Mycoplasma superantigen is a CDR3-dependent ligand for the T cell antigen receptor

Abstract

Superantigens are defined as proteins that activate a large number of T cells through interaction with the Vbeta region of the T cell antigen receptor (TCR). Here we demonstrate that the superantigen produced by Mycoplasma arthritidis (MAM), unlike six bacterial superantigens tested, interacts not only with the Vbeta region but also with the CDR3 (third complementarity-determining region) of TCR-beta. Although MAM shares typical features with other superantigens, direct interaction with CDR3-beta is a feature of nominal peptide antigens situated in the antigen groove of major histocompatibility complex (MHC) molecules rather than superantigens. During peptide recognition, Vbeta and Valpha domains of the TCR form contacts with MHC and the complex is stabilized by CDR3-peptide interactions. Similarly, recognition of MAM is Vbeta-dependent and is apparently stabilized by direct contacts with the CDR3-beta region. Thus, MAM represents a new type of ligand for TCR, distinct from both conventional peptide antigens and other known superantigens.

Figure 1

Proliferative response of human T cell clones to bacterial superantigens. The shaded boxes represent examples of clones differing in MAM reactivity despite shared Vβ usage (Vβ5.1, 8, 12.2 and 17.1). Vβ13.1 and 13.2 differ primarily in the HVR4 region, a component of the lateral TCR surface that interacts with superantigens (24, 37). We confirm that TCRs expressing the two closely related Vβ13 gene segments have few superantigen reactivities in common. Key to symbols: −, no reactivity with 1 ng/ml or any concentration; +, reactivity with 10⁻¹ ng/ml or greater; ++, reactivity with 10⁻² ng/ml or greater; +++, reactivity with 10⁻³ ng/ml or greater. See Table 1 for example of raw data.

Figure 2

TCR α and β chains from human T cell clones with different MAM reactivity. The sequences represent MAM-reactive clones (+), MAM nonreactive clones (−), and dominant clonal sequences (SEL) obtained from three polyclonal cell lines (K3, J5, D3) that were repeatedly stimulated with MAM, respectively, 3×, 5×, or 3× consecutively at 2-wk invervals. The ratio shown after the name of the cell line indicates the number of identical CDR3-β sequences over the total number of sequences obtained for the indicated Vβ subset. Vα and Vβ nomenclature is according to reference , and Jα is according to EMBL/Genbank/ DDBJ accession No. M94081.

Figure 3

Reactivity to MAM is not MHC-restricted. Four lymphoblastoid cell lines, 2P68 (HLA DR4), LS (HLA DR5,2), RF (HLA DR2,6), and 8866P (HLA DR15), were used for presentation of MAM, SEC2, and SEE to MAM⁺ and MAM⁻ T cell clones. In each group of T cell clones (Vβ17, Vβ8, Vβ12, and Vβ5) specific reactivity with the tested superantigens was not dependent on the HLA alleles expressed. MAM⁺ clones remained reactive regardless of the APCs used and MAM⁻ clones remained nonreactive.

Figure 4

Mapping of the TCR residues required for reactivity with MAM. Mutations based on alignment between N17 and J17 map the positions involved in interaction with MAM (I and Y). Deletions in the middle of the CDR3 loop (Δ) did not affect reactivity, as well as several mutations in the HVR4 region. Constructs marked with a * were transfected into both DS23.27 (mouse Vα2) and YLβ- (mouse Vα3) cells, and had identical reactivity regardless of the α chain used. 10-fold dilutions of superantigens were tested in IL-2 assays as described in reference , (see also Table 2). Reactivity was scored as the minimal concentration of the tested superantigens required for an IL-2 response and the key to the symbols is: +++, 0.1 ng/ml, ++, 1 ng/ml, and +, 10 ng/ml.

Figure 5

Linear model of TCR-β indicating residues implicated as superantigen (SAG) interaction sites by mutation studies. Typically, superantigens interact with solvent-exposed residues of CDR1, CDR2, and HVR4 encoded by Vβ genes (, –67). The interaction of MAM with TCR-β is clearly distinct, as it interacts both with the Vβ region because of its Vβ specificity (the exact site is not known), and with two residues located at the base of the CDR3-β loop.