The Constitutive Tyrosine Phosphorylation of CD3ζ Results from TCR-MHC Interactions That Are Independent of Thymic Selection¹ Free

T cell development and effector functions are controlled by TCR interactions with peptide-self-MHC complexes. In developing thymocytes, TCR recognition of a select set of self-peptides embedded in MHC molecules can support both positive and negative selection processes (1). These selection processes ensure the development of mature T cells that are tolerant to self-peptide/self-MHC and responsive to foreign peptides presented by self-MHC molecules. Even before selection, the TCR appears to have an intrinsic bias toward the recognition of MHC molecules (2, 3).

Many thymocyte and peripheral T cell functions are controlled by TCR-mediated increases in the phosphorylation of intracellular proteins (reviewed in Ref. 1). These TCR-regulated phosphorylation cascades are initiated by at least three distinct families of protein tyrosine kinases, Src, Syk, and Tec. The functions of these kinases coordinate around the ITAMs, a signaling motif that is present in 10 copies in the invariant subunits of the TCR complex (CD3γ, -δ, -ε, and -ζ) (reviewed in Refs. 4 and 5). TCR interactions with peptide-MHC complexes result in a transient phosphorylation of two tyrosine residues in each ITAM (YxxLx_6–8YxxL), which allows the Zap70/Syk protein tyrosine kinases to bind via their tandem SH2 domains. The ITAM pathway not only provides the basis for TCR signaling but is also involved in BCR and certain Fc- and activating NK cell receptors (reviewed in Refs. 6 and 7).

The TCR complex is distinct from all other ITAM-containing receptors because it contains 10 ITAMs, whereas most other receptors contain one or two (8). Six of the ten TCR ITAMs are located in the CD3ζ homodimer (three ITAMs per chain). After TCR engagement, CD3ζ appears as two distinct tyrosine-phosphorylated intermediates of 21 and 23 kDa, respectively (p21 and p23) (9). p21 is constitutively phosphorylated in thymocytes and peripheral T cells and can complex with an inactive population of Zap70 molecules (10, 11). The constitutive phosphorylation of CD3ζ results from TCR interactions with MHC molecules, given that p21 levels are reduced 5- to 15-fold in MHC class II/β₂-microglobulin (β₂m)³ double-knockout mice and are almost absent in TCRα-deficient mice (11, 12). In addition, p21 is reduced or absent in Lck- and Zap70-deficient mice (13, 14). The p21 that is present in developing thymocytes maintains TCR expression at a low level, in part through its interactions with the SH2 domain containing Src-like adaptor protein in association with Cbl (15, 16). Elevated levels of p21 have been correlated with autoimmune diseases and/or T cell hyperactivity (17, 18). How the constitutive phosphorylation of CD3ζ (p21) influences T cell functions is controversial. For example, p21 was reported to enhance the sensitivity of naive T cells to foreign Ag by ∼5- to 10-fold (19). In contrast, partial phosphorylation of CD3ζ (p21-like) reduced TCR-mediated functions, eliciting T cell anergy (20, 21). Still other studies have shown that p21 is not required for positive selection, TCR signal transmission, or T cell anergy (22, 23, 24). Yet, p21 has been reported to influence the effectiveness of negative selection (25). These results leave unanswered the physiological contribution of p21 to T cell biology.

To better understand the functional role(s) of p21 in thymocytes, we have analyzed the regulation of ζ phosphorylation. We report herein that the constitutive tyrosine phosphorylation of CD3ζ was maintained in thymocytes isolated from both class I- and class II-restricted TCR-transgenic lines when the cells developed in either a selecting or a nonselecting MHC environment. These findings suggested that the intrinsic capacity of the TCR to recognize MHC molecules before repertoire selection resulted in TCR-mediated intracellular signals that contributed to the constitutive phosphorylation of CD3ζ. Biochemical signaling assays and intracellular staining procedures indicated that the presence of p21 had little impact on signals induced through the TCR. These findings suggest that tonic TCR interactions with MHC molecules elicit a constitutive phosphorylation of CD3ζ that, under normal physiological conditions, is functionally inert.

