Adoptively transferred p14 CD8+ T cells coexpressed CD44, PD-1 and IL-7Rα as analyzed by FACS analysis of blood (Fig. 2G, Supporting Information Fig. 2C) and spleens (data not shown) 5 days after transfer. Thus, CML-specific CTL display an activated phenotype but retain IL-7Rα Afatinib purchase expression. The fact that specific CTL downregulate IL-7Rα expression in the presence of a chronic infection but maintain IL-7Rα expression in the presence of CML expressing the same viral antigen was surprising and led

to the question if IL-7 production is increased in CML mice. To analyze this, we compared IL-7 expression in mRNA isolated from spleen of CML and naïve C57BL/6 mice by RT-PCR. The thymus as organ with documented high IL-7 production served as a positive control. IL-7 mRNA was detectable in the spleen of CML and of naïve C57BL/6 mice (Fig. 3A and Supporting Information Fig. 3). Next, we analyzed whether IL-7 mRNA is detectable in CML granulocytes and in control granulocytes. We therefore quantitatively compared IL-7 mRNA production of sorted GFP+ granulocytes from CML mice with sorted granulocytes from C57BL/6 mice.

Surprisingly, IL-7 mRNA was detectable in both malignant and control granulocytes (Fig. 3B). Moreover, this experiment revealed that IL-7 mRNA was not differently expressed in malignant and in normal granulocytes. However, the total number of granulocytes in the spleen of mice with CML is three to four-fold higher than that found in C57BL/6 control mice (Fig. 3C). These findings were confirmed by quantification of IL-7 protein levels per Metformin order milligram spleen of naïve C57BL/6 mice and CML mice (Fig. 3D). Furthermore, IL-7 was detectable by intracellular staining of brefeldin-treated malignant (GFP+) and normal (GFP−) granulocytes but not in granulocytes from IL-7-deficient mice (MFI increase of IL-7 in Cyclooxygenase (COX) GFP− granulocytes (12.4±2.9%) and GFP+ granulocytes (11.4±2.9%)

(Fig. 3E and F)). Taken together, the malignant granulocytes produce IL-7 and are increased in numbers in secondary lymphoid organs such as the spleen. To study the role of IL-7 produced by leukemic cells in more detail, H8×IL-7-deficient mice were used as bone marrow donors (H8×IL-7−/−-CML mice) to establish CML disease in C57BL/6 recipients. In this experiment, the leukemic cells will not produce IL-7. However, stromal and epithelial cells of the recipient mouse are capable of IL-7 secretion. Purified p14 CD8+ T cells (CD45.1+CD8+Vα2+) were adoptively transferred to H8×IL-7−/−-CML mice, H8-CML and naïve C57BL/6 mice. P14 CD8+ T cells expanded similarly in H8×IL-7−/−-CML mice and in H8-CML mice (Fig. 4A). However, significantly more p14 CTL survived long term in H8-CML mice than H8×IL-7−/−-CML mice (analyzed in blood: H8-CML: 8.2±3.7%; H8×IL-7−/−-CML: 1.2±0.6%; p=0.04).