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n2:pmcid: PMC0
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Subject Item: _:vb47651
rdf:type: n8:Section
dc:title: materials and methods
n8:contains: _:vb47656 _:vb47652 _:vb47653 _:vb47654 _:vb47655

Subject Item: _:vb47652
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rdf:value: Splenic DC were used instead of the DC that can be obtained from cytokine-stimulated bone marrow cultures, to minimize potential presentation of FCS serum proteins that could lead to background detection of FCSspecific T cells (>>10<<).
n2:mentions: n3:1460426

Subject Item: _:vb47653
rdf:type: n2:Context
rdf:value: DC purity was confirmed by flow cytometry as described by Levin et al. (>>11<<). In brief, purified DC (105) were incubated with FITC-labeled anti-FcR mAb 2.4G2 (PharMingen, San Diego, CA), FITC-labeled anti-B220 mAb RA3-6B2 (PharMingen), biotin-labeled anti-class II MHC mAb M5/114, and PE-labeled streptavidin (SA)
n2:mentions: n3:7903097

Subject Item: _:vb47654
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rdf:value: Cells were then fixed in 2% formaldehyde, and permeabilized with 0.5% saponin, as previously described (>>12<<, 13). A PE-labeled anti–IL-2 mAb S4B6 (PharMingen) or isotype control mAb R35-95 (PharMingen) was then added to detect cytosolic IL-2. After washing, 1,000–3,000 CD4+, KJ1-26+ or CD4+, KJ1-26− events were collected and analyzed.
n2:mentions: n3:7910138

Subject Item: _:vb47655
rdf:type: n2:Context
rdf:value: Cells were then fixed in 2% formaldehyde, and permeabilized with 0.5% saponin, as previously described (12, >>13<<). A PE-labeled anti–IL-2 mAb S4B6 (PharMingen) or isotype control mAb R35-95 (PharMingen) was then added to detect cytosolic IL-2. After washing, 1,000–3,000 CD4+, KJ1-26+ or CD4+, KJ1-26− events were collected and analyzed.
n2:mentions: n3:7595206

Subject Item: _:vb47656
rdf:type: n2:Context
rdf:value: Confocal microscopy and image analyses were performed as previously described by Brelje et al. (>>14<<). In brief, sections were analyzed using a confocal microscope equipped with a krypton/argon laser (MRC-1000; Bio-Rad Life Science Group, Hercules, CA). Separate green and red images were collected for each section analyzed. Final image
n2:mentions: n3:8246789

Subject Item: _:vb47657
rdf:type: n8:Section
dc:title: results
n8:contains: _:vb47658 _:vb47659 _:vb47660 _:vb47661

Subject Item: _:vb47658
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rdf:value: Using immunohistochemical detection with the KJ1-26 mAb, we previously showed that DO11.10 T cells were present in the T cell–rich paracortical regions of all lymph nodes within 24 h of intravenous injection (>>5<<). Similarly, intravenously injected, CMFDA-labeled DO11.10 T cells were found in the paracortical regions of the lymph nodes (Fig.
n2:mentions: n3:7889419

Subject Item: _:vb47659
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rdf:value: Approximately 75% of the CD4+ cells purified from the DO11.10 mice express the DO11.10 TCR-α and TCR-β chains (>>6<<, 15). Because of incomplete allelic exclusion, the remaining 25% express the DO11.10 TCR-β chain with an endogenous TCR-α chain, and are specific for antigens other than OVA (15).
n2:mentions: n3:2125367

Subject Item: _:vb47660
rdf:type: n2:Context
rdf:value: Approximately 75% of the CD4+ cells purified from the DO11.10 mice express the DO11.10 TCR-α and TCR-β chains (6, >>15<<). Because of incomplete allelic exclusion, the remaining 25% express the DO11.10 TCR-β chain with an endogenous TCR-α chain, and are specific for antigens other than OVA (15).
n2:mentions: n3:8955176

Subject Item: _:vb47661
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rdf:value: Because of incomplete allelic exclusion, the remaining 25% express the DO11.10 TCR-β chain with an endogenous TCR-α chain, and are specific for antigens other than OVA (>>15<<). DO11.10 mice were backcrossed with SCID mice, which produce endogenous TCR-α chains only at very low levels, to exclude the possibility that endogenous TCR-α chain–expressing T cells were interacting with the DC. Flow cytometric
n2:mentions: n3:8955176

