The CD3γ di-leucine-based motif plays a central role in TCR down-regulation. However, little is understood about the role of the CD3γ di-leucine-based motif in physiological T cell responses. In this study, we show that the expansion in numbers of virus-specific CD8+ T cells is impaired in mice with a mutated CD3γ di-leucine-based motif. The CD3γ mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic molecule Bcl-2. This resulted in a 2-fold reduction in the clonal expansion of virus-specific CD8+ T cells during the acute phase of vesicular stomatitis virus and lymphocytic choriomeningitis virus infections. These results identify an important role of CD3γ-mediated TCR down-regulation in virus-specific CD8+ T cell responses.

Ag-specific CD8+ T cells undergo massive expansion in numbers during the acute phase of viral infections. This expansion serves to generate a large number of effector cells that contribute to the clearance of the pathogen and to memory cells that confer increased levels of protection on re-exposure to the pathogen. Before infection, the precursor frequency of CD8+ T cells specific for any particular Ag is in the range of 1 in 200,000 (1). Following infection, the precursor cells undergo a period of extensive expansion, dividing as many as 15–20 times and increasing up to 50,000-fold in number (1, 2, 3). The magnitude of the clonal expansion is determined by the recruitment of Ag-specific T cells, the division rate of the recruited cells and their apoptosis rate (4). In general, there are three major classes of signals necessary for T cell recruitment and expansion: signal 1 from Ag, signal 2 from costimulation, and signal 3 from cytokines (5, 6, 7).

The TCR is the key receptor that recognizes Ag and subsequently delivers signal 1 to the interior of the cell. The TCR is a multichain receptor composed of the Ag-recognizing αβ heterodimer and the tightly associated signal-transducing CD3γε, CD3δε, and CD3ζζ dimers (8, 9). Like many other cell surface receptors, the TCR is down-regulated following ligand triggering. Ligand-induced receptor down-regulation is often caused by increased endocytosis and has traditionally been considered a mechanism to desensitize receptor signaling and thereby to prevent harmful hyperstimulation that might lead to autoimmunity or malignant cell transformation (10, 11, 12, 13). For a great number of receptors, endocytosis is mediated by receptor-sorting motifs within the cytosolic domains of the receptors (14). Most of these motifs consist of short, linear sequences of amino acid residues. Some motifs are referred to as tyrosine-based sorting motifs and conform to the YXXØ or NPXY consensus motifs (single letter amino acid code, where X represents any amino acid and Ø represents hydrophobic amino acids). Other motifs are referred to as di-leucine-based (diL)5 motifs and fit to the [D/E]XXXL[L/I] consensus motif. These receptor-sorting motifs are recognized by components of the protein coats associated with the cytosolic face of membranes (15). In some cases, phosphorylation events regulate the activity/accessibility of diL motifs (14, 15, 16).

Interestingly, at least two distinct pathways exist for TCR down-regulation (17, 18). One pathway is dependent on protein tyrosine kinase (PTK) activity and leads to TCR ubiquitination and degradation (19, 20). The other pathway is dependent on protein kinase C (PKC)-mediated activation of the diL motif found in the CD3γ-chain of the TCR and leads to TCR recycling (16, 21, 22). It is puzzling why T cells have two distinct pathways for TCR down-regulation at their disposal. In accordance with the standing paradigm that receptor down-regulation serves to prevent harmful hyperstimulation, studies have shown that blocking of the PTK/ubiquitin-dependent TCR down-regulation pathway leads to loss of TCR desensitization and to hyperresponsive T cells (12, 13). Thus, Naramura et al. (12) found that T cells from c-Cbl Cbl-b double knock-out (KO) mice showed impaired TCR down-regulation and TCR degradation after ligand engagement and were hyperresponsive to anti-CD3 stimulation. These mice showed heavy signs of autoimmunity and died at a young age between 12–16 wk. Likewise, Lee et al. (13) found that T cells from CD2AP KO mice were hyperresponsive to Ag stimulation and that this hypersensitivity correlated with reduced TCR down-regulation and degradation. In contrast to PTK-dependent TCR down-regulation, the role of CD3γ diL motif-mediated TCR down-regulation is still not known.

To investigate the role of CD3γ diL motif-mediated TCR down-regulation during the acute phase of viral infections, we generated CD3γLLAA knock-in mice homozygous for a double leucine to alanine mutation in the CD3γ diL motif. In vivo studies demonstrated that clonal expansion of virus-specific CD8+ CD3γLLAA T cells was impaired compared with the clonal expansion of virus-specific CD8+ wild-type (WT) T cells. The CD3γLLAA mutation did not impair early TCR signaling or compromise recruitment or proliferation of the virus-specific T cells, but instead it increased their apoptosis rate by increasing the down-regulation of the antiapoptotic molecule Bcl-2. These results identify a significant role of CD3γ diL motif-mediated TCR down-regulation in virus-specific T cell responses.

A mouse genomic clone encompassing the CD3γ gene was isolated from a 129SVJ phage library (Stratagene) as previously described (23). To construct the targeting vector, the 1.6-kb BamHI/HindIII fragment comprising CD3γ exons 5–7 was subcloned into plasmid pBS-SKII (Stratagene) to obtain pBS-exon 5–7. The LL to AA mutation was created by overlap extension PCR using primer LLAA-A (5′-CGCTCTAGAACTAGTGG-ATCC), LLAA-B (5′-ACAGCTGTTCATTTTGAGCGGCCGTCTGCTTGTCTG-AAGCTGC), LLAA-C (5′-TCAGACAAGCAGACGGCCGCTCAAAATGAACAG-CTGTACCAG), and LLAA-D (5′-GCGGTGCAGAGCCTGCAGGGCACT) and the pBS-exon 5–7 as template. Primer LLAA-A was complementary to the XbaI/SpeI/BamHI restriction site in the multiple cloning site of pBS-SKII. LLAA-B and C was complementary to exon 5 containing the diL motif except for the underlined sequences that introduced the LL to AA mutation and a new EagI site. LLAA-D was complementary to the intron region 3′ of exon 6 that comprised the PstI site. The final PCR product was digested with BamHI and PstI and subcloned into the BamHI/PstI fragment of pBS-exon 5–7 to obtain pBS-exon 5–7 LLAA. The mutation was verified by DNA sequencing. Next, the HindIII/XhoI fragment containing the 3′ part of exon 7 and the downstream sequence was subcloned into pBS-exon 5–7 LLAA to create pBS-exon 5–7-down LLAA. Subsequently, the XbaI/BamHI fragment containing exon 3 and 4 was subcloned into pBS-exon 5–7-down LLAA to create pBS-exon 3–7-down LLAA. Finally, a 2.2 kb hygromycin cassette flanked by loxP sites was subcloned into pBS-exon 3–7-down LLAA to obtain the final targeting vector. The 11.0 kb XhoI fragment of the targeting vector was electroporated into the E14 embryonic stem cell line derived from 129OLA mice. Clones resistant to hygromycin (Life Technologies) were individually screened by Southern blot analysis for homologous recombination events using SacI-digested DNA and a probe located outside the targeting construct that recognizes a 10.6 kb WT fragment and a 12.8 kb recombinant fragment. Four homologous recombinants were identified out of 192 colonies tested. One clone was selected and injected into C57BL/6 blastocysts to generate chimeric mice. Male chimeric mice were subsequently crossed to female FVB mice that transgenically expressed Cre. Offspring with germline transmission that had recombined out the hygromycin cassette was identified by Southern blot as described above in combination with CD3γWT and CD3γLLAA-specific PCR. CD3γWT was detected using the upstream primer E5WT-UP (5′-CTTCAGACAAGCAGACTCTGTTG) and the common downstream primer E6-DOWN (5′-GGGCTGAAGAGGACAATAGC). CD3γLLAA was detected using the upstream primer E5LLAA-UP (5′-GACAAGCAGACGGCCGCT) and the common downstream primer E6-DOWN. Heterozygous mice were backcrossed for five generations to C57BL/6 mice. Finally, heterozygous mice were intercrossed to produce homozygous CD3γLLAA mice and homozygous WT littermates. The WT littermates were used as controls in all experiments. TCR transgenic P14 mice (24) were crossed to CD3γLLAA mice to obtain P14LLAA mice. The animal experiments were approved by the Animal Experiments Inspectorate, The Danish Ministry of Justice (approval number 2002/561-540).

