T Cell Antagonism is Functionally Uncoupled from the 21- and 23-kDa Tyrosine-Phosphorylated TCR ζ Subunits

Lisa A. Pitcher, Pamela S. Ohashi and Nicolai S. C. van Oers
J Immunol July 15, 2003, 171 (2) 845-852; DOI: https://doi.org/10.4049/jimmunol.171.2.845

Abstract

The functional effects of altered peptide ligands on T cells is proposed to involve differential intracellular signaling mediated by the 21- and 23-kDa tyrosine-phosphorylated derivatives of the TCR ζ subunit (p21 and p23). To understand the functional contribution of p21 and p23 to T cell development and T cell antagonism, we generated selected TCR ζ transgenic mice maintained on the P14 αβ TCR transgenic line such that p23 or both p21 and p23 were selectively eliminated. Importantly, one line (YF1,2) retains the constitutively tyrosine-phosphorylated p21 in the complete absence of inducible p23. We determined that T cell development was uncoupled from p21 and/or p23. Using a series of agonist, weak agonist, and antagonist peptides, we analyzed the role of each of the phosphorylated forms of TCR ζ on T cell activation and antagonism. In this study, we report that the proliferative responses of αβ P14 T cells to agonist peptides and the inhibition of proliferation resulting from antagonist peptide treatments was functionally uncoupled from p21 and/or p23. These results suggest that the mechanism of T cell antagonism is independent of the two phosphorylated TCR ζ derivatives.

The αβ TCR is a multisubunit complex consisting of the Ag-specific αβ heterodimer that is noncovalently associated with the TCR ζ homodimer and CD3 invariant chains (δε, γε). Ligation of the αβ TCR with its cognate peptide/MHC ligand expressed on APCs is translated into a series of intracellular signals through specific sequences in the cytoplasmic portions of the TCR ζ and CD3 chains (1). These sequences, termed immunoreceptor tyrosine-based activation motifs (ITAMs),3 are present in one or more copies in the CD3 and TCR ζ-chains and contain a conserved signaling sequence, YxxL x6–8 YxxL (2). TCR interactions with appropriate ligands result in the rapid phosphorylation of the two tyrosine residues present in the ITAMs of the TCR ζ- and CD3 chains. These biphosphorylated ITAMs complex to the Syk/ζ-associated protein-70 (ZAP-70) family of protein tyrosine kinases (PTKs), resulting in the subsequent activation of downstream effector molecules (3). The ultimate outcome of these signaling events is the induction of cellular responses, such as differentiation, proliferation, cytokine secretion, or cytolytic functions (1).

The TCR complex has an amazing capacity to discriminate between small differences in the peptide ligands bound to the MHC. In fact, single amino acid substitutions in particular peptides can dramatically alter the biological responses of T cells, resulting in either positive or negative selection for thymocytes, and T cell activation or T cell anergy for peripheral T cells (4, 5). Several studies have suggested that the distinct phosphorylated derivatives of the TCR ζ subunit can directly affect these developmental and functional decisions (6). The TCR ζ-chain is proposed to represent one of two independent signaling modules in the TCR complex, contributing six of the 10 possible ITAMs in the complex (7, 8). TCR ζ is one of the first and more heavily tyrosine-phosphorylated proteins following receptor ligation and has been detected as two stable phosphorylated forms of 21- and 23-kDa (p21 and p23, respectively) (reviewed in Ref. 9). p21 is constitutively phosphorylated in thymocytes and peripheral T cells as a consequence of TCR interactions with self-peptide/MHC complexes (10, 11, 12). This phosphorylated form of TCR ζ (p21) is complexed to an inactive population of ZAP-70 molecules (11). Although many studies have focused on the functions of the individual ITAMs of the TCR ζ subunit, the specific contribution of p21 and/or p23 to T cell development and signaling has remained elusive (7). The TCR ζ ITAMs clearly contribute to T cell development in TCR transgenic mice expressing low-avidity TCRs (13). It has also been suggested that the phosphorylated TCR ζ subunits function as molecular sensors, capable of discriminating between agonist and antagonist peptide ligands complexed to the same MHC molecule (14, 15). This notion began with the characterization of altered peptide ligands, variants of agonist peptides that specifically inhibit T cell responses (reviewed in Ref.4). The interaction between T cells and APCs expressing an agonist peptide/MHC complex results in the initiation of TCR-mediated intracellular signals. Consequently, the TCR ζ subunit develops into two heavily phosphorylated intermediates of 21- and 23- kDa, and the ZAP-70 PTK is subsequently recruited to these phosphorylated subunits and becomes catalytically active (14, 15). In contrast, the stimulation of T cells with antagonist peptide results in a very weak induction of p23 relative to p21. This contributes to the recruitment and association of an inactive population of ZAP-70 molecules (14, 15, 16). Based on these findings, it was hypothesized that the relative ratios of p23 to p21 could determine whether T cells were activated or antagonized (14, 15, 16). In fact, the constitutively phosphorylated p21 expressed in peripheral T cells was proposed to contribute an inhibitory signaling environment (17).

