Peptide-Induced Positive Selection of TCR Transgenic Thymocytes in a Coreceptor-Independent Manner

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,† Tak W. Mak,* ‡ and Pamela S. Ohashi* * Ontario Cancer Institute Departments of Medical Biophysics and Immunology Toronto, Ontario M5G 2M9 Canada † R. W. Johnson Pharmaceutical Research Institute 3535 General Atomics Ct. San Diego, California 92121 ‡ Amgen Institute 620 University Ave. Toronto, Ontario M5G 2C1 Canada

Summary T cell receptor (TCR) transgenic thymocytes specific for the LCMV gp peptide are normally positively selected to the CD8 lineage. Transgenic thymocyte development was substantially reduced in the absence of these CD8 coreceptors. However, efficient positive selection was restored when TCR transgenic CD82/2 fetal thymic lobes were cultured with a peptide variant of the wild-type ligand. These mature thymocytes were functional, as shown by their ability to respond against strong peptide agonists. Additional experiments demonstrated that transgenic positive selection was peptide-specific. These results prove that CD8 does not possess essential signaling properties that are necessary for T cell development. In addition, the unilateral commitment of transgenic thymocytes to mature CD42TCRhi T cells expressing intracellular perforin suggests that there must be some instructive component to CD4 down-regulation and lineage commitment during thymocyte selection. Introduction T cell maturation actively selects for functional thymocytes that can interact with self–major histocompatibility complex (MHC) while tolerizing potentially autoreactive T cells. Concurrent with this selection process, CD41CD81 double-positive thymocytes differentiate into CD41 or CD81 single-positive T cells. This lineage commitment strongly correlates with helper or cytotoxic T cell function, respectively. The CD4 and CD8 coreceptors have been implicated in all of these stages of thymocyte development, although the extent of their participation remains unclear. The protein tyrosine kinase p56lck, which is thought to be involved in early T cell receptor (TCR)–induced intracellular signaling and thymocyte development (Molina et al., 1992; Levin et al., 1993), has been shown to bind noncovalently to a common CXCP motif found in the cytoplasmic domain of both of these coreceptors (Shaw et al., 1990; Turner et al., 1990). Models have proposed that coengagement

of the TCR and coreceptor to an MHC molecule conveys lck to other kinases associated with the TCR complex, ultimately leading to downstream signaling. However, TCR transgenic mice expressing mutant CD8 molecules that are unable to associate with lck are still able to undergo thymocyte selection (Chan et al., 1993), suggesting that CD8–lck interactions are not essential for T cell maturation. Similar results were obtained using mutant CD4 molecules (Killeen and Littman, 1993). Additional experiments using TCR transgenic mice lacking the CD8a cytoplasmic domain have shown a defect in MHC class I–restricted positive selection, suggesting that an intracellular portion of CD8a is involved in thymocyte development (Fung-Leung et al., 1993). Recent 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 et al., 1994). Therefore, the exact function of CD8 during thymocyte selection remains unresolved. In addition to a potential role in signaling, experiments have demonstrated that coreceptors contribute to thymocyte– stromal cell avidity (Knobloch et al., 1992; Lee et al., 1992; Robey et al., 1992; Sherman et al., 1992), which may in turn affect T cell development. The importance of coreceptors during thymocyte maturation was further illustrated using transgenic mice expressing mutant MHC class I molecules that did not associate with CD8 (Aldrich et al., 1991; Ingold et al., 1991; Killeen et al., 1992). Positive and negative selection was disrupted in these experiments, indicating that coreceptors are a key component of thymocyte development. Another significant aspect of thymocyte ontogeny deals with coreceptor down-regulation (for reviews, see Chan et al., 1994; Davis and Littman, 1994; Robey, 1994; Fowlkes and Schweighoffer, 1995; von Boehmer, 1996). An instructive model proposes that TCR MHC restriction determines lineage commitment. T cells expressing MHC class I– or class II–restricted TCRs down-regulate CD4 or CD8, respectively. Alternatively, coreceptor down-regulation could be an arbitrary event. According to a stochastic model, initial TCR stimulation leads to random coreceptor down-regulation. Continued maturation is dependent on a properly matched MHCrestricted TCR and coreceptor. Early experiments using TCR transgenic mice showed a strong correlation between MHC-restriction and lineage commitment (Kisielow et al., 1988; Sha et al., 1988; Teh et al., 1988; Berg et al., 1989; Kaye et al., 1989; Scott et al., 1989), arguing in favour of an instructive model for thymocyte development. These findings were further supported by studies using mice transgenic for both TCRs and coreceptors. For example, HY-specific TCR1CD8tg transgenic mice did not rescue CD41 thymocytes as would be expected according to a stochastic model (Borgulya et al., 1991; Robey et al., 1991). However, additional experiments using transgenic mice expressing different TCRs were able to inefficiently rescue mismatched coreceptors, suggesting that the stochastic model may in fact be valid (Davis et al., 1993; Corbella et al., 1994; Itano et al., 1994b). Further evidence was found by examining

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thymocyte development in MHC deficient mice. A transitional stage of CD41CD8loTCRint thymocytes were found in mice lacking class II, suggesting that lineage commitment 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 commitment, 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 thymocyte–stromal cell avidity affects positive and negative selection (Ashton-Rickardt et al., 1994; Sebzda et al., 1994). Therefore, we wanted to determine whether peptide variants could contribute significantly to thymocyte avidity in order to compensate for absent coreceptors during T cell ontogeny. The system used in these experiments 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 coreceptors during thymocyte maturation.

