marker appeared to be an indicator of stimulatory activity, but incubating the cells with PNA to bind the cell surface ligand did not interfere with the MLR. GC B cells were also capable of stimulating a syngeneic MLR in most experiments although this was not consistently obtained. It appears that germinal centers represent a unique in vivo microenvironment that provides the necessary signals for B cells to become highly effective antigen‐presenting cells.

A Subpopulation of Germinal Center B Cells Differentiate Directly into Antibody Forming Cells upon Secondary Immunization

In the present study we sought to test the hypothesis that GC B cells may be stimulated to differentiate into AFCs during the induction of the secondary antibody response. To test this we gave HRP immune mice a booster immunization and looked for AFCs in the GCs making anti-HRP specific antibody. Within 3 days cells in the GCs were making anti-HRP. Most of the cells were just starting to make immunoglobulin in the rough endoplasmic reticulum but some were nearly mature plasma cells like those in the medullary cords. Furthermore, we isolated GC B cells after a boost with OVA and tested their ability to differentiate directly into AFCs. Data obtained using an OVA specific RIA indicated that purified GC B cells would differentiate and produce specific antibody in the absence of added T cells or antigen. Examination of the cells after 5 days in culture indicated that many of them had differentiated into large plasma cells. These results obtained both in vivo and in vitro demonstrate that shortly after booster immunization GC B cells receive the appropriate antigen and T cell signals in vivo to differentiate and become AFCs.

Germinal Center B Cells Present Antigen Obtained in vivo to T Cells in vitro and Stimulate Mixed Lymphocyte Reactions

In the present study we sought to test the ability of GC B cells to activate T cells by antigen presentation and mixed lymphocyte reactions. The first set of experiments demonstrated that GC B cells obtained three or more weeks after immunization could induce IL 2 production by T cells only by adding antigen to the cell cultures. In the next (table; see text) series, it was demonstrated that GC B cells isolated as early as 1 day and for over a week following an antigenic challenge with OVA (i.e. where they obtained antigen from the FDC-derived iccosomes in vivo), were able to stimulate high levels of IL 2 production in the absence of exogenous OVA. This response was maximal on Day 5 which corresponded precisely with the keinetics of the ultrastructural studies which document the uptake of antigen by GC B cells in vivo (see Szakal et. al, this volume). Furthermore, it was shown that the FDC-derived antigen was remarkably immunogenic when compared with exogenous antigen. The data from the MLRs demonstrated that GC B cells, which are low density cells, are potent stimulators of T cells compared to the resting, low density B cells. In addition, GC B cells even stimulated a significant syngeneic response. These results indicate that GC B cells can present antigen and interact with T cells efficiently. This is believed to be important in the germinal center reaction and in the induction and maintenance of the immune responses.

Antigenic Phenotype of Rodent Follicular Dendritic Cells in Vitro and in Situ

Follicular dendritic cells (FDCs) were first described at the transmission electron microscopic level in the lymph node by Nossal et al. (1968) and in the spleen by Szaka1 and Hanna (1968). Until recently, the study of FDCs was limited to in situ investigations due to the lack of adequate isolation and enrichment techniques. In 1982, Schnizlein et al. developed a procedure in our laboratory which produced a single cell suspension specifically enriched for FDCs from lymph nodes of mice. This procedure provided an opportunity to establish diagnostic morphological features of intact FDCs in vitro, using Nomarski optics, scanning EM (SEM) and transmission EM (TEM). The SEM of isolated FDCs revealed cells with a “sunburst-like” appearance due to numerous long thin dendrites emanating from a spherical or ovoid cell body. Nomarski optics and TEM confirmed the presence of immune complexes on the processes of FDCs by using the histochemically detectable antigen (Ag), horseradish peroxidase (HRP), and revealed features consistent with those observed in lymphoid sections. The cytological evaluation of FDCs in vitro provided functionally supported morphological criteria by which these cells could be distinguished from leukocytes and other dendritic cells (Szakal, Gieringer, Kosco and Tew, 1984).