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Persistence of peptide-induced CD4+ T cell anergy in vitro.

Ryan KR, Evavold BD - J. Exp. Med. (1998)

Bottom Line: The anergic state was characterized by blunted proliferation and interleukin (IL) 2 production upon restimulation with Hb(64-76), and was not the result of impaired TCR/CD3 downmodulation.Interestingly, persistence of the anergic phenotype was observed despite biweekly restimulation with antigen, APCs, and IL-2.Thus, T cell unresponsiveness induced by a peptide produced a stable, persistent anergic state in a Th0 clone that was not reversible by stimulation with IL-2 or -12.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, USA.

ABSTRACT
Clonal T cell unresponsiveness, or anergy, has been proposed as a mechanism of peripheral tolerance in vivo, and as a potential means of curbing unwanted T cell responses. In this study, anergy was induced in a T helper cell (Th) clone reactive to hemoglobin (Hb) peptide 64-76 by coculture of the T cells with live antigen-presenting cells (APCs) and 74L, a peptide analog of Hb(64-76) that contains a single amino acid substitution of leucine for glycine at position 74, or with a low concentration of the agonist ligand. The anergic state was characterized by blunted proliferation and interleukin (IL) 2 production upon restimulation with Hb(64-76), and was not the result of impaired TCR/CD3 downmodulation. The addition of exogenous IL-12 transiently restored proliferation of the anergic lines, but removal of IL-12 from culture returned the T cells to their nonproliferative state. Interestingly, persistence of the anergic phenotype was observed despite biweekly restimulation with antigen, APCs, and IL-2. Thus, T cell unresponsiveness induced by a peptide produced a stable, persistent anergic state in a Th0 clone that was not reversible by stimulation with IL-2 or -12.

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(a) The unresponsive clones do not proliferate to Hb(64–76) after one passage in culture. The anergized clones were “passed” once in culture (as  described in Materials and Methods) and proliferation was again assessed (as described in Materials and Methods). (b) The anergic clones I + 10 μM 74L and I  + 0.1 μM wt remain unresponsive to wt antigen after a period of 5 mo in culture. The clones were maintained in culture through a series of biweekly restimulations with antigen and APCs. 5 mo later, the clones were stimulated in vitro with Hb(64–76) and APCs. Proliferation was measured in a proliferation assay, as described.
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Figure 4: (a) The unresponsive clones do not proliferate to Hb(64–76) after one passage in culture. The anergized clones were “passed” once in culture (as described in Materials and Methods) and proliferation was again assessed (as described in Materials and Methods). (b) The anergic clones I + 10 μM 74L and I + 0.1 μM wt remain unresponsive to wt antigen after a period of 5 mo in culture. The clones were maintained in culture through a series of biweekly restimulations with antigen and APCs. 5 mo later, the clones were stimulated in vitro with Hb(64–76) and APCs. Proliferation was measured in a proliferation assay, as described.

Mentions: It has been proposed that an anergic Th0 would differentiate into a Th2 as a result of the loss of IL-2 (14). To test the postanergic phenotype, the unresponsive Th0 lines were maintained in culture through biweekly restimulation with antigen, APCs, and IL-2. We did not observe a Th2 phenotype, but, intriguingly, the passaged Th0 cells still displayed an anergic phenotype. This was apparent after one cycle in culture (Fig. 4 A), and again at the end of a 5-mo period (Fig. 4 B), despite the presence of IL-2 (40–50 U), agonist peptide, and competent APCs during restimulation. Anergy was also seen at several intervals leading up to 5 mo (data not shown). Clonal unresponsiveness of the Th0 cells thus persisted through long-term maintenance in culture.


Persistence of peptide-induced CD4+ T cell anergy in vitro.

Ryan KR, Evavold BD - J. Exp. Med. (1998)

(a) The unresponsive clones do not proliferate to Hb(64–76) after one passage in culture. The anergized clones were “passed” once in culture (as  described in Materials and Methods) and proliferation was again assessed (as described in Materials and Methods). (b) The anergic clones I + 10 μM 74L and I  + 0.1 μM wt remain unresponsive to wt antigen after a period of 5 mo in culture. The clones were maintained in culture through a series of biweekly restimulations with antigen and APCs. 5 mo later, the clones were stimulated in vitro with Hb(64–76) and APCs. Proliferation was measured in a proliferation assay, as described.
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Related In: Results  -  Collection

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Figure 4: (a) The unresponsive clones do not proliferate to Hb(64–76) after one passage in culture. The anergized clones were “passed” once in culture (as described in Materials and Methods) and proliferation was again assessed (as described in Materials and Methods). (b) The anergic clones I + 10 μM 74L and I + 0.1 μM wt remain unresponsive to wt antigen after a period of 5 mo in culture. The clones were maintained in culture through a series of biweekly restimulations with antigen and APCs. 5 mo later, the clones were stimulated in vitro with Hb(64–76) and APCs. Proliferation was measured in a proliferation assay, as described.
Mentions: It has been proposed that an anergic Th0 would differentiate into a Th2 as a result of the loss of IL-2 (14). To test the postanergic phenotype, the unresponsive Th0 lines were maintained in culture through biweekly restimulation with antigen, APCs, and IL-2. We did not observe a Th2 phenotype, but, intriguingly, the passaged Th0 cells still displayed an anergic phenotype. This was apparent after one cycle in culture (Fig. 4 A), and again at the end of a 5-mo period (Fig. 4 B), despite the presence of IL-2 (40–50 U), agonist peptide, and competent APCs during restimulation. Anergy was also seen at several intervals leading up to 5 mo (data not shown). Clonal unresponsiveness of the Th0 cells thus persisted through long-term maintenance in culture.

Bottom Line: The anergic state was characterized by blunted proliferation and interleukin (IL) 2 production upon restimulation with Hb(64-76), and was not the result of impaired TCR/CD3 downmodulation.Interestingly, persistence of the anergic phenotype was observed despite biweekly restimulation with antigen, APCs, and IL-2.Thus, T cell unresponsiveness induced by a peptide produced a stable, persistent anergic state in a Th0 clone that was not reversible by stimulation with IL-2 or -12.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, USA.

ABSTRACT
Clonal T cell unresponsiveness, or anergy, has been proposed as a mechanism of peripheral tolerance in vivo, and as a potential means of curbing unwanted T cell responses. In this study, anergy was induced in a T helper cell (Th) clone reactive to hemoglobin (Hb) peptide 64-76 by coculture of the T cells with live antigen-presenting cells (APCs) and 74L, a peptide analog of Hb(64-76) that contains a single amino acid substitution of leucine for glycine at position 74, or with a low concentration of the agonist ligand. The anergic state was characterized by blunted proliferation and interleukin (IL) 2 production upon restimulation with Hb(64-76), and was not the result of impaired TCR/CD3 downmodulation. The addition of exogenous IL-12 transiently restored proliferation of the anergic lines, but removal of IL-12 from culture returned the T cells to their nonproliferative state. Interestingly, persistence of the anergic phenotype was observed despite biweekly restimulation with antigen, APCs, and IL-2. Thus, T cell unresponsiveness induced by a peptide produced a stable, persistent anergic state in a Th0 clone that was not reversible by stimulation with IL-2 or -12.

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