Results T Cell Maturation Induced in a Coreceptor-Independent Manner The role of the CD8 coreceptor during positive selection was examined using TCR transgenic mice that expressed receptors (Va2/Vb8.1) specific for the lymphocytic choriomeningitis virus (LCMV) peptide (KAVYNFATC) presented by H-2Db. Thymocyte maturation in this mouse model was characterized by a skewing to the CD81 lineage and high surface expression of the transgenic TCR (Figure 1A). Peripheral CD81 T cells also expressed the transgenic TCR (Table 1). In contrast, very few TCRhi thymocytes were detected in TCR CD82/ 2 mice, although a few CD42CD82 or CD41CD82 TCRhi T 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 al., 1992; Sherman et al., 1992). Therefore, we wanted to determine whether peptide variants could increase thymocyte avidity sufficiently to overcome the requirement for CD8 molecules during positive selection. Fetal thymic lobes from TCR CD82/2 mice were cultured with a control, H-2D b–restricted adenovirus peptide, AV (SGPSNTPPEI). Compared to thymocyte maturation in TCR CD81 fetal thymic organ cultures (FTOCs), these CD8-deficient lobes had a substantially reduced CD42Va2hi transgenic cell population (Figure 1B). However, TCRhi transgenic thymocytes were recovered when TCR CD82/2 fetal thymic lobes were cultured in the presence of the peptide variant, A4Y (KAVANFATM), which has previously been shown to positively select TCR CD81 thymocytes (Sebzda et al., 1996). These results indicate that CD8 coreceptors are an important, but not mandatory, component of T cell development. To ensure that these CD42Va2hi thymocytes had undergone positive selection, T cells from TCR CD82/ 2 organ cultures treated with A4Y or from TCR CD81

Figure 1. TCR Transgenic Thymocyte Development Can Occur in the Absence of CD8 Coreceptors (A) Positive selection of TCR transgenic thymocytes is greatly reduced in CD8-deficient mice. Thymocytes from TCR CD81/1 and TCR CD82/2 mice were triple stained with antibodies specific for CD8, CD4, and Va2. Total thymocytes are shown for dot plots and histograms. (B) The peptide A4Y efficiently induces transgenic positive selection in TCR CD82/2 FTOC. TCR CD82/2 thymic lobes were cultured with AV (control H-2Db–restricted adenovirus peptide) or A4Y and then stained with antibodies specific for CD4 and Va2. TCR CD81/1 lobes were used as a positive control. This experiment was repeated eight times, with similar results.

controls were stained with the maturity markers, heatstable antigen (HSA) and peanut agglutinin (PNA). CD42Va2hi thymocytes from A4Y-treated or positive control FTOCs expressed low levels of HSA and PNA, characteristic of mature thymocytes (Figure 2). In contrast, CD41Va2int cells from these cultures had high surface expression of HSA and PNA, similar to that found on wild-type CD41CD81 double-positive thymocytes. CD42Va2int thymocytes expressed intermediate levels of maturity markers relative to CD41Va2 int and CD42Va2hi cells, suggesting that transgenic thymocytes progressed from a CD41 to a CD42 stage prior to becoming mature, CD42Va2hi cells. Together, these results confirmed that CD42Va2hi thymocytes were a mature T cell population that had undergone positive selection. The CD42Va2hi cell numbers summarized in Table 2 demonstrate the relative importance of CD8 during positive selection. Thymic lobes lacking this coreceptor had a reduced ability to produce mature thymocytes expressing the transgenic TCR. Addition of A4Y compensated for the absent coreceptor and provided adequate

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Table 1. Transgenic Splenocyte Numbers from TCR CD81 and TCR CD82/2 Littermates CD81Va2hi CD81

TCR TCR CD82/2

CD41Va2hi

1.9 6 0.3 3 10 0

7

CD82CD42Va2hi

1.2 6 0.2 3 10 5.6 6 0.5 3 106

1.2 6 0.3 3 106 4.1 6 0.4 3 106

6

n54 n54

Data are shown 6 SD.

