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

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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,† of the TCR and coreceptor to an MHC molecule conveys Tak W. Mak,*‡ 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 † 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 CD8␣ cytoplasmic domain have shown a defect in ‡ Amgen Institute MHC class I–restricted positive selection, suggesting 620 University Ave. that an intracellular portion of CD8␣ is involved in thymo- Toronto, Ontario M5G 2C1 cyte development (Fung-Leung et al., 1993). Recent Canada work has also implicated the CD8␤ 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 CD8Ϫ/Ϫ 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). CD4ϪTCRhi 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, CD4ϩCD8ϩ double-positive thymocytes differenti- low et al., 1988; Sha et al., 1988; Teh et al., 1988; Berg ate into CD4ϩ or CD8ϩ 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 TCRϩCD8tg transgenic mice participation remains unclear. The protein tyrosine ki- did not rescue CD4ϩ 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 CD4ϩCD8loTCRint thymocytes were found in mice lacking class II, suggesting that lineage commitment was a random event (Chan et al., 1993). Likewise, a CD4loCD8ϩTCRint 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 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 (V␣2/V␤8.1) specific for the lympho- duced in CD8-deficient mice. Thymocytes from TCR CD8ϩ/ϩ and cytic choriomeningitis virus (LCMV) peptide (KAVYN- TCR CD8Ϫ/Ϫ mice were triple stained with antibodies specific for FATC) presented by H-2Db. Thymocyte maturation in CD8, CD4, and V␣2. Total thymocytes are shown for dot plots and this mouse model was characterized by a skewing to histograms. the CD8ϩ lineage and high surface expression of the (B) The peptide A4Y efficiently induces transgenic positive selection Ϫ/Ϫ Ϫ/Ϫ transgenic TCR (Figure 1A). Peripheral CD8ϩ 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 V␣2. TCR CD8ϩ/ϩ lobes hi Ϫ/Ϫ very few TCR thymocytes were detected in TCR CD8 were used as a positive control. This experiment was repeated eight mice, although a few CD4ϪCD8Ϫ or CD4ϩCD8Ϫ 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 CD4ϪV␣2hi 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 CD8Ϫ/Ϫ mice were cultured trast, CD4ϩV␣2int 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 CD4ϩCD8ϩ double-positive thymocytes. TCR CD8ϩ fetal thymic organ cultures (FTOCs), these CD4ϪV␣2int thymocytes expressed intermediate levels CD8-deficient lobes had a substantially reduced of maturity markers relative to CD4ϩV␣2int and CD4ϪV␣2hi CD4ϪV␣2hi transgenic cell population (Figure 1B). How- cells, suggesting that transgenic thymocytes pro- ever, TCRhi transgenic thymocyteswere recovered when gressed from a CD4ϩ to a CD4Ϫ stage prior to becoming TCR CD8Ϫ/Ϫ fetal thymic lobes were cultured in the pres- mature, CD4ϪV␣2hi cells. Together, these results con- ence of the peptide variant, A4Y (KAVANFATM), which firmed that CD4ϪV␣2hi thymocytes were a mature T cell has previously been shown to positively select TCR population that had undergone positive selection. CD8ϩ thymocytes (Sebzda et al., 1996). These results The CD4ϪV␣2hi cell numbers summarized in Table 2 indicate that CD8 coreceptors are an important, but not demonstrate the relative importance of CD8 during posi- mandatory, component of T cell development. tive selection. Thymic lobes lacking this coreceptor had To ensure that these CD4ϪV␣2hi thymocytes had un- a reduced ability to produce mature thymocytes ex- dergone positive selection, T cells from TCR CD8Ϫ/Ϫ pressing the transgenic TCR. Addition of A4Y compen- organ cultures treated with A4Y or from TCR CD8ϩ sated for the absent coreceptor and provided adequate Coreceptor-Independent T Cell Selection 645

Table 1. Transgenic Splenocyte Numbers from TCR CD8ϩ and TCR CD8Ϫ/Ϫ Littermates

CD8ϩV␣2hi CD4ϩV␣2hi CD8ϪCD4ϪV␣2hi TCR CD8ϩ 1.9 Ϯ 0.3 ϫ 107 1.2 Ϯ 0.2 ϫ 106 1.2 Ϯ 0.3 ϫ 106 n ϭ 4 TCR CD8Ϫ/Ϫ 0 5.6 Ϯ 0.5 ϫ 106 4.1 Ϯ 0.4 ϫ 106 n ϭ 4

