4). As expected, the percentage of CFSElow cells — that is those that had divided in the host

— was higher in the BM than in spleen and LNs of B6 mice (Fig. 4A). In both IL-15 KO and IL-15Rα KO mice, the percentage of CFSElow cells was low, without differences among the three organs examined (Fig. 4A). A pronounced CD127 downmodulation by donor WT CFSE+ cells was observed only in the BM of B6 mice (Fig. 4B). To investigate whether in B6 mice the lower CD127 membrane expression by BM CD44high CD8+ T cells was related with a higher fraction of proliferating cells in this organ [[10-12]], we performed a more detailed analysis on CFSElow and CFSEhigh cells (Supporting Information Fig. 2 and Fig. 4C). Within each organ, we found that CFSElow cells had a lower

signaling pathway CD127 MFI as compared with CFSEhigh cells. More importantly, within each of the two populations, BM cells had a lower CD127 membrane expression as compared with those in either spleen or LNs (Fig. 4C). Our results on genetically deficient mice show that IL-15 is required for homeostatic proliferation and CD127 downmodulation in the BM by conventional WT CD44high CD8+ T cells. Our analysis on adoptive transfers into WT mice shows that both undivided cells (CFSEhigh) and cells which had recently divided (CFSElow) Ibrutinib have a lower CD127 membrane expression in BM than in spleen and LNs. Our next question was whether low membrane CD127 expression by BM CD44high CD8+ T cells was due to decreased CD127 mRNA level [[6]]. We performed real-time PCR analysis of CD127 mRNA expression by fluorescence-activated cell sorter (FACS)-sorted highly purified CD44high CD8+ T cells from either spleen or BM

of WT mice and found that CD127 mRNA amount was lower in the BM (Fig. 5). In this group of experiments, cells from LNs were not included due to low cell yields. As a control for suppression of CD127 mRNA transcription, Idelalisib solubility dmso we incubated purified splenic CD8+ T cells with either medium or IL-15 for an overnight (Fig. 5). Real-time PCR results were in agreement with northern blot analysis on purified spleen and BM CD8+ T cells (data not shown). We were unable to perform similar analysis in IL-15 KO mice due to low cell yields (average percentages ± SD of BM TCR+CD8+ cells were 0.30 ± 0.12 in IL-15 KO and 2.59 ± 0.53 in WT, N = 5 per group, p ≤ 0.01). To directly address the molecular mechanisms regulating CD127 gene expression, we used a CD127 genetically modified mouse strain (CD127tg) generated by the Ashwell’s laboratory (National Institutes of Health, Bethesda, MD, USA) [[30]]. This strain has a CD127 transgene under the control of human CD2 promoter, leading to CD127 transgene high expression in T cells and unresponsiveness to the normal transcriptional regulation acting on the endogenous gene. We confirmed that CD127tg is a suitable tool for our experiments by showing that CD127tg CD8+ T cells are unresponsive to IL-15 effect on CD127 expression.