Frequency and stability of populations of CD4+ and CD4+CD25+Foxp3+CD127lo Treg in healthy adults defined by cytometry using monoclonal antibodies to T cell associated molecules.

Monitoring subpopulations of CD4⁺ T cells in blood, especially regulatory CD4⁺CD25⁺Foxp3⁺CD127^loT cells, has the potential to identify tolerance to transplants and defects that cause autoimmunity. CD45RA is expressed by naïve/resting CD4⁺, not by activated cells. Staining CD45RA with CD25 or Foxp3 identifies five populations of CD4⁺ T cells, three Treg, and two CD4⁺Foxp3^-T cells. CD25 is induced on activation of effector cells and is constitutively expressed by Treg. Examining Foxp3⁺ cells in CD4⁺CD25⁺CD127^lo, identified three Treg populations. It is not known how stable these populations of CD4⁺T cells are within individuals and between individuals. Repeated estimations over 3 years in 10 HV showed the proportion of cells in the three Treg populations was stable, whereas the two Foxp3^- populations varied. In a larger cohort of 110 samples, females had higher numbers of CD4⁺ cells than males. As a percentage of lymphocytes, there was no sex difference in the proportion of cells in the five populations. With age, there were fewer total Treg, with fewer resting Treg but an increase in activated Treg. Activation of both naïve CD4⁺ T cells and Treg induces expression of chemokine receptors associated with Th1, Th17, and Th2 responses that promote their infiltration into sites of inflammation. Activated Treg expressed CCR4 and, in addition, expressed CXCR3 (Th1), CCR6 (Th17), or neither CXCR3 nor CCR6 (Th2). Some Treg expressed both CCR6 and CXCR3. HLA-DR and CD39 were expressed by activated Treg, and many cells expressed both. There was low PD-1 expression. The stability of the major Treg populations suggested it could be feasible to establish normal ranges for the three Treg populations. Staining for chemokine receptors and Treg effector molecules in activated Treg populations may detect changes in immune homeostasis and tolerance.