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Comment on "MHC Class II Expression Identifies Functionally Distinct Human Regulatory T Cells"

Department of Pulmonary Diseases Research, Genomics Group, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

In the April 15, 2006 issue, Baecher-Allan et al. (1) demonstrated that HLA-DR expression on human CD4⁺CD25^high cells identifies a mature and highly active regulatory T cell (Treg) population. The authors (1) used an anti-HLA-DR mAb (L243; BD Pharmingen) to sort and to identify highly suppressive CD4⁺CD25^high regulatory T cells and suggested a specific role of HLA-DR expression in the homeostatic maintenance of Tregs in vivo. Monoclonal L243 Ab, as stated by the manufacturer, reacts with a nonpolymorphic HLA-DR epitope and should not cross-react with other MHC class II molecules. Binding of mAb L243 on the surface of the T cells indicates its reactivity toward N-terminal sequence of HLA, representing type I membrane protein.

In our hands, mAb L243 recognized approximately 20% of Foxp3⁺CD4⁺CD25^high T cells (Fig. 1), thus confirming results of Baecher-Allan et al. With the aim of comparing the specificities of a variety of class II MHC mAbs, we tested binding specificity of the L243 by epitope mapping on the HLA-DP β-chain (HLA-DPβ) peptide scan. Surprisingly, the peptide scan results revealed that L243 binds to the HLA-DP-specific linear motif (LERYIYNREEFA) in the N terminus of the protein (Fig. 2A, left panel). In contrast, we could not observe any reactivity of mAb L243 toward HLA-DR peptide of the corresponding N-terminal sequence (Fig. 2A, right panel). Since HLA-DP and HLA-DR share only 58% sequence identity in the region corresponding to the mapped L243 epitope, a potential cross-reactivity appears very unlikely. These results were confirmed by competition assay using a blocking peptide corresponding to the HLA-DPβ epitope. Preincubation of L243 with blocking peptide clearly reduced its specificity toward human Tregs (Fig. 2B), unambiguously proving its anti-HLA-DP specificity.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. mAb L243 recognizes a surface Ag expressed on human peripheral blood CD4+CD25highFoxp3+ Tregs. Human CD4+CD25high T cells were isolated from peripheral blood and analyzed by flow cytometry for the expression of CD25 (M-A251), Foxp3 (PCH101), and L243. Individual populations expressing L243 Ag and/or CD25 and Foxp3, including their relative ratios in percentages, are shown.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. A, Left panel, Mapping of L243 epitope. A total of 83 overlapping peptides, covering the entire HLA-DPβ-chain sequence, were synthesized and immobilized on cellulose membrane (PepSpots; JPT Peptide Technologies) and subsequently incubated with mAb L243. The boxes mark mAb-specific epitopes; recognized amino acid residues and their position in HLA-DPβ sequence are listed. Right panel, Specificity of mAb L243 was tested against synthesized peptide corresponding to the N-terminal sequence in the β-chain of HLA-DP or HLA-DR. Absence of the signal for HLA-DR reactivity is indicated by the circle. B, Demonstration of mAb specificity using an epitope-derived blocking peptide. Isotype control and mAb L243 were preincubated with 10x stoichiometric excess of the LERYIYNREEFA epitope peptide (ePEP) before the staining of human CD4+CD25high Tregs. Positive cells were gated and their percentages were calculated, demonstrating that L243 binding to Tregs was significantly reduced in the presence of HLA-DP-specific epitope peptide. For control experiments, mAb L243 were preincubated with 10x stoichiometric excess of scrambled control peptide (control) before staining.

References

1. Baecher-Allan, C., E. Wolf, D. A. Hafler. 2006. MHC class II expression identifies functionally distinct human regulatory T cells. J. Immunol. 176: 4622-4631. [Abstract/Free Full Text]

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				C. Baecher-Allan and D. A. Hafler Response to Comment on "MHC Class II Expression Identifies Functionally Distinct Human Regulatory T Cells" J. Immunol., March 15, 2008; 180(6): 3626 - 3626. [Full Text] [PDF]

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