Antagonist peptides may also be required for positive selection processes in the thymus (reviewed in Ref.18). By inference, the induction of p21would be necessary for thymocyte positive selection, whereas the induction of p23 would lead to negative selection. Despite these dramatic conclusions, several studies have reported that T cell selection and T cell antagonism are not dependent on the phosphorylation states of the TCR ζ subunits (13, 19, 20, 21, 22). However, in many of these studies, the constitutively tyrosine-phosphorylated p21 was not maintained and the induction of p23 was difficult to assess. These issues leave unresolved the functional contribution of the two predominant phosphorylated forms of ζ on T cell development and T cell antagonism.

We have previously mapped the tyrosine residues that are phosphorylated in both p21 and p23 (23). Using these phosphorylation maps, we have generated a unique series of TCR ζ transgenic mice in which selected tyrosine residues were substituted with phenylalanine. The tyrosine substitutions result in the selective elimination of just p23, or both p21 and p23 in the transgenic mice (9, 23). It is important to note that one of these lines (YF1,2) is the only TCR ζ-substituted line reported to date that maintains the constitutive phosphorylation of p21 without any evident p23 before or after receptor ligation. Each transgenic line was mated to the P14 lymphochoriomeningitis virus (LCMV)-specific αβ TCR transgenic mice. The various P14/TCR ζ transgenic lines allowed a definitive assessment of the functional contribution of both p21 and p23 on T cell development and T cell antagonism. In this report, we provide evidence that the positive selection of the P14 transgenic line is functionally uncoupled from the 21- and 23-kDa ζ-chains. Furthermore, using a series of agonist, weak agonist, and antagonist peptides, we show that T cell antagonism is independent of p21 and/or p23. Significantly, these results provide the first direct demonstration refuting a dominant role for p21 in inhibiting T cell functions.

Materials and Methods

Abs and cell lines

Abs with the following specificities were used: 145-2C11, anti-CD3ε (American Type Culture Collection, Manassas, VA); 4G10, anti-phosphotyrosine and goat anti-mouse conjugated to HRP (Upstate Biotechnology, Lake Placid, NY); and fluorescein-conjugated anti-CD8α and anti-CD3ε, PE-conjugated anti-CD4, and anti-B220 (BD PharMingen, San Diego, CA). Vα2- and Vβ8-specific Abs were purchased from BD Pharmingen (San Diego, CA). mAbs and polyclonal antisera to the TCR ζ subunit (6B10.2) and ZAP-70 (1E7.2 or 1222-12) have been described previously (24, 25). Anti-B220-conjugated magnetic beads were purchased from Dynal Biotech (Lake Success, NY). APCs used for the peptide stimulation experiments included the H2Db dendritic cell line, DC2.4, obtained from K. Rock (Dana-Farber Cancer Institute, Boston, MA) and the H2Db macrophage line, IC21, obtained from American Type Culture Collection.

TCR ζ transgenic mice

TCR ζ transgenic mice containing select tyrosine-to-phenylalanine substitutions in the TCR ζ ITAMs were generated using the VA-CD2 transgenic cassette in C57BL/6 mice as previously described (23, 26). These lines included the YF1,2 and YF5,6 lines and a new YF1–6 line reported in this study. The various transgenic founders were backcrossed to TCR ζ-null mice and typed by either Southern blotting and/or PCR for the presence of the transgenic and absence of the endogenous TCR ζ gene. All mice were maintained on a C57BL/6 background. TCR ζ transgenic mice were also crossed to P14 TCR transgenic mice obtained from The Jackson Laboratory (Bar Harbor, ME) and typed by PCR for the P14 TCR, TCR ζ transgene, and endogenous TCR ζ subunit. All mice were maintained in the specific pathogen-free animal facility on the north campus at the University of Texas Southwestern Medical Center.