The following Abs were used: 145-2C11, anti-CD3ε (American Type Culture Collection); 4G10, anti-phosphotyrosine and goat anti-mouse conjugated to HRP (Upstate Biotechnology). Biotinylated anti-CD4, anti-CD8, anti-CD44, and anti-L-selectin (CD62L); fluorescein-conjugated anti-CD8α, anti-CD3ε, anti-B220; PE-conjugated anti-CD4 and anti-B220, and APC-anti-CD4 were obtained from BD Biosciences. Vα2-, Vβ5-, and Vβ8-specific Abs were also purchased from BD Biosciences. Unlabeled or Alexa Fluor 647 anti-CD3ζ (pY142, fifth of six tyrosines in ζ) was obtained from BD Biosciences (see manufacturer’s instructions). mAbs and/or polyclonal antisera to the TCRζ subunit (6B10.2) and Zap70 (1E7.2 mAb or 1222-12, 1225-14, or 1600 polyclonal antisera) were described elsewhere (26, 27). Anti-phospho-Zap70 antisera (Y493 and Y319) were obtained from Cell Signaling Technologies. A mAb recognizing the HY TCR (T3.70) (provided by Dr. Hung-Sia The, University of British Columbia, Vancouver, Canada) was labeled with fluorescein.

CD3ζ-transgenic mice (also referred to as TCRζ) containing select tyrosine to phenylalanine substitutions in the ζ ITAMs, defined as YF1,2; YF5,6; and YF1–6, were described previously (23, 28). The YF1,2 and YF5,6 lines have equivalent ITAM numbers (8 of 10), but only the YF1,2 line retains p21 phosphorylation. MHC class-I-restricted αβ TCR transgenic lines included HY (H-2^b), P14 (H-2^b), OT-I (H-2^b), and C10.4 (MHC class Ib molecule, H2-M3), whereas the MHC class II-restricted mice consisted of OT-II (H-2^b), D011.10 (H-2^d), and 5C.C7 (H-2^k). HY/Rag on H-2^b and H-2^d backgrounds, 5CC7/Rag on H-2^k and H-2^b backgrounds were obtained either from Taconic Farms or through the National Institute of Allergy and Infectious Diseases/Taconic Farms emerging models program. All the mice were contained in the Specific Pathogen Free Facility on the North campus at University of Texas Southwestern Medical Center (Dallas, TX). The procedures were undertaken with Institutional Animal Care and Use Committee-approved protocols.

Thymocytes, splenocytes, and lymph node cells were isolated from wild-type C57BL/6 mice and/or the various CD3ζ- and TCR-transgenic lines. The thymocytes and peripheral T cells were processed and analyzed using previously outlined procedures (23, 28). Quantitation of proteins was done with an Alpha Innotech gel documentation system. FACScan or FACSCalibur flow cytometers were used. Data analysis was performed using CellQuest or CellQuest Pro software. Cell sorting was undertaken with a high speed Mo-Flo cell sorter (Dako Cytomation).

Four-week-old mice were thymectomized under Institutional Animal Care and Use Committee-approved procedures. Nine weeks after thymectomy, the mice were euthanized. Lymphoid organs were obtained from these mice and analyzed by flow cytometry. Littermates that had been mock thymectomized and/or mice that still retained one of two thymic lobes after the thymectomy served as controls.

In most T cell lines, TCR ligation is required for the induction of two tyrosine-phosphorylated derivates of CD3ζ, which are defined by their two distinct molecular masses of 21-kDa (p21) and 23-kDa (p23) (Fig. 1,A, lane 2 vs lane 1) (28). p21 is phosphorylated on all four tyrosines in the two-membrane distal ITAMs, whereas p23 is fully phosphorylated on all three ITAMs (28). Unlike cultured T cell lines, p21 is constitutively tyrosine phosphorylated in both thymocytes and peripheral T cells due to TCR interactions with MHC molecules (10, 28, 29). This was evident after Western blot analysis of CD3ζ precipitates prepared from unstimulated thymocyte lysates, which revealed an abundant constitutive tyrosine phosphorylation of CD3ζ (p21) (Fig. 1,A, lane 3). p23 was detected after TCR cross-linking, although it was present at much lower levels than p21 (Fig. 1 A, lane 4). Prolonged exposures did reveal some p23 expression in unstimulated samples (data not shown).

