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The Journal of Immunology, 1999, 163: 599-602.
Copyright © 1999 by The American Association of Immunologists

Differential CD3{zeta} Phosphorylation Is Not Required for the Induction of T Cell Antagonism by Altered Peptide Ligands1

Haiyan Liu*,{dagger} and Dario A. A. Vignali2,*,{ddagger}

* Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and {dagger} Graduate Program in Pathology and {ddagger} Department of Pathology, University of Tennessee Medical Center, Memphis, TN 38163


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cells recognize foreign Ags in the form of short peptides bound to MHC molecules. Ligation of the TCR:CD3 complex gives rise to the generation of two tyrosine-phosphorylated forms of the CD3 {zeta}-chain, pp21 and pp23. Replacement of residues in MHC-bound peptides that alter its recognition by the TCR can generate altered peptide ligands (APL) that antagonize T cell responses to the original agonist peptide, leading to altered T cell function and anergy. This biological process has been linked to differential CD3{zeta} phosphorylation and generation of only the pp21 phospho-species. Here, we show that T cells expressing CD3{zeta} mutants, which cannot be phosphorylated, exhibit a 5-fold reduction in IL-2 production and a 30-fold reduction in sensitivity following stimulation with an agonist peptide. However, these T cells are still strongly antagonized by APL. These data demonstrate that: 1) the threshold required for an APL to block a response is much lower than for an agonist peptide to induce a response, 2) CD3{zeta} is required for full agonist but not antagonist responses, and 3) differential CD3{zeta} phosphorylation is not a prerequisite for T cell antagonism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The TCR:CD3 complex consists of an Ag-specific {alpha}ß heterodimer and six associated CD3 chains ({epsilon}{gamma}, {epsilon}{delta}, and {zeta}{zeta}), which are required for correct assembly and transport to the cell surface (1, 2). Signal transduction through this complex is mediated by immunoreceptor tyrosine-based activation motifs (ITAM)3 [(D/E)X2YX2(L/I)X7YX2(L/I)], which are phosphorylated upon TCR ligation and recruit SH2-containing proteins (3, 4, 5). While some studies have shown that all three CD3 {zeta}-chain ITAMs are required for maximal T cell function, others suggest that signal transduction proceeds normally in their absence (6, 7, 8). Although T cell development and function is normal in CD3{zeta}-/- mice reconstituted with a CD3{zeta} mutant lacking ITAMs, the selection of T cells expressing certain TCR transgenes is altered and autoreactive T cells have been identified (9, 10, 11, 12).

One of the first demonstrable biochemical events following TCR ligation by MHC:peptide complexes is the tyrosine phosphorylation of the TCR-associated CD3{zeta} homodimer (13). This gives rise to the recruitment of ZAP-70, a tyrosine kinase that has been shown to be a key component of the signaling cascade (14, 15, 16). Differential phosphorylation of the ITAMs on CD3{zeta} leads to the generation of two distinct m.w. species by SDS-PAGE, a lower pp21 band and an upper pp23 band; the latter is only seen following full T cell activation (17, 18, 19). Recent studies have suggested that the pp21 form contains two or three monophosphorylated ITAMs, while pp23 has all the ITAM tyrosines phosphorylated (20). The former observation is surprising given that ZAP-70 is constitutively associated with pp21 and that ZAP-70 will only bind to doubly phosphorylated ITAMs (21, 22, 23).

Presentation of altered peptide ligands (APL) has been shown to induce only the lower pp21 CD3{zeta} phospho-species, essentially no phosphorylated CD3{epsilon}, and weak association of unphosphorylated ZAP-70 that lacks stable kinase activity (17, 18, 19, 24, 25, 26, 27, 28). A similar phenotype has also been seen with T cells stimulated with nonmitogenic anti-CD3 mAbs (29). Thus, a correlation has been drawn between differential CD3{zeta} phosphorylation and lack of active ZAP-70, and the induction of T cell antagonism, anergy, and altered T cell function.

