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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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Proliferative response of lymph node (A) and spleen (B) cells of  double versus single transgenic mice upon antigenic stimulation in vitro.  2 ×105 sIg−cells were cultured with 5 ×105 irradiated Balb/c splenocytes and stimulated with different doses of peptide (10 and 1 μg/ml),  with coated 6.5 mAb (100 μg/ml) or with medium alone. 3H was added  to the culture 48 h afterwards and left for an additional 18 h. The number  of 6.5+ cells per well was determined and proliferation values shown correspond to cpm/ 103 6.5+ cells. Values correspond to one set of experiments that included two HA+6.5+ mice and one HA−6.5+ mouse. Similar data were obtained in separate experiments.
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Figure 2: Proliferative response of lymph node (A) and spleen (B) cells of double versus single transgenic mice upon antigenic stimulation in vitro. 2 ×105 sIg−cells were cultured with 5 ×105 irradiated Balb/c splenocytes and stimulated with different doses of peptide (10 and 1 μg/ml), with coated 6.5 mAb (100 μg/ml) or with medium alone. 3H was added to the culture 48 h afterwards and left for an additional 18 h. The number of 6.5+ cells per well was determined and proliferation values shown correspond to cpm/ 103 6.5+ cells. Values correspond to one set of experiments that included two HA+6.5+ mice and one HA−6.5+ mouse. Similar data were obtained in separate experiments.

Mentions: We first analyzed the fate of class II restricted cells in double transgenic mice where the antigen would be constantly present at relatively high concentrations both in the thymus and in the periphery. For that purpose, 6.5+ TCR transgenic mice were crossed to mice transgenic for the HA gene under control of the immunoglobulin κ promoter. In the latter mice the antigen is expressed on hemopoietic cells and their spleen cells stimulate a good response of unprimed T cells bearing the transgenic TCR. In double transgenic mice (HA+6.5+), the absolute number of thymocytes was about half that of HA−6.5+ littermates and, as shown in Fig. 1, the percentage of 6.5+ thymocytes among the CD4+CD8− and CD4−CD8+ subpopulations was drastically decreased. Note that in peripheral lymphoid tissue 6.5+ cells express less of the transgenic receptor than thymocytes. This is in part due to the fact that the peripheral T cells in these mice express in general slightly lower TCR levels as judged by staining with the TCR-β antibodies and analysis of RAG−/−6.5+ mice and in part due to the fact that 6.5+ cells in the transgenic mice express endogenous TCR-α chains (not shown). The percentages of double-negative, double-positive, and singlepositive thymocytes were not very different from that of HA transgene negative littermates in part because other receptors generated by endogenous TCR α rearrangements were selected. Likewise, the absolute number of peripheral T cells was not greatly affected in HA+6.5+ mice and the percentage of B and T cells in the spleen was similar to that observed in 6.5+HA− littermates (data not shown). Surprisingly, however, 6.5+ cells could be detected in the periphery of the double transgenic mice and the frequency of splenic 6.5+ cells was comparable to that of 6.5+HA− mice (Fig. 1). There was no evidence for TCR or coreceptor downregulation among peripheral 6.5+ cells from double transgenic mice that had escaped deletion, but these cells differed from T cells from 6.5+HA− mice in that they had upregulated CD69 and downregulated CD62-L and CD45RB levels (not shown). When splenic or lymph node T cells from the double transgenic mice were stimulated in vitro either with antigen at different concentrations or with the clonotypic antibody, the 6.5+ cells were unresponsive unlike those from 6.5+HA− mice which proliferated well without prior immunization (Fig. 2, A and B). There was no response when cells from normal, non-transgenic BALB/c mice were stimulated either by antigen or the clonotypic antibody indicating that 6.5+ cells were essential for a proliferative response as well as cytokine production (see below). Addition of IL2 did not restore the response. These data demonstrate that tolerance in class II restricted cells can be induced by an antigen expressed in hemopoietic cells and that the mechanisms may involve deletion of immature cells as well as anergy of peripheral lymphocytes. The anergy may have in fact been induced after thymic emigration of the 6.5+ cells since at birth we cannot detect in the thymus HA that can stimulate 6.5+ cells in vitro (not shown).