Immunoprecipitation and Western blotting

Thymocytes, splenocytes, and lymph node cells were isolated from the wild-type P14 or the various P14/TCR ζ transgenic lines, and the phosphorylation patterns of the TCR ζ subunit were determined as previously described (23). Briefly, peripheral T cells were prepared by pooling splenocytes and lymph node cells and depleting these mixtures of B cells by adding anti-B220 magnetic beads as suggested by the manufacturer’s instructions (Dynal Biotech). Approximately 1 × 108 thymocytes or 2 × 107 peripheral T cells (from spleen and lymph node) were left untreated or incubated with the peptide-pulsed APCs for the indicated times. Following these incubation periods, the cells were pelleted and subsequently lysed in 1.0 ml of lysis buffer for 20 min on ice. The lysis buffer consisted of a Tris-based buffer (20 mM Tris-Cl (pH 7.6), 150 mM NaCl, and 2.0 mM EDTA supplemented with protease and phosphatase inhibitors) containing 1% Triton X-100. Cellular debris from the lysis procedure was removed by centrifugation for 10–15 min at 14,000 rpm. The cleared lysates were directly immunoprecipitated for 2–4 h at 4°C by adding 2–4 μg of an anti-TCR ζ mAb (6B10.2) or 2–4 μl of polyclonal antisera to ZAP-70 in combination with 20–40 μl of Gammabind G-Sepharose (Amersham, Piscataway, NJ). The precipitates were washed four to six times in lysis buffer, boiled in SDS-sample buffer, and resolved on 12.5% SDS-PAGE. Gels were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) for 2 h, blocked with 4% BSA (Serologicals, Norcross, GA) in a TBS solution containing 0.5% Tween 20. The membranes were blotted with anti-phosphotyrosine (4G10) followed by detection using ECL procedures (Amersham). In certain cases, the blots were stripped in SDS/2-ME-containing solutions and reblotted with either anti-TCR ζ (6B10.2) or anti-ZAP-70 (1E7.2) mAbs.

Peptides and stimulations

Peptides used for the P14 stimulation experiments were previously defined and described in detail as control (AV), agonist (p33), antagonist (S4Y), and weak agonist (A4Y) (27). The peptides were purchased from AnaSpec (San Jose, CA) or the Howard Hughes Biopolymer Facility (University of Texas Southwestern Medical Center): AV, SGPSNTPPEI; p33, KAVYNFATM; S4Y, KAVSNFATM; and A4Y, KAVANFATM; these peptides were previously shown to equivalently bind H2Db (27). For the induction of TCR ζ phosphorylation, 3 × 106 murine dendritic cells (DC2.4) were prepulsed with 3 μM the indicated peptides for 2 h at 37°C. The cells were then washed and incubated with 1 × 108 thymocytes or 2 × 107 peripheral T cells for 5 min at 37°C. The stimulated cells were lysed with the Triton X-100-containing buffer.

Proliferation and antagonist assays

Spleens were isolated from a TCR ζ-null mouse, and a single-cell suspension was prepared and depleted of RBCs. The splenocytes were used as feeder cells following irradiation with 1500 rad. The splenocytes were plated at 5 × 104 cells/well. These cells were used as APCs and pulsed with varying concentrations of control, agonist, or antagonist peptides for 2 h at 37°C. The splenocytes were washed twice and incubated for 48 h at 37°C with P14 or P14/TCR ζ transgenic cells harvested from the lymph nodes of these mice at 1 × 105 P14-specific T cells/well. For the last 16 h of culture, cells were incubated with 1 μCi/well [3H]thymidine. Cells were harvested onto glass-fiber filter mats, dried, and counted in a scintillation counter. The RBC-depleted TCR ζ-null splenocytes were also used as APCs in the antagonist assays. In brief, these cells were prepulsed with a suboptimal concentration (10−8 M) of the agonist peptide p33 for 2 h at 37°C. The cells were then washed twice and incubated for 1 h with varying concentrations of the antagonist peptide S4Y. The splenocytes were subsequently washed and transferred to a plate with 1 × 105 P14-specific T cells/well. Cells were incubated for 48 h at 37°C and then pulsed for 16 h with 1 μCi/well [3H]thymidine.

Results

P14 αβ TCR transgenic T cells develop normally in the presence of the constitutively tyrosine-phosphorylated TCR ζ subunit

To definitively address the functional contribution of the 21- and 23-kDa tyrosine-phosphorylated forms of TCR ζ on P14 T cell development, we made use of a series of TCR ζ transgenic mice that we had previously developed (23). These TCR ζ lines, termed YF1,2 and YF5,6 (Fig. 1⇓A), have targeted substitutions of particular tyrosine residues with phenylalanine. We also generated an additional transgenic line, denoted YF1–6, in which all six tyrosines in the TCR ζsubunit have been substituted with phenylalanine residues (Fig. 1⇓A). The mice have a selective elimination of just p23, or both p21 and p23, with the YF1,2 line maintaining the constitutively tyrosine-phosphorylated p21 (9, 23). Each line was bred onto the P14 TCR transgenic mice (maintained on a ζ-null background). The P14 mice contain αβ T cells that specifically recognize a peptide derived from LCMV, termed p33, which is presented by the MHC class I molecule H2Db (28).

FIGURE 1.
FIGURE 1.

The development of P14 TCR transgenic thymocytes is normal in the presence of the 16- or 21-, or 21- and 23-kDa tyrosine-phosphorylated forms of the TCR ζ subunit. A, Specific tyrosine-to-phenylalanine substitutions were made in the TCR ζ subunit, and these were designated YF1,2, YF5,6, and YF1–6. A schematic of the TCR complex in these different transgenic lines is illustrated. B, Thymocytes from P14 and P14/TCR-ζ null mice were compared with mice expressing the YF1,2, YF5,6, or YF1–6 TCR ζ constructs on the P14/TCR-ζ-null background. Single-cell suspensions were prepared and stained with mAbs to CD4 and CD8 (B) or Vβ8 and Vα2 (C) and analyzed by flow cytometry. Results are representative of seven independent experiments.

