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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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Downmodulation of the TCR in response to antigenic stimulation occurs to equivalent degrees in I and the unresponsive clones. The  T cell clones were stimulated for 7 h in vitro with doses of Hb(64–76)  and Con A–activated peritoneal macrophages. The cells were then stained  with an α-CD3 ab (2C11) labeled with FITC, and surface TCR levels  were assessed using flow cytometric analysis.
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Figure 5: Downmodulation of the TCR in response to antigenic stimulation occurs to equivalent degrees in I and the unresponsive clones. The T cell clones were stimulated for 7 h in vitro with doses of Hb(64–76) and Con A–activated peritoneal macrophages. The cells were then stained with an α-CD3 ab (2C11) labeled with FITC, and surface TCR levels were assessed using flow cytometric analysis.

Mentions: Several studies have drawn a correlation between the level of CD4+ T cell proliferation and the amount of TCR/CD3 complexes downmodulated from the cell surface in response to peptide– MHC ligand recognition (20, 23). In particular, significant downmodulation occurs at high doses of agonist peptide when the T cells are fully activated, and ligands that only partially activate the T cell cause significantly less downmodulation (20). Similarly, our partial agonist, 74L, caused only slight or no TCR downmodulation (data not shown), which correlates with the proliferative response (Fig. 1). To investigate whether the lack of proliferation in the anergic Th0 cells correlated with a failure to downmodulate their TCR/CD3 in response to antigen, flow cytometric analysis was used to assess the relative level of surface TCR/CD3 expression of the anergic cells. Anergic T cells retained an ability to downmodulate their receptors (Fig. 5, B and C) to a similar degree as the proliferative parent clone I (Fig. 5 A). TCR downmodulation peaks at 100 μM because APC were used that were prepulsed with antigen rather than APC that were continually in the presence of antigen, as was the case in Fig. 1.


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

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

Downmodulation of the TCR in response to antigenic stimulation occurs to equivalent degrees in I and the unresponsive clones. The  T cell clones were stimulated for 7 h in vitro with doses of Hb(64–76)  and Con A–activated peritoneal macrophages. The cells were then stained  with an α-CD3 ab (2C11) labeled with FITC, and surface TCR levels  were assessed using flow cytometric analysis.
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Related In: Results  -  Collection

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Figure 5: Downmodulation of the TCR in response to antigenic stimulation occurs to equivalent degrees in I and the unresponsive clones. The T cell clones were stimulated for 7 h in vitro with doses of Hb(64–76) and Con A–activated peritoneal macrophages. The cells were then stained with an α-CD3 ab (2C11) labeled with FITC, and surface TCR levels were assessed using flow cytometric analysis.
Mentions: Several studies have drawn a correlation between the level of CD4+ T cell proliferation and the amount of TCR/CD3 complexes downmodulated from the cell surface in response to peptide– MHC ligand recognition (20, 23). In particular, significant downmodulation occurs at high doses of agonist peptide when the T cells are fully activated, and ligands that only partially activate the T cell cause significantly less downmodulation (20). Similarly, our partial agonist, 74L, caused only slight or no TCR downmodulation (data not shown), which correlates with the proliferative response (Fig. 1). To investigate whether the lack of proliferation in the anergic Th0 cells correlated with a failure to downmodulate their TCR/CD3 in response to antigen, flow cytometric analysis was used to assess the relative level of surface TCR/CD3 expression of the anergic cells. Anergic T cells retained an ability to downmodulate their receptors (Fig. 5, B and C) to a similar degree as the proliferative parent clone I (Fig. 5 A). TCR downmodulation peaks at 100 μM because APC were used that were prepulsed with antigen rather than APC that were continually in the presence of antigen, as was the case in Fig. 1.

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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