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A Redundant Role of the CD3-Immunoreceptor Tyrosine-Based Activation Motif in Mature T Cell Function¹

Mariëlle C. Haks^*, Tanina A. Cordaro^*, Jeroen H. N. van den Brakel^*, John B. A. G. Haanen^*, Evert F. R. de Vries, Jannie Borst, Paul Krimpenfort and Ada M. Kruisbeek^2,*

^* Division of Immunology, Division of Cellular Biochemistry, and Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3, CD3, and CD3) or three (CD3) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3 chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3- module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3-, -, and -ITAMs regulate TCR signaling. We here report on the role of the CD3 chain and the CD3-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3 null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3-ITAM. Loss of integrity of the CD3-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3 and/or the CD3 chains endow the TCR with qualitatively unique signaling functions.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
During their development, T cells encounter several Ag receptor-driven checkpoints that are critical for their further maturation. The earliest checkpoint thymocytes encounter occurs early in intrathymic development at the transition from the CD4^-CD8^- double negative to the CD4⁺CD8⁺ double-positive (DP)³ stage. This transition is a control point for productive TCR- rearrangements and is mediated by the pre-TCR (1, 2, 3, 4, 5). The pre-TCR is formed by a TCR- polypeptide, a nonrearranging pT-chain, as well as noncovalently linked invariant CD3 subunits (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Functioning of the pre-TCR critically depends on the signal transduction capacity of the CD3 complex.

The second major Ag receptor-dependent checkpoint in intrathymic T cell development is at the transition from the CD4⁺CD8⁺ DP to the CD4 or CD8 single-positive stage. After completion of TCR- rearrangements, the pre-TCR on DP thymocytes is replaced by a mature clonotypic TCR complex and cells are subjected to positive and negative selection events. Thymocytes surviving this selection process will shut off expression of either the CD4 or CD8 coreceptors and eventually exit the thymus to populate the peripheral lymphoid organs (21, 22).

In the periphery, T cells require an Ag receptor-driven signal to become activated, proliferate, and exert their effector function. These events are mediated by the mature clonotypic TCR complex (23). This TCR complex is composed of a TCR- heterodimer, recognizing Ag in the context of MHC class I and II molecules (24, 25, 26), and the CD3 complex, which plays a key role in transmitting signals after TCR engagement (27, 28, 29, 30). Each TCR- heterodimer is linked to at least four different monomorphic CD3 components, termed CD3, , , and . According to current knowledge, per complex, two copies of the CD3 and CD3 chains are present, yet only a single copy of the highly homologous CD3 and CD3 chains (31, 32, 33). However, the relative contribution of the several CD3 components to mature TCR-mediated signaling has not been completely elucidated.

CD3 components may have partially overlapping functions, because all CD3 components encompass one or several conserved immunoreceptor tyrosine-based activation motifs (ITAMs) (YxxL/Ix_6–8YxxL/I) within their cytoplasmic domains that fully account for their individual signal transduction capacity (30, 34). In support of the view that multiple ITAMs may provide the capacity to amplify signals generated by a single mature TCR complex, a direct relationship between the number of CD3-ITAMs and the efficiency of both positive and negative selection was observed (35). Furthermore, the intensity of the induction of NF-AT activity by chimeric CD3-ITAM-containing polypeptide chains also depended on the number of ITAMs present, suggesting a quantitative function of the several ITAMs contained within the CD3 chain (36). In addition, crippling of the CD3-ITAMs did not result in obvious abnormalities in the spectrum of activation events and effector functions of CD8⁺ peripheral T cells, indicating that at least the CD3-ITAMs have no exclusive role in T cell activation (37). Instead, these findings suggest that the ITAMs in the CD3- module are sufficient for qualitatively normal signaling of the TCR (37). Therefore, the different ITAMs may be functionally redundant.

Alternatively, several reports suggest specialized functions for individual ITAMs (38, 39), presumably as a result of substantial variability in the amino acids flanking the phosphotyrosine residues within the ITAMs, predicting interactions with distinct SH2-domain-containing cytosolic mediators (40, 41). Indeed, ITAMs derived from distinct CD3 chains bind with varying affinities to downstream adaptors and enzymes such as Syk, ZAP-70, p59^fyn, Lyn, Shc, Grb-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase (30, 42, 43, 44, 45, 46, 47, 48, 49). Moreover, signaling by rCD3-ITAM- or CD3-ITAM-containing receptors resulted in differences in substrate tyrosine phosphorylation patterns (27), induction of apoptosis (38), and mobilization of intracellular free calcium (39), suggesting that they may couple to distinct signaling pathways. The observation that these recombinant receptors containing the cytoplasmic tail of CD3 or CD3 were equally capable of inducing early and late T cell activation events (27, 37, 50) already indicates that both options (qualitative vs quantitative differences between individual ITAMs) are not mutually exclusive and may operate simultaneously during TCR-mediated signaling (38).

