Using the rationale that the mechanisms responsible for the generation and maintenance of a SIgA response is critical to resurrecting mucosal homeostasis following HIV infection, this investigator plans to: 1) study the role human Peyer Patch and intestinal lamina propria microenviroments play in the development of memory IgA B cells (i.e. cells capable of giving rise to clones secreting exclusively IgA upon antigenic stimulation) and to identify signals responsible for the generation of memory B cells that specifically traffic to mucosal sites. To do this, the PI, will characterize PP B cell populations in regard to their ability to proliferate and mature to IgA secreting cells; and determine the effects of extravasation molecules and dendritic cells from the intestinal LP on the isotype potential and/or proliferative capacity of PP B cell populations. PP biopsies will be obtained from relatively healthy individuals and mIgM+IgD-, mIgA-, and mIgA+ cells extracted from GCs using immunologic gating with PNL binding , dendritic cells will be obtained and Dr Graham will provide LP cells. mIgM+IgD-ZRG-5-populations will be stimulated with Cdw32L cells, anti-CD40, and sham antigenic stimulation (anti-kappa and anti-gamma antibodies), IL-10 +/- TGF-B. The IgA secreted will be measured by ELISA. mIgA+ and-cells will cultured as just described to see if plasma or memory cells develop; secreted IgA will be measured in addition to CD38, CD20, cytoplasmic Ig, and morphology described. To confirm the memory phenotype, B7.1 and B7.2 expression using FACS will be measured. IgA- cells will also be cultured as above with or without anti-CD40 with IL-2 and IL-10. Ig isotype will be measured by ELISA and alphatranscripts will be measured using RT-PCR. mIgA+, mIgM+, mIgD- PP populations will be isolated, alpha4beta7 expression confirmed by FACS. The B populations will be stimulated with irradiated Cdw32L and anti-alpha4beta7 for varying lengths of time; appropriate control cells will be used. The cells will then be re-washed and stimulated under standard procedure. Total Ig secretion and isotypes will be measured by ELISA. Similar experiments will be performed using isolated dendritic cells. 2) a) identify signals responsible for the generation of mucosal intergrins on memory B cells in PP germinal centers (GCs). PP GC cells will be stimulated to memory phenotype using system above and alpha4beta7 expression sought. Additional cytokines will be added as necessary to produce expression. b) determine if the pattern of the integrins is intrinsic to GC cells from mucosal or peripheral lymphoid tissues. PP and peripheral LNs will be evaluated as above for alpha4beta7 and alpha4beta1 expression.
| Guilliano, M J; Foxx-Orenstein, A E; Lebman, D A (2001) The microenvironment of human Peyer's patches inhibits the increase in CD38 expression associated with the germinal center reaction. J Immunol 166:2179-85 |
