Pediatric Thymic Development of T Cells and Tregs

Abstract

RationaleThymic development of T cells and Tregs has not been well studied in human when compared to murine. It is not fully known whether markers seen in peripheral Tregs are expressed during thymic development and the association of Helios, BCL2 and BCL10 in survival.MethodsFresh thymi obtained from cardiac surgery of pediatric patients (n=66) from 1 week to 14 years old with congenital heart defects were processed into cell suspension for FACS analysis. Patients with syndromes associated with immunodeficiency were excluded. Intracellular transcription factor staining was performed with eBioscience fix/perm buffers and protocol.ResultsThere was a major change in the frequencies of CD4/CD8 double negative (DN), double positive (DP) and single positive (SP) at <2weeks (n=18) vs. >2weeks (n=48) old. Foxp3-: DN* (5 vs. 2.4%), DP* (34 vs. 73%), CD4SP* (45 vs. 16%) and CD8SP* (13 vs 7%). Foxp3+: DN (1.4 vs. 1.4%), DP* (2.6 vs 0.8%), CD4SP (12 vs. 11%) and CD8SP (2.7 vs. 2.7%). >90% of Tregs were Helios+ with the greatest expression at the DP stage and unchanged with age. High expression of BCL2 and BCL10 was seen in Foxp3+DP, Foxp3+CD4SP, Foxp3-CD4SP and CD8SP, while Foxp3-DP had low expression. Tregs expressed CD39, CD134, CD137, CD278, CD279, CD25, CD152 and CD127 at both DP and SP stage. *p<0.001.ConclusionsHelios, BCL2 and BCL10 are upregulated at the DP stage for Tregs, suggesting a survival and selection advantage. Early in the DP stage, Tregs expressed phenotypic markers. This study provides more insights into thymic development in human. RationaleThymic development of T cells and Tregs has not been well studied in human when compared to murine. It is not fully known whether markers seen in peripheral Tregs are expressed during thymic development and the association of Helios, BCL2 and BCL10 in survival. Thymic development of T cells and Tregs has not been well studied in human when compared to murine. It is not fully known whether markers seen in peripheral Tregs are expressed during thymic development and the association of Helios, BCL2 and BCL10 in survival. MethodsFresh thymi obtained from cardiac surgery of pediatric patients (n=66) from 1 week to 14 years old with congenital heart defects were processed into cell suspension for FACS analysis. Patients with syndromes associated with immunodeficiency were excluded. Intracellular transcription factor staining was performed with eBioscience fix/perm buffers and protocol. Fresh thymi obtained from cardiac surgery of pediatric patients (n=66) from 1 week to 14 years old with congenital heart defects were processed into cell suspension for FACS analysis. Patients with syndromes associated with immunodeficiency were excluded. Intracellular transcription factor staining was performed with eBioscience fix/perm buffers and protocol. ResultsThere was a major change in the frequencies of CD4/CD8 double negative (DN), double positive (DP) and single positive (SP) at <2weeks (n=18) vs. >2weeks (n=48) old. Foxp3-: DN* (5 vs. 2.4%), DP* (34 vs. 73%), CD4SP* (45 vs. 16%) and CD8SP* (13 vs 7%). Foxp3+: DN (1.4 vs. 1.4%), DP* (2.6 vs 0.8%), CD4SP (12 vs. 11%) and CD8SP (2.7 vs. 2.7%). >90% of Tregs were Helios+ with the greatest expression at the DP stage and unchanged with age. High expression of BCL2 and BCL10 was seen in Foxp3+DP, Foxp3+CD4SP, Foxp3-CD4SP and CD8SP, while Foxp3-DP had low expression. Tregs expressed CD39, CD134, CD137, CD278, CD279, CD25, CD152 and CD127 at both DP and SP stage. *p<0.001. There was a major change in the frequencies of CD4/CD8 double negative (DN), double positive (DP) and single positive (SP) at <2weeks (n=18) vs. >2weeks (n=48) old. Foxp3-: DN* (5 vs. 2.4%), DP* (34 vs. 73%), CD4SP* (45 vs. 16%) and CD8SP* (13 vs 7%). Foxp3+: DN (1.4 vs. 1.4%), DP* (2.6 vs 0.8%), CD4SP (12 vs. 11%) and CD8SP (2.7 vs. 2.7%). >90% of Tregs were Helios+ with the greatest expression at the DP stage and unchanged with age. High expression of BCL2 and BCL10 was seen in Foxp3+DP, Foxp3+CD4SP, Foxp3-CD4SP and CD8SP, while Foxp3-DP had low expression. Tregs expressed CD39, CD134, CD137, CD278, CD279, CD25, CD152 and CD127 at both DP and SP stage. *p<0.001. ConclusionsHelios, BCL2 and BCL10 are upregulated at the DP stage for Tregs, suggesting a survival and selection advantage. Early in the DP stage, Tregs expressed phenotypic markers. This study provides more insights into thymic development in human. Helios, BCL2 and BCL10 are upregulated at the DP stage for Tregs, suggesting a survival and selection advantage. Early in the DP stage, Tregs expressed phenotypic markers. This study provides more insights into thymic development in human.