In all Ag receptor-driven checkpoints, the CD3 chain may play a crucial role, in particular because it was shown that the CD3- module endows the TCR with normal signaling function (37). We previously reported that mice lacking CD3, due to targeted gene disruption, display serious defects in T cell development (16). The transition from the CD4^-CD8^- double negative to the CD4⁺CD8⁺ DP stage is severely impaired, indicating that CD3 is required for the earliest Ag receptor-driven control point (16). Furthermore, the nearly complete absence of CD69-positive DP thymocytes in mice lacking CD3 (51) suggests that CD3 also plays a role during the second major Ag receptor-dependent control point, because expression of CD69 is normally up-regulated by DP thymocytes that have been positively selected. Here we explore the consequences of CD3 deficiency and CD3-ITAM deficiency for the functional capacity of peripheral T cells in vitro and in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of CD3-ITAM mutant mice

A mouse genomic clone encompassing the CD3 gene was isolated from a 129SVJ phage library (Stratagene, La Jolla, CA). To construct the targeting vector CD3-I, a 10.2-kb fragment comprising CD3 exons 3–7 was subcloned into plasmid pBS-SKII (Stratagene). A 1.3-kb PvuII-NsiI fragment encompassing the CD3-ITAM was replaced by two complementary oligonucleotides encoding 5'-CTGTGAGTGTCCCCCCTTCTATCCAGCACCCAGAATCAAAACAATGCA-3' and 5'-TTGTTTTGATTCTGGGTGCTGGATAGAAGGGGGGACACTCACAG-3', restoring the PvuII site in exon 5 and introducing a stop codon immediately after this particular PvuII site. The oligonucleotide sequence directly 3' of the stop codon restores the deleted nucleotide sequence of exon 7 including the NsiI site. In addition, a 1.1-kb SacI-SacI fragment contained within intron 4 was replaced by a 3.0-kb pgk-HPRT cassette flanked by loxP sites. The CD3-I targeting vector was electroporated into the HM-1 ES cell line derived from 129OLA mice. Clones resistant to HAT (Life Technologies, Paisley, U.K.) were individually screened by Southern blot analysis for homologous recombination events, using a probe located outside the targeting construct, which recognizes a 10.6-kb wild-type (wt) fragment and a 9.3-kb recombinant fragment in case DNA is digested with SacI and an 8.2-kb wt fragment and an 11.2-kb recombinant fragment when PstI-digested DNA is used. Two homologous recombinants were identified of 600 colonies tested. Subsequently, clones were transiently transfected with Cre recombinase to remove the pgk-HPRT cassette from the embryonic stem (ES) cell genome. Clones surviving counterselection by 6-Thioguanine (Sigma, St. Louis, MO) were individually screened by Southern blot analysis for loss of the pgk-HPRT cassette, using EcoRV-digested DNA and a probe located inside the targeting construct recognizing an 11.0-kb wt fragment, a 3.9-kb recombinant fragment still containing the pgk-HPRT cassette, and an 8.6-kb recombinant fragment after removal of the pgk-HPRT cassette. One clone was selected of 150 colonies tested and injected into C57BL/6 blastocysts to generate chimeric mice. Male chimeric mice were subsequently crossed to female FVB mice. Germline transmission was obtained and heterozygous mice were intercrossed to produce homozygous CD3^I/I mice.

Mice

Mice were maintained under specific pathogen-free conditions in the animal colony of The Netherlands Cancer Institute and analyzed at 8–12 wk of age. Mice deficient for CD3 and mice expressing the F₅-TCR transgenes have been described in detail elsewhere (16, 52).

Virus infection

Purified recombinant influenza A virus strain A/NT/60/68 was kindly provided by Dr. R. Consalves (National Institute of Medical Research, London, U.K.). A/NT/60/68 was grown and tested for hemagglutination activity and infectious titers in the Department of Virology, Erasmus University Rotterdam, The Netherlands.

Mice were anesthetized and subsequently infected intranasally with 50 µl of PBS with or without A/NT/60/68 virus (25 Hau for a primary infection and 250 Hau for a secondary infection). Draining mediastinal lymph nodes (MLNs), lungs, and spleens were analyzed at the indicated days postinfection.

Flow cytometry

Preparation of samples for flow cytometry analysis was performed as described (16). Cells were analyzed on a BD Biosciences FACSCalibur (Mountain View, CA). Forward- and side-scatter gating and/or propidium iodide gating was used to exclude dead cells from the analysis.

Biotinylated, FITC-, PE-, or APC-conjugated Abs specific for murine CD4 (clone RM4-5), CD8 (clone 53-6.7), CD8 (clone 53-5.8), CD25 (clone 7D4), CD62L (L-selectin; clone MEL-14), CD69 (clone H1.2F3), CD90.1 (Thy-1.1; clone HIS51), CD90.2 (Thy-1.2; clone 30-H12), TCR- (clone GL3), and TCR- (clone H57-597) were obtained from BD PharMingen (San Diego, CA). R-PE anti-mouse CD4 (clone CT-CD4) was purchased from Caltag (South San Francisco, CA). Where appropriate, streptavidin-Tricolor or streptavidin-PE (Caltag) was used as second-step reagent.

Preparation of PE-conjugated H-2D^b-tetramers containing the nucleoprotein peptide (NP_{366–374(NT)}; ASNENMDAM) of the influenza A virus strain A/NT/60/68 has been described previously (53).

