Components of the ligand for a Ni++ reactive human T cell clone

Abstract

The major histocompatibility complex (MHC) restriction element for a human Ni(2+) reactive T cell, ANi-2.3, was identified as DR52c. A series of experiments established that the functional ligand for this T cell was a preformed complex of Ni(2+) bound to the combination of DR52c and a specific peptide that was generated in human and mouse B cells, but not in fibroblasts nor other antigen processing-deficient cells. In addition, ANi-2.3 recognition of this complex was dependent on His81 of the MHC beta chain, suggesting a role for this amino acid in Ni(2+) binding to MHC. We propose a general model for Ni(2+) recognition in which betaHis81 and two amino acids from the NH(2)-terminal part of the MHC bound peptide coordinate Ni(2+) which then interacts with some portion of the Valpha CDR1 or CDR2 region.

Figure 1.

Identification of DR52c as the Ni²⁺ presenting element for ANi-2.3. (A) Fluorescent staining with mAb's was used to assess the surface expression of DR13 and DR52c on the surface of HO301 cells. Three mAbs were used: L243, specific for the common DRα chain; L227, specific for all DRB1 β chains (in this case DRB1*1302); and FK7.3, specific for DR52c β chain. HO301 cells were incubated with biotinylated versions of the antibodies, which were then detected with phycoerythrin streptavidin (unfilled histograms). The negative controls (filled histograms) were HO301 cells incubated with only phycoerythrin streptavidin. (B) The same set of anti-DR mAb's was used in an attempt to inhibit IL-2 production by either ANi-2.3 stimulated by Ni²⁺ or AL-8.1 stimulated by pTT presented by HO301. The antibodies (10 μg/ml) were added at the initiation of the IL-2 production cultures. (C) The DRB1*0101 homozygous EBV transformed B cell line, LG2, was tested before and after transduction with the gene for DR52c β chain for its ability to present Ni²⁺ to ANi-2.3.

Figure 2.

pH-dependent, reversible binding of Ni²⁺ to fixed HO301 cells. HO301 cells were fixed with paraformaldehyde and then incubated with 250 μM Ni²⁺ overnight at 37°C. The cells were washed to remove unbound Ni²⁺ and then incubated at various pHs for 40 min at 37°C. The cells were then centrifuged and washed three times with BSS. The treated cells were used as antigen presenting cells for ANi-2.3, alone (squares), plus 100 μM Ni²⁺ (circles), or plus 50 ng/ml SEB (triangles).

Figure 3.

Natural DR52c purified from HO301 cells can present Ni²⁺. Various amounts of papain treated DR52c, immunopurified from HO301 cells were immobilized by absorption to plastic tissue culture wells. The immobilized MHCII was then used to present either Ni²⁺ (100 μM, left panel) or SEB (50 ng/ml, right panel) to ANi-2.3. As a control, HO301 cells (X) were used for antigen presentation.

Figure 4.

Ni²⁺ presentation requires DR52c expression in a professional APC. A number of human and murine cell lines lacking DR expression were transduced with the genes for the common DR α chain and the DR52c β chain. Surface expression of DR52c on the transductants was monitored by staining with FK-7.3 (mean channel fluorescence shown in parentheses). Cells were used before (white bar) and after (black bar) transduction to present Ni²⁺ (100 μM) to ANi-2.3. Untransduced HO301 cells were used as a control APC.

Figure 5.

Peptides isolated from HO301 produced DR52c can transfer Ni²⁺ presenting ability to other DR52c bearing cells. Peptides were acid stripped and purified from DR52c immunopurified from HO301 cells. An aliquot of the peptides (3 × 10⁸ cell equivalents) were added to either DR52c expressing DAP cells or pH3-treated fixed HO301 cells. The cells were used either before (white bar) or after (black bar) peptide exposure to present Ni²⁺ (100μM) to ANi-2.3 cells. As negative controls (hatched bar), peptides (pTu, pDRA, pλ) known to bind well to DR52c were used (100 μg/ml). Presentation by untreated HO301 cells served as the positive control.

Figure 6.

DR52c βHis81 is important for ANi-2.3 recognition of DR52c presented Ni²⁺. LG2 cells were transduced with the gene for the DR52c β chain in which the codon for βHis81 was changed to that for Gln. Presentation of Ni²⁺ to ANi-2.3 cells by the LG2 cells transduced with the mutant β chain (hatched bar) was compared with that seen with untransduced LG2 cells (white bar) or LG2 cells transduced with the wild type β chain (black bar).

Figure 7.

Possible model for Ni²⁺ coordination by MHCII βHis81 and amino acids at positions p-1 and p2 of the peptide. In the left panel a portion of the structure of the DR1 (DRA*0101, DRB1*0101) bound to a peptide from influenza hemagglutinin (reference 38) is shown in the vicinity of βHis81. The conserved hydrogen bond between βHis81 and the peptide backbone is shown as a dashed line. In the right panel, using Swiss PDB Viewer, peptide amino acids at position p-1 and p2 were changed to Asp and His, respectively. These amino acids and βHis81 were given rotamers that create the correct geometry for three ligands for potential tetragonal coordination (dashed lines) of a Ni²⁺ ion (sphere).