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Novel genetic regulation of T helper 1 (Th1)/Th2 cytokine production and encephalitogenicity in inbred mouse strains.

Conboy IM, DeKruyff RH, Tate KM, Cao ZA, Moore TA, Umetsu DT, Jones PP - J. Exp. Med. (1997)

Bottom Line: A splenic APC reduced IFN-gamma and TNF-alpha production by established Th1 MBP-specific Ak-restricted B10.BR TCL and by a Th1 KLH-specific, Ek-restricted B10.BR T cell clone.The nature of the downregulatory activity of B10.A APC on IFN-gamma and TNF-alpha production was explored. 2-hour supernatants from antigen-activated B10.A APC/TCL cultures or from B10.A APC activated by LPS had the same inhibitory effects on IFN-gamma and TNF-alpha production by B10.BR TCL.The downregulatory effects of B10.A APC are independent of TNF-alpha, IL-4, IL-10, IL-12p40, IFN-gamma, IL-13, TGF-beta, and PGE2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, California 94305-5020, USA.

ABSTRACT
Development of T helper cell (Th)1 or Th2 cytokine responses is essential for effector and regulatory functions of T helper cells. We have compared cytokine profiles of myelin basic protein (MBP) Ac1-16 peptide-specific T helper cells from inbred mouse strains expressing identical k haplotype-derived MHC class II molecules B10.A and B10.BR, B10.BR T cell lines (TCL) produced Th1 cytokines (including high levels of TNF-alpha) and induced experimental autoimmune encephalomyelitis after adoptive transfer. In contrast, B10.A TCL produced Th2 cytokines (including low levels of TNF-alpha) and were poorly encephalitogenic. The contributions of the genetic origin of the T cells and the APC were explored. Serial restimulations of the B10.BR TCL with B10.A or (B10.A x B10.BR) F1 splenic antigen presenting cells (APC) during the establishment of TCL markedly reduced both Th1 cytokine production and encephalitogenicity. In addition, a single restimulation with B10. A splenic APC reduced IFN-gamma and TNF-alpha production by established Th1 MBP-specific Ak-restricted B10.BR TCL and by a Th1 KLH-specific, Ek-restricted B10.BR T cell clone. These studies suggest that B10.A and B10.BR APC differ in their ability to stimulate IFN-gamma and TNF-alpha production by mature Th1 cells and also influence their Th1/Th2 commitment in vivo. The nature of the downregulatory activity of B10.A APC on IFN-gamma and TNF-alpha production was explored. 2-hour supernatants from antigen-activated B10.A APC/TCL cultures or from B10.A APC activated by LPS had the same inhibitory effects on IFN-gamma and TNF-alpha production by B10.BR TCL. The downregulatory effects of B10.A APC are independent of TNF-alpha, IL-4, IL-10, IL-12p40, IFN-gamma, IL-13, TGF-beta, and PGE2. Thus, genetic difference(s) between B10.A and B10.BR APC appear(s) to control the production or activity of a novel soluble cytokine regulatory factor that influences Th1/Th2 commitment and controls production of IFN-gamma and TNF-alpha by mature Th1 cells.

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Downregulation of IFN-γ and TNF-α levels is not affected  when 2-h B10.A culture supernatant is depleted of known Th1/Th2 cytokine regulators. (A and B) B10.BR or B10.A T cells (TCL 4) were restimulated with mMBP Ac1-16 and syngeneic splenic APC for 2 h. Neutralizing antibodies specific for mouse IL-4, IL-10, TNF-α, IFN-γ, TGF-β,  IL-13, and IL-12p40 were added to these culture supernatants for 30 min,  and antibody–cytokine complexes were removed using protein G–coated  agarose beads. Cytokine depleted 2-h supernatants were then used as media in which B10.BR TCL 4 were restimulated with mMBP Ac1-16 and  B10.BR splenic APC for 24 h. IFN-γ (A) and TNF-α (B) levels were  measured as above. Lower levels of TNF-α after treatment with anti– TNF-α antibody and protein G beads were due to the presence of residual anti–TNF-α antibody, which has low affinity for protein G in medium with serum. Neutralizing antibodies were used at concentrations  equal to (not shown) and twice what were recommended by the manufacturers for complete neutralization of cytokine levels produced by high  expressing cells, giving similar results. Data presented are means and standard deviations of triplicate wells for each sample (n = 3). (C) B10.BR  TCL 4 cells were restimulated with B10.BR splenic APC and mMBP  Ac1-16 in 2-h culture supernatants from B10.A or B10.BR TCL 4, used  either undiluted or at serial 1:2 dilutions in RPMI, 10% FCS. TNF-α  levels are means and standard deviations of triplicate wells for each sample  (n = 3).
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Figure 5: Downregulation of IFN-γ and TNF-α levels is not affected when 2-h B10.A culture supernatant is depleted of known Th1/Th2 cytokine regulators. (A and B) B10.BR or B10.A T cells (TCL 4) were restimulated with mMBP Ac1-16 and syngeneic splenic APC for 2 h. Neutralizing antibodies specific for mouse IL-4, IL-10, TNF-α, IFN-γ, TGF-β, IL-13, and IL-12p40 were added to these culture supernatants for 30 min, and antibody–cytokine complexes were removed using protein G–coated agarose beads. Cytokine depleted 2-h supernatants were then used as media in which B10.BR TCL 4 were restimulated with mMBP Ac1-16 and B10.BR splenic APC for 24 h. IFN-γ (A) and TNF-α (B) levels were measured as above. Lower levels of TNF-α after treatment with anti– TNF-α antibody and protein G beads were due to the presence of residual anti–TNF-α antibody, which has low affinity for protein G in medium with serum. Neutralizing antibodies were used at concentrations equal to (not shown) and twice what were recommended by the manufacturers for complete neutralization of cytokine levels produced by high expressing cells, giving similar results. Data presented are means and standard deviations of triplicate wells for each sample (n = 3). (C) B10.BR TCL 4 cells were restimulated with B10.BR splenic APC and mMBP Ac1-16 in 2-h culture supernatants from B10.A or B10.BR TCL 4, used either undiluted or at serial 1:2 dilutions in RPMI, 10% FCS. TNF-α levels are means and standard deviations of triplicate wells for each sample (n = 3).