stimulation to positively select CD42Va2hi thymocytes. The efficiency of A4Y-induced positive selection was examined by testing this peptide in TCR CD82/2 FTOCs at various concentrations. A titration profile of A4Y (Figure 3) showed that positive selection occurred over a broad concentration range, extending from 1025 to 1029 M. Together, these results demonstrated that A4Y efficiently induced positive selection of transgenic thymocytes in a coreceptor-independent manner. Peptide-Specific Positive Selection of TCR CD8 2/ 2 Thymocytes Although A4Y positively selected TCR CD82/ 2 thymocytes, it was possible that this peptide was simply compensating for lost avidity due to a lack of coreceptors, and not providing a unique TCR ligand that could promote positive selection. For this reason, we searched for peptide variants that were similar in nature to A4Y to determine whether these peptides could efficiently induce positive selection. Initially we tested potential peptides to ensure that they bound to H-2D b molecules. Using TAP-2 (transporter associated with antigen processing type 2)–deficient RMA-S cells, type 2, we were able to demonstrate that each of the relevant peptides rescued H-2D b surface expression (Figure 4A). Since these peptides could bind to H-2Db, we further tested these ligands for their ability to stimulate transgenic T cells. Previous experiments revealed that A4Y, an alanine-variant of the wild-type peptide, was an agonist with no detectable antagonist properties (Sebzda et al., 1996). As shown in Figure 4B, antigen-presenting cells (APCs) pulsed with various concentrations of this modified peptide induced TCR CD81 splenocyte proliferation. Similar concentrations of the strong peptide agonist, p33 (KAVYNFATM), resulted in increased proliferation. However, transgenic splenocytes stimulated with the peptide variant, A3V (KAAYNFATM), proliferated at rates common to both peptides. At high

peptide concentrations (1024–10 25 M), A3V induced proliferation to the same degree as p33. At lower concentrations (1027–10211 M), A3V and A4Y induced almost identical amounts of proliferation. To determine the relative importance of CD8 in peptide-specific interactions, TCR transgenic splenocytes were stimulated with peptide-coated APCs in the presence of anti-CD8 monoclonal antibodies (Figure 4B). High antibody concentrations inhibited T cell proliferation in response to all of the peptides, although not to the same extent. A4Y did not induce T cell proliferation. In contrast, reduced proliferation was seen with p33 and A3V. Notably, A3V did not seem to be as affected by the blocking antibody as p33. Similar proliferation patterns were observed with lower concentrations of anti-CD8. In general, anti-CD8 antibody inhibited transgenic T cell proliferation in response to all of the peptides tested; however, A3V and p33 were much more efficient at stimulating CD8-independent proliferation than A4Y. These assays indicated that TCR–A3V/MHC interactions were stronger and less CD8-dependent than those involving A4Y. Hence, these results suggested that A3V was at least as potent an agonist peptide as A4Y. To determine whether A3V could induce positive selection in the absence of CD8, this peptide was tested in TCR CD82/2 FTOCs. As shown in Figure 5A, TCR CD82/2 thymocytes cultured with high concentrations of p33 or A3V (1026 M) underwent clonal deletion. This was characterized by a dramatic reduction in the percentage of CD42Va2hi and CD41 Va2int thymocytes and a decrease in absolute cell numbers. Previous experiments have demonstrated that clonal deletion occurs during high-avidity TCR–peptide/MHC interactions. Therefore, these current results suggest that the TCR– A3V/H-2D b interaction is strong enough to provide sufficient avidity for clonal deletion to occur at high peptide concentrations. If A4Y-induced positive selection was due simply to

Figure 2. Thymocytes Expressing High Levels of the Transgenic Receptor Are Mature Positive control TCR CD81/1 or TCR CD82/2 thymic lobes cultured with A4Y were stained with antibodies specific for CD4, Va2, and HSA or PNA. HSA and PNA profiles are shown from gated CD41Va2int, CD42Va2int, or CD42Va2hi cells. Similar results were obtained when these experiments were repeated three times.

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Table 2. Transgenic Thymocyte Numbers from FTOCs Treated with or without A4Y

CD81

TCR TCR CD82/2 no peptide TCR CD82/2 A4Y (1026 M)

Total Thymocyte Number

CD42Va2hi

7.4 6 0.5 3 10 7.3 6 1.7 3 10 5 10 6 1.7 3 10 5

1.9 6 0.1 3 10 5 1.0 6 0.2 3 10 5 2.4 6 0.4 3 10 5

5

n55 n56 n55

Data are shown 6 SD.

the peptide increasing thymocyte–stromal cell avidity to compensate for absent coreceptors, then nondeleting concentrations of A3V should have had a similar effect. Instead, as shown in Figure 5B, the highest concentration of A3V (1028 M) that did not induce detectable amounts of clonal deletion, had no effect on T cell development. Low concentrations of A3V neither positively nor negatively selected transgenic thymocytes. This is in marked contrast to similar concentrations of A4Y, which resulted in positive selection. Even lower concentrations of A3V (1029 –10212 M) also had no effect on thymocyte selection (data not shown), demonstrating that potential TCR transgenic positive selection was not being masked by clonal deletion. For this reason, we suggest that TCR CD82/2 thymocyte maturation is peptide specific. Specific Proliferative Response of Positively Selected TCR CD82/ 2 Thymocytes Proliferation assays were conducted to determine whether TCR CD82/2 thymocytes selected in the presence of A4Y were functional. Mature CD42Va2hi T cells from TCR CD82/ 2 thymic lobes cultured with A4Y and TCR CD81 controls both proliferated in response to

Figure 3. The Peptide A4Y Induces Positive Selection of TCR CD82/2 Thymocytes over a Wide Concentration Range TCR CD82/2 thymic lobes were cultured with different concentrations of A4Y and analyzed with antibodies specific for CD4 and Va2. CD42Va2hi cell numbers are included for this experiment. These data are representative of three experiments.

