T lymphocyte activation evokes distinct changes in cell surface O-glycans. CD8+ T cells undergo an elimination of sialic acid on core 1 O-glycans and an induction of core 2 O-glycans until either apoptotic death or differentiation into memory cells. We find that the ST3Gal-I sialyltransferase is required for core 1 O-glycan sialylation and its deficiency induces core 2 O-glycan biosynthesis. Apoptosis ensues with the loss of peripheral CD8+ T cells in the absence of immune stimulation. Cell surface ligation of the ST3Gal-I substrate CD43 recapitulates this phenotype by a caspase 3-independent mechanism. Control of core 1 O-glycan sialylation in T lymphocytes by ST3Gal-I comprises a homeostatic mechanism that eliminates CD8+ T cells by apoptosis while facilitating the production of viable CD8+ memory T cells.

Teleost fish are among the most ancient vertebrates possessing an adaptive immune system with B and T lymphocytes that produce memory responses to pathogens. Most bony fish, however, have only 2 types of B lymphocytes, in contrast to the 4 types available to mammals. To better understand the evolution of adaptive immunity, we generated transgenic zebrafish in which the major immunoglobulin M (IgM(+)) B-cell subset expresses green fluorescence protein (GFP) (IgM1:eGFP). We discovered that the earliest IgM(+) B cells appear between the dorsal aorta and posterior cardinal vein and also in the kidney around 20 days postfertilization. We also examined B-cell ontogeny in adult IgM1:eGFP;rag2:DsRed animals, where we defined pro-B, pre-B, and immature/mature B cells in the adult kidney. Sites of B-cell development that shift between the embryo and adult have previously been described in birds and mammals. Our results suggest that this developmental shift occurs in all jawed vertebrates. Finally, we used IgM1:eGFP and cd45DsRed;blimp1:eGFP zebrafish to characterize plasma B cells and investigate B-cell function. The IgM1:eGFP reporter fish are the first nonmammalian B-cell reporter animals to be described. They will be important for further investigation of immune cell evolution and development and host-pathogen interactions in zebrafish

SummaryThe interactions between CD40 on B cells and its ligand gp39 on activated T helper cells are known to be essential for the development of thymus-dependent humoral immunity. However, CD40 is also functionally expressed on thymic epithelial cells and dendritic cells, suggesting that gp39-CD40 interactions may also play a role in thymic education, the process by which self-reactive cells are deleted from the T cell repertoire. Six systems of negative selection were studied for their reliance on gp39-CD40 interactions to mediate negative selection. In all cases, when the antigen/superantigen was endogenously expressed (in contrast to exogenously administered), negative selection was blocked by loss of gp39 function. Specifically, blockade of gp39-CD40 interactions prevented the deletion of thymocytes expressing V[33, V[311, and V[312, specificities normally deleted in BALB/c mice because of the endogenous expression of minor lymphocyte-stimulating determinants. Independent verification of a role of gp39 in negative selection was provided by studies in gp39-deficient mice where alterations in T cell receptor (TCP,) V[3 expression were also observed. Studies were also performed in the AND TCR. transgenic (Tg) mice, which bear the Vti11,V[33 TCR. and recognize both pigeon cytochrome c (PCC)/IE k and H-2A s. Neonatal administration ofanti-gp39 to AND TCP, Tg mice that endogenously express H-2A s or endogenously produce PCC prevented the deletion of TCR Tg T cells. In contrast, deletion mediated by high-dose PCC peptide antigen (administered exogenously) in AND TCI< mice was unaltered by administration ofanti-gp39. In addition, deletion by Staphylococcus enterotoxin B in conventional mice was also unaffected by antigp39 administration, gp39 expression was induced on thymocytes by mitogens or by antigen on TCR Tg thymocytes. Immunohistochemical analysis of B7-2 expression in the thymus indicated that, in the absence of gp39, B7-2 expression was substantially reduced. Taken together, these data suggest that gp39 may influence negative selection through the regulation of costimulatory molecule expression. Moreover, the data support the hypothesis that, for negative selection to some endogenously produced antigens, negative selection may be dependent on TCR. engagement and costimulation.

