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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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Co-administration of  CD4 antibody plus HA-Ig antigen. 6.5+ mice received i.v. 50  μg of YTA3.1.2 one day and 50  μg of YTA plus 0.1 mg of HAIg or PBS the next day. Other  mice received antigen (0.1 mg  HA-Ig) alone. Between two and  four mice per group were analyzed at different timepoints after  treatment. The absolute numbers  of CD4+6.5+ cells in the periphery is shown (CD8+6.5+ cells remained relatively constant). 3H  incorporation is shown for LN  cells stimulated in vitro with 1  μg/ml of peptide (B). The response to higher doses of peptide  (10 μg/ml) was similarly decreased in treated animals. Proliferation values for day 21 were  obtained from thymectomized  mice (Tx) treated with PBS,  anti-CD4 or anti-CD4/0.1 mg  HA-Ig. Percentages of 6.5+ cells  among total T cells were determined for each mouse (D). IFN-γ  values were determined by ELISA  from the supernatant of spleen T  cells stimulated in vitro with 10  μg/ml of peptide (C).
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Figure 5: Co-administration of CD4 antibody plus HA-Ig antigen. 6.5+ mice received i.v. 50 μg of YTA3.1.2 one day and 50 μg of YTA plus 0.1 mg of HAIg or PBS the next day. Other mice received antigen (0.1 mg HA-Ig) alone. Between two and four mice per group were analyzed at different timepoints after treatment. The absolute numbers of CD4+6.5+ cells in the periphery is shown (CD8+6.5+ cells remained relatively constant). 3H incorporation is shown for LN cells stimulated in vitro with 1 μg/ml of peptide (B). The response to higher doses of peptide (10 μg/ml) was similarly decreased in treated animals. Proliferation values for day 21 were obtained from thymectomized mice (Tx) treated with PBS, anti-CD4 or anti-CD4/0.1 mg HA-Ig. Percentages of 6.5+ cells among total T cells were determined for each mouse (D). IFN-γ values were determined by ELISA from the supernatant of spleen T cells stimulated in vitro with 10 μg/ml of peptide (C).

Mentions: It has been shown by several groups that administration of CD4 and/or CD8 antibodies can be helpful in the establishment of peripheral tolerance (26, 27, for review see reference 28). Again, the precise mechanisms are not known. To study the effect of CD4 antibodies on responsiveness to HA we adopted a protocol described by Bushell et al. (29) with the aim of (a) decreasing CD4 cell numbers and (b) potentially providing negative signals that may favor the induction of unresponsiveness. The CD4 antibody (YTA3.1.2) depleted 60–70% of peripheral CD4 cells at day 7, as previously described (18, 29). Mice were injected i.v. with 50 μg of the antibody YTA3.1.2 and 1 d later with 50 μg of YTA3.1.2 plus 0.1 mg of HA-Ig. Control mice received either only antigen or only CD4 antibody. Mice were killed at days 3, 7, and 14 and the number and functional capacity of the 6.5+ cells was determined. As can be seen in Fig. 5, combining anti-CD4 and antigen considerably decreased cell numbers and induced non-responsiveness in terms of proliferation as well as of IFN-γ production when analyzed 7 d later. At day 14, the cells began to regain responsiveness. This could have been due to the fact that newly produced 6.5 cells, which had not been in contact with antigen, were entering secondary lymphoid tissues from the thymus. This seemed, however, not to be the case since 6.5+ cells from mice thymectomized before treatment and analyzed 21 d after treatment also recovered responsiveness. Therefore, the protocol proved to be efficient in induction, but not in maintenance of tolerance. It is also clear that anti-CD4 on its own had an effect on the proliferative capacity of 6.5+ cells, suggesting that part of this anergy at that point in time was not antigen specific. No effect of anti-CD4 alone was observed in terms of IFN-γ production, however, suggesting that the decrease in cytokine production observed at day 7 in anti-CD4 plus antigen treated mice depended on antigen. Such a decrease in IFN-γ was not due to a Th1 to Th2 shift among the antigen-specific cells since no IL4 production was detected. The lack of an effect of anti-CD4 on IFN-γ production and the decreased IFN-γ production after anti-CD4 plus antigen may be due to the fact that antigen is required to stop IFN-γ production by the TCR expressing CD8+ T cells.


Conditions that induce tolerance in mature CD4+ T cells.

