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Subject Item: _:vb36845
rdf:type: n7:Section
dc:title: materials and methods
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Subject Item: _:vb36846
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rdf:value: Cells were washed again on day 10 and 105 cells were restimulated in 96-well round-bottom plates for 24 h with plate-bound anti-CD3 antibodies (clone TR66; reference >>19<<) to measure IFN-γ and IL-4 in culture supernatants by ELISA assays (provided by H.
n2:mentions: n3:3102250

Subject Item: _:vb36847
rdf:type: n2:Context
rdf:value: Gallati, F. Hoffmann-La Roche AG, Basel, Switzerland; reference >>20<<). To test the effects of type I and type II interferons on T helper cell differentiation, T cell lines were generated by stimulating cord blood leukocytes with PHA in the presence of the indicated cytokines or anti-cytokine antibodies as
n2:mentions: n3:3140597

Subject Item: _:vb36848
rdf:type: n2:Context
rdf:value: CD45RA+ T cells were isolated by two rounds of immunomagnetic negative selection with a mixture of the following monoclonal antibodies: anti-CD16 (B73.1; reference >>21<<), anti-CD45RO (UCHL-1; reference 22) and anti–HLA-DR (1-1C4; reference 23).
n2:mentions: n3:6833758

Subject Item: _:vb36849
rdf:type: n2:Context
rdf:value: CD45RA+ T cells were isolated by two rounds of immunomagnetic negative selection with a mixture of the following monoclonal antibodies: anti-CD16 (B73.1; reference 21), anti-CD45RO (UCHL-1; reference >>22<<) and anti–HLA-DR (1-1C4; reference 23).
n2:mentions: n3:2422219

Subject Item: _:vb36850
rdf:type: n2:Context
rdf:value: T cells were isolated by two rounds of immunomagnetic negative selection with a mixture of the following monoclonal antibodies: anti-CD16 (B73.1; reference 21), anti-CD45RO (UCHL-1; reference 22) and anti–HLA-DR (1-1C4; reference >>23<<). The suspension was incubated with goat anti–mouse IgG-coated Dynabeads (Dynal, Inc., Geat Neck, NY) and exposed to a magnetic field using a magnetic particle concentrator (Dynal, Inc.
n2:mentions: n3:1574119

Subject Item: _:vb36851
rdf:type: n2:Context
rdf:value: Purified CD45RA+ T cells were stimulated with plate-bound anti-CD3 mAbs (TR66; reference >>19<<) without the addition of cytokines or in the presence of IL-12 (2 ng/ml) or IL-4 (200 U/ml).
n2:mentions: n3:3102250

Subject Item: _:vb36852
rdf:type: n2:Context
rdf:value: Th1 and Th2 cells generated from cord blood lymphocytes were cloned by limiting dilution as described (>>24<<). In brief, 0.3 cells/well were plated in 96-well plates in the presence of irradiated (5,000 rads) allogeneic PBMCs, PHA (2 μg/ml), and IL-2 (100 U/ml).
n2:mentions: n3:1722274

Subject Item: _:vb36853
rdf:type: n2:Context
rdf:value: A 327-bp DNA fragment encompassing the cytoplasmic region of the human IL-12R β2 subunit (>>25<<) and a 553-bp EcoRI/BamHI fragment derived from the IL-12R β1 cDNA (26) were subcloned into pGEM 3Z.
n2:mentions: n3:8943050

Subject Item: _:vb36854
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rdf:value: A 327-bp DNA fragment encompassing the cytoplasmic region of the human IL-12R β2 subunit (25) and a 553-bp EcoRI/BamHI fragment derived from the IL-12R β1 cDNA (>>26<<) were subcloned into pGEM 3Z. The constructs were linearized with EcoRI and radiolabeled antisense transcripts were synthesized with SP6 polymerase and a commercial kit according to the manufacturer's protocol (Promega Corp., Madison, WI).
n2:mentions: n3:7911493

