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An instructive component in T helper cell type 2 (Th2) development mediated by GATA-3.

Farrar JD, Ouyang W, Löhning M, Assenmacher M, Radbruch A, Kanagawa O, Murphy KM - J. Exp. Med. (2001)

Bottom Line: We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model.Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate.These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Center for Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Although interleukin (IL)-12 and IL-4 polarize naive CD4(+) T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

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Redirection of committed non–IL-4–producing progenitors is IL-4 dependent and requires Stat6. (a) Comparison of predictions of selective and instructive models. Cells that have either committed to an IL-4–producing phenotype (shaded circles) or a non–IL-4–producing phenotype (open circles) are separately reactivated in a secondary stimulation. The instructive differentiation model allows cytokine-dependent redirection, whereas the selective model does not. (b) Wild-type or Stat6-deficient (Stat6−/−) DO11.10 cells were activated by OVA in the presence of anti–IL-12, anti–IFN-γ, and anti–IL-4 for 7 d as described (reference 17; A). Cells were reactivated and purified by cellular affinity matrix technology and flow cytometric sorting into IL-4–nonsecreting (IL-4 Negative; B) and IL-4–secreting (IL-4 Positive; C) populations. After sorting, cells were returned to culture and were reactivated with OVA and irradiated BALB/c splenocytes either in the presence of IL-4 (100 U/ml; E, G, I, K, M, O, Q, and S) or neutralizing anti–IL-4 mAb (11B11; D, F, H, J, L, N, P, and R). After 7 d, cells were stimulated with PMA and ionomycin for 4 h followed by analysis of mCD4 expression and IL-4 (D to G and L to O) and IFN-γ (H to K and P to S) production by intracellular staining. Analysis gates exclude dead cells. Quadrants were set based on isotype control staining as described (reference 26), and the percentage in the top right quadrant indicates the frequency of cells positive for cytokine production. These experiments were performed three times with consistent results.
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Figure 2: Redirection of committed non–IL-4–producing progenitors is IL-4 dependent and requires Stat6. (a) Comparison of predictions of selective and instructive models. Cells that have either committed to an IL-4–producing phenotype (shaded circles) or a non–IL-4–producing phenotype (open circles) are separately reactivated in a secondary stimulation. The instructive differentiation model allows cytokine-dependent redirection, whereas the selective model does not. (b) Wild-type or Stat6-deficient (Stat6−/−) DO11.10 cells were activated by OVA in the presence of anti–IL-12, anti–IFN-γ, and anti–IL-4 for 7 d as described (reference 17; A). Cells were reactivated and purified by cellular affinity matrix technology and flow cytometric sorting into IL-4–nonsecreting (IL-4 Negative; B) and IL-4–secreting (IL-4 Positive; C) populations. After sorting, cells were returned to culture and were reactivated with OVA and irradiated BALB/c splenocytes either in the presence of IL-4 (100 U/ml; E, G, I, K, M, O, Q, and S) or neutralizing anti–IL-4 mAb (11B11; D, F, H, J, L, N, P, and R). After 7 d, cells were stimulated with PMA and ionomycin for 4 h followed by analysis of mCD4 expression and IL-4 (D to G and L to O) and IFN-γ (H to K and P to S) production by intracellular staining. Analysis gates exclude dead cells. Quadrants were set based on isotype control staining as described (reference 26), and the percentage in the top right quadrant indicates the frequency of cells positive for cytokine production. These experiments were performed three times with consistent results.

Mentions: However, the instructive and selective models differ in their predictions of how committed cells should respond to subsequent IL-4 exposure. In the selective model, commitment is permanent, with subsequent cytokine exposure altering cell growth, but not differentiation. In the instructive model, commitment results from cytokine-derived signals, which could be delivered even after initial activation. These predictions can be distinguished by testing responses of committed progenitors to the polarizing effects of IL-4. To test this experimentally, cells initially committed to an IL-4–producing phenotype or IL-4–nonproducing phenotype would need to be separated and analyzed independently for differentiation when exposed to IL-4 (Fig. 2 a). To separate IL-4–producing from IL-4–nonproducing committed cells, we used the novel cellular affinity matrix technology for purifying live cells based on their IL-4 secretion 17. Wild-type and Stat6-deficient (Stat6−/−) DO11.10 naive T cells were activated in the presence of anti–IL-4, anti–IL-12, and anti–IFN-γ. On day 7, cells were reactivated with PMA and ionomycin (Fig. 2 b, A), and CD4+ T cells were sorted into IL-4–nonsecreting cells (Fig. 2 b, B) and IL-4–secreting cells (Fig. 2 b, C). Each population was then divided and restimulated either in the presence of IL-4 or anti–IL-4 mAb (11B11) for 7 d. Upon restimulation, IL-4 and IFN-γ production by individual cells was measured by intracellular staining (Fig. 2 b, D–S).


