Ellen A. Robey

Development of many vertebrate tissues involves long-range cell migrations. In most cases, these migrations have been inferred from analysis of single time points and the migration process has not been directly observed and quantitated in real time. In the mammalian adult thymus, immature CD4⁺CD8⁺ double-positive (DP) thymocytes are found in the outer cortex, whereas after T cell antigen receptor (TCR) repertoire selection, CD4⁺CD8^– and CD4^–CD8⁺ single-positive (SP) thymocytes are found in the central medulla. Here we have used two-photon laser-scanning microscopy and quantitative analysis of four-dimensional cell migration data to investigate the movement of thymocytes through the cortex in real time within intact thymic lobes. We show that prior to positive selection, cortical thymocytes exhibit random walk migration. In contrast, positive selection is correlated with the appearance of a thymocyte population displaying rapid, directed migration toward the medulla. These studies provide our first glimpse into the dynamics of developmentally programmed, long-range cell migration in the mammalian thymus.

The intracellular parasiteToxoplasma gondii, the causative agent of toxoplasmosis, induces a protective CD8 T-cell response in its host; however, the mechanisms by whichT. gondiiproteins are presented by the class I major histocompatibility complex remain largely unexplored.T. gondiiresides within a specialized compartment, the parasitophorous vacuole, that sequesters the parasite and its secreted proteins from the host cell cytoplasm, suggesting that an alternative cross-priming pathway might be necessary for class I presentation ofT. gondiiantigens. Here we used a strain ofT. gondiiexpressing yellow fluorescent protein and a secreted version of the model antigen ovalbumin to investigate this question. We found that presentation of ovalbumin secreted by the parasite requires the peptide transporter TAP (transporter associated with antigen processing) and occurs primarily in actively infected cells rather than bystander cells. We also found that dendritic cells are a major target ofT. gondiiinfection in vivo and account for much of the antigen-presenting activity in the spleen. Finally, we obtained evidence that Cre protein secreted byT. gondiican mediate recombination in the nucleus of the host cell. Together, these results indicate thatToxoplasmaproteins can escape from the parasitophorous vacuole into the host cytoplasm and be presented by the endogenous class I pathway, leading to direct recognition of infected cells by CD8 T cells.

Although stable repression of CD4 and CD8 genes is a central feature of T cell lineage commitment, we lack detailed information about the timing and mechanism of this repression. Stable gene repression has been linked to the position of genes within the nucleus. Therefore, information about the nuclear position of CD4 and CD8 genes during T cell development could provide insights into both the mechanism of regulation of CD4 and CD8 genes, and the process of lineage commitment. Here, we report that lineage-specific repression of CD4 and CD8 genes is associated with the repositioning of alleles close to heterochromatin. We also provide evidence that the relocalization of CD4 and CD8 genes to heterochromatin can occur as an early response to positive selection signals. We discuss our results in terms of our current knowledge of CD4 and CD8 gene regulation and CD4 versus CD8 lineage commitment.