American Society of Hematology

Figure 6

$Figure 6. Expression of chemokine receptors and identification of cytokine-producing cells. (A-B) Representative flow cytometric analysis of CXCR4, CCR4, CCR7, and CCR9 on fractionated OT-I DPs cultured with unpulsed (A) or EIINFEKL-pulsed (B) OP9 cells without cytokine supplementation. Black lines represent isotype controls. (C) qPCR analysis of IL-7 and IL-13 transcripts in CD45+ (black bars) and CD45− (open bars) cells sorted from the thymi of C57BL/6 mice; transcript abundance in CD45+ versus CD45− cells was different. *P < .05. (D) Flow cytometric analysis of IL-21 expression in hematopoietic (CD45+), nonhematopoietic (CD45−), and epithelial (EpCAM+CD45−) thymic cells. (E) Immunolocalization of PLZF+ cells in the thymus. Thymi from 8-week-old WT C57BL/6 mice were fixed in 10% formalin, permeabilized with Triton X-100 0.2%, and mounted in paraffin. Sections were stained with PLZF-specific primary Ab and secondary anti–rabbit Alexa Fluor 488, and then scanned using the NanoZoomer Digital Pathology system and NPD.scan Version 2.3.4 software (Hamamatsu) as described previously (40×/1.3 numerical aperture oil objective).19 Data are representative of 3 separate experiments.$

Expression of chemokine receptors and identification of cytokine-producing cells. (A-B) Representative flow cytometric analysis of CXCR4, CCR4, CCR7, and CCR9 on fractionated OT-I DPs cultured with unpulsed (A) or EIINFEKL-pulsed (B) OP9 cells without cytokine supplementation. Black lines represent isotype controls. (C) qPCR analysis of IL-7 and IL-13 transcripts in CD45⁺ (black bars) and CD45⁻ (open bars) cells sorted from the thymi of C57BL/6 mice; transcript abundance in CD45⁺ versus CD45⁻ cells was different. *P < .05. (D) Flow cytometric analysis of IL-21 expression in hematopoietic (CD45⁺), nonhematopoietic (CD45⁻), and epithelial (EpCAM⁺CD45⁻) thymic cells. (E) Immunolocalization of PLZF⁺ cells in the thymus. Thymi from 8-week-old WT C57BL/6 mice were fixed in 10% formalin, permeabilized with Triton X-100 0.2%, and mounted in paraffin. Sections were stained with PLZF-specific primary Ab and secondary anti–rabbit Alexa Fluor 488, and then scanned using the NanoZoomer Digital Pathology system and NPD.scan Version 2.3.4 software (Hamamatsu) as described previously (40×/1.3 numerical aperture oil objective).¹⁹ Data are representative of 3 separate experiments.

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