An instructive component in T helper cell type 2 (Th2) development mediated by GATA-3.

Farrar JD, Ouyang W, Löhning M, Assenmacher M, Radbruch A, Kanagawa O, Murphy KM - J. Exp. Med. (2001)

Redirection of committed non–IL-4–producing progenitors is IL-4 dependent and requires Stat6. (a) Comparison of predictions of selective and instructive models. Cells that have either committed to an IL-4–producing phenotype (shaded circles) or a non–IL-4–producing phenotype (open circles) are separately reactivated in a secondary stimulation. The instructive differentiation model allows cytokine-dependent redirection, whereas the selective model does not. (b) Wild-type or Stat6-deficient (Stat6−/−) DO11.10 cells were activated by OVA in the presence of anti–IL-12, anti–IFN-γ, and anti–IL-4 for 7 d as described (reference 17; A). Cells were reactivated and purified by cellular affinity matrix technology and flow cytometric sorting into IL-4–nonsecreting (IL-4 Negative; B) and IL-4–secreting (IL-4 Positive; C) populations. After sorting, cells were returned to culture and were reactivated with OVA and irradiated BALB/c splenocytes either in the presence of IL-4 (100 U/ml; E, G, I, K, M, O, Q, and S) or neutralizing anti–IL-4 mAb (11B11; D, F, H, J, L, N, P, and R). After 7 d, cells were stimulated with PMA and ionomycin for 4 h followed by analysis of mCD4 expression and IL-4 (D to G and L to O) and IFN-γ (H to K and P to S) production by intracellular staining. Analysis gates exclude dead cells. Quadrants were set based on isotype control staining as described (reference 26), and the percentage in the top right quadrant indicates the frequency of cells positive for cytokine production. These experiments were performed three times with consistent results.
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Related In: Results  -  Collection

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Figure 2: Redirection of committed non–IL-4–producing progenitors is IL-4 dependent and requires Stat6. (a) Comparison of predictions of selective and instructive models. Cells that have either committed to an IL-4–producing phenotype (shaded circles) or a non–IL-4–producing phenotype (open circles) are separately reactivated in a secondary stimulation. The instructive differentiation model allows cytokine-dependent redirection, whereas the selective model does not. (b) Wild-type or Stat6-deficient (Stat6−/−) DO11.10 cells were activated by OVA in the presence of anti–IL-12, anti–IFN-γ, and anti–IL-4 for 7 d as described (reference 17; A). Cells were reactivated and purified by cellular affinity matrix technology and flow cytometric sorting into IL-4–nonsecreting (IL-4 Negative; B) and IL-4–secreting (IL-4 Positive; C) populations. After sorting, cells were returned to culture and were reactivated with OVA and irradiated BALB/c splenocytes either in the presence of IL-4 (100 U/ml; E, G, I, K, M, O, Q, and S) or neutralizing anti–IL-4 mAb (11B11; D, F, H, J, L, N, P, and R). After 7 d, cells were stimulated with PMA and ionomycin for 4 h followed by analysis of mCD4 expression and IL-4 (D to G and L to O) and IFN-γ (H to K and P to S) production by intracellular staining. Analysis gates exclude dead cells. Quadrants were set based on isotype control staining as described (reference 26), and the percentage in the top right quadrant indicates the frequency of cells positive for cytokine production. These experiments were performed three times with consistent results.
Mentions: However, the instructive and selective models differ in their predictions of how committed cells should respond to subsequent IL-4 exposure. In the selective model, commitment is permanent, with subsequent cytokine exposure altering cell growth, but not differentiation. In the instructive model, commitment results from cytokine-derived signals, which could be delivered even after initial activation. These predictions can be distinguished by testing responses of committed progenitors to the polarizing effects of IL-4. To test this experimentally, cells initially committed to an IL-4–producing phenotype or IL-4–nonproducing phenotype would need to be separated and analyzed independently for differentiation when exposed to IL-4 (Fig. 2 a). To separate IL-4–producing from IL-4–nonproducing committed cells, we used the novel cellular affinity matrix technology for purifying live cells based on their IL-4 secretion 17. Wild-type and Stat6-deficient (Stat6−/−) DO11.10 naive T cells were activated in the presence of anti–IL-4, anti–IL-12, and anti–IFN-γ. On day 7, cells were reactivated with PMA and ionomycin (Fig. 2 b, A), and CD4+ T cells were sorted into IL-4–nonsecreting cells (Fig. 2 b, B) and IL-4–secreting cells (Fig. 2 b, C). Each population was then divided and restimulated either in the presence of IL-4 or anti–IL-4 mAb (11B11) for 7 d. Upon restimulation, IL-4 and IFN-γ production by individual cells was measured by intracellular staining (Fig. 2 b, D–S).

Bottom Line: We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model.Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate.These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Center for Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Although interleukin (IL)-12 and IL-4 polarize naive CD4(+) T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

Show MeSH