Surface marker, proliferation, and intracellular signal transduction analyses of human CRLF2_r and CRLF2_wt leukemias. (A) Flow cytometry analysis of primary human CRLF2_r and CRLF2_wt ALL samples (N = 70; 4 representative samples shown) for the TSLPR and the IL-7Rα chain (CD127). CD10⁺CD19⁺ ALL cells with CRLF2 alterations demonstrated increased surface staining of the TSLPR regardless of the mechanism of CRLF2 rearrangement. No increased TSLPR staining was observed for leukemias without CRLF2 alterations. CD127 staining was similar in CRLF2_r and CRLF2_wt ALL cells. Shaded histograms indicate isotype control, and open histograms, stained cells. (B) Surface marker analysis of human B-precursor CRLF2_r JAK_mut ALL cell line MUTZ5 and CRLF2_wt JAK_wt cell line RCH-ACV. MUTZ5 cells stained uniformly for TSLPR, but were intermediate for CD132 (the common γ chain). RCH-ACV demonstrated bright CD127, absent TSLPR, and intermediate-to-bright CD132, suggesting the presence of the normal heterodimeric IL-7R. (C) Incubation of cells with human TSLP for 72 hours resulted in dose-dependent proliferation of MUTZ5, but not of RCH-ACV, measured by MTT assay. Red indicates 1 ng/mL; yellow, 10 ng/mL; green, 25 ng/mL; and blue, 100 ng/mL of TSLP. Data are normalized to the medium-only condition at 72 hours for each cell line. (D) Primary CRLF2_r ALL cells were exposed to increasing concentrations of human TSLP for 30 minutes, then fixed and permeabilized for flow cytometric analysis of pSTAT5. Near-maximal STAT5 phosphorylation was elicited with 25 ng/mL of TSLP, the concentration used in the in vitro studies.