The phenotype and fate of the antibody‐forming cells of the splenic foci

Abstract

The first product of the humoral response to antigen is low-affinity antibody, produced by extrafollicular foci of antibody-forming cells (AFC) in organs such as spleen and lymph node. These cells proliferate rapidly but then undergo an equally rapid decline, so that they are present in only small numbers 14 days after immunization. We have used 6-parameter flow cytometry to isolate and examine the characteristics of (4-hydroxy-5-nitrophenyl)acetyl-specific AFC, looking in particular for those markers that might differentiate them from cells of the intrafollicular (germinal center) arm of the T-dependent immune response. At day 7 of the primary response, most AFC were found to express surprisingly low levels of B220, high levels of syndecan, and retain significant levels of surface IgG1. We then used enzyme-linked immunospot assays to demonstrate that the rapid decline of these cells was not likely to be due to migration to organs such as the bone marrow. Their decline could, however, be explained by apoptosis in situ, which was demonstrated immunohistologically by nick-end labeling.

Correlation of Tissue Distribution, Developmental Phenotype, and Intestinal Homing Receptor Expression of Antigen-Specific B Cells During the Murine Anti-Rotavirus Immune Response

“…Detailed FACS surface phenotype analysis and correlation with ASC function of Ag-specific murine B cells has been conducted with hapten adjuvant models using a FACS strategy similar to ours (18,20). In these studies Ag-specific B cells were identified as being IgM Ϫ IgG1 ϩ cells and not IgD Ϫ cells as we found.…”

“…Subsequently, RV-specific GC B cells were detected (Fig. 3, A-C, upper panels); these cells were shown to be IgD Ϫ and express high levels of B220 (18,20). ASC derived from GC are IgD Ϫ and FIGURE 2.…”

“…3, which shows examples of the three types of RV-specific B cells identified in this study. Using a similar FACS strategy, the first hapten-specific B cells detected during a primary immune response are extrafollicular cells and were found to be large IgD Ϫ cells with low levels of B220 expression, secreting class-switched, nonsomatically hypermutated Ab, and to arise before the formation of germinal centers (GC) (18,20).…”

“…4A, upper panel). As mentioned above, this phenotype has been used to identify the first Ag-specific ASC (class-switched, non-GC B cells) appearing following immunization (18,20). The frequency of these cells peaked in both PP and MLN on day 7 (see Fig.…”

“…The overwhelming majority (Ͼ95%) of Ag-specific Syndecan-1 ϩ B cells have been shown to be ASC (defined by Ag-specific ELISPOT assays), and Syndecan-1 is thus a reliable and selective marker for ASC (18,20). To determine whether RV-specific B cells express Syndecan-1, we substituted an anti-Syndecan-1 Ab for the anti-␣ 4 ␤ 7 Ab used in the experiments illustrated in Fig.…”