Recent studies have highlighted an immunogenic role for Galectin-10 (Gal10) in type 2 5 inflammation1, 2. Activated eosinophils release Gal10, which can auto-crystallize to form 6 Charcot-Leyden Crystals (CLCs) 2, 3. Importantly, the dissolution of CLCs by crystal-dissolving 7 antibodies may provide a novel therapeutic target for the treatment of severe asthma and 8 eosinophil-mediated inflammation1, 2. Concomitantly, FOXP3+ regulatory T cells (Tregs) play a 9 crucial role in controlling allergic diseases4. Interestingly, Gal10 has been reported as being 10 significantly overexpressed in Tregs and actively involved in their function5. Given the potential 11 therapeutic application of CLC-dissolving antibodies in eosinophilic disorders and the essential 12 immunoregulatory function of Tregs, we have investigated the role of Gal10 and the effects of 13 Gal10 crystals and an anti-Gal10 antibody capable of CLC dissolution in human T cell subsets 14 in detail. 15

A previous study reported almost exclusive Gal10 expression in CD25+-enriched Tregs 16 compared to CD4+CD25-conventional T cells (Tconvs) 5. Although no evidence of Gal10 17 secretion or crystal formation was observed, Gal10 downregulation in Tregs enhanced IFNγ 18 and TNF-α secretion and inhibited their suppressive capacity5. We have re-examined Gal10 19 expression in highly pure FACS-sorted CD4+CD25-CD127+Tconvs and CD4+CD25+CD127-20 Tregs (FigureS1A). However, we observed only low Gal10 mRNA expression in both T cell 21 subsets using two sets of previously described primers5, 6 (Figure1A). Following in vitro 22 stimulation, Gal10 expression only increased in Tconvs but not Tregs (Figure1A). To 23 independently confirm our data, we reanalyzed published transcriptomic datasets of Tregs and 24 observed again in contrast to a previous study5 lower Gal10 than FOXP3 mRNA, and 25 comparable low Gal10 expression between different Tconv and Treg subsets (FigureS1B-F). 26 Moreover, epigenetic analysis of the Gal10 locus in Tconvs and Tregs predicted 27 quiescent/weak expression of this gene, while expectedly weak or strong FOXP3 expression 28 was predicted in Tconvs or Tregs respectively (FigureS1G-H). 29