Antigen-independent pathways of T-cell activation are functional in human immature thymocytes

The signal requirements for proliferation of CD1⁺CD3^- immature thymocytes have been studied in order to define whether this immature cell population could function despite the lack of the CD3/T-cell receptor complex. We found that CD1⁺CD3^- cells proliferate upon stimulation with anti-CD28 monoclonal antibody as well as with a pair of anti-CD2 monoclonal antibodies in the presence of low doses (0.5 ng/ml) of phorbol-13-myristate-12-acetate and/or recombinant interleukin-2. A minor fraction of CD3⁺ cells (15%-20%) was also present in the proliferating cell population originating from CD1⁺CD3^- thymocytes stimulated with phorbol-13-myristate-12-acetate and recombinant interleukin-2, either in the presence or in the absence of specific monoclonal antibodies. We further observed that the anti-CD3 monoclonal antibody did not induce the proliferation of CD1⁺CD3^- cells, as expected, and efficiently triggered unfractionated or CD1⁺CD3⁺ thymocytes only if exogeneous recombinant interleukin-2 was provided. Unexpectedly, we noted that highly purified (>99%), CD1⁺CD3^- immature thymocytes could mobilize calcium via CD3, besides CD2 and CD28 surface molecules, suggesting that a minor undetectable fraction (+ cells was still present in the purified CD3^- population. Nevertheless, the preferential expansion of CD3^-CD8⁺ cells (about one-third of proliferating cells) after triggering via CD28, and to a lesser extent via CD2, support the notion that the alternative pathways of T-cell activation are actually functional in CD1⁺CD3^- immature thymocytes.