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Functional inactivation in the whole population of human V gamma 9/V delta 2 T lymphocytes induced by a nonpeptidic antagonist.

Bürk MR, Carena I, Donda A, Mariani F, Mori L, De Libero G - J. Exp. Med. (1997)

Bottom Line: We report that one of these ligands, 2,3-diphosphoglyceric acid (DPG), which induces expansion of V gamma 9/V delta T cells ex vivo, antagonizes the same cell population after repetitive activation.These findings show that TCR antagonism is a general phenomenon of T cells.However, in contrast to the clonal specificity of altered peptides antagonizing alpha beta T cells, all the tested V gamma 9/V delta 2 polyclonal cell lines and clones become unresponsive, a fact that may be relevant for the regulation of their response in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Research, University Hospital, Basel, Switzerland.

ABSTRACT
Nonpeptidic compounds stimulate human T cells bearing the TCR-gamma delta in the absence of major histocompatibility complex restriction. We report that one of these ligands, 2,3-diphosphoglyceric acid (DPG), which induces expansion of V gamma 9/V delta T cells ex vivo, antagonizes the same cell population after repetitive activation. Stimulation with DPG results in partial early protein tyrosine phosphorylation and a prolonged, but reversible, state of unresponsiveness to agonist ligands in V gamma 9/V delta 2, but not in other T cells. These findings show that TCR antagonism is a general phenomenon of T cells. However, in contrast to the clonal specificity of altered peptides antagonizing alpha beta T cells, all the tested V gamma 9/V delta 2 polyclonal cell lines and clones become unresponsive, a fact that may be relevant for the regulation of their response in vivo.

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DPG rapidly induces a prolonged, but reversible, state of unresponsiveness to IPP. (A) The Vγ9/Vδ2 cell line BCI 31 was incubated  with DPG for the time indicated on the x-axis. After extensive washing,  the TNF release stimulated by 50 μM IPP (▪) or 1 μg/ml PHA (•) was  measured. (B) Vγ9/Vδ2 clone BCI 49 (2 × 106 cells/ml) was incubated  in IL-2–free medium with 1 mM DPG overnight and washed extensively. The cells were then rested in medium for the time indicated on the  x-axis. Proliferative responses to medium (♦), 100 μM IPP (▪), or 20  U/ml IL-2, (○) are shown. □ indicates control proliferation to IPP (100  μM) of the same cells not preincubated with DPG. Results obtained in  four independent experiments using different Vγ9/Vδ2 clones showed  recovery of responsiveness after 1 to 5 d. Preincubation with DPG did  not affect the responsiveness of CD4+ TCR αβ cells to their specific peptide (data not shown).
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Figure 3: DPG rapidly induces a prolonged, but reversible, state of unresponsiveness to IPP. (A) The Vγ9/Vδ2 cell line BCI 31 was incubated with DPG for the time indicated on the x-axis. After extensive washing, the TNF release stimulated by 50 μM IPP (▪) or 1 μg/ml PHA (•) was measured. (B) Vγ9/Vδ2 clone BCI 49 (2 × 106 cells/ml) was incubated in IL-2–free medium with 1 mM DPG overnight and washed extensively. The cells were then rested in medium for the time indicated on the x-axis. Proliferative responses to medium (♦), 100 μM IPP (▪), or 20 U/ml IL-2, (○) are shown. □ indicates control proliferation to IPP (100 μM) of the same cells not preincubated with DPG. Results obtained in four independent experiments using different Vγ9/Vδ2 clones showed recovery of responsiveness after 1 to 5 d. Preincubation with DPG did not affect the responsiveness of CD4+ TCR αβ cells to their specific peptide (data not shown).

Mentions: To investigate whether stimulation by DPG could result in a state of altered responsiveness, Vγ9/Vδ2 T cells were preincubated with DPG, and then washed and challenged with IPP. Preincubation with DPG blocks the subsequent ability of IPP to stimulate TNF release and cell proliferation (Fig. 3) indicating that DPG induces a state of unresponsiveness which persists after removal of the ligand. This prolonged unresponsiveness is not due to inadequate washout of the antagonist, since control cells are fully reactive in the presence of equal numbers of DPG-preincubated cells (data not shown). Incubation with DPG for only 5 min is sufficient to block IPP-mediated TNF release (Fig. 3 A). The rapidity with which DPG exerts its inhibitory effect is consistent with observations that phosphorylated nonpeptidic ligands induce Ca2+ fluxes in Vγ9/Vδ2 T cells within 2 min (11, 12). The IPP-unresponsiveness induced by DPG is not a consequence of cell death since the number of viable cells recovered is the same (70–100%) as controls, and the cells remain responsive to low doses (20 U/ml) of IL-2 (Fig. 3 B). Furthermore, once a state of unresponsiveness is induced, it lasts for 1–5 d (using different cell lines and clones), after which the cells regain their ability to respond to the agonist (Fig. 3 B). The induction of unresponsiveness by DPG differs from the induction of T cell refractoriness by repetitive stimulation with agonist ligands. Indeed, (a) γδ cells stimulated by DPG become unresponsive without induction of effector functions, and (b) the induction of unresponsiveness by DPG has a dominant effect even over simultaneous stimulation by IPP (Fig. 2).


