CXCR4-Dependent Repulsion of Mature Single-Positive CD4 Cells Mediates Emigration from Thymus.

Developing thymocytes undergo maturation during their passage through the cortex to the medulla of the thymus before emigrating to peripheral lymphoid organs. Chemokines control T cell migration within and away from the thymus. We have previously shown that the chemokine SDF-1, produced by thymic stroma, repels mature thymocytes away from thymus via a CXCR4 mediated mechanism. We proposed that CXCR4 plays a specific role in thymic emigration. We evaluated the emigration of mature thymocytes away from thymus in a modified fetal thymic organ culture (FTOC) system in fetal thymi from CXCR4+/− (Control) and CXCR4 −/− (KO) mice at day 15.5. Fetal thymic emigrants were collected using a transwell system with a polycarbonate membrane (3 μm pore size). The immunophenotype of thymic emigrants was distinct from that of thymocytes resident in the thymus in both control and CXCR4-KO thymi. From day 9 of culture onwards, only mature SP cells were able to move away from thymus into the lower chamber of the transwell. Only SP CD4 thymocytes expressing CD62L^high were found among emigrants, whereas two distinct populations of SP CD8 cells expressing CD62L^high and CD62L^low were present in the emigrant fraction. Incubation of FTOC with pertussis toxin significantly inhibited emigration of mature SP thymocytes out of the thymus in control CXCR4 mice (p=0.002). The total number of emigrants collected from CXCR4-KO thymi was always significantly reduced compared to emigrants from control thymi. In addition, the total number of SP CD4 emigrants from CXCR4-KO thymi were strikingly reduced in comparison to control SP CD4 emigrants (p=0.035). There was no statistically significant difference in the maturation of intrathymic thymocytes from CXCR4-KO as compared to control FTOC. Analysis of CD62L expression in CXCR4-KO thymocytes showed retention of SP CD4 cells in the thymus with the immunophenotypic characteristic normally associated with recent thymic emigrants (p<0.05). Treatment of FTOC with the specific CXCR4 antagonist, AMD3100, significantly increased the total number of SP CD4 cells retained in the thymus compared to untreated FTOC (p=0.002). Treatment with AMD3100 significantly reduced the absolute number of emigrants compared to untreated FTOC (p=0.015) and again SP CD4 cells were most affected in their ability to migrate away from the thymus. Treatment of FTOC with sphingosine-1-phosphate (S1P) resulted in an increase in the emigration of SP CD4 cells from CXCR4-KO thymi (p=0.02). In conclusion these data support the view that CXCR4-mediated repulsion of mature thymocytes away from the medullary chemokine SDF-1, termed fugetaxis, is a key mechanism controlling T cell egress from the thymus. These data support the existence of a CXCR4 dependent mechanism governing thymic emigration which may itself have S1P dependent and/or independent components.