Data are shown Ϯ SD.

stimulation to positively select CD4ϪV␣2hi thymocytes. peptide concentrations (10Ϫ4–10Ϫ5 M), A3V induced pro- The efficiency of A4Y-induced positive selection was liferation to the same degree as p33. At lower concentra- examined by testing this peptide in TCR CD8Ϫ/Ϫ FTOCs tions (10Ϫ7–10Ϫ11 M), A3V and A4Y induced almost identi- at various concentrations. A titration profile of A4Y (Fig- cal amounts of proliferation. ure 3) showed that positive selection occurred over a To determine the relative importance of CD8 in pep- broad concentration range, extending from 10Ϫ5 to 10Ϫ9 tide-specific interactions, TCR transgenic splenocytes M. Together, these results demonstrated that A4Y effi- were stimulated with peptide-coated APCs in the pres- ciently induced positive selection of transgenic thymo- ence of anti-CD8 monoclonal antibodies (Figure 4B). cytes in a coreceptor-independent manner. High antibody concentrations inhibited T cell proliferation in response to all of the peptides, although not to Peptide-Specific Positive Selection the same extent. A4Y did not induce T cell proliferation. of TCR CD8Ϫ/Ϫ Thymocytes In contrast, reduced proliferation was seen with p33 and Although A4Y positively selected TCR CD8Ϫ/Ϫ thymo- A3V. Notably, A3V did not seem to be as affected by the cytes, it was possible that this peptide was simply com- blocking antibody as p33. Similar proliferation patterns pensating for lost avidity due to a lack of coreceptors, were observed with lower concentrations of anti-CD8. and not providing a unique TCR ligand that could pro- In general, anti-CD8 antibody inhibited transgenic T cell mote positive selection. For this reason, we searched proliferation in response to all of the peptides tested; for peptide variants that were similar in nature to A4Y however, A3V and p33 were much more efficient at stim- to determine whether these peptides could efficiently ulating CD8-independent proliferation than A4Y. These induce positive selection. Initially we tested potential assays indicated that TCR–A3V/MHC interactions were peptides to ensure that they bound to H-2Db molecules. stronger and less CD8-dependent than those involving Using TAP-2 (transporter associated with antigen pro- A4Y. Hence, these results suggested that A3V was at cessing type 2)–deficient RMA-S cells, type 2, we were least as potent an agonist peptide as A4Y. able to demonstrate that each of the relevant peptides To determine whether A3V could induce positive se- rescued H-2Db surface expression (Figure 4A). lection in the absence of CD8, this peptide was tested Since these peptides could bind to H-2Db, we further in TCR CD8Ϫ/Ϫ FTOCs. As shown in Figure 5A, TCR tested these ligands for their ability to stimulate CD8Ϫ/Ϫ thymocytes cultured with high concentrations transgenic T cells. Previous experiments revealed that of p33 or A3V (10Ϫ6 M) underwent clonal deletion. This A4Y, an alanine-variant of the wild-type peptide, was an was characterized by a dramatic reduction in the per- agonist with no detectable antagonist properties (Seb- centage of CD4ϪV␣2hi and CD4ϩ V␣2int thymocytes and zda et al., 1996). As shown in Figure 4B, antigen-pres- a decrease in absolute cell numbers. Previous experi- enting cells (APCs) pulsed with various concentrations ments have demonstrated that clonal deletion occurs of this modified peptide induced TCR CD8ϩ splenocyte during high-avidity TCR–peptide/MHC interactions. proliferation. Similar concentrations of the strong pep- Therefore, these current results suggest that the TCR– tide agonist, p33 (KAVYNFATM), resulted in increased A3V/H-2Db interaction is strong enough to provide suffi- proliferation. However, transgenic splenocytes stimu- cient avidity for clonal deletion to occur at high peptide lated with the peptide variant, A3V (KAAYNFATM), pro- concentrations. liferated at rates common to both peptides. At high If A4Y-induced positive selection was due simply to

Figure 2. Thymocytes Expressing High Levels of the Transgenic Receptor Are Mature Positive control TCR CD8ϩ/ϩ or TCR CD8Ϫ/Ϫ thymic lobes cultured with A4Y were stained with antibodies specific for CD4, V␣2, and HSA or PNA. HSA and PNA profiles are shown from gated CD4ϩV␣2int, CD4ϪV␣2int, or CD4ϪV␣2hi cells. Similar results were obtained when these experiments were repeated three times. Immunity 646

