Limits...
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.

Show MeSH
Kinetics of commitment to IL-2 gene transcription and TCR down-modulation by naive T cells. CD4 T cells from DO11.IL-2P/GFP Tg mice were cultured with purified splenic DCs and 5 μg/ml OVAp after centrifugation onto the bottom of culture wells. At the indicated times, CD4 T cells were recovered by magnetic sorting after mechanical disruption of the T cell–APC clusters, and were returned to culture without DCs for a total of 20 h. Expression of CD4, transgenic TCR (KJ1–26), and IL-2P/GFP transgene was assessed by flow cytometry. Control cultures were as follows: APC and T cells without OVAp (“No Ag”) and an unseparated culture (OVAp/ “20 h”). Cytometric analyses of cells not recovered by magnetic sorting established that >92% of the total CD4+GFP+ and >87% of the CD4+GFP− T cells were recovered for subsequent culture (see also Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20022230/DC1). Red circles denote contaminating DCs.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196090&req=5

fig2: Kinetics of commitment to IL-2 gene transcription and TCR down-modulation by naive T cells. CD4 T cells from DO11.IL-2P/GFP Tg mice were cultured with purified splenic DCs and 5 μg/ml OVAp after centrifugation onto the bottom of culture wells. At the indicated times, CD4 T cells were recovered by magnetic sorting after mechanical disruption of the T cell–APC clusters, and were returned to culture without DCs for a total of 20 h. Expression of CD4, transgenic TCR (KJ1–26), and IL-2P/GFP transgene was assessed by flow cytometry. Control cultures were as follows: APC and T cells without OVAp (“No Ag”) and an unseparated culture (OVAp/ “20 h”). Cytometric analyses of cells not recovered by magnetic sorting established that >92% of the total CD4+GFP+ and >87% of the CD4+GFP− T cells were recovered for subsequent culture (see also Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20022230/DC1). Red circles denote contaminating DCs.

Mentions: For the T cell–DC contact time determinations, CD4 T cells were separated after the indicated time of culture with DCs by mechanical disruption of the aggregates followed by purification using anti-CD4 magnetic beads (Dynal). The isolated CD4 cells were further incubated for a total of 20 h before measurement of GFP expression. Greater than 96% of the recovered T cells at each time point were CD4hi and negative for expression of CD11c and MHC class II (see Figs. 2 and S1 and unpublished data).


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)

Kinetics of commitment to IL-2 gene transcription and TCR down-modulation by naive T cells. CD4 T cells from DO11.IL-2P/GFP Tg mice were cultured with purified splenic DCs and 5 μg/ml OVAp after centrifugation onto the bottom of culture wells. At the indicated times, CD4 T cells were recovered by magnetic sorting after mechanical disruption of the T cell–APC clusters, and were returned to culture without DCs for a total of 20 h. Expression of CD4, transgenic TCR (KJ1–26), and IL-2P/GFP transgene was assessed by flow cytometry. Control cultures were as follows: APC and T cells without OVAp (“No Ag”) and an unseparated culture (OVAp/ “20 h”). Cytometric analyses of cells not recovered by magnetic sorting established that >92% of the total CD4+GFP+ and >87% of the CD4+GFP− T cells were recovered for subsequent culture (see also Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20022230/DC1). Red circles denote contaminating DCs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196090&req=5

fig2: Kinetics of commitment to IL-2 gene transcription and TCR down-modulation by naive T cells. CD4 T cells from DO11.IL-2P/GFP Tg mice were cultured with purified splenic DCs and 5 μg/ml OVAp after centrifugation onto the bottom of culture wells. At the indicated times, CD4 T cells were recovered by magnetic sorting after mechanical disruption of the T cell–APC clusters, and were returned to culture without DCs for a total of 20 h. Expression of CD4, transgenic TCR (KJ1–26), and IL-2P/GFP transgene was assessed by flow cytometry. Control cultures were as follows: APC and T cells without OVAp (“No Ag”) and an unseparated culture (OVAp/ “20 h”). Cytometric analyses of cells not recovered by magnetic sorting established that >92% of the total CD4+GFP+ and >87% of the CD4+GFP− T cells were recovered for subsequent culture (see also Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20022230/DC1). Red circles denote contaminating DCs.
Mentions: For the T cell–DC contact time determinations, CD4 T cells were separated after the indicated time of culture with DCs by mechanical disruption of the aggregates followed by purification using anti-CD4 magnetic beads (Dynal). The isolated CD4 cells were further incubated for a total of 20 h before measurement of GFP expression. Greater than 96% of the recovered T cells at each time point were CD4hi and negative for expression of CD11c and MHC class II (see Figs. 2 and S1 and unpublished data).

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