We wanted to determine whether p21 phosphorylation resulted from TCR interactions with self-peptide/self-MHC molecules involved in positive and/or negative selection, and whether the TCR density influenced its formation (11, 12, 13). To answer these questions, thymocytes from several different TCR-transgenic lines were analyzed for the levels of p21 and the 16-kDa nonphosphorylated form of CD3ζ (p16). Consistent with previous reports, p21 was detected in unstimulated thymocytes from C57BL/6 mice and all class I (HY, P14, OT-I, C10.4)-and class II (OTII, 5C.C7, D011.10)-restricted TCR-transgenic lines examined (Fig. 1,A, lanes 5, 7, and 9; data not shown and Refs. 10 , 29 , and 30). Furthermore, stimulation of the thymocytes with agonist peptide-loaded APCs induced p23 (Fig. 1,A, lanes 6, 8, and 10). Using a normalized value of 1 for the p21:p16 ratio in C57BL/6 thymocytes, the p21:p16 values ranged from 0.49 ± 0.08 (n = 3) for the HY mice to 1.14 ± 0.18 (n = 3) for the P14 line (Fig. 1,B and Table I). Thymocytes from the various mice were also analyzed by flow cytometry for the cell surface expression of CD4, CD8, and CD3 (Fig. 1,C). There was a wide range in the percentage of positively selected cells, from 15.1 ± 1.8% (P14) to 49.4 ± 3.0% (5C.C7) (Table I). The TCR density was also markedly varied, ranging from an MFI of 34 (C57BL/6) to 201 (5C.C7) (Table I). In thymocytes from wild-type C57BL/6 mice, quantitative measurements indicated that p21 represented 4.0 ± 3.1% (n = 4) of the total available pool of CD3ζ, with most phospho-ζ associated with the TCR (Fig. 2, A and B). A comparison of the p21/p16 ratios, the percentage of positively selected thymocytes, and TCR density revealed no obvious relationship between any of these parameters, leaving unresolved the mechanism by which p21 is constitutively present (Table I).

Several alternative explanations have been proposed to account for the constitutive phosphorylation of CD3ζ including coreceptor interactions with MHC molecules and/or in vitro experimental manipulations (10, 31). To address these possibilities, we characterized the phosphorylation state of CD3ζ in mice wherein coreceptor interactions were eliminated. The levels of p21 in thymocytes isolated from wild-type and CD4-deficient mice were determined. P21 was maintained at equivalent levels in thymocytes from CD4-null mice when compared with wild type mice (Fig. 2,C, lane 1 vs lane 2). In addition, the constitutive phosphorylation of p21 was analyzed in thymocytes from TCR-transgenic male and female HY mice. In male HY mice, >85–95% of thymocytes are CD4⁻CD8⁻ because of the deletion processes. p21 was constitutively present in these cells, suggesting that neither CD4 nor CD8 coreceptor molecules were necessary for its formation (Fig. 2,C, lanes 3 and 4). This is consistent with earlier findings that p21 is present in CD4/β₂m double-knockout mice, a condition that eliminates all coreceptor interactions with MHC (11). To examine the contribution of temperature-regulated in vitro manipulations of CD3ζ phosphorylation, one thymic lobe isolated from a mouse was immediately lysed and processed by Dounce homogenization, eliminating any in vitro temperature and cell suspension variability, whereas the second lobe was processed under standard conditions. p21 was evident in both samples (Fig. 2 C, lanes 5 and 6). Overall, these findings indicated that direct TCR interactions with MHC molecules were primarily responsible for the formation of p21.

