Multiple sclerosis: Molecular mimicry of an antimyelin HLA class I restricted T-cell receptor

Geraldine Rühl; Anna G. Niedl; Atanas Patronov; Katherina Siewert; Stefan Pinkert; Maria Kalemanov; Manuel A. Friese; Kathrine E. Attfield; Iris Antes; Reinhard Hohlfeld; Klaus Dornmair

doi:10.1212/NXI.0000000000000241

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Figure 1 T-cell receptor 2D1 recognizes human leukocyte antigen (HLA)–A2–restricted octameric and nonameric synthetic peptides
Left: Fluorescence microscope images from coculture of 58α⁻β⁻ T hybridoma cells expressing TCR 2D1, human CD8αβ molecules, and sGFP under the control of nuclear factor of activated T cells (58-2D1-CD8-sGFP) and COS-7 cells expressing HLA-A*02:01(COS-7-A2) cells preincubated with synthetic peptides. Green cells indicate activation of 58-2D1-CD8-sGFP cells. The names of the peptides are given as inserts. Scale bars: 100 μm. Right: Secreted interleukin (IL)–2 as measured by ELISA of the coculture's supernatants. In the top row (A–D), octameric peptides were used. Only glycerolphosphatidylcholine phosphodiesterase 1 (GPCPD1) (15-22) weakly activated 58-2D1-CD8-sGFP cells. In the second row (E–H), the same octameric peptides were used but all carried an additional methionine residue at their N-terminus. All of them strongly activated 58-2D1-CD8-sGFP cells. In the third row (I–L), the same octameric peptides were used but all carried their original amino acid residue at their N-terminus. Only GPCPD1(14-22) activated 58-2D1-CD8-sGFP cells. Lowest row: (M) The entire domain of GPCPD1(1-118) that harbors the antigenic peptide GPCPD1(14-22) did not activate 58-2D1-CD8-sGFP cells when transfected into COS-7-A2 cells. When no peptide (N) or the irrelevant peptide T-lymphotrophic virus-2 protein (TAX) (11-19) (O) was added, 58-2D1-CD8-sGFP cells were not activated. Error bars represent standard deviation of the mean. DMXL2 = Dmx-like-2; EML5 = echinoderm microtubule associated protein-like 5; NCAN = Neurocan.
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Figure 2 In vitro refolded human leukocyte antigen (HLA)–A2:peptide complexes activate TZR 2D1
(A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis of HLA-A*02:01 (HLA-A2) refolded from recombinant heavy chain and β2m in the presence of either the octameric glycerolphosphatidylcholine phosphodiesterase 1 (GPCPD1) (15-22) or the nonameric M-GPCPD1(15-22) under reducing and nonreducing conditions. Lanes 1 and 6, protein standard. Molecular masses are given in kDa at the left; lane 2, HLA-A2, β2m, and M-GPCPD1(15-22) under reducing conditions; lane 3, HLA-A2, β2m, and GPCPD1(15-22) under reducing conditions; lane 4, HLA-A2, β2m, and M-GPCPD1(15-22) under nonreducing conditions; lane 5, HLA-A2, β2m, and GPCPD1(15-22) under nonreducing conditions. (B) Activation of 58α⁻β⁻ T hybridoma cells expressing TCR 2D1, human CD8αβ molecules, and sGFP under the control of nuclear factor of activated T cells (58-2D1-CD8-sGFP) cells by in vitro refolded trimeric HLA-A2:β2m:peptide complexes as measured by fluorescence microscopy. Left panel: 58-2D1-CD8-sGFP cells were incubated with refolded and biotinylated HLA-A2, β2m, and GPCPD1(15-22). Right panel: As left panel, but with M-GPCPD1(15-22). Scale bars: 100 μm. (C) IL-2 concentration measured in the supernatant from the experiments shown in (B). Error bars represent SD of the mean.
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Figure 3 The T-cell receptor (TCR) Vα2Jα21-chain determines cross-reactivity of 2D1 cells
Activation of TCR-transfected T hybridoma cells by human leukocyte antigen (HLA)–transfected COS-7 cells and plasmid-encoded peptides. Adherent COS-7 cells expressing HLA-A*02:01 (COS-7-A2) (shaded bars) or COS-7 cells expressing HLA-A*03:01 (COS-7-A3) (white bars) were transfected with plasmids coding for proteolipid protein (PLP) (45-53), glycerolphosphatidylcholine phosphodiesterase 1 (GPCPD1) (15-22), or empty plasmid (NC). Transfected COS-7 cells were superposed with 58α-β-cells that were transfected with CD8, the TCR Vβ15.1Jβ2.7 β-chain, and (A) both α-chains (Vα2Jα21 and Vα7Jα11), (B) the Vα2Jα21-chain alone, or (C) the Vα7Jα11-chain alone. T-cell activation was analyzed by measurement of secreted IL-2 by ELISA. Error bars represent SD of the mean.
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Figure 4 Molecular models of antigenic human leukocyte antigen (HLA)–A2:peptide complexes by molecular dynamics simulations
(A) Aligned structures of the peptides glycerolphosphatidylcholine phosphodiesterase 1 (GPCPD1) (14-22):LLPGEVFAI (orange), M-GPCPD1(15-22):MLPGEVFAI (yellow), and GPCPD1(15-22):LPGEVFAI (cyan) bound to HLA-A*02:01 (HLA-A2) after 10 ns molecular dynamics run. HLA-A2 with bound nonamers GPCPD1(14-22) and M-GPCPD1(15-22) is shown in light gray and HLA-A2 with bound octamer GPCPD1(15-22) is shown in dark gray. The HLA:peptide complexes are shown from the top, i.e., from the position of the TCR. (B) The 3 peptides (see A) shown for clarity in greater magnification without the HLA molecules. The diverse courses of the polypeptide backbones and the different positions of the amino acid side chains are evident. These differences are particularly pronounced in the region around F20, which is facing towards the HLA floor for the nonamers but faces upward in the octamer. (C) View from the N-terminus of HLA-A2 with bound nonameric GPCPD1(14-22) (orange). The residues Y171, Y159, and Y7 have stabilizing effect on the bound nonamer and are shown in red. This tyrosine triad interacts with the N-terminal leucine of the bound peptide by forming hydrogen bonds that anchor the peptides tightly. (D) View from the N-terminus of the HLA-A2 with bound octameric GPCPD1(15-22) (cyan). The bound octamer does not form stabilizing hydrogen bonds with the tyrosine residues, thus appearing as more flexible during the molecular dynamics simulations.

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Multiple sclerosis

Molecular mimicry of an antimyelin HLA class I restricted T-cell receptor

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