Development of Ly-1+ B cells in immunodeficient CBA/N mice.

Spleen cells from CBA/N mice developing a systemic autoimmune disease after daily injection of CsA during an autologous bone marrow reconstitution were transferred into unmanipulated syngeneic recipients. Adoptive transfer allowed the development of Ly-1+ B cells, which shared Mac-1 differentiation antigen expression with the myelomonocytic lineage. Interestingly, expansion of formerly absent Ly- 1+ B cells was paralleled by a severe reduction in common, Ly-1-, B cell development in the recipient. We conclude that precursors for Ly- 1+ B lineage do exist in CBA/N mice.

continuously from the date of irradiation . Control mice were treated with CsA alone (Csmice) or irradiated and saline injected (X-mice), as described before (7) . Here, spleens from irradiated and CsA-treated mice (thereafter referred to as CsX-CBA/N), as well as controls, were used as a source of donor cells 4-8 wk after irradiation . Recipients were unmanipulated adult CBA/N mice who were injected intravenously with 5 X 106 total spleen cells from each group of donor mice .
Cell Staining and Flow-cytometry Analysis. Bone marrow, lymph nodes, and spleen cells from transferred mice were examined for surface expression of differentiation antigens after immunofluorescence staining with the indicated antibodies, as described elsewhere (7) . All the procedure was performed at 4°C in PBS with 0 .1% sodium azide . The frequencies of Ly-1 + B cells were obtained from two-color stainings of cells with fluoresceinated anti-IgM and biotinated anti-Ly-1 antibodies plus PE . Isotype-matched antibodies of irrelevant specificity and either the fluoresceinated second-step reagent or PE were used to ensure specificity and standardize the background settings (3,7) . A similar procedure was used to define Mac-1 + B cells . Immunofluorescence of 5 X 10 4 live cells was analyzed with an Epics-C flow cytometer, and the frequencies were estimated using the consort computer statistical package V2 .2A (Coulter Electronics Inc ., Hialeah, FL) .

Results
Previous studies (8)   Bone marrow precursors for the lymphocytes and myelomonocytes can be discriminated by means of their light scatter properties, and thus can be conveniently gated in forward light scatter (FLS) X 90" light scatter (90' LS), twoparameter, displays for an independent analysis (7). As shown in Fig. 3A, the studies revealed a marked depletion of cells of the lymphoid lineage in the bone marrow of transferred mice, as opposed to control groups of sham-transferred mice, revealing a striking similarity to the findings in donor mice . In agreement, pre-B cells defined as s1g B220 + precursors were again virtually absent (reference 7, data not shown) . In fact, the multiparameter analyses of the residual population demonstrate that the few B cells present mostly belong to the Ly-1' lineage. Furthermore, B cells, rare (<3%) in lymph nodes from these mice, are again Ly-1 + B cells (Fig . 3B).
The numbers of Mac-I + cells in the spleen of the recipient mice were markedly augmented when compared with the control groups (Fig . 1) . Interestingly, spleen B cells from the recipient, besides being Ly-1' s1g+ , also coexpress the Mac-1 antigen (Fig . 3 C) . Although Mac-1 (CD 11 b) is currently defined as a differentiation antigen specifically expressed in myelomonocytic lineage (9), its presence in some Ly-1 + pre-B and B cell lines has been reported (1).

Discussion
The most straightforward interpretation of our results would be that Ly-1 + B cells, abnormally present in CsX-CBA/N mice, selectively expand in the recipient 80 7 CBA/N microenvironment . This would imply that the lack of Ly-1 + B cells in immunodeficient CBA/N mice is not the consequence of a primary defect in their precursors . Rather, our findings suggest a failure, in xid strains, of the regulatory mechanisms allowing the initial expansion of Ly-1 + B cells in neonates, and their maintenance in adult peritoneum and spleen (1). Alternatively, the existence of a suppressive mechanism, released by Cs treatment, specifically blocking Ly-1 + B cell development in xid mice may be proposed . However, the mechanism would be expected to operate in the unmanipulated recipient CBA/N. Here, only 5 X 106 spleen cells were inoculated in a syngeneic system, making this explanation unlikely . Interestingly, the existence of hypercycling, T cell-mediated mechanisms in the expansion of idiotypically defined B cell subpopulations has been proposed (10) . We have shown that the influence of B cells on T cell repertoires is exerted mostly in the neonatal period (11), when Ly-1 + B cells are a major subpopulation (1). We therefore suggest that distinctive T cells expanded in CsX-CBA/N mice (7) would select Ly-l + B cell precursors from the differentiating pool, which in turn would then be engaged in mutual selection . In agreement with this, we have recently observed a bias of primordial T cell repertoires for interactions with B cells in the Ly-1 + lineage (Marcos, M. A. R ., A . de la Hera, P. Pereira, M. L. Toribio, A. Coutinho, and C. Martinez-A, manuscript submitted for publication) which may contribute to the preferential expansion of this subset .
Primary immunodeficiency defects in CBA/N mice have formerly been placed at the B cell level . Novel formulations of B cell development pathways should take into consideration present evidence for interplay among T and B lymphocyte subpopulations within the two lineages of differentiation (10)(11)(12). Besides, the differentiation of common B cell precursors in CBA/N mice shows a strong T cell dependence (13)(14)(15). Thus, in addition to the defects of the major subpopulation I spontaneously caused by xid genes (3), the alterations in intrathymic development present in CsX-CBA/N and transferred mice (7) (1). It suggests that direct interactions among lymphocytes in distinct lineages might play an essential role in the physiology and pathology of the immune system .

Summary
Spleen cells from CBA/N mice developing a systemic autoimmune disease after daily injection of CsA during an autologous bone marrow reconstitution were transferred into unmanipulated syngeneic recipients . Adoptive transfer allowed the development of Ly-l + B cells, which shared Mac-I differentiation antigen expression with the myelomonocytic lineage . Interestingly, expansion of formerly absent Ly-1 + B cells was paralleled by a severe reduction in common, Ly-1 -, B cell development in the recipient . We conclude that precursors for Ly-1 + B lineage do exist in CBA/N mice.