Thymocytes, spleen, and lymph node (LN) cells were collected using standard protocols. To analyze expression of various surface markers, cells were incubated with fluorochrome-conjugated anti-CD4 (RM4–5), anti-CD8a (53–6.7), anti-TCRβ (H57–597), anti-CD3 (145.2C11), anti-Vα2 (B20.1), anti-Vβ8 (MR5.2), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD49d (VLA-4, R1–2), or anti-IFN-γ (XMG1.2) mAbs, all from BD Biosciences. For identification of Ag-specific CD8+ T cells, cells were incubated with fluorochrome-labeled MHC Dextramers class I (MHC Dextramer H-2Kb(VSV-NP52–59)) containing H-2Kb and the immunodominant peptide epitope (np52–59) from vesicular stomatitis virus (VSV) nucleoprotein (25), (MHC Dextramer H-2Db(LCMV-np396–404)) containing H-2Db and the peptide epitope (np396–404) from lymphocytic choriomeningitis virus (LCMV) nucleoprotein or (MHC Dextramer H-2Db(LCMV-gp33–41)) containing H-2Db and the peptide epitope (gp33–41) from LCMV glycoprotein (provided by C. Jespersgaard and J. Schøller, DakoCytomation, Denmark). Following incubation for 30 min at 4°C, mAbs against surface molecules were added and the cells were incubated for a further 30 min at 4°C. Cells were subsequently analyzed on a FACSCalibur (BD Biosciences) with CellQuest software. For intracellular Bcl-2 staining, 1 × 106 cells fixed by incubations with 1% para-formaldehyde for 10 min on ice. The cells were subsequently washed, permeabilized by treatment with 0.5% saponin for 10 min at room temperature, and then resuspended in 20 μl PE-conjugated anti-Bcl-2 (BD Biosciences). Following incubation for 20 min at room temperature the cells were washed and analyzed on a FACSCalibur. To determine apoptotic cells, 1 × 106 cells were stained with fluorochrome-conjugated Abs as described above, washed twice in cold PBS, and resuspended in 1 × Annexin V binding buffer (BD Biosciences). Annexin V (BD Biosciences) was added and the cells were vortexed and incubated for 15 min at room temperature in the dark. Subsequently, 400 μl of 1 × Annexin V binding buffer was added and the samples analyzed by flow cytometry within 1 h.

For surface biotinylation, 1 × 108 lymph node cells were washed twice in PBS and resuspended in a freshly prepared solution of 0.5 mg/ml sulfo-NHS-Biotin/PBS (Pierce). The cells were incubated on ice for 30 min and gently mixed. Subsequently, the cells were washed twice in PBS, lysed in lysis buffer (50 mM Tris-base (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 mg/ml pefabloc) plus 1% digitonin, precleared with protein A-agarose beads (Kem-En-Tec) and immunoprecipitated with anti-CD3ε mAb (145–2C11) and protein A-agarose beads. The beads were washed five times, resuspended in incubation buffer (20 mM Na3PO4 (pH 7.5), 0.02% NaN3, 0.1% SDS, 50 mM 2-ME), and boiled for 5 min. The supernatant was divided in two aliquots and transferred to new tubes. Nonidet P-40 was added to a final concentration of 0.75% and one of the samples was treated with N-Glycanase F (Genzyme). The tubes were incubated overnight at 37°C, boiled for 5 min in sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.01% bromophenol, 2% 2-ME), and run on a 8–15% gradient polyacrylamide gel. The proteins were transferred to nitrocellulose sheets (Amersham Biosciences) and Western blotting was performed using HRP-conjugated streptavidin (Pierce) following visualization with ECL (Amersham Biosciences).

To analyze constitutive TCR endocytosis LN cells from P14 and P14LLAA mice were isolated and incubated with anti-Vα2 mAb at 10°C for 1 h in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% FCS, 0.5 IU/l penicillin, 500 mg/l streptomycin, and 50 μM 2-ME) in 96-well round-bottom plates. We used the anti-Vα2 mAb as marker for the transgenic TCR, as our initial studies demonstrated that, in contrast to anti-Vβ8, anti-TCRβ, and anti-CD3 mAbs, which all elicited ligand-induced TCR down-regulation, anti-Vα2 mAb did not induce TCR endocytosis by its own. After labeling, the cells were shifted to 37°C to allow spontaneous TCR trafficking. At various time points, the amounts of endocytosed anti-Vα2 mAb were determined by FACS analyses of cell samples treated with low pH buffer to strip of anti-Vα2 mAb from the cell surface and subsequently stained with anti-CD4 and anti-CD8 mAb.

For activation-induced TCR down-regulation, LN or spleen cells were isolated and plated at 4 × 105 cells/well in a final volume of 200 μl complete RPMI 1640 medium in 96-well round-bottom plates with the indicated concentrations of either phorbol 12,13-dibutyrate (PDB) or gp33–41 for the time indicated at 37°C. Subsequently, the cells were analyzed by flow cytometry and the TCR mean fluorescence intensity (MFI) determined. TCR down-regulation was calculated as ((MFI of untreated cells minus MFI of treated cells)/MFI of untreated cells) × 100%.

Eight- to ten-week-old WT and CD3γLLAA mice were injected i.v. with 1 × 106 PFU of VSV of the Indiana strain or 1 × 104 PFU of LCMV of the Armstrong 53b strain. Spleen cells were isolated at the indicated days after infection and stained with the indicated fluorochrome-conjugated mAbs and with VSV-np52–59/Kb dextramers for VSV infections and LCMV-np396–404/H-2Db or LCMV-gp33–41/H-2Db dextramers for LCMV infections and analyzed by flow cytometry. To determine the ability of virus-specific T cells to produce IFN-γ, spleen cells were cultured for 5 h at 37°C in complete RPMI 1640 medium supplemented with 1 μg/ml of the VSV-np52–59 peptide, murine rIL-2 (50 U/ml), and monensin (3 μM). IFN-γ expression and virus titers were determined as previously described (24, 25).

For analysis of tyrosine phosphorylation, spleen and LN cells were obtained from P14 and P14LLAA mice. The RBC were lysed with Gey’s solution (26) followed by two washes in RPMI 1640. The CD8+ T cells were subsequently isolated using a Mouse CD8 Negative Isolation Kit according to the manufacturer (Dynal Biotech). The purified CD8+ cells were starved in serum-free medium for 30 min at 37°C, 5% CO2. The cells were adjusted to 2 × 106 cells/800 μl serum free medium and stimulated with gp33–41 at a final concentration of 100 ng/ml for the time indicated. The cells were lysed in lysis buffer (50 mM Tris-base (pH 7.5), 150 mM NaCl, and 1 mM MgCl2) supplemented with 1 mg/ml Pefabloc SC, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 5 mM EDTA, 10 mM NaF, 1 mM Na3VO4, and 1% Triton X-100 and run on 10% polyacrylamide gels. The proteins were transferred to nitrocellulose sheets (Amersham Biosciences), and visualized by primary Abs: anti-linker for activation of T cells (LAT) (Upstate Biotechnology), anti-LAT-pY171 (Cell Signaling Technology), anti-Zap70 (Cell Signaling Technology), anti-Zap70-pY319 (Cell Signaling Technology), anti-PLCγ (Upstate Biotechnology), and anti-PLCγ-pY783 (Biosource) followed by HRP-conjugated swine anti-rabbit Ig or HRP-conjugated rabbit anti-mouse Ig using ECL (Amersham Biosciences) technology.

For measurement of intracellular calcium flux, LN cells from P14 and P14LLAA mice were loaded with 5 μM Fura-Red AM and 2 μM Fluo-3 AM (Invitrogen/Molecular Probes) in serum-free medium at 37°C for 30 min. The cells were plated in aliquots of 1 × 106 in 96-well plates, washed twice in medium without FCS, and subsequently labeled with anti-CD8-allophycocyanin at room temperature for 30 min. The cells were washed twice and analyzed on a FACSCalibur flow cytometer. Each cell sample was preheated to 37°C before analysis. Baseline measurements were achieved by running the sample without stimulation for 30 s. At 30 s, gp33–41 peptide was added to a final concentration of 100 or 10 ng/ml. The cells were spun down for 5 s and the flow cytometer analysis resumed. Data was collected for 512 s. At 465 s, ionomycin (Sigma-Aldrich) was added to a final concentration of 500 ng/ml as calcium flux control. Samples were kept at 37°C during the entire flow cytometer analysis by using a coil-heated Falcon tube. The software FlowJo (Tree Star) was subsequently used for ratiometric analysis.

For expression of activation markers, equivalent numbers of congenic P14 and P14LLAA cells were mixed and stimulated with gp33–41 peptide at a final concentration of 0.1 ng/ml for 24 h. The cells were subsequently analyzed on a FACSCalibur flow cytometer and the expression of CD25 and CD69 determined on the transgenic Vα2+CD8+ cells.

Cell recruitment and division were analyzed by labeling spleen cells with CFSE (Molecular Probes). In brief, cells were incubated with 0.5 μM CFSE in PBS for 10 min at 37°C. FCS was added to a final concentration of 5% and the cells were subsequently washed twice in RPMI 1640 medium before they were plated at 4 × 105 cells/well in a final volume of 200 μl complete RPMI 1640 medium in 96-well round-bottom plates containing varying concentrations of the gp33–41 peptide. The cells were stimulated for 32 or 48 h at 37°C and subsequently analyzed for CFSE labeling of Vα2+CD8+ T cells on a FACSCalibur. The fraction of dividing Ag-specific cells and the division rates were calculated as previously described (27).

