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Regulation of the interleukin (IL)-12R beta 2 subunit expression in developing T helper 1 (Th1) and Th2 cells.

Szabo SJ, Dighe AS, Gubler U, Murphy KM - J. Exp. Med. (1997)

Bottom Line: Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation.IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production.Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) beta 2 subunit expression. To determine the basis for this selective loss, we examined IL-12R beta 2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R beta 2 is not expressed by naive resting CD4+ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation. IL-4 inhibited IL-12R beta 2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production. Thus, IFN-gamma may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.

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IFN-γ restores the IL-12 signaling pathway in developing  Th2 cells. FACS®-sorted naive CD4+ DO11.10 T cells were cultured  with OVA peptide and irradiated BALB/c splenocytes under the conditions indicated. In the primary cultures, IFN-γ levels were either unaltered (none), neutralized with 30 μg/ml of the anti–IFN-γ mAb H22  (anti–IFN-γ) or 100 U/ml of IFN-γ (IFN-γ) were added as indicated.  On day 5 after primary antigen activation whole cell lysates from developing T cells (20 × 106) were prepared after incubation for 25 min with  10 U/ml IL-12. Lysates were immunoprecipitated with Stat4 antiserum,  separated by SDS-PAGE (7% gel), transferred to nitrocellulose, and probed  with the anti-phosphotyrosine reagent RC20. After exposure, blots were  stripped and reprobed with anti-Stat4 antisera.
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Figure 4: IFN-γ restores the IL-12 signaling pathway in developing Th2 cells. FACS®-sorted naive CD4+ DO11.10 T cells were cultured with OVA peptide and irradiated BALB/c splenocytes under the conditions indicated. In the primary cultures, IFN-γ levels were either unaltered (none), neutralized with 30 μg/ml of the anti–IFN-γ mAb H22 (anti–IFN-γ) or 100 U/ml of IFN-γ (IFN-γ) were added as indicated. On day 5 after primary antigen activation whole cell lysates from developing T cells (20 × 106) were prepared after incubation for 25 min with 10 U/ml IL-12. Lysates were immunoprecipitated with Stat4 antiserum, separated by SDS-PAGE (7% gel), transferred to nitrocellulose, and probed with the anti-phosphotyrosine reagent RC20. After exposure, blots were stripped and reprobed with anti-Stat4 antisera.

Mentions: We suspected that IL-12 was not the primary stimulus for induction of the IL-12R β2 subunit, since a cytokine cannot signal unless its receptor is already expressed at some level on the cell surface. Previously, IFN-γ has been shown to be required for IL-12–induced Th1 development from naive BALB/c T cells (7, 34). IL-12 is known to induce IFN-γ production from CD4+ and CD8+ T cells, as well as NK cells. Since these cells are present in the irradiated splenocytes used as APCs, we suspected that IFN-γ could be inducing the expression of the IL-12R β2 subunit. To test this possibility, combinations of IL-12, IFN-γ and IL-4, or the corresponding neutralizing antibodies were added during primary T cell activation and the IL-12 responsiveness of these developing Th cells was analyzed by measuring IL-12–induced Stat4-phosphorylation on day 5 after activation (Fig. 4). Th cells activated in the presence of both IL-12 and IL-4 maintained IL-12-induced Stat4 phosphorylation (Fig. 4, lane 4). The maintenance of IL-12 responsiveness on these cells was completely dependent on the production of endogenous IFN-γ during primary activation, since neutralization of IFN-γ abolished IL-12–induced Stat4 phosphorylation (Fig. 4, lane 5). Under Th2 inducing conditions (IL-4 and anti–IL-12), Th2 cells lost the ability to phosphorylate Stat4 in response to IL-12 (Fig. 4, lane 7). However, addition of IFN-γ to this culture restored IL-12induced Stat4 phosphorylation (Fig. 4, lane 9). Finally, under Th1 inducing conditions (IL-12 and anti–IL-4), T cells retained the ability to phosphorylate Stat4 in response to IL-12 in a manner that was independent of IFN-γ (Fig. 4, lanes 1–3). In summary, (a) the presence of IL-4 during primary activation inhibits IL-12 signaling in developing Th cells, and (b) IFN-γ is able to override this inhibition and restore IL-12 signaling in early Th cells.


