T cell activation by antigen-presenting cells (APC) is regulated by positive and negative costimulatory molecules in the B7 family. Here we describe a novel addition in this family, designated as B7S1, which is uniquely anchored to the cell membrane via a GPI linkage. B7S1 is expressed on professional APC and widely distributed in nonlymphoid tissues. A soluble B7S1-Ig fusion protein binds to activated but not naive T cells. B7S1-Ig inhibits T cell activation and IL-2 production. A monoclonal antibody that blocks binding of B7S1 to its receptor enhances T cell proliferation in vitro and exacerbates experimental autoimmune encephalomyelitis in vivo. This study identifies a novel negative regulator of T cell activation and further reveals complex costimulatory regulation of immune responses.

T cell activation and immune function are regulated by costimulatory molecules of the B7 superfamily. Human B7-H3 is a recent addition to this family and has been shown to mediate T cell proliferation and IFN-γ production. In this work we describe the identification of the mouse B7-H3 homolog, which is ubiquitously expressed in a variety of tissues. Activated CD4 and CD8 T cells express a putative receptor that can be recognized by soluble mouse B7-H3-Ig molecules. While the mouse B7-H3 gene was found to contain a single copy, we discovered a novel isoform of human B7-H3 (named as B7-H3b hereafter) with four Ig-like domains that results from gene duplication and differential splicing. B7-H3b is the major isoform expressed in several tissues. This structural information suggests a genetic variation of the B7-H3 gene in mammalian species.

Notch1 plays a critical role in regulating T lineage commitment during the differentiation of lymphoid precursors. The physiological relevance of Notch1 signaling during subsequent stages of T cell differentiation has been more controversial. This is due in part to conflicting data from studies examining the overexpression or targeted deletion of Notch1 and to difficulties in distinguishing between the activities of multiple Notch family members and their ligands, which are expressed in the thymus. We employed a polyclonal antiserum against the extracellular domain of Notch1 to study surface expression during thymopoiesis. We found high levels of Notch1 on the cell surface only on double negative (DN) stage 2 through the immature single-positive stage of thymocyte development, before the double-positive (DP) stage. The Notch signaling pathway, as read out by Deltex1 expression levels, is highly active in DN thymocytes. When an active Notch1 transgene, Notch1IC, is exogenously introduced into thymocytes of recombinase-activating gene 2-deficient mice, it promotes proliferation and development to the DP stage following anti-CD3 treatment without apparently affecting the intensity of pre-TCR signaling. In addition, a stromal cell line expressing the Notch ligand, Delta-like-1, promotes the in vitro expansion of wild-type DN3 thymocytes in vitro. Consistent with other recent reports, these data suggest a role for Notch1 during the DN to DP stage of thymocyte maturation and suggest a cellular mechanism by which Notch1IC oncogenes could contribute to thymoma development and maintenance.

Superoxide and its derivatives have been implicated as secondary messenger molecules that influence signaling cascades in non-phagocytes. B lymphocytes produce superoxide after BCR ligation. We found that these ROS regulate B-cell signaling and entry into the cell cycle. B cells from mice deficient in the gp91 phox subunit of the NADPH oxidase complex are unable to generate ROS after BCR ligation. However, after BCR stimulation, more gp91 phox KO B cells enter the G1 stage of the cell cycle and proliferate than WT B cells. BCR ligation leads to a more rapid decrease in p27 Kip1 levels in gp91 phox KO B cells. Gp91 phox KO mice display enhanced T-cell-independent type 2, but normal T-dependent Ab responses. ROS-dependent regulation of BCR-induced proliferation may help modulate the size of the humoral response to T-cell-independent type 2 Ag immunization.

Summary: B cells are induced to enter the cell cycle by stimuli including ligation of the B‐cell receptor (BCR) complex and Toll‐like receptor (TLR) agonists. This review discusses the contribution of several molecules, which act at distinct steps in B‐cell activation. The adapter molecule Bam32 (B‐lymphocyte adapter of 32 kDa) helps promote BCR‐induced cell cycle entry, while the secondary messenger superoxide has the opposite effect. Bam32 and superoxide may fine tune BCR‐induced activation by competing for the same limited resources, namely Rac1 and the plasma membrane phospholipid PI(3,4)P2. The co‐receptor CD22 can inhibit BCR‐induced proliferation by binding to novel CD22 ligands. Finally, regulators of B‐cell survival and death also play roles in B‐cell transit through the cell cycle. Caspase 6 negatively regulates CD40‐ and TLR‐dependent G1 entry, while acting later in the cell cycle to promote S‐phase entry. Caspase 6 deficiency predisposes B cells to differentiate rather than proliferate after stimulation. Bim, a pro‐apoptotic Bcl‐2 family member, exerts a positive regulatory effect on cell cycle entry, which is opposed by Bcl‐2. New insights into what regulates B‐cell transit through the cell cycle may lead to thoughtful design of highly selective drugs that target pathogenic B cells.

Purpose: To investigate the contribution of BCR-induced superoxides to BCR-dependent signaling outcomes. Methods: gp91phoxiKO mice were utilized. Purified B cells from WT or gp91phoxiKO mice were loaded with CFSE, and stimulated via the B cell receptor (BCR). Proliferation was determined by CFSE dilution and G1 entry by Pyronin-Y staining. Western blotting of BCR-stimulated B cells was performed to measure cell cycle proteins. WT and gp91phoxiKO mice were immunized with TI-2 and TD antigens and serum Ab titers measured using ELISA. Results: More gp91phoxiKO B cells entered G1 and knockout B cells proliferated more after BCR stimulation. Reduction in levels of p27Kip1 occurred more quickly in BCR-stimulated gp91phoxiKO B cells. gp91phoxiKO mice displayed enhanced Ag-specific Ab production after TI-2 immunization. Conclusions: Superoxide derivatives negatively regulate signaling pathways leading to BCR-dependent proliferation. The perturbation in signaling in ROS-deficient B cells lies downstream of calcium signaling but upstream of p27Kip1. These studies supported by a Cancer Research Institute Training Grant and NIH grants GM37905 and AI44257.