The CD3γ diL motif is encoded by exon 5 (28) and consists of the amino acids 127DKQTLL132 immediately preceded by S126 (amino acid numbering according to Krissansen et al. (29)). We generated CD3γLLAA mutant mice by gene targeting in mouse embryonic stem cells. The targeting vector comprised 8.8 kb of the CD3γ allele with the 131LL132 to 131AA132 mutation in exon 5 and a 2.2 kb hygromycin cassette inserted at the BamHI site just 5′ to exon 5 (Fig. 1,A). We identified successful targeting of the gene by Southern blot analysis using DNA from targeted embryonic stem cells. The hygromycin cassette was recombined out by crossing male chimeric mice to female mice that transgenically expressed Cre. Offspring was tested by a combination of Southern blot and PCR specific for WT and CD3γLLAA (Fig. 1,B). The amino acid sequence of the cytoplasmic tail of CD3γ from WT and CD3γLLAA mice is shown in Fig. 1 C.

FIGURE 1.

Mutation of the CD3γ diL motif by homologous recombination. A, Partial organization of the CD3γWT allele, the targeting vector, and the mutated CD3γ allele following homologous recombination and deletion of the hygromycin cassette. The probe used for hybridization and the predicted fragment sizes generated by the endogenous (10.6 kb) and targeted (12.8 kb) alleles after SacI digestion are depicted. Exons are denoted as filled boxes and numbered 3–7. Restriction enzymes sites (B, BamHI; Ea, EagI, EV, EcoRV; H, HindIII; N, NsiI; P, PstI; Pv, PvuII; S, SacI; X, XhoI). B, PCR analysis of tail DNA derived from WT (+/+), heterozygous CD3γLLAA (+/−), and homozygous CD3γLLAA mice (−/−) using CD3γWT (WT) and CD3γLLAA (LLAA) specific primers. C, Schematic presentation of the amino acids in the cytoplasmic tail of CD3γ from WT and CD3γLLAA mice.

FIGURE 1.

Mutation of the CD3γ diL motif by homologous recombination. A, Partial organization of the CD3γWT allele, the targeting vector, and the mutated CD3γ allele following homologous recombination and deletion of the hygromycin cassette. The probe used for hybridization and the predicted fragment sizes generated by the endogenous (10.6 kb) and targeted (12.8 kb) alleles after SacI digestion are depicted. Exons are denoted as filled boxes and numbered 3–7. Restriction enzymes sites (B, BamHI; Ea, EagI, EV, EcoRV; H, HindIII; N, NsiI; P, PstI; Pv, PvuII; S, SacI; X, XhoI). B, PCR analysis of tail DNA derived from WT (+/+), heterozygous CD3γLLAA (+/−), and homozygous CD3γLLAA mice (−/−) using CD3γWT (WT) and CD3γLLAA (LLAA) specific primers. C, Schematic presentation of the amino acids in the cytoplasmic tail of CD3γ from WT and CD3γLLAA mice.

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The CD3γLLAA mice were viable and fertile and appeared normal. To assess the development and presence of T cells, we stained thymocytes and spleen cells from 8–12 wk old WT and CD3γLLAA mice for surface expression of CD4 and CD8 and analyzed them by flow cytometry. Thymi from CD3γLLAA mice contained reduced numbers of thymocytes (72 × 106 for CD3γLLAA mice vs 91 × 106 for WT mice, p = 0.038) but similar proportions of double-negative, double-positive and single-positive subpopulations compare with their WT littermates (Fig. 2,A). Likewise, we found a reduced number of total cells in the spleens from CD3γLLAA mice compared with spleens from WT mice. The percentage of CD4+ cells was slightly increased and the percentage of CD8+ cells was slightly decreased in the spleens of CD3γLLAA mice (Fig. 2 A). This resulted in equal numbers of CD4+ cells but decreased numbers of CD8+ cells in CD3γLLAA spleens compared with WT spleens (6.4 × 106 CD8+ cells for CD3γLLAA mice vs 7.8 × 106 CD8+ cells for WT mice, p = 0.047). The reduction in CD8+ T cells affected the naive CD44low subset whereas the number of CD8+CD44high subset was unaffected (data not shown). Thus, although not dramatic, the CD3γLLAA mutation affected thymic cellularity and caused a minor reduction in the number of naive CD8+ T cells.

FIGURE 2.

T cell phenotype and TCR expression in CD3γLLAA mice. A, Thymocytes (upper panel) and spleen cells (lower panel) of 8–12-wk-old WT and CD3γLLAA mice were enumerated and analyzed by flow cytometry for the expression of CD4 and CD8. The total cell numbers are shown above the corresponding dot plots as mean ± SEM. The mean percentage of cells within each quadrant is indicated. The data show values obtained from four independent experiments with 14 mice in each group. B, Biochemical analysis of TCR cell surface expression on T cells from WT and CD3γLLAA mice. Lysates from biotinylated LN cells were immunoprecipitated with anti-CD3ε mAb and either treated with N-glycanase (+) or left untreated (−). Immunoprecipitates were analyzed by SDS-PAGE under nonreducing conditions on 12% acrylamide gels. The position of the glycosylated and deglycosylated (d) TCR chains is indicated. C, TCR expression levels in P14 and P14LLAA T cells. LN cells from P14 and P14LLAA mice were isolated and stained with mAb against CD8 and Vα2. The Vα2 expression levels were subsequently determined on CD8+Vα2+ T cells. Shaded histograms represent gated cells from P14 mice and unshaded histograms gated cells from P14LLAA mice. The Vα2 mean fluorescence intensity for each of the cell populations and the relative TCR expression level on P14LLAA cells are given at the upper left corner. Data are representative of four independent experiments. D, Constitutive TCR endocytosis. LN cells from P14 and P14LLAA mice were isolated and incubated with anti-Vα2 mAb at 10°C for 1 h. Subsequently, the cells were shifted to 37°C to allow constitutive TCR endocytosis. At various time points, the amount of endocytosed anti-Vα2 mAb was determined by FACS analysis on CD8+ (left histogram) and CD4+ (right histogram) T cells. Data are representative of three independent experiments. E, PKC-induced TCR down-regulation. LN cells from WT and CD3γLLAA mice were isolated and plated with 100 nM of PDB at 37°C for the times indicated. The cells were subsequently analyzed by flow cytometry and the degree of TCR down-regulation calculated. Data are shown for CD8+ cells and are representative of three independent experiments.

FIGURE 2.

T cell phenotype and TCR expression in CD3γLLAA mice. A, Thymocytes (upper panel) and spleen cells (lower panel) of 8–12-wk-old WT and CD3γLLAA mice were enumerated and analyzed by flow cytometry for the expression of CD4 and CD8. The total cell numbers are shown above the corresponding dot plots as mean ± SEM. The mean percentage of cells within each quadrant is indicated. The data show values obtained from four independent experiments with 14 mice in each group. B, Biochemical analysis of TCR cell surface expression on T cells from WT and CD3γLLAA mice. Lysates from biotinylated LN cells were immunoprecipitated with anti-CD3ε mAb and either treated with N-glycanase (+) or left untreated (−). Immunoprecipitates were analyzed by SDS-PAGE under nonreducing conditions on 12% acrylamide gels. The position of the glycosylated and deglycosylated (d) TCR chains is indicated. C, TCR expression levels in P14 and P14LLAA T cells. LN cells from P14 and P14LLAA mice were isolated and stained with mAb against CD8 and Vα2. The Vα2 expression levels were subsequently determined on CD8+Vα2+ T cells. Shaded histograms represent gated cells from P14 mice and unshaded histograms gated cells from P14LLAA mice. The Vα2 mean fluorescence intensity for each of the cell populations and the relative TCR expression level on P14LLAA cells are given at the upper left corner. Data are representative of four independent experiments. D, Constitutive TCR endocytosis. LN cells from P14 and P14LLAA mice were isolated and incubated with anti-Vα2 mAb at 10°C for 1 h. Subsequently, the cells were shifted to 37°C to allow constitutive TCR endocytosis. At various time points, the amount of endocytosed anti-Vα2 mAb was determined by FACS analysis on CD8+ (left histogram) and CD4+ (right histogram) T cells. Data are representative of three independent experiments. E, PKC-induced TCR down-regulation. LN cells from WT and CD3γLLAA mice were isolated and plated with 100 nM of PDB at 37°C for the times indicated. The cells were subsequently analyzed by flow cytometry and the degree of TCR down-regulation calculated. Data are shown for CD8+ cells and are representative of three independent experiments.

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To analyze whether the composition of the TCR was affected in the CD3γLLAA mice, we immunoprecipitated the TCR from surface-biotinylated LN cells lysed in digitonin buffer. All of the TCR chains, including the CD3γ-chain, were precipitated from CD3γLLAA T cells with similar proportions as for WT cells (Fig. 2 B), indicating that the stoichiometry of the TCR expressed at the surface of CD3γLLAA T cells was unaffected by the CD3γLLAA mutation.

