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Conditions that induce tolerance in mature CD4+ T cells.

Lanoue A, Bona C, von Boehmer H, Sarukhan A - J. Exp. Med. (1997)

Bottom Line: Mature CD4+6.5+ cells that were transferred into antigen-containing recipients went through an initial phase of expansion after which most cells were deleted and those remaining became unresponsive, as previously described for CD8+ cells.It was only after decreasing cell numbers by CD4 antibody treatment and by repeatedly reintroducing antigen thereafter that unresponsiveness of 6.5+ cells was achieved and maintained.In no case could we observe the appearance of antigen-specific T cells with a Th2 cytokine profile among the remaining cells and therefore conclude that deletion and anergy represent the major mechanisms of tolerance in our studies.

View Article: PubMed Central - PubMed

Affiliation: Unité Institut National de la Santé et de la Recherche Médicale 373, Institut Necker, Paris.

ABSTRACT
Establishment of antigen-specific tolerance among mature T cells has been a long debated, yet poorly understood issue. In this study we have used transgenic mice bearing a class II--restricted TCR specific for the hemmagglutinin of the influenza virus in order to test the behavior of CD4+ T cells upon exposure to antigen in different forms and doses. We first studied the fate of T cells expressing the transgenic TCR (6.5) in double transgenic mice where HA was expressed as a self antigen by hemapoietic cells. In these mice, we found some mature T cells in periphery that had escaped thymic deletion and that showed signs of activation but which were anergic. Mature CD4+6.5+ cells that were transferred into antigen-containing recipients went through an initial phase of expansion after which most cells were deleted and those remaining became unresponsive, as previously described for CD8+ cells. Inducing tolerance in CD4+6.5+ cells in situ in single transgenic mice proved a difficult task: classical protocols using single doses of soluble or deaggregated antigen as well as feeding antigen all failed to induce antigen-specific unresponsiveness. It was only after decreasing cell numbers by CD4 antibody treatment and by repeatedly reintroducing antigen thereafter that unresponsiveness of 6.5+ cells was achieved and maintained. In no case could we observe the appearance of antigen-specific T cells with a Th2 cytokine profile among the remaining cells and therefore conclude that deletion and anergy represent the major mechanisms of tolerance in our studies.

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Recovery of 6.5+ cells after transfer into antigen-positive and  antigen-negative recipients. 2.5 ×106 LN cells from a 6.5+/+RAG−/−  mouse were injected i.v. into sublethally irradiated HA+ or HA− littermates. Recipients were killed at different timepoints and single cell suspensions from lymph nodes and spleen were used for three-color staining.  The absolute number of 6.5+ cells recovered from the periphery (lymph  nodes plus spleen) was determined. Values correspond to one set of experiments that included two antigen-positive mice and one antigen-negative control analyzed at each timepoint. Separate transfer experiments  gave similar results.
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Figure 3: Recovery of 6.5+ cells after transfer into antigen-positive and antigen-negative recipients. 2.5 ×106 LN cells from a 6.5+/+RAG−/− mouse were injected i.v. into sublethally irradiated HA+ or HA− littermates. Recipients were killed at different timepoints and single cell suspensions from lymph nodes and spleen were used for three-color staining. The absolute number of 6.5+ cells recovered from the periphery (lymph nodes plus spleen) was determined. Values correspond to one set of experiments that included two antigen-positive mice and one antigen-negative control analyzed at each timepoint. Separate transfer experiments gave similar results.

Mentions: We next addressed the question whether mature naive CD4+ cells, when exposed to high doses of antigen, would follow the same fate as previously described for CD8+ cells (2, 13) . For this purpose, lymph node cells from RAG−/−6.5+/+ mice were injected i.v. into x-irradiated HA+ mice or HA negative littermates and recipients were analyzed at different time points after transfer. As can be seen in Fig. 3, during the first 4 d 6.5+ cells expanded vigorously upon transfer into antigen-positive recipients and cell numbers declined thereafter. In contrast, when 6.5+ cells were transferred into HA− recipients, there was no significant expansion of these cells during the period of observation though about five percent of the injected cells could be recovered from these recipients. 6.5+ cells recovered at day 4 after transfer into HA-containing recipients were capable of responding to antigenic stimulation in vitro, though their response was much reduced when compared to 6.5+ naive cells. Curiously, contrary to 6.5+ cells from single TCR transgenic mice, the recovered cells proliferated better upon stimulation with the 6.5+ mAb than upon stimulation with the peptide. At 6 or 8 d after transfer, the remaining 6.5+ cells gave an even lower response than on day 4 to either the peptide or the clonotypic antibody (Table 1) and their anergic state could not be reversed by exogenous IL-2. Cells recovered from HA+ recipients at day 4 after transfer were still capable of expanding in vivo when retransferred into another HA+ recipient, while cells recovered 21 d after transfer were no longer capable of doing so (data not shown). Cytokine production by the 6.5+ cells recovered at different timepoints after transfer into antigen-positive recipients was also studied: IFN-γ and IL-4 were produced at significant levels when cells were stimulated in vitro at early timepoints after transfer. At day 30 after transfer, no proliferation and no IFN-γ secretion by remaining cells could be detected (data not shown).


