This R21 application is submitted in response to PAS-02-160 (Application of Exploratory/Developmental Technologies to NIAID-Funded Research) on the basis of our RO1 grant # AI 49660-01A1. A central problem for the study of human B-cell tolerance is the lack of suitable experimental systems. We have approached this critical problem by establishing that a subset of autoreactive human B-cells (VH4.34 B-cells) is strictly censored in healthy subjects from participating in productive germinal center (GC) reactions. In addition, we have shown that the regulatory mechanisms acting upon VH4.34 B-cells in healthy subjects are faulty in SLE patients, where VH4.34 B-cells are frequently found in the germinal centers as well as in the IgG memory and PC compartments and contribute substantially to the pathogenic autoantibody repertoire. Another major problem in this field has to do with the complex nature of the regulatory processes responsible for the maintenance of B-cell tolerance. Indeed, the fate of autoreactive B-cells depends on the balance of multiple signaling (both positive and negative), survival and migration programs with the ability to mutually regulate one another through modulatory and feedback circuits. While targeted biochemical, cellular and genetic approaches are useful to reveal the role of specific factors and pathways, we submit that a functional genomics approach can provide unique insights into the role and interaction of known players and may also identify previously unsuspected elements. We have recently been funded through the RO1 mechanism to study different aspects of human B-cell tolerance using VH4.34 B-cells as an experimental system. In this R21 application, we propose to extend these studies by using DNA microarray analysis to compare the gene expression profiles of naive autoreactive VH4.34 B-cells in normal individuals with those of control naive B-cells. In addition, we shall also study the gene expression profiles of SLE VH4.34 B-cells before and during their transition to germinal center B-cells. Our experimental approach has the potential to identify genes generally associated with SLE as well as genes specifically involved in the maintenance of B-cell tolerance. In addition, although the questions asked are hypothesis-driven, the approach proposed is highly exploratory and, as such, is responsive to this RFA.
| Jenks, Scott A; Palmer, Elise M; Marin, Elides Y et al. (2013) 9G4+ autoantibodies are an important source of apoptotic cell reactivity associated with high levels of disease activity in systemic lupus erythematosus. Arthritis Rheum 65:3165-75 |