Regulation of the interleukin (IL)-12R beta 2 subunit expression in developing T helper 1 (Th1) and Th2 cells.

Szabo SJ, Dighe AS, Gubler U, Murphy KM - J. Exp. Med. (1997)

IFN-γ restores the IL-12 signaling pathway in developing  Th2 cells. FACS®-sorted naive CD4+ DO11.10 T cells were cultured  with OVA peptide and irradiated BALB/c splenocytes under the conditions indicated. In the primary cultures, IFN-γ levels were either unaltered (none), neutralized with 30 μg/ml of the anti–IFN-γ mAb H22  (anti–IFN-γ) or 100 U/ml of IFN-γ (IFN-γ) were added as indicated.  On day 5 after primary antigen activation whole cell lysates from developing T cells (20 × 106) were prepared after incubation for 25 min with  10 U/ml IL-12. Lysates were immunoprecipitated with Stat4 antiserum,  separated by SDS-PAGE (7% gel), transferred to nitrocellulose, and probed  with the anti-phosphotyrosine reagent RC20. After exposure, blots were  stripped and reprobed with anti-Stat4 antisera.
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Related In: Results  -  Collection

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Figure 4: IFN-γ restores the IL-12 signaling pathway in developing Th2 cells. FACS®-sorted naive CD4+ DO11.10 T cells were cultured with OVA peptide and irradiated BALB/c splenocytes under the conditions indicated. In the primary cultures, IFN-γ levels were either unaltered (none), neutralized with 30 μg/ml of the anti–IFN-γ mAb H22 (anti–IFN-γ) or 100 U/ml of IFN-γ (IFN-γ) were added as indicated. On day 5 after primary antigen activation whole cell lysates from developing T cells (20 × 106) were prepared after incubation for 25 min with 10 U/ml IL-12. Lysates were immunoprecipitated with Stat4 antiserum, separated by SDS-PAGE (7% gel), transferred to nitrocellulose, and probed with the anti-phosphotyrosine reagent RC20. After exposure, blots were stripped and reprobed with anti-Stat4 antisera.
Mentions: We suspected that IL-12 was not the primary stimulus for induction of the IL-12R β2 subunit, since a cytokine cannot signal unless its receptor is already expressed at some level on the cell surface. Previously, IFN-γ has been shown to be required for IL-12–induced Th1 development from naive BALB/c T cells (7, 34). IL-12 is known to induce IFN-γ production from CD4+ and CD8+ T cells, as well as NK cells. Since these cells are present in the irradiated splenocytes used as APCs, we suspected that IFN-γ could be inducing the expression of the IL-12R β2 subunit. To test this possibility, combinations of IL-12, IFN-γ and IL-4, or the corresponding neutralizing antibodies were added during primary T cell activation and the IL-12 responsiveness of these developing Th cells was analyzed by measuring IL-12–induced Stat4-phosphorylation on day 5 after activation (Fig. 4). Th cells activated in the presence of both IL-12 and IL-4 maintained IL-12-induced Stat4 phosphorylation (Fig. 4, lane 4). The maintenance of IL-12 responsiveness on these cells was completely dependent on the production of endogenous IFN-γ during primary activation, since neutralization of IFN-γ abolished IL-12–induced Stat4 phosphorylation (Fig. 4, lane 5). Under Th2 inducing conditions (IL-4 and anti–IL-12), Th2 cells lost the ability to phosphorylate Stat4 in response to IL-12 (Fig. 4, lane 7). However, addition of IFN-γ to this culture restored IL-12induced Stat4 phosphorylation (Fig. 4, lane 9). Finally, under Th1 inducing conditions (IL-12 and anti–IL-4), T cells retained the ability to phosphorylate Stat4 in response to IL-12 in a manner that was independent of IFN-γ (Fig. 4, lanes 1–3). In summary, (a) the presence of IL-4 during primary activation inhibits IL-12 signaling in developing Th cells, and (b) IFN-γ is able to override this inhibition and restore IL-12 signaling in early Th cells.

Bottom Line: Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation.IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production.Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) beta 2 subunit expression. To determine the basis for this selective loss, we examined IL-12R beta 2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R beta 2 is not expressed by naive resting CD4+ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation. IL-4 inhibited IL-12R beta 2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production. Thus, IFN-gamma may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.

Show MeSH
Related in: MedlinePlus