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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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TCR downmodulation and cytokine production. T cell clone 3C6 was cultured with P13.9 + PCC88-104 (10 μM) in the presence of  monensin. After 5 h of incubation, surface TCR Vβ3, as well as intracellular IFN-γ and IL-2, were analyzed. (A) IFN-γ and IL-2 production by 3C6.  R1 = cells producing both IFN-γ and IL-2; R2 = cells producing IFN-γ alone; R3 = cells producing neither IFN-γ nor IL-2. (B) TCR expression of  each cell subpopulation after incubation with 10 μM PCC88-104 + P13.9. Gates R1, R2, and R3 are as in A. The histogram for cells in R2 largely  overlaps that for cells in R3. (C) Ungated TCR expression of 3C6 incubated with either with 10 μM PCC88-104 (solid line) or without peptide (dotted  line) + P13.9. The gates set to analyze cytokine expression by the cells with the 15% lowest (TCRlow) and 15% highest (TCRhi) receptor levels after incubation are indicated. (D) IFN-γ and IL-2 production profiles of TCRlow and TCRhi populations as detailed in C. The percentage of total cells and the  mean intensity of cytokine staining are shown for cells in the respective quadrants of the two-parameter histograms.
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Figure 5: TCR downmodulation and cytokine production. T cell clone 3C6 was cultured with P13.9 + PCC88-104 (10 μM) in the presence of monensin. After 5 h of incubation, surface TCR Vβ3, as well as intracellular IFN-γ and IL-2, were analyzed. (A) IFN-γ and IL-2 production by 3C6. R1 = cells producing both IFN-γ and IL-2; R2 = cells producing IFN-γ alone; R3 = cells producing neither IFN-γ nor IL-2. (B) TCR expression of each cell subpopulation after incubation with 10 μM PCC88-104 + P13.9. Gates R1, R2, and R3 are as in A. The histogram for cells in R2 largely overlaps that for cells in R3. (C) Ungated TCR expression of 3C6 incubated with either with 10 μM PCC88-104 (solid line) or without peptide (dotted line) + P13.9. The gates set to analyze cytokine expression by the cells with the 15% lowest (TCRlow) and 15% highest (TCRhi) receptor levels after incubation are indicated. (D) IFN-γ and IL-2 production profiles of TCRlow and TCRhi populations as detailed in C. The percentage of total cells and the mean intensity of cytokine staining are shown for cells in the respective quadrants of the two-parameter histograms.

Mentions: TCR downmodulation serves as a convenient single cell measure of TCR occupancy (10, 18). The same cell populations analyzed for cytokine production by intracellular staining were therefore also analyzed for TCR expression before and after antigen exposure for 5 h, a time point we find to be near the plateau of TCR downmodulation under these conditions (Itoh, Y., and R.N. Germain, manuscript in preparation). For 3C6, both the majority of cells producing only IFN-γ and many of the IFN-γ–negative cells have a similar TCR level, lower than that on cells not exposed to specific antigen (Fig. 5 B). These data indicate that a subpopulation of these cloned T cells engage their TCR, but fail to make either of the studied cytokines. In contrast, the TCR expression of IL-2– producing cells is lower than that of IL-2–negative cells (Fig. 5 B), suggesting that the former have undergone more TCR downmodulation, and have therefore received more antigen-dependent signals.


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)

TCR downmodulation and cytokine production. T cell clone 3C6 was cultured with P13.9 + PCC88-104 (10 μM) in the presence of  monensin. After 5 h of incubation, surface TCR Vβ3, as well as intracellular IFN-γ and IL-2, were analyzed. (A) IFN-γ and IL-2 production by 3C6.  R1 = cells producing both IFN-γ and IL-2; R2 = cells producing IFN-γ alone; R3 = cells producing neither IFN-γ nor IL-2. (B) TCR expression of  each cell subpopulation after incubation with 10 μM PCC88-104 + P13.9. Gates R1, R2, and R3 are as in A. The histogram for cells in R2 largely  overlaps that for cells in R3. (C) Ungated TCR expression of 3C6 incubated with either with 10 μM PCC88-104 (solid line) or without peptide (dotted  line) + P13.9. The gates set to analyze cytokine expression by the cells with the 15% lowest (TCRlow) and 15% highest (TCRhi) receptor levels after incubation are indicated. (D) IFN-γ and IL-2 production profiles of TCRlow and TCRhi populations as detailed in C. The percentage of total cells and the  mean intensity of cytokine staining are shown for cells in the respective quadrants of the two-parameter histograms.
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Related In: Results  -  Collection

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Figure 5: TCR downmodulation and cytokine production. T cell clone 3C6 was cultured with P13.9 + PCC88-104 (10 μM) in the presence of monensin. After 5 h of incubation, surface TCR Vβ3, as well as intracellular IFN-γ and IL-2, were analyzed. (A) IFN-γ and IL-2 production by 3C6. R1 = cells producing both IFN-γ and IL-2; R2 = cells producing IFN-γ alone; R3 = cells producing neither IFN-γ nor IL-2. (B) TCR expression of each cell subpopulation after incubation with 10 μM PCC88-104 + P13.9. Gates R1, R2, and R3 are as in A. The histogram for cells in R2 largely overlaps that for cells in R3. (C) Ungated TCR expression of 3C6 incubated with either with 10 μM PCC88-104 (solid line) or without peptide (dotted line) + P13.9. The gates set to analyze cytokine expression by the cells with the 15% lowest (TCRlow) and 15% highest (TCRhi) receptor levels after incubation are indicated. (D) IFN-γ and IL-2 production profiles of TCRlow and TCRhi populations as detailed in C. The percentage of total cells and the mean intensity of cytokine staining are shown for cells in the respective quadrants of the two-parameter histograms.
Mentions: TCR downmodulation serves as a convenient single cell measure of TCR occupancy (10, 18). The same cell populations analyzed for cytokine production by intracellular staining were therefore also analyzed for TCR expression before and after antigen exposure for 5 h, a time point we find to be near the plateau of TCR downmodulation under these conditions (Itoh, Y., and R.N. Germain, manuscript in preparation). For 3C6, both the majority of cells producing only IFN-γ and many of the IFN-γ–negative cells have a similar TCR level, lower than that on cells not exposed to specific antigen (Fig. 5 B). These data indicate that a subpopulation of these cloned T cells engage their TCR, but fail to make either of the studied cytokines. In contrast, the TCR expression of IL-2– producing cells is lower than that of IL-2–negative cells (Fig. 5 B), suggesting that the former have undergone more TCR downmodulation, and have therefore received more antigen-dependent signals.

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