It has recently been suggested that the lack of IL-2 production, rather than an altered pattern of TCR-mediated phosphorylation, is the crucial factor controlling anergy induction (24). While an altered pattern of CD3{epsilon} and CD3{zeta} phosphorylation was observed in T cells anergized by exposure to APL, this was not observed when anergy was induced by a lack of costimulation. However, it is not clear whether the biochemical events that lead to reduced IL-2 production are the same in the two systems. In addition, several groups have observed a correlation between differential CD3{zeta} phosphorylation and antagonism induced by APL in murine T cell hybridomas, despite their lack of autocrine dependency on IL-2 (28, 30). Thus, the relative importance of differential CD3{zeta} phosphorylation remains unclear, and there has been no direct molecular examination of this issue. Resolution of this question is important given current interest in the use of APL as a strategy for treating autoimmune disease.

In this study, we have utilized CD3{zeta}-loss variants of the hen egg lysozyme (HEL) 48-62-specific, H-2Ak-restricted murine T cell hybridoma 3A9 to determine whether differential CD3{zeta} phosphorylation is a prerequisite for, or a consequence of, T cell antagonism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hybridomas and CD3{zeta} mutants

CD3{zeta}-loss variants of 3A9 were cloned by using FACS of cells stained with Abs against CD3 and CD4 (PharMingen, San Diego, CA), as previously described (17, 31). CD3{zeta} mutants were made by recombinant PCR, using a murine CD3{zeta} cDNA as a template (gift from Larry Samelson, National Institutes of Health, Bethesda, MD), as previously described (32, 33). Details of the oligos used are available on request. Mutants were verified by sequencing and cloned into one of two eukaryotic expression vectors, pCIneo (Promega, Madison, WI) or pHßApr-1neo (33). CD3{zeta} loss variants were transfected by electroporation, selected with G418, and either cloned or bulk sorted by FACS (32, 33).

Ag presentation and antagonism assays

Ag presentation assays were performed essentially as described elsewhere (32, 34). Briefly, T cell hybridomas were stimulated with synthetic peptides (Center for Biotechnology CORE facility at St. Jude Children’s Research Hospital, Memphis, TN) at the concentrations indicated, together with LK35.2 as APC (murine B cell lymphoma, H-2Akd). After 24 h, supernatants were removed for the estimation of IL-2 secretion against a recombinant murine IL-2 standard (Genzyme, Cambridge, MA) by culturing with the IL-2-dependent T cell line CTLL-2. Antagonism assays were set up in the same way, except that the APC were first prepulsed with the agonist for 6 h, washed three times, pulsed with the antagonist peptide for 2 h, and then T cell hybridomas added (28).

CD3{zeta} tyrosine phosphorylation analysis.

CD3{zeta} tyrosine phosphorylation analysis was determined as previously described (17). Briefly, LK35.2 cells pulsed with 3 µM peptide were mixed with T cell hybridomas and incubated at 37°C for 5 min. The cell pellet was lysed in 1% Brij 97 (polyoxyethylene 10 oleyl ether; Sigma, St. Louis, MO) at room temperature for 1 h, and immunoprecipitated with a rabbit anti-CD3{zeta} antisera (2 µl, No. 551; gift from David Weist, Fox Chase Cancer Center, Philadelphia, PA) for 2 h at room temperature followed by incubation with 25 µl protein A-Sepharose (Pharmacia, Piscataway, NJ) for 1 h at room temperature (unlike other commonly used detergents, Brij97 precipitates at 4°C and so has to be used at room temperature). Eluted proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Schleicher & Schuell, Keene, NH). Blots were blocked with 5% BSA (Boehringer Mannheim, Indianapolis, IN) in TBST, and tyrosine phosphorylation was detected with biotinylated 4G10 (0.1 µg/ml; Upstate Biotechnology, Lake Placid, NY) (90 min at room temperature), followed by 1:12,000 dilution of streptavidin-HRP (Amersham, Arlington Heights, IL) (60 min at room temperature). Blots were developed using ECLplus (Amersham). To detect the original protein, blots were stripped in 100 mM 2-ME (Bio-Rad, Hercules, CA), 2% SDS, 62.5 mM Tris-HCl (pH 6.7) for 30 min at 50°C, washed three times and blocked with 5% nonfat dry milk in TBST at 4°C overnight. Blots were probed with an anti-CD3{zeta} mAb, H146 (1:4 for 60 min at room temperature; gift from Ralph Kubo, Cytel, San Diego, CA), followed by protein A-HRP (1:12,000; Amersham) (60 min at room temperature). Blots were developed as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Significant contribution of the CD3{zeta} ITAMs in IL-2 production

