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Single cell analysis reveals regulated hierarchical T cell antigen receptor signaling thresholds and intraclonal heterogeneity for individual cytokine responses of CD4+ T cells.

Itoh Y, Germain RN - J. Exp. Med. (1997)

Bottom Line: T cell receptor (TCR) recognition of peptide-major histocompatibility complex antigens can elicit a diverse array of effector activities.Finally, although the IFN-gamma/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-gamma and not IL-2, and the amount of IFN-gamma exceeds that in double producers.Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.

ABSTRACT
T cell receptor (TCR) recognition of peptide-major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4(+) cell population. Low concentrations of TCR ligand elicit only interferon-gamma (IFN-gamma) production. Increasing ligand recruits more cells into the IFN-gamma+ pool, increases IFN-gamma produced per cell, and also elicits IL-2, but only from cells already making IFN-gamma. Most cells producing only IFN-gamma show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-gamma synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-gamma/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-gamma and not IL-2, and the amount of IFN-gamma exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.

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Effect of CD28 costimulation on response threshold, the number of responding cells, and the response per cell for IL-2 and IFN-γ induced  by anti-CD3 antibody. A.E7 cells were precultured without (top) or with (bottom) anti-CD28 antibody and then added for 5 h to the wells of plates precoated with the indicated concentration of anti-CD3 antibody. The cells were then stained for intracellular IL-2 and IFN-γ.
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Figure 7: Effect of CD28 costimulation on response threshold, the number of responding cells, and the response per cell for IL-2 and IFN-γ induced by anti-CD3 antibody. A.E7 cells were precultured without (top) or with (bottom) anti-CD28 antibody and then added for 5 h to the wells of plates precoated with the indicated concentration of anti-CD3 antibody. The cells were then stained for intracellular IL-2 and IFN-γ.

Mentions: The above experiments were conducted using APCs with a fixed CD80 and ICAM-1 display. Costimulatory signals produced by CD28 interaction with CD80 or CD86 markedly augment the total amount of IL-2 secreted at a given level of offered TCR ligand, with a lesser effect on other cytokines such as IL-3 and IFN-γ (4, 12, 13). The quantitative aspects of the hierarchical relationships just described might thus be modified by changing the costimulatory environment in which the T cell is signaled, an idea for which some support already exists (9, 11, 24, 25, 30). DCEK Hi7 is a transfected APC that has the same I-Ek levels as P13.9, but expresses low CD80 and little or no ICAM-1 (29, 31). At 10 μM peptide, DCEK induces a similar fraction of the cells to produce IFN-γ as does P13.9 (Fig. 6 B), although the amount of IFN-γ per cell is somewhat lower than with the CD80+ cells. However, the difference between P13.9 and DCEK is most remarkable looking at IL-2 production. P13.9 and 10 μM of PCC88-104 induce numerous IL-2– positive cells, whereas DCEK + PCC88-104 stimulate only very few IL-2–producing cells at the same antigen dose, despite promoting the same level of TCR downmodulation (Fig. 6, B and C). These data directly demonstrate that CD28- and ICAM-1–mediated costimulation can change the relative TCR occupancy thresholds required for elicitation of different cytokines. This effect on the functional response to TCR signaling occurs without substantially altering the extent of TCR engagement, indicating an intracellular site of action rather than a change in the efficiency of ligand recognition. Fig. 6 also shows that the change in threshold upon removal of CD28 costimulation reveals itself not in a grossly different amount of IL-2 produced per cell, but in a smaller number of cells producing IL-2 at all. A similar result is obtained if the peptide ligand is replaced by anti-CD3 and CD80 signaling replaced by anti-CD28 antibody, to reduce the possibility that variations in cell–cell contact contribute to these findings (Fig. 7). Thus, the threshold change appears to affect the cells individually in an all-or-none fashion, as also observed when examining the antigen-dose response in the presence of fixed costimulation (Fig. 2).


