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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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Similar stimulation thresholds for IFN-γ and CD40L production. 3C6 cells were stimulated for 8 h with P13.9 APC alone (left) or  with P13.9 pulsed with 10 μM PCC88-104 (right) in the absence of monensin, then stained surface CD40L expression and intracellular IFN-γ  content.
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Figure 3: Similar stimulation thresholds for IFN-γ and CD40L production. 3C6 cells were stimulated for 8 h with P13.9 APC alone (left) or with P13.9 pulsed with 10 μM PCC88-104 (right) in the absence of monensin, then stained surface CD40L expression and intracellular IFN-γ content.

Mentions: Multiparameter flow cytometric assays for intracellular cytokine levels were conducted to examine whether these ELISA findings reflect the properties of individual cells in a population. In accord with the lower antigen dose requirement for IFN-γ production seen by ELISA, staining of activated A.E7 for IFN-γ and IL-2 after low level (0.1 nM) antigen exposure reveals only two populations, IFN-γ single producers and cells making neither IFN-γ nor IL-2 at detectable levels (Fig. 2). As the antigen concentration is increased, cells producing IL-2 become apparent, but these IL-2 producers also make IFN-γ. Both the number of cells making IFN-γ and the amount of this cytokine per cell increase with antigen concentration. In contrast, IL-2 production remains more constant per cell as ligand increases, with the primary change being in the number of cells making this cytokine at a fixed level. 3C6 shows a similar pattern as antigen dose is increased, although somewhat more cells making IL-2 with low accompanying IFN-γ production are observed at a high antigen concentration (Fig. 2). These single cell data thus confirm the results of the ELISA, showing that IFN-γ is made by the cells at lower offered antigen doses than those required for IL-2. They further show that this hierarchical relationship reflects the behavior of individual cells in the population, each of which makes either IFN-γ or both IFN-γ and IL-2, but not IL-2 alone. Each cell thus shows a higher response threshold for IL-2 production, rather than there being two different sets of cells in the population each making either IL-2 or IFN-γ, and each with distinct sensitivities to TCR signals. In contrast to this relationship for IL-2 and IFN-γ, a similar analysis examining single cell IFN-γ and CD40L responses shows a different result (Fig. 3). Virtually no cells make only one or the other cytokine, and only a small fraction of cells makes responses strongly biased to one or the other mediator. Instead, the majority of cells make similar levels of both cytokines, indicating that for these two responses, the triggering thresholds are similar.


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)

Similar stimulation thresholds for IFN-γ and CD40L production. 3C6 cells were stimulated for 8 h with P13.9 APC alone (left) or  with P13.9 pulsed with 10 μM PCC88-104 (right) in the absence of monensin, then stained surface CD40L expression and intracellular IFN-γ  content.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Similar stimulation thresholds for IFN-γ and CD40L production. 3C6 cells were stimulated for 8 h with P13.9 APC alone (left) or with P13.9 pulsed with 10 μM PCC88-104 (right) in the absence of monensin, then stained surface CD40L expression and intracellular IFN-γ content.
Mentions: Multiparameter flow cytometric assays for intracellular cytokine levels were conducted to examine whether these ELISA findings reflect the properties of individual cells in a population. In accord with the lower antigen dose requirement for IFN-γ production seen by ELISA, staining of activated A.E7 for IFN-γ and IL-2 after low level (0.1 nM) antigen exposure reveals only two populations, IFN-γ single producers and cells making neither IFN-γ nor IL-2 at detectable levels (Fig. 2). As the antigen concentration is increased, cells producing IL-2 become apparent, but these IL-2 producers also make IFN-γ. Both the number of cells making IFN-γ and the amount of this cytokine per cell increase with antigen concentration. In contrast, IL-2 production remains more constant per cell as ligand increases, with the primary change being in the number of cells making this cytokine at a fixed level. 3C6 shows a similar pattern as antigen dose is increased, although somewhat more cells making IL-2 with low accompanying IFN-γ production are observed at a high antigen concentration (Fig. 2). These single cell data thus confirm the results of the ELISA, showing that IFN-γ is made by the cells at lower offered antigen doses than those required for IL-2. They further show that this hierarchical relationship reflects the behavior of individual cells in the population, each of which makes either IFN-γ or both IFN-γ and IL-2, but not IL-2 alone. Each cell thus shows a higher response threshold for IL-2 production, rather than there being two different sets of cells in the population each making either IL-2 or IFN-γ, and each with distinct sensitivities to TCR signals. In contrast to this relationship for IL-2 and IFN-γ, a similar analysis examining single cell IFN-γ and CD40L responses shows a different result (Fig. 3). Virtually no cells make only one or the other cytokine, and only a small fraction of cells makes responses strongly biased to one or the other mediator. Instead, the majority of cells make similar levels of both cytokines, indicating that for these two responses, the triggering thresholds are similar.

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