T cell purification

Single-cell suspensions of total lymph node cells were prepared in IMDM (Life Technologies) supplemented with 10% FCS (PAA Laboratories GmbH, Linz, Austria), 2 x 10^-5 M 2-ME (Merck, Darmstadt, Germany), 100 U/ml penicillin, and 100 µg/ml streptomycin. Erythrocytes were lysed by incubating the cells in a hypotonic buffer (0.14 M NH₄Cl, 0.017 M Tris pH 7.2) for 5 min on ice. Subsequently, cells were washed twice and passaged over a nylon wool column. To remove remaining B cells and other MHC class II expressing cells, cells were incubated with saturating concentrations of anti-MHC class II (clone M5/114) mAb for 30 min at 4°C. Cells were washed once and MHC class II-positive cells were negatively selected twice using a mixture of goat anti-mouse IgG-coated (Advanced Magnetics, Cambridge, MA) and sheep anti-rat IgG-coated magnetic beads (Dynal, Oslo, Norway) in a 5:1 ratio. This procedure resulted in >97% Thy-1.1/Thy-1.2 positive T cells as determined by flow cytometry.

T cell activation, TCR down-regulation, and proliferation assay

Purified lymph node T cells (2 x 10⁵) were cocultured with 4 x 10⁵ (in case flow cytometry was performed as a readout system: expression analysis of activation markers and TCR down-regulation) or 1 x 10⁵ (in case [³H]thymidine incorporation was measured as a readout system: proliferation assay) irradiated syngeneic spleen cells as a source of APCs in a total volume of 200 µl per round-bottom microtiter well. Cultures were incubated in a 37°C, 5% CO₂ humidified incubator. For immobilized, plate-bound Ab induced stimulation, anti-CD3 (clone 145-2C11) (54) or control hamster IgG (Jackson ImmunoResearch, West Grove, PA) was diluted in PBS, and 50 µl of diluted Ab was added per well. Plates were incubated for 6 h at 37°C and washed three times with PBS before use. After 24 h, cells were stained for flow cytometric analysis. Alternatively, 100 µl of culture supernatant was collected at indicated time points for cytokine analysis and cultures were pulsed with 0.5 µCi/well [³H]thymidine. Cultures were harvested 18 h later, and [³H]thymidine incorporation was measured using liquid scintillation counting. The analysis was performed in triplicate and SDs were <10% of the mean.

IL-2 assay

IL-2 production was analyzed using the IL-2-responsive cell line CTLL-2 (American Type Culture Collection, Manassas, VA). After three washes with complete medium, 5 x 10³ CTLL-2 cells/well were cultured in a flat-bottom microtiter plate with 75 µl supernatant in a total volume of 100 µl. Human rIL-2 (Cetus, Emeryville, CA) was used as a control. At indicated time points, cultures were pulsed with 0.5 µCi/well [³H]thymidine for 18 h and thymidine incorporation was measured by liquid scintillation counting. All assays were performed in triplicate and SDs were <10% of the mean.

Mixed lymphocyte reaction

For primary MLRs, 2 x 10⁵ purified lymph node T cells (for suboptimal conditions: 1 x 10⁵ purified lymph node T cells) were cocultured with indicated numbers of irradiated MHC-mismatched SJL/J spleen cells in a round-bottom microtiter plate in a total volume of 200 µl. After 4 days, cultures were pulsed with 0.5 µCi/well [³H]thymidine for 18 h and thymidine incorporation was measured by liquid scintillation counting. All assays were performed in triplicate and SDs were <10% of the mean.

CTL effector function assay

Polyclonal induction of CTL activity was established by coculturing 2 x 10⁶ purified lymph node T cells with 4 x 10⁶ irradiated MHC-mismatched SJL/J spleen cells (for suboptimal conditions: 1 x 10⁶ purified lymph node T cells with 2 x 10⁶ irradiated MHC-mismatched SJL/J spleen cells) in a 24-well plate in a total volume of 2 ml. After 4 days, effector T cells were harvested and CTL activity was assayed as described (54). Briefly, effector cells were cocultured with ⁵¹Cr-labeled (Amersham, Little Chalfont, U.K.) non-Ag-bearing, Fc receptor-positive K562 cells for 18 h in the presence or absence of 5 µg/ml of soluble anti-CD3 (clone 145-2C11). The percentage of specific lysis was calculated as the ratio of 100 x (cpm experimental release - cpm spontaneous release)/(cpm 1% Triton X-100 release - cpm spontaneous release).

Generation of anti-peptide sera

Peptides corresponding to the C-terminal regions of CD3 (EYDQYSHLQGNQLRKK) and CD3 (TQYSRLGGNWPRNKKS) were synthesized and coupled to cationized BSA using the Imject Activated SuperCarrier System according to the manufacturer’s protocol (Pierce, Rockford, IL). The peptide-cationized BSA conjugates were used to immunize rabbits.

