Isopentenyl Pyrophosphate, a Mycobacterial Non-peptidic Antigen, Triggers Delayed and Highly Sustained Signaling in Human γδ T Lymphocytes without Inducing Down-modulation of T Cell Antigen Receptor

The Vγ9Vδ2 T cell subset, which represents up to 90% of the circulating γδ T cells in humans, was shown to be activated, via the T cell receptor (TcR), by non-peptidic phosphorylated small organic molecules. These phosphoantigens, which are not presented by professional antigen-presenting cells, induce production of high amounts of interferon-γ and tumor necrosis factor (TNF-α). To date, the specific signals triggered by these antigens have not been characterized. Here we analyze proximal and later intracellular signals triggered by isopentenyl pyrophosphate (IPP), a mycobacterial antigen that specifically stimulates Vγ9Vδ2 T cells, and compare these to signals induced by the non-physiological model using an anti-CD3 antibody. During antigenic stimulation we noticed that, except for the proximal p56lck signal, which is triggered early, the signals appear to be delayed and highly sustained. This delay, which likely accounts for the delay observed in TNF-α production, is discussed in terms of the ability of the antigen to cross-link and recruit transducing molecules mostly anchored to lipid rafts. Moreover, we demonstrate that, in contrast to anti-CD3 antibody, IPP does not induce down-modulation of the TcR·CD3 complex, which likely results in the highly sustained signaling and release of high levels of TNF-α. The Vγ9Vδ2 T cell subset, which represents up to 90% of the circulating γδ T cells in humans, was shown to be activated, via the T cell receptor (TcR), by non-peptidic phosphorylated small organic molecules. These phosphoantigens, which are not presented by professional antigen-presenting cells, induce production of high amounts of interferon-γ and tumor necrosis factor (TNF-α). To date, the specific signals triggered by these antigens have not been characterized. Here we analyze proximal and later intracellular signals triggered by isopentenyl pyrophosphate (IPP), a mycobacterial antigen that specifically stimulates Vγ9Vδ2 T cells, and compare these to signals induced by the non-physiological model using an anti-CD3 antibody. During antigenic stimulation we noticed that, except for the proximal p56lck signal, which is triggered early, the signals appear to be delayed and highly sustained. This delay, which likely accounts for the delay observed in TNF-α production, is discussed in terms of the ability of the antigen to cross-link and recruit transducing molecules mostly anchored to lipid rafts. Moreover, we demonstrate that, in contrast to anti-CD3 antibody, IPP does not induce down-modulation of the TcR·CD3 complex, which likely results in the highly sustained signaling and release of high levels of TNF-α. T cell receptor tumor necrosis factor α isopentenyl pyrophosphate peripheral blood mononuclear cells extracellular regulated kinase mitogen-activated protein kinase antibody monoclonal antibody fluorescein isothiocyanate recombinant interleukin-2 immunoreceptor tyrosine-based activation motif zeta-associated protein-70 phosphoinositide 3-kinase linker for activation of T cells protein kinase B polyacrylamide gel electrophoresis major histocompatibility complex fetal calf serum enzyme-linked immunosorbent assay phenylmethylsulfonyl fluoride 1,4-piperazinediethanesulfonic acid immunoprecipitated T cells expressing the γδ T cell receptor (TcR)1 represent in humans a relatively low T lymphocyte population and, particularly in peripheral blood, these cells account for only 1–5% of the circulating T cells (see Ref. 1Haas W. Pereira P. Tonegawa S. Annu. Rev. Immunol. 1993; 11: 637-685Crossref PubMed Scopus (799) Google Scholar for review). 