Functional inactivation in the whole population of human V gamma 9/V delta 2 T lymphocytes induced by a nonpeptidic antagonist.

Bürk MR, Carena I, Donda A, Mariani F, Mori L, De Libero G - J. Exp. Med. (1997)

DPG rapidly induces a prolonged, but reversible, state of unresponsiveness to IPP. (A) The Vγ9/Vδ2 cell line BCI 31 was incubated  with DPG for the time indicated on the x-axis. After extensive washing,  the TNF release stimulated by 50 μM IPP (▪) or 1 μg/ml PHA (•) was  measured. (B) Vγ9/Vδ2 clone BCI 49 (2 × 106 cells/ml) was incubated  in IL-2–free medium with 1 mM DPG overnight and washed extensively. The cells were then rested in medium for the time indicated on the  x-axis. Proliferative responses to medium (♦), 100 μM IPP (▪), or 20  U/ml IL-2, (○) are shown. □ indicates control proliferation to IPP (100  μM) of the same cells not preincubated with DPG. Results obtained in  four independent experiments using different Vγ9/Vδ2 clones showed  recovery of responsiveness after 1 to 5 d. Preincubation with DPG did  not affect the responsiveness of CD4+ TCR αβ cells to their specific peptide (data not shown).
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Figure 3: DPG rapidly induces a prolonged, but reversible, state of unresponsiveness to IPP. (A) The Vγ9/Vδ2 cell line BCI 31 was incubated with DPG for the time indicated on the x-axis. After extensive washing, the TNF release stimulated by 50 μM IPP (▪) or 1 μg/ml PHA (•) was measured. (B) Vγ9/Vδ2 clone BCI 49 (2 × 106 cells/ml) was incubated in IL-2–free medium with 1 mM DPG overnight and washed extensively. The cells were then rested in medium for the time indicated on the x-axis. Proliferative responses to medium (♦), 100 μM IPP (▪), or 20 U/ml IL-2, (○) are shown. □ indicates control proliferation to IPP (100 μM) of the same cells not preincubated with DPG. Results obtained in four independent experiments using different Vγ9/Vδ2 clones showed recovery of responsiveness after 1 to 5 d. Preincubation with DPG did not affect the responsiveness of CD4+ TCR αβ cells to their specific peptide (data not shown).
Mentions: To investigate whether stimulation by DPG could result in a state of altered responsiveness, Vγ9/Vδ2 T cells were preincubated with DPG, and then washed and challenged with IPP. Preincubation with DPG blocks the subsequent ability of IPP to stimulate TNF release and cell proliferation (Fig. 3) indicating that DPG induces a state of unresponsiveness which persists after removal of the ligand. This prolonged unresponsiveness is not due to inadequate washout of the antagonist, since control cells are fully reactive in the presence of equal numbers of DPG-preincubated cells (data not shown). Incubation with DPG for only 5 min is sufficient to block IPP-mediated TNF release (Fig. 3 A). The rapidity with which DPG exerts its inhibitory effect is consistent with observations that phosphorylated nonpeptidic ligands induce Ca2+ fluxes in Vγ9/Vδ2 T cells within 2 min (11, 12). The IPP-unresponsiveness induced by DPG is not a consequence of cell death since the number of viable cells recovered is the same (70–100%) as controls, and the cells remain responsive to low doses (20 U/ml) of IL-2 (Fig. 3 B). Furthermore, once a state of unresponsiveness is induced, it lasts for 1–5 d (using different cell lines and clones), after which the cells regain their ability to respond to the agonist (Fig. 3 B). The induction of unresponsiveness by DPG differs from the induction of T cell refractoriness by repetitive stimulation with agonist ligands. Indeed, (a) γδ cells stimulated by DPG become unresponsive without induction of effector functions, and (b) the induction of unresponsiveness by DPG has a dominant effect even over simultaneous stimulation by IPP (Fig. 2).

Bottom Line: We report that one of these ligands, 2,3-diphosphoglyceric acid (DPG), which induces expansion of V gamma 9/V delta T cells ex vivo, antagonizes the same cell population after repetitive activation.These findings show that TCR antagonism is a general phenomenon of T cells.However, in contrast to the clonal specificity of altered peptides antagonizing alpha beta T cells, all the tested V gamma 9/V delta 2 polyclonal cell lines and clones become unresponsive, a fact that may be relevant for the regulation of their response in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Research, University Hospital, Basel, Switzerland.

ABSTRACT
Nonpeptidic compounds stimulate human T cells bearing the TCR-gamma delta in the absence of major histocompatibility complex restriction. We report that one of these ligands, 2,3-diphosphoglyceric acid (DPG), which induces expansion of V gamma 9/V delta T cells ex vivo, antagonizes the same cell population after repetitive activation. Stimulation with DPG results in partial early protein tyrosine phosphorylation and a prolonged, but reversible, state of unresponsiveness to agonist ligands in V gamma 9/V delta 2, but not in other T cells. These findings show that TCR antagonism is a general phenomenon of T cells. However, in contrast to the clonal specificity of altered peptides antagonizing alpha beta T cells, all the tested V gamma 9/V delta 2 polyclonal cell lines and clones become unresponsive, a fact that may be relevant for the regulation of their response in vivo.

Show MeSH
Related in: MedlinePlus