Radiolabeling and immunoprecipitation

Lactoperoxidase-catalyzed surface labeling of 10⁸ purified lymph node T cells with Na¹²⁵I (Amersham) was performed and cells were subsequently lysed in lysis buffer (1% Brij96, 150 mM NaCl, 10 mM triethanolamine (pH 7.8), 5 mM EDTA, 1 mM PMSF, 20 µg/ml trypsin inhibitor, 20 µg/ml leupeptin, and 20 µg/ml TLCK) for 30 min on ice. Nuclear debris was removed by centrifugation at 14.000 rpm for 15 min at 4°C. Cell lysates were extensively precleared by incubation with normal hamster serum or normal rabbit serum and protein A-Sepharose-CL4B beads (Pharmacia, Uppsala, Sweden). Subsequently, extracts were immunoprecipitated with Abs specific for CD3, CD3, or CD3 (54) in the presence of protein A-Sepharose-CL4B beads for 2 h at 4°C. After five to six washes in lysis buffer, immunoprecipitates were resuspended in 30 µl of sample buffer (10% glycerol, 3% SDS, 62.5 mM Tris pH 6.8, 0.005% Bromophenol blue, and, if applicable, 5% 2-ME), heated for 5 min at 95°C, cooled to room temperature, and loaded on one-dimensional 10–15% gradient SDS-PAGE gels or first on a 12.5% SDS polyacrylamide tube gel under nonreducing conditions, followed by a 12.5% SDS-PAGE slab gel under reducing conditions. After fixation and drying of the gels, signals were visualized and quantified by phospho-imaging or autoradiography at -70°C.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subunit composition of the mature TCR complex expressed by CD3-deficient peripheral T cells

In contrast to peripheral T cells derived from wt mice, the few peripheral T cells that are exported to the periphery in mice lacking CD3 display severely reduced expression levels of CD3 and TCR-, reaching only 5–10% of the levels observed on control populations (16). This finding suggests that in the absence of CD3, assembly and/or efficient transport of the mature TCR complex to the cell surface is severely impaired. Indeed, biochemical analysis of the mature TCR complex after surface radio-iodination confirmed that lymph node T cells lacking CD3 express strongly reduced levels of both the TCR- heterodimer as well as CD3 components compared with their wt counterparts (Fig. 1A). It should be noted, in this respect, that the exposure time of the CD3-deficient TCRs shown in Fig. 1A is three times as long as the exposure time of the wt peripheral TCRs. Furthermore, only anti-CD3 and anti-CD3 but not anti-CD3 Abs immunoprecipitated the TCR complex expressed on lymph node T cells from CD3-deficient mice, while all three antisera used could easily immunoprecipitate the TCR complex expressed by control T cells (Fig. 1A). These data indicate that with the exception of CD3, lymph node T cells derived from CD3 null-mutant mice display the normal complement of TCR subunits. In agreement with these results, two-dimensional nonreduced/reduced SDS-PAGE revealed once more the presence of CD3 and CD3 proteins in addition to the disulfide-linked TCR- heterodimer at the cell surface of T cells lacking CD3 (Fig. 1B).

View larger version (76K): [in this window] [in a new window]   FIGURE 1. Biochemical analysis of the clonotypic TCR complex expressed on peripheral T cells lacking CD3. Lymph node T cells of 8- to 12-wk-old CD3+/+, CD3I/I, and CD3-/- mice were surface iodinated, lysed, and immunoprecipitated with the indicated Abs. Immunoprecipitates were analyzed (A) under nonreducing conditions by SDS-PAGE on a 10–15% gradient gel or (B) by 2-D nonreduced/reduced SDS-PAGE. The positions of TCR-, CD3, CD3, CD3, and CD3 chains are indicated. Poor visualization of the CD3- dimers is due to inefficient labeling by surface radio-iodination of this particular chain.

T cell development in CD3-ITAM mutant mice

Because the CD3 protein is clearly required for efficient surface expression of the TCR, besides endowing the TCR with signaling capacity, quantitative or qualitative causes for any signaling defect must be distinguished. For this purpose, CD3-ITAM mutant mice lacking only the ITAM of the CD3 chain (Fig. 2A) were generated by gene targeting in ES cells, using CD3-I as a targeting vector containing a pgk-HPRT cassette flanked by loxP sites (Fig. 2B). Two homologous recombinants were identified of 600 colonies tested and subsequently transiently transfected with Cre recombinase to remove the pgk-HPRT cassette from the ES cell genome. One clone was selected of 150 colonies tested and used for the generation of chimeric mice. Germline transmission was obtained and heterozygous mice were intercrossed to produce mice homozygous for the CD3-ITAM mutation (Fig. 2C).

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Disruption of the CD3-ITAM by homologous recombination. A, Schematic representation of the intracellular tail of the wt CD3 chain (top) and the CD3-ITAM chain (bottom). B, Partial organization of the CD3 locus (top), structure of the targeting vector CD3-I (middle), and structure of the disrupted CD3 gene following homologous recombination and transient transfection of the Cre recombinase (bottom). The probes used for the hybridizations and the predicted fragment sizes generated by the endogenous and targeted alleles after EcoRV digestion are depicted. Exons are denoted as filled boxes and numbered 3–7. The location of the CD3-ITAM is indicated by Y LYL above exons 5 and 6, respectively. Restriction enzymes: B, BamHI; EV, EcoRV; N, NsiI; P, PstI; Pv, PvuII; S, SacI. C, Southern blot analysis of EcoRV-digested tail DNA derived from wt (CD3+/+), heterozygous mutant (CD3+/I), and homozygous mutant (CD3I/I) mice.

