Abstract
The ordered migration of immature thymocytes through thymic microenvironments generates both adaptive MHC restricted αβT-cells and innate CD1d-restricted iNKT-cells. While several chemokine receptors and ligands control multiple stages of this process, their involvement during early thymocyte development often precludes direct analysis of potential roles during later developmental stages. For example, because of early lethality of CXCR4−/− mice, and stage-specific requirements for CXCR4 in thymus colonisation and pre-TCR mediated selection, its role in thymic positive selection is unclear. Here we have examined CXCR4-CXCL12 interactions during the maturation of CD4+CD8+ thymocytes, including downstream stages of iNKT and αβT-cell development. We show CXCL12 expression is a common feature of cortical thymic epithelial cells, indicating widespread availability throughout the cortex. Moreover, CXCR4 expression by CD4+CD8+ pre-selection thymocytes is progressively downregulated following both MHC and CD1d-restricted thymic selection events. However, using CD4Cre-mediated deletion to bypass its involvement in CD4−CD8− thymocyte development, we show CXCR4 is dispensable for the maintenance and intrathymic positioning of CD4+CD8+ thymocytes, and their ability to generate mature αβT-cells and CD1d-restricted iNKT-cells. Collectively, our data define dynamic changes in CXCR4 expression as a marker for intrathymic selection events, and show its role in T-cell development is restricted to pre-CD4+CD8+ stages.
Highlights
Progenitors and contribute to their entry into the thymus[11,12,13,14,15]
During the intrathymic development of αβT-cells, DP thymocytes that reside in the cortex undergo thymic selection events determined by the specificities of the αβTCR they express
While conventional αβT-cells are selected via recognition of self-peptide/MHC molecules expressed by cortical thymic epithelial cells (cTEC), DP-DP thymocyte interactions in the thymic cortex can result in the generation of CD1d-restricted iNKT-cells
Summary
Progenitors and contribute to their entry into the thymus[11,12,13,14,15]. Interestingly, chemokine receptor expression is highly dynamic during thymocyte development, suggesting specific roles at particular developmental stages. The role of CXCR4 appears to extend beyond the positional regulation of immature thymocytes, with CXCR4 exerting a critical impact on DN thymocyte proliferation and survival during β-selection via co-stimulatory interplay with the pre-TCR28 These p56lckCreCXCR4fl/fl models preclude analysis of later developmental stages due to impaired DN to DP transition. Unlike CCR9, pre-selection DP CD69−CXCR4hi cells undergo rapid CXCR4 downregulation following positive selection initiation to generate DP CD69+CXCR4low cells, followed by CD4+ and CD8P+ SP thymocytes that lack detectable CXCR4 expression Despite this significant shift in chemokine receptor expression and the maintenance of CXCR4 expression by positive selection intermediates, we show that in both steady state and competitive conditions, conventional and CD1d-restricted αβT-cell development occurs normally in CD4CreCXCR4fl/fl mice. While differing levels of CXCR4 expression by DP thymocytes serve as an indicator of cells undergoing TCR-mediated thymic selection, we show that unlike for the DN compartment, CXCR4 is not required for survival/homeostasis and downstream development of the pre-selection DP compartment
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