Two CD3{zeta}-loss variants of 3A9 (3A9{zeta}-.4 and 3A9{zeta}-.7) were transfected with either wild-type CD3{zeta} (CD3{zeta}.WT), a CD3{zeta} mutant lacking functional ITAM motifs (CD3{zeta}.{Delta}ITAM), or a truncated form of CD3{zeta} lacking most of the cytoplasmic domain (CD3{zeta}.{Delta}CY) (Fig. 1GoA). All three molecules restored TCR:CD3 expression to levels comparable to the parental TCR + 3A9 T cell hybridoma (Fig. 1GoB, and data not shown). Western blot analysis demonstrated that the amount of CD3{zeta}.WT, CD3{zeta}.{Delta}CY, and CD3{zeta}.{Delta}ITAM expressed by the transfectants was similar (Fig. 1GoC). Furthermore, no parental wild-type CD3{zeta} could be detected in the CD3{zeta}.{Delta}CY transfectants or the loss mutants.



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  		FIGURE 1. Characteristics of T cell hybridomas expressing mutant and wild-type CD3{zeta}. A, Schematic depicting the CD3{zeta} molecules expressed by the T cell hybridomas. The extracellular domain is on the left, and the transmembrane region is in black. In CD3{zeta}.{Delta}ITAM, the tyrosine (Y) residues in the ITAMs have been substituted with phenylalanine (F). In CD3{zeta}.{Delta}CY, residues 46–129 have been deleted. CY, cytoplasmic tail. B, Flow cytometric analysis of TCR expression by the 3A9 CD3{zeta} loss mutants and their transfectants, as indicated by surface staining with an anti-CD3{epsilon} Ab. C, Western blot analysis of T cell hybridomas expressing mutant and wild-type CD3{zeta}. The untransfected CD3{zeta} loss mutants are included as controls.

 
The CD3{zeta}.WT T cell transfectants were phenotypically and functionally indistinguishable from the original 3A9 T cell hybridoma in all assays performed in this study (data not shown). The CD3{zeta}.{Delta}CY and CD3{zeta}.{Delta}ITAM T cell transfectants, however, produced 5 times less IL-2 than the CD3{zeta}.WT control, and required 30 times more peptide to induce IL-2 production (Fig. 2GoA). Thus, the loss of CD3{zeta} ITAMs reduces T cell sensitivity and strength of response. Some clones of CD3{zeta}.WT.3A9{zeta}-.4 were isolated that produced lower levels of IL-2 comparable to the T cells expressing mutant CD3{zeta} (Fig. 2GoA). These served as useful controls for subsequent experiments.



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  		FIGURE 2. T cell function in cells expressing mutant CD3{zeta}. A, IL-2 production by T cell hybridomas following stimulation with HEL 48-63 presented by LK35.2 B cells. IL-2 concentration was determined using CTLL-2 cells. Data are representative of three experiments. B, Only wild-type CD3{zeta} is tyrosine phosphorylated following T cell stimulation. T cell hybridomas transfected with wild-type and mutant CD3{zeta}, and the CD3{zeta}- control, were incubated with or without HEL 48–63 and the degree of CD3{zeta} phosphorylation determined. 3A9{zeta}-.4 transfectants gave identical results.

 
As expected, stimulation of the CD3{zeta}.WT, but not the CD3{zeta}.{Delta}CY and CD3{zeta}.{Delta}ITAM T cell transfectants with HEL 48–63-pulsed B cells induced the tyrosine phosphorylation of CD3{zeta} (Fig. 2GoB). These data confirmed that the mutant CD3{zeta} molecules could not be phosphorylated and that these T cell transfectants had not re-expressed wild-type CD3{zeta}.

T cell antagonism in the absence of the CD3{zeta} cytoplasmic domain

Using T cells from 3A9.TCR transgenic mice, we have shown that HEL 48-Q57A-63 is a strong peptide antagonist, HEL 48-L56A-63 is a weak peptide antagonist, and HEL 48-L56A-61AA is a null peptide (R. T. Carson and D. A. A. Vignali, unpublished observations). These substitutions do not affect peptide affinity (17, 35, 36). Stimulation of the CD3{zeta}.WT T cell transfectants with HEL 48-63 induced the tyrosine phosphorylation of CD3{zeta} and generation of both the pp21 and pp23 forms (Fig. 3GoA). The strong antagonist, HEL 48-Q57A-63, induced only the lower pp21 phosphorylated form of CD3{zeta}. This was consistent with previous studies that had demonstrated a correlation between differential CD3{zeta} phosphorylation and the induction of anergy (18, 19). However, no CD3{zeta} phosphorylation above basal levels was observed with HEL 48-L56A-63 and 48-L56A-61AA in the CD3{zeta}.WT transfectants.



