Requirements for activation of human peripheral blood T cells by mouse monoclonal antibodies to CD3

Abstract

The present study was undertaken in an attempt to reconcile the conflicting results concerning the signals required for the activation of human resting T cells by antibodies to the T-cell receptor/CD3 complex ( Ti CD3 ). For this purpose we have used highly purified peripheral blood T cells, depleted of monocytes and of preactivated Ia + T cells, to the extent that they were unable to proliferate to interleukin 2 (IL-2) alone or to optimal doses of phytohemagglutinin (PHA). To further minimize the contribution of contaminating monocytes, we used the anti-CD3 mAb, Leu-4, and cells from Leu-4 nonresponder subjects, whose monocytes we show completely fail to bind the Leu-4 mAb. The parameters of T-cell activation which we measured were rises in intracellular free calcium ion [Ca 2+] i, IL-2 receptor expression IL-2 production, and cell proliferation. Our results indicate that induction of proliferation of resting T cells requires at least two signals. Signal one is best delivered by multivalent anti-CD3 mAb, such as Leu-4 mAb covalently linked to Sepharose 4B (Seph-Leu-4), or with Leu-4 mAb and antimouse IgG. These reagents crosslink the CD3 receptor complex on the T cell, and result in a rise in intracellular [Ca 2+] i, in expression of receptors for IL-2, and in proliferation upon addition of IL-2. In contrast, purified T cells exposed to soluble Leu-4 mAb do not exhibit a rise in intracellular [Ca 2+] i, do not express receptors for IL-2, and do not proliferate upon addition of IL-2, indicating that the valency of anti-CD3 mAb is critical for the delivery of the first activation signal to the T cell. The essential step of crosslinking of CD3 antigens on T cells by anti-CD3 mAb is normally mediated by monocytes which have bound anti-CD3 mAbs via their Fc receptors. Monocytes from Leu-4 nonresponder subjects, which we show fail to bind Leu-4 mAb, fail to crosslink CD3 antigens on T cells, resulting in failure of T-cell activation. The second signal needed for the proliferation of T cells whose Ti >CD3 complexes are crosslinked is IL-2. IL-2 production by such T cells required a monocyte delivered signal, which must be delivered to these T cells simultaneously with the crosslinking of their Ti CD3 antigens. This IL-2-inductive signal can be delivered by both Leu-4 nonresponder and Leu-4 responder monocytes, indicating that delivery of this IL-2 inductive signal is independent of anti-CD3 mAb binding by monocytes. The IL-2-inductive signal differed from IL-1 as neither purified nor recombinant IL-1 substituted for monocytes in inducing IL-2 production in T cells.