Single cell analysis reveals regulated hierarchical T cell antigen receptor signaling thresholds and intraclonal heterogeneity for individual cytokine responses of CD4+ T cells.

Itoh Y, Germain RN - J. Exp. Med. (1997)

Effect of CD28 costimulation on response threshold, the number of responding cells, and the response per cell for IL-2 and IFN-γ induced  by anti-CD3 antibody. A.E7 cells were precultured without (top) or with (bottom) anti-CD28 antibody and then added for 5 h to the wells of plates precoated with the indicated concentration of anti-CD3 antibody. The cells were then stained for intracellular IL-2 and IFN-γ.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199012&req=5

Figure 7: Effect of CD28 costimulation on response threshold, the number of responding cells, and the response per cell for IL-2 and IFN-γ induced by anti-CD3 antibody. A.E7 cells were precultured without (top) or with (bottom) anti-CD28 antibody and then added for 5 h to the wells of plates precoated with the indicated concentration of anti-CD3 antibody. The cells were then stained for intracellular IL-2 and IFN-γ.
Mentions: The above experiments were conducted using APCs with a fixed CD80 and ICAM-1 display. Costimulatory signals produced by CD28 interaction with CD80 or CD86 markedly augment the total amount of IL-2 secreted at a given level of offered TCR ligand, with a lesser effect on other cytokines such as IL-3 and IFN-γ (4, 12, 13). The quantitative aspects of the hierarchical relationships just described might thus be modified by changing the costimulatory environment in which the T cell is signaled, an idea for which some support already exists (9, 11, 24, 25, 30). DCEK Hi7 is a transfected APC that has the same I-Ek levels as P13.9, but expresses low CD80 and little or no ICAM-1 (29, 31). At 10 μM peptide, DCEK induces a similar fraction of the cells to produce IFN-γ as does P13.9 (Fig. 6 B), although the amount of IFN-γ per cell is somewhat lower than with the CD80+ cells. However, the difference between P13.9 and DCEK is most remarkable looking at IL-2 production. P13.9 and 10 μM of PCC88-104 induce numerous IL-2– positive cells, whereas DCEK + PCC88-104 stimulate only very few IL-2–producing cells at the same antigen dose, despite promoting the same level of TCR downmodulation (Fig. 6, B and C). These data directly demonstrate that CD28- and ICAM-1–mediated costimulation can change the relative TCR occupancy thresholds required for elicitation of different cytokines. This effect on the functional response to TCR signaling occurs without substantially altering the extent of TCR engagement, indicating an intracellular site of action rather than a change in the efficiency of ligand recognition. Fig. 6 also shows that the change in threshold upon removal of CD28 costimulation reveals itself not in a grossly different amount of IL-2 produced per cell, but in a smaller number of cells producing IL-2 at all. A similar result is obtained if the peptide ligand is replaced by anti-CD3 and CD80 signaling replaced by anti-CD28 antibody, to reduce the possibility that variations in cell–cell contact contribute to these findings (Fig. 7). Thus, the threshold change appears to affect the cells individually in an all-or-none fashion, as also observed when examining the antigen-dose response in the presence of fixed costimulation (Fig. 2).

Bottom Line: T cell receptor (TCR) recognition of peptide-major histocompatibility complex antigens can elicit a diverse array of effector activities.Finally, although the IFN-gamma/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-gamma and not IL-2, and the amount of IFN-gamma exceeds that in double producers.Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.

ABSTRACT
T cell receptor (TCR) recognition of peptide-major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4(+) cell population. Low concentrations of TCR ligand elicit only interferon-gamma (IFN-gamma) production. Increasing ligand recruits more cells into the IFN-gamma+ pool, increases IFN-gamma produced per cell, and also elicits IL-2, but only from cells already making IFN-gamma. Most cells producing only IFN-gamma show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-gamma synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-gamma/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-gamma and not IL-2, and the amount of IFN-gamma exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.

Show MeSH
Related in: MedlinePlus