CD1d-restricted T cell activation by nonlipidic small molecules

Abstract

In addition to NK T cells expressing invariant Valpha14 or Valpha24 T cell receptors (TCRs), the CD1d-restricted T cell repertoire is comprised of T cells with diverse TCRs that mediate inflammation during autoimmune and infectious disease. Here we describe the isolation of human Valpha24(-) T cells that are activated by antigen and CD1d. Mass spectrometric and NMR studies revealed that the stimulatory compounds were neither peptidic nor lipidic but instead were composed of sulfur and aromatic hydrocarbon rings, corresponding to the general structure of phenyl pentamethyldihydrobenzofuran sulfonates. Studies of the molecular mechanism of T cell activation showed that a clonotypic Valpha2/Vbeta21 TCR transmitted activating signals, which were highly specific for hydroxylation and methylation patterns at the terminal structures of stimulatory compounds. These studies provide evidence for noninvariant CD1d-restricted T cells in humans and identify the complete molecular structure of a nonlipidic small molecule that activates T cells through an alphabeta TCR.

Fig. 1.

T cell activation is CD1d-restricted and antigen (Ag)-dependent. (a and b) Proliferation of ABd T cells stimulated with DC and antigen mixtures was measured in the presence of mAb against CD1a, CD1b, CD1c, CD1d, or isotype-matched control (P3) (20 μg/ml). (c) C1R cells transfected with CD1a, CD1b, CD1c, CD1d, or vector alone (mock) were treated with the antigen mixture at the indicated concentration (in μg/ml). Blocking mAb and polyclonal ABd T cells were added, and proliferation was measured.

Fig. 2.

Isolation of PPBF as T cell antigen. (a) Mixtures of stimulating compounds were resolved by using reversed-phase HPLC and monitored by UV (214 nm) absorbance (solid line). Each fraction was normalized for area under the UV absorbance curve and tested for its ability to stimulate ABd in the presence of DC. (b) Fraction 9, the only fraction that stimulated ABd, was subjected to electrospray ionization-tandem MS. The experiment showed an ion at m/z 347.1, which with collisional activation gave rise to prominent product ions that are interpreted as shown in c.

Fig. 3.

T cell fine specificity for synthetic structural analogs of PPBF. (a) By using reversed-phase HPLC and mass spectrometric ion monitoring at m/z 347, two products of the PPBF synthesis mixture with a m/z 347 were recovered. The later eluting compound showed nearly identical retention to fraction 9 from the stimulatory compounds (347) and an identical collisional mass spectrum. The early eluting compound was assigned as containing a C–S linkage as indicated. Both compounds were normalized for absorbance at 290 nm and tested for their ability to induce proliferation of ABd. Ag, antigen. (b) PPBF analogs were synthesized by coupling phenol or any of three forms of methyl phenol (cresol) with 2,2,4,6,7-pentamethyldihydrobenzofuransulfonate (PBF). After normalization for absorbance at 290 nm, the ability of PPBF analogs to induce IL-2 release by ABd was tested in the presence of C1R cells transfected with CD1d.

Fig. 4.

PPBFs do not stimulate NK T cells. (a and b) The T cell line ABd and the Vα24⁺ T cell clone J3N.5 were stimulated with DC and a PPBF-containing antigen (Ag) mixture or αGalCer, and IL-2 release was measured after 24 h. (c) PPBF, 3-methyl PPBF and the indicated anilinonapthalene sulfonic acid (ANS) compounds were incubated in molar excess with αGalCer and washed, and then NK T cells were added for 14 h before measuring IL-2 release.

Fig. 5.

PPBF recognition is mediated by variable regions of the TCR α and β chains. (a) J.RT3-T3.5 cells were transfected with the ABd TCR Vα2 and Vβ21 chains (J.RT3.ABd) or with empty vectors (J.RT3.mock) and tested for the ability to release IL-2 upon stimulation with PPBF. (b) To determine whether PPBF could activate through intact CD3 complexes that were reconstituted by transfection of a TCR with irrelevant antigen specificity, J.RT3-T3.5 cells transfected with the TCR α and β chains of the CD8-2 cell line (J.RT3.CD8-2) that recognizes DDM were tested for IL-2 release upon stimulation with DDM or PPBF.