Antigen-induced activation of T cells can be specifically inhibited by antigen analogs that have been termed T-cell receptor peptide antagonists. These antagonists appear to act by inducing the formation of nonstimulatory or partially stimulatory complexes between T-cell receptors and the major histocompatibility complex molecules presenting the peptides.Herein, we have investigated the effect of T-cell receptor peptide antagonists on thymocyte negative selection. First, peptide antagonists were identified for the cytochrome c-specific T-cell clone AD1O. These peptides were then tested for their ability to induce negative selection in an in vitro model system using thymocytes from mice transgenic for the AD1O T-cell receptor. Though unable to induce mature T-cell activation, the T-cell receptor peptide antagonists induced deletion of CD4+ CD8+ thymocytes. These results suggest that negative selection of CD4+ CD8+ thymocytes can be induced by T-cell receptor interactions of a lower affinity than those required for mature T-cell activation.Mature T cells are activated to proliferate and secrete lymphokines by antigenic peptides bound to major histocompatibility complex (MHC) proteins. It has been shown recently that mature T-cell activation can be blocked by antigen analogs that are substituted at sites that contact the T-cell receptor for antigen (TCR) (1)(2)(3)(4)(5). These antigen analogs block T-cell activation at least 20-fold more efficiently than do unrelated peptides that still bind MHC proteins but have no specificity for the TCR (1). Thus, it has been proposed that these analogs act as TCR antagonists by forming an inactive complex between the TCR and the MHC protein presenting the peptide (1, 2). The mechanism by which TCR antagonists block T-cell activation is unknown. Ruppert et al. (6) found that TCR peptide antagonists do not block T cell-antigenpresenting cell (APC) conjugate formation. Instead, the antagonistic peptides blocked antigen-induced increases in phosphatidylinositol breakdown and calcium in the T cells. Thus, the TCR antagonists may act by blocking early transmembrane signaling events. Alternatively, antigen analogs that partially activate T cells may act by impeding delivery of required costimulatory signals from the APC to the T cell (4, 5).The effect of antigen analogs on T-cell maturation in the thymus has not been explored. Thymocyte development is frequently analyzed by following surface expression of the TCR and the CD4 and CD8 accessory proteins. Early thymocyte immigrants are CD4-CD8-and also lack expression of the TCR. These double negative (DN) cells then proceed to a CD4+ CD8+ [double positive (DP)] stage, where they begin to express the TCR (7,8). It is at this stage that thymocytes can undergo negative selection due to interaction with self-antigen or positive selection to become mature T cells (reviewed in ref. 9). Positive selection is mediated, in part, by an interaction of the TCR with MHC molecules on epithelial cells ofthe thymic stroma. This interaction ind...

Autoreactive thymocytes can be deleted at an immature stage of their development by Ag-induced apoptosis or negative selection. In addition to Ag, negative selection also requires costimulatory signals from APC. We recently used a fetal thymus organ culture system to show that CD5, CD28, and TNF cooperatively regulate deletion of autoreactive thymocytes. Although these experiments provided strong evidence for the action of several costimulators in negative selection, we wished to demonstrate a role for these molecules in a physiologically natural model where thymocytes are deleted in vivo by endogenously expressed Ags. Accordingly, we examined thymocyte deletion in costimulator-null mice in three models of autoantigen-induced negative selection. We compared CD5−/− CD28−/− mice to CD40L−/− mice, which exhibited a profound block in negative selection in all three systems. Surprisingly, only one of the three models revealed a requirement for the CD5 and CD28 costimulators in autoantigen-induced deletion. These results suggest that an extraordinarily complex array of costimulators is involved in negative selection. We predict that different sets of costimulators will be required depending on the timing of negative selection, the Ag, the signal strength, the APC, and whether Ag presentation occurs on class I or class II MHC molecules.