Acetylcholine-producing CD4 T cells regulate vasculature in humans

In addition to the nervous system, many non-neuronal cells can produce and release the neurotransmitter acetylcholine (ACh). Included among these nonneuronal sources of ACh are T cells from the immune system (1). Expression of the enzyme choline acetyltransferase (Chat) and production of ACh were first identified in human leukemic cell lines (2), and after the development of a reporter mouse (3), confirmed in mouse primary T cells (4). With mouse models, the signals that drive Chat expression in T cells and the biological role of chat-expressing T cells are rapidly being unraveled. In mice, chat-expressing T cells provide protection during sepsis by responding to vagus nerve stimulation (4). The ACh released from these cells tempers the inflammatory response by signaling to macrophages (5). These ACh-producing T cells are also critical in vascular control, both during homeostasis and infection. Mice with a specific deletion of chat in T cells have higher blood pressure under homeostasis (6) and do not undergo vasodilation in infected tissues during disseminated viral infection (7). These studies have all found that signaling through the T cell receptor is required but ultimately not sufficient for chat expression in T cells. In addition to antigen-specific signaling, the cytokine IL-21 is also required for chat expression in T cells responding to viral infection (7), and the transcription factor Bhlhe40 is critical for chat induction in Th17-polarized CD4 T cells in vitro (8).

A new study by Tarnawski et al. is the first to identify chat expression in human primary T cells, and investigate how chat is regulated in CD4 T cells.

Despite these detailed studies in mice, the investigation of whether primary human T cells express chat and what signals could induce its expression has been hampered by technical challenges. A new study by Tarnawski et al. (9) is the first to identify chat expression in human primary T cells and investigate how chat is regulated in CD4 T cells. As identified in mice, expression of chat by human T cells requires signaling through the T cell receptor (9). Interestingly, the authors found striking variation in the ability of T cells from individual donors to express chat after in vitro stimulation (9). The authors found that the chat locus is methylated, and activation of the cells results in demethylation of the locus and expression (Fig. 1). The wide variation of chat expression and methylation in human samples is intriguing and could implicate previous T cell activation to a recent or subclinical infection increasing the pool of T cells with partial demethylation of the chat locus. It will be interesting in future work to look at T cell responses to described antigens, such as after vaccination, to determine when the chat locus becomes demethylated after in vivo activation, and for how long after challenge this locus remains accessible.

Fig. 1.

Activation of CD4 T cells results in demethylation of the chat locus and expression of chat. (A) In circulating CD4 T cells from healthy donors, the chat promoter is methylated in a REST-dependent manner, preventing chat expression. (B) Activation of CD4 T cells induces downregulation of REST. This downregulation, in combination with phosphoinositide 3-kinase (PI3K) signaling, results in demethylation of the chat locus and expression of chat mRNA. (C) Chat-expressing T cells release acetylcholine, which induces vasodilation and improves vascular integrity by signaling through muscarinic acetylcholine receptors on vascular endothelial cells. (D) The presence of these chat-expressing T cells correlates with better organ perfusion in critically ill patients on Veno-Arterial Extracoporeal Life Support (VA-ECLS), possibly by enhancing vasodilation and improving vascular integrity through acetylcholine release in vivo. Figure created with Biorender.com.

Alternatively, the diversity in chat methylation between different donors could be reflective of natural variability in the population. In this case, it would be interesting to discern if a greater degree of methylation in the chat locus is associated with greater susceptibility to hypertension or vascular diseases. Previous work has demonstrated that mice with a genetic deletion of chat in the T cell lineage have higher blood pressure (6), and Tarnawski et al have found that activated human T cells can induce vasodilation and preserve vascular endothelial integrity via cholinergic signaling (9). Furthermore, a greater frequency of chat-expressing T cells in critically ill patients correlated with better outcomes and improved markers of organ perfusion by the vascular system. Therefore, changes in the ability to induce chat in responding T cells could have impacts on susceptibility to vascular disease and hypertension.

Methylation of the chat locus in human T cells appears to be dependent on the transcriptional regulator RE-1 Silencing Transcription factor (REST). Knockdown of REST in a human T cell line resulted in increased chat expression (9). In late-onset Alzheimer’s disease patients, peripheral blood mononuclear cells (PBMCs) have reduced REST expression compared to healthy controls (10). In this limited patient sample, there was also a reduction in hypertension in the Alzheimer’s patients compared to the healthy controls, although this reduction was not significant. It would be interesting to determine in a larger patient subset if loss of REST expression in PBMCs correlated with a lower blood pressure, better organ perfusion, and increased chat expression in T cells.

These findings are also of interest in the context of cancer, as getting immune cells into tumors is critical for immune-mediated rejection. During viral infection in mice, chat-expressing T cells induce vasodilation, which facilitates T cell entry into infected tissues (7). In cancer patients receiving checkpoint inhibitor blockade to promote T cell responses, an increase in serum choline 7 wk after treatment predicted which patients went on to have progression-free survival (11). While this study did not directly measure chat expression in T cells in these patients, Tarnawski et al have similarly found that chat expression in T cells associated with improved markers of organ blood perfusion and survival in critically ill patients. Together, these studies indicate that chat-expressing T cells could be important in cancer treatment, infection, and vascular diseases.