Costimulatory Signals Are Required for Induction of Transcription Factor Nur77 During Negative Selection of CD4 CD8 Thymocytes
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Proc. Natl. Acad. Sci. USA Vol. 96, pp. 622–627, January 1999 Immunology Costimulatory signals are required for induction of transcription factor Nur77 during negative selection of CD41CD81 thymocytes DERK AMSEN*, CONCHI REVILLA CALVO*†,BARBARA A. OSBORNE‡, AND ADA M. KRUISBEEK*§ *Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; and ‡Department of Veterinary and Animal Sciences, University of Massachusetts, 304 Paige Lab, Amherst, MA 01003 Edited by Philippa Marrack, National Jewish Medical and Research Center, Denver, CO, and approved November 19, 1998 (received for review September 25, 1998) ABSTRACT A major question in end-stage T cell devel- presenting cells (APCs) to induce either positive or negative opment is how T cell receptor(TCR) ligation on immature selection may reflect in part their localization in different CD41CD81 double positive thymocytes is translated into spatial and temporal compartments. In addition, variables such either survival (positive selection) or apoptotic (negative as MHC expression levels and expression of adhesion mole- selection) signals. Because different types of antigen- cules may also contribute to the outcome of the interaction presenting cells (APCs) induce positive or negative selection between DP thymocytes and thymic APCs. Finally, the dif- in the thymus and express different costimulatory molecules, ferential ability of thymic presenting cells to induce positive or involvement of such costimulatory molecules in determining negative selection may reflect cell type-specific expression of cell fate of DP thymocytes is considered here. If TCR- costimulatory molecules. Such molecules may, through en- generated signals are modulated by APCs, this should be gagement of their counter-receptors on DP thymocytes, qual- reflected in the activation of distinct biochemical pathways. itatively influence the signals induced by the TCR, leading to We here demonstrate that costimulatory signals involved in induction of either apoptotic or survival pathways. negative selection also are required for induction of protein In support of a contribution of cell-derived costimulatory expression of Nur77 and its family members. These transcrip- signals, clonal deletion of thymocytes was indeed shown to tion factors are critically involved in negative but not positive occur only if, next to signals through the TCR, additional selection. In contrast, the signals that costimulate negative APC-derived signals are provided (11–14). Whether thymo- selection are not required for induction of several molecular cytes also use the molecular interactions that mature T cells events associated with positive selection. These include acti- exploit to generate costimulatory signals has been controver- vation of the immediate early gene Egr-1, the mitogen- sial. On the one hand, several investigators have reported a activated protein kinase ERK2, and surface expression of the lack of a phenotype with respect to negative selection in CD69 marker. Thus, costimulation for negative selection CD282y2 mice (15–17). On the other hand, apoptosis of selectively provides signals for activation of apoptotic medi- thymocytes induced in suspension can be costimulated by ators. These data provide molecular insights into how TCR- crosslinking the CD28 receptor, either by using antibodies (12, engagement by ligands on different thymic APCs can deter- 18, 19) or by its natural ligands CD80 and CD86 (13, 14, 20). mine cell fate. Moreover, these findings have been corroborated in the intact thymus in fetal thymic organ culture and recently also in The random process that creates T cell receptor (TCR) CD28-deficient mice (13, 21), providing formal support for a diversity generates a large pool of thymocytes, of which only a role for CD28 in clonal deletion in vivo. While acknowledging few will migrate to the periphery. Within the population of that other molecules than CD28 can costimulate deletion (refs. 1 1 thymocytes that has arrived at the CD4 CD8 double positive 11–17; present study), these findings document that CD28– (DP) stage of development, positive and negative selection CD80y86 interactions can serve to provide prototypical co- processes determine which cells are allowed to develop into stimulatory signals for thymocyte deletion. Supportive evi- mature CD4 or CD8 TCRab cells. Both of these selection dence for this notion stems from the observation that thymic processes depend on ligation of the TCR by peptide-loaded medullary expression of CD80 is correlated with preferential MHC molecules present in the thymus but result in diamet- deletion of self-reactive thymocytes in the medulla (22, 23). rically