In resting T cells, the TCR is constitutively endocytosed from the cell surface and recycled back to the cell surface (18, 22, 30, 31). Studies on T cell lines have indicated that the CD3γ diL motif plays a central role in constitutive TCR endocytosis (32). As the TCR exocytosis rate is constant, the rate of TCR endocytosis determines the ratio of cell surface-expressed TCR to intracellular located TCR (22). Thus, if the CD3γ diL motif plays a role in constitutive TCR endocytosis in primary T cells and no compensatory mechanisms are found in CD3γLLAA cells, increased TCR expression levels would be expected at the cell surface of CD3γLLAA T cells. To compare expression levels of TCR with identical specificity, we crossed CD3γLLAA mice with the P14 TCR transgenic mice that express a TCR (Vα2+Vβ8+) specific for LCMV glycoprotein (gp33–41) bound to H-2Db (33). We found that the expression levels of the transgenic TCR were increased 8–15% in CD8+ P14LLAA cells compared with P14 cells (Fig. 2 C), indicating that constitutive TCR endocytosis is impaired in CD3γLLAA T cells.

To investigate directly whether constitutive TCR endocytosis was affected in CD3γLLAA T cells, we labeled the cell surface-expressed transgenic TCR on P14 and P14LLAA cells with anti-Vα2 mAb at 10°C. After labeling, the cells were shifted to 37°C to allow spontaneous TCR trafficking. At various time points, we determined the amount of endocytosed anti-Vα2 mAb. These experiments demonstrated that constitutive TCR endocytosis was reduced in both Vα2+CD8+ and Vα2+CD4+ T cells from CD3γLLAA mice (Fig. 2 D).

In addition to its central role for constitutive TCR endocytosis, studies in T cell lines have shown that the CD3γ diL motif also plays important roles in activation-induced TCR down-regulation (15, 16, 34). To examine whether the CD3γ diL motif plays similar roles in primary T cells, we incubated LN cells from WT and CD3γLLAA mice with the PKC activator PDB and subsequently analyzed for TCR expression. We found that PKC-induced TCR down-regulation was completely abolished in both CD8+ and CD4+ CD3γLLAA T cells. In most experiments, we actually observed a minor TCR up-regulation in T cells from CD3γLLAA mice following PDB treatment. (Fig. 2 E and data not shown).

These data indicated that the mutation completely abolished PKC-induced TCR down-regulation and caused a reduction in the constitutive TCR endocytosis rate resulting in increased TCR expression levels in resting CD3γLLAA T cells.

The results outlined above suggested that the CD3γ diL motif might play important regulatory roles during the induction and differentiation of in vivo immune responses. To test this hypothesis, we explored the ability of Ag-specific CD8+ T cells to respond during the acute phase of a viral infection. T cell responses to acute infection can generally be divided in three phases: the expansion, the contraction, and the memory phase (4). We infected mice i.v. with 1 × 106 PFU of VSV strain Indiana. VSV infection in mice is transient and often used as a model for acute, nonpersistent viral infections. To directly assess the number of Ag-specific CD8+ T cells during the expansion and contraction phases, we isolated spleen cells 6, 7, 9, and 15 days after infection and stained them with MHC Dextramers class I (VSV-np52–59/Kb). We observed a significant impairment in the expansion of np52–59-specific CD8+ T cells in spleens from CD3γLLAA mice (Fig. 3, A and B). At day 6, CD3γLLAA mice had <50% Ag-specific CD8+ T cells compared with their WT littermates. In contrast, the contraction phase did not seem to be affected by the CD3γLLAA mutation, and at day 15, equal numbers of np52–59-specific CD8+ T cells were found in spleens from CD3γLLAA and WT mice. We assessed CD8+ T cell differentiation further by measuring the number of Ag-specific IFN-γ-producing CD8+ cells. We found a reduced number of CD8+ T cells that produced IFN-γ during the expansion phase in CD3γLLAA mice compared with WT mice. At day 6, CD3γLLAA mice had a >60% reduction in the number of np52–59-specific CD8+ T cells that produced IFN-γ compared with their WT littermates (Fig. 3, C and D). Again, the contraction phase did not seem to be affected by the CD3γLLAA mutation, and at day 15, equal numbers of IFN-γ-producing CD8+ T cells were found in spleens from CD3γLLAA and WT mice. These experiments demonstrated that the CD3γ diL motif is required for normal expansion of VSV-specific CD8+ T cells.

FIGURE 3.

Impaired expansion of virus-specific CD8+ T cells in CD3γLLAA mice. A–D, WT and CD3γLLAA mice were injected i.v. with 1 × 106 PFU of VSV of the Indiana strain, and their spleens were collected 6, 7, 9, and 15 days later. A, Total number of VSV-np52–59/Kb+CD8+ spleen cells from uninfected and infected mice. B, The percentage of Ag-specific CD8+ T cells was assessed by staining spleen cells with VSV-np52–59/Kb MHC Dextramers and Abs to CD8 and CD44 followed by flow cytometry. The plots show CD44 vs VSV-np52–59/Kb staining of CD8+ cells. The total number of CD8+ T cells in the spleens of WT and CD3γLLAA mice is shown above the plots. The percentage of CD8+ cells staining positive for VSV-np52–59/Kb is given in the upper right quadrant. Only data for day 6 are shown. C, Total number of IFN-γ+CD8+ spleen cells from uninfected and infected mice. A and C, The data show the mean value ± SEM obtained from at least two independent experiments with at least six WT and seven CD3γLLAA mice for each time point. Statistical differences (∗, p < 0.05; ∗∗∗, p < 0.0005) between the WT and CD3γLLAA were determined using Student’s t test. D, Expression of IFN-γ vs CD44 in CD8+ spleen cells from WT and CD3γLLAA mice. Spleen cells from infected mice were stimulated with VSV-np52–59 for 5 h in vitro and analyzed for IFN-γ, CD8, and CD44 expression. The total number of CD8+ T cells in the spleens of WT and CD3γLLAA mice is given above each plot. The percentage of CD8+ cells staining positive for IFN-γ is given in the upper right quadrant. Only data for day 6 are shown. E–G, WT and CD3γLLAA mice were injected i.v. with 1 × 104 PFU of LCMV of the Armstrong strain, and their spleens were collected 4, 6, 8, and 10 days later. E, Total number of LCMV-np396–404/Db+CD8+ spleen cells from uninfected and infected mice. F, Total number of LCMV-gp33–41/Db+CD8+ spleen cells from uninfected and infected mice. G, Spleen virus titers given as PFU/g spleen at day 6. E–G, The data show the mean value ± SEM obtained from at least two independent experiments with at least eight WT and eight CD3γLLAA mice for each time point. Statistical differences (∗, p < 0.05; ∗∗, p < 0.005) between the WT and CD3γLLAA were determined using Student’s t test.

FIGURE 3.

Impaired expansion of virus-specific CD8+ T cells in CD3γLLAA mice. A–D, WT and CD3γLLAA mice were injected i.v. with 1 × 106 PFU of VSV of the Indiana strain, and their spleens were collected 6, 7, 9, and 15 days later. A, Total number of VSV-np52–59/Kb+CD8+ spleen cells from uninfected and infected mice. B, The percentage of Ag-specific CD8+ T cells was assessed by staining spleen cells with VSV-np52–59/Kb MHC Dextramers and Abs to CD8 and CD44 followed by flow cytometry. The plots show CD44 vs VSV-np52–59/Kb staining of CD8+ cells. The total number of CD8+ T cells in the spleens of WT and CD3γLLAA mice is shown above the plots. The percentage of CD8+ cells staining positive for VSV-np52–59/Kb is given in the upper right quadrant. Only data for day 6 are shown. C, Total number of IFN-γ+CD8+ spleen cells from uninfected and infected mice. A and C, The data show the mean value ± SEM obtained from at least two independent experiments with at least six WT and seven CD3γLLAA mice for each time point. Statistical differences (∗, p < 0.05; ∗∗∗, p < 0.0005) between the WT and CD3γLLAA were determined using Student’s t test. D, Expression of IFN-γ vs CD44 in CD8+ spleen cells from WT and CD3γLLAA mice. Spleen cells from infected mice were stimulated with VSV-np52–59 for 5 h in vitro and analyzed for IFN-γ, CD8, and CD44 expression. The total number of CD8+ T cells in the spleens of WT and CD3γLLAA mice is given above each plot. The percentage of CD8+ cells staining positive for IFN-γ is given in the upper right quadrant. Only data for day 6 are shown. E–G, WT and CD3γLLAA mice were injected i.v. with 1 × 104 PFU of LCMV of the Armstrong strain, and their spleens were collected 4, 6, 8, and 10 days later. E, Total number of LCMV-np396–404/Db+CD8+ spleen cells from uninfected and infected mice. F, Total number of LCMV-gp33–41/Db+CD8+ spleen cells from uninfected and infected mice. G, Spleen virus titers given as PFU/g spleen at day 6. E–G, The data show the mean value ± SEM obtained from at least two independent experiments with at least eight WT and eight CD3γLLAA mice for each time point. Statistical differences (∗, p < 0.05; ∗∗, p < 0.005) between the WT and CD3γLLAA were determined using Student’s t test.