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

Total Thymocyte Number CD4ϪV␣2hi TCR CD8ϩ 7.4 Ϯ 0.5 ϫ 105 1.9 Ϯ 0.1 ϫ 105 n ϭ 5 TCR CD8Ϫ/Ϫ no peptide 7.3 Ϯ 1.7 ϫ 105 1.0 Ϯ 0.2 ϫ 105 n ϭ 6 TCR CD8Ϫ/Ϫ A4Y (10Ϫ6 M) 10 Ϯ 1.7 ϫ 105 2.4 Ϯ 0.4 ϫ 105 n ϭ 5

Data are shown Ϯ SD.

the peptide increasing thymocyte–stromal cell avidity to APCs coated with p33 (Figure 6A). Positively selected compensate for absent coreceptors, then nondeleting TCRhi CD8Ϫ/Ϫ thymocytes did not respond as vigorously concentrations of A3V should have had a similar effect. as the TCR CD8ϩ controls, consistent with the previous Instead, as shown in Figure 5B, the highest concentra- anti-CD8 mAb proliferation experiments. Neither V␣2hi tion of A3V (10Ϫ8 M) that did not induce detectable CD8ϩ nor V␣2hi CD8Ϫ/Ϫ thymocytes proliferated when amounts of clonal deletion, had no effect on T cell devel- cultured with a control H-2Db–restricted adenovirus pep- opment. Low concentrations of A3V neither positively tide, AV, demonstrating that proliferation was peptide nor negatively selected transgenic thymocytes. This is specific. As expected, APCs coated with A4Y induced in marked contrast to similar concentrations of A4Y, moderate levels of TCRhi CD8ϩ proliferation. In contrast, which resulted in positive selection. Even lower concen- TCR CD8Ϫ/Ϫ thymocytes that were positively selected trations of A3V (10Ϫ9–10Ϫ12 M) also had no effect on with this peptide did not respond against A4Y. To deter- thymocyte selection (data not shown), demonstrating mine whether TCR proliferative responses were depen- that potential TCR transgenic positive selection was not dent on CD8, proliferation assays were done using being masked by clonal deletion. For this reason, we splenocytes from TCR CD8Ϫ/Ϫ mice. Mature TCR CD8Ϫ/Ϫ suggest that TCR CD8Ϫ/Ϫ thymocyte maturation is pep- T cells could proliferate in response to p33 but not A4Y tide specific. (Figure 6B), indicating that A4Y-induced proliferation was CD8 dependent. Therefore, a lack of TCR CD8Ϫ/Ϫ Specific Proliferative Response of Positively thymocyte proliferation was expected when stimulated Selected TCR CD8Ϫ/Ϫ Thymocytes with A4Y. As a result, we could not distinguish between Proliferation assays were conducted to determine inherent unresponsiveness due to a lack of coreceptors whether TCR CD8Ϫ/Ϫ thymocytes selected in the pres- and tolerance induced by the selecting ligand. However, ence of A4Y were functional. Mature CD4ϪV␣2hi T cells we conclude that A4Y promoted TCR CD8Ϫ/Ϫ thymocyte from TCR CD8Ϫ/Ϫ thymic lobes cultured with A4Y and maturation of functional T cells. TCR CD8ϩ controls both proliferated in response to Mature TCR CD8Ϫ/Ϫ Thymocytes Were Committed to the Cytotoxic Lineage Having established that TCR CD8Ϫ/Ϫ 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 CD8ϩ cytotoxic thymocytes characteristi- cally expressed high levels of perforin, in contrast to the low levels expressed by mature CD4ϩ 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 CD4ϪV␣2hi T cells from fetal thymic lobes cultured in the presence of A4Y positively stained for perforin. Likewise, V␣2hi thymocytes from an adult positive control TCR RAGϪ/Ϫ thymus expressed similar high levels of intracellular perforin. Mature thymocytes from a CD8Ϫ/Ϫ thymus, which lack a significant cytotoxic T cell population (Fung-Leung et al., 1991), were used as Figure 3. The Peptide A4Y Induces Positive Selection of TCR a negative control. These thymocytesclearly lacked high CD8Ϫ/Ϫ Thymocytes over a Wide Concentration Range levels of perforin. Since positively selected CD4ϪV␣2hi TCR CD8Ϫ/Ϫ thymic lobes were cultured with different concentrations of A4Y and analyzed with antibodies specific for CD4 and V␣2. thymocytes expressed perforin, this experiment demon- CD4ϪV␣2hi cell numbers are included for this experiment. These strated these thymocytes were committed to the cyto- data are representative of three experiments. toxic lineage in the absence of CD8. Coreceptor-Independent T Cell Selection 647

