Lack of Foxp3+ macrophages in both untreated and B16 melanoma-bearing mice.

Foxp3 Tregs are essential for maintaining immune tolerance in mice and men. Except for expression in a minor population of CD8 T cells,1 Foxp3 is currently believed to be restricted to CD4 Tregs in mice, because widespread nonhematopoietic Foxp3 expression and its putative implication in tolerance have been refuted.2,3 Thus, the employment of Foxp3 reporter mice, including Foxp3DTR-eGFP (DEREG) mice,4 has been valuable for the investigation of Foxp3 Treg biology. Recently, we demonstrated that the depletion of Foxp3 Tregs unleashes potent therapeutic tumor-specific immunity,5,6 unlike less selective agents, eg, targeting CD25. The recent postulation of immunoregulatory Foxp3 macrophages7 implies that macrophage deletion in DEREG mice could contribute to the striking effects of Foxp3 cell ablation as cancer immunotherapy. We thus specifically investigated this issue. Anti– Foxp3-PE stainings of WT spleens revealed a CD11b Foxp3 Treg population as expected, whereas the CD11b fraction contained a clearly weaker PE signal (Figure 1A). The latter was identified as autofluorescence since it persisted in isotype stainings (Figure 1A) or unstained samples (not shown), in contrast to CD11b Foxp3 Tregs. To further assess if the CD11b PElow population actively transcribes the foxp3 locus, we analyzed spleens of DT-treated DEREG mice. Strikingly, CD11b PElow cells persisted on DT administration, whereas CD11b Foxp3 Tregs were depleted (Figure 1A), suggesting that the autofluorescent CD11b population does not express Foxp3DTR-eGFP. Similar results were obtained with BM (not shown). In line with these