Requirement for a complex array of costimulators in the negative selection of autoreactive thymocytes in vivo.

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.

The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis.

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.

Cutting edge: thymic selection and autoreactivity are regulated by multiple coreceptors involved in T cell activation.

Immune responses are shaped by several processes that promote responses to pathogens and hinder responses to self. One mechanism that contributes to this polarization in response is negative selection, in which thymocytes that can respond to self-peptide/MHC complexes are deleted from the T cell repertoire. I found here that several coreceptors known to contribute to mature T cell activation also participate in negative selection. Interestingly, these molecules appeared to act in a cooperative fashion. Blocking the contribution of these molecules in fetal thymus organ culture not only prevented negative selection in the CD4+ lineage, but also induced the appearance of autoreactive thymocytes. This is the first demonstration that blocking coreceptor interactions during thymic development can produce autoreactive T cells. The contribution of negative selection to the mature T cell repertoire and to autoimmunity is discussed in light of these results.

Assurance opportunities for local health departments.

CONTEXT: Widespread changes in health care in the United States, especially the growth in the number and membership of health maintenance organizations (HMOs), provide increasing opportunities for collaboration, especially through contracts, between local health departments (LHDs) and HMOs to achieve public health goals. OBJECTIVES: (1) To identify, through a review of the literature, the areas in which HMOs and LHDs can collaborate to their mutual benefit and (2) for the leadership of an LHD in Florida to engage HMOs in its county in Florida in collaboration. DESIGN: University faculty reviewed the relevant literature; the Director of Medical Services for the Duval County Health Department developed the relationships with Duval County HMOs. SETTING: The setting for the study was Duval County. PARTICIPANTS: The participants in the study included faculty from the College of Public Health at the University of South Florida, State and county public health officials, and representatives of 10 HMOs. MAIN OUTCOME MEASURES: The main outcome measures were a literature review to identify feasible areas for collaboration and successful collaboration with HMOs in areas of mutual interest. RESULTS: The director of medical services for the county health department initiated a variety of collaborative activities with 10 HMOs. These led to agreements, participation on committees and task forces, provision of services, and improved communication. CONCLUSIONS: Collaboration between LHDs and HMOs to achieve public health assurance goals is feasible. Other organizations can encourage such collaboration through public and private policies.

TNF receptor-deficient mice reveal striking differences between several models of thymocyte negative selection.

Central tolerance depends upon Ag-mediated cell death in developing thymocytes. However, the mechanism of induced death is poorly understood. Among the known death-inducing proteins, TNF was previously found to be constitutively expressed in the thymus. The role of TNF in thymocyte negative selection was therefore investigated using TNF receptor (TNFR)-deficient mice containing a TCR transgene. TNFR-deficient mice displayed aberrant negative selection in two models: an in vitro system in which APC are cultured with thymocytes, and a popular in vivo system in which mice are treated with anti-CD3 Abs. In contrast, TNFR-deficient mice displayed normal thymocyte deletion in two Ag-induced in vivo models of negative selection. Current models of negative selection and the role of TNFR family members in this process are discussed in light of these results.

Role of Thy-1 in T cell development.

Tolerance to self proteins is accomplished in part by elimination of autoreactive immature T cells as they develop in the thymus. Although many investigators have studied the cellular interactions that regulate this important process, the specific molecules involved in negative selection are still not well understood. Thy-1 is a glycosyl-phosphatidylinositol-linked protein that is expressed at high levels on immature thymocytes, and recent evidence suggests that it is involved in thymocyte apoptosis. Correspondingly, we have found that Abs to Thy-1 block Ag-dependent thymocyte deletion in an in vitro culture system. Thus, we investigated the role of Thy-1 in T cell development by using Thy-1 -deficient mice containing a TCR transgene specific for a class II MHC-restricted Ag. With this system, the role of Thy-1 in Ag-specific self-restriction and self-tolerance could be analyzed. Thy-1-null mice were found to undergo normal negative selection in three different models: the in vitro culture system, anti-CD3-induced thymocyte deletion in vivo, and Ag-induced thymocyte deletion in vivo. Self-restriction to MHC class II also appeared to occur normally in Thy-1-null mice. These results therefore suggest that Thy-1 is not essential for either self-restriction or self-tolerance to MHC class II-restricted Ags. This finding is discussed in light of recent data regarding the role of other glycosyl-phosphatidylinositol-linked proteins in thymocyte development.

