The observation that has been made is that xid B-cells are short lived and that bcl-2 is low in xid B-cells. The goals of this application are to understand B-cell survival in xid cells, to correlate survival with bcl-2 and Bax expression and to determine how overexpression of bcl-2 or targeted disruption of p53 alter survival. The first set of experiments is to analyze susceptibility to apoptosis and bcl-2 expression in vitro. Bcl-2 expression will be quantitated in normal and xid cells over time in culture. Inhibitors of apoptosis will be added, PMA, PMA and ionophore, CD40L, and IL-4. These have differing effects on normal and xid B-cells, with PMA and ionophor blocking apoptosis in xid cells while the other treatments lead to only partial protection. Bcl-2 and bcl-XL will be assayed to see if expression of these proteins correlates with protection. The phosphorylation state of bcl-2 will be determined. PKA and PKC inhibitors as well as phosphatase inhibitors will be added to cells and survival and bcl-2 status measured. This will be done in irradiated normal and xid B-cells, as these cell populations both undergo rapid apoptosis but differ with respect to bcl-2 expression. These inhibitors will also be added to cultures of xid B-cells expressing a bcl-2 transgene. Finally, xid cells will be transfected with altered bcl-2 constructs lacking phosphorylation sites and survival of cells will be measured. In this first set of in vitro experiments Bax expression will also be quantitated. Dr. Woodland has identified a 16-18 Kd protein recognized by anti-Bax antibodies, and the expression of this protein will also be evaluated. Pulse chase experiments will determine if the 16-18 Kd species derives from Bax and if another short 13 Kd form is also a degradation product. All of the experiments described above will be repeated to assess Bax expression. In addition, Bax expression will be assessed in xid cells transfected with wild type or mutant bcl-2. Bax expression will also be assessed in p53 knock-out mice.
The second aim i s to study cell survival in vivo. The hypothesis is that interaction with the microenvironment upregulates bcl-2 and permits B-cells to be long lived. Lack of bcl-2 upregulation leads to short lived cells. This hypothesis will be tested in a number of adoptive transfer experiments, using allotype congenic B-cells, normal, xid, xid bcl-2, and p53 knock-out. In addition, xid B-cells do not display an appropriate fetal repertoire. It will be determined if xid/bcl-2 mice have a stabilization of the normal fetal to adult progression of VH repertoire. VH repertoire will be examined in p53 knock-out mice also. These studies are in collaboration with Judy Teale. The last set of experiments is to correct the xid mutation in vitro and in vivo. Xid B-cells will be transfected with TSK, and ultimately with mutant TSK. TSK is the T cell analog of BTK and is used because of the concern that the xid BTK may function as a dominant negative. Transfected cells will be assayed for survival, bcl-2 and bax expression in all the situations described in Aim 1. Finally a TSK transgenic mouse will be made with TSK under an Ig promoter.
| Cancro, M P; Sah, A P; Levy, S L et al. (2001) xid mice reveal the interplay of homeostasis and Bruton's tyrosine kinase-mediated selection at multiple stages of B cell development. Int Immunol 13:1501-14 |
| Schmidt, M R; Piekos, B; Cabatingan, M S et al. (2000) Expression of a human coxsackie/adenovirus receptor transgene permits adenovirus infection of primary lymphocytes. J Immunol 165:4112-9 |
| Woodland, R T; Schmidt, M R; Korsmeyer, S J et al. (1996) Regulation of B cell survival in xid mice by the proto-oncogene bcl-2. J Immunol 156:2143-54 |
| Schmidt, M R; Gravel, K A; Woodland, R T (1995) Progression of a vesicular stomatitis virus infection in primary lymphocytes is restricted at multiple levels during B cell activation. J Immunol 155:2533-44 |
| Woodland, R T; Schmidt, M R; Riggs, J E et al. (1995) Radiation-induced apoptosis is differentially regulated in primary B cells from normal mice and mice with the CBA/N X-linked immunodeficiency. J Immunol 155:3453-63 |