Figure 4. The H-2Db–Binding Peptide A3V Can Activate Mature T cells and Induce Inter- mediate Levels of Proliferation Relative to p33 and A4Y (A) Peptides were tested for their ability to bind and rescue H-2Db surface expression on RMA-S cells relative to a control peptide, LCMV nucleoprotein 118–127 (H-2Dd restricted). RMA-S cells were cultured with se- rial 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 al., 1992; Fung-Leung et al., 1993; Penninger et al., 1995) and the development of helper or cytotoxic effector Various experiments have demonstrated that the CD4 functions (Fung-Leung et al., 1991; Rahemtulla et al., and CD8 coreceptors play an important role during T 1991; Locksley et al., 1993; Rahemtulla et al., 1994; Dal- cell maturation. Although the coreceptors have an im- loul et al., 1996) can occur independently of coreceptors. mediate effect on many aspects of thymocyte develop- For this reason, we wanted to determine whether MHC ment, the possibility remains that these molecules are class I–restricted transgenic thymocytes could be posi- not absolutely required for lineage commitment or thy- tively selected in a peptide-specific manner without CD8 mocyte selection. Indeed, previous reports have shown coreceptors. TCR transgenic thymocytes specific for that processes such as negative selection (Wallace et LCMV in the context of H-2Db were positively selected Immunity 648

Figure 6. TCR CD8Ϫ/Ϫ Thymocytes Selected in the Presence of A4Y Proliferate in Response to Strong Agonists (A) Positively selected CD4ϪV␣2hi thymocytes from TCR CD8Ϫ/Ϫ thymic lobes cultured with A4Y (open bars) or CD8ϩV␣2hi cells from TCR CD8ϩ/ϩ 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 CD8Ϫ/Ϫ mice cannot proliferate in response to A4Y. Spleen cells from TCR CD8Ϫ/Ϫ mice were Figure 5. The Peptide A3V Can Induce Clonal Deletion but Not Posi- stimulated with APCs prepulsed with p33 (squares) or A4Y (trian- tive Selection of TCR CD8Ϫ/Ϫ Thymocytes gles). Background proliferation was 800 cpm. (A) TCR CD8Ϫ/Ϫ thymocytes were clonally deleted using high concentrations of A3V or p33. Thymocytes from TCR CD8Ϫ/Ϫ fetal lobes Ϫ/Ϫ cultured with or without p33 or A3V were stained with antibodies A3V induced TCR CD8 clonal deletion, further dem- specific for CD4 and V␣2. CD4ϪV␣2hi thymocyte numbers are in- onstrating that CD8 is not required for negative selec- cluded for this experiment. Similar results were obtained when this tion. Therefore, these experiments demonstrate that experiment was repeated three times. CD8 coreceptors are not absolutely required to govern (B) Positive selection of transgenic thymocytes is detected in the peptide-specific MHC class I–restricted thymocyte se- presence of A4Y but not A3V. TCR CD8Ϫ/Ϫ thymocytes cultured in the presence of A3V or A4Y were stained with CD4 and V␣2 specific lection. antibodies. Again, CD4ϪV␣2hi thymocyte numbers are shown. Data One of the repercussions of these findings concerns are representative of five experiments. early intracellular signaling following TCR engagement during thymocyte development. The cytoplasmic protein tyrosine kinase p56lck has been implicated in the to the CD8 lineage. In the absence of this coreceptor, initiation of kinase signaling pathways (Weiss and Litt- mature transgenic thymocyte numbers were greatly di- man, 1994). A member of the Src family of tyrosine ki- minished, emphasizing the role of CD8 during positive nases, lck noncovalently associates with the CD8␣ selection. However, when TCR CD8Ϫ/Ϫ thymic lobes chain. Models predicted that coengagement of the TCR were cultured with the peptide A4Y, efficient positive and CD8 by an MHC molecule would result in the recruit- selection of transgenic thymocytes was restored. High ment of lck to the TCR complex, ultimately leading to the expression of intracellular perforin confirmed that these phosphorylation and activation of associated tyrosine cells were committed to the cytotoxic lineage. In addi- kinases and downstream signaling. However, the pres- tion, these positively selected TCR CD8Ϫ/Ϫ thymocytes ent work supports previous findings which have sug- were able to respond against the strong agonist p33, gested that coreceptor–lck association is not essential confirming that these cells were functional. Fetal thymic for T cell development. Although lck has an important organ cultures containing high concentrations of p33 or role during thymocyte ontogeny, it does not have to be Coreceptor-Independent T Cell Selection 649