We initially characterized these different mice for the development of LCMV-specific αβ T cells. Thymus, lymph nodes, and the spleen were isolated from the different mice. Single-cell suspensions were prepared, and an aliquot of cells was stained with fluorochrome-labeled mAbs to CD4 and CD8, Vα2 and Vβ8, or CD3 and B220, and analyzed by flow cytometry. Consistent with previous reports, the development of CD4−CD8+ thymocytes expressing the transgenic αβ TCR is severely impaired in TCR ζ-deficient animals (Fig. 1⇑, B and C) (13, 22). Reconstitution of the P14/TCR ζ-null mice with the YF1,2 and the YF5,6 TCR ζ subunits completely restored development of normal numbers and percentages of CD4−CD8+ P14 (12 and 11%, respectively) thymocytes expressing the αβ transgene (Fig. 1⇑, B and C). On the basis of seven independent experiments, we found no significant differences in the numbers and percentages of thymocyte subsets in the P14, P14/YF1,2, and P14/YF5,6 lines. However, we did note reduced numbers of cells in the P14/YF1–6 line. Several groups had previously reported that the development of mature P14+CD8+ T cells proceeds without the involvement of the TCR ζ ITAMs (13, 22). Importantly, the YF1,2 line is the only TCR ζ transgenic line generated to date that selectively maintains the constitutively tyrosine-phosphorylated p21. This form was never maintained in any of the TCR ζ mutant transgenic lines described in those studies. Our results clearly demonstrate that αβ T cell development can proceed normally in the presence of the constitutively tyrosine-phosphorylated p21 form and in the complete absence of p21 and p23. The YF1–6 TCR ζ molecule also facilitated the development of CD4−CD8+ P14 TCR transgenic thymocytes. Taken together, these results clearly demonstrate that the development of P14 LCMV-specific TCR transgenic thymocytes is independent of the phosphorylation state of the TCR ζ subunit, although the efficiency is reduced slightly in the YF1–6 line.

We subsequently analyzed the lymph nodes for the appearance of the CD4−CD8+ P14 TCR transgenic T cells. Single-cell suspensions were stained for various cell surface markers and analyzed by flow cytometry. As shown in Fig. 2⇓, the CD4−CD8+ T cells from P14 TCR transgenic mice comprise ∼50–55% of the lymph node cells (Fig. 2⇓, A and B). Almost no CD4−CD8+ T cells are detected in the absence of the TCR ζ subunit. Consistent with the findings in the thymus, the appearance of CD4−CD8+ TCR transgenic T cells are re-established once the YF1,2 or YF5,6 subunits are expressed. Although the YF1–6 construct restored the appearance of P14 TCR transgenic CD4−CD8+ T cells in the peripheral lymphoid organs, the numbers of these cells was always reduced 1.5- to 2.0-fold relative to those of wild-type mice. In fact, the number of CD4+CD8− T cells was enhanced in the P14/YF1–6 transgenic line (22 vs 8% in a wild-type P14 mouse). Overall, these results indicate that the P14 mice that maintain a constitutive tyrosine-phosphorylated p21 develop as efficiently as mice without any evidence of constitutive TCR ζ phosphorylation, and that positive selection is not affected or antagonized by the constitutively phosphorylated p21.

FIGURE 2.
FIGURE 2.

Normal numbers of P14 TCR transgenic lymph node T cells develop in the presence of the constitutively tyrosine-phosphorylated TCR ζ subunit. A, Lymph node cells were isolated from P14, P14/TCR-ζ-null, P14/TCR ζYF1,2, P14/TCR ζ YF5,6, and P14/TCR ζ YF1–6 mice. Single-cell suspensions were prepared and stained with mAbs to CD4 and CD8 (A) or Vβ8 and Vα2 (B) and analyzed by flow cytometry. Results are representative of six independent experiments.

