The ability to respond to the T cell- independent type 2 (TI-2) antigen TNP-Ficoll arises later in ontogeny than the response to the TI-1 antigen TNP- Brucella abortus (TNP-BA). TI-2 responses are correlated with B cells which express low levels of surface IgM (sIgM), the minor lymphocyte-stimulating antigen (Mls), and the """"""""maturational"""""""" antigens Lyb 3 and Lyb 5. The X-linked immunodeficient (xid) CBA/N mouse has B cells which express the antigens (low sIgM, Mls-, Lyb 3-, Lyb 5-) of neonatal B cells. These xid B cells are unable to make in vitro plaque forming responses to sheep erythrocytes (SRBC) and TNP-Ficoll (TI-2), but do make responses to TNP-BA (TI-1). In addition, xid B cells are not receptive to several T cell-derived lymphokines, including T cell replacing factor (TRF) and B cell growth factor I and II (BCGF-I and BCGF-II). Taken together, this evidence indicates the xid defect causes B cells to arrest at an early stage in maturation. We have analyzed the B cells in Peyer's patches (PP) of xid mice and found a population of low sIgM cells which are capable of in vitro PFC responses to both SRBC and TNP-Ficoll. These B cells were shown to be fully mature in experiments which demonstrated the expression of the Mls and Lyb 5 maturational antigens. Our recent results indicate that cocultures of PP T cells and xid B cells are capable of making PFC responses against SRBC and TNP-Ficoll. This mature-type response requires the function of an Lyt 1+ 2- PP T cell, or the 24 hr Con A-induced supernatent (Con A sup) from PP cells. We propose to further investigate the cellular and molecular requirements for the support of mature-type responses by xid B cells. Our approach will be divided into three major categories: 1) Further characterization of the T cell subset(s) required to support SRBC and TNP-Ficoll responses by xid B cells. These studies will include evaluation of Lyt 1+ 2+ cells as precursors to the effector T cells and determination of any involvement by T contrasuppressor cells, 2) Purification of the active factors from PP Con A sup will be performed by sequential column chromatography and chromatofocusing steps. One and 2-dimensional PAGE will be used to assess purity, and 3) The functional phenotypic changes induced in xid B cells by the PP factor will be evaluated. These include changes in the expression of sIgM, sIgD, Lyb 3, Lyb 5 and receptivity to TRF, BCGF-I and BCGF-II.