CD8Ϫ/Ϫ 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 TCR- mediated 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. Antago- nist 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. Figure 7. Positively Selected TCR CD8Ϫ/Ϫ Thymocytes Express Per- Although these data are compatible with an efficacy forin model, previous studies conflict with this interpretation CD4ϪV␣2hi thymocytes from TCR CD8Ϫ/Ϫ thymic lobes cultured with of thymocyte development. Using the LCMV-specific ϩ hi Ϫ/Ϫ ϩ hi Ϫ/Ϫ A4Y or CD8 V␣2 thymocytes from TCR RAG and CD4 V␣2 TCR ␤2m mouse model, we have demonstrated that thymocytes from CD8Ϫ/Ϫ mice were stained with perforin-specific A4Y can positively select thymocytes at concentrations mAb. This experiment was repeated three times. ϩ Ϫ/Ϫ sufficient to induce TCR ␤2m or TCR ␤2m T cell activity (Sebzda et al., 1996; E. S., unpublished data). Therefore, there is no direct correlation between the recruited via CD8 to participate in TCR-induced tyrosine ability of A4Y to induce positive selection and its ability phosphorylation pathways. Indeed, the effective selec- to stimulate T cell activity. These results are further sup- tion of transgenic thymocytes in the absence of CD8 ported by experiments showing that positively selecting demonstrated that this coreceptor does not contain in- ligands do not have to act as antagonists (Ignatowicz et trinsic properties that are crucial for T cell maturation. al., 1996). In addition, several groups have demonstrated Furthermore, since the CD8␣ chain is required for CD8␤ that antagonist peptides can block thymocyte selection surface expression, this study demonstrates that thymo- (Spain et al., 1994; Williams et al., 1996) or promote cyte development can occur in the absence of both the clonal deletion (Page et al., 1994). For these reasons, we CD8 ␣ and ␤ chains. Therefore, CD8 does not play an do not favor an efficacy model of thymocyte selection. obligatory role in intracellular signaling or lineage com- An alternative interpretation of these experiments is mitment during thymocyte development. that, unlike A3V, A4Y was capable of inducing a confor- If the CD8 coreceptor is not strictly required for intra- mational change within the transgenic TCR that was cellular signaling during thymocyte development, then optimal for positive selection. It has been postulated perhaps its main function is to stabilize TCR–MHC class that this conformational change stimulates incomplete I interactions. Efficient TCR transgenic positive selection TCR-mediated signaling, resulting in positive selection required CD8 binding in addition to endogenous pep- (Janeway, 1994, 1995). However, experiments have tide–TCR interactions, suggesting that natural endoge- demonstrated that A4Y can activate mature TCR nous ligands had too low an affinity or were present at transgenic T cells, suggesting that this ligand induces too low a concentration for transgenic thymocytes to a TCR conformation capable of providing complete sig- survive without additional avidity provided by corecep- nals. Since high concentrations of A4Y can promote tors. Theoretically, A4Y could have induced transgenic TCR transgenic positive selection (Sebzda et al., 1996), positive selection by increasing the number of TCR– this argues against a conformational model that relies peptide/MHC interactions and therefore restoring thy- on incomplete TCR signaling for thymocyte develop- mocyte avidity. However, experiments using A3V sug- ment. At the same time, we do not rule out the possibility gested that this was not the case. This peptide variant that A4Y has some unique features distinct from its had sufficient affinity to induce clonal deletion, and yet properties as a weak agonist. lower concentrations of A3V did not induce efficient Instead, we suggest that TCR-peptide/MHC affinity positive selection. If peptide-induced positive selection and thymocyte–stromal cell avidity are at least two com- was simply a means of compensating for lost avidity ponents that regulate T cell development. Recent stud- associated with the CD8 coreceptor, then low concen- ies demonstrated that specific transgenic TCRs have a trations of A3V should have promoted TCR CD8Ϫ/Ϫ mat- low affinity for positively selecting ligands (Alam et al., uration. Since A3V could not produce a similar pheno- 1996). Therefore, it is possible that efficient T cell matu- type as A4Y, these results argue that efficient TCR ration could not be detected using A3V because of a Immunity 650