different outcomes—survival and maturation (positive Also, CD86 expression in the medullary region of the thymus selection) or apoptosis (negative selection) (reviewed in ref. 1). is substantially reduced in gp39-deficient mice (24), a pheno- The critical issue in understanding end-stage T cell devel- type correlated with reduced deletion of thymocytes specific opment is how TCR occupancy can be perceived in such for endogenously expressed self-antigens (24). markedly different ways by DP thymocytes. Whether positive Thus far, only two molecular signaling modules have spe- or negative selection is induced appears to depend in part on cifically been linked to either positive or negative selection. the affinityyavidity of the interaction between thymocyte Activation of the mitogen-activated protein kinase (MAPK) TCRs and major histocompatibility complex (MHC)–peptide cascade is required only for positive selection: transgenically complexes on presenting cells in the thymus (2, 3). However, expressed dominant negative mutants of Ras, Raf1, and a different model of thymocyte selection contends that in fact MEK-1 each reduce positive selection (25–27), whereas neg- the type of thymic cell presenting MHC–peptide ligands to ative selection is unaffected by perturbations in the MAPK developing thymocytes dictates the outcome of selection. cascade. By contrast, a specific role in negative selection has Support for this model has been provided by many different experimental systems (4–10) and is difficult to ignore. This This paper was submitted directly (Track II) to the Proceedings office. differential ability of different types of thymic antigen- Abbreviations: EMSA, electrophoretic mobility-shift assay; APC, antigen-presenting cell; DP, double positive; MHC, major histocom- The publication costs of this article were defrayed in part by page charge patibility complex; GST, glutathione S-transferase; TCR, T cell re- ceptor; ECDI, 1-ethyl-3-(39-dimethylaminopropyl) carbodiimide; payment. This article must therefore be hereby marked ‘‘advertisement’’ in MAPK, mitogen-activated protein kinase. accordance with 18 U.S.C. §1734 solely to indicate this fact. †Present address: Centro de Investigaciones Biolo´gicas, Madrid, Spain. PNAS is available online at www.pnas.org. §To whom reprint requests should be addressed. e-mail: [email protected]. 622 Downloaded by guest on September 25, 2021 Immunology: Amsen et al. Proc. Natl. Acad. Sci. USA 96 (1999) 623 been documented for members of the Nur77 family of orphan purified and unfractionated thymocytes and yielded similar steroid receptors. Nur77 is expressed in an immediate early results. fashion in response to apoptotic TCR triggering in thymocytes In Vitro Deletion Assay. Thymocytes were brought into (28, 29), and transgenic expression of Nur77 or its relative single-cell suspension in complete medium. Thymocytes (2– Nor1 in thymocytes leads to massive apoptosis (30). Moreover, 4 3 105) were cultured in 96-well flat-bottom plates overnight mice expressing a transgene that encodes dominant negative with 1 3 105 APCs. After 14–18 hr, cells were harvested and Nur77 lacking its transactivation domain exhibit severely im- prepared for flow cytometry analysis. Fixation of DCEK cells paired ability to induce clonal deletion (31, 32), whereas was carried out as described (47). positive selection proceeds undisturbed. These findings docu- Flow Cytometry. Cells were resuspended in PBS containing ment that transactivation of as-yet-unidentified pro-apoptotic 0.5% BSA and 0.02% sodium azide and incubated with target genes by Nur77 and closely related factors is a prereq- antibodies on ice for 30 min. Incubation with biotin-labeled uisite step in thymocyte clonal deletion but not in positive anti-CD69 (PharMingen) was followed by incubation with selection. The precise identity of these pro-apoptotic target avidin–fluorescein isothiocyanate (FITC) (Sigma). For mea- genes is currently unclear. It has been suggested that Nur77 surement of deletion, thymocytes were (after surface staining m y functions upstream from the CD95 ligand–CD95 interaction. for CD8 expression) exposed to 1 g ml ethidium bromide 3 4 3 5 Thus, the massive apoptosis occurring in thymuses from Nur77 (Sigma) as described (48). Cells (1 10 to 2.5 10 per transgenic mice was reported to be reduced by introduction of sample) were analyzed on a Becton Dickinson FACSCAN by the gld mutation (30). However, these results were recently using LYSIS 2 software. APCs were segregated electronically on contradicted by another study (33) in which such rescue was the basis of forward scatter/side scatter pattern. not detected. As expression of dominant negative Nur77 yields Stimulation of Cells and Separation from APCs. APC lines a much more pronounced block in negative selection (31, 32) were grown