Quantitative and functional analysis of CD69(+) NKG2D(+) T regulatory cells in healthy subjects.

T regulatory (Treg) cells have a key role in immune homeostasis and the pathogenesis of chronic inflammatory and autoimmune diseases. CD69 is an early leukocyte activation molecule that under steady state conditions is detected in a small proportion of lymphocytes in peripheral blood and lymphoid tissues. Although it has been reported that a subset of CD69(+) T cells behaves as Treg lymphocytes, the possible relationship between CD69(+) Treg cells and CD4(+)NKG2D(+) T lymphocytes, which also exert immunosuppressive activity, has not been explored. In this study, we analyzed the expression of CD69 and NKG2D by T lymphocytes from the peripheral blood of twenty-five healthy subjects by multi-parametric flow cytometry analysis, and their suppressive activity by an assay of inhibition of lymphocyte activation (CD40L expression) and proliferation (carboxyfluorescein partition assay). We found a very small percentage of CD4(+)CD69(+)NKG2D(+) T cells (median 0.002%, Q1-Q3, 0.001-0.004%), which also expressed TGF-ß (Latency Associated Peptide or LAP) and IL-10, in all samples analyzed. These cells exerted an important in vitro suppressive effect on both activation and proliferation of T effector cells. Our data suggest that at very small numbers, CD4(+)CD69(+)NKG2D(+) lymphocytes seem to exert a relevant functional immune-regulatory role in healthy subjects.

Influenza virus infection but not H1N1 influenza virus immunization is associated with changes in peripheral blood NK cell subset levels.

The innate immune system constitutes the first line of defense against viral agents, and NK cells seem to have an important protective role during the early phases of influenza virus infections. We decided to assess the levels of NK and NKT lymphocytes and the expression levels of different membrane receptors (NKp44, NKp46, NKG2A, killer cell immune-like receptor [KIR] 3DL1/DS1, KIR2DL1/DS1, and CD161) in peripheral blood samples of patients with influenza (n = 17) and healthy individuals immunized against this virus (seasonal and [H1N1]pdm2009 influenza vaccines; n = 15 and 12, respectively). Blood samples were obtained from all individuals, and NK and NKT cell subsets were analyzed by multiparametric flow cytometry. We found that the patients with severe influenza (n = 9) showed significant increases in the percentages of NKp46(+) NKp44(+) NK cells and the proportions of NK and NKT lymphocytes expressing KIR2DL1 and KIR3DL1 and reductions in the percentages of NKp46(+) NKp44(-) NK cells compared to those in the healthy controls (n = 27). In contrast, influenza immunization, against either the seasonal or the pandemic H1N1 virus, was not associated with important changes in the levels of NK and NKT lymphocytes or the expression levels of the different receptors by these cells. Our data suggest that severe influenza is associated with important and complex alterations on NK cells, which might contribute to the pathogenesis of this condition.