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To determine whether the impaired expansion of virus-specific CD8+ T cells in CD3γLLAA mice was an isolated finding only found subsequent to VSV infections or whether CD3γLLAA mice in general had reduced expansion of pathogen-specific CD8+ T cells, we next studied the expansion of virus-specific CD8+ T cells following LCMV infections. Murine infection with LCMV is a widely used model to study CD8+ T cell responses. LCMV infects a range of host cells and stimulates a powerful CD8+ T cell response (35, 36). Acute infection of mice with LCMV results in rapid viral growth that causes little host damage because LCMV is noncytopathic. We infected mice i.v. with 1 × 104 PFU of LCMV strain Armstrong. To directly assess the clonal expansion of two different LCMV-specific CD8+ T cell populations, we isolated spleen cells 4, 6, 8, and 10 days after infection and stained them with either MHC Dextramers class I (LCMV-np396–404/Db) or (LCMV-gp33–41/Db). We observed a significant impairment in the expansion of both np396–404- and gp33–41-specific CD8+ T cells in spleens from CD3γLLAA mice (Fig. 3, E and F). In agreement, we found a minor but statistical significant increase in virus titers in CD3γLLAA mice at day 6 (Fig. 3 G).

Taken together, these results suggested that clonal expansion of virus-specific CD8+ T cells was impaired in CD3γLLAA mice.

Our previous results indicated that, before infection, CD3γLLAA mice have a small reduction in the number of peripheral CD8+ T cells compared with their WT littermates. To determine whether the reduced expansion of virus-specific CD8+ T cells in CD3γLLAA mice was due to a reduced number of virus-specific precursor cells, we performed adoptive transfer experiments. We mixed equivalent numbers of donor Vα2+CD8+ TCR transgenic cells from congenic CD45.1+ P14 and CD45.2+ P14LLAA mice and transferred them i.v. to untreated CD45.2+ C57BL/6 mice at day −1. At day 0, we infected the recipient mice i.v. with 1 × 104 PFU of LCMV. To assess the clonal expansion of the Ag-specific Vα2+CD8+ donor cell populations, we isolated spleen cells 3, 4, and 5 days after infection and determined the number of P14 and P14LLAA donor Vα2+CD8+ cells. We observed a significant impairment in the expansion of Vα2+CD8+ P14LLAA TCR transgenic donor cells compared with Vα2+CD8+ P14 TCR transgenic donor cells (Fig. 4 A). These experiments suggested that a reduced number of Ag-specific precursor cells was not the cause of the impaired expansion of virus-specific CD8+ T cells in CD3γLLAA mice. Furthermore, the mechanism behind the deficient clonal expansion seemed to be intrinsic to the Ag-specific CD3γLLAA CD8+ T cells and independent of the environment as Ag-specific CD8+ P14 T cells expanded more efficiently than CD8+ P14LLAA T cells in the same host.

FIGURE 4.

Impaired expansion of virus-specific CD8+ CD3γLLAA T cells in adoptive transfer and bone marrow chimeric experiments. A, Adoptive transfer. Equivalent numbers (5 × 104/recipient) of donor Vα2+CD8+ TCR transgenic cells from congenic CD45.1+ P14 and CD45.2+ P14LLAA mice were mixed and transferred to untreated CD45.2+ C57BL/6 mice at day −1. At day 0, the recipient mice were infected i.v. with 1 × 104 PFU of LCMV. The total number of P14 and P14LLAA donor Vα2+CD8+ cells in the spleen was determined 3, 4, and 5 days after infection The data show the mean value ± SEM obtained from two independent experiments with four recipient mice for each time point. Statistical differences (∗, p < 0.05; ∗∗, p < 0.005) between P14 and P14LLAA donor cells were determined using Student’s t test. B, Bone marrow chimeras. Equivalent numbers of bone marrow cells from congenic WT and CD3γLLAA mice were mixed and transferred to lethally irradiated C57BL/6. Ten weeks after bone marrow transplantation, the recipient chimeric mice were infected i.v. with 1 × 106 PFU of VSV. Six days after infection, spleen cells were isolated and the total number of np52–59-specific CD8+ IFN-γ producing cells was determined. The data show the mean values ± SEM obtained from two independent experiments with eight chimeric mice. Statistical differences (∗, p < 0.05) between WT and CD3γLLAA cells were determined using Student’s t test.

FIGURE 4.

Impaired expansion of virus-specific CD8+ CD3γLLAA T cells in adoptive transfer and bone marrow chimeric experiments. A, Adoptive transfer. Equivalent numbers (5 × 104/recipient) of donor Vα2+CD8+ TCR transgenic cells from congenic CD45.1+ P14 and CD45.2+ P14LLAA mice were mixed and transferred to untreated CD45.2+ C57BL/6 mice at day −1. At day 0, the recipient mice were infected i.v. with 1 × 104 PFU of LCMV. The total number of P14 and P14LLAA donor Vα2+CD8+ cells in the spleen was determined 3, 4, and 5 days after infection The data show the mean value ± SEM obtained from two independent experiments with four recipient mice for each time point. Statistical differences (∗, p < 0.05; ∗∗, p < 0.005) between P14 and P14LLAA donor cells were determined using Student’s t test. B, Bone marrow chimeras. Equivalent numbers of bone marrow cells from congenic WT and CD3γLLAA mice were mixed and transferred to lethally irradiated C57BL/6. Ten weeks after bone marrow transplantation, the recipient chimeric mice were infected i.v. with 1 × 106 PFU of VSV. Six days after infection, spleen cells were isolated and the total number of np52–59-specific CD8+ IFN-γ producing cells was determined. The data show the mean values ± SEM obtained from two independent experiments with eight chimeric mice. Statistical differences (∗, p < 0.05) between WT and CD3γLLAA cells were determined using Student’s t test.

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Arguments have been presented that modeling endogenous T cell responses in adoptive transfer experiments using relative high numbers of donor TCR transgenic cells might substantially alter the kinetics and magnitude of the clonal expansion (37). Therefore, we decided to analyze clonal expansion of virus-specific WT and CD3γLLAA cells in bone marrow chimeric mice. We transplanted lethally irradiated C57BL/6 mice with bone marrow from congenic WT and CD3γLLAA mice in a 1/1 ratio. Ten weeks after bone marrow transplantation, chimeric mice had normal levels of T cells with equal numbers of WT and CD3γLLAA T cells. We infected the chimeric mice i.v. with 1 × 106 PFU of VSV. Six days after infection, we isolated spleen cells and assessed the expansion of virus-specific CD8+ T cells by measuring the number of np52–59-specific CD8+ IFN-γ producing cells. We found a 60% reduction in number of Ag-specific CD3γLLAA CD8+ T cells that produced IFN-γ compared with WT CD8+ T cells (Fig. 4 B). These results conclusively demonstrated that the mechanism behind the deficient clonal expansion is intrinsic to the Ag-specific CD3γLLAA CD8+ T cells and independent of the host environment.

To determine the mechanisms by which the CD3γ diL motif optimizes virus-specific CD8+ T cell expansion, we next analyzed whether the CD3γLLAA mutation affected TCR signaling. First, we examined early TCR signaling as manifested by tyrosine phosphorylation upon TCR triggering. We stimulated transgenic CD8+ T cells from P14 and P14LLAA mice with gp33–41 and analyzed for tyrosine phosphorylation of ZAP-70, LAT, and PLC-γ1. Phosphotyrosine blots revealed no major differences between P14 and P14LLAA T cells; however, in most experiments the intensity of the phospho-specific bands was slightly increased in P14LLAA T cells compared with P14 cells (Fig. 5,A). Next, we examined intracellular calcium flux following TCR triggering. Again, we observed no major differences in intracellular calcium flux responses between P14 and P14LLAA cells but noticed a minor tendency toward higher calcium flux in P14LLAA cells (Fig. 5,B). Likewise, the fraction of cells that became positive for the activation markers CD25 and CD69 and the expression level of these markers were slightly increased on P14LLAA T cells compared with P14 cells following TCR triggering (Fig. 5 C).

FIGURE 5.

Increased early TCR signaling in CD3γLLAA T cells. A, Tyrosine phosphorylation. Purified CD8+ T cells from P14 and P14LLAA mice were stimulated with gp33–41 at a final concentration of 100 ng/ml for the times indicated. We subsequently lysed the cells and directly analyzed for specific tyrosine phosphorylation using phosphospecific p-ZAP-70, p-LAT, and p-PLC-γ1 Abs. Protein loading was quantified by probing the stripped membranes for total ZAP-70, LAT, and PLC-γ1. Results are representative of three independent experiments. B, Calcium flux. P14 (gray lines) and P14LLAA cells (black lines) were loaded with Fura-Red and Fluo-3 and surface stained with anti-CD8 mAb. Calcium flux was analyzed by flow cytometry over time. At 30 s, cells were stimulated with 100 (upper panel) or 10 (lower panel) ng/ml gp33–41. At 465 s, ionomycin was added. The histograms represent calcium flux for gated CD8+ T cells. The abscissa gives the time in s. The ordinate gives the Fluo-3/Fura-Red fluorescence emission ratio. Results are representative of two independent experiments. C, Activation markers. Equivalent numbers of spleen cells from P14 and P14LLAA mice were mixed and either left unstimulated (unfilled histograms) or stimulated (black filled histograms) with gp33–41 for 24 h. Expression of CD25 (left panel) and CD69 (right panel) was subsequently determined on the Vα2+CD8+ cells. The abscissa gives the fluorescence intensity of the indicated activation marker and the ordinate the relative cell number. The fraction of cells positive for the indicated activation marker and the MFI are given in each histogram.

