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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.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.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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Hierarchical cytokine production thresholds detected by  ELISA. 3C6 and A.E7 were cultured with P13.9 and PCC88-104. Supernatants were collected 24 h later and IL-2, IL-3, and IFN-γ secretion was  determined by ELISA. The results were converted into cytokine units using standard curves and expressed as the percentage of the maximum response obtained for each cytokine at plateau. Absolute cytokine production representing the maximal response on this scale ranged in various  experiments from 50 to 200 U/ml for IL-2, 6,000 to 19,000 U/ml for  IFN-γ for both cells, 5 to 25 U/ml for 3C6, and 80 to 700 /ml for A.E7  for IL-3.
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Figure 1: Hierarchical cytokine production thresholds detected by ELISA. 3C6 and A.E7 were cultured with P13.9 and PCC88-104. Supernatants were collected 24 h later and IL-2, IL-3, and IFN-γ secretion was determined by ELISA. The results were converted into cytokine units using standard curves and expressed as the percentage of the maximum response obtained for each cytokine at plateau. Absolute cytokine production representing the maximal response on this scale ranged in various experiments from 50 to 200 U/ml for IL-2, 6,000 to 19,000 U/ml for IFN-γ for both cells, 5 to 25 U/ml for 3C6, and 80 to 700 /ml for A.E7 for IL-3.

Mentions: We have shown previously that the ligand concentration needed to elicit 50% of the maximum response by the 3C6 Th1 clone for each of two cytokines (IL-2 and IL-3) can differ by up to 100–fold, and that this relationship depends in large measure on the availability of CD28 costimulation (9). These data were interpreted as evidence for hierarchical TCR occupancy thresholds for triggering individual effector responses. Similar findings have been made in studies using human T cells, which show that induction of IFN-γ production and proliferation require substantially more antigen-induced TCR downmodulation than cytotoxicity (10). To expand on these previous observations, ELISA was used to measure cytokine secretion in response to agonist peptide–MHC class II ligand presented by transfected cells whose CD80/CD86-mediated costimulatory function does not change in response to T cell signals. Fig. 1 shows IFN-γ, IL-2, and IL-3 production by two different Th1 clonal cell populations (3C6 and A.E7), displayed as percentage of maximum response to allow direct comparison of the fractional response for each mediator at each antigen concentration. The ligand amounts eliciting the minimally measurable and maximal responses are different for each cytokine, with the overall hierarchy being IFN-γ > IL-2 > IL-3 for 3C6 and IFN-γ > IL-3 = IL-2 for A.E7. These findings confirm earlier data suggesting that the numbers of TCR ligands needed to elicit a fixed fractional response for different cytokines are distinct. They also demonstrate that among individual clones, some effector responses (IL-3 in this case) may vary in their relative position in this dose- response hierarchy.


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)

Hierarchical cytokine production thresholds detected by  ELISA. 3C6 and A.E7 were cultured with P13.9 and PCC88-104. Supernatants were collected 24 h later and IL-2, IL-3, and IFN-γ secretion was  determined by ELISA. The results were converted into cytokine units using standard curves and expressed as the percentage of the maximum response obtained for each cytokine at plateau. Absolute cytokine production representing the maximal response on this scale ranged in various  experiments from 50 to 200 U/ml for IL-2, 6,000 to 19,000 U/ml for  IFN-γ for both cells, 5 to 25 U/ml for 3C6, and 80 to 700 /ml for A.E7  for IL-3.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199012&req=5

Figure 1: Hierarchical cytokine production thresholds detected by ELISA. 3C6 and A.E7 were cultured with P13.9 and PCC88-104. Supernatants were collected 24 h later and IL-2, IL-3, and IFN-γ secretion was determined by ELISA. The results were converted into cytokine units using standard curves and expressed as the percentage of the maximum response obtained for each cytokine at plateau. Absolute cytokine production representing the maximal response on this scale ranged in various experiments from 50 to 200 U/ml for IL-2, 6,000 to 19,000 U/ml for IFN-γ for both cells, 5 to 25 U/ml for 3C6, and 80 to 700 /ml for A.E7 for IL-3.
Mentions: We have shown previously that the ligand concentration needed to elicit 50% of the maximum response by the 3C6 Th1 clone for each of two cytokines (IL-2 and IL-3) can differ by up to 100–fold, and that this relationship depends in large measure on the availability of CD28 costimulation (9). These data were interpreted as evidence for hierarchical TCR occupancy thresholds for triggering individual effector responses. Similar findings have been made in studies using human T cells, which show that induction of IFN-γ production and proliferation require substantially more antigen-induced TCR downmodulation than cytotoxicity (10). To expand on these previous observations, ELISA was used to measure cytokine secretion in response to agonist peptide–MHC class II ligand presented by transfected cells whose CD80/CD86-mediated costimulatory function does not change in response to T cell signals. Fig. 1 shows IFN-γ, IL-2, and IL-3 production by two different Th1 clonal cell populations (3C6 and A.E7), displayed as percentage of maximum response to allow direct comparison of the fractional response for each mediator at each antigen concentration. The ligand amounts eliciting the minimally measurable and maximal responses are different for each cytokine, with the overall hierarchy being IFN-γ > IL-2 > IL-3 for 3C6 and IFN-γ > IL-3 = IL-2 for A.E7. These findings confirm earlier data suggesting that the numbers of TCR ligands needed to elicit a fixed fractional response for different cytokines are distinct. They also demonstrate that among individual clones, some effector responses (IL-3 in this case) may vary in their relative position in this dose- response hierarchy.

Bottom Line: T cell receptor (TCR) recognition of peptide-major histocompatibility complex antigens can elicit a diverse array of effector activities.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.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