View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Immunity, Vol. 6, 643±653, May, 1997, Copyright 1997 by Cell Press Peptide-Induced Positive Selection of TCR Transgenic Thymocytes in a Coreceptor-Independent Manner Eric Sebzda,* Mabel Choi,* Wai Ping Fung-Leung,² of the TCR and coreceptor to an MHC molecule conveys Tak W. Mak,*³ and Pamela S. Ohashi* lck to other kinases associated with the TCR complex, *Ontario Cancer Institute ultimately leading to downstream signaling. However, Departments of Medical Biophysics TCR transgenic mice expressing mutant CD8 molecules and Immunology that are unable to associate with lck are still able to Toronto, Ontario M5G 2M9 undergo thymocyte selection (Chan et al., 1993), sug- Canada gesting that CD8±lck interactions are not essential for ² R. W. Johnson Pharmaceutical T cell maturation. Similar results were obtained using Research Institute mutant CD4 molecules (Killeen and Littman, 1993). Addi- 3535 General Atomics Ct. tional experiments using TCR transgenic mice lacking San Diego, California 92121 the CD8a cytoplasmic domain have shown a defect in ³ Amgen Institute MHC class I±restricted positive selection, suggesting 620 University Ave. that an intracellular portion of CD8a is involved in thymo- Toronto, Ontario M5G 2C1 cyte development (Fung-Leung et al., 1993). Recent Canada work has also implicated the CD8b chain in signaling during T cell maturation (Crooks and Littman, 1994; Fung-Leung et al., 1994; Itano et al., 1994a; Nakayama Summary et al., 1994). Therefore, the exact function of CD8 during thymocyte selection remains unresolved. In addition to T cell receptor (TCR) transgenic thymocytes specific a potential role in signaling, experiments have demon- for the LCMV gp peptide are normally positively se- strated that coreceptors contribute to thymocyte± lected to the CD8 lineage. Transgenic thymocyte de- stromal cell avidity (Knobloch et al., 1992; Lee et al., 1992; Robey et al., 1992; Sherman et al., 1992), which velopment was substantially reduced in the absence may in turn affect T cell development. The importance of these CD8 coreceptors. However, efficient positive of coreceptors during thymocyte maturation was further selection was restored when TCR transgenic CD82/2 illustrated using transgenic mice expressing mutant fetal thymic lobes were cultured with a peptide variant MHC class I molecules that did not associate with CD8 of the wild-type ligand.These mature thymocytes were (Aldrich et al., 1991; Ingold et al., 1991; Killeen et al., functional, as shown by their ability to respond against 1992). Positive and negative selection was disrupted in strong peptide agonists. Additional experiments dem- these experiments, indicating that coreceptors are a key onstrated that transgenic positive selection was pep- component of thymocyte development. tide-specific. These results prove that CD8 does not Another significant aspect of thymocyte ontogeny possess essential signaling properties that are neces- deals with coreceptor down-regulation (for reviews, see sary for T cell development. In addition, the unilateral Chan et al., 1994; Davis and Littman, 1994; Robey, 1994; commitment of transgenic thymocytes to mature Fowlkes and Schweighoffer, 1995; von Boehmer, 1996). CD42TCRhi T cells expressing intracellular perforin An instructive model proposes that TCR MHC restriction suggests that there must be some instructive compo- determines lineage commitment. T cells expressing nent to CD4 down-regulation and lineage commitment MHC class I± or class II±restricted TCRs down-regulate during thymocyte selection. CD4 or CD8, respectively. Alternatively, coreceptor down-regulation could be an arbitrary event. According Introduction to a stochastic model, initial TCR stimulation leads to random coreceptor down-regulation. Continued matu- T cell maturation actively selects for functional thy- ration is dependent on a properly matched MHC- mocytes that can interact with self±major histocom- restricted TCR and coreceptor. Early experiments using patibility complex (MHC) while tolerizing potentially TCR transgenic mice showed a strong correlation be- autoreactive T cells. Concurrent with this selection pro- tween MHC-restriction and lineage commitment (Kisie- cess, CD41CD81 double-positive thymocytes differenti- low et al., 1988; Sha et al., 1988; Teh et al., 1988; Berg ate into CD41 or CD81 single-positive T cells. This lin- et al., 1989; Kaye et al., 1989; Scott et al., 1989), arguing eage commitment strongly correlates with helper or in favour of an instructive model for thymocyte develop- cytotoxic T cell function, respectively. The CD4 and CD8 ment. These findings were further supported by studies coreceptors have been implicated in all of