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This aspect is of great importance for the possible pharmacological control of TNF-α release (24Badger A.M. Bradbeer J.N. Votta B. Lee J.C. Adams J.L. Griswold D.E. J. Pharmacol. Exp. Ther. 1996; 279: 1453-1461PubMed Google Scholar, 25Wadsworth S.A. Cavender D.E. Beers S.A. Lalan P. Schafer P.H. Malloy E.A. Wu W. Fahmy B. Olini G.C. Davis J.E. Pellegrino-Gensey J.L. Wachter M.P. Siekierka J.J. J. Pharmacol. Exp. Ther. 1999; 291: 680-687PubMed Google Scholar) by these cells, because an overproduction of this cytokine could result in immunopathology (26Kabelitz D. Wesch D. Hinz T. Spring. Semin. Immunopathol. 1999; 21: 55-75PubMed Google Scholar). In αβ T cells, it is well established that activation occurs as a result of multimolecular interactions between T cells and antigen-presenting cells (27Lanzavecchia A. Sallusto F. Curr. Opin. Immunol. 2000; 12: 92-98Crossref PubMed Scopus (129) Google Scholar, 28Acuto O. Cantrell D. Ann. Rev. 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Med. 1990; PubMed Scopus Google which with or is as with αβ T cells, of these which and are for the of the which the and of the (27Lanzavecchia A. Sallusto F. Curr. Opin. Immunol. 2000; 12: 92-98Crossref PubMed Scopus (129) Google Scholar, A. C. Davis M.L. Science. 1999; PubMed Scopus Google Scholar, M.L. Immunol. 2000; PubMed Scopus Google Scholar). we TNF-α release by Vγ9Vδ2 T cells with a monoclonal antibody the complex or with the mycobacterial isopentenyl pyrophosphate V. J. A. J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that TNF-α production does not as is the in αβ T cells, Moreover, we noticed that the cytokine production in Vγ9Vδ2 T cells was highly delayed the cells activated by a of anti-CD3 monoclonal antibody This delayed cytokine production could be the result of a delayed triggered signaling or of the of signaling molecules to the In the we the signals triggered in Vγ9Vδ2 T cells upon stimulation with isopentenyl a non-peptidic mycobacterial to be specifically for Vγ9Vδ2 T cells Y. Sano S. Nieves E. De Libero G. Rosa D. Modlin R.L. Brenner M.B. Bloom B.R. Morita C.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8175-8179Crossref PubMed Scopus (368) Google Scholar, 15Tanaka Y. Morita C.T. Tanaka Y. Nieves E. Brenner M.B. Bloom B.R. Nature. 1995; 375: 155-158Crossref PubMed Scopus (891) Google and these to the signals induced by anti-CD3 that the of the signals triggered upon IPP stimulation is from that of the signals induced upon anti-CD3 the signals are delayed the cells are with the non-peptidic antigen with induced upon anti-CD3 activation except for this delay be to the of Moreover, we that the majority of the in contrast to the are highly sustained and for This cell signaling observed with IPP stimulation is to the of of TcR·CD3 down-modulation that we demonstrate was from isopentenyl pyrophosphate and from and from antibody and from was from and recombinant protein from and from monoclonal antibody and or from blood mononuclear cells from T purified from by using and with T cells specifically activated in the of IPP and T purified from by using and with T cells specifically activated in the of and peripheral γδ T as and in with fetal calf serum and in for or γδ T cells in in with serum in a of cells with for with IPP or and for TNF-α using a TNF-α to the cells by or IPP for the cells in of and of by with of from cells by to and with the and with the cells p56lck or cells and in of cell or immunoprecipitated from with of of or of using protein with for and by or with and with specific kinase assay kinase p56lck kinase in of specific kinase and of p56lck was in the of of and for p56lck was by of the in of kinase assay in the of of of and kinase in of kinase in the of of recombinant of and for The by of and to and by of the phosphorylated in the results has been using a by by or IPP in for in and with of fluorescein isothiocyanate in with fetal calf in a of the cells in and by a with established that high amounts of TNF-α are by Vγ9Vδ2 T cells with anti-CD3 or with the antigen IPP V. J. A. J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in upon anti-CD3 TNF-α is with production induced by IPP only occurs This delay between the stimulation could a of signals or a in the of the triggered To these we the of the extracellular regulated kinase and mitogen-activated protein kinase in TNF-α production by Vγ9Vδ2 T cells with anti-CD3 or with IPP V. J. A. J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Vγ9Vδ2 T cells with anti-CD3 or with an of IPP a and activation of was shown in of upon anti-CD3 activation is and a high for the is to a low it In the cells are with the of to be and a which for the to it have shown that IPP cell and cytokine release in T cells not in of γδ T cells (13Constant P. Davodeau F. Peyrat M.