APCs coated with p33 (Figure 6A). Positively selected TCRhi CD82/2 thymocytes did not respond as vigorously as the TCR CD81 controls, consistent with the previous anti-CD8 mAb proliferation experiments. Neither Va2hi CD81 nor Va2 hi CD82/2 thymocytes proliferated when cultured with a control H-2D b–restricted adenovirus peptide, AV, demonstrating that proliferation was peptide specific. As expected, APCs coated with A4Y induced moderate levels of TCRhi CD81 proliferation. In contrast, TCR CD82/2 thymocytes that were positively selected with this peptide did not respond against A4Y. To determine whether TCR proliferative responses were dependent on CD8, proliferation assays were done using splenocytes from TCR CD82/2 mice. Mature TCR CD82/2 T cells could proliferate in response to p33 but not A4Y (Figure 6B), indicating that A4Y-induced proliferation was CD8 dependent. Therefore, a lack of TCR CD82/2 thymocyte proliferation was expected when stimulated with A4Y. As a result, we could not distinguish between inherent unresponsiveness due to a lack of coreceptors and tolerance induced by the selecting ligand. However, we conclude that A4Y promoted TCR CD82/ 2 thymocyte maturation of functional T cells. Mature TCR CD8 2/ 2 Thymocytes Were Committed to the Cytotoxic Lineage Having established that TCR CD82/2 thymocytes positively selected in the presence of A4Y were functional, we wanted to determine whether these transgenic cells were committed to the cytotoxic lineage. Previous in vivo experiments have demonstrated that single-positive mature CD81 cytotoxic thymocytes characteristically expressed high levels of perforin, in contrast to the low levels expressed by mature CD41 helper cells (Vandekerckhove et al., 1994). Therefore, we examined mature transgenic thymocytes for the presence of intracellular perforin. To ensure that only positively selected transgenic thymocytes were being analyzed, thymic T cells were initially sorted for high surface expression of the transgenic TCR. These lymphocytes were then stained for intracellular perforin. As shown in Figure 7, the majority of CD42Va2hi T cells from fetal thymic lobes cultured in the presence of A4Y positively stained for perforin. Likewise, Va2hi thymocytes from an adult positive control TCR RAG2/2 thymus expressed similar high levels of intracellular perforin. Mature thymocytes from a CD82/ 2 thymus, which lack a significant cytotoxic T cell population (Fung-Leung et al., 1991), were used as a negative control. These thymocytes clearly lacked high levels of perforin. Since positively selected CD42Va2hi thymocytes expressed perforin, this experiment demonstrated these thymocytes were committed to the cytotoxic lineage in the absence of CD8.

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Figure 4. The H-2Db–Binding Peptide A3V Can Activate Mature T cells and Induce Intermediate Levels of Proliferation Relative to p33 and A4Y (A) Peptides were tested for their ability to bind and rescue H-2D b surface expression on RMA-S cells relative to a control peptide, LCMV nucleoprotein 118–127 (H-2Dd restricted). RMA-S cells were cultured with serial dilutions of p33 (squares), A3V (circles), A4Y (triangles), or AV (diamonds). (B) The proliferative response of mature spleen cells from TCR transgenic mice was measured when stimulated with H-2b spleen cells pulsed with various concentrations of p33 (squares), A3V (circles), or A4Y (triangles) in the presence or absence of anti-CD8 blocking antibody. Background proliferation was less than 100 cpm. This experiment was repeated four times, with similar results.

Discussion Various experiments have demonstrated that the CD4 and CD8 coreceptors play an important role during T cell maturation. Although the coreceptors have an immediate effect on many aspects of thymocyte development, the possibility remains that these molecules are not absolutely required for lineage commitment or thymocyte selection. Indeed, previous reports have shown that processes such as negative selection (Wallace et

al., 1992; Fung-Leung et al., 1993; Penninger et al., 1995) and the development of helper or cytotoxic effector functions (Fung-Leung et al., 1991; Rahemtulla et al., 1991; Locksley et al., 1993; Rahemtulla et al., 1994; Dalloul et al., 1996) can occur independently of coreceptors. For this reason, we wanted to determine whether MHC class I–restricted transgenic thymocytes could be positively selected in a peptide-specific manner without CD8 coreceptors. TCR transgenic thymocytes specific for LCMV in the context of H-2Db were positively selected

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Figure 6. TCR CD82/2 Thymocytes Selected in the Presence of A4Y Proliferate in Response to Strong Agonists