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  		FIGURE 3. The absence of a functional CD3 {zeta}-chain has no effect on the ability of T cells to be antagonized. A, Differential CD3{zeta} phosphorylation in response to peptide antagonists. 3A9{zeta}-.7 CD3{zeta}.WT T cells were stimulated with LK35.2 cells pulsed with 3 µM agonist (48-63) and APL (48-Q57A-63, 48-L56A-63, 48-L56A-61AA) as indicated, and tyrosine phosphorylation of CD3{zeta} determined. Blots were stripped and reprobed with anti-CD3{zeta} to demonstrate comparable loading. 3A9{zeta}-.4 CD3{zeta}. WT transfectants gave comparable results. B, The ability of three APL to antagonize the response of T cell hybridomas expressing mutant and wild-type CD3{zeta} to the agonist, HEL 48-63, was tested. Percentages on the right-hand side represent the reduction in IL-2 release induced by the agonist following addition of 10 µM of the APL indicated. Data are representative of nine experiments.

 
The ability of these three APL to antagonize T cell responses to the agonist, HEL 48-63, was assessed. A 30-fold molar excess of the HEL 48-Q57A-63 APL over agonist resulted in a substantial reduction in IL-2 secretion by the CD3{zeta}.WT T cell transfectants (3A9{zeta}-.7, 97%; 3A9{zeta}-.4, 86%: Fig. 3GoB). Both HEL 48-L56A-63 and HEL 48-L56A-61AA manifested reduced levels of antagonism. Three conclusions can be drawn from these data. 1) The ability of APL to modulate responses of 3A9 T cell hybridomas and T cells from 3A9.TCR transgenic mice was comparable. 2) A low level of T cell antagonism could be obtained with only basal levels of CD3{zeta} phosphorylation. 3) The ability of HEL 48-Q57A-63 and 48-L56A-63 to antagonize CD3{zeta}.WT transfectants of 3A9{zeta}-.7 and 3A9{zeta}-.4 was comparable, suggesting that differences in sensitivity and IL-2 production in response to the agonist has little effect on the ability of T cells to be antagonized by APL.

To our surprise, the CD3{zeta}.{Delta}CY and CD3{zeta}.{Delta}ITAM T cell transfectants were also antagonized with HEL 48-Q57A-63 and 48-L56A-63 to a level comparable to the CD3{zeta}.WT T cell transfectant (Fig. 3GoB). Furthermore, antagonism was observed despite the low IL-2 production by, and reduced sensitivity of, T cell transfectants expressing mutant CD3{zeta} in response to the agonist HEL 46-63. This phenotype was comparable to the 3A9{zeta}-.4 CD3{zeta}.WT transfectant, which was also less sensitive to the agonist (Fig. 2GoA). The ability of HEL 48-Q57A-63 and 48-L56A-63 to antagonize T cell responses was consistent and highly reproducible among all the clones and bulk transfectants tested. Although it is not clear why results with the HEL 48-L56A-61AA peptide were variable, the results do show that mutation of the CD3 {zeta}-chain has no effect on the ability of T cells to be antagonized.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data highlight two important observations. First, differential phosphorylation of CD3{zeta} is not a prerequisite for the induction of T cell antagonism. Indeed the complete loss of signal transduction through CD3{zeta} has no effect, implying that the critical biochemical events that underlie T cell antagonism can be efficiently mediated via CD3{epsilon}, CD3{gamma}, and/or CD3{delta}. It remains to be determined whether T cell antagonism could occur in a TCR:CD3 complex in which CD3{zeta} was the only component possessing an intact cytoplasmic tail. Second, despite a significant reduction in sensitivity to Ag and quantity of IL-2 released, T cells expressing TCRs that have lost 6 of 10 ITAMs can still be potently antagonized. This suggests that the threshold required for an antagonist to block a response is much lower than for an agonist to induce a response. This is synonymous with the observation that the absence of CD4 has a significant effect on the response of a T cell hybridoma to agonist peptides, but has little effect on the ability of APL to induce T cell antagonism (37).