Cellular and biochemical requirements for thymocyte negative selection.

Developing T cells which recognize self-proteins are specifically deleted by the process of negative selection in the thymus. This review summarizes data from both in-vitro and in-vivo systems on the cellular and biochemical requirements that play roles in this process. We first consider the involvement of co-receptors and antigen presenting cells in negative selection. Next, we discuss the involvement of various signalling pathways in thymocyte deletion, particularly under conditions of limiting stimulation. Finally, these data are discussed in terms of how positive and negative selection are regulated during T-cell development.

An essential role for gp39, the ligand for CD40, in thymic selection.

The 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 beta 3, V beta 11, and V beta 12, 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 (TCR) V beta expression were also observed. Studies were also performed in the AND TCR transgenic (Tg) mice, which bear the V alpha 11, V beta 3 TCR and recognize both pigeon cytochrome c (PCC)/IEk and H-2As. Neonatal administration of anti-gp39 to AND TCR Tg mice that endogenously express H-2As 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 TCR mice was unaltered by administration of anti-gp39. In addition, deletion by Staphylococcus enterotoxin B in conventional mice was also unaffected by anti-gp39 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.

Negative selection of CD4+ CD8+ thymocytes by T-cell receptor peptide antagonists.

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 AD10. 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 AD10 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.

Two signals are required for negative selection of CD4+CD8+ thymocytes.

Recent results indicate that two signals are required for activation of mature T cells. The first is delivered through the TCR, and the second is delivered through receptors that bind various ligands expressed on APC. For example, it has been shown that B7/BB1, which is expressed on many APC, can costimulate T cell activation by binding to CD28 or CTLA4, which are expressed on mature T cells. In contrast, little is known of the signals required for negative selection of autoreactive thymocytes. Thus, we have investigated this issue by using an in vitro culture system in which thymocytes from mice that are transgenic for a class II MHC-restricted TCR are cultured with murine fibroblast lines that express class II MHC. Under these conditions, CD4+CD8+ (DP) thymocytes undergo an Ag-dependent programmed cell death, which likely represents the negative selection of autoreactive thymocytes that would occur in an intact thymus. Using this culture system, we first found that both TCR- and APC-dependent stimuli were required in order to induce deletion of DP thymocytes. Anti-TCR antibodies alone did not cause deletion of DP cells, but merely induced a decrease in their expression of CD4 and CD8 to produce a DPdull phenotype. Addition of APC was then required for deletion of these DPdull cells. One obvious candidate for the costimulatory signal expressed by these APC was B7. Three different experimental approaches indicated, however, that B7 was not the APC-dependent signal required for deletion of DP thymocytes. Thus, these results suggest that negative selection of autoreactive thymocytes is a two-step process in which stimulation of the TCR causes downregulation of CD4 and CD8 on DP thymocytes, and then an unknown ligand expressed on APC stimulates a receptor on DP thymocytes to induce their deletion.

Mutational analysis of antigen receptor regulation of B lymphocyte growth. Evidence for involvement of the phosphoinositide signaling pathway.

Stimulation of the antigen receptor of WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-IgM) induces irreversible growth arrest. Anti-IgM stimulates two kinds of transmembrane signaling events, phosphorylation of proteins on tyrosyl residues and breakdown of inositol phospholipids, which results in increases of inositol phosphates, diacylglycerol, and calcium. The roles of these reactions in mediating the growth arrest of the B lymphoma cells have not been established. To examine this issue, we took a genetic approach. Mutants of WEHI-231 cells were isolated that were resistant to anti-IgM-induced growth arrest. Five out of seven independent mutants analyzed had normal cell-surface expression of antigen receptors. Although each of these five mutants had tyrosine protein phosphorylation patterns comparable to wild-type cells, they exhibited alterations in the phosphoinositide signaling pathway. Four of the mutants had decreased phosphoinositide breakdown, probably due to an alteration in phospholipase C. Decreased second messenger production may be responsible for the growth-resistant phenotype. Full growth arrest was restored upon addition of the calcium ionophore ionomycin, suggesting that the limiting second messenger was intracellular free calcium. The final mutant appeared to be altered in a component(s) that responds to diacylglycerol and calcium. Taken together, these results provide further evidence that the phosphoinositide pathway is at least partly responsible for mediating antigen receptor regulation of B lymphoma cell growth.