Subject Item: _:vb47662
rdf:type: n8:Section
dc:title: discussion
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Subject Item: _:vb47663
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rdf:value: Our results are consistent with those of others who showed that clusters of proliferating T cells are found in proximity to T cell zone DC after injection of superantigens (>>2<<) or allogeneic cells (3).
n2:mentions: n3:9053455

Subject Item: _:vb47664
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rdf:value: Our results are consistent with those of others who showed that clusters of proliferating T cells are found in proximity to T cell zone DC after injection of superantigens (2) or allogeneic cells (>>3<<). The capacity of individual OVA peptide-pulsed DC to simultaneously interact with many antigen-specific T cells in vivo is reminiscent of the in vitro studies of Steinman and coworkers (16–19), and provides a possible explanation for the
n2:mentions: n3:9034155

Subject Item: _:vb47665
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rdf:value: The capacity of individual OVA peptide-pulsed DC to simultaneously interact with many antigen-specific T cells in vivo is reminiscent of the in vitro studies of Steinman and coworkers (>>16<<–19), and provides a possible explanation for the ability of small numbers of tumor peptide-pulsed DC to induce cancer immunity (20, 21).
n2:mentions: n3:2962742 n3:6206192 n3:2935590 n3:2835312

Subject Item: _:vb47666
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rdf:value: many antigen-specific T cells in vivo is reminiscent of the in vitro studies of Steinman and coworkers (16–19), and provides a possible explanation for the ability of small numbers of tumor peptide-pulsed DC to induce cancer immunity (>>20<<, 21). It should also be noted that DO11.10 T cells formed clusters around endogenous DC following soluble OVA injection indicating that indeed DC play an important role in antigen presentation in vivo.
n2:mentions: n3:7489412

Subject Item: _:vb47667
rdf:type: n2:Context
rdf:value: antigen-specific T cells in vivo is reminiscent of the in vitro studies of Steinman and coworkers (16–19), and provides a possible explanation for the ability of small numbers of tumor peptide-pulsed DC to induce cancer immunity (20, >>21<<). It should also be noted that DO11.10 T cells formed clusters around endogenous DC following soluble OVA injection indicating that indeed DC play an important role in antigen presentation in vivo.
n2:mentions: n3:8690042

Subject Item: _:vb47668
rdf:type: n2:Context
rdf:value: It is possible that these transient interactions are stabilized if the DC display the appropriate peptide–MHC complexes and activate the interacting T cells to upregulate the activity of their adhesion molecules (>>22<<). The subsequent stable binding between antigen-specific T cells and antigen-presenting DC would allow sustained TCR signaling, which has been shown to be required for T cell commitment to lymphokine production (23). An alternative
n2:mentions: n3:2477710

Subject Item: _:vb47669
rdf:type: n2:Context
rdf:value: The subsequent stable binding between antigen-specific T cells and antigen-presenting DC would allow sustained TCR signaling, which has been shown to be required for T cell commitment to lymphokine production (>>23<<). An alternative possibility is that naive T cells are first activated by an APC from the recipient before binding to the OVA peptide-pulsed, labeled DC. This is unlikely because it would require transfer of OVA peptide from the labeled
n2:mentions: n3:7836913

Subject Item: _:vb47670
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rdf:value: CD40 signaling has been shown to stimulate cytokine production and costimulatory molecule expression in DC, and the activated DO11.10 T cells present in the clusters would be expected to express CD40 ligand (>>24<<). However, if dye metabolism due to DC activation was the only explanation, then many DO11.10 T cell clusters lacking a labeled DC should have been observed. This was not the case; many fewer clusters were present at 48 h but most
n2:mentions: n3:8630727

Subject Item: _:vb47671
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rdf:value: CD4+ T cell killing of the cognate APC has been described in several cases (>>25<<, 26). In addition, a recent study by De Smedt et al. (27) showed that LPS also induces the activation and then disappearance of DC in the spleen. Therefore, DC activation by inflammatory cytokines or cognate interactions with
n2:mentions: n3:2228021

Subject Item: _:vb47672
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rdf:value: CD4+ T cell killing of the cognate APC has been described in several cases (25, >>26<<). In addition, a recent study by De Smedt et al. (27) showed that LPS also induces the activation and then disappearance of DC in the spleen. Therefore, DC activation by inflammatory cytokines or cognate interactions with antigen-specific
n2:mentions: n3:7603571

Subject Item: _:vb47673
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rdf:value: In addition, a recent study by De Smedt et al. (>>27<<) showed that LPS also induces the activation and then disappearance of DC in the spleen.
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