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The Ets-1 proto-oncogene is a prototype member of Ets family of transcription factors. It is preferentially expressed in lymphoid cells, where it is essential for the maintenance of the normal pool of resting T and B cells. We have investigated the protein expression of the Ets-1 transcription factor during the activation and apoptosis of T and B cells by flow cytometry and confocal microscopy. Cells of the thymus, spleen and bursa expressed high levels of Ets-1 protein, while resting peripheral blood mononuclear cells had lower Ets-1 expression. Activation and proliferation of T cells induced the upregulation of Ets-1. alphabeta-T cells were found to upregulate Ets-1 expression more than gammadelta-T cells. Increased Ets-1 protein expression was located predominantly in the perinuclear area. In contrast, during apoptosis, Ets-1 expression was downregulated. Collectively, our results indicate that Ets-1 expression can be accurately determined by flow cytometry and confocal microscopy. Ets-1 expression level and distribution are differentially controlled in resting, activated and apoptotic lymphocytes.

During B-cell development in the avian bursa of Fabricius most of the developing B cells die by apoptosis and only a minority survive to emigrate into the periphery. Recently, it has been shown that when developing bursal cells become mature and ready to migrate they start to express chL12 antigen. The expression of this cell-surface molecule was found to be associated with the survival of the bursal cells both after in vitro culture and after in vivo cyclophosphamide (CY) treatment. The frequency of early apoptotic cells in freshly isolated bursal cells was found to be high. The high susceptibility of these cells to apoptosis is in line with the finding of low bcl-2 mRNA expression. We conclude that expression of avian chL12 antigen is associated with the survival of bursal cells.

In the chicken, the B cells develop in a specialized organ, the bursa of Fabricius. Earlier it was shown that neonatal bursal cells treated with polyclonal anti-chicken immunoglobulin antibodies are not able to recolonize the bursa when transferred into cyclophosphamide-treated chicks. In this study, 4-day-old bursal cells were treated with different polyclonal and monoclonal anti-immunoglobulin antibodies and transferred into 4-day-old cyclophosphamide-treated chickens. Two monoclonal anti-chicken IgM antibodies, CVI-59.7 and 21-2B2, recognizing distinct epitopes of the mu heavy chain, were inhibitory. Incubation of cells with 21-2B2 antibody caused about 90% inhibition of bursal recolonization. After incubation with CVI-59.7 antibody the inhibition was 50%. The high inhibition by 21-2B2 antibody was also seen when F(ab')2 fragments of the antibody were used. These results suggest that the entry of the cells needed for bursal recolonization is inhibited almost totally by 21-2B2 antibody, or that this antibody blocks further proliferation of the cells in bursal follicles. In conclusion, we have shown that a mu heavy chain epitope is intimately involved in the recolonization of bursal follicles, and distinct epitopes of the mu heavy chain are not equally important in this process.

Allogeneic yolk sac-embryo chimaeras were constructed by association of B15B15 yolk sac and B2B2 embryo on day 2 of incubation. Five days later yolk sac cells from the chimaeras were injected intravenously into 14-day-old irradiated embryos, using recipients of B2B2 and B15B15 genotypes. One week after hatching, cells in the bursa of Fabricius and peripheral blood erythrocytes were studied for Ia-like antigens and B alloantigens, respectively, to determine whether they were derived from the embryo or yolk sac part of the chimaera. The results obtained demonstrate that prebursal and erythropoietic stem cells migrate from the early embryo to the yolk sac during the 2nd to the 7th day of incubation. They also exclude the de novo generation of prebursal stem cells in the yolk sac.

In-vitro-cultured bursal epithelium (BE) and BE-conditioned medium (BECM) induce B-L antigen on chicken intraembryonic cells. Cells prepared from 9-day-old intraembryonic mesoderm were fractionated in accordance with cell size by linear albumin gradient sedimentation at 1 g. Two cell types could be distinguished on which expression of the B-L antigen was altered after a 6-h incubation with the bursal epithelial component. One fraction contained small mononuclear cells with low sedimentation velocity (less than or equal to 3 mm/h) and low spontaneous proliferation activity. These cells responded strongly to BECM and showed a slight but not significant response to BE (index after incubation with BECM 3.8, with BE 1.7, as compared with RPMI medium control). The other fraction was composed of large mononuclear cells with sedimentation velocity greater than 9 mm/h and with high spontaneous proliferation. These cells showed a response of equal magnitude to both BE and BECM (index after incubation with BE 2.0, with BECM 2.3). These results suggest that the bursa of Fabricius has influence on two different cell types: a large, probably primitive undifferentiated cell, responding equally to bursal cellular contacts and BE culture supernatant, and a small mononuclear cell type, probably more mature, responding more clearly to the bursal humoral factor.

MHC class-I antigens on peripheral blood macrophages and bursa cells were analysed after adoptive bursa cell transfer to cyclophosphamide-treated immunodeficient chickens. We studied the expression of B-F locus-encoded antigens (B-F is homologous to mouse H-2K, D) on macrophages and B cells and found that macrophage B-F antigens are of host origin, whereas bursa cells express only donor-type B-F antigens. Both syngeneic and allogeneic bursa cells restored IgM antibody production to a T-cell-independent antigen, Brucella, but only syngeneic bursa cells could restore the IgG antibody response to a thymus-dependent antigen, sheep erythrocytes. The results indicate that B-cell maturation and macrophage-B-cell interaction are not MHC-restricted but that a restriction exists in T-cell-B-cell collaboration.

Maturation of bursal stem cells in an allogeneic environment was studied. The occurrence of surface IgM, surface IgG, and Ia-like antigens on B cells after differentiatoin within an allogeneic bursa was studied. Furthermore, the immediate capacity of B cells to form germinal centres after differentiation in an allogeneic bursa was studied by injecting them with histocompatible T cells into "test-tube" birds. The results indicate that the switch to surface-IgG-bearing cells occurs in an allogeneic bursa in a similar manner as in a syngeneic bursa. Studies of the occurrence of Ia-like antigens on bursa cells demonstrate that they retain their original Ia-like antigens, even during maturation in an allogeneic host. Third, the results demonstrate that after differentiation within an allogeneic environment bursa cells have acquired the ijmediate capacity to cooperate with histocompatible T cells as measured by germinal centre formation in "test-tube" birds.

We have studied the expression of immunoglobulin genes in the chicken B-cell precursors, and of a B-cell surface marker (Bu-1) on the bursal and peripheral B cells during normal ontogeny. Since there is no way of distinguishing the precursor cells from the more mature bursal lymphocytes on the basis of surface markers, we chose to study the total bursal lymphocyte population at ages when the numbers of the various precursor cells (bursal, early post-bursal, and post-bursal stem cells) in the bursa are estimated to be at their highest. Thereafter, comparisons with the more mature lymphocytes in the peripheral organs were made. As a result, levels of the lambda and mu transcripts and expression of Bu-1 antigen in the chicken B-cell precursors were found to be unchanged during the post-hatching period. In the light of these experiments, the later events of B-cell differentiation, i.e. the development from the bursal to post-bursal B lymphocytes, occurs without the lambda, mu, and Bu-1 gene loci involved. On the other hand, the higher level of lambda and mu expression in the splenic B lymphocytes indicates that the post-bursal stem cells mature into highly active plasma cells after seeding to the peripheral organs.