Mentions: To assess whether downregulation of IFN-γ and TNF-α by B10.A APC is mediated by or depends on any of the known Th1/Th2 regulators, 2-h culture supernatants derived from B10.BR or B10.A TCL/APC cultures were depleted of IL-4, IL-10, IL-12p40, IL-13, TNF-α, IFN-γ, and TGF-β and tested for their effects on IFN-γ and TNF-α production by B10.BR TCL 4 restimulated with B10.BR APC and mMBP peptide. Cytokine depletion did not diminish the downregulatory activity of the B10.A culture supernatant (Fig. 5, A and B). Serial dilutions of B10.A and B10.BR 2-h culture supernatants (Fig. 5 C) demonstrated that the downregulatory activity of B10.A-derived factor is rapidly lost by dilution, indicating that any depletion of the downregulatory cytokine would have been detected. Essentially identical results were obtained when the anti-cytokine antibodies were also used at half the concentrations (not shown).


Novel genetic regulation of T helper 1 (Th1)/Th2 cytokine production and encephalitogenicity in inbred mouse strains.

Conboy IM, DeKruyff RH, Tate KM, Cao ZA, Moore TA, Umetsu DT, Jones PP - J. Exp. Med. (1997)

Downregulation of IFN-γ and TNF-α levels is not affected  when 2-h B10.A culture supernatant is depleted of known Th1/Th2 cytokine regulators. (A and B) B10.BR or B10.A T cells (TCL 4) were restimulated with mMBP Ac1-16 and syngeneic splenic APC for 2 h. Neutralizing antibodies specific for mouse IL-4, IL-10, TNF-α, IFN-γ, TGF-β,  IL-13, and IL-12p40 were added to these culture supernatants for 30 min,  and antibody–cytokine complexes were removed using protein G–coated  agarose beads. Cytokine depleted 2-h supernatants were then used as media in which B10.BR TCL 4 were restimulated with mMBP Ac1-16 and  B10.BR splenic APC for 24 h. IFN-γ (A) and TNF-α (B) levels were  measured as above. Lower levels of TNF-α after treatment with anti– TNF-α antibody and protein G beads were due to the presence of residual anti–TNF-α antibody, which has low affinity for protein G in medium with serum. Neutralizing antibodies were used at concentrations  equal to (not shown) and twice what were recommended by the manufacturers for complete neutralization of cytokine levels produced by high  expressing cells, giving similar results. Data presented are means and standard deviations of triplicate wells for each sample (n = 3). (C) B10.BR  TCL 4 cells were restimulated with B10.BR splenic APC and mMBP  Ac1-16 in 2-h culture supernatants from B10.A or B10.BR TCL 4, used  either undiluted or at serial 1:2 dilutions in RPMI, 10% FCS. TNF-α  levels are means and standard deviations of triplicate wells for each sample  (n = 3).
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Figure 5: Downregulation of IFN-γ and TNF-α levels is not affected when 2-h B10.A culture supernatant is depleted of known Th1/Th2 cytokine regulators. (A and B) B10.BR or B10.A T cells (TCL 4) were restimulated with mMBP Ac1-16 and syngeneic splenic APC for 2 h. Neutralizing antibodies specific for mouse IL-4, IL-10, TNF-α, IFN-γ, TGF-β, IL-13, and IL-12p40 were added to these culture supernatants for 30 min, and antibody–cytokine complexes were removed using protein G–coated agarose beads. Cytokine depleted 2-h supernatants were then used as media in which B10.BR TCL 4 were restimulated with mMBP Ac1-16 and B10.BR splenic APC for 24 h. IFN-γ (A) and TNF-α (B) levels were measured as above. Lower levels of TNF-α after treatment with anti– TNF-α antibody and protein G beads were due to the presence of residual anti–TNF-α antibody, which has low affinity for protein G in medium with serum. Neutralizing antibodies were used at concentrations equal to (not shown) and twice what were recommended by the manufacturers for complete neutralization of cytokine levels produced by high expressing cells, giving similar results. Data presented are means and standard deviations of triplicate wells for each sample (n = 3). (C) B10.BR TCL 4 cells were restimulated with B10.BR splenic APC and mMBP Ac1-16 in 2-h culture supernatants from B10.A or B10.BR TCL 4, used either undiluted or at serial 1:2 dilutions in RPMI, 10% FCS. TNF-α levels are means and standard deviations of triplicate wells for each sample (n = 3).
Mentions: To assess whether downregulation of IFN-γ and TNF-α by B10.A APC is mediated by or depends on any of the known Th1/Th2 regulators, 2-h culture supernatants derived from B10.BR or B10.A TCL/APC cultures were depleted of IL-4, IL-10, IL-12p40, IL-13, TNF-α, IFN-γ, and TGF-β and tested for their effects on IFN-γ and TNF-α production by B10.BR TCL 4 restimulated with B10.BR APC and mMBP peptide. Cytokine depletion did not diminish the downregulatory activity of the B10.A culture supernatant (Fig. 5, A and B). Serial dilutions of B10.A and B10.BR 2-h culture supernatants (Fig. 5 C) demonstrated that the downregulatory activity of B10.A-derived factor is rapidly lost by dilution, indicating that any depletion of the downregulatory cytokine would have been detected. Essentially identical results were obtained when the anti-cytokine antibodies were also used at half the concentrations (not shown).