Figure 5. The Peptide A3V Can Induce Clonal Deletion but Not Positive Selection of TCR CD82/2 Thymocytes (A) TCR CD82/2 thymocytes were clonally deleted using high concentrations of A3V or p33. Thymocytes from TCR CD82/2 fetal lobes cultured with or without p33 or A3V were stained with antibodies specific for CD4 and Va2. CD42Va2hi thymocyte numbers are included for this experiment. Similar results were obtained when this experiment was repeated three times. (B) Positive selection of transgenic thymocytes is detected in the presence of A4Y but not A3V. TCR CD82/2 thymocytes cultured in the presence of A3V or A4Y were stained with CD4 and Va2 specific antibodies. Again, CD42Va2hi thymocyte numbers are shown. Data are representative of five experiments.

to the CD8 lineage. In the absence of this coreceptor, mature transgenic thymocyte numbers were greatly diminished, emphasizing the role of CD8 during positive selection. However, when TCR CD82/2 thymic lobes were cultured with the peptide A4Y, efficient positive selection of transgenic thymocytes was restored. High expression of intracellular perforin confirmed that these cells were committed to the cytotoxic lineage. In addition, these positively selected TCR CD82/2 thymocytes were able to respond against the strong agonist p33, confirming that these cells were functional. Fetal thymic organ cultures containing high concentrations of p33 or

(A) Positively selected CD42Va2hi thymocytes from TCR CD82/2 thymic lobes cultured with A4Y (open bars) or CD81Va2hi cells from TCR CD81/1 thymic lobes cultured in media alone (closed bars) were stimulated with peptide-coated APC. Background proliferation was 200 cpm. These results are representative of four experiments. (B) Mature transgenic T cells from TCR CD82/2 mice cannot proliferate in response to A4Y. Spleen cells from TCR CD82/2 mice were stimulated with APCs prepulsed with p33 (squares) or A4Y (triangles). Background proliferation was 800 cpm.

A3V induced TCR CD82/2 clonal deletion, further demonstrating that CD8 is not required for negative selection. Therefore, these experiments demonstrate that CD8 coreceptors are not absolutely required to govern peptide-specific MHC class I–restricted thymocyte selection. One of the repercussions of these findings concerns early intracellular signaling following TCR engagement during thymocyte development. The cytoplasmic protein tyrosine kinase p56lck has been implicated in the initiation of kinase signaling pathways (Weiss and Littman, 1994). A member of the Src family of tyrosine kinases, lck noncovalently associates with the CD8a chain. Models predicted that coengagement of the TCR and CD8 by an MHC molecule would result in the recruitment of lck to the TCR complex, ultimately leading to the phosphorylation and activation of associated tyrosine kinases and downstream signaling. However, the present work supports previous findings which have suggested that coreceptor–lck association is not essential for T cell development. Although lck has an important role during thymocyte ontogeny, it does not have to be

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Figure 7. Positively Selected TCR CD82/2 Thymocytes Express Perforin CD42Va2hi thymocytes from TCR CD82/2 thymic lobes cultured with A4Y or CD81Va2hi thymocytes from TCR RAG2/2 and CD41Va2hi thymocytes from CD82/2 mice were stained with perforin-specific mAb. This experiment was repeated three times.

recruited via CD8 to participate in TCR-induced tyrosine phosphorylation pathways. Indeed, the effective selection of transgenic thymocytes in the absence of CD8 demonstrated that this coreceptor does not contain intrinsic properties that are crucial for T cell maturation. Furthermore, since the CD8a chain is required for CD8b surface expression, this study demonstrates that thymocyte development can occur in the absence of both the CD8 a and b chains. Therefore, CD8 does not play an obligatory role in intracellular signaling or lineage commitment during thymocyte development. If the CD8 coreceptor is not strictly required for intracellular signaling during thymocyte development, then perhaps its main function is to stabilize TCR–MHC class I interactions. Efficient TCR transgenic positive selection required CD8 binding in addition to endogenous peptide–TCR interactions, suggesting that natural endogenous ligands had too low an affinity or were present at too low a concentration for transgenic thymocytes to survive without additional avidity provided by coreceptors. Theoretically, A4Y could have induced transgenic positive selection by increasing the number of TCR– peptide/MHC interactions and therefore restoring thymocyte avidity. However, experiments using A3V suggested that this was not the case. This peptide variant had sufficient affinity to induce clonal deletion, and yet lower concentrations of A3V did not induce efficient positive selection. If peptide-induced positive selection was simply a means of compensating for lost avidity associated with the CD8 coreceptor, then low concentrations of A3V should have promoted TCR CD82/2 maturation. Since A3V could not produce a similar phenotype as A4Y, these results argue that efficient TCR

