Single Cell Analysis Reveals Regulated Hierarchical T Cell Antigen Receptor Signaling Thresholds and Intraclonal Heterogeneity for Individual Cytokine Responses of CD4(+) T Cells

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-γ (IFN-γ) production. Increasing ligand recruits more cells into the IFN-γ(+) pool, increases IFN-γ produced per cell, and also elicits IL-2, but only from cells already making IFN-γ. Most cells producing only IFN-γ 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-γ 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-γ/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-γ and not IL-2, and the amount of IFN-γ 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.