Conditions that induce tolerance in mature CD4+ T cells.

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

Proliferative response of lymph node (A) and spleen (B) cells of  double versus single transgenic mice upon antigenic stimulation in vitro.  2 ×105 sIg−cells were cultured with 5 ×105 irradiated Balb/c splenocytes and stimulated with different doses of peptide (10 and 1 μg/ml),  with coated 6.5 mAb (100 μg/ml) or with medium alone. 3H was added  to the culture 48 h afterwards and left for an additional 18 h. The number  of 6.5+ cells per well was determined and proliferation values shown correspond to cpm/ 103 6.5+ cells. Values correspond to one set of experiments that included two HA+6.5+ mice and one HA−6.5+ mouse. Similar data were obtained in separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Proliferative response of lymph node (A) and spleen (B) cells of double versus single transgenic mice upon antigenic stimulation in vitro. 2 ×105 sIg−cells were cultured with 5 ×105 irradiated Balb/c splenocytes and stimulated with different doses of peptide (10 and 1 μg/ml), with coated 6.5 mAb (100 μg/ml) or with medium alone. 3H was added to the culture 48 h afterwards and left for an additional 18 h. The number of 6.5+ cells per well was determined and proliferation values shown correspond to cpm/ 103 6.5+ cells. Values correspond to one set of experiments that included two HA+6.5+ mice and one HA−6.5+ mouse. Similar data were obtained in separate experiments.
Mentions: We first analyzed the fate of class II restricted cells in double transgenic mice where the antigen would be constantly present at relatively high concentrations both in the thymus and in the periphery. For that purpose, 6.5+ TCR transgenic mice were crossed to mice transgenic for the HA gene under control of the immunoglobulin κ promoter. In the latter mice the antigen is expressed on hemopoietic cells and their spleen cells stimulate a good response of unprimed T cells bearing the transgenic TCR. In double transgenic mice (HA+6.5+), the absolute number of thymocytes was about half that of HA−6.5+ littermates and, as shown in Fig. 1, the percentage of 6.5+ thymocytes among the CD4+CD8− and CD4−CD8+ subpopulations was drastically decreased. Note that in peripheral lymphoid tissue 6.5+ cells express less of the transgenic receptor than thymocytes. This is in part due to the fact that the peripheral T cells in these mice express in general slightly lower TCR levels as judged by staining with the TCR-β antibodies and analysis of RAG−/−6.5+ mice and in part due to the fact that 6.5+ cells in the transgenic mice express endogenous TCR-α chains (not shown). The percentages of double-negative, double-positive, and singlepositive thymocytes were not very different from that of HA transgene negative littermates in part because other receptors generated by endogenous TCR α rearrangements were selected. Likewise, the absolute number of peripheral T cells was not greatly affected in HA+6.5+ mice and the percentage of B and T cells in the spleen was similar to that observed in 6.5+HA− littermates (data not shown). Surprisingly, however, 6.5+ cells could be detected in the periphery of the double transgenic mice and the frequency of splenic 6.5+ cells was comparable to that of 6.5+HA− mice (Fig. 1). There was no evidence for TCR or coreceptor downregulation among peripheral 6.5+ cells from double transgenic mice that had escaped deletion, but these cells differed from T cells from 6.5+HA− mice in that they had upregulated CD69 and downregulated CD62-L and CD45RB levels (not shown). When splenic or lymph node T cells from the double transgenic mice were stimulated in vitro either with antigen at different concentrations or with the clonotypic antibody, the 6.5+ cells were unresponsive unlike those from 6.5+HA− mice which proliferated well without prior immunization (Fig. 2, A and B). There was no response when cells from normal, non-transgenic BALB/c mice were stimulated either by antigen or the clonotypic antibody indicating that 6.5+ cells were essential for a proliferative response as well as cytokine production (see below). Addition of IL2 did not restore the response. These data demonstrate that tolerance in class II restricted cells can be induced by an antigen expressed in hemopoietic cells and that the mechanisms may involve deletion of immature cells as well as anergy of peripheral lymphocytes. The anergy may have in fact been induced after thymic emigration of the 6.5+ cells since at birth we cannot detect in the thymus HA that can stimulate 6.5+ cells in vitro (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