FIGURE 5.

Increased early TCR signaling in CD3γLLAA T cells. A, Tyrosine phosphorylation. Purified CD8+ T cells from P14 and P14LLAA mice were stimulated with gp33–41 at a final concentration of 100 ng/ml for the times indicated. We subsequently lysed the cells and directly analyzed for specific tyrosine phosphorylation using phosphospecific p-ZAP-70, p-LAT, and p-PLC-γ1 Abs. Protein loading was quantified by probing the stripped membranes for total ZAP-70, LAT, and PLC-γ1. Results are representative of three independent experiments. B, Calcium flux. P14 (gray lines) and P14LLAA cells (black lines) were loaded with Fura-Red and Fluo-3 and surface stained with anti-CD8 mAb. Calcium flux was analyzed by flow cytometry over time. At 30 s, cells were stimulated with 100 (upper panel) or 10 (lower panel) ng/ml gp33–41. At 465 s, ionomycin was added. The histograms represent calcium flux for gated CD8+ T cells. The abscissa gives the time in s. The ordinate gives the Fluo-3/Fura-Red fluorescence emission ratio. Results are representative of two independent experiments. C, Activation markers. Equivalent numbers of spleen cells from P14 and P14LLAA mice were mixed and either left unstimulated (unfilled histograms) or stimulated (black filled histograms) with gp33–41 for 24 h. Expression of CD25 (left panel) and CD69 (right panel) was subsequently determined on the Vα2+CD8+ cells. The abscissa gives the fluorescence intensity of the indicated activation marker and the ordinate the relative cell number. The fraction of cells positive for the indicated activation marker and the MFI are given in each histogram.

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Taken together, these experiments indicated that TCR signaling was slightly increased in T cells with the CD3γLLAA mutation.

The magnitude of the clonal expansion of Ag-specific T cells is determined by 1) the fraction of dividing Ag-specific T cells, 2) the division rate of the dividing cells, and 3) the apoptosis rate of the dividing cells (4). To determine whether the fraction of dividing Ag-specific CD8+ T cells or their division rate were affected by the CD3γLLAA mutation, we mixed equivalent numbers of spleen cells from congenic P14 and P14LLAA mice, labeled them with CFSE, and incubated them with gp33–41. After stimulation for 32 and 48 h, we determined the fraction of dividing Ag-specific CD8+ T cells, the division rate for all Ag-specific CD8+ precursors, and the division rate for dividing Ag-specific CD8+ precursors. From these experiments, in which P14 and P14LLAA T cells were stimulated under exactly the same conditions, we found that the fraction of dividing Ag-specific CD8+ cells and their division rates were the same for P14 and P14LLAA T cells (Fig. 6).

FIGURE 6.

Normal division rates and fraction of dividing Ag-specific CD8+ CD3γLLAA T cells. Equivalent numbers of spleen cells from P14 and P14LLAA mice were mixed, loaded with CFSE, and stimulated with the indicated concentrations of gp33–41 for 32 h (A, C, E, and G) or 48 h (B, D, F, and H). A and B, The histograms represent CFSE staining of isotype-gated Vα2+CD8+ T cells. The abscissa gives the CFSE staining intensity. The ordinate gives the arbitrary cell number. C and D, The fraction of dividing Vα2+CD8+ T cells following stimulation for 32 or 48 h. E and F, Mean cell division rates for all precursor cells following stimulation for 32 or 48 h. G and H, Mean cell division rates for dividing precursor cells following stimulation for 32 or 48 h. Results are representative of three independent experiments.

FIGURE 6.

Normal division rates and fraction of dividing Ag-specific CD8+ CD3γLLAA T cells. Equivalent numbers of spleen cells from P14 and P14LLAA mice were mixed, loaded with CFSE, and stimulated with the indicated concentrations of gp33–41 for 32 h (A, C, E, and G) or 48 h (B, D, F, and H). A and B, The histograms represent CFSE staining of isotype-gated Vα2+CD8+ T cells. The abscissa gives the CFSE staining intensity. The ordinate gives the arbitrary cell number. C and D, The fraction of dividing Vα2+CD8+ T cells following stimulation for 32 or 48 h. E and F, Mean cell division rates for all precursor cells following stimulation for 32 or 48 h. G and H, Mean cell division rates for dividing precursor cells following stimulation for 32 or 48 h. Results are representative of three independent experiments.

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The observations that 1) the fraction of dividing Ag-specific cells and 2) the division rate of the dividing cells were unaffected by the CD3γLLAA mutation suggested that the impairment in clonal expansion of pathogen-specific T cells in CD3γLLAA mice was caused by an increased apoptosis rate of the dividing cells. Naive CD8+ T cells express basal levels of the antiapoptotic molecule Bcl-2 and the proapoptotic Bcl-2 family member Bim (38). Following activation, the balance between Bcl-2 and Bim determines whether the cell will stay alive or die by apoptosis (39, 40, 41, 42). Before apoptosis of activated Ag-specific T cells in vivo, the Bcl-2 level becomes down-regulated, and this allows Bim to trigger apoptosis (40, 43, 44, 45). To determine Bcl-2 levels in activated virus-specific CD8+ T cells, we infected bone marrow chimeric mice i.v. with 1 × 106 PFU of VSV. Six days after infection, we isolated spleen cells and measured the Bcl-2 levels in activated (CD44high) and naive (CD44low) CD8+ WT and CD3γLLAA cells. We found that the levels of Bcl-2 were identical in naive CD8+CD44low WT and CD3γLLAA cells. Compared with naive cells, activated CD8+CD44high cells separated into a major subpopulation that had down-regulated Bcl-2 and a minor subpopulation that had up-regulated Bcl-2. A larger fraction of the CD8+CD44high CD3γLLAA cells had down-regulated Bcl-2 expression compared with CD8+CD44high WT cells. In agreement, a larger fraction of the CD8+CD44high WT cells had up-regulated Bcl-2 expression compared with the CD8+CD44high CD3γLLAA cells. Furthermore, the expression levels of Bcl-2 were reduced in CD8+CD44high CD3γLLAA cells with up-regulated Bcl-2 compared with CD8+CD44high cells with up-regulated Bcl-2 (Fig. 7, A and B).

FIGURE 7.

Increased down-regulation of Bcl-2 in activated virus-specific CD8+ CD3γLLAA T cells. A, Bone marrow chimeric mice were infected i.v. with VSV. The levels of Bcl-2 were determined in naive CD8+CD44low (upper panel) and activated CD8+CD44high (lower panel) WT (black lines) and CD3γLLAA (gray dotted lines) T cells 6 days after infection. The staining profiles obtained with an irrelevant mAb are given as black filled histograms. The MFI of Bcl-2 in naive CD8+CD44low T cells was identical for WT and CD3γLLAA and is given in the upper panel. The Bcl-2 MFI for activated CD8+CD44high T cells with up-regulated Bcl-2 levels is given in the lower panel. The fractions of activated CD8+CD44high WT and CD3γLLAA T cells with up-regulated and down-regulated Bcl-2 levels are indicated with the markers M1 and M2, respectively. B, The data show the mean M1 and M2 values ± SEM obtained from two independent experiments with eight chimeric mice. Statistical differences (∗, p < 0.05) between WT and CD3γLLAA cells were determined using Student’s t test. C, Equivalent numbers of donor Vα2+CD8+ TCR transgenic cells from congenic CD45.1+ P14 and CD45.2+ P14LLAA mice were mixed and transferred to untreated CD45.2+ C57BL/6 mice at day −1. At day 0, the recipient mice were infected i.v. with LCMV. The levels of Bcl-2 were determined in P14 (black lines) and P14LLAA (gray dotted lines) donor Vα2+CD8+ spleen cells 3, 4, and 5 days after infection. D, The data show the relative mean Bcl-2 MFI value ± SEM obtained from two independent experiments with four recipient mice for each time point. Statistical differences (∗, p < 0.05; ∗∗, p < 0.005) between P14 and P14LLAA donor cells were determined using Student’s t test.

FIGURE 7.

