Defense of the clone: antibody 259D effectively labels human FOXP3 in a variety of applications

To the editor:

Three related articles in Blood have recently highlighted an issue with a significant impact on the study of regulatory T cells (Tregs), namely the specificity and affinity of the antibodies used to detect forkhead box P3 (FOXP3).^1-3  There is phenomenal interest in Tregs and controversial hypotheses derived from conflicting data have been relatively common. This often reflects differences between model systems, diseases, and species, variability between individual patients, and the choice of experimental reagents.

The retention of only a partial clone name on commercialization of our anti-FOXP3 monoclonal antibodies, and the tendency not to sufficiently emphasize the original publication describing antibody production and characterization in product literature, may have caused some confusion. While the authors^1-3  purchased 236A/E7, 206D, and 259D from different companies, each of these 3 different hybridoma cell lines was originally generated from the same fusion.⁴  Pillai and Karandikar's²  suggestion that 259D is conclusively a less sensitive detector of total FOXP3 conflicts not only with the data from Tran and Shevach,^1,3  but also with our own data.⁴  Our initial experience was that 259D/C7 exhibited strong reactivity with FOXP3, both by immunohistochemistry and flow cytometry of FOXP3 transfectants.⁴  Here we present immunolabeling data that demonstrates that antibody 259D/C7 detects tonsillar FOXP3⁺ Tregs in routinely fixed tissue as effectively as any of our FOXP3 antibodies (Figure 1A). Antibodies 150D/E4 and 206D/B1 showed less labeling, as previously reported,⁴  although no immunolabeling data were originally presented. Furthermore, using Biolegend's (San Diego, CA) commercially prepared Alexa 488 direct fluorochrome conjugates, 259D exhibited comparable labeling of CD4⁺ peripheral blood cells to 206D and 150D, by intracellular flow cytometric staining.⁵ 

Pillai and Karandikar²  propose that differences may arise through FOXP3 antibodies recognizing different epitopes. We agree that this is a valid issue, particularly because human Tregs express an additional, smaller isoform of the FOXP3 protein that derives from a transcript lacking the second coding exon.⁶  Immunohistochemical labeling of truncated recombinant FOXP3 proteins demonstrated that all 7 antibodies recognized epitopes within the first 235 amino acids (aa) of the FOXP3 protein (Figure 1B). The absence of reactivity to the highly conserved FOXP3 forkhead domain is consistent with their lack of cross-reactivity with the related FOXP proteins.⁴  Antibodies157B/F4 and 86D/D6 recognized an epitope encoded by exon 1 (aa 1-70), 150D/A3 an epitope encoded by exon 2 (aa 70-105) and thus, 206D/B1, 221D/D3, 236A/E7 and 259D/C7 bind within the remaining N-terminus (aa 105-235). Antibodies 259D/C7 and 206D/B1 both labeled FOXP3 from CD4⁺CD25⁺ Tregs equally effectively by Western blotting. As expected, the smaller FOXP3 isoform lacking exon 2 was not recognized by 150D/A3 (Figure 1C). We therefore conclude that 259D/C7 represents a sensitive detector of FOXP3 protein expression.

We strongly advocate the use of well-validated mono-clonal antibodies to FOXP3 and the confirmation of unexpected or conflicting data with additional antibody reagents. Furthermore, as FOXP3 mRNA can be detected in the absence of FOXP3 protein expression,^7,8  we recommend caution in interpreting data where FOXP3 gene expression is presented as the sole corroborating evidence to support discordant protein data.