Tight regulation of transcription factors, such as PU.1, is crucial for generation of all hematopoietic lineages. We previously reported that mice with a deletion of an upstream regulatory element (URE) of the gene encoding PU.1 (Sfpi1) developed acute myeloid leukemia. Here we show that the URE has an essential role in orchestrating the dynamic PU.1 expression pattern required for lymphoid development and tumor suppression. URE deletion ablated B2 cells but stimulated growth of B1 cells in mice. The URE was a PU.1 enhancer in B cells but a repressor in T cell precursors. TCF transcription factors coordinated this repressor function and linked PU.1 to Wnt signaling. Failure of appropriate PU.1 repression in T cell progenitors with URE deletion disrupted differentiation and induced thymic transformation. Genome-wide DNA methylation assessment showed that epigenetic silencing of selective tumor suppressor genes completed PU.1-initiated transformation of lymphoid progenitors with URE deletion. These results elucidate how a single transcription factor, PU.1, through the cell context-specific activity of a key cis-regulatory element, affects the development of multiple cell lineages and can induce cancer.

The TCR signals are essential for T cell activation and proliferation, primarily through the induction of cytokine and cytokine receptors. Several transcription factor families, including NF-kappaB/Rel, have been implicated in the regulation of cytokine gene expression in T cells in response to antigen, cytokine and mitogenic stimulation. In this study, we show that the mice with a null mutation in the lymphoid-specific c-Rel gene have normal development of lymphoid tissues and T cell compartment. However, T cells derived from the c-Rel knockout mice have several functional abnormalities. The c-Rel-deficient T lymphocytes fail to respond to activation and proliferation signals mediated by the TCR and mitogens in vitro. This is attributed to an impaired production of cytokines IL-2, IL-3 and granulocyte macrophage colony stimulating factor. In addition, the induction of IL-2R alpha chain is impaired in the c-Rel(-/-) T cells. The poor expression of cytokines and IL-2R alpha chain correlates with a reduced nuclear translocation of NF-kappaB components in c-Rel(-/-) T cells. Since activation is prerequisite for differentiation into effector cells, c-Rel(-/-) T cells failed to differentiate into cytotoxic T cells or Th cells without rescuing cytokines. However, upon supplement with exogenous IL-2, the c-Rel(-/-) cytotoxic T lymphocytes are able to execute cytotoxicity and the c-Rel(-/-) Th cells are capable of providing help to normal B cells. These data suggest that c-Rel is important for inducible cytokine and cytokine receptor expression, and a key regulator of early activation and proliferation in T cells.

B1 B cells are the major source of natural antibody that is essential for innate immunity. The B1 repertoire is skewed toward production of phosphatidylcholine (PtC)-binding V H 11 and V H 12 immunoglobulin that plays a key role in immune defense against bacterial infection. Programmed death-ligand 2 (PD-L2) is a ligand for the immunosuppressive receptor programmed death-1 (PD-1). It has been reported that expression of PD-L2 is restricted to dendritic cells and macrophages in mice. Here we show that 50-70% of resting peritoneal B1 cells express PD-L2, which is not present or inducible on conventional B2 B cells or PD-L2 -B1 cells. Although PD-L2 + and PD-L2 -B1 cells are similar in proliferative responses and spontaneous immunoglobulin secretion, PD-L2 + B1 cells are highly enriched for expression of V H 11 and V H 12 genes and encompass the bulk of PtC-binding B1 cells. These findings extend the range of known PD-L2 expression to B cells and show that B1 cells identified by this marker express a specific repertoire associated with anti-bacterial immunity. IntroductionB1 B cells comprise a unique subset of B cells distinguished phenotypically, ontogenetically, and functionally from conventional (B2) B cells [1][2][3]. B1 B cells are the major source of natural immunoglobulin in normal unimmunized mice, providing a vital front line defense against bacterial and viral infection [4][5][6][7][8]. This function is associated with a skewed repertoire containing autoreactive specificities, one manifestation of which is expression of phosphatidylcholine (PtC)-binding V H 11 and V H 12 by a substantial portion of peritoneal B1 cells in unimmunized mice [9][10][11]. PtC is the polar headgroup of common membrane phospholipids. Reconstitution of anti-PtC IgM in sIgM-deficient mice substantially reduced the bacterial load in a cecal ligation and puncture model [5]. Thus, V H 11/V H 12-expressing B1 cells play an important role in immunity; considering that PtC-binding B1 cells recognize protease-treated autologous erythrocytes, they may play a role in hemolytic autoimmune dyscrasias as well [10,12].B cells, like T cells, express programmed death-1 (PD-1), engagement of which negatively regulates B cell activity in vitro [13]. Surface levels of PD-1 are upregulated by BCR stimulation and down-regulated by Results and discussionPeritoneal B1 cells uniquely express PD-L2To evaluate PD-L1 and PD-L2 expression, cells from normal peritoneal washout fluids and spleens were stained with fluorescent antibodies to identify B1 cells, B2 cells and T cells and were counterstained to detect PD-L1 and PD-L2. Like B2 cells and T cells, B1 cells expressed a basal level of PD-L1, although to a much greater extent (Fig. 1A). Surprisingly, we found that a significant portion of peritoneal B1 cells (50-70%) expressed PD-L2, whereas B2 cells and T cells failed to do so (Fig. 1A). A smaller and less distinct population of splenic B1 cells also expressed PD-L2. To exclude the possibility of staining artifacts, we sorted PD-L2 + and PD-L2 -periton...