Increased down-regulation of Bcl-2 in activated virus-specific CD8+ CD3γLLAA T cells. A, Bone marrow chimeric mice were infected i.v. with VSV. The levels of Bcl-2 were determined in naive CD8+CD44low (upper panel) and activated CD8+CD44high (lower panel) WT (black lines) and CD3γLLAA (gray dotted lines) T cells 6 days after infection. The staining profiles obtained with an irrelevant mAb are given as black filled histograms. The MFI of Bcl-2 in naive CD8+CD44low T cells was identical for WT and CD3γLLAA and is given in the upper panel. The Bcl-2 MFI for activated CD8+CD44high T cells with up-regulated Bcl-2 levels is given in the lower panel. The fractions of activated CD8+CD44high WT and CD3γLLAA T cells with up-regulated and down-regulated Bcl-2 levels are indicated with the markers M1 and M2, respectively. B, The data show the mean M1 and M2 values ± SEM obtained from two independent experiments with eight chimeric mice. Statistical differences (∗, p < 0.05) between WT and CD3γLLAA cells were determined using Student’s t test. C, Equivalent numbers of donor Vα2+CD8+ TCR transgenic cells from congenic CD45.1+ P14 and CD45.2+ P14LLAA mice were mixed and transferred to untreated CD45.2+ C57BL/6 mice at day −1. At day 0, the recipient mice were infected i.v. with LCMV. The levels of Bcl-2 were determined in P14 (black lines) and P14LLAA (gray dotted lines) donor Vα2+CD8+ spleen cells 3, 4, and 5 days after infection. D, The data show the relative mean Bcl-2 MFI value ± SEM obtained from two independent experiments with four recipient mice for each time point. Statistical differences (∗, p < 0.05; ∗∗, p < 0.005) between P14 and P14LLAA donor cells were determined using Student’s t test.

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To study Bcl-2 expression levels in a more homogeneous system, we next performed an adoptive transfer experiment. We transferred equivalent numbers of donor Vα2+CD8+ TCR transgenic cells from congenic P14 and P14LLAA mice to untreated C57BL/6 mice at day −1. At day 0, we infected the recipient mice i.v. with 1 × 104 PFU of LCMV. At day 3, 4, and 5, we isolated spleen cells and determined the Bcl-2 expression levels in Vα2+CD8+ P14 and P14LLAA donor cells, respectively. We found a significantly increased down-regulation of Bcl-2 in P14LLAA cells compared with P14 cells (Fig. 7, C and D).

Taken together, these experiments demonstrated that during the acute phase of viral infections, virus-specific CD8+ T cells with the CD3γLLAA mutation down-regulated Bcl-2 more than WT cells did. These results supported the suggestion that an increased apoptosis rate must be the reason of the impaired clonal expansion of pathogen-specific T cells in CD3γLLAA mice.

Following TCR triggering, a critical signaling threshold must be reached to initiate the cell cycle program in Ag-specific T cells. Increasing the signaling intensity above this threshold does not further augment cell cycling and may even have a negative impact on cell survival (46, 47, 48). In addition, it has been shown that TCR signaling is dependent on the number of TCR expressed at the T cell surface (49). To determine how the CD3γLLAA mutation affected TCR expression levels on cycling Ag-specific CD8+ T cells, we mixed equivalent numbers of spleen cells from congenic P14 and P14LLAA mice, labeled them with CFSE, and incubated them with gp33–41. After stimulation for 48 h, we determined the TCR expression levels on Ag-specific CD8+ WT and CD3γLLAA T cells that had undergone 0, 1, 2, 3, and 4 cell divisions, respectively. We found that following the first cell division, Ag-specific CD8+ CD3γLLAA T cells expressed ∼50% higher levels of TCR at their cell surface than WT cells (Fig. 8, A–C).

FIGURE 8.

Increased TCR expression and apoptosis in activated virus-specific CD8+ CD3γLLAA T cells. A–C, Equivalent numbers of spleen cells from P14 and P14LLAA mice were mixed, loaded with CFSE, and stimulated with a final gp33–41 concentration of 1.0 ng/ml for 48 h. A, The histograms represent CFSE staining of isotype-gated Vα2+CD8+ P14 (shaded histogram) and P14 LLAA (unshaded histogram) T cells. The abscissa gives the CFSE staining intensity. The ordinate gives the arbitrary cell number. The number of cell divisions for each subpopulation is indicated. B, Based on the CFSE histogram shown in A, the cells were gated according to the number of cell divisions they had undergone. The TCR expression levels was subsequently determined for each subpopulation of Vα2+CD8+ P14 (shaded histogram) and P14 LLAA (unshaded histogram) T cells. The abscissa gives the TCR staining intensity. The ordinate gives the arbitrary cell number. The TCR MFI for P14 and P14 LLAA cells and the relative TCR expression levels on P14LLAA cells are given in each histogram. C, Bar histogram showing the TCR expression levels (MFI) as a function of number of cell divisions. Results are representative of three independent experiments. D, Bone marrow chimeric mice were infected i.v. with VSV. The fraction of apoptotic cells was determined by analysis of forward scatter vs side scatter of CD8+ cells 6 days after infection. E, WT and CD3γLLAA mice were infected i.v. with VSV. The fraction of apoptotic cells was determined by annexin V staining 6 days after infection. D and E, The fractions of apoptotic cells in the CD8+ WT and CD3γLLAA cell populations are shown. The data show the mean values ± SEM obtained from two independent experiments with eight mice in each group. Statistical differences (∗∗, p < 0.005; ∗∗∗, p < 0.0005) between WT and CD3γLLAA cells were determined using Student’s t test.

FIGURE 8.

Increased TCR expression and apoptosis in activated virus-specific CD8+ CD3γLLAA T cells. A–C, Equivalent numbers of spleen cells from P14 and P14LLAA mice were mixed, loaded with CFSE, and stimulated with a final gp33–41 concentration of 1.0 ng/ml for 48 h. A, The histograms represent CFSE staining of isotype-gated Vα2+CD8+ P14 (shaded histogram) and P14 LLAA (unshaded histogram) T cells. The abscissa gives the CFSE staining intensity. The ordinate gives the arbitrary cell number. The number of cell divisions for each subpopulation is indicated. B, Based on the CFSE histogram shown in A, the cells were gated according to the number of cell divisions they had undergone. The TCR expression levels was subsequently determined for each subpopulation of Vα2+CD8+ P14 (shaded histogram) and P14 LLAA (unshaded histogram) T cells. The abscissa gives the TCR staining intensity. The ordinate gives the arbitrary cell number. The TCR MFI for P14 and P14 LLAA cells and the relative TCR expression levels on P14LLAA cells are given in each histogram. C, Bar histogram showing the TCR expression levels (MFI) as a function of number of cell divisions. Results are representative of three independent experiments. D, Bone marrow chimeric mice were infected i.v. with VSV. The fraction of apoptotic cells was determined by analysis of forward scatter vs side scatter of CD8+ cells 6 days after infection. E, WT and CD3γLLAA mice were infected i.v. with VSV. The fraction of apoptotic cells was determined by annexin V staining 6 days after infection. D and E, The fractions of apoptotic cells in the CD8+ WT and CD3γLLAA cell populations are shown. The data show the mean values ± SEM obtained from two independent experiments with eight mice in each group. Statistical differences (∗∗, p < 0.005; ∗∗∗, p < 0.0005) between WT and CD3γLLAA cells were determined using Student’s t test.

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The increased numbers of TCR at the cell surface of cycling CD3γLLAA T cells would be expected to result in increased and potentially harmful TCR signaling intensity in these already activated cells (49, 50). To directly determine whether the CD3γLLAA mutation affected apoptosis of activated virus-specific CD8+ T cells, we determined the fraction of apoptotic CD8+ T cells during VSV infections. In the first set of experiments, we infected bone marrow chimeric mice i.v. with 1 × 106 PFU of VSV. Six days after infection, we isolated spleen cells and measured the fraction of apoptotic CD8+ T cells by forward/side scatter gating. We found a significantly increased fraction of apoptotic cells in CD8+ CD3γLLAA cells compared with CD8+ WT cells (Fig. 8,D). In the second set of experiments, we infected WT and CD3γLLAA mice i.v. with 1 × 106 PFU of VSV. Six days after infection, we isolated spleen cells and measured the fraction of apoptotic CD8+ T cells by annexin V staining. In accordance with the first set of experiments, we found a significantly increased fraction of apoptotic cells in CD8+ CD3γLLAA cells compared with CD8+ WT cells (Fig. 8 E).

This is the first study that describes the role of TCR down-regulation mediated by the CD3γ diL receptor-sorting motif during physiological immune responses. We report that the CD3γ diL motif plays a critical role for the expansion in numbers of virus-specific CD8+ T cells. Compared with the dramatic effect on T cell development and function observed in some KO mouse models (51, 52, 53, 54), the observed reduction in expansion of virus-specific T cells in the present study might not appear impressive at first sight. However, a 50–60% reduction in expansion of pathogen-specific T cells might well be crucial in the race between invading pathogens and the immune system during some infections. Taken into consideration that our model is a knock-in mouse, and that the only difference between CD3γLLAA and WT mice is a subtle mutation of the CD3γ diL motif, we find that our results convincingly demonstrate an important role of the CD3γ diL motif in physiological T cell responses. The significant role of the CD3γ diL motif in T cell biology is further supported by the observation that this motif has been conserved for more than 350 million years of evolution and that it is even found in the common ancestor of the CD3γ- and δ-chains in amphibians (55).

