CD69 is an activation marker for neonatal lymphocytes

The CD69 antigen is an early activation marker that may be induced on the suriaca of lymphocytes and granulocytes. It is linked to the activation of protein kinase C but its natural ligand is unknown. We assessed the ability of mononuclear cells (MNC) from normal newborns (cord blood) to express CD69 and the effects of adherence and cytokines. The percentage of fresh neonatal MNC staining for CD69 was <3%. The MNC were allowed to adhere to culture dishes for 24 hr. in the presence of interleukin-2 (IL-2; 1000 u/ml) and then non-adherent cells were removed; this method had been previously used to enrich for natural killer (NK) cells. Other MNC were set up in suspension culture (10⁶/ml). The cells were cultured for 7 days. Flow cytometry results are shown below. TABLE: EFFECTS OF ADHERENCE AND CYTOKINES ON CD69 AND CD58 Conditions Markers Cytokines Added IL-21100 u/ml) IL-2(10u/ml) IL-12 (1 ng/mg) IL-2 (100 u/ml) + IL-12 Suspension(n=7) CD69 16.3±3.1 14.9±5.5 12.8±4.0 14.1±11.7 CD 56 14.6±4.9 8.5±7.5 8.5±1.8 9.2±3.8 Adherent CD(n=4) 69 71.9±18.1 60.6±9.4 47.7±4.6 38.9±13.7 CD56 53.3±9.6 48.4±7.4 48.8±2.2 20.5±10.1 As shown above, the adherence step greatly increased (approximately 4-fold) the percentage of cells staining with CD69. IL-12, which stimulates NK cell function, tended to decrease the percentage of CD69+ cells, although it did increase CD69 expression in over-night culture (not shown). Also shown is data on CD66, an NK marker, and the percentage of CD66 cells was again much higher (3 to 4 fold) in the adherent population. When these adherent cells were tested in NK cytotoxicity assays using K562 target cells, the greatest NK acitvity occurred in those populations with the greatest percentage of CD69+ cells. Thus, newborn MNC can be induced to express CD69 with IL-2 stimulation and adherence, and greater expression appears to correlate with increased NK activity.