cultured with A4Y-coated APCs (Figure 4B). There was little variation in terms of absolute proliferation over an extended range of A4Y peptide concentrations. To- gether, 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. Thymo- cytes bearing MHC class I–specific TCRs down-regulate CD4 expression while MHC class II–specific T cells ter- minate CD8 expression. An alternative model postulates that the initial signal through the TCR prompts thymo- Figure 8. Potential Relationship between Thymocyte Stimulation and Increasing Concentrations of Specific Peptides, in an Affinity/ cytes to down-regulate randomly one of the corecep- Avidity Model of Thymocyte Selection tors. According to this stochastic model, properly Positive and negative selection thresholds are defined by the accu- matched TCRs and coreceptors allows thymocytes to mulation of TCR-mediated signaling, which in turn is primarily gov- survive. If this model was correct and thymocytes ran- erned by thymocyte–stromal cell avidity. The rate at which thymo- domly down-regulate coreceptors, then we should have cyte signaling increases is subject to the affinity of the specific been able to detect a substantial number of CD4ϩV␣2hi peptide. High-affinity ligands promote high rates of thymocyte stim- T cells. Instead, we were only able to observe a mature ulation at low peptide concentrations. This results in a narrow range Ϫ hi of peptide concentrations during which positive selection can occur. CD4 V␣2 thymocyte population, suggesting that CD4 In contrast, low-affinity peptides can induce positive selection over coreceptors were actively being down-regulated in an a larger range of peptide concentrations because of the reduced instructive manner. A recent study using MHC class rate of thymocyte signaling generated by this ligand. II–restricted transgenic T cells has shown that under certain circumstances, mature cells may be selected from a CD4ϪCD8Ϫ population without passing through relatively high-affinity interaction with the transgenic a double-positive stage (Liu et al., 1996). However, using TCR. In contrast, A4Y had sufficiently low affinity to this LCMV-specific TCR transgenic mouse model, it is promote positive selection. Current affinity/avidity mod- possible to follow thymocyte maturation through the up- els postulate that positive and negative selection events regulation of the transgenic TCR (Ohashi et al., 1990) have defined avidity thresholds and that higher-avidity The lack of a consistent, substantial CD4Ϫ population conditions are required for clonal deletion than positive continuously increasing its transgenic TCRs versus the selection (Sprent et al., 1988). The accumulation of sig- presence of such a population expressing CD4 suggests nals within a certain time frame, which is primarily gov- that in this system, thymocytes proceeded through a erned by thymocyte avidity, define these thresholds. CD4ϩ stage prior to maturation (Figure 1B). These find- High concentrations of A3V or p33 contributed enough ings correlate with experiments following thymocyte avidity to induce negative selection. At low peptide con- maturity markers. Thymocytes stained with HSA and centrations, this threshold was not met, resulting in an PNA indicated that CD4ϪV␣2int thymocytes were more absence of clonal deletion. Previous studies have shown mature than CD4ϩV␣2int cells, suggesting that T cells that p33 induced positive selection at lower peptide down-regulated CD4 as they progressed towards a concentrations and over a much narrower range than CD4ϪV␣2hi stage (Figure 2). At the same time, we cannot A4Y (Ashton-Rickardt et al., 1994; Sebzda et al., 1994, conclusively rule out the possibility that at least some 1996), consistent with the view that the rate of transgenic thymocytes bypassed a CD4ϩ stage during T cell matu- thymocyte stimulation is subject to the affinity of specific ration. Forthose cells that did express CD4, it is possible peptides (Figure 8). Since A3V had a relatively high affin- that TCR transgenic thymocytes either directly signaled ity, as demonstrated in anti-CD8 proliferation assays, CD4 termination through MHC class I–restricted TCRs this ligand was expected to promote positive selection or, alternatively, down-regulated these coreceptors as over a limited span of peptide concentrations. It is likely a result of CD4 not being able to participate during linage that this potential window for positive selection was commitment. These results support recent studies that too small for detection in FTOC, possibly because of have proposed an instructive mechanism to CD4 down- background levels of thymocyte maturation induced by regulation (Suzuki et al., 1995; Lucas et al., 1996). Since endogenous ligands. This contrasts with A4Y-induced this instructive mechanism did not require coengage- positive selection which was easily observed over a ment of CD8 with the TCR, these current findings sug- broad range of peptide concentrations. This low-affinity gest that MHC class I– restricted T cells down-regulate ligand did not change thymocyte stimulation as dramati- CD4 expression in accordance with a coreceptor-inde- cally as A3V and thus was able to provide a positively pendent instructive model. selecting environment over a larger range of peptide These experiments demonstrate that positive and concentrations. This interpretation is supported by the negative selection can occur in the absence of CD8, proliferative response of TCR transgenic spleen cells raising into question the exact role of this coreceptor Coreceptor-Independent T Cell Selection 651