The 21- and 23-kDa forms of TCR ζ can be elicited by antagonist peptides

Two peptides related to p33, termed A4Y and S4Y, have been described based on their ability to partially activate or antagonize T cell effector functions to p33, respectively (27, 29). To carefully examine the effect of these peptides on the induction of different phosphorylated forms of TCR ζ in the P14 TCR transgenic system, thymocytes or peripheral T cells isolated from the P14 TCR transgenic mice were stimulated with p33, S4Y, A4Y, or a control peptide. Briefly, DC2.4 dendritic cells (H2Db) were prepulsed with 3 μM the indicated peptides and then cocultured with T cells for 5 min. The T cells were subsequently lysed, and the phosphorylated TCR ζ subunits associating with the ZAP-70 PTK were coimmunoprecipitated with anti-ZAP-70 polyclonal antisera. In the absence of peptide stimulation, p21 was constitutively expressed and associated with ZAP-70 (Fig. 3⇓A, lane 1). In addition, a small amount of p23 was detected in these cells (Fig. 3⇓A, lane 1). Stimulation of the P14 thymocytes with a control peptide, AV, caused a slight increase in the levels of p23 (Fig. 3⇓A, lane 2). Stimulation with the strong agonist peptide, p33, induced a 10-fold increase in p23 (Fig. 3⇓A, lane 3). The antagonist peptide, S4Y, also induced an increase in p23, but at levels markedly less than the wild-type agonist peptide (Fig. 3⇓A, lane 4). Identical stimulation experiments were repeated with T cells isolated from spleen and lymph node. Again, p21 was constitutively expressed in peripheral P14 T cells (Fig. 3⇓B, lane 1). Stimulation of these cells with agonist peptide also resulted in a reproducible increase in p23 (Fig. 3⇓B, lane 3). This fully phosphorylated TCR ζ subunit was not as readily detected when the cells were stimulated with the control or antagonist peptides (lanes 2 and 5). Taken together, these results demonstrated that the p23 form of TCR ζ is easily detected following agonist peptide stimulations. However, the ability of weak agonist and antagonists to induce p23 is more difficult to assess, particularly in the peripheral T cells. As such, these experiments leave unresolved the contribution of p21 and/or p23 on T cell proliferation and T cell antagonism.

FIGURE 3.
FIGURE 3.

The 21- and 23-kDa tyrosine-phosphorylated forms of TCR ζ are induced upon stimulation with antagonist peptides. Dendritic cells (H2Db) were pulsed with 3 μM control peptide (AV, lane 2), the agonist peptide (p33, lane 3), an antagonist peptide (S4Y, lane 4), or a weak agonist peptide (A4Y, lane 5). These peptide-loaded dendritic cells were incubated with thymocytes (A) or peripheral T cells (B) from the P14 TCR transgenic mice for a 5-min period. Following the incubation period, the cells were lysed, and the ZAP-70 PTK was immunoprecipitated. The precipitates were resolved on 12.5% SDS-PAGE and blotted with an anti-phosphotyrosine mAb.

The two membrane-distal ITAMs in TCR ζ are essential for maintaining the constitutive tyrosine-phosphorylated 21-kDa form in P14 TCR transgenic mice

To rigorously confirm that the various YF transgenic lines resulted in the selective elimination of p23 alone or both p21 and p23 in the P14 system, we examined the phosphorylation state of ζ before and after agonist stimulation. Thus, thymocytes or peripheral T cells from the P14, P14/YF1,2, P14/YF5,6, and P14/YF1–6 transgenic lines were isolated and incubated with unloaded or p33 peptide-loaded dendritic cells. Five minutes after stimulation, the cells were lysed, and the TCR ζ subunit was directly immunoprecipitated with an anti-ζ mAb in the presence of 0.1% SDS. These experiments were performed in the presence of SDS to ensure that the TCR ζ subunit was isolated in the absence of any associating proteins or coprecipitating CD3 subunits. The ζ immunoprecipitates were resolved on a 12.5% SDS-PAGE gel and subsequently immunoblotted with an anti-phosphotyrosine Ab (4G10). Thymocytes from P14 TCR transgenic mice constitutively express p21 and, following stimulation, can be induced to express p23 (Fig. 4⇓A, lanes 1 and 2). Although the constitutively expressed p21 was maintained in the P14/YF1,2 double transgenic line, there was no detectable p23 following p33 stimulation (Fig. 4⇓A, lanes 3 and 4). In the P14/YF5,6 line, there was no evidence of any constitutive or inducible p21 or p23 (Fig. 4⇓A, lanes 5 and 6). Instead, several very weakly phosphorylated species were detected with molecular masses ranging from 19 to 20 kDa following peptide stimulation (Fig. 4⇓A, lanes 5 and 6). As expected, there were no detectable phosphorylated intermediates in the P14/YF1–6 line (Fig. 4⇓A, lanes 7 and 8). Despite the selective elimination of p21 and p23 subunit, there is no alteration in the nonphosphorylated 16-kDa form, because it was readily detected in all of the transgenic lines characterized (Fig. 4⇓B). It should be noted that treatment of the YF1,2 cells with the general phosphatase inhibitor, pervanadate, also fails to elicit any p23 (23). Moreover, stimulation of the YF5,6 line with pervanadate results in the formation of several heavily phosphorylated TCR ζ intermediates, all with molecular masses of <20 kDa. Similar results were found with lymph node T cells (data not shown). For the YF1–6 line, there is some evidence of TCR ζ degradation that we have been unable to prevent with protease inhibitors (data not shown).

FIGURE 4.
FIGURE 4.

