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. 1999 Feb 16;96(4):1547-52.
doi: 10.1073/pnas.96.4.1547.

Multivalent structure of an alphabetaT cell receptor

G Fernández-Miguel  1 , B Alarcón, A Iglesias, H Bluethmann, M Alvarez-Mon, E Sanz, A de la Hera
Affiliations

Multivalent structure of an alphabetaT cell receptor

G Fernández-Miguel et al. Proc Natl Acad Sci U S A. .

Abstract

Whether there is one or multiple alphabetaT cell antigen receptor (TCR) recognition modules in a given TCR/CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCRbeta chains incorporate at least two alphabetaTCRs in a single TCR/CD3 complex. Evidence for bispecific alphabetaTCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by fluorescence resonance energy transfer and comodulation assays by using antibodies specific for TCRbeta-variable regions. Such (alphabeta)2TCR/CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro. T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen/major histocompatibility complex ligand. The implications of a multivalent (alphabeta)2TCR/CD3 complex stoichiometry for the ordered docking of specific antigen/major histocompatibility complex, CD4, or CD8 coreceptors and additional TCRs are discussed.

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Figures

Figure 1
Figure 1
Identification of bispecific αβTCRs on the surface of T cells from Vβ2×Vβ8 double transgenic mice. Immunoprecipitations of TCRβ from transgenic T cells were done with Vβ domain-specific mAbs or control reagents (−) and immunoblotted with mAbs to Vβ2, Vβ8, or Cα. Cell sources were either spleen T cells expanded in culture for 2–3 weeks with interleukin 2 (A–C) or freshly isolated single-cell suspensions from spleen and thymus (D) (10). Organs were either from Vβ2 or Vβ8 single transgenic mice or Vβ2×Vβ8 double transgenic F1 mice (–19), as indicated (A). In additional control immunoprecipitations, lines labeled as C in D, Vβ2 and Vβ8 single transgenic cells were mixed in a 1:1 ratio before the immunoprecipitation with TCRVβ mAb, which had the reciprocal specificity to those used in immunoblotting. Double transgenic cells were used in the remaining experiments (B–D). Two-dimensional SDS/PAGE (C) of immunoprecipitates with mAb to Vβ2 (Left) were blotted with mAb to Vβ8 (Upper) and Cα (Lower), whereas immunoprecipitates with mAb to Vβ8 (Right) where blotted with anti-Vβ2 (Upper) and control (Lower) mAbs. Immunoprecipitates were digested (B) in the absence (−) or presence of endoglycosidase H (H) or N-glycosidase F (N) to assess the TCR glycosylation pattern (29). Sizes before and after the removal of N-linked sugars are indicated by open arrowheads and asterisks, respectively. Cells were lysed in 1% of either Nonidet P-40 (first three lines from the left in A) or Brij96 lysis buffer in the remaining experiments shown. Associations were also evident in the presence of two other detergents (0.25% saturated digitonin and 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, data not shown; ref. 16). Neat coprecipitations were attained in 30 independent experiments, each with different transgenic mice. Ticks indicate migration of molecular size markers (Bio-Rad), given in kDa: 101, 83, 50.6, and 35.5 kDa (A), 148, 60, 42, and 30 kDa (B), 60 and 42 kDa (C), and 145, 83, 60, 50, and 35 kDa (D).
Figure 2
Figure 2
The two distinct TCRβ chains are neighbors in the plasma membrane of live T cells bearing dual αβTCRs. Spleen T cell lines from Vβ2×Vβ8 double transgenic mice were stained (Upper) with mAb to Vβ2 conjugated with FITC alone or followed by PE-labeled mAb against either Vβ8, CD3, CD4, or CD8. Alternatively (Lower), cells were stained with FITC-conjugated anti-Vβ8 mAb alone or followed by PE-labeled mAb against either Vβ2, CD3, CD11a, or CD45. Flow cytometry analyses of average donor quenching (10, 31) measure the putative reduction in the green FITC fluorescence emission on the surface of viable T cells promoted by neighbor PE-acceptor molecules (10, 31). In cells stained with the two anti-TCRVβ mAbs (A and D), the FITC fluorescence distribution was reduced as indicated by the shift to the left in the presence of the quenching, reciprocal anti-TCRVβ mAb (shaded histograms). Both TCRβ chains were also in the vicinity of CD3 subunits (shaded histograms in C and F). Histograms for TCRVβ staining in the presence and absence of the PE-labeled mAb against CD4, CD8, CD11a, and CD45 instead are superimposed. The ratio of Vβ2 to Vβ8 mean fluorescence intensity ranged from 1:1 to 2:3 in samples from several mice. Similar results were observed for tetraplicate samples in three independent experiments as well as in analyses of freshly isolated spleen and thymus cells (data not shown).
Figure 3
Figure 3
Comodulation of unstimulated TCRs upon down-regulation of specific TCR/CD3 complexes in dual-receptor T cells. Immunofluorescence distribution profiles of spleen T cell lines from Vβ2×Vβ8 double transgenic mice submitted to different TCR down-regulation protocols and stained with FITC-labeled mAb against TCRVβ8 (A) or TCRVβ2 (B). Left histograms in both A and B represent the background staining with anti-Ig fluoresceinated control reagents that was superimposable to the autofluorescence (data not shown). Shaded histograms show the comodulation of the stained, unstimulated TCRs promoted after down-regulation of the reciprocal TCRβ, done by incubation at 37°C for 4 h in plates coated with 5 μg/ml of mAb specific for the reciprocal TCRVβ. Down-modulation is not promoted either in replicate plates kept in parallel at 4°C (dotted line) or by a control mAb [OKT3, which promotes TCR/CD3 comodulation in human CD3 transgenic T cells (10)] (continuous line), as shown in the right histograms. The mAbs do not promote modulation of the reciprocal TCRβ in single transgenic mice (data not shown). Results are representative of four experiments.
Figure 4
Figure 4
Hypothetical model of (αβTCR)2/CD3 complex. (A) Packing of two αβTCR in a single TCR/CD3 complex. Disulfide bridges in each αβTCR heterodimer or in the ζ homodimer are depicted as black squares. The blue rectangles represent two antiparallel MHC class II molecules presenting the specific peptides (yellow strands). The surface of the αβTCR interface opposite to the CD3 pocket is flat; for simplicity, ovals have been used in most cases. Different isoforms of TCR have been reported with regard to the CD3/ζ module usage, which is reviewed in ref. . For simplicity, only the three most common transduction modules are included. (B) The hexameric antigen/MHC packing arises after homotypical aggregation of TCRs and coreceptors, as observed from the T cell nuclei looking toward the APC. Oligomerization occurs in the two dimensions of the T cell and APC membrane after the TCR/CD3 complexes have engaged specific antigen/MHC complexes. The specific TCR ligands are monomers that are rare and occur naturally dispersed on the APC surface among a majority population of irrelevant antigen/MHC complexes but may be concentrated to the T cell/APC interface by the constitutively divalent TCRs.
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