B6-Sp6 transgenic mice carry fully rearranged (BALB/c-derived, Igh-Ca allotype) mu heavy chain and kappa light chain transgenes, specific for trinitrophenyl, on a C57BL background (Igh-Cb allotype). FACS analyses show that the majority of B cells in peripheral lymphoid organs and bone marrow (BM) express transgenic IgM exclusively. A small proportion of the B cells, however, express endogenous IgM, usually concomitant with transgenic IgM. Three criteria establish that the endogenous IgM expressing B cells belong to the B-1 cell lineage. (i) Endogenous IgM expressing B cells in B6-Sp6 mice have the same localization pattern as B-1 cells from normal animals: they are enriched in the peritoneal cavity. (ii) The endogenous IgM+ B cells have the phenotype of B-1 cells: the endogenous IgM+ peritoneal B cells express Mac-1 (CD11b) and low levels of IgD, and most also express CD5 (Ly-1). (iii) B6-Sp6 BM poorly reconstitutes endogenous IgM+ B cells, just as adult BM from normal mice poorly reconstitutes B-1 cells. In contrast, B cells which only express the transgene are readily reconstituted by B6-Sp6 BM. The few endogenous IgM+ cells in the B6-Sp6 BM recipients are located in the peritoneal cavity and have the phenotype of B-1b cells (previously the Ly-1 B sister population), which are known to be reconstituted by adult BM. Two-color immunofluorescence staining of tissue sections from the gut and from isolated gut lamina propria cells shows the presence of many IgA containing cells, about one-third of which simultaneously express cytoplasmic (transgenic) IgM. The C-region of this IgA is produced by endogenous C alpha genes, because the transgene encodes only for C mu. Furthermore, the majority of gut IgA containing cells do not express the idiotype of the transgene, indicating that most of the gut IgA cells are encoded by endogenous VH genes and thus the result of an isotype switch from endogenous IgM expressing B cells. Since the endogenous IgM+ cells are B-1 cells (both B-1a and B-1b), the data strongly indicate that the intestinal IgA plasma cells also belong to the B-1 cell lineage.

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Identification of those cells within the thymic stroma which are responsible for tolerance induction remains controversial. Evidence derived from studies of bone marrow chimeras or thymus transplants attributed this function to cells of haematopoietic origin, usually class II positive medullary dendritic cells (DC). Recent data suggest, however, that a stromal element located in the thymus cortex might be involved in negative selection. To further explore this issue we used immunohistology and immunocytology with a combination of allotype and cell type specific monoclonal antibodies (MoAb) to study the turnover of thymic stromal cells of haematopoietic origin in different rat models of allogeneic and congenic bone marrow (BM) radiation chimeras. Use of CFU-GM cultured BM inoculum for congenic recipients allowed us to distinguish between direct homing of donor myeloid cells and the delayed migration of the donor stem cell progeny after the post-irradiation recovery of the recipient. Our data indicate a heterogeneity in the turnover rate of thymic mobile stromal cells. While DC and a subset of macrophages located in the cortex as well as in the medulla (ED1+), within 4 weeks were virtually all of donor type, cortical macrophages detected by ED2 MoAbs were still incompletely replaced after a period as long as 20 weeks. Slow turnover, location and variable class II expression may imply a role for thymic cortical macrophages in (self-) tolerance induction.

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