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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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Cytokine production by anergized T cells. A.E7 was cultured with P13.9 alone (A) or P13.9 with 50 μM MCC97I (B) for 24 h. After separation from APC and resting for a week, cells were restimulated with P13.9 and 10 μM PCC88-104 in the presence of monensin, then stained with  anti–IFN-γ, IL-2, and TCR antibodies. IFN-γ production and TCR expression are shown after the second incubation in C and D, respectively. Cells  precultured with P13.9 and MCC97I (—) or P13.9 without peptide (----) were restimulated with P13.9 and PCC88-104. Cells without antigen restimulation were shown (·····) for both cell populations.
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Figure 8: Cytokine production by anergized T cells. A.E7 was cultured with P13.9 alone (A) or P13.9 with 50 μM MCC97I (B) for 24 h. After separation from APC and resting for a week, cells were restimulated with P13.9 and 10 μM PCC88-104 in the presence of monensin, then stained with anti–IFN-γ, IL-2, and TCR antibodies. IFN-γ production and TCR expression are shown after the second incubation in C and D, respectively. Cells precultured with P13.9 and MCC97I (—) or P13.9 without peptide (----) were restimulated with P13.9 and PCC88-104. Cells without antigen restimulation were shown (·····) for both cell populations.

Mentions: If Th1 cells are first exposed to TCR ligand and deprived of IL-2, subsequent stimulation in the presence of CD80/86+ APC reveals a relatively selective loss of IL-2 production (proliferative anergy) (4, 12, 13). The predominant effect on IL-2 has been attributed to a specific signaling lesion affecting IL-2 gene induction, possibly mediated through a block in the ras-raf-ERK pathway (32, 33). Like elimination of CD28 costimulation, this may also represent a shift in the relative TCR occupancy thresholds for individual cytokine responses. To explore this possibility, A.E7 cells were rendered anergic using the partial agonist peptide 97I (31), then rested. The recovered cells were restimulated using CD80+/ICAM-1+ APC and agonist PCC88-104 peptide. Cells precultured with antigen-free APC show the expected IFN-γ only and IFN-γ + IL-2 double-producing populations upon restimulation (Fig. 8 A). In contrast, T cells precultured with APC bearing 97I show very few cells producing IL-2, and the population of high IFN-γ only producers requiring extensive TCR engagement under normal conditions is also diminished (Fig. 8, B and C). Cells from both treatment groups have the same TCR levels at the beginning and end of the assay culture (Fig. 8 D), indicating that there is no defect in occupancy-induced downmodulation in the 97I-exposed (anergic) cells. Thus, the cytokine response of Th1 cells rendered anergic resembles that of cells exposed to low levels of antigen or deprived of CD28 costimulation (compare Figs. 2, 6, 7, and 8 B).


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)

Cytokine production by anergized T cells. A.E7 was cultured with P13.9 alone (A) or P13.9 with 50 μM MCC97I (B) for 24 h. After separation from APC and resting for a week, cells were restimulated with P13.9 and 10 μM PCC88-104 in the presence of monensin, then stained with  anti–IFN-γ, IL-2, and TCR antibodies. IFN-γ production and TCR expression are shown after the second incubation in C and D, respectively. Cells  precultured with P13.9 and MCC97I (—) or P13.9 without peptide (----) were restimulated with P13.9 and PCC88-104. Cells without antigen restimulation were shown (·····) for both cell populations.
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Related In: Results  -  Collection

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Figure 8: Cytokine production by anergized T cells. A.E7 was cultured with P13.9 alone (A) or P13.9 with 50 μM MCC97I (B) for 24 h. After separation from APC and resting for a week, cells were restimulated with P13.9 and 10 μM PCC88-104 in the presence of monensin, then stained with anti–IFN-γ, IL-2, and TCR antibodies. IFN-γ production and TCR expression are shown after the second incubation in C and D, respectively. Cells precultured with P13.9 and MCC97I (—) or P13.9 without peptide (----) were restimulated with P13.9 and PCC88-104. Cells without antigen restimulation were shown (·····) for both cell populations.
Mentions: If Th1 cells are first exposed to TCR ligand and deprived of IL-2, subsequent stimulation in the presence of CD80/86+ APC reveals a relatively selective loss of IL-2 production (proliferative anergy) (4, 12, 13). The predominant effect on IL-2 has been attributed to a specific signaling lesion affecting IL-2 gene induction, possibly mediated through a block in the ras-raf-ERK pathway (32, 33). Like elimination of CD28 costimulation, this may also represent a shift in the relative TCR occupancy thresholds for individual cytokine responses. To explore this possibility, A.E7 cells were rendered anergic using the partial agonist peptide 97I (31), then rested. The recovered cells were restimulated using CD80+/ICAM-1+ APC and agonist PCC88-104 peptide. Cells precultured with antigen-free APC show the expected IFN-γ only and IFN-γ + IL-2 double-producing populations upon restimulation (Fig. 8 A). In contrast, T cells precultured with APC bearing 97I show very few cells producing IL-2, and the population of high IFN-γ only producers requiring extensive TCR engagement under normal conditions is also diminished (Fig. 8, B and C). Cells from both treatment groups have the same TCR levels at the beginning and end of the assay culture (Fig. 8 D), indicating that there is no defect in occupancy-induced downmodulation in the 97I-exposed (anergic) cells. Thus, the cytokine response of Th1 cells rendered anergic resembles that of cells exposed to low levels of antigen or deprived of CD28 costimulation (compare Figs. 2, 6, 7, and 8 B).

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