We considered two hypotheses to account for the constitutive presence of p21 in thymocytes. First, p21 could result from TCR interactions with MHC complexes independent of positive and/or negative selection. This would be consistent with the proposed structural capacity of the TCR to recognize MHC molecules independent of the peptides bound in the peptide-binding cleft (2, 3). Alternatively, p21 could result from TCR interactions with the diverse array of positively and negatively selecting peptides bound to MHC molecules (1). To address these possibilities, the phosphorylation state of CD3ζ was compared in thymocytes isolated from several distinct TCR-transgenic lines maintained on selecting or nonselecting MHC backgrounds. All the mice were on a Rag-deficient background to ensure that TCR/MHC interactions in the thymus resulted from the transgenic TCR, with no contributions from endogenous TCRα gene products (32, 33). In initial experiments, 6-wk-old TCR-transgenic female HY/Rag mice were obtained on either selecting (H-2^b) or nonselecting (H-2^d) MHC backgrounds. At this age, only small numbers of thymocytes are positively selected (34). Mature CD4⁻CD8⁺ T cells developed only in the H-2^b female mice, while most thymocytes were blocked at the CD4⁺CD8⁺ stage in an H-2^d environment (Fig. 3,A) (32, 33). There were no T cells detected in the lymph nodes of female HY/Rag/H2^d mice (data not shown). The phosphorylation state of CD3ζ was examined in equivalent numbers of thymocytes from the two sets of mice. p21 was detected in thymocytes isolated from both the female HY/Rag/H2^b and HY/Rag/H2^d mice (Fig. 3 B). By normalizing the p21:p16 ratio to 1 in the HY/Rag/H2^b mice (1.00 ± 0.02, n = 3), we determined that the p21:p16 ratio was higher in thymocytes not undergoing positive selection (1.47 ± 0.11, n = 3). This finding strongly supported the idea that p21 resulted from TCR interactions with MHC molecules, independent of positive and/or negative selection, and that this occurred in developing CD4⁺CD8⁺ thymocytes.

We next characterized the phosphorylation state of CD3ζ in a class II-restricted TCR-transgenic line (5C.C7/Rag) where the thymocytes developed in selecting (IE^k) or nonselecting MHC backgrounds (IE^b). Upwards of 55% of the thymocytes from the 5CC7/Rag/IE^k line were positively selected into the CD4⁺CD8⁻ subset (Fig. 3,C). Mature CD4⁺CD8⁻ thymocytes (0.5%) and lymph node T cells were completely absent in the 5CC7/Rag/IE^b line (Fig. 3,C, data not shown). Again, CD3ζ was constitutively phosphorylated in both sets of thymocytes, although the levels of p21 were higher in the thymocytes that developed in a selecting environment (Fig. 3,D). This increase may have been a consequence of the extremely high TCR density in the 5CC7/Rag/IE^k mice (Fig. 3 C). Using a normalized p21:p16 ratio of 1 in the 5CC7/Rag/IE^k line (1.00 ± 0.17, n = 3), we determined that the ratio was 4-fold higher in thymocytes from the nonselecting environment (4.49 ± 2.58).

Based on these results, we concluded that the constitutive phosphorylation of CD3ζ (p21) was independent of the selecting ligands available in the thymus. The data support the concept that the TCR has a structural design facilitating MHC interactions that are independent of the self-peptide/self-MHC molecules responsible for positive or negative selection.

We next addressed the functional consequences of the constitutive phosphorylation of p21 on TCR signal transmission. Biochemical studies have revealed that Zap70 is complexed to p21, with the tandem SH2 domains of Zap70 stabilizing p21 (28). The p21-associated Zap70 kinase is catalytically inactive, as defined by its weak to nonexistent tyrosine phosphorylation state. Given the high levels of p21 present in thymocytes, we wanted to determine whether the presence of p21 and associated Zap70 enhanced TCR signaling, as measured by Zap70 activation. We compared TCR signal transduction with different CD3ζ-transgenic lines that have selected substitutions of tyrosine residues in the ITAMs (9). These lines are called CD3ζ YF1,2, YF5,6, and YF1–6. The YF1,2 and YF5,6 lines are matched for TCR density, ITAM number, and single-positive and double-positive thymocyte percentages (24). T cells from the YF1,2 line retain p21 and the associated Zap70 but cannot generate p23. Cells from the YF5,6 line contain weak phospho-ζ forms of 19 and 20 kDa that are detected after TCR stimulation and have no pre-existing phospho-ζ/Zap70 complexes. All tyrosine residues in CD3ζ are substituted with phenylalanine in the YF1–6 line, resulting in no phospho-ζ intermediates (23).