Conditions that induce tolerance in mature CD4+ T cells.

Lanoue A, Bona C, von Boehmer H, Sarukhan A - J. Exp. Med. (1997)

Recovery of 6.5+ cells after transfer into antigen-positive and  antigen-negative recipients. 2.5 ×106 LN cells from a 6.5+/+RAG−/−  mouse were injected i.v. into sublethally irradiated HA+ or HA− littermates. Recipients were killed at different timepoints and single cell suspensions from lymph nodes and spleen were used for three-color staining.  The absolute number of 6.5+ cells recovered from the periphery (lymph  nodes plus spleen) was determined. Values correspond to one set of experiments that included two antigen-positive mice and one antigen-negative control analyzed at each timepoint. Separate transfer experiments  gave similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196030&req=5

Figure 3: Recovery of 6.5+ cells after transfer into antigen-positive and antigen-negative recipients. 2.5 ×106 LN cells from a 6.5+/+RAG−/− mouse were injected i.v. into sublethally irradiated HA+ or HA− littermates. Recipients were killed at different timepoints and single cell suspensions from lymph nodes and spleen were used for three-color staining. The absolute number of 6.5+ cells recovered from the periphery (lymph nodes plus spleen) was determined. Values correspond to one set of experiments that included two antigen-positive mice and one antigen-negative control analyzed at each timepoint. Separate transfer experiments gave similar results.
Mentions: We next addressed the question whether mature naive CD4+ cells, when exposed to high doses of antigen, would follow the same fate as previously described for CD8+ cells (2, 13) . For this purpose, lymph node cells from RAG−/−6.5+/+ mice were injected i.v. into x-irradiated HA+ mice or HA negative littermates and recipients were analyzed at different time points after transfer. As can be seen in Fig. 3, during the first 4 d 6.5+ cells expanded vigorously upon transfer into antigen-positive recipients and cell numbers declined thereafter. In contrast, when 6.5+ cells were transferred into HA− recipients, there was no significant expansion of these cells during the period of observation though about five percent of the injected cells could be recovered from these recipients. 6.5+ cells recovered at day 4 after transfer into HA-containing recipients were capable of responding to antigenic stimulation in vitro, though their response was much reduced when compared to 6.5+ naive cells. Curiously, contrary to 6.5+ cells from single TCR transgenic mice, the recovered cells proliferated better upon stimulation with the 6.5+ mAb than upon stimulation with the peptide. At 6 or 8 d after transfer, the remaining 6.5+ cells gave an even lower response than on day 4 to either the peptide or the clonotypic antibody (Table 1) and their anergic state could not be reversed by exogenous IL-2. Cells recovered from HA+ recipients at day 4 after transfer were still capable of expanding in vivo when retransferred into another HA+ recipient, while cells recovered 21 d after transfer were no longer capable of doing so (data not shown). Cytokine production by the 6.5+ cells recovered at different timepoints after transfer into antigen-positive recipients was also studied: IFN-γ and IL-4 were produced at significant levels when cells were stimulated in vitro at early timepoints after transfer. At day 30 after transfer, no proliferation and no IFN-γ secretion by remaining cells could be detected (data not shown).

Bottom Line: Mature CD4+6.5+ cells that were transferred into antigen-containing recipients went through an initial phase of expansion after which most cells were deleted and those remaining became unresponsive, as previously described for CD8+ cells.It was only after decreasing cell numbers by CD4 antibody treatment and by repeatedly reintroducing antigen thereafter that unresponsiveness of 6.5+ cells was achieved and maintained.In no case could we observe the appearance of antigen-specific T cells with a Th2 cytokine profile among the remaining cells and therefore conclude that deletion and anergy represent the major mechanisms of tolerance in our studies.

View Article: PubMed Central - PubMed

Affiliation: Unité Institut National de la Santé et de la Recherche Médicale 373, Institut Necker, Paris.

ABSTRACT
Establishment of antigen-specific tolerance among mature T cells has been a long debated, yet poorly understood issue. In this study we have used transgenic mice bearing a class II--restricted TCR specific for the hemmagglutinin of the influenza virus in order to test the behavior of CD4+ T cells upon exposure to antigen in different forms and doses. We first studied the fate of T cells expressing the transgenic TCR (6.5) in double transgenic mice where HA was expressed as a self antigen by hemapoietic cells. In these mice, we found some mature T cells in periphery that had escaped thymic deletion and that showed signs of activation but which were anergic. Mature CD4+6.5+ cells that were transferred into antigen-containing recipients went through an initial phase of expansion after which most cells were deleted and those remaining became unresponsive, as previously described for CD8+ cells. Inducing tolerance in CD4+6.5+ cells in situ in single transgenic mice proved a difficult task: classical protocols using single doses of soluble or deaggregated antigen as well as feeding antigen all failed to induce antigen-specific unresponsiveness. It was only after decreasing cell numbers by CD4 antibody treatment and by repeatedly reintroducing antigen thereafter that unresponsiveness of 6.5+ cells was achieved and maintained. In no case could we observe the appearance of antigen-specific T cells with a Th2 cytokine profile among the remaining cells and therefore conclude that deletion and anergy represent the major mechanisms of tolerance in our studies.

Show MeSH
Related in: MedlinePlus