Selection elimination of the 21- and/or 23-kDa tyrosine-phosphorylated forms of TCR ζ in P14/TCR ζ transgenic mice stimulated with agonist peptides. Thymocytes (A and B) were isolated from P14 mice expressing a wild-type TCR ζ subunit (lanes 1 and 2) or TCR ζ-chains containing various tyrosine-to-phenylalanine substitutions designated YF1,2 (lanes 3 and 4), YF5,6 (lanes 5 and 6), and YF1–6 (lanes 7 and 8). These cells were incubated with dendritic cells (H2Db) that had been untreated or prepulsed with 3 μM strong agonist peptide (p33). Following a 5-min incubation period, the cells were lysed, and the TCR ζ subunit was directly immunoprecipitated in the presence of 0.1% SDS. The precipitates were resolved on 12.5% SDS-PAGE and blotted with an anti-phosphotyrosine mAb (A) followed by an anti-TCR ζ mAb (B).

These results clearly indicate that we have effectively eliminated the p23 or both p21 and p23 phosphorylated forms of TCR ζ in the various P14/TCR ζ transgenic lines.

The constitutively phosphorylated 21-kDa TCR ζ subunit promotes full activation of T cells by agonist peptide

Using the P14/TCR ζ transgenic lines described above, we were in a unique position to examine the effects of p21 and p23 on T cell activation and T cell antagonism. Transgenic T cells from the various mice were isolated and incubated with varying doses of peptide-pulsed APCs. Proliferative responses were measured by [3H]thymidine incorporation during the last 16 h of culture. In response to increasing doses of agonist peptide (10−10–10−4 M), the P14 TCR transgenic T cells exhibited a nearly 5-fold increase in proliferation (Fig. 5⇓A). This dose-response curve was nearly identical when the P14/YF1,2 T cells were stimulated in an identical manner. These results indicate that p21, expressed independent of p23, does not inhibit proliferative responses. A very similar dose-response curve was revealed when the P14/YF5,6 transgenic line was compared with wild-type cells (Fig. 5⇓B). These results suggest that neither p21 nor p23 is contributing to proliferative responses. The ability of the YF1–6 T cells, which lack all tyrosine-phosphorylated TCR ζ subunits, was slightly impaired vs wild-type P14 T cells in response to agonist p33 peptide, with only a 3-fold increase in proliferation over the dose range used (Fig. 5⇓C). The response to the weak agonist peptide, A4Y, was equivalent in the YF1,2 and YF5,6 transgenic lines compared with the P14 wild-type cells (data not shown). When the various P14/TCR ζ thymocytes were compared with the P14 line for the induction of phosphoproteins, equivalent patterns of phosphorylation of ZAP-70, Src homology 2 domain-containing leukocyte protein-76, and linker for activation of T cells were detected following peptide/MHC stimulation.4 Taken together, these results directly demonstrate that the phosphorylated 21- and 23-kDa forms of TCR ζ are uncoupled from activation of membrane-proximal signals and the proliferative response of the P14 T cells. More specifically, the constitutive 21-kDa form of TCR ζ does not interfere with or antagonize T cell activation by agonist peptides in this system or in a non-TCR transgenic system.4

FIGURE 5.
FIGURE 5.

Agonist peptide-induced proliferation in the P14 T cells is independent of the phosphorylated TCR ζ subunits. Lymph node cells (105) were isolated from wild-type P14 mice or P14 mice bearing selected tyrosine-to-phenylalanine substitutions in the TCR ζ-chain designated P14/ζYF1,2 (A), P14/ζYF5,6 (B), and P14/ζYF1–6 (C). These cells were cultured with varying concentrations of agonist peptide (p33)-pulsed APCs (5 × 104) for 48 h. Proliferation was measured by adding 1 μCi of [3H]thymidine for the last 16 h of culture. These results are representative of three independent experiments.

T cell antagonism is uncoupled from the 21- and 23-kDa tyrosine-phosphorylated forms of TCR ζ

T cell proliferative responses vs an agonist peptide can be effectively inhibited by the addition of select antagonist peptides (17, 29). The various P14/TCR ζ transgenic lines that selectively express either p21, or both p21 and p23, were used to directly analyze whether the 21-kDa form of TCR ζ correlated with T cell antagonism. The predominant expression of p21 was previously reported to actively inhibit T cell responses and induce anergy (17). This finding would suggest that the YF1,2 cells in our system should have enhanced antagonist responses. T cells isolated from lymph nodes of P14/TCR ζ mice were cocultured with APCs that had been prepulsed with a suboptimal (10−8 M) concentration of the p33 agonist peptide, followed by increasing concentrations of antagonist peptides in the range from 10−7 to 10−4 M. P14 T cells were effectively inhibited by the addition of high concentrations of the antagonist peptide, S4Y, with a 3-fold decrease in thymidine uptake compared with the suboptimal concentration of agonist peptide (Fig. 6⇓A). Notably, the P14/ζYF1,2 transgenic T cells that express only the constitutively phosphorylated p21 exhibited an equivalent magnitude of antagonism over the entire dose of peptide used (Fig. 6⇓A). Likewise, the P14/ζYF5,6 transgenic T cells that lack p21 and p23 also show an equivalent level of T cell antagonism compared with wild-type P14 T cells (Fig. 6⇓B). These results provide direct evidence that the 21- and/or 23-kDa tyrosine-phosphorylated TCR ζ subunits are completely uncoupled from T cell antagonism. Interestingly, we were unable to detect T cell antagonism in the absence of any tyrosine-phosphorylated TCR ζ intermediates in the YF1–6 line (Fig. 6⇓C). The implications of this finding are discussed below.

