Differences in Expression Level of Helios and Neuropilin-1 Do Not Distinguish Thymus-Derived from Extrathymically-Induced CD4+Foxp3+ Regulatory T Cells

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Abstract
Helios transcription factor and semaphorin receptor Nrp-1 were originally described as constitutively expressed at high levels on CD4+Foxp3+ T regulatory cells of intrathymic origin (tTregs). On the other hand, CD4+Foxp3+ Tregs generated in the periphery (pTregs) or induced ex vivo (iTregs) were reported to express low levels of Helios and Nrp-1. Soon afterwards the reliability of Nrp-1 and Helios as markers discriminating between tTregs and pTregs was questioned and until now no consensus has been reached. Here, we used several genetically modified mouse strains that favor pTregs or tTregs formation and analyzed the TCR repertoire of these cells. We found that Tregs with variable levels of Nrp-1 and Helios were abundant in mice with compromised ability to support natural differentiation of tTregs or pTregs. We also report that TCR repertoires of Treg clones expressing high or low levels of Nrp-1 or Helios are similar and more alike repertoire of CD4+Foxp3+ than repertoire of CD4+Foxp3- thymocytes. These results show that high vs. low expression of Nrp-1 or Helios does not unequivocally identify Treg clones of thymic or peripheral origin.

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