At face value, T cell development proceeds undisturbed in the absence of the CD3-ITAM. Indeed, as illustrated in Fig. 3, A and B, by CD4 vs CD8 staining (top), mature T cells can easily be detected in normal numbers in the thymus and lymph nodes of CD3^I/I mice. Consistent with our previous report, mice lacking the complete CD3 chain exhibit strongly reduced thymic and lymph node cellularity and reduced CD3 and TCR- surface expression on mature T cells (Fig. 3, A and B) (16). The expression level of the mature TCR complex on T cells from CD3-ITAM mice, in contrast, is similar to that on control T cells (Fig. 3, A and B, bottom). We also analyzed to which extent loss of the CD3-ITAM affects development of the TCR- lineage and find apparently normal maturation of this T cell lineage as well, quite in contrast to the situation in CD3-deficient mice (Fig. 3C) (16). The absolute numbers of TCR--positive T cells that could be detected in the thymus as well as in the periphery of CD3-ITAM mutant and wt mice are comparable, and equivalent levels of the TCR complex are expressed in wt and CD3-ITAM mutant T cells (Fig. 3C; data not shown). Importantly, these data indicate that in CD3-deficient mice, the observed defects in and T cell development are not the result of impaired CD3-ITAM-specific signaling, but of defective assembly of the pre-TCR in case of maturation of T cells, and impaired assembly of a pre-TCR--type structure or the mature TCR- complex in case of T cell differentiation.

View larger version (55K): [in this window] [in a new window]   FIGURE 3. Development of both the and T cell lineage is unaffected in CD3-ITAM mice. (A) Thymocytes and (B) lymph node cells of 8- to 12-wk-old CD3+/+, CD3I/I, and CD3-/- mice were analyzed by three-parameter flow cytometry for the expression of CD4-PE vs CD8-Biotin plus SA-Tricolor (top) and for the expression of TCR-FITC (gray) or an irrelevant mAb (white) in the distinct cell populations that are characterized, based on the differential expression of CD4 vs CD8 (bottom). The percentage of cells within each quadrant is indicated. The absolute number of cells detected in the different genotypes is depicted above the corresponding dot display. C, Total thymocytes were analyzed for the expression of TCR-FITC vs CD3-PE. The percentage of cells within the depicted gates is indicated in each dot display.

Expression of surface activation markers is impaired in CD3^-/- as well as CD3-ITAM peripheral T cells albeit to different extents

To investigate the functional capacity of primary T cells deficient for CD3 or expressing CD3 subunits devoid of a functional ITAM, lymph node T cells from wt, CD3-ITAM, and CD3 null-mutant mice were stimulated with plate-bound anti-CD3 or control hamster IgG and stained for CD25, CD69, or CD62L (Fig. 4). Within 24 h, 75–95% of the wt peripheral T cells express CD25 or CD69 and down-regulate CD62L in response to anti-CD3 treatment. In the absence of an intact CD3-ITAM, the percentage of T cells exhibiting an activated phenotype drops to 50–65%, suggesting a quantitative contribution of the CD3-ITAM with respect to the induction or reduction in expression of these particular activation markers. Moreover, only a relatively small fraction (30–35%) of the CD3-deficient T cells display a similar activated phenotype. This reduced number is likely to be related to the recent report that T cell activation requires a certain number of TCRs to be triggered, irrespective of the nature of the triggering ligand (55). The capacity to reach this activation threshold is severely compromised by a reduction in the number of TCRs expressed at the cell surface. Therefore, the reduced TCR surface expression on lymph node T cells lacking CD3 (16) (Figs. 1A and 3B) will impair the ability to reach the optimal activation threshold and may provide an explanation for the small proportion of activated CD3^-/- T cells detected after exposure to anti-CD3 mAb. Importantly, the activated CD3^-/- T cell population expresses normal levels of CD25 and CD69 (Fig. 4). This suggests that, at least with respect to the expression levels of these cell surface markers, T cells that finally have reached the activation threshold can become fully activated in the absence of CD3.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Regulation of expression of activation markers is impaired in CD3-deficient as well as CD3-ITAM-deficient peripheral T cells, albeit to different extents. Lymph node T cells of 8- to 12-wk-old CD3+/+, CD3I/I, and CD3-/- mice were analyzed by flow cytometry for the expression of CD25, CD69, and CD62L (open areas) or an irrelevant mAb (gray areas) in response to stimulation with 10 µg/ml plate-bound control hamster IgG (thin line) or 145–2C11 (anti-CD3) (fat line) for 24 h. In several histograms, the staining with irrelevant mAb and the specific staining after stimulation with control hamster IgG overlap.

Proliferation and cytokine secretion is delayed only in CD3-deficient peripheral T cells