during T cell development. Clearly CD8 is important dur- Flow Cytometry ing positive selection since mature transgenic thymo- All flow cytometric analysis was performed on a FACScan instru- cyte numbers are greatly reduced in the absence of this ment (Becton Dickinson). Samples were gated for live cells based on forward and side scatter parameters (10,000 events/sample) and coreceptor. However, these studies have shown that it analyzed using Lysis II software (Becton Dickinson). is possible to reestablish a substantial transgenic thymocyte population by culturing fetal lobes with a posi- Peptides tively selecting peptide, indicating that CD8 is not essen- The peptides p33 (KAVYNFATM), A3V (KAAYNFATM), A4Y (KAVAN- tial for thymocyte development or lineage commitment. FATM), and adenovirus peptide AV (SGPSNTPPEI) were synthe- Instead, this coreceptor functions as an adaptor mole- sized, purified, and characterized at the Amgen Institute as pre- cule, providing a means for maturing thymocytes to ad- viously described (Sebzda et al., 1996). just to their thymic environment. CD8 molecules may help stabilize low-affinity TCR–endogenous peptide/ Peptide-Binding Assay MHC interactions, providing a suitable threshold for In peptide-pulsing experiments, 106 RMA-S cells, which were pre- ‘‘natural’’ thymocyte selection. In the absence of these viously cultured overnight at 29ЊC in RPMI plus 10% FCS, were incubated with various concentrations of peptide at 29ЊC for 30 min. coreceptors, thymocyte selection is limited to a small These cells were then transferred to a 37ЊC incubator for 3 hr, after subset of ligands capable of efficient TCR stimulation. which time the cells were washed and stained with anti-H-2Db mAb Therefore, CD8 coreceptors expand the TCR repertoire from tissue culture supernatant (B22.249) (Hammerling et al., 1979; by extending the potential body of positively selecting Allen et al., 1986) and then FITC-conjugated rat anti-mouse immuno- ligands to include peptides with transient TCR interac- globulin (Sigma Chemical). RMA-S cells were incubated with LCMV tions. The CD8 coreceptor also expands the potential nucleoprotein 118–127 (H-2d restricted) (von Herrath et al., 1994) to determine background H-2Db expression. antigen repertoire, allowing T cells to respond against low-affinity peptides. Although CD8 molecules are not strictly required for transgenic T cell maturation or differ- Proliferation Assays TCR transgenic spleen cells (105/well), cultured previously with or entiation, the coreceptors enhance the efficiency of without various dilutions of anti-CD8 mAb (YTS 169.4) for 1 hr at these processes. 37ЊC, were incubated in triplicate in 96-well flat-bottom plates with 2 ϫ 104/well irradiated C57B1/6J (H-2b) splenocytes that had been Experimental Procedures prepulsed with various concentrations of peptide for 3 hr at 37ЊC. After 48 hr of cocultivation, the cells were pulsed with 1 ␮Ci of Mice [3H]thymidine (Amersham, Arlington Heights, IL) for 16 hr. Cells were TCR-transgenic mice were previously generated using ␣ and ␤ harvested and counted on a direct ␤ counter (Matrix96, Canberra chains isolated from CTL clone P14, which recognized the LCMV Packard Canada, Mississauga, Ontario, Canada). glycoprotein (peptide p33–41) presented by H-2Db. This line was backcrossed with H-2b CD8Ϫ/Ϫ mice to obtain TCR CD8Ϫ/Ϫ (H-2b) FTOC Proliferation Assay animals. Progeny were typed for transgenic TCR and homozygous Cultured thymic lobes were teased apart and cultured with 1 ml CD8 disruption by staining peripheral blood with mAbs at 4ЊCin of anti-HSA (J11d) and 400 ␮l of Low-Tox-M rabbit complement phosphate-buffered saline (PBS) containing 2% NaN , and 20 mM 3 (Cedarlane Laboratories, Hornby, Ontario, Canada) for 45 min at EDTA. TCR-transgenic cells were detected with rat anti-mouse 37ЊC. Irradiated spleen cells from a C57B1/6Jmouse were prepulsed V␤8.1 (KJ16) mAb followed by fluorescein isothiocyanate (FITC)– with 10Ϫ8 M AV, A4Y, or p33 for 3 hr at 37ЊC; washed; and distributed conjugated goat anti-rat Mab (Tago, Burlingame, CA). CD8Ϫ/Ϫ mice in triplicate on a flat-bottom 96-well plate at a concentration of 105 were screened using rat anti-mouse CD8 (YTS 169.4) mAb followed cells/well. Mature thymocytes (3 ϫ 104/well) resuspended in IMDM, by FITC-conjugated goat anti-rat mAb. After the second antibody 10% FCS, penicillin, streptomycin, and 5 ϫ 10Ϫ5 M 2-mercaptoetha- incubation period, red blood cells were lysed using 1ϫ FACS lysing nol were then added to these wells. The cells were cultured at 37ЊC solution (Becton Dickinson, Mountain View, CA). TCR RAGϪ/Ϫ (H-2b) for 72 hr, pulsed with 1 ␮Ci of [3H]thymidine for 16 hr, and harvested mice were obtained as described previously (Kawai and Ohashi, as described. 1995).

