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Restricted clonal expression of IL-2 by naive T cells reflects differential dynamic interactions with dendritic cells.

Hurez V, Saparov A, Tousson A, Fuller MJ, Kubo T, Oliver J, Weaver BT, Weaver CT - J. Exp. Med. (2003)

Bottom Line: T cells destined to produce IL-2 required prolonged interactions with DCs, whereas most T cells established only transient interactions with DCs and were activated, but did not express IL-2.Extended conjugation of T cells with DCs was not always sufficient to initiate IL-2 expression.Thus, there is intrinsic variability in clonal T cell populations that restricts IL-2 commitment, and prolonged engagement with mature DCs is necessary, but not sufficient, for IL-2 gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, AL 35294, USA.

ABSTRACT
Limited frequencies of T cells express IL-2 in primary antigenic responses, despite activation marker expression and proliferation by most clonal members. To define the basis for restricted IL-2 expression, a videomicroscopic system and IL-2 reporter transgenic model were used to characterize dendritic cell (DC)-T cell interactions. T cells destined to produce IL-2 required prolonged interactions with DCs, whereas most T cells established only transient interactions with DCs and were activated, but did not express IL-2. Extended conjugation of T cells with DCs was not always sufficient to initiate IL-2 expression. Thus, there is intrinsic variability in clonal T cell populations that restricts IL-2 commitment, and prolonged engagement with mature DCs is necessary, but not sufficient, for IL-2 gene transcription.

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Limited complete activation of naive T cell population is not due to APC exhaustion. Naive CD4 T cells isolated from DO11.RAG-2−/− mice were labeled with an FL2-detectable vital dye (“DO11.10 T cells” in red) before culture with purified splenic DCs and OVAp as described in Fig. 2. 4 h after initiation of culture, a second aliquot of DO11.RAG-2−/− T cells bearing the IL-2P/GFP transgene (“DO11.IL-2P/GFP” in green), which was not dye-labeled, was added to the initial culture, with (middle) or without (bottom) fresh splenic DCs. Incubation was continued for an additional 20 h, and cells were recovered and analyzed for GFP expression in lymphocyte or dye-negative lymphocyte gates. A culture that contained only DO11.IL-2P/GFP and DCs for the duration of the total incubation period was included as a positive control for reporter induction. The percentage of GFP+ cells in the unlabeled, DO11.IL-2P/GFP fraction is indicated in the histograms (right).
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fig3: Limited complete activation of naive T cell population is not due to APC exhaustion. Naive CD4 T cells isolated from DO11.RAG-2−/− mice were labeled with an FL2-detectable vital dye (“DO11.10 T cells” in red) before culture with purified splenic DCs and OVAp as described in Fig. 2. 4 h after initiation of culture, a second aliquot of DO11.RAG-2−/− T cells bearing the IL-2P/GFP transgene (“DO11.IL-2P/GFP” in green), which was not dye-labeled, was added to the initial culture, with (middle) or without (bottom) fresh splenic DCs. Incubation was continued for an additional 20 h, and cells were recovered and analyzed for GFP expression in lymphocyte or dye-negative lymphocyte gates. A culture that contained only DO11.IL-2P/GFP and DCs for the duration of the total incubation period was included as a positive control for reporter induction. The percentage of GFP+ cells in the unlabeled, DO11.IL-2P/GFP fraction is indicated in the histograms (right).

Mentions: Having established the minimal DC–T cell conjugation time required for maximal IL-2 recruitment, we sought to determine whether T cells that engaged antigen-bearing DCs early in the antigenic response had a competitive advantage over those that engaged DCs late. A two-step culture system was devised, wherein splenic DCs were preincubated with DO11.RAG-2−/− CD4 T cells and OVAp for 4 h before the addition of fresh DO11.IL-2P/GFP.RAG-2−/− T cells or fresh DO11.IL-2P/GFP.RAG-2−/− T cells, plus fresh DCs (Fig. 3). The addition of the second T cell population was delayed 4 h after initiation of the preculture to allow formation of DC–T cell conjugates sufficient to fully activate the first wave of T cells, and the cultures were continued an additional 20 h before analysis. Because only the T cells added late to the cultures bore the GFP reporter transgene, IL-2 expression could be uniquely detected in this population. To further distinguish the “early” and “late” T cell populations to permit calculation of IL-2P/GFP transgene expression frequencies, DO11.RAG-2−/− T cells were prelabeled with a red fluorescent dye that identified them.