FIGURE 6.
FIGURE 6.

T cell antagonism does not involve the 21-kDa tyrosine-phosphorylated TCR ζ subunit. Lymph node cells (105) from the various P14 single and double transgenic lines were cultured with APCs that had been prepulsed with suboptimal concentrations of agonist peptide (10−8 M p33) followed by increasing concentrations of antagonist peptide (10−7–10−4 M S4Y). P14 and P14/ζYF1,2 (A), P14 and P14/ζYF5,6 (B), or P14 and P14/ζYF1–6 (C) were compared. After 48 h, proliferation of T cells was measured by pulsing cultures with 1 μCi of [3H]thymidine for the last 16 h of culture. These results are representative of three independent experiments. The magnitude of proliferation was less in the YF1–6 line in an independent experiment.

Discussion

The TCR ζ subunit is one of the earliest and the most heavily tyrosine-phosphorylated proteins detected in T cells following TCR ligation (reviewed in Ref.30). In fact, two distinct tyrosine-phosphorylated derivatives of 21- and 23-kDa are commonly revealed upon agonist peptide- or Ab-mediated activation of T cells (9). p21 is specifically phosphorylated on the four tyrosine residues in the two membrane-distal ITAMs, whereas p23 is fully phosphorylated on the six tyrosines dispersed among all three ITAMs (23). Notably, p21 appears as a constitutively phosphorylated protein in thymocytes and peripheral T cells and is associated with an inactive pool of ZAP-70 molecules (11). The constitutive phosphorylation of p21 results from TCR interactions with self-peptide/MHC complexes in the thymus and peripheral lymphoid organs (10, 11, 12, 31, 32). A plethora of publications have supported a functional role for both p21 and p23 in predicating the outcomes of T cell selection, T cell antagonism, and peripheral T cell survival (for reviews, see Refs.4, 7 , and 9). Yet, an equally impressive number of publications have refuted roles for these phosphorylated derivatives in regulating these biological events (for reviews, see Refs.4 , 7 , and 9). Despite these publications, not one study has examined the consequences of maintaining p21 in the absence of p23.

In this report, we definitively demonstrate that positive selection and T cell antagonism in the P14 TCR transgenic system are completely uncoupled from the presence of p23 or both p21 and p23. First, we used a series of TCR ζ transgenic mice that contain substitutions at several distinct tyrosine residues that result in the selective elimination of just p23 or both p21 and p23 (23). Importantly, the YF1,2 line is the only transgenic line developed to date that maintains p21 without being able to form p23. When introduced onto the P14 TCR transgenic system, we determined that positive selection was independent of p23 and was unaffected by the presence of the constitutively tyrosine-phosphorylated p21 form. Second, we used a series of peptide analogs previously defined as agonist, weak agonist, and antagonist peptides in the P14 system to show that T cell antagonism is not influenced by p21 and/or p23. One prediction of the earlier model concerning T cell antagonism is that the level of antagonism would increase if p21 were the only phosphorylated intermediate. In fact, we find an equivalent dose-response curve for antagonist peptide-mediated inhibition of T cell proliferation when comparing P14 and the P14/YF1,2 mice. Moreover, we determined that the magnitude of T cell antagonism is equivalent in wild-type mice and in mice lacking both the 21- and 23-kDa derivatives of TCR ζ. It had been proposed that the constitutively phosphorylated p21 present in resting T cells provides a predominant inhibitory signal for T cells, and that this may explain T cell anergy (6, 8, 14, 15, 17, 33). Our results clearly refute a role for p21 in dominantly affecting T cell antagonism. An earlier report suggested that monophosphorylated ITAMs were the basis of the 21-kDa structure (34). Importantly, our mapping studies demonstrated that the 21-kDa form of TCR ζ consists solely of two biphosphorylated ITAMs (23). We cannot rule out the possibility that monophosphorylated ITAMs, distinct from the 21-kDa form and not detected in our system, may actually contribute to T cell antagonism. For example, Allen and coworkers (17) have demonstrated, using CD8/ζ chimeric molecules, that monophosphorylated ITAMs can promote T cell antagonism. We have found that the YF1–6 TCR ζ molecule was unable to support antagonism and this molecule remains completely nonphosphorylated before and following TCR cross-linking. As reported elsewhere, phosphorylated TCR ζ molecules can contribute to T cell selection with low-avidity TCRs (13). It is conceivable that the high-avidity P14 line used in our study requires some phosphorylated ζ molecules distinct from p21 and/or p23 for effective antagonism. For example, a previous study using the P14 TCR transgenic system in which TCR ζ was selectively substituted at the first, third, and fifth tyrosine residues (called a1−b1−c1−) revealed that antagonism could only be established when using weak agonists such as AV, and not p33 (22). Yet, the proliferative responses to p33 were equivalent in those mice when compared with wild-type P14 mice. Because the YF5,6 line used in our study is weakly phosphorylated in the P14 system following agonist stimulation, a single tyrosine within the second ITAM of TCR ζ could potentially mediate antagonism. However, these possibilities seem unlikely for the following reasons. First, we have shown that T cell proliferation occurs in the YF1–6 line, which lack any functional ITAMs in the ζ-chain. Second, we have previously used a TCR ζ construct in which tyrosines 3 and 4 were substituted with phenylalanine (YF3,4) (23). T cell lines expressing this TCR ζ construct exhibit T cell response identical with that of wild-type T cells, eliminating the third and fourth tyrosine residues as potential inhibitory phosphorylation sites (23). Third, Malissen and coworkers (22) have generated TCR ζ transgenic lines with specific substitutions at the first, third, and fifth tyrosine residues. Notably, T cells expressing this TCR ζ molecule could be antagonized again, suggesting that no specific individual tyrosine residue is mediating antagonism. In another report, T cell antagonism was evident with fully truncated TCR ζ molecules expressed in TCR ζ-null T cell hybridomas (20). However, no constitutive tyrosine-phosphorylated p21 was evident in this system, precluding an assessment of p21. Because this hybridoma system involves a TCR distinct from the P14 system, the avidity of the receptor for particular peptides may be sufficiently high to preclude any phosphorylated TCR ζ involvement. Current experiments are underway to address these issues.

