Two Functionally Distinct Subsets of Mast Cells Discriminated By IL-2–Independent CD25 Activities

Abstract

We identified two mast cell subsets characterized by the differential expression of surface CD25 (IL-2Rα) and by different abilities to produce cytokines and to proliferate, both in vitro and in vivo. CD25 can be expressed on the surface of immune cells in the absence of the other chains of the IL-2R, which are indispensable for IL-2 signaling. We show that functional differences between the two mast cell populations were dependent on CD25 itself, which directly modulated proliferation and cytokine responses. These effects were completely independent from IL-2 or the expression of the other chains of the high-affinity IL-2R, indicating an autonomous and previously unappreciated role for CD25 in regulating cell functions. Cells genetically ablated for CD25 completely recapitulated the CD25-negative phenotype and never acquired the properties characteristic of CD25-positive mast cells. Finally, adoptive transfer experiments in the mouse demonstrated a different impact of these populations in models of anaphylaxis and contact sensitivity. Our findings indicate a general role for CD25 in contexts where IL-2 signaling is not involved, and may have important implications for all mast cell-related diseases, as well as in all cell types expressing CD25 independently of its IL-2–related functions.

TET2 Regulates Mast Cell Differentiation and Proliferation through Catalytic and Non-catalytic Activities

“…First, we evaluated cell survival in response to withdrawal of IL-3, which was unaffected by Tet2 deletion (Figure S2A). Conversely, by assessing BrdU incorporation in response to IL-3 treatment (Figure S2B) (Deho' et al, 2014), we found that Tet2 −/− mast cells showed significant hyperproliferation compared to their Tet2 +/+ counterparts, while Tet2 +/− cells displayed an intermediate phenotype, pointing again toward a gene-dosage effect of Tet2 in regulating mast cellproliferation (Figures 1D and 1E). …”

“…Cell differentiation was monitored by surface staining of relevant mast cell and myeloid markers (Figure 1A). In these culture conditions, more than 90% of WT ( Tet2 +/+ ) cells differentiated to mast cells (Kit + FcεRIα + ) by the end of the third week of culture (Figures 1A–1C) (Deho' et al, 2014; Mayoral et al, 2011; Mayoral and Monticelli, 2010; Rusca et al, 2012); however, Tet2 −/− cultures showed reduced percentages of mast cells, as well as aberrant expression of markers for other myeloid cells (Mac1 and Ly6G) (Figure 1A), suggesting delayed or altered differentiation. Cultures from heterozygous littermates showed an intermediate phenotype, pointing toward a gene-dosage effect of Tet2 expression.…”

“…Mast cells were differentiated from the bone marrow of Tet2 −/− mice (Ko et al, 2011) and their Tet2 +/+ and Tet2 +/− littermates in the presence of IL-3 as described (Deho' et al, 2014; Mayoral and Monticelli, 2010). Mice were 8-12 weeks of age and both male and female, although for the majority of the experiments male cells were used.…”

“…Intracellular cytokine staining was performed exactly as described (Deho' et al, 2014; Rusca et al, 2012). Briefly, cells were stimulated with 1 μg/ml IgE-anti-DNP and 0.2 μg/ml HSA-DNP (both from Sigma) for 3 hr, with the addition of 10 μg/ml brefeldin A in the last 2 hr of stimulation.…”

“…The murine Cebpa lentiviral expression vector was purchased from ABM (pLentiGIII-EF1α; NCBI: NM_007678) and either used as is or moved into the pScalps lentiviral vector (Deho' et al, 2014). The murine Cebpe gene (NCBI: NM_207131) was cloned by PCR from murine LSK (Lin − Sca1 + Kit + ) hematopoietic progenitors isolated from the bone marrow.…”