Subject Item: _:vb36855
rdf:type: n2:Context
rdf:value: RNA was extracted from Th1 and Th2 lines or clones using Ultraspec total RNA extraction reagent (Biotecx Laboratories Inc., Houston, TX) as described (>>27<<). The antisense RNA probes were hybridized to 10 μg total RNA and ribonuclease protection assays were performed with a commercial kit (Ambion Inc., Austin, TX) according to the company's protocol. Products were resolved on 6% denaturing
n2:mentions: n3:7568143

Subject Item: _:vb36856
rdf:type: n2:Context
rdf:value: [125I]IL-12 binding to human Th1 and Th2 cell lines was determined as described previously (>>28<<). Specific binding was analyzed using the LIGAND program (29) and plotted by the Scatchard method.
n2:mentions: n3:1578138

Subject Item: _:vb36857
rdf:type: n2:Context
rdf:value: Specific binding was analyzed using the LIGAND program (>>29<<) and plotted by the Scatchard method.
n2:mentions: n3:3840547

Subject Item: _:vb36858
rdf:type: n7:Section
dc:title: results and discussion
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Subject Item: _:vb36859
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rdf:value: Using a murine TCR transgenic system, Szabo et al. observed that IL-12 selectively activated the Janus kinase Jak2, and the signal transducers and activators of transcription Stat3 and Stat4 in Th1, but not in differentiated Th2 cells (>>30<<). To ascertain whether human Th1 and Th2 subsets also differ in IL-12 signaling, we examined Stat4 expression and tyrosine phosphorylation in response to IL-12 in Th1 and Th2 lines and clones.
n2:mentions: n3:7796298

Subject Item: _:vb36860
rdf:type: n2:Context
rdf:value: 2, bottom) required for IL-12 signaling (>>31<<, 32). Similarly, after IL-12 stimulation, Jak2 (33), which was also expressed at comparable levels in Th1 and Th2 cells, was phosphorylated only in Th1 cells (data not shown).
n2:mentions: n3:7638186

Subject Item: _:vb36861
rdf:type: n2:Context
rdf:value: 2, bottom) required for IL-12 signaling (31, >>32<<). Similarly, after IL-12 stimulation, Jak2 (33), which was also expressed at comparable levels in Th1 and Th2 cells, was phosphorylated only in Th1 cells (data not shown).
n2:mentions: n3:7722452

Subject Item: _:vb36862
rdf:type: n2:Context
rdf:value: Similarly, after IL-12 stimulation, Jak2 (>>33<<), which was also expressed at comparable levels in Th1 and Th2 cells, was phosphorylated only in Th1 cells (data not shown).
n2:mentions: n3:7528775

Subject Item: _:vb36863
rdf:type: n2:Context
rdf:value: IL-12R β1 or β2, independently expressed in COS cells, bind human IL-12 with low affinity (K D = 3–5 nM), whereas coexpression of IL-12R β1 and β2 in COS cells gives rise to high affinity binding sites (K D = 50 pm) (>>25<<, 26). These studies have also shown that the IL-12R β2 is the signaling component of the IL-12R, in agreement with the fact that the IL-12R β2, in contrast to the IL-12R β1, contains tyrosine residues in its cytoplasmic domain (25). We
n2:mentions: n3:8943050

Subject Item: _:vb36864
rdf:type: n2:Context
rdf:value: IL-12R β1 or β2, independently expressed in COS cells, bind human IL-12 with low affinity (K D = 3–5 nM), whereas coexpression of IL-12R β1 and β2 in COS cells gives rise to high affinity binding sites (K D = 50 pm) (25, >>26<<). These studies have also shown that the IL-12R β2 is the signaling component of the IL-12R, in agreement with the fact that the IL-12R β2, in contrast to the IL-12R β1, contains tyrosine residues in its cytoplasmic domain (25). We
n2:mentions: n3:7911493