It remains unclear what signals are involved in the antagonism of T cell responses. One possibility is that a threshold of signaling must be reached for cells to be fully activated. We have some preliminary evidence suggesting that the CD3 subunits may form the predominant signaling module in T cells.4 Furthermore, there is some correlative data indicating that the degree of CD3ε phosphorylation may regulate T cell responsiveness vs antagonism (14). We are also exploring the possibility that the level of ZAP-70 PTK activation may be the primary determinant in regulating T cell antagonism, because ZAP-70 is generally not detected as a phosphoprotein following the stimulation of T cells with antagonist peptides. Other mechanisms have been proposed for the different T cell responses to agonist vs antagonist peptides (reviewed in Ref.4). These include conformational changes in the TCR complex upon ligand binding, the relative affinity of the receptor for the peptide/MHC molecule ligand, and/or the kinetics of activation. Alternatively, a negative regulator may be involved in generating an antagonist response (35). A paradigm for this model has been established with the identification of the Src homology 2 domain-containing suppressor of cytokine signaling family of inhibitors for cytokine/cytokine receptor signaling (36). Notably, the expression of several viral ITAM-containing proteins in lymphocytes can directly attenuate subsequent signal transduction through the Ag receptors (37, 38, 39, 40). Similar mechanisms may operate to maintain T cell antagonism. However, several studies have again refuted this possibility, because cross-antagonism is not seen with T cells expressing two distinct TCRs (41, 42).

Overall, our studies have shown that the development and function of T cells from the P14 TCR transgenic line are separate from the two major tyrosine-phosphorylated derivatives of the TCR ζ subunit. Current experiments are focusing on the functional contribution of p21 to peripheral T cell survival.

In summary, we have developed a number of TCR ζ transgenic lines, one of which is the only TCR ζ transgenic line ever described that successfully permits expression of the constitutively tyrosine-phosphorylated p21 form of TCR ζ without any available p23. This YF1,2 line has uniquely enabled us to provide definitive and unequivocal results pertaining to the p21 form of TCR ζ. With these mice as the basis for our investigations, we definitively show that p21, on its own, does not attenuate T cell development nor contribute to T cell antagonism, refuting the generally accepted theory that p21 plays a dominant inhibitory role.

Acknowledgments

We thank Meredith A. Mathis and Philip C. Wrage for technical support and Shirley Hall for the generation of transgenic mice.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grant AI 42953 to N.S.C.v.O.

  • ↵2 Address correspondence and reprint requests to Dr. Nicolai S. C. van Oers, NA7.201, Center for Immunology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9093. E-mail address: nicolai. vanoers{at}utsouthwestern.edu

  • ↵3 Abbreviations used in this paper: ITAM, immunoreceptor-based tyrosine activation motif; PTK, protein tyrosine kinase; LCMV, lymphochoriomeningitis virus; ZAP-70, ζ-associated protein-70.

  • ↵4 L. A. Pitcher, M. A. Mathis, M. C. Durham, L. M. DeFord, and N. S. C. van Oers. The CD3 subunits promote normal T cell activation regardless of phosphorylated TCR ζ. Submitted for publication.

  • Received February 27, 2003.
  • Accepted May 14, 2003.

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