CD82/2 positive selection was in fact a peptide-specific process that occurred independently of coreceptors. We believe that A4Y was not simply “helping” a variety of naturally occurring CD8-dependent positively selecting peptides but was instead providing distinct ligands which promoted transgenic thymocyte maturation. The positively selecting peptide A4Y was able to interact with the transgenic TCR in a manner that A3V was not able to duplicate. One interpretation of these results incorporates an efficacy model of thymocyte selection (Mannie, 1991). A peptide that is able to generate a TCRmediated biological function is described as having efficacy. According to this model, positively selecting ligands lack efficacy, whereas negatively selecting peptides are capable of stimulating T cell activity. Antagonist peptides, which by definition lack efficacy, have been shown to induce positive selection (Hogquist et al., 1994; Jameson et al., 1994; Hogquist et al., 1995). The current study suggested that the peptide A4Y did not have efficacy in the absence of CD8, yet was able to mediate positive selection. A3V was able to stimulate T cell proliferation in a relatively CD8-independent manner and induced clonal deletion at high concentrations. Although these data are compatible with an efficacy model, previous studies conflict with this interpretation of thymocyte development. Using the LCMV-specific TCR b2m2/2 mouse model, we have demonstrated that A4Y can positively select thymocytes at concentrations sufficient to induce TCR b2 m1 or TCR b2m2/ 2 T cell activity (Sebzda et al., 1996; E. S., unpublished data). Therefore, there is no direct correlation between the ability of A4Y to induce positive selection and its ability to stimulate T cell activity. These results are further supported by experiments showing that positively selecting ligands do not have to act as antagonists (Ignatowicz et al., 1996). In addition, several groups have demonstrated that antagonist peptides can block thymocyte selection (Spain et al., 1994; Williams et al., 1996) or promote clonal deletion (Page et al., 1994). For these reasons, we do not favor an efficacy model of thymocyte selection. An alternative interpretation of these experiments is that, unlike A3V, A4Y was capable of inducing a conformational change within the transgenic TCR that was optimal for positive selection. It has been postulated that this conformational change stimulates incomplete TCR-mediated signaling, resulting in positive selection (Janeway, 1994, 1995). However, experiments have demonstrated that A4Y can activate mature TCR transgenic T cells, suggesting that this ligand induces a TCR conformation capable of providing complete signals. Since high concentrations of A4Y can promote TCR transgenic positive selection (Sebzda et al., 1996), this argues against a conformational model that relies on incomplete TCR signaling for thymocyte development. At the same time, we do not rule out the possibility that A4Y has some unique features distinct from its properties as a weak agonist. Instead, we suggest that TCR-peptide/MHC affinity and thymocyte–stromal cell avidity are at least two components that regulate T cell development. Recent studies demonstrated that specific transgenic TCRs have a low affinity for positively selecting ligands (Alam et al., 1996). Therefore, it is possible that efficient T cell maturation could not be detected using A3V because of a

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Figure 8. Potential Relationship between Thymocyte Stimulation and Increasing Concentrations of Specific Peptides, in an Affinity/ Avidity Model of Thymocyte Selection Positive and negative selection thresholds are defined by the accumulation of TCR-mediated signaling, which in turn is primarily governed by thymocyte–stromal cell avidity. The rate at which thymocyte signaling increases is subject to the affinity of the specific peptide. High-affinity ligands promote high rates of thymocyte stimulation at low peptide concentrations. This results in a narrow range of peptide concentrations during which positive selection can occur. In contrast, low-affinity peptides can induce positive selection over a larger range of peptide concentrations because of the reduced rate of thymocyte signaling generated by this ligand.

relatively high-affinity interaction with the transgenic TCR. In contrast, A4Y had sufficiently low affinity to promote positive selection. Current affinity/avidity models postulate that positive and negative selection events have defined avidity thresholds and that higher-avidity conditions are required for clonal deletion than positive selection (Sprent et al., 1988). The accumulation of signals within a certain time frame, which is primarily governed by thymocyte avidity, define these thresholds. High concentrations of A3V or p33 contributed enough avidity to induce negative selection. At low peptide concentrations, this threshold was not met, resulting in an absence of clonal deletion. Previous studies have shown that p33 induced positive selection at lower peptide concentrations and over a much narrower range than A4Y (Ashton-Rickardt et al., 1994; Sebzda et al., 1994, 1996), consistent with the view that the rate of transgenic thymocyte stimulation is subject to the affinity of specific peptides (Figure 8). Since A3V had a relatively high affinity, as demonstrated in anti-CD8 proliferation assays, this ligand was expected to promote positive selection over a limited span of peptide concentrations. It is likely that this potential window for positive selection was too small for detection in FTOC, possibly because of background levels of thymocyte maturation induced by endogenous ligands. This contrasts with A4Y-induced positive selection which was easily observed over a broad range of peptide concentrations. This low-affinity ligand did not change thymocyte stimulation as dramatically as A3V and thus was able to provide a positively selecting environment over a larger range of peptide concentrations. This interpretation is supported by the proliferative response of TCR transgenic spleen cells