Antigen receptor-induced cell cycle arrest in WEHI-231 B lymphoma cells depends on the duration of signaling before the G1 phase restriction point.

Stimulation of antigen receptors on WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-immunoglobulin M [IgM]) causes irreversible growth arrest. This may be a model for antigen-induced tolerance to self components in the immune system. Antigen receptor stimulation also causes inositol phospholipid hydrolysis, producing diacylglycerol, which activates protein kinase C, and inositol 1,4,5-trisphosphate, which causes release of calcium from intracellular stores. To better understand the nature of the antigen receptor-induced growth arrest of WEHI-231 cells, we have examined the basis for it. WEHI-231 cells in various phases of the cell cycle were isolated by centrifugal elutriation, and their response was evaluated following treatment with either anti-IgM or pharmacologic agents that raise intracellular free calcium levels and activate protein kinase C. Treatment with anti-IgM or the pharmacologic agents did not lengthen the cell cycle. Instead, growth inhibition was solely the result of arrest in the G1 phase. The efficiency of G1 arrest increased with the length of time during which the cells received signaling before reaching the G1 phase arrest point. Maximum efficiency of arrest was achieved after approximately one cell cycle of receptor signaling. These results imply that anti-IgM causes G1 arrest of WEHI-231 cells by slowly affecting components required for S phase progression, rather than by rapidly inhibiting such components or by rapidly activating a suicide mechanism. Antigen receptor stimulation was twice as effective as stimulation via the mimicking reagents phorbol dibutyrate and ionomycin. Thus, although the phosphoinositide second messengers diacylglycerol and calcium probably play roles in mediating the effects of anti-IgM on WEHI-231 cells, other second messengers may also be involved.

Role of phosphoinositide-derived second messengers in mediating anti-IgM-induced growth arrest of WEHI-231 B lymphoma cells.

Anti-IgM irreversibly inhibits the growth of WEHI-231 B lymphoma cells and induces phosphoinositide hydrolysis--producing diacylglycerol, which activates protein kinase C, inositol 1,4,5-trisphosphate, which induces the release of calcium from intracellular storage sites into the cytoplasm, and other inositol polyphosphates. The roles of two of the possible second messengers, cytoplasmic free calcium and diacylglycerol, in mediating the action of anti-IgM on WEHI-231 cells were assessed by elevating [Ca2+]i with ionomycin and by activating protein kinase C with phorbol 12,13-dibutyrate (PdBu). The combination of 250 nM ionomycin and 4 to 7 nM PdBu was found to cause growth arrest and cell volume decrease responses in WEHI-231 cells which were similar to those caused by anti-IgM, although clearly slower. Both anti-IgM and the combination of mimicking reagents induced growth arrest of WEHI-231 cells in the G1 phase of the cell cycle. In both cases, this growth arrest was mitigated by addition of bacterial LPS. Moreover, 250 nM ionomycin plus 4 to 7 nM PdBu did not inhibit the growth of two other murine B lymphoma cell lines, each of which did exhibit increased phosphoinositide hydrolysis but not growth arrest in response to anti-Ig. Taken together, these results suggest that ionomycin and PdBu, at the concentrations used, did not inhibit WEHI-231 growth by general toxicity, but rather by mimicking the effects of the natural second messengers generated from Ag receptor cross-linking. Thus, the phosphoinositide-derived second messengers Ca2+i and diacylglycerol are capable of playing important roles in mediating the action of anti-IgM on WEHI-231 B lymphoma cells. However, the response of WEHI-231 cells to anti-IgM could not be fully reproduced with ionomycin and phorbol diester. These results suggest that another second messenger induced by anti-IgM may also play an important role in mediating the growth arrest of these cells.