We next evaluated the capacity of peripheral T cells that developed in CD3^+/+, CD3^I/I, or CD3^-/- mice to proliferate and secrete IL-2 in response to anti-CD3-mediated cross-linking (Fig. 5). Lymph node T cells harboring signaling-defective CD3 chains displayed dose-response curves almost superimposable to those obtained using control peripheral T cells (Fig. 5A), clearly indicating that the ITAM of the CD3 subunit is dispensable for induction of these late T cell activation events. In contrast, peripheral T cells lacking CD3 display a delayed onset of proliferation and IL-2 production in response to CD3-mediated triggering (Fig. 5A). Two days after stimulation, CD3-deficient T cells show a strongly impaired capacity to proliferate and to produce IL-2 compared with control T cells. However, 3 days poststimulation, CD3-deficient T cells clearly have started to proliferate and to secrete levels of IL-2 comparable to those observed in wt and CD3^I/I mice (Fig. 5A). These data are in accordance with the view that only a certain proportion of the CD3-deficient T cells gets activated in response to CD3-mediated cross-linking and would suggest that also with respect to proliferation and IL-2 production, CD3-deficient T cells are not dramatically hampered in their functional ability, once they get activated by anti-CD3 mAb triggering. Another possibility is that 3 days after stimulation with anti-CD3 mAb, all CD3-deficient T cells have become activated. However, also this latter explanation would suggest that once activated, CD3-deficient T cells are not defective in the execution of these late T cell activation events. In contrast to anti-CD3 exposure, stimulation with PMA, bypassing external triggering of the TCR complex, results in comparable proliferation and IL-2 production between CD3-deficient and CD3-ITAM or wt peripheral T cells (Fig. 5B). These results indicate that the observed defects in inducing activation are primarily at the level of initiation of the signal-transduction pathway by the TCR complex expressed at the cell surface.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Proliferation and IL-2 secretion are delayed only in CD3-deficient peripheral T cells. Proliferation and IL-2 production were analyzed in lymph node T cells of 8- to 12-wk-old CD3+/+ (), CD3I/I (), and CD3-/- (•) mice in response to (A) stimulation with 145-2C11 (anti-CD3) for 48 h (top) or 72 h (bottom), or (B) treatment with PMA for 24 h (top) or 48 h (bottom).

TCR down-regulation is completely abrogated in CD3-deficient T cells

In addition to the ITAM, a di-leucine motif has been identified in the cytoplasmic domain of the CD3 chain (56, 57, 58). Studies in cell lines have suggested that this motif may be involved at least in protein kinase C (PKC)-mediated TCR down-regulation but is irrelevant for TCR down-modulation resulting from exposure to anti-CD3 (59, 60). After T cell activation, TCR internalization ultimately results in extinction of the signaling process, allowing the cells to calibrate the response to the level of the stimulus. Consistent with the view that the CD3 di-leucine motif is important for PKC-induced internalization, stimulation of lymph node T cells of control mice with PMA resulted in a clear-cut down-modulation of the TCR, whereas T cells derived from mice lacking CD3 were completely defective in this respect (Fig. 6A, top), despite the fact that 90% of the CD3-deficient lymph node T cells were activated according to the expression of the early activation marker CD69 (Fig. 6A, bottom). In sharp contrast, the CD3-ITAM seemed dispensable for efficient internalization of the TCR in response to PMA treatment, because the dose-response curves of TCRs deprived of a functional CD3-ITAM and their wt counterparts were overlapping (Fig. 6A).

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Induction of TCR down-regulation is defective only in CD3-deficient T cells. Lymph node T cells of 8- to 12-wk-old CD3+/+ (), CD3I/I (), and CD3-/- (•) mice were analyzed by flow cytometry for the expression of TCR-FITC (top) or CD69-biotin plus SA-PE (bottom) in response to (A) treatment with PMA for 5 h or (B) stimulation with anti-CD3 for 24 h. In both A and B, 100% value corresponds to the mean fluorescence of the TCR- staining on unstimulated peripheral T cells.

Cytotoxic T cell responses are affected in T cells lacking CD3 when stimulated under suboptimal conditions

To examine the cytolytic capability of effector T cells deficient for CD3 or expressing CD3 subunits lacking ITAMs, polyclonal induction of CTL activity was established by a primary MLR. T cells deficient for CD3 or T cells lacking the CD3-ITAM displayed a similar proliferative response induced in MLR to those induced in T cells encompassing the normal complement of CD3 ITAMs (Fig. 7A, top), suggesting that at least this aspect of the induction phase of CTL activity did not depend on the presence of an intact CD3 chain. CTL activity of the effector T cells was assayed by coculturing these cells with ⁵¹Cr-labeled non-Ag-bearing, Fc receptor-positive K562 cells in the presence or absence of soluble anti-CD3. In this assay, Abs directed against the TCR complex trigger the lytic function by binding to the TCR complex on the effector T cells and provide cell-cell contact with the target K562 cells through Fc receptor binding (54). As expected, in the absence of anti-CD3, cytolytic activity was below detection levels in control as well as CD3^I/I and CD3-deficient effector T cells (data not shown). In the presence of anti-CD3, cytolytic activity could easily be detected in all effector T cells tested, even in the complete absence of a CD3 chain, reaching 50–60% specific lysis at the highest E:T ratios used (Fig. 7B, top). However, polyclonal induction of CTL activity under suboptimal conditions (by decreasing both the absolute number of stimulator and responder cells) resulted in a reduction in the proliferative response of CD3^-/- T cells compared with wt and CD3-ITAM T cells in MLR and completely abolished cytolytic activity of only the CD3-deficient effector T cells (Fig. 7, A and B, bottom). Therefore, more stringent culture conditions reveal that loss of the CD3 chain does result in a severe loss of the capacity to generate effector T cells. Nevertheless, this is not a consequence of a unique contribution of CD3-ITAM.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. The effector function of cytotoxic T cells is affected in T cells lacking CD3. A, Proliferation was assayed in a primary mixed lymphocyte reaction by coculturing lymph node T cells derived from 8- to 12-wk-old CD3+/+ (), CD3I/I (), and CD3-/- (•) mice with indicated numbers of MHC-mismatched SJL/J spleen cells under optimal conditions (top) or suboptimal conditions (bottom) for 4 days. B, Effector T cells of 8- to 12-wk-old CD3+/+ (), CD3I/I (), and CD3-/- (•) mice generated in an MLR under optimal conditions (top) or suboptimal conditions (bottom) for 4 days were analyzed for their cytolytic capacity in a 51Cr-release assay in the presence of 145-2C11 (anti-CD3).

