Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression
- Miguel Muñoz-Ruiz†1, 2,
- Verónica Pérez-Flores†1,
- Beatriz Garcillán1, 2,
- Alberto C Guardo1,
- Marina S Mazariegos1, 2,
- Hidetoshi Takada3,
- Luis M Allende4,
- Sara S Kilic5,
- Ozden Sanal6,
- Chaim M Roifman7,
- Eduardo López-Granados8,
- María J Recio1, 2,
- Eduardo Martínez-Naves1,
- Edgar Fernández-Malavé1, 2 and
- José R Regueiro1, 2Email author
© Muñoz-Ruiz et al; licensee BioMed Central Ltd. 2013
Received: 21 September 2012
Accepted: 16 January 2013
Published: 21 January 2013
The T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αβ or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single null or leaky mutation in CD3G (γ+/−) or CD3D (δ+/−, δ+/leaky) with that of normal controls.
Although the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/− individuals, whereas CD3δ+/− and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αβ TCR expression.
The results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.
KeywordsT cells CD3 Haploinsufficiency
The human T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion . First, variable αβ or γδ heterodimers bind to invariant CD3δε heterodimers, then to CD3γε heterodimers and finally to CD247 (or TCRζ) homodimers, resulting in surface TCR complexes with equal amounts of the two different, albeit highly homologous, CD3 heterodimers. In contrast, mouse αβ and γδ TCR differ drastically in their stoichiometry, since the γδ TCR does not incorporate any CD3δε dimers but, rather, two CD3γε dimers . This finding begs the question as to whether the human variable αβ and γδ chains show identical affinity for both CD3 heterodimers.
We reasoned that, if both the αβ and the γδ TCR isotypes use identical amounts of CD3γε and CD3δε, then decreased availability of CD3γ or CD3δ proteins, as observed in heterozygous carriers of mutations in CD3 genes , would be expected to have a similar impact on the assembly and surface expression of both αβ and γδ TCR isotypes. To test this hypothesis, we compared TCR surface levels of primary αβ and γδ T cells from healthy haploinsufficient donors carrying null or leaky mutations in CD3G (γ+/−) or CD3D (δ+/−, δ+/leaky). The results did not support the hypothesis of a similar impact on both TCR isotypes, but rather suggested a differential CD3γε and CD3δε usage scheme for each TCR isotype.
Reduced surface αβ and γδ TCR expression in CD3γ+/−, CD3δ+/− or CD3δ+/leaky human T lymphocytes
Lymphocyte studies in haploinsufficient individuals a
Number of subjects
46 ± 10
33 ± 1
37 ± 12
LYMPHOCYTE IMMUNOPHENOTYPE (%)
60 ± 6
66 ± 1
62 ± 5
45 ± 4
39 ± 5
26 ± 1
18 ± 1
26 ± 6
16 ± 7
18 ± 5
12 ± 2
17 ± 3
17 ± 2
9 ± 6
T cell proliferation (% of control max)b
3 ± 1
8 ± 2
4 ± 3
74 ± 4
84 ± 6
61 ± 5
60 ± 4
Serum Ig (mg/dl)
790 ± 319
306 ± 54
47 ± 29
611 ± 37
165 ± 222
30 ± 6
9 ± 6
NK cell cytotoxicity (% lysis)
52 ± 6
80 ± 5
Serial dilutions of CD3 mAb further confirmed the findings above (Figure 2D), since the reduced binding to γ+/− T cells persisted in saturation conditions, but it was gradually lost near the endpoint, supporting the existence of less CD3 binding sites .
From these results we conclude that human CD3G or D haploinsufficient donors show reduced binding of CD3-specific mAb to the TCR of their γδ and αβ T cells.
Discordant reduction of surface αβ and γδ TCR expression in CD3γ+/− but not CD3δ+/− or CD3δ+/leaky human T lymphocytes
Human and mouse TCR complexes are assembled into octamers following common cues provided by transmembrane ionizable aminoacids in each dimer, with CD3γε and CD3δε ectodomains contributing additional extracellular interactions for their ordered incorporation into the complex . The extracellular interactions are quite specific, as the mouse γδ TCR does not incorporate otherwise available CD3δε dimers, but rather two copies of the highly homologous CD3γε dimer. In sharp contrast, the human γδ TCR incorporates both . Mammalian CD3γ, but not CD3δ, has a uniquely kinked ectodomain which fits into an asymmetrical loop in TCRβ for optimal αβ TCR assembly and expression, and cannot be easily replaced by CD3δ due to steric hindrance, with functional consequences . This likely leads to the conserved structural asymmetry shared by the human and mouse αβ TCR. In contrast, TCRγ lacks the asymmetrical loop of its TCRβ homologue and seems to allow a less stringent (i.e., with less affinity) CD3 dimer usage in the γδ TCR, which may explain its disparate stoichiometry in the two species.
Further studies are required to demonstrate a direct link between CD3γ or δ availability and TCR assembly and surface expression. However, the paucity of CD3 haploinsufficient individuals might hamper these studies in humans. A flow cytometry-based approach as illustrated in Figure 2B could help to identify such individuals.
Lastly, since carriers of CD3G or CD3D mutations showed affected TCR expression (Figure 2) and T-cell selection (Figure 1), which seemed in turn to impair to some extent T-cell function (Table 1)  the question of whether they also have increased risk of immunological dysfunction deserves further investigation.