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

Co-administration of  CD4 antibody plus HA-Ig antigen. 6.5+ mice received i.v. 50  μg of YTA3.1.2 one day and 50  μg of YTA plus 0.1 mg of HAIg or PBS the next day. Other  mice received antigen (0.1 mg  HA-Ig) alone. Between two and  four mice per group were analyzed at different timepoints after  treatment. The absolute numbers  of CD4+6.5+ cells in the periphery is shown (CD8+6.5+ cells remained relatively constant). 3H  incorporation is shown for LN  cells stimulated in vitro with 1  μg/ml of peptide (B). The response to higher doses of peptide  (10 μg/ml) was similarly decreased in treated animals. Proliferation values for day 21 were  obtained from thymectomized  mice (Tx) treated with PBS,  anti-CD4 or anti-CD4/0.1 mg  HA-Ig. Percentages of 6.5+ cells  among total T cells were determined for each mouse (D). IFN-γ  values were determined by ELISA  from the supernatant of spleen T  cells stimulated in vitro with 10  μg/ml of peptide (C).
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Related In: Results  -  Collection

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Figure 5: Co-administration of CD4 antibody plus HA-Ig antigen. 6.5+ mice received i.v. 50 μg of YTA3.1.2 one day and 50 μg of YTA plus 0.1 mg of HAIg or PBS the next day. Other mice received antigen (0.1 mg HA-Ig) alone. Between two and four mice per group were analyzed at different timepoints after treatment. The absolute numbers of CD4+6.5+ cells in the periphery is shown (CD8+6.5+ cells remained relatively constant). 3H incorporation is shown for LN cells stimulated in vitro with 1 μg/ml of peptide (B). The response to higher doses of peptide (10 μg/ml) was similarly decreased in treated animals. Proliferation values for day 21 were obtained from thymectomized mice (Tx) treated with PBS, anti-CD4 or anti-CD4/0.1 mg HA-Ig. Percentages of 6.5+ cells among total T cells were determined for each mouse (D). IFN-γ values were determined by ELISA from the supernatant of spleen T cells stimulated in vitro with 10 μg/ml of peptide (C).
Mentions: It has been shown by several groups that administration of CD4 and/or CD8 antibodies can be helpful in the establishment of peripheral tolerance (26, 27, for review see reference 28). Again, the precise mechanisms are not known. To study the effect of CD4 antibodies on responsiveness to HA we adopted a protocol described by Bushell et al. (29) with the aim of (a) decreasing CD4 cell numbers and (b) potentially providing negative signals that may favor the induction of unresponsiveness. The CD4 antibody (YTA3.1.2) depleted 60–70% of peripheral CD4 cells at day 7, as previously described (18, 29). Mice were injected i.v. with 50 μg of the antibody YTA3.1.2 and 1 d later with 50 μg of YTA3.1.2 plus 0.1 mg of HA-Ig. Control mice received either only antigen or only CD4 antibody. Mice were killed at days 3, 7, and 14 and the number and functional capacity of the 6.5+ cells was determined. As can be seen in Fig. 5, combining anti-CD4 and antigen considerably decreased cell numbers and induced non-responsiveness in terms of proliferation as well as of IFN-γ production when analyzed 7 d later. At day 14, the cells began to regain responsiveness. This could have been due to the fact that newly produced 6.5 cells, which had not been in contact with antigen, were entering secondary lymphoid tissues from the thymus. This seemed, however, not to be the case since 6.5+ cells from mice thymectomized before treatment and analyzed 21 d after treatment also recovered responsiveness. Therefore, the protocol proved to be efficient in induction, but not in maintenance of tolerance. It is also clear that anti-CD4 on its own had an effect on the proliferative capacity of 6.5+ cells, suggesting that part of this anergy at that point in time was not antigen specific. No effect of anti-CD4 alone was observed in terms of IFN-γ production, however, suggesting that the decrease in cytokine production observed at day 7 in anti-CD4 plus antigen treated mice depended on antigen. Such a decrease in IFN-γ was not due to a Th1 to Th2 shift among the antigen-specific cells since no IL4 production was detected. The lack of an effect of anti-CD4 on IFN-γ production and the decreased IFN-γ production after anti-CD4 plus antigen may be due to the fact that antigen is required to stop IFN-γ production by the TCR expressing CD8+ T cells.

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