Background:  Interleukin-7 (IL-7) signaling is important for CD8 T cell homeostasis and function and we have previously shown decreased expression of the IL-7R alpha-chain (CD127) on CD8 T cells in HIV⁺ patients. Suppression of CD127 is mediated by both the HIV tat protein and IL-7, both of which are elevated during HIV infection. We show here by flow cytometry, Western blot analysis, and confocal microscopy that IL-7 induces the rapid internalization of CD127 by endocytosis and subsequent degradation by the proteasome.

Methods:  CD8 T cells from healthy donors were incubated with IL-7 at concentrations ranging from 10-10 000 pg/mL. Surface CD127 expression was determined by flow cytometry and total protein was determined by Western blot. Intracellular protein expression was visualized by confocal microscopy.

Results:  IL-7 decreases surface CD127 expression on CD8 T cells in a dose- and time-dependent manner with initial suppression as early as 20 minutes and maximal suppression at 12 hours. This decrease at the cell surface is followed by loss of total CD127 protein within the cell as demonstrated by Western blot. IL-7 induces internalization of surface CD127 through endocytic vesicles. The endocytosis inhibitors Filipin, and Dynasore both prevent IL-7 induced down-regulation of CD127 at the cell membrane. Consistent with this we have shown by confocal microscopy that in resting CD8 T cells CD127 is distributed evenly throughout the cell. After treatment with IL-7, CD127 forms multiple intracellular punctae with increased co-localization with clathrin by 5 minutes followed by co-localization with the early endosomal marker EEA1 after 30 minutes. By 2 hours CD127 staining associates with the late endosome marker RAB7 and the proteasomal 20S subunit, while at the same time showing decreased co-localization with the lysosomal marker LAMP1. Interestingly, the proteasome inhibitors lactacystin and MG132 both blocked IL-7’s ability to down-regulate CD127.

Conclusions:  IL-7, a potent immunomodulatory cytokine, is currently being investigated as a potential therapy and vaccine adjuvant in the treatment of HIV infection. In order to maximize these therapeutic strategies, we must first understand how the IL-7 receptor is regulated. We show here that IL-7 down-regulates CD127 protein in CD8 T cells. IL-7 binding to its receptor induces aggregation of CD127 into lipid rafts, internalization through endosomes, and ultimately degradation via the proteasome.