Association of HLA-DR1 with the allergic response to the major mugwort pollen allergen: molecular background

Abstract

Background: Mugwort pollen allergens represent the main cause of pollinosis in late summer. The major allergen, Art v 1, contains only one single immunodominant, solely HLA-DR-restricted T cell epitope (Art v 125-36). The frequency of HLA-DRB1*01 is highly increased in mugwort-allergic individuals and HLA-DR1 serves as restriction element for Art v 125-36. However, Art v 125-36 also binds to HLA-DR4 with high affinity and DR1-restricted Art v 125-36 -specific T cell receptors can be activated by HLA-DR4 molecules. To understand the predominance of HLA-DR1 in mugwort allergy in spite of the degeneracy in HLA/peptide-binding and TCR-recognition, we investigated the molecular background of Art v 125-36 /MHC/TCR interactions in the context of HLA-DR1 compared to -DR4.

Results: The majority of Art v 125-36 -specific T cell lines and clones from HLA-DR1 carrying, mugwort pollen-allergic donors reacted to synthetic and naturally processed Art v 1-peptides when presented by HLA-DR1 or HLA-DR4 expressing antigen presenting cells. However, at limiting peptide concentrations DR1 was more effective in T cell stimulation. In addition, the minimal epitope for 50% of Art v 125-36 -specific T cells was shorter for DR1 than for DR4. In vitro binding assays of Art v 125-36 mutant peptides to isolated DR1- and DR4-molecules indicated similar binding capacities and use of the same register. In silico simulation of Art v 125-36 binding to HLA-DR1 and -DR4 suggested similar binding of the central part of the peptide to either molecule, but a higher flexibility of the N- and C-terminal amino acids and detachment at the C-terminus in HLA-DR1.

Conclusions: The predominance of HLA-DR1 in the response to Art v 125-36 may be explained by subtle conformation changes of the peptide bound to DR1 compared to DR4. Computer simulation supported our experimental data by demonstrating differences in peptide mobility within the HLA-DR complex that may influence TCR-binding. We suggest that the minor differences observed in vitro may be more relevant in the microenvironment in vivo, so that only presentation by HLA-DR1, but not -DR4 permits successful T cell activation.

Figure 1

Presentation of Art v 1_25-36by APC expressing HLA-DR1 or -DR4. A. HLA-DR expression on the EBV cell lines used as APC. B. Stimulation of TCL (n = 9) and TCC (n = 17) derived from 5 different subjects with an optimum concentration of Art v 1_25-36 peptide (3 μM). Box plots indicating the median and quartile ranges are shown. Background proliferations for DR1 or DR4 plus T cells ranged from 1,002-27,612 cpm and 2406-21054 cpm for TCL and from 1525-4264 cpm and 370-4270 cpm for TCC). C. T cell reactivity to different concentrations of Art v 1_25-36 presented by HLA-DR1 or -DR4 T cells were stimulated for 3 days with DR1- or DR4-expressing APC and proliferation was measured by ³H-thymidine uptake during the last 16 hours. Background proliferations were 2796/3056 cpm for Pat 2, 2247/1318 cpm for Pat 8.

Figure 2

Mapping of minimal T cell epitopes and critical amino acids within Art v 1_25-36presented by HLA-DR1 or -DR4. T cell reactivity was tested with truncated and single mutant Art v 1_25-36 –peptides. T cells were stimulated for 3 days with DR1- or DR4-expressing APC and proliferation was measured by ³H-thymidine uptake during the last 16 hours; (+) denotes >50% and (–) >70% reduction of proliferation induced by KCIEWEKAQHGA (dpm). Background proliferations for DR1 or DR4 plus T cells ranged from 1,002-27,612 cpm and 2406-21054 cpm for TCL and from 1525-4264 cpm and 370-4270 cpm for TCC.

Figure 3

Presentation of naturally processed Art v 1-peptides by DR1- or DR4-expressing APC to Art v 1_25-36-specific TCL and TCC. T cells were stimulated for 3 days and proliferation was measured by ³H-thymidine uptake during the last 16 hours.

Figure 4

Comparison of the spatial dynamics of HLA-DRB1*01:01 and HLA-DRB1*04:01. (A and B) Cartoon representation of HLA-DRB1*01:01. Blue: MHC alpha chain; Red: MHC beta chain; Yellow: Equally distributed snapshots of the peptide over the whole simulation time; Green: Residues differing between HLA-DRB1*01:01 and HLA-DRB1*04:01 which are within 10 Å of the peptide. (C) RMSF of Art v 1_25-36 bound to HLA-DRB1*01:01 and HLA-DRB1*04:01. (D) Difference in average SASA between the two complexes. (E) RMSF of the single residues of the helical segment of the MHC alpha chain. (F) RMSF of the single residues of the helical segment of the MHC beta chain.

Figure 5

Visualisation of Gag p24 34-48 PEVIPMFSALSEGAT bound to HLA-DRB1*0101 [PDB:1sje] against a sub state of the Art v 1_25-36/HLA-DRB1*01:01 simulation. Blue: MHC alpha chain; Red: MHC beta chain; Yellow: Equally distributed snapshots of Art v 1_25-36 between nanoseconds 25 and 28; Green: Initial conformation of Art v 1_25-36 at the beginning of the simulation. Black: peptide conformation of the x-ray structures of [PDB:1sjh] Gag p24 34-46 (13mer peptide) and Gag p24 34-48 [PDB:1sje] (15mer peptide).