CD3 deficiency and CD3-ITAM deficiency have opposite effects on the appearance of NP_366–374-specific CD8 T cells in response to influenza infection

Collectively, the results shown this far indicate that CD3-deficient T cells function relatively normal in some respects (induction of cytokine production and proliferation), although they are defective in others. Particularly notable is their inability to perform cytolytic effector function under suboptimal conditions. Because this response is dependent on CD8 T cells, we explored the ability of Ag-specific CD8 T cells to respond in vivo to a viral infection by expansion. Mice were infected intranasally with influenza A virus strain A/NT/60/68 and at the peak of the infection (8 days postinfection), 4–7% of the CD8-positive T cells obtained from inflamed lung tissue derived from control mice stained positive for H-2D^b-NP_{366–374(NT)} tetramers (Fig. 8A, top). NP_366–374-specific CD8 T cells could also be detected in the spleens and draining MLNs of these mice, albeit at lower frequencies (1.5–2.5% and 0.5–1.5%, respectively) (data not shown) (53). In sharp contrast, NP_366–374-specific CD8 T cells were entirely undetectable in the lungs, spleens, and MLNs of mice deficient for CD3 (Fig. 8A, top; data not shown). This is not simply due to a delay in the response, because even at 21 days postinfection, NP_366–374-specific CD8 T cells are still undetectable in the lungs of influenza A infected CD3-deficient mice (Fig. 8A, bottom). Moreover, even a primary infection with influenza A virus followed 8 wk later by induction of a memory T cell response by a second infection with influenza A virus did not result in any detectable Ag-specific CD8 T cells in the lungs of CD3-deficient mice, while, in the lungs of control mice, Ag-specific CD8 T cells now comprised 25–30% of the total CD8 T cell population (Fig. 8B). Thus, although the in vitro proliferative response induced by anti-CD3 or MLR in CD3-deficient T cells is slightly delayed (only in the case of anti-CD3) but otherwise comparable in magnitude (Fig. 5A; Fig. 7A), the in vivo expansion of an Ag-specific CD8 T cell population is completely abrogated by lack of CD3 (Fig. 8, A and B).

View larger version (71K): [in this window] [in a new window]   FIGURE 8. CD3 deficiency and CD3-ITAM deficiency have opposite effects on the appearance of NP366–374-specific CD8 T cells in response to influenza infection. CD3+/+, CD3I/I, and CD3-/- mice were infected with influenza A virus strain A/NT/60/68. At the indicated days postinfection electronically gated CD8-APC-positive T cells from the lung were stained with H-2Db-NP366–374(NT)-PE conjugated tetramers (A, B, and D) or TCR-FITC (C). E, DP thymocytes derived from CD3-deficient mice and F5-TCR transgenic CD3-deficient mice were stained with H-2Db-NP366–374(NT)-PE-conjugated tetramers (top) or TCR-FITC (bottom). F, Single-cell suspensions from influenza infected lung tissue were stained with CD8-APC plus H-2Db-NP366–374(NT)-PE-conjugated tetramers.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study explores the consequences of CD3 deficiency and CD3 ITAM deficiency for TCR-induced activation and differentiation of peripheral T cells. These experiments are particularly relevant in view of the recent finding (35, 37) that the CD3- module is sufficient for endowing the TCR with qualitatively normal signaling capabilities. The spectrum of T cell activation, proliferation, and differentiation events that was examined in this report did not depend on the integrity of the CD3-ITAM, with the exception of the expression of activation markers in which a slight reduction could be observed. In contrast, expression of activation markers and induction of proliferation, IL-2 secretion, and TCR down-regulation were all affected in lymph node T cells lacking the complete CD3 chain. The small fraction of T cells up-regulating CD25 and CD69 and down-regulating CD62L in CD3-deficient T cells, as well as the delayed onset of proliferation and IL-2 secretion, is presumably the result of impaired cell surface expression of the TCR complex (16) (Fig. 1A). In accordance with this view, it has recently been shown that T cells count the number of triggered TCRs on their cell surface and respond only when a certain threshold is reached (55). Because reduced TCR expression levels will compromise the capacity to reach this activation threshold, this predicts that fewer peripheral T cells in CD3^-/- mice will be activated in response to a certain stimulus. Importantly, the normal expression levels of CD25 and CD69 and the massive proliferation and IL-2 secretion observed at later time points after stimulation suggest that, once activated, these in vitro parameters of T cell activation become independent of an intact CD3 chain.

Studies using (transformed) T cell lines derived from CD3-deficient patients revealed that whereas proliferation was completely normal in these cells, IL-2 secretion was severely impaired (61, 62). However, in these studies, a kinetic analysis of the response was not performed, complicating the interpretation of the results and providing an explanation for the differences between our observations and these earlier data. Furthermore, the response of a (transformed) T cell line to a certain stimulus may not be representative of the response of a polyclonal peripheral T cell population. For example, the diversity in the expression of activation markers in response to CD3-mediated cross-linking (Fig. 4) can never be mimicked by a T cell line.