cultured with A4Y-coated APCs (Figure 4B). There was little variation in terms of absolute proliferation over an extended range of A4Y peptide concentrations. Together, these results suggest that both TCR-peptide/ MHC affinity and avidity regulate T cell development, consistent with an affinity/avidity model of thymocyte selection. The ability to positively select transgenic thymocytes in a coreceptor-independent manner has direct implications for lineage commitment. An instructive model proposes that TCR engagement initiates distinct intracellular signals that direct thymocytes to the appropriate lineage according to the restricting element. Thymocytes bearing MHC class I–specific TCRs down-regulate CD4 expression while MHC class II–specific T cells terminate CD8 expression. An alternative model postulates that the initial signal through the TCR prompts thymocytes to down-regulate randomly one of the coreceptors. According to this stochastic model, properly matched TCRs and coreceptors allows thymocytes to survive. If this model was correct and thymocytes randomly down-regulate coreceptors, then we should have been able to detect a substantial number of CD41Va2hi T cells. Instead, we were only able to observe a mature CD42Va2hi thymocyte population, suggesting that CD4 coreceptors were actively being down-regulated in an instructive manner. A recent study using MHC class II–restricted transgenic T cells has shown that under certain circumstances, mature cells may be selected from a CD42CD82 population without passing through a double-positive stage (Liu et al., 1996). However, using this LCMV-specific TCR transgenic mouse model, it is possible to follow thymocyte maturation through the upregulation of the transgenic TCR (Ohashi et al., 1990) The lack of a consistent, substantial CD42 population continuously increasing its transgenic TCRs versus the presence of such a population expressing CD4 suggests that in this system, thymocytes proceeded through a CD41 stage prior to maturation (Figure 1B). These findings correlate with experiments following thymocyte maturity markers. Thymocytes stained with HSA and PNA indicated that CD42Va2 int thymocytes were more mature than CD41Va2int cells, suggesting that T cells down-regulated CD4 as they progressed towards a CD42Va2hi stage (Figure 2). At the same time, we cannot conclusively rule out the possibility that at least some thymocytes bypassed a CD41 stage during T cell maturation. For those cells that did express CD4, it is possible that TCR transgenic thymocytes either directly signaled CD4 termination through MHC class I–restricted TCRs or, alternatively, down-regulated these coreceptors as a result of CD4 not being able to participate during linage commitment. These results support recent studies that have proposed an instructive mechanism to CD4 downregulation (Suzuki et al., 1995; Lucas et al., 1996). Since this instructive mechanism did not require coengagement of CD8 with the TCR, these current findings suggest that MHC class I– restricted T cells down-regulate CD4 expression in accordance with a coreceptor-independent instructive model. These experiments demonstrate that positive and negative selection can occur in the absence of CD8, raising into question the exact role of this coreceptor

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during T cell development. Clearly CD8 is important during positive selection since mature transgenic thymocyte numbers are greatly reduced in the absence of this coreceptor. However, these studies have shown that it is possible to reestablish a substantial transgenic thymocyte population by culturing fetal lobes with a positively selecting peptide, indicating that CD8 is not essential for thymocyte development or lineage commitment. Instead, this coreceptor functions as an adaptor molecule, providing a means for maturing thymocytes to adjust to their thymic environment. CD8 molecules may help stabilize low-affinity TCR–endogenous peptide/ MHC interactions, providing a suitable threshold for ‘‘natural’’ thymocyte selection. In the absence of these coreceptors, thymocyte selection is limited to a small subset of ligands capable of efficient TCR stimulation. Therefore, CD8 coreceptors expand the TCR repertoire by extending the potential body of positively selecting ligands to include peptides with transient TCR interactions. The CD8 coreceptor also expands the potential antigen repertoire, allowing T cells to respond against low-affinity peptides. Although CD8 molecules are not strictly required for transgenic T cell maturation or differentiation, the coreceptors enhance the efficiency of these processes. Experimental Procedures Mice TCR-transgenic mice were previously generated using a and b chains isolated from CTL clone P14, which recognized the LCMV glycoprotein (peptide p33–41) presented by H-2Db. This line was backcrossed with H-2b CD82/2 mice to obtain TCR CD82/2 (H-2b) animals. Progeny were typed for transgenic TCR and homozygous CD8 disruption by staining peripheral blood with mAbs at 48C in phosphate-buffered saline (PBS) containing 2% NaN3, and 20 mM EDTA. TCR-transgenic cells were detected with rat anti-mouse Vb8.1 (KJ16) mAb followed by fluorescein isothiocyanate (FITC)– conjugated goat anti-rat Mab (Tago, Burlingame, CA). CD82/2 mice were screened using rat anti-mouse CD8 (YTS 169.4) mAb followed by FITC-conjugated goat anti-rat mAb. After the second antibody incubation period, red blood cells were lysed using 13 FACS lysing solution (Becton Dickinson, Mountain View, CA). TCR RAG2/2 (H-2b) mice were obtained as described previously (Kawai and Ohashi, 1995). FTOCs Timed breedings were established between TCR CD82/2 H-2b males and CD82/2 H-2b females. At day 16 of gestation, females were sacrificed, and thymic lobes were removed from the fetuses. DNA was extracted from embryonic tails so that transgenic fetuses could be determined using primers specific for the Va2 TCR transgene (Sebzda et al., 1996). The fetal thymic lobes were placed on 0.8 mm polycarbonate filters (Costar, Cambridge, MA), which floated on 1 ml of Iscove’s modified Dulbecco’s medium (IMDM), 13 NutridomaSP (Boehringer Mannheim, Indianapolis, IN), 5 3 10 25 M 2-mercaptoethanol, penicillin, streptomycin, 2 mM glutamine, and designated peptides. These lobes were then cultured for 6 days at 378C, during which time the medium was changed daily. After this incubation period, the thymic lobes were teased apart and stained with mAbs at 48C in PBS containing 2% fetal calf serum (FCS) and 0.2% NaN3. Three-color analysis was done with rat anti-mouse FITC-conjugated anti-CD4 (PharMingen, San Diego, CA), phycoerythrin (PE)– conjugated anti-Va2 (B20.1) (PharMingen), and biotinylated antiHSA (M1/69) (PharMingen), or FITC-conjugated anti-PNA (Sigma Chemical, St. Louis, MO), PE-conjugated anti-CD4 (PharMingen), and biotinylated anti-Va2 (B20.1) (PharMingen). Biotinylated antibodies were detected with streptavidin-red 670 (GIBCO–BRL, Gaitherburg, MD).

