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

Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

In the current issue of The Journal of Immunology, the letter from Swiatek-de Lange et al. (1) presents data confirming our observation that class II expressing CD25^high human natural regulatory T cells (nTregs) express the highest levels of FoxP3. We welcome additional studies into this nTreg subset because we believe that the class II-expressing nTregs (DR⁺ Treg) are an important and functionally distinct subset that functions as end-stage effector nTregs, as they induce a strong and rapid suppression and exhibit low in vitro expansion capabilities (2).

The purpose of the report by Swiatek-de Lange et al., however, is to suggest that the subset of cells that we had referred to as DR⁺ Treg should be referred to as DP⁺ Treg. This is based on their contention that the anti-HLA-DR mAb, clone L324, which we had used to FACS sort the DR⁺ Treg subset, is specific for HLA-DP peptide. We believe their claim, that the L243 Ab does not bind DR but instead binds DP, is based on flawed experimental design and ignores a large body of literature. The simplest explanation for their observation that the L243 mAb binds DP is that they are detecting a low-level cross reactivity of the Ab on denatured linear epitopes spotted at high density, which in other studies also has resulted in the demonstration of Ab polyspecificity (3). However, the biological reality of denatured linear peptides is questionable. Furthermore, in seminal work Gorga et al. (4) demonstrated that the L243 mAb specifically recognized native, highly pure HLA-DR molecules that had been immunoprecipitated and ultimately crystallized and that it did not cross-react with purified native HLA-DP molecules. Thus, there is little doubt that L243 recognizes native DR molecules; that the Ab can recognize linear denatured peptides on a solid medium would appear to have little biologic relevance.

We are pleased that Swiatek-de Lange and coworkers have begun to examine the interesting class II + (DR⁺ Treg) subpopulation of human nTregs. However, owing to the preponderance of data demonstrating the DR specificity of mAb L234 and because of our ability to amplify HLA-DR transcripts from DR⁺ Tregs, we do not agree with their conclusion that the subpopulation should be referred to as DP⁺ Treg.

References

1. Swiatek-de Lange, M., W. Rist, H. F. Stahl, A. Weith, M. C. Lenter. 2008. Comment on "MHC class II expression identifies functionally distinct human regulatory T cells.". J. Immunol. 180: 3625[Free Full Text]
2. 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]
3. Kramer, A., T. Keitel, K. Winkler, W. Stocklein, W. Hohne, J. Schneider-Mergener. 1997. Molecular basis for the binding promiscuity of an anti–p24 (HIV-1) monoclonal antibody. Cell 91: 799-809. [Medline]
4. Gorga, J. C., V. Horejsi, D. R. Johnson, R. Raghupathy, J. L. Strominger. 1987. Purification and characterization of class II histocompatibility antigens from a homozygous human B cell line. J. Biol. Chem. 262: 16087-16094. [Abstract/Free Full Text]

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