Subject Item: _:vb36865
rdf:type: n2:Context
rdf:value: These studies have also shown that the IL-12R β2 is the signaling component of the IL-12R, in agreement with the fact that the IL-12R β2, in contrast to the IL-12R β1, contains tyrosine residues in its cytoplasmic domain (>>25<<). We analyzed the mRNA expression levels for the IL-12R β1 and β2 subunits by ribonuclease protection assays in both Th1 and Th2 lines at different time points after priming and in Th1 and Th2 clones at day 13 after restimulation. Whereas
n2:mentions: n3:8943050

Subject Item: _:vb36866
rdf:type: n2:Context
rdf:value: This difference in IL-12 binding behavior in the mouse and human systems may reflect the dominant role mouse IL-12R β1 plays in binding IL-12 (>>34<<), whereas in humans, both IL-12R β1 and β2 appear to contribute equally to IL-12 binding (25).
n2:mentions: n3:7594587

Subject Item: _:vb36867
rdf:type: n2:Context
rdf:value: This difference in IL-12 binding behavior in the mouse and human systems may reflect the dominant role mouse IL-12R β1 plays in binding IL-12 (34), whereas in humans, both IL-12R β1 and β2 appear to contribute equally to IL-12 binding (>>25<<). These results demonstrate that the IL-12R β2 subunit is selectively expressed in Th1 cells where it is induced upon T cell activation.
n2:mentions: n3:8943050

Subject Item: _:vb36868
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rdf:value: The IL-12–induced transient upregulation of the IL-12R β2 transcripts in Th2 clones may account for the transient period of IFN-γ production in cells with an established Th2 phenotype (>>35<<, 36).
n2:mentions: n3:7908322

Subject Item: _:vb36869
rdf:type: n2:Context
rdf:value: The IL-12–induced transient upregulation of the IL-12R β2 transcripts in Th2 clones may account for the transient period of IFN-γ production in cells with an established Th2 phenotype (35, >>36<<).
n2:mentions: n3:7947460

Subject Item: _:vb36870
rdf:type: n2:Context
rdf:value: The block is complete if IL-12 is not present in the priming culture, but incomplete if IL-12 is present (>>9<<). Consistent with this observation, we found that cord blood lymphocytes primed in the presence of IL-4 and IL-12 together developed into cells secreting both IL-4 (1.1 ng/ml) and IFN-γ (0.9 ng/ml) (Fig. 6 A). These cells phosphorylate
n2:mentions: n3:7901851

Subject Item: _:vb36871
rdf:type: n2:Context
rdf:value: In addition to IL-12, IFN-α and IFN-γ have also been shown to influence Th1 cell development (>>37<<, 38). We therefore examined the effect of these cytokines on the regulation of the IL-12R β2 expression.
n2:mentions: n3:8605940

Subject Item: _:vb36872
rdf:type: n2:Context
rdf:value: In addition to IL-12, IFN-α and IFN-γ have also been shown to influence Th1 cell development (37, >>38<<). We therefore examined the effect of these cytokines on the regulation of the IL-12R β2 expression.
n2:mentions: n3:8568246

Subject Item: _:vb36873
rdf:type: n2:Context
rdf:value: Our findings differ from previous reports showing that in the mouse IFN-γ but not IFN-α influences Th1 cell development (>>38<<). Our data, together with the evidence that IFN-α in vitro favors the development of human allergen-specific T cells into a Th1 phenotype (37), suggest that type I and type II IFNs could have different functional effects on human as
n2:mentions: n3:8568246

Subject Item: _:vb36874
rdf:type: n2:Context
rdf:value: Our data, together with the evidence that IFN-α in vitro favors the development of human allergen-specific T cells into a Th1 phenotype (>>37<<), suggest that type I and type II IFNs could have different functional effects on human as compared to mouse T cells.
n2:mentions: n3:8605940

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