-A. Poquet Y. Puzo G. Bonneville M. Fournié J.-J. Science. 1994; 264: 267-270Crossref PubMed Scopus (646) Google Scholar, 14Tanaka Y. Sano S. Nieves E. De Libero G. Rosa D. Modlin R.L. Brenner M.B. Bloom B.R. 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However, we could not the that, IPP was not to in γδ T cells, which do not the TcR complex, it could intracellular To we in T cells are subset of γδ T cells in shown in anti-CD3 stimulation a strong and activation in T however, IPP is not to activation in these cells or the observed delay in activation could be to a to shown in with a protein does not the activation of induced by In we that protein the in these experiments not activation of kinase in Vγ9Vδ2 T cells that have been with anti-CD3 or with that, as for to be delayed in IPP stimulation with anti-CD3 The are to that of with the activation in IPP stimulation and as a for In anti-CD3 the is and the activated for we have shown for IPP does not activation in T cells and does not activation in T cells the delay observed for and kinase which are later signals in the could be to a in the of a to TcR·CD3 that we activation of the kinase which represents of the in αβ T cell that the immunoprecipitated in the of a in anti-CD3 which to the of p56lck to an phosphorylated T. 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However, of the TcR by anti-CD3 of which to of of and It that of the signaling the TcR·CD3 proximal signaling molecules are that the small non-peptidic which likely does not have and is not presented to TcR by or is not to and cross-link with the TcR·CD3 complex and the lipid rafts. a possible the for the of a of TcR·CD3 to with the anchored transducing molecules is that in anti-CD3 to a delay in cell signaling and TNF-α the results we are in results with purified Vγ9Vδ2 T cells, and it be that in vivo the antigens could be presented by cell cell molecules not to an the of the triggered signals could be To this we TNF-α production by Vγ9Vδ2 T cells by IPP in the of This was to IPP to bind to cell molecules in possible to γδ not in the or in the amounts of TNF-α upon stimulation with IPP or with IPP in the of not Moreover, we that non-peptidic antigens are the of do not as a antigen and could TcR·CD3 in this the of the triggered signals could be To this we the of TNF-α production by Vγ9Vδ2 T cells induced by a non-peptidic antigen as IPP and by a to as a a of which we have shown a non-peptidic antigen that is to T cells F. J. A. F. J. Immunology. 2000; PubMed Scopus Google Scholar). TNF-α production that we was in from cells with bacteria from with IPP to the of non-peptidic antigen the of the bacteria with the IPP that we the of TNF-α production was not One of the is that are highly sustained with induced by anti-CD3 or with in αβ T cells with anti-CD3 or antigens presented in the of molecules. It has been shown that sustained signaling be to TcR S. M. K. H. A. J. Exp. Med. 1995; PubMed Scopus Google Scholar). Here we that, in contrast to anti-CD3 IPP does not induce down-modulation of the TcR·CD3 a that the sustained signals observed in Vγ9Vδ2 T cells by IPP results from a between the antigen and the T cell antigen This could account for the high and production of TNF-α in activated Vγ9Vδ2 T cells and which has been shown in to result in immunopathology (26Kabelitz D. Wesch D. Hinz T. Spring. Semin. Immunopathol. 1999; 21: 55-75PubMed Google Scholar).