RESUMEN
CD27 and CD28 have emerged as indicators demarcating the transition of thymocytes through beta-selection. We found that CD28 exhibits a greater dynamic range of expression during this phase, thus it was employed to further parse the DN/CD44(-) compartment in order to assess IL-7 signaling during the beta-selection process. Plotting CD28 versus CD25 expression revealed six DN/CD44(-) populations. OP9-DL1 stromal cell co-culture was used to demonstrate a developmental linkage from DN3a (CD25(+)CD28(-/lo)) to DN3b (CD25(+)CD28(+)) to DN3c (CD25(int)CD28(+)) to DN4a (CD25(-)CD28(+)) to double positive (DP) and showed the DN4b (CD25(-)CD28(hi)) and DN4c (CD25(-)CD28(-/lo)) populations to be inefficient in producing DP cells. Using CD69 as an additional marker to further parse the DN4a population, we found the pre-DP cells to be the CD44(-)CD25(-)CD28(int)CD69(-)CD4(-/lo)CD8(-/lo) subset. Using this refined developmental scheme, IL-7R alpha expression was found to be transiently up-regulated post-beta-selection in the DN3b and DN3c subsets; however, this increase did not confer enhanced responsiveness over that observed in the DN3a population. CD28 messenger RNA expression was up-regulated in post-beta-selected cells, whereas transcripts for CD27, IL-7R alpha and Bcl-2 were lower than that observed in the DN3a population. This study refines the current thymocyte differentiation scheme to allow for more detailed evaluation of events controlling early T-cell development, specifically surrounding the beta-selection checkpoint.
Asunto(s)
Antígenos CD28/genética , Antígenos CD28/inmunología , Diferenciación Celular , Linfocitos T/citología , Timo/citología , Animales , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/genética , Diferenciación Celular/inmunología , Técnicas de Cocultivo , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Humanos , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/inmunología , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Receptores de Interleucina-7/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Timo/inmunologíaRESUMEN
Murine thymocytes down-regulate IL-7 responsiveness following beta-selection and reacquire sensitivity after positive selection. To assess the potential consequences of IL-7 signaling during this phase of development, transgenic IL-7 receptor alpha (IL-7Ralpha) mice were evaluated for IL-7 responsiveness as gauged by STAT-5 phosphorylation. Transgenic IL-7Ralpha expression increased the percentage of thymocytes responsive to IL-7 yet resulted in a decrease in total thymic cellularity. Aberrant thymocyte development in transgenic mice was first manifested by a reduction of DN3 thymocytes that correlated with lower Bcl-2 expression. Surprisingly, transgenic restoration of Bcl-2 expression did not correct thymic hypocellularity induced by IL-7Ralpha overexpression. These findings demonstrate that failure to appropriately downregulate IL-7Ralpha expression interferes with thymocyte development past the pro-T stage resulting in significantly lower levels of mature thymocytes.