Several studies have implicated the CD3 di-leucine motif specifically in PKC-mediated TCR internalization (57) and not in ligand- or anti-CD3-induced TCR down-modulation (59, 60, 63). Ligand-induced TCR down-modulation has been suggested to depend on p56^lck and p59^fyn (59, 64). These Src family protein tyrosine kinases are involved in phosphorylation of the ITAMs after ligation of the TCR complex (29). Interestingly, it has been suggested using chimeric TAC/CD3 or CD3 cytoplasmic domain-containing polypeptide chains that besides the di-leucine motif also ITAMs may be involved in TCR down-modulation (56). Moreover, clathrin-coated vesicles mediate endocytosis of trans-membrane receptors (65), and ITAM regions contain a tyrosine-based sorting signal able to interact with a subunit of the AP-2 clathrin-associated protein complex (66). Together, these studies predict that in mice lacking CD3 as well as in CD3-ITAM mutant mice, there will be no TCR down-modulation due to lack of the di-leucine motif and/or the CD3-ITAM, respectively. In contrast to these predictions, internalization of the TCR complex after exposure to anti-CD3 mAb occurred irrespective of the presence of intact CD3-ITAMs, excluding a unique role for this particular ITAM in internalization (Fig. 6B).

The in vitro activation studies with T cells lacking CD3 indicate a deficiency mainly in induction of cytolytic activity, while induction of cytokine secretion and proliferation are delayed but otherwise intact. These findings of only mild defects contrast sharply with the dramatic immune deficiency syndrome observed in CD3-deficient patients (67, 68). Therefore, it was surprising to find that the ability of CD3-deficient mice to mount an in vivo response to an antigenic challenge is completely abrogated. This was not predicted by their in vitro behavior, and several possible (not mutually exclusive) explanations for the absence of a response to the main NP-epitope of influenza virus in CD3-deficient mice can be put forward. First, in the absence of CD3, positive and/or negative selection may be disturbed, resulting in absence of an NP-specific T cell repertoire. Second, influenza A virus infection may be unable to activate and/or expand NP-specific CD8 T cells in the absence of CD3. Third, CD3-deficient NP-specific CD8 T cells may be activated by influenza A virus, but due to a delayed expansion (Fig. 5A), they may remain below detection level. However, the observations that NP-specific CD8 T cells can still not be detected in the lungs of CD3-deficient mice at later time points or in the memory response render this explanation rather unlikely. Fourth, activated NP-specific CD8 T cells lacking CD3 may be unable to home to the lung. This also seems unlikely because CD3-deficient T cells that do get activated express normal levels of activation markers and homing receptors, such as LFA-1 (Fig. 4 and data not shown) and an influx of CD8 T cells can be observed in the lungs of influenza infected CD3-deficient mice (Fig. 8F). Finally, CD3^-/- NP-specific CD8 T cells may get activated in vivo but then die more quickly due to activation-induced cell death than normal CD8 T cells. Because CD3-deficient T cells are defective in down-regulation of the TCR, the extinction of the signaling process may be defective and make the cells more prone to activation-induced cell death. Several of these issues will be addressed in further analyses of F₅-TCR transgenic mice expressing a TCR specific for influenza nucleoprotein (52) crossed to the CD3^-/-RAG^-/- background. The augmentation in the percentage of NP_366–374-specific CD8 T cells in the lungs of influenza A-infected mice lacking a CD3-ITAM compared with control mice might indicate a defect in activation-induced cell death, because the lungs are the site for accumulation of previously activated apoptotic T cells. This option will also be the subject of further investigation.

Overall, these studies indicate that proper TCR functioning does require the CD3 chain but not the CD3-ITAM, at least not in the presence of the full collection of other ITAMs. Both quantitative and qualitative differences may exist between the various CD3-ITAMs with respect to their ability to interact with distinct kinases and adaptors (69, 70), and the present data predict that CD3 contributes primarily in a quantitative manner to TCR signaling. It remains to be investigated whether a TCR lacking the CD3-ITAM couples differentially to cytosolic substrates and signaling pathways. If it does, dissecting how distinct biochemical events couple the TCR to similar functions will also require further study.

	Acknowledgments

 
We thank Hergen Spits for critically reviewing the manuscript; B. Malissen for providing the genomic probe used to screen the phage library; M. Toebes, M. Hoffmann, and M. Beumkes for their excellent technical assistance; and M. A. van Halem for help in preparing the manuscript.

	Footnotes

 
¹ This work was supported by Grant 901-07-178 from The Netherlands Organization for Scientific Research and Grant RG0335/1998-M from the Human Frontier Science Program Organization (to M.C.H.), and, in part, by Grant 901-02-095 from the Netherlands Organization for Scientific Research (to P.K.).

² Address correspondence and reprint requests to Dr. Ada M. Kruisbeek, Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 C. X. Amsterdam, The Netherlands.

³ Abbreviations used in this paper: DP, double positive; ITAM, immunoreceptor tyrosine-based activation motif; wt, wild type; ES, embryonic stem; PKC, protein kinase C; MLN, mediastinal lymph node.

Received for publication July 14, 2000. Accepted for publication November 9, 2000.

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