Natural regulatory T cells (Tregs) counterbalance immunity by suppressing cell proliferation, survival, maturation, cytokine and/or chemokine production, and release of cytotoxic components from granules. This broad suppressive capacity is likely exerted by different mechanisms at different stages of immune activation (1
Fundamental to initial T cell activation is the receipt of signals that promote cytokine production, cell proliferation and cell survival. IL-2 is the first cytokine produced by naïve T cells and is critical for successful adaptive immunity (3
). Upon TCR engagement the nuclear accumulation of NFAT, NFκB, and AP-1, in concert, drives early IL-2 transcription (4
). CD28 co-stimulatory signaling quantitatively changes TCR signaling: enhancing NFκB and AP-1 to promote transcription and stabilizing IL-2 mRNA (6
). Tregs suppress T cell activation by inhibiting cell proliferation and cytokine production, in particular early IL-2 production (8
). We have mapped Treg suppression of IL-2 at the transcript and protein level to a tight kinetic window 6–10 hours after initial CD4 T cell activation (9
). However the mechanism by which Tregs specifically abort IL-2 production remains unknown.
Tregs could negatively regulate T cell signals for IL-2 via CTLA-4/B7 (10
), cAMP (11
) or potentially by utilizing E3 ligases (12
). Alternatively, recent visualization of Treg suppressive events suggest Tregs could terminate T cell signals by disrupting the stability or duration of CD4 T cell-antigen presenting cell interactions (14
). To define the changes in T cell signaling in target CD4 T cells activated in the presence of Tregs we used multispectral imaging flow cytometry (Amnis Imagestream) to quantify the frequency of CD4 T cells with specific transcription factor (TF) nuclear accumulation. Tregs did not terminate T cell signaling at the time of IL-2 inhibition. Rather, signaling in targeted CD4 T cells was selectively modified by attenuation of nuclear NFκB but not NFAT and AP-1, through an APC-independent mechanism.