Selective Reduction of Post-Selection CD8 Thymocyte Proliferation in IL-15Rα Deficient Mice

Peripheral CD8+ T cells are defective in both IL-15 and IL-15Rα knock-out (KO) mice; however, whether IL-15/IL-15Rα deficiency has a similar effect on CD8 single-positive (SP) thymocytes remains unclear. In this study, we investigated whether the absence of IL-15 transpresentation in IL-15Rα KO mice results in a defect in thymic CD8 single positive (SP) TCRhi thymocytes. Comparison of CD8SP TCRhi thymocytes from IL-15Rα KO mice with their wild type (WT) counterparts by flow cytometry showed a significant reduction in the percentage of CD69− CD8SP TCRhi thymocytes, which represent thymic premigrants. In addition, analysis of in vivo 5-bromo-2-deoxyuridine (BrdU) incorporation demonstrated that premigrant expansion of CD8SP TCRhi thymocytes was reduced in IL-15Rα KO mice. The presence of IL-15 transpresentation-dependent expansion in CD8SP TCRhi thymocytes was assessed by culturing total thymocytes in IL-15Rα-Fc fusion protein-pre-bound plates that were pre-incubated with IL-15 to mimic IL-15 transpresentation in vitro. The results demonstrated that CD8SP thymocytes selectively outgrew other thymic subsets. The contribution of the newly divided CD8SP thymocytes to the peripheral CD8+ T cell pool was examined using double labeling with intrathymically injected FITC and intravenously injected BrdU. A marked decrease in FITC+ BrdU+ CD8+ T cells was observed in the IL-15Rα KO lymph nodes. Through these experiments, we identified an IL-15 transpresentation-dependent proliferation process selective for the mature CD8SP premigrant subpopulation. Importantly, this process may contribute to the maintenance of the normal peripheral CD8+ T cell pool

Estimating average cellular turnover from 5-bromo-2'-deoxyuridine (BrdU) measurements.

Cellular turnover rates in the immune system can be determined by labelling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) or deuterated glucose ((2)H-glucose). To estimate the turnover rate from such measurements one has to fit a particular mathematical model to the data. The biological assumptions underlying various models developed for this purpose are controversial. Here, we fit a series of different models to BrdU data on CD4(+) T cells from SIV(-) and SIV(+) rhesus macaques. We first show that the parameter estimates obtained using these models depend strongly on the details of the model. To resolve this lack of generality we introduce a new parameter for each model, the 'average turnover rate', defined as the cellular death rate averaged over all subpopulations in the model. We show that very different models yield similar estimates of the average turnover rate, i.e. ca. 1% day(-1) in uninfected monkeys and ca. 2% day(-1) in SIV-infected monkeys. Thus, we show that one can use BrdU data from a possibly heterogeneous population of cells to estimate the average turnover rate of that population in a robust manner