We compared the TCR-induced changes in Zap70 phosphorylation among the wild-type, YF1,2-transgenic, and YF5,6-transgenic lines. Before stimulation, p21 coprecipitated with Zap70 in the wild-type and YF1,2 but not the YF5,6 line (Fig. 4,A, lanes 1, 5, and 9). TCR ligation induced the tyrosine phosphorylation of Zap70 at 10 and 30 min in all three lines, which diminished by 90 min (Fig. 4,A, lanes 2–4, 6–8, and 10–12). Some phospho-CD3δ and -CD3ε were detected in the Zap70 immunoprecipitates at the 10-min stimulation point (Fig. 4,A, lanes 2, 6, and 10). The magnitude and kinetics of Zap70 phosphorylation were similar when comparing all three distinct lines (Fig. 4,A, lanes 2, 3, 6, 7, 10, and 11). This was consistent with our earlier findings that agonist peptide-stimulated thymocytes isolated from HY, HY/YF1,2 and HY/YF5,6 TCR-transgenic female mice had very similar patterns and kinetics of Zap70 and SLP-76 phosphorylation (24). The experiments suggested that the pre-existing p21/Zap70 complex failed to enhance the magnitude of TCR signaling in thymocytes. We further explored this possibility by examining the amounts of p21 associated with activated Zap70 before and after receptor ligation. If the constitutively phosphorylated CD3ζ/Zap70 population contributes to TCR signaling, then the population of activated Zap70 detected after TCR engagement should include the majority of p21. If p21/Zap70 is excluded from newly engaged TCRs, then very little should coprecipitate with activated Zap70. To address these possibilities, we analyzed signaling events in thymocytes from class I- or class II-restricted TCR-transgenic lines (HY and 5C.C7). Cells from the HY mice were left untreated or stimulated for 10 min with agonist peptide-loaded APCs. Phospho-Zap70 was precipitated with antisera directed against two phosphotyrosine residues that are only phosphorylated when Zap70 is catalytically active (phosphotyrosine residues 319 and 493). These antisera do not detect nonphosphorylated Zap70, as evidenced by the absence of Zap70 in Fig. 4,B, lanes 1 and 2. In unstimulated lysates, only 9 ± 5% (n = 4) of total p21 coprecipitated with phospho-Zap70, and there was very little detectable phospho-Zap70 (Fig. 4,B, lane 1). After receptor triggering, both p21 and p23 detectably coprecipitated with active Zap70 (Fig. 4,B, lane 2). The amount of p21 that coprecipitated with phospho-Zap70 increased to 13 ± 7% of the total pool of p21, suggesting that the amounts of new p21 induced after receptor ligation were small. After immunodepletion of active Zap70, the remaining pool of total Zap70 was immunoprecipitated (Fig. 4,B, lanes 3 and 4). The amount of phospho-Zap70 was very low in these sequential immunoprecipitates, indicating that the majority of active Zap70 was precipitated with the anti-phospho-active mAb. A significant amount of p21 coprecipitated with nonphosphorylated Zap70 (Fig. 4,B, lanes 3 and 4). This indicated that the majority of the pre-existing p21/Zap70 complex was not activated after TCR cross-linking. CD3ζ was immunoprecipitated next (Fig. 4 B, lanes 5 and 6). Most p21 remained in the lysates after Zap70 depletion, and additional experiments indicated that 30–40% of p21 was not associated with Zap70 (data not shown).

