In brief, C57BL/6 mice received 200 l of commercial olive oil via oral gavage 60 minutes prior to lymph collection to allow for easier visualization of the lymphatic ducts

In brief, C57BL/6 mice received 200 l of commercial olive oil via oral gavage 60 minutes prior to lymph collection to allow for easier visualization of the lymphatic ducts. After vaccination, humoral immune responses begin by B cells binding with their receptors to cognate antigen, followed by the formation of germinal centers (GCs) where these cells undergo proliferation and affinity maturation, leading to the production of high-affinity antibodies against the target antigen (1C3). Factors determining the makeup of the eventual affinity-matured polyclonal antibody response remain incompletely understood, although the precursor frequency of antigen-specific B cells, the affinity of precursors for the antigen, antigen complexity, follicular helper T cell-derived signals, and antibody feedback all contribute (4C8). Rabbit polyclonal to BIK.The protein encoded by this gene is known to interact with cellular and viral survival-promoting proteins, such as BCL2 and the Epstein-Barr virus in order to enhance programed cell death. In addition, the duration Chitinase-IN-2 of antigen exposure and the amount of antigen available to B cells plays an important role (9, 10). We hypothesized that this structural integrity of the antigen in vivo is an additional important factor. To elicit protective responses, antigens need to present neutralizing epitopes that are faithful structural mimics of the target pathogen, which are often complex three-dimensional surfaces (11). Disruption of these epitopes could not only limit the activation Chitinase-IN-2 of B cells with the capacity to produce neutralizing antibodies but might also produce distracting de novo epitopes irrelevant for protective immunity. It has been reported that model protein antigens can be rapidly proteolyzed as they reach the subcapsular sinus (SCS) of lymph nodes (LNs), and this antigen cleavage was linked to protease activity in serum and interstitial fluid (12). Such pathways of antigen breakdown might at least partially explain the substantial proportion of B cells that enter GC reactions Chitinase-IN-2 but do not detectably bind to the immunizing antigen (7, 9). By contrast, several lines of evidence suggest that antigen trapped on dendrites of follicular dendritic cells (FDCs) may remain intact over extended time periods. Early studies showed that FDC-bound antigen recovered from LNs after 12 weeks could be recognized by epitope-sensitive monoclonal antibodies (mAbs) and were eluted in size exclusion chromatography in a manner suggesting gross antigen integrity (13). HIV virions deposited on FDCs in mice can be extracted from LNs and functional viral particles recovered over several months, although the quantitative proportion of particles that are infective is not clear (14). FDCs have also been shown to cyclically internalize and recycle trapped antigen, which may protect it from extracellular degradation (15). These data collectively suggest that the follicles and the FDC networks in particular may be sites within LNs where antigens are protected from degradation, whereas regions such as the sinuses may be areas of high proteolytic activity. To our knowledge, however, the nature of protease activity in lymphoid organs has not been studied, and how antigen proteolysis affects the immune response to vaccines is poorly understood. To shed light on the fate of antigens during the Chitinase-IN-2 primary immune response, we developed a FRET-based approach to track the integrity of antigens after subunit vaccine immunization, and analyzed the spatial pattern of protease expression and activity in LNs. Unexpectedly, we found a pronounced spatial variation in protease activity, with high levels of antigen breakdown and protease expression in extrafollicular regions of mouse and human lymphoid tissues, but low levels of protease activity and high retention of antigen integrity over time within the FDC network of B cell follicles. Prompted by these findings, we evaluated the impact of antigen localization on the specificity of GC B cell responses, and found evidence that FDC-targeted protein immunizations achieve substantially greater proportions of antigen-specific B cell responses targeting conformationally intact epitopes compared with traditional bolus vaccination. RESULTS Monitoring antigen integrity using FRET analysis To investigate vaccine antigen stability after immunization, we labeled immunogens with paired small-molecule dyes capable of undergoing fluorescence resonance energy transfer (FRET) to detect gross disruptions of antigen structure in tissues. Chitinase-IN-2 We hypothesized that antigen proteolysis would lead to the separation of FRET donor and acceptor dyes, lowering FRET signals in proportion to the degree of antigen degradation, a process that can be tracked by microscopy or flow cytometry (Fig. 1A). For microscopy-based analysis, we used the acceptor photobleaching method (16), in which the emission of a donor dye (Cy3).