these stages using mice transgenic for both TCRs and coreceptors. of thymocyte development, although the extent of their For example, HY-specific TCR1CD8tg transgenic mice participation remains unclear. The protein tyrosine ki- did not rescue CD41 thymocytes as would be expected nase p56lck, which is thought to be involved in early according to a stochastic model (Borgulya et al., 1991; T cell receptor (TCR)±induced intracellular signaling and Robey et al., 1991). However, additional experiments thymocyte development (Molina et al., 1992; Levin et using transgenic mice expressing different TCRs were al., 1993), has been shown to bind noncovalently to a able to inefficiently rescue mismatched coreceptors, common CXCP motif found in the cytoplasmic domain suggesting that the stochastic model may in fact be of both of these coreceptors (Shaw et al., 1990; Turner valid (Davis et al., 1993; Corbella et al., 1994; Itano et et al., 1990). Models have proposed that coengagement al., 1994b). Further evidence was found by examining Immunity 644 thymocyte development in MHC deficient mice. A transi- tional stage of CD41CD8loTCRint thymocytes were found in mice lacking class II, suggesting that lineage commit- ment was a random event (Chan et al., 1993). Likewise, a CD4loCD81TCRint thymocyte population was found in class I±deficient mice (van Meerwijk and Germain, 1993). Although mistakes have been found in lineage commit- ment, the significance of these cell populations remains unclear. To understand more fully the role of coreceptors during thymocyte ontogeny, we studied MHC class I±restricted transgenic T cell maturation in the absence of CD8. Previous work has demonstrated that thymo- cyte±stromal cell avidity affects positive and negative selection (Ashton-Rickardt et al., 1994; Sebzda et al., 1994). Therefore, we wanted to determine whether pep- tide variants could contribute significantly to thymocyte avidity in order to compensate for absent coreceptors during T cell ontogeny. The system used in these experi- ments allowed us to analyze thymocyte development in a coreceptor-independent manner, and thus provided us with a unique ability to address unresolved problems concerning lineage commitment and the role of core- ceptors during thymocyte maturation. Results T Cell Maturation Induced in a Coreceptor-Independent Manner The role of the CD8 coreceptor during positive selection Figure 1. TCR Transgenic Thymocyte Development Can Occur in the Absence of CD8 Coreceptors was examined using TCR transgenic mice that ex- (A) Positive selection of TCR transgenic thymocytes is greatly re- pressed receptors (Va2/Vb8.1) specific for the lympho- duced in CD8-deficient mice. Thymocytes from TCR CD81/1 and cytic choriomeningitis virus (LCMV) peptide (KAVYN- TCR CD82/2 mice were triple stained with antibodies specific for FATC) presented by H-2Db. Thymocyte maturation in CD8, CD4, and Va2. Total thymocytes are shown for dot plots and this mouse model was characterized by a skewing to histograms. the CD81 lineage and high surface expression of the (B) The peptide A4Y efficiently induces transgenic positive selection 2/2 2/2 transgenic TCR (Figure 1A). Peripheral CD81 T cells also in TCR CD8 FTOC. TCR CD8 thymic lobes were cultured with AV (control H-2Db±restricted adenovirus peptide) or A4Y and then expressed the transgenic TCR (Table 1). In contrast, stained with antibodies specific for CD4 and Va2. TCR CD81/1 lobes hi 2/2 very few TCR thymocytes were detected in TCR CD8 were used as a positive control. This experiment was repeated eight mice, although a few CD42CD82 or CD41CD82 TCRhi T times, with similar results. cells were detected in the periphery. Coreceptors have been shown to contribute to thymocyte avidity and affect T cell development (Lee et al., 1992; Robey et controls were stained with the maturity markers, heat- al., 1992; Sherman et al., 1992). Therefore, we wanted stable antigen (HSA) and peanut agglutinin (PNA). to determine whether peptide variants could increase CD42Va2hi thymocytes from A4Y-treated or positive thymocyte avidity sufficiently to overcome the require- control FTOCs expressed low levels of HSA and PNA, ment for CD8 molecules during positive selection. characteristic of mature thymocytes (Figure 2). In con- Fetal thymic lobes from TCR CD82/2 mice were cultured trast, CD41Va2int cells from these cultures had high with a control, H-2Db±restricted adenovirus peptide, AV surface expression of HSA and PNA, similar to that found (SGPSNTPPEI). Compared to thymocyte maturation in on wild-type CD41CD81 double-positive thymocytes. TCR CD81 fetal thymic organ cultures (FTOCs), these CD42Va2int thymocytes expressed intermediate levels CD8-deficient lobes had a substantially reduced