The experiments were repeated with thymocytes from the 5C.C7 TCR-transgenic line. In unstimulated cells, 14 ± 3% (n = 2) of total p21 coprecipitated with phospho-Zap70 (Fig. 4,C, lane 1). After TCR engagement, both p21 and p23 detectably coprecipitated with phospho-Zap70, with 24 ± 5% of total p21 present in these samples (Fig. 4,C, lane 2). The 11% increase in p21 detected after TCR stimulation could have developed from the pre-existing population of constitutively phosphorylated CD3ζ and/or newly formed p21 from the nonphosphorylated pool of ζ. Such experiments also suggested that more p21 and p23 are generated as more mature thymocytes are used, likely due to higher surface expression of the TCR (Fig. 4 C, lanes 1–6). Taken together, the findings suggest that the constitutive phosphorylation of p21 and its associated pool of Zap70 do not enhance TCR signal transmission.

To examine whether only a subset of thymocytes contained p21, we performed intracellular staining with a mAb that specifically recognizes the fifth phosphotyrosine residue in CD3ζ (pY-142). This tyrosine is constitutively phosphorylated in p21 (28). In both unstimulated and TCR-stimulated CD4⁺CD8⁺ cells from either the C57BL/6 or YF1–6 line, pY-142 (p21) was not detected by intracellular staining (Fig. 5,A). In fact, pY-142 (p21) was detected at appreciable levels only in the unstimulated CD4⁺CD8⁻ thymocytes, and these levels increased after TCR cross-linking (Fig. 5,A). There were very minimal changes in the levels of phospho-pY142 in the CD4⁺CD8⁻ thymocytes from the YF1–6 line (Fig. 5,A). These experiments suggested that the constitutively phosphorylated CD3ζ was inaccessible to the anti-phospho-ζ mAb in immature thymocytes, the levels of p21 were below the detection limits, and/or the tyrosine at position 142 was not phosphorylated. To examine these possibilities, thymocytes from C57BL/6 mice were sorted into CD4⁺CD8⁺ and CD4⁺CD8⁻ subsets and lysed. The samples were then immunoblotted with the same anti-pY142 mAb (unlabeled version) as that used for intracellular staining (Fig. 5,B). The samples were also immunoblotted with anti-phosphotyrosine mAbs (Fig. 5,C). p21 was detected in lysates of CD4⁺CD8⁺ cells that were isolated from wild-type but not YF1–6 mice (Fig. 5 C). Taken together, the experiments suggested that p21 is inaccessible or partially blocked from detection in CD4⁺CD8⁺ thymocytes when using intracellular staining techniques. This could account for the inability of p21 to enhance TCR signaling. Second, most of the pre-existing p21/Zap70 pool was excluded from agonist peptide or Ab induced TCR signaling, which could elicit new p21 and p23 from the previously nonphosphorylated pool of CD3ζ.

The function of p21 in peripheral T cells has yet to be determined. For peripheral T cells, the constitutive phosphorylation of p21 has been correlated with peripheral T cell survival (35). In contrast, a distinct report has shown that CD4⁺ T cells decline after their adoptive transfer into an MHC class II-deficient environment independent of p21 (30).

To address the role of p21 in regulating peripheral T cell survival, we analyzed the percentage of CD4⁺CD8⁻ and CD4⁻CD8⁺ T cells in control and thymectomized mice 9 wk postthymectomy. Thymectomies were used to eliminate the presence of thymic emigrants to the peripheral T cell pool. Mice used for these assays included C57BL/6 mice and the CD3ζ-transgenic lines YF1,2, YF5,6, and YF1–6. Upon thymectomy, the number of CD4⁺CD8⁻ T cells in the lymph node was reduced by 11% in C57BL/6 mice relative to mock-treated controls (p < 0.001; Fig. 6,A). A similar decrease occurred in mice that contained p21 but lacked p23 (YF1,2), and in mice that lacked both p21 and p23 (YF5,6). In mice that lacked all phosphorylated CD3ζ forms (YF1–6), the number of CD4⁺CD8⁻ T cells decreased by 19%. An analysis of the CD4⁻CD8⁺ T cells revealed a decrease in the percentage of cells similar to that shown for the CD4⁺CD8⁻ T cells (Fig. 6,B). There was, however, no significant decrease in the number of CD4⁻CD8⁺ T cells from the thymectomized YF1–6 line, as had been observed for the CD4⁺CD8⁻ cells (p > 0.05; Fig. 6 B).