The results indicate that the dimer assembly scheme of the human TCR complex is different in αβ versus γδ T cells, as revealed by their discordant behaviour when confronted with limiting amounts of CD3γ, but not of the homologous CD3δ chain. The novel data allow proposing a modified version of the prevailing TCR assembly model.
After obtaining informed consent following IRB authorization (Ethics Committee for Clinical Investigation of Clínico San Carlos Hospital, Madrid), we studied nine healthy heterozygous carriers of mutations in CD3γ (γ+/−) [3, 7] of Spanish or Turkish descent and six healthy heterozygous carriers of mutations in CD3δ (δ+/−, δ+/leaky) [8, 16, 17] of Japanese, Mennonite or Colombian descent. Normal donors (termed+/+) were used as controls. Their lymphocyte immunophenotype and functional features are summarized in Table 1. PBL were isolated by Ficoll-Hypaque (GE Healthcare) gradients and resuspended in RPMI 1640 medium (Gibco) supplemented with 10% FCS (PAA Laboratories), 1% L-glutamine and antibiotics-antymitotic (100 units/ml of penicillin G, 100 μg/ml of streptomycin sulfate, and 0.25 μg/ml of amphotericin B) from Gibco.
Antibodies and flow cytometry
The expression of different surface markers was studied by flow cytometry using standard procedures in fresh whole blood or isolated PBL . For intracellular stainings cells were fixed and permeabilized as previously reported .
The following anti-human mAb were used: anti-CD3ε (S4.1) from Caltag Laboratories (now Invitrogen), anti-CD3εγ/εδ (UCHT-1), anti-TCR αβ (BMA031), and anti-TCRγδ (Immu510) from Beckman Coulter, anti-CD3ε (SK7), anti-CD4 (Leu2a), anti-CD8 (Leu3a), anti-TCRγδ (11F2), and anti-CD8 (SK1) from BD Biosciences. Anti-CD3εγ/εδ (F101.01) hybridoma supernatant and anti-CD3δ (APA1/2) ascitic fluid were a generous gift from Dr. B. Rubin (CHU de Purpan, France). TG5 (an anti-CD3γ rabbit antiserum raised against the CD3γ C-terminal peptide GLQGNQLRRN) was kindly provided by D. Alexander (Babraham Institute, U.K.). The mAb were FITC-, PE-Cy5 or PE-conjugated, or purified, and for the latter a PE-conjugated goat anti-mouse IgG (H + L) or anti-rabbit (H + L) from Caltag Laboratories was used as a secondary reagent. Background fluorescence was defined with an isotype-matched irrelevant mAb from Caltag Laboratories. For comparative stainings we used the mean fluorescence intensity (MFI), defined as the average fluorescence value of the corresponding mAb referred to the logarithmic scale of fluorescence intensity along the x-axis of the histograms. Data were analyzed with FlowJo software (TreeStar).
1×105 PBLs were placed in round-bottom microtitre wells and stimulated with 10 μg/ml anti-human CD3 (UCHT-1) or 10 μg/ml PHA. After 3 days of in vitro culture, wells were individually pulsed with 1 μCi of 3H-TdR (Amersham, Buckinghamshire, U.K.) for another 16 to 18 h and harvested onto glass fiber filters. Thymidine incorporation into cellular DNA was evaluated as cpm in a scintillation βcounter (Packard, Meriden, CT).
For CFSE (carboxyfluorescein diacetate succinimidyl ester) dilution experiments, cells were labeled with 1 μM CFSE in PBS for 10 min at 37°C at day 0, washed twice in cold PBS, plated and stimulated as above. CFSE dilution was subsequently determined by flow cytometry within CD3+ lymphocytes.
Cytotoxicity was measured using the nonradioactive Cytotoxicity Detection kit LDH (Roche). Cells were cocultured in a 96 V-well plates for 4 h at 25: 1 (Effector: Target) ratios and the percentage of specific lysis was determined from the amount of lactate dehydrogenase activity detected in culture supernatants.
Student’s t-tests were performed using SPSS 11.5.1 statistical program software (Chicago, IL). Only p values below 0.05 were considered significant. Data are presented as mean ± SEM (standard error of the mean) or ± SD (standard deviation).
E. Fernández-Malavé and J.R. Regueiro are joint senior authors.
Peripheral blood lymphocytes
Mean flourescence intensity
T cell antigen receptor
Fetal calf serum
Bent Rubin provided continuous support and comments. Elena M. Busto, Joaquín Caspistegui, Juana Gil, Miguel Fdez-Arquero and Jesús Reiné provided technical help. Brenda Reid, Sandra Mendonca and Linda Pires from the Hospital for Sick Children in Toronto are greatefully acknowledged for their excellent collaboration to obtain and ship blood samples. This work was supported by grants from the Ministerio de Educación (BFU2005-01738/BMC and SAF2011-24235), Comunidad Autónoma de Madrid (CAM) (GR/SAL/0570/2004 and S2011/BMD-2316), Fundación Lair, Instituto de Salud Carlos III (ISCIII PI080921, PI060057 and RIER RD08-0075-0002) and the Hospital 12 de Octubre Health Research Institute. MMR was supported by the Universidad Complutense de Madrid and ISCIII and VPF was supported by Ministerio de Educación.
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