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99 HIV-1 Tat Protein Induces Polyclonal Stimulation of Human T Cells
M. Tuzova*, A. Nath, and J. Woodward
Univ. of Kentucky, Lexington, USA
Background: Tat protein of human immunodeficiency virus type-1 (HIV-1) exerts a pleiotropic effect on the survival and proliferation of different cell types in vivo and in vitro. Many of these effects are due to the action of extracellular Tat, suggesting that this form of Tat may play an important role in AIDS pathogenesis. Extracellular Tat has been shown to activate human T cells, but the literature are conflicting on whether Tat alone can induce polyclonal T-cell stimulation and the extent to which these effects occur at physiological concentrations.
Methods: T lymphocytes were purified from heparinized blood drawn from 20 healthy donors by E-rosetteing and isolation on density gradients. Different forms of Tat proteins were purified from E. coli that express Tat fused to a cleavable biotinylation site by avidin affinity chromatography. Proliferation assays were performed with anti-CD3, anti-CD28, and different concentrations of Tat protein.
Results: We observed strong T-cell proliferation in response to Tat at concentrations in the nanomolar to picomolar range. The response was comparable to anti-CD3-stimulation of T cells in vitro. Not all donors responded to Tat: 11 donors showed a high response, 3 donors showed a low response, and 6 donors showed no response. The high responders responded to Tat 1-72, Tat 1-86, a His-13 to Arg mutant, and a delta 31-61 deletion mutant. No synergy or antagonism was observed when Tat was combined with anti-CD3. Interestingly, 2 of the nonresponding donors showed a high response when Tat was combined with anti-CD28.
Conclusions: Our data show, for the first time, that highly purified Tat induces polyclonal stimulation of purified resting human T cells. The fact that this stimulation occurs at nanomolar to picomolar concentrations suggests that this response is physiologically relevant. The fact that Tat does not synergize with anti-CD3, but does synergize with anti-CD28 is consistent with the possibility that Tat is acting through the T-cell receptor. Finally, the use of Tat mutants demonstrates that this activity is not dependent on exon 2, the trans-activation domain, or the intracellular targeting sequence. Thus, Tat may exert its immunosuppressive effects through polyclonal T-cell stimulation, in a manner analogous to bacterial and viral superantigens.
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