Next, we analyzed the survival of naive CD4⁺ T cells, as defined by the expression of CD62L (Fig. 6 C). The number of naive CD62L⁺CD4⁺ T cells in the lymph node declined by 12% in C57BL/6 mice 9 wk after thymectomy (p < 0.05). There was a 7% reduction of these cells in the YF1,2 line. A more substantial reduction of 17% was noticed in the YF5,6 line (p < 0.01). The greatest reduction of naive cells was noted in the YF1–6 mice, where a 27% reduction in T cells was detected (p < 0.001). We also analyzed the percentage of peripheral T cells in the spleens of control and thymectomized mice. The results were comparable with that noted in the lymph node (data not shown). We also undertook adoptive transfer experiments using CSFE-labeled T cells from the YF lines into C57BL/6 recipients. Again, we were unable to detect statistically significant differences in the survival span of the YF1,2 vs YF5,6 T cells (data not shown). In summary, p21 did not appear to be critical for regulating T cell survival based on the comparisons of the YF1,2 and YF5,6 lines. The substantial changes seen in the YF1–6 mice, however, suggested that the number of ITAMs available in the TCR were important for T cell survival in the peripheral lymphoid organs.

The CD3ζ subunit is unique among the ITAM-containing proteins in the TCR, BCR, Fc, and NK cell receptor complexes as it exists as a constitutively tyrosine-phosphorylated protein (p21) when isolated ex vivo (reviewed in Ref. 6). There has been a considerable effort to define the functional role of the different phosphorylated forms of CD3ζ, in particular p21 (reviewed in Ref. 36). We provide evidence that the constitutive tyrosine phosphorylation of p21 resulted from TCR interactions with MHC molecules in the thymus, independent of the specificity of the TCR for self-peptide/self-MHC molecules. p21 appeared to be functionally inert, given that the development of mature thymocytes and the biochemical changes in Zap70 activation assessed after TCR triggering were not significantly enhanced or inhibited by its presence. Intracellular staining with anti-phospho-ζ-specific mAbs suggested that p21 was either inaccessible or at levels too low to detect when present in a constitutively phosphorylated form in immature thymocytes.

The constitutive phosphorylation of CD3ζ was dependent on TCR interactions with MHC molecules, with a 5- to 15-fold reduction in the magnitude of p21 phosphorylation evident in thymocytes isolated from MHC II-β₂m double-deficient mice (11). The presence of some p21 in these mice was consistent with the fact that a low level of MHC class I expression, particularly H-2D^b, is apparent in β₂m-deficient mice (37). That TCR interactions with peptide-MHC molecules elicit p21 formation was supported by analyses of TCRα- and Zap70-deficient mice, wherein p21 phosphorylation was almost absent (12, 13). It is assumed that the positive and/or negative selecting peptides that shape the T cell repertoire are responsible for the constitutive phosphorylation of CD3ζ. Our current experiments suggest that the TCR interactions with selecting self-peptides/self-MHC are not required for the generation of p21. p21 was evident in thymocytes expressing either class I- or class II-restricted TCRs that were unable to interact with the selecting ligands. We also noted that CD4⁻CD8⁻ thymocytes from HY TCR-transgenic male mice and cells from CD4-deficient mice contained p21 (11, 29). Given these findings, we propose the following mechanism for the constitutive tyrosine phosphorylation of CD3ζ. TCR interactions with MHC molecules, independent of the selection environment, activate intracellular signals involving the Src and Syk families of kinases. This is consistent with the reported ability of the TCR to recognize disparate MHC molecules before repertoire selection (2, 3). These interactions may cause an activation and/or redistribution of Lck (and to a lesser extent, Fyn), which phosphorylates the four distal tyrosine residues in the ITAMs of CD3ζ. Upon their tyrosine phosphorylation, the two C-terminal ITAMs in CD3ζ are complexed by the tandem SH2 domains of Zap70, which stabilize p21. CD4 interactions with MHC class II interactions are hypothesized to activate Lck, which in turn phosphorylates CD3ζ in immature thymocytes (10, 38). In addition, CD8 can bind to MHC class I molecules in immature thymocytes, implicating a possible role for CD8-associated Lck to phosphorylated CD3ζ (39). Our findings indicate that p21 is independent of CD4 and/or CD8 coreceptor interactions with MHC molecules. However, both CD4 and CD8 could contribute to p21 by stabilizing appropriate TCR/MHC encounters. It remains very interesting why the Zap70 that is precomplexed to p21 does not become activated. Current experiments are addressing this issue.