FTOCs Intracellular Perforin Staining Timed breedings were established between TCR CD8Ϫ/Ϫ H-2b males Thymic cell suspensions were initially stained with rat anti-mouse and CD8Ϫ/Ϫ H-2b females. At day 16 of gestation, females were PE-conjugated anti-V␣2 (B20.1) (PharMingen). These cells were then sacrificed, and thymic lobes were removed from the fetuses. DNA sorted using a FACStar Plus instrument (Becton Dickinson) to collect was extracted from embryonic tails so that transgenic fetuses could V␣2hi thymocytes. The thymocytes were fixed in 4 ml 70% ethanol be determined using primers specific for the V␣2 TCR transgene (Ϫ20ЊC) and incubated on ice for 30 min. Following this incubation

(Sebzda et al., 1996). The fetal thymic lobes were placed on 0.8 ␮m period, the cells were pelleted and resuspended in 0.5% HIO3. Ten polycarbonate filters (Costar, Cambridge, MA), which floated on 1 minutes later, the cells were washed and incubated with 1% bovine ml of Iscove’s modified Dulbecco’s medium (IMDM), 1ϫ Nutridoma- serum albumin–PBS for 30 min. The cells were then washed and SP (Boehringer Mannheim, Indianapolis, IN), 5 ϫ 10Ϫ5 M2-mercapto- stained with rat anti-mouse perforin antibody (PI-8 ascitic fluid ethanol, penicillin, streptomycin, 2 mM glutamine, and designated 1:500) (kindly provided by Dr. K. Okumura) for 1 hr. The thymocytes peptides. These lobes were then cultured for 6 days at 37ЊC, during were subsequently washed and stained with FITC-conjugated goat which time the medium was changed daily. After this incubation anti-rat mAb (Tago, Burlingame, CA) for 1 hr. period, the thymic lobes were teased apart and stained with mAbs at 4ЊC in PBS containing 2% fetal calf serum (FCS) and 0.2% NaN . 3 Acknowledgments Three-color analysis was done with rat anti-mouse FITC-conjugated anti-CD4 (PharMingen, San Diego, CA), phycoerythrin (PE)– We thank Dr. K. Okumura for the rat anti-mouse perforin antibody conjugated anti-V␣2 (B20.1) (PharMingen), and biotinylated anti- and D. Bouchard for cell sorting. This work was supported by the HSA (M1/69) (PharMingen), or FITC-conjugated anti-PNA (Sigma Medical Research Council (MRC) of Canada. P. S. O. is a recipient Chemical, St. Louis, MO), PE-conjugated anti-CD4 (PharMingen), of an MRC Scholarship. and biotinylated anti-V␣2 (B20.1) (PharMingen). Biotinylated antibodies were detected with streptavidin-red 670 (GIBCO–BRL, Gaith- erburg, MD). Received February 4, 1997; revised April 7, 1997. Immunity 652

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