Introduction.  T regulatory cells are a major mediator of alloantigen specific transplant tolerance.  Mediators of alloantigen specific transplant tolerance can be identified by their CD4⁺CD25⁺ phenotype shared with naïve non antigen specific Treg, and markers of T cells activation such as low molecular weight isoforms of CD45 and Class II MHC expression.  An ability to monitor activated Treg may be of use in protocols to induce transplant tolerance.           Miyara et al, identified that CD4⁺CD25⁺CD127^loFoxp3⁺T cells were distributed in three subpopulations, based on expression of CD45RA and high or low expression of CD25 or Foxp3.  Population I (Pop I), which expresses CD45RA and normal levels of Foxp3 or CD25, identifies naïve/resting Treg.   Pop II is CD45RA^‑ and CD25^hi/ Foxp3^hi and identifies highly activated Treg.    Pop III, which is CD45RA^- and have normal Foxp3 and CD25 expression identifies a mixed population of activated Treg and T effector cells.     CCR7 is expressed by Pop I whereas CXCR3 is expressed by Pop II and III. In this study, we examined in PBMC of healthy volunteers (HV) the proportion of Treg in the three populations and the stability of this frequency distribution over time.  

Methods.  44 HV (17 male and 27 female) had PBMC analysed for Treg subsets with a total of 110 individual estimates of Treg in HV.  10 HV had repeat studies over three years.  PBMC were stained with panels of monoclonal antibodies to CD4, CD25, CD127, CD45RA, Foxp3, CXCR3 and CCR7. Data was acquired on BD FACS Canto II using FACS DIVA (V8.0) and analysed using FlowJo.  CD4 and Treg phenotypes and subpopulations were analysed after FSC vs SSC and doublets exclusion.  

Results.   The CD4⁺CD25⁺CD127^lo and CD4⁺CD25⁺CD127^loFoxp3⁺ were nearly always <10% of CD4⁺T cells.  A significant minority of HV had >10% of CD4⁺ cells stained with Foxp3.  The Proportion of cells in Population I, II and III was quite similar, in samples taken from the same HV at intervals for 4 years. The proportion of CD4⁺CD25⁺CD127^lo cells and of CD4⁺CD25⁺CD127^loFoxp3⁺ cells was very stable over the three years of the study.   The proportion of CD4⁺ cells in lymphocytes had variation.   However, the proportion of Pop I, II, related to either CD4⁺T cells or CD4⁺Foxp3⁺CD127^loTreg was very stable over time, as were Pop IV and V in CD4.   The frequency of Treg in Population III was more variable. The proportion of cells in all five Population was similar in males and females.  With age, the proportion of CD4⁺CD25⁺CD127⁺Foxp3⁺T cells overall declined due to a fall in Pop I, whereas Pop II and Pop III had an increase (p<0.0001 in linear regression). Pop I and V expressed mainly CCR7 whereas Pop II, II and IV expressed CXCR3.

Conclusions.  These findings suggest in healthy individuals the proportion of Treg in various states of activation is stable.  Establishment of normal ranges, with age variations may allow detection of changes in diseases or in transplant tolerance.