Three cellular fates are classically defined for developing thymocytes: death by neglect if the TCR cannot recognize self-peptide/self-MHC; positive selection; or negative selection (reviewed in Ref. 1). Our results suggest an additional step during thymopoiesis, wherein T cell recognition of MHC, independent of positive or negative selection and/or coreceptor expression, induces a biochemical signal resulting in CD3ζ phosphorylation. This likely reflects the structural capacity of the TCR in the prerepertoire population of thymocytes to recognize peptide/MHC complexes. The consequence of this interaction leading to phospho-CD3ζ is currently being elucidated but is also likely to contribute to some as yet unidentified peripheral T cell function.

The constitutively phosphorylated p21 could not be detected in CD4⁺CD8⁺ thymocytes by intracellular staining with anti-phospho-ζ-specific mAbs. Attempts to detect this constitutive pool with TAT fusion proteins containing the tandem SH2 domains of Zap70 linked to GFP or pure 2SH2-GFP were also unsuccessful (Ref. 40 and data not shown). Only after TCR triggering or by Western immunoblotting was phospho-ζ detected. These results suggested that p21 was modified, preventing its detection. This could be a result of its distinct intracellular distribution within the cytoskeleton (41, 42). Alternatively, the p21 could be interacting with distinct SH2 domain containing proteins such as SHP-1, Shc, Sts-1, Sts-2, and/or Src-like adaptor protein in addition to Zap70 (42, 43). The idea that p21 is excluded from de novo signaling events is consistent with the inability of p21 to enhance TCR signal transduction elicited by Abs or peptides. A recently proposed model suggests that endogenous and agonist peptide/MHC complexes function cooperatively in amplifying TCR signal transduction and subsequent effector functions (44, 45). Our findings also imply that the constitutively phosphorylated CD3ζ subunit does not contribute to the agonist response. These interpretations are extremely divergent from that published for peripheral T cells where it was proposed that the presence of p21 enhanced peripheral T cell responses 10-fold over T cells wherein p21 was reduced (19, 46). Additional experiments are necessary to account for these discrepancies but may relate to the particular TCR-transgenic line used and/or the in vivo Ab manipulations. Finally, p21 has been linked to peripheral T cell survival in some but not all studies. Using thymectomy procedures, we were unable to identify a role for p21 in T cell survival (30, 35). However, such experiments are limited because recent reports have indicated the presence of more than one functional thymus in mice (47). Transfer of the various YF series of mice into appropriate MHC-deficient hosts that express or lack the selecting allele could be another way in which to address this question.

In summary, the constitutive phosphorylation of p21 occurs in thymocytes and peripheral T cells in a mechanism that does not require selecting ligands and/or coreceptor molecules. Under certain disease conditions or autoimmune diseases, changes in the levels of p21 could contribute signals that enhance or inhibit autoimmune progression (17, 48).

We appreciate technical help from Angela Mobley for cell sorting with the Mo-Flow cell sorter, Jennifer Young for critically reading the manuscript, and Jessica Murphy for mouse typing. We appreciate the efforts of Dr. B. J. Fowlkes and Charles Mainhart (National Institutes of Health) for their suggestions and assistance in obtaining the 5CC7/Rag/IE^b line from the National Institute of Allergy and Infectious Diseases/Taconic emerging models program. We also thank Drs. Kui Liu and Chandra Mohan (OTII, OTII/Rag), Rance Berg (C10.5, OTI), Jose Canseco (5C.C7), and David Farrar (D011.10) for access to the various TCR-transgenic lines.

The authors have no financial conflict of interest.