Thymocyte differentiation from lentivirus-marked CD34(+) cells in infant and adult human thymus

Changes in thymic function and immune system homeostasis associated with HIV infection or chemotherapy have significant effects on the ability of patients to maintain a complete T cell receptor repertoire. Therefore, the development of in vitro systems to evaluate thymic function in children and adults may aid in the understanding of thymopoiesis and the development of new therapies to improve thymic output. Here we use a lentivirus-based gene transfer system to mark CD34(+) cells with EGFP and follow their differentiation into CD4(+) and CD8(+) single positive thymocytes in human thymic organ cultures. Lentivirus-marked cells entered the thymus and were detected in both the cortex and medulla. Pretreatment of the thymus with 2-deoxyguanosine depleted resident thymocytes and significantly increased the percentage of EGFP(+) thymocytes. High frequency gene transfer into CD34(+) cells and maintained expression throughout differentiation allows for the in vitro assessment of thymic function. In thymuses ranging in age from fetal to adult we observed EGFP(+) thymocytes at all stages of development suggesting that thymuses of all ages are capable of accepting new T cell progenitors and contributing to the maintenance of T cell homeostasis.     

Deep sequencing of the TCR‐β repertoire of human forkhead box protein 3 (FoxP3)+ and FoxP3– T cells suggests that they are completely distinct and non‐overlapping

Maintenance of peripheral tolerance requires a balance between autoreactive conventional T cells (T_conv) and thymically derived forkhead box protein 3 (FoxP3)⁺ regulatory T cells (tT_regs). Considerable controversy exists regarding the similarities/differences in T cell receptor (TCR) repertoires expressed by T_conv and tT_regs. We generated highly purified populations of human adult and cord blood T_conv and tT_regs based on the differential expression of CD25 and CD127. The purity of the sorted populations was validated by intracellular staining for FoxP3 and Helios. We also purified an overlap group of CD4 T cells from adult donors to ensure that considerable numbers of shared clonotypes could be detected when present. We used deep sequencing of entire TCR‐β CDR3 sequences to analyse the TCR repertoire of T_conv and tT_regs. Our studies suggest that both neonatal and adult human T_conv and tT_reg cells are, in fact, entirely distinct CD4 T cell lineages.