Flow Cytometry All flow cytometric analysis was performed on a FACScan instrument (Becton Dickinson). Samples were gated for live cells based on forward and side scatter parameters (10,000 events/sample) and analyzed using Lysis II software (Becton Dickinson). Peptides The peptides p33 (KAVYNFATM), A3V (KAAYNFATM), A4Y (KAVANFATM), and adenovirus peptide AV (SGPSNTPPEI) were synthesized, purified, and characterized at the Amgen Institute as previously described (Sebzda et al., 1996). Peptide-Binding Assay In peptide-pulsing experiments, 10 6 RMA-S cells, which were previously cultured overnight at 298C in RPMI plus 10% FCS, were incubated with various concentrations of peptide at 298C for 30 min. These cells were then transferred to a 378C incubator for 3 hr, after which time the cells were washed and stained with anti-H-2D b mAb from tissue culture supernatant (B22.249) (Hammerling et al., 1979; Allen et al., 1986) and then FITC-conjugated rat anti-mouse immunoglobulin (Sigma Chemical). RMA-S cells were incubated with LCMV nucleoprotein 118–127 (H-2d restricted) (von Herrath et al., 1994) to determine background H-2Db expression. Proliferation Assays TCR transgenic spleen cells (105 /well), cultured previously with or without various dilutions of anti-CD8 mAb (YTS 169.4) for 1 hr at 378C, were incubated in triplicate in 96-well flat-bottom plates with 2 3 104 /well irradiated C57B1/6J (H-2b) splenocytes that had been prepulsed with various concentrations of peptide for 3 hr at 378C. After 48 hr of cocultivation, the cells were pulsed with 1 mCi of [ 3H]thymidine (Amersham, Arlington Heights, IL) for 16 hr. Cells were harvested and counted on a direct b counter (Matrix96, Canberra Packard Canada, Mississauga, Ontario, Canada). FTOC Proliferation Assay Cultured thymic lobes were teased apart and cultured with 1 ml of anti-HSA (J11d) and 400 ml of Low-Tox-M rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) for 45 min at 378C. Irradiated spleen cells from a C57B1/6J mouse were prepulsed with 10 28 M AV, A4Y, or p33 for 3 hr at 378C; washed; and distributed in triplicate on a flat-bottom 96-well plate at a concentration of 10 5 cells/well. Mature thymocytes (3 3 104/well) resuspended in IMDM, 10% FCS, penicillin, streptomycin, and 5 3 10 25 M 2-mercaptoethanol were then added to these wells. The cells were cultured at 378C for 72 hr, pulsed with 1 mCi of [3 H]thymidine for 16 hr, and harvested as described. Intracellular Perforin Staining Thymic cell suspensions were initially stained with rat anti-mouse PE-conjugated anti-Va2 (B20.1) (PharMingen). These cells were then sorted using a FACStar Plus instrument (Becton Dickinson) to collect Va2hi thymocytes. The thymocytes were fixed in 4 ml 70% ethanol (2208C) and incubated on ice for 30 min. Following this incubation period, the cells were pelleted and resuspended in 0.5% HIO 3. Ten minutes later, the cells were washed and incubated with 1% bovine serum albumin–PBS for 30 min. The cells were then washed and stained with rat anti-mouse perforin antibody (PI-8 ascitic fluid 1:500) (kindly provided by Dr. K. Okumura) for 1 hr. The thymocytes were subsequently washed and stained with FITC-conjugated goat anti-rat mAb (Tago, Burlingame, CA) for 1 hr. Acknowledgments We thank Dr. K. Okumura for the rat anti-mouse perforin antibody and D. Bouchard for cell sorting. This work was supported by the Medical Research Council (MRC) of Canada. P. S. O. is a recipient of an MRC Scholarship. Received February 4, 1997; revised April 7, 1997.

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