Bottom Line: A splenic APC reduced IFN-gamma and TNF-alpha production by established Th1 MBP-specific Ak-restricted B10.BR TCL and by a Th1 KLH-specific, Ek-restricted B10.BR T cell clone.The nature of the downregulatory activity of B10.A APC on IFN-gamma and TNF-alpha production was explored. 2-hour supernatants from antigen-activated B10.A APC/TCL cultures or from B10.A APC activated by LPS had the same inhibitory effects on IFN-gamma and TNF-alpha production by B10.BR TCL.The downregulatory effects of B10.A APC are independent of TNF-alpha, IL-4, IL-10, IL-12p40, IFN-gamma, IL-13, TGF-beta, and PGE2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, California 94305-5020, USA.

ABSTRACT
Development of T helper cell (Th)1 or Th2 cytokine responses is essential for effector and regulatory functions of T helper cells. We have compared cytokine profiles of myelin basic protein (MBP) Ac1-16 peptide-specific T helper cells from inbred mouse strains expressing identical k haplotype-derived MHC class II molecules B10.A and B10.BR, B10.BR T cell lines (TCL) produced Th1 cytokines (including high levels of TNF-alpha) and induced experimental autoimmune encephalomyelitis after adoptive transfer. In contrast, B10.A TCL produced Th2 cytokines (including low levels of TNF-alpha) and were poorly encephalitogenic. The contributions of the genetic origin of the T cells and the APC were explored. Serial restimulations of the B10.BR TCL with B10.A or (B10.A x B10.BR) F1 splenic antigen presenting cells (APC) during the establishment of TCL markedly reduced both Th1 cytokine production and encephalitogenicity. In addition, a single restimulation with B10. A splenic APC reduced IFN-gamma and TNF-alpha production by established Th1 MBP-specific Ak-restricted B10.BR TCL and by a Th1 KLH-specific, Ek-restricted B10.BR T cell clone. These studies suggest that B10.A and B10.BR APC differ in their ability to stimulate IFN-gamma and TNF-alpha production by mature Th1 cells and also influence their Th1/Th2 commitment in vivo. The nature of the downregulatory activity of B10.A APC on IFN-gamma and TNF-alpha production was explored. 2-hour supernatants from antigen-activated B10.A APC/TCL cultures or from B10.A APC activated by LPS had the same inhibitory effects on IFN-gamma and TNF-alpha production by B10.BR TCL. The downregulatory effects of B10.A APC are independent of TNF-alpha, IL-4, IL-10, IL-12p40, IFN-gamma, IL-13, TGF-beta, and PGE2. Thus, genetic difference(s) between B10.A and B10.BR APC appear(s) to control the production or activity of a novel soluble cytokine regulatory factor that influences Th1/Th2 commitment and controls production of IFN-gamma and TNF-alpha by mature Th1 cells.

Show MeSH
Related in: MedlinePlus