Effect of in vitro stimulation.

In vitro stimulation leads to an increased proportion of CD4^lo cells and to decreased expression of CD4. Peripheral blood lymphocytes of 4 healthy individuals (donor a, 34 years; donor b, 39 years; donor c, 47 years; and donor d, 60 years old) were stimulated in vitro with immobilized anti-CD3 (OKT-3) as described in “Materials and methods”; stained with anti-CD4, anti-CD3, and anti-CD28 antibodies; and analyzed by flow cytometry. (A) Lymphocytes were stimulated for 48 hours, and the proportion of CD4^lo cells was compared in the control and OKT-3–treated CD3⁺CD4⁺ populations. (B) The number of CD4 molecules was determined by means of Quantibrite beads on CD4^lo (black symbols) and CD4⁺ (open symbols) T cells before (control) and after 48 hours' stimulation with anti-CD3 (OKT-3). (C) (D) In a separate experiment, peripheral blood lymphocytes of a healthy 47-year-old man were stimulated with OKT-3 for 8 days, stained with anti-CD4 and anti-CD28, and analyzed. The proportion of the CD4^lo cells showing simultaneous decrease of CD28 expression was greatly increased compared with the CD4⁺population (compare Figure 5A).