Although exogenous IL-2 has been shown to break anergy, the biochemical basis for the lack of IL-2 production by antagonized and anergic T cells remains unresolved. It has been argued that the lack of IL-2 production is the primary factor controlling anergy induction, rather than an altered pattern of TCR-mediated phosphorylation (24). This conclusion was based on the observation that anergy induced by a lack of costimulation failed to give the altered pattern of CD3{epsilon} and CD3{zeta} phosphorylation that was observed in T cells anergized by exposure to APL. However, in the former, there was still a clear difference in the amount of ZAP-70 phosphorylation, and this could have given rise to the same biochemical phenotype as in the APL-stimulated T cells (24) (Fig. 2GoC). Furthermore, no indication was provided for the relative amount of ZAP-70 kinase activity in the two systems. In addition, we and others have demonstrated strong T cell antagonism in T cell hybridomas despite their lack of autocrine dependency on IL-2 (28, 30 , and the present study).

Several groups have shown a correlation between the induction of T cell antagonism and anergy and a novel CD3{zeta} and CD3{epsilon} tyrosine phosphorylation pattern (18, 19, 24, 25, 26, 27, 28). This leads to a transient association of unphosphorylated ZAP-70, which lacks stable kinase activity. This has lead to the suggestion that differential CD3{zeta} phosphorylation may be a key biochemical event in the induction of T cell anergy (19, 20). Our data suggest that while this may be a phenotype of peptide antagonism (or anergy), it may not be a prerequisite. Indeed, the loss of a functional CD3{zeta} cytoplasmic tail clearly has a significant effect on IL-2 production but no effect on the ability of APL to induce antagonism.

Recent studies have suggested that successive, ordered CD3{zeta} phosphorylation occurs following TCR ligation of MHC:peptide complexes (20). The authors suggest that this process sets thresholds that determine whether interaction with a TCR ligand is sufficient to result in T cell activation. APL give rise to incomplete ITAM phosphorylation, thus allowing for the recruitment of single SH2 domain-containing signaling molecules, but not ZAP-70. Given that the T cells used in our study can be potently antagonized in the complete absence of CD3{zeta} phosphorylation, it is probable that such multistep CD3{zeta} phosphorylation plays no role in the induction of T cell antagonism. While we cannot rule out the possibility that this could be mediated by phosphorylation of only one tyrosine in CD3{epsilon}, CD3{gamma}, and/or CD3{delta}, it is important to point out that such differential phosphorylation has not been directly demonstrated for these CD3 molecules. Although the loss of CD3{epsilon} phosphorylation has also been shown as a hallmark of APL-induced TCR signaling (18, 24, 25), it is unclear if this represents partial or no phosphorylation. This could be determined using phosphopeptide-specific Abs against each of the CD3{epsilon}, CD3{gamma}, and CD3{delta} tyrosine motifs, as was recently used to analyze CD3{zeta} phosphorylation (20). While it is likely that the controlled genetic manipulation of all the CD3 components will be required to elucidate the biochemical events that lead to T cell antagonism, it is clear that the differential phosphorylation of CD3{zeta} is not a prerequisite.


   Acknowledgments
 
We thank Kate Vignali for assistance with phosphorylation analysis, David Wiest for the anti-CD3{zeta} antisera 551, Ralph Kubo for the H146 anti-CD3{zeta} mAb, and Larry Samelson for the CD3{zeta} cDNA. We also thank Paula Arnold, Dharmesh Desai, David Woodland, and Creg Workman for their critical review of the manuscript.


   Footnotes
 
1 This work was supported by the National Institutes of Health Grant AI-39480, Cancer Center Support CORE Grant 5 P30 CA21765-17, and the American Lebanese Syrian Associated Charities (ALSAC). Back

2 Address correspondence and reprint request to Dr. Dario Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. E-mail address: Back

3 Abbreveations used in the paper: ITAM, immunoreceptor tyrosine-based activation motif; APL, altered peptide ligand; HEL, hen egg lysozyme. Back

Received for publication January 4, 1999. Accepted for publication April 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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