Recent data from clinical transplantations suggest a critical role for alloantibodies in acute and chronic rejection. A role has also been hypothesized for alloreactive B cells as antigen-presenting cells, stimulating alloreactive T cells via the indirect pathway. However, the diversity of the B cell repertoire makes it difficult to study the behavior of alloreactive B cells in vivo. We hypothesize that a mouse with a monoclonal population of MHC Class I-specific B cells allow in-depth studies into the role of alloreactive B cells in allograft rejection. In accordance with the stated intent of an R03 award of developing new reagents and model systems, the goal of this application is to generate an alloreactive BCR-knock-in (anti-BALB/c) mouse on a C57BL/6 background. We have identified an H-2Kd- and H-2Kk-specific HB-24 hybridoma, and have cloned and sequenced the genes encoding the light and heavy chains. BCR-transgenic (Tg) mice expressing both the light and heavy chain transgenes have been generated in C57BL/6 mice using standard transgenesis techniques. Alloreactive BCR-transgenic (Tg) B cells are enriched in HB-24 BCR-Tg C57BL/6 mice, and respond to immunization with Kd- or Kk-expressing cells by secreting antibodies of HB-24 specificity. We now propose to generate HB-24 BCR-knock-in mice. The specific experiments are organized into two Specific Aims.
Specific Aim I : Generate anti-H2-KdDk BCR-knock-in mice.
Specific Aim 2 : Perform phenotypic and functional characterizations of the B cells in the anti-H2-KdDk BCR-knock-in mice. The successful generation of alloreactive BCR-knock-in mice will facilitate the investigation of several central questions in transplant immunology, including the role of B cells in rejection and tolerance. Recent data from clinical transplantation suggest a critical role for allo-reactive B cells and antibodies in acute and chronic rejection. We propose to generate a mouse expressing a high frequency of alloreactive B cells to study the fate of these B cells after allograft transplantation. Such studies with this new mouse model may lead to the identification of new therapies for controlling alloreactive B cell responses and facilitating the acquisition of long-term allograft tolerance.