The CD3γ diL motif has been carefully characterized at the molecular level and its role in TCR trafficking in transfected T cell lines has previously been described (15, 16, 18, 56). In this study, we establish the critical role of the CD3γ diL motif for constitutive TCR endocytosis and activation-induced TCR down-regulation in primary T cells. We found that constitutive TCR endocytosis was severely reduced in naive T cells from CD3γLLAA mice, and in accordance, that TCR expression levels on naive T cells from CD3γLLAA mice were increased by ∼10% compared with TCR expression on WT T cells. If no compensatory mechanisms were found, a higher TCR expression level would be expected to result in increased TCR signaling (49). In agreement, we found that early TCR signaling in CD3γLLAA T cells was slightly increased compared with TCR signaling in WT T cells.

Taking into account that CD3γLLAA T cells had increased TCR expression and were slightly hypersensitive to TCR triggering, an impaired expansion in numbers of Ag-specific CD8+ CD3γLLAA T cells following VSV infections was at first sight surprising. However, we consistently found a significant reduction in expansion of a variety of virus-specific CD8+ T cells in CD3γLLAA mice following infections with both VSV and LCMV. By studying antiviral CD8+ T cell responses in mixed adoptive transfer experiments, we found that the reduced expansion of virus-specific CD3γLLAA CD8+ T cells was not due to a reduced number of Ag-specific precursor T cells in CD3γLLAA mice. Furthermore, our studies in bone marrow chimeric mice demonstrated that the impaired expansion of pathogen-specific CD8+ CD3γLLAA T cells was cell intrinsic and independent on the host environment.

So why is the expansion in number of virus-specific CD8+ T cells in CD3γLLAA mice impaired? The magnitude of the expansion of Ag-specific T cells is dependent on three factors, namely recruitment (i.e., fraction of dividing Ag-specific cells), division, and apoptosis of the Ag-specific cells (4). We found that Ag-specific CD8+ CD3γLLAA T cells were normally recruited and had similar division rates as Ag-specific CD8+ WT T cells. This indicated that an increased apoptosis rate caused the impaired expansion of Ag-specific CD8+ CD3γLLAA T cells. Indeed, we found decreased levels of the antiapoptotic molecule Bcl-2 and an ∼5% increased fraction of apoptotic cells in activated virus-specific CD8+ CD3γLLAA T cells compared with activated virus-specific CD8+ WT T cells. The probability of apoptosis (α) during the cell cycle relates to signal 1 from Ag, signal 2 from costimulation, and signal 3 from cytokines (5, 6, 7, 57, 58) and α has been estimated to be in the range of 0.07–0.25 (27, 59, 60). For P14 cells, α has been estimated to 0.09 (27). Given that the number of precursors, the recruitment, and the division rates of virus-specific CD8+ T cells were equal in WT and CD3γLLAA mice, but that α was increased with 5% from 0.09 to 0.14 in CD3γLLAA cells, this would result in an ∼50% reduction in the expansion in numbers of Ag-specific CD3γLLAA T cells compared with WT T cells following 12–13 cell divisions (Fig. 9). Thus, a minor increase of α in activated, pathogen-specific CD8+ CD3γLLAA T cells could fully explain our observations. In accordance, an increased α has recently been identified as reason for the impaired expansion of Ag-specific T cells deficient of the type I IFN receptor (57). In this study, a significant impairment in the expansion in numbers of type I IFN receptor deficient CD8+ T cells was observed following LCMV infections. The impairment in expansion was not caused by a compromised recruitment or division rate but exclusively by an increased apoptosis rate of the activated LCMV-specific CD8+ T cells during the expansion phase (57).

FIGURE 9.

Correlation between T cell expansion and the probability of apoptosis (α) during cell cycling. A, The equation gives the theoretical number of two Ag-specific T cell populations during the expansion phase of an acute infection. Importantly, only α differ between the two cell populations (0.09 vs 0.14). The initial numbers of Ag-specific precursors, the recruitment of Ag-specific precursors, and the division rate are identical for the two populations. The first column gives the number of cell divisions (d), the second and third column give the absolute number (A) of the Ag-specific cell populations given that α is 0.09 and 0.14, respectively. The fourth column gives the ratio of the number of Ag-specific cells with α = 0.14 to the number of Ag-specific cells with α = 0.09. B, Schematic presentation of the absolute cell numbers during the expansion phase of two Ag-specific T cell populations with α = 0.09 and α = 0.14, respectively.

FIGURE 9.

Correlation between T cell expansion and the probability of apoptosis (α) during cell cycling. A, The equation gives the theoretical number of two Ag-specific T cell populations during the expansion phase of an acute infection. Importantly, only α differ between the two cell populations (0.09 vs 0.14). The initial numbers of Ag-specific precursors, the recruitment of Ag-specific precursors, and the division rate are identical for the two populations. The first column gives the number of cell divisions (d), the second and third column give the absolute number (A) of the Ag-specific cell populations given that α is 0.09 and 0.14, respectively. The fourth column gives the ratio of the number of Ag-specific cells with α = 0.14 to the number of Ag-specific cells with α = 0.09. B, Schematic presentation of the absolute cell numbers during the expansion phase of two Ag-specific T cell populations with α = 0.09 and α = 0.14, respectively.

Close modal

The CD3γLLAA mutation affected the expansion phase but did not seem to affect the contraction phase of the immune responses in the present study. This is in good agreement with the observation that the CD3γLLAA mutation affected TCR expression and thereby TCR signaling (49), and that the expansion phase is partially dependent on signal 1 delivered by the TCR following Ag recognition, whereas the contraction phase is independent of Ag/TCR signaling (3, 5). Previous studies have demonstrated that a certain signaling threshold must be reached to initiate cell cycling of Ag-specific T cells. Signaling above this threshold does not augment cell cycling, but on the contrary may increase apoptosis of the cycling cells (46, 47, 48). We found that cycling virus-specific CD8+ CD3γLLAA T cells expressed ∼50% more TCR at their cell surface than cycling virus-specific CD8+ WT T cells. Thus, as long as Ag is present, it should be expected that increased TCR signaling takes place in cycling virus-specific CD8+ CD3γLLAA T cells compared with cycling virus-specific CD8+ WT T cells. As the signaling threshold for cell cycling already has been reached in both cycling CD8+ CD3γLLAA and WT T cells, the sustained high intensity TCR signaling in CD3γLLAA cells could potential be harmful to the cells (48, 49, 50). TCR signaling increases the level of the proapoptotic molecule Bim and down-regulates the level of the antiapoptotic molecule Bcl-2, and the balance between these pro- and antiapoptotic molecules determine whether the activated virus-specific T cells survive or die by apoptosis (38, 40, 41, 42). We found increased down-regulation of Bcl-2 in activated virus-specific CD8+ CD3γLLAA T cells and in accordance a higher fraction of apoptotic cells in virus-specific CD8+ CD3γLLAA T cells than in virus-specific CD8+ WT T cells.

At first sight, our results seem to be in discordance with previous studies comparing T cell responsiveness and ligand-induced TCR down-regulation (12, 13). These studies found that reduced TCR down-regulation and degradation correlated with loss of desensitization and appearance of hyperresponsive T cells. However, importantly these studies also found that the modified KO T cells had normal constitutive TCR internalization, which demonstrated that CD3γ-mediated TCR trafficking was intact in these cells. In the present study, we analyzed the role of the CD3γ diL motif-dependent TCR down-regulation pathway, whereas Naramura and Lee (12, 13) investigated the role of the PTK-dependent pathway. Thus, our results are not in contrast with the results of Naramura and Lee as different pathways and mechanisms for TCR down-regulation were analyzed in the respective studies. We propose that the CD3γ diL motif serves to regulate TCR expression and thereby to fine-tune TCR signaling and optimize T cell expansion in activated T cells by balancing survival and death signals. The diverse functional outcome of the two distinct pathways for TCR down-regulation might explain why it is necessary for T cells to maintain both pathways.

In conclusion, this study identified the CD3γ diL receptor-sorting motif as a key player in obtaining optimal TCR expression levels and signaling in activated T cells to attain maximum pathogen-specific T cell expansion during acute viral infections.

The technical help of Martin Weiss Nielsen and Stephanie Geisler Crone is acknowledged. MHC Dextramers were provided by C. Jespersgaard and J. Schøller, DakoCytomation, Denmark.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from The Danish Medical Research Council, The Novo Nordisk Foundation, The A.P. Møller Foundation for the Advancement of Medical Sciences, The Agnes and Poul Friis Foundation, and The Astrid Thaysen Foundation for Basic Medical Sciences.

5

Abbreviations used in this paper: diL, di-leucine-based; PTK, protein tyrosine kinase; PKC, protein kinase C; KO, knock-out; WT, wild type; LN, lymph node; VSV, vesicular stomatitis virus; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; PDB, phorbol 12,13-dibutyrate; LAT, linker for activation of T cells.

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