Restricted clonal expression of IL-2 by naive T cells reflects differential dynamic interactions with dendritic cells.

Hurez V, Saparov A, Tousson A, Fuller MJ, Kubo T, Oliver J, Weaver BT, Weaver CT - J. Exp. Med. (2003)

Limited complete activation of naive T cell population is not due to APC exhaustion. Naive CD4 T cells isolated from DO11.RAG-2−/− mice were labeled with an FL2-detectable vital dye (“DO11.10 T cells” in red) before culture with purified splenic DCs and OVAp as described in Fig. 2. 4 h after initiation of culture, a second aliquot of DO11.RAG-2−/− T cells bearing the IL-2P/GFP transgene (“DO11.IL-2P/GFP” in green), which was not dye-labeled, was added to the initial culture, with (middle) or without (bottom) fresh splenic DCs. Incubation was continued for an additional 20 h, and cells were recovered and analyzed for GFP expression in lymphocyte or dye-negative lymphocyte gates. A culture that contained only DO11.IL-2P/GFP and DCs for the duration of the total incubation period was included as a positive control for reporter induction. The percentage of GFP+ cells in the unlabeled, DO11.IL-2P/GFP fraction is indicated in the histograms (right).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196090&req=5

fig3: Limited complete activation of naive T cell population is not due to APC exhaustion. Naive CD4 T cells isolated from DO11.RAG-2−/− mice were labeled with an FL2-detectable vital dye (“DO11.10 T cells” in red) before culture with purified splenic DCs and OVAp as described in Fig. 2. 4 h after initiation of culture, a second aliquot of DO11.RAG-2−/− T cells bearing the IL-2P/GFP transgene (“DO11.IL-2P/GFP” in green), which was not dye-labeled, was added to the initial culture, with (middle) or without (bottom) fresh splenic DCs. Incubation was continued for an additional 20 h, and cells were recovered and analyzed for GFP expression in lymphocyte or dye-negative lymphocyte gates. A culture that contained only DO11.IL-2P/GFP and DCs for the duration of the total incubation period was included as a positive control for reporter induction. The percentage of GFP+ cells in the unlabeled, DO11.IL-2P/GFP fraction is indicated in the histograms (right).
Mentions: Having established the minimal DC–T cell conjugation time required for maximal IL-2 recruitment, we sought to determine whether T cells that engaged antigen-bearing DCs early in the antigenic response had a competitive advantage over those that engaged DCs late. A two-step culture system was devised, wherein splenic DCs were preincubated with DO11.RAG-2−/− CD4 T cells and OVAp for 4 h before the addition of fresh DO11.IL-2P/GFP.RAG-2−/− T cells or fresh DO11.IL-2P/GFP.RAG-2−/− T cells, plus fresh DCs (Fig. 3). The addition of the second T cell population was delayed 4 h after initiation of the preculture to allow formation of DC–T cell conjugates sufficient to fully activate the first wave of T cells, and the cultures were continued an additional 20 h before analysis. Because only the T cells added late to the cultures bore the GFP reporter transgene, IL-2 expression could be uniquely detected in this population. To further distinguish the “early” and “late” T cell populations to permit calculation of IL-2P/GFP transgene expression frequencies, DO11.RAG-2−/− T cells were prelabeled with a red fluorescent dye that identified them.

Bottom Line: T cells destined to produce IL-2 required prolonged interactions with DCs, whereas most T cells established only transient interactions with DCs and were activated, but did not express IL-2.Extended conjugation of T cells with DCs was not always sufficient to initiate IL-2 expression.Thus, there is intrinsic variability in clonal T cell populations that restricts IL-2 commitment, and prolonged engagement with mature DCs is necessary, but not sufficient, for IL-2 gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, AL 35294, USA.

ABSTRACT
Limited frequencies of T cells express IL-2 in primary antigenic responses, despite activation marker expression and proliferation by most clonal members. To define the basis for restricted IL-2 expression, a videomicroscopic system and IL-2 reporter transgenic model were used to characterize dendritic cell (DC)-T cell interactions. T cells destined to produce IL-2 required prolonged interactions with DCs, whereas most T cells established only transient interactions with DCs and were activated, but did not express IL-2. Extended conjugation of T cells with DCs was not always sufficient to initiate IL-2 expression. Thus, there is intrinsic variability in clonal T cell populations that restricts IL-2 commitment, and prolonged engagement with mature DCs is necessary, but not sufficient, for IL-2 gene transcription.

Show MeSH