Choline acetyltransferase-expressing T cells are required to control chronic viral infection

The prototypic neurotransmitter acetylcholine (ACh) was the first neurotransmitter identified (1, 2). ACh has numerous physiological roles, including mediating skeletal and smooth muscle contraction, communication between neurons, and induction of vasodilation (1–3). In addition to neurons, a population of CD4⁺ T cells and B cells express the enzyme choline acetyltransferase (ChAT) (4, 5), which catalyzes the rate-limiting step of ACh production. Although these ChAT-expressing T cells have a demonstrated impact on blood pressure (6) and the release of inflammatory cytokines (4), the biological role of immune-derived ACh during infection has not been elucidated. In this study, we have determined that Chat is induced by IL-21 in T cells during infection to facilitate T cell entry into infected tissues, thereby genetically identifying the function of T cell-derived ACh during an immune response.

Chat⁺ CD4⁺ T cells uniformly exhibit an “antigen-experienced” phenotype (4). Yet, the signals that drive Chat expression in T cells are undefined. We infected Chat-green fluorescent protein (GFP) reporter mice (7) with the rapidly cleared Armstrong strain of lymphocytic choriomeningitis virus (LCMV-Arm). There was a massive increase in Chat-GFP expression in both CD4⁺ and CD8⁺ T cells 8 days postinfection (Fig. 1A). In splenic virus-specific T cells, expression rapidly declined after LCMV-Arm clearance (Fig. 1B), yet Chat-GFP expression was retained in both virus-specific CD4⁺ and CD8⁺ T cells from mice chronically infected with LCMV clone 13 (LCMV-Cl13) (Fig. 1B). GFP expression correlated with Chat mRNA in T cells (Fig. 1C). In CD4⁺ T cells, Chat-GFP was expressed by all subsets; however, expression was highest in T follicular helper (T_FH) cells (fig. S1, A to D). In CD8⁺ T cells, there was no correlation with either memory precursor or short-lived effector phenotypes (fig. S1E). Furthermore, Chat-GFP was induced in germinal center (GC) B cells in the spleen, although Chat expression was not retained in this population during persistent infection (fig. S1, F and G). Chat-GFP was also induced in both CD4⁺ and CD8⁺ T cells after vesicular stomatitis virus infection (fig. S1H).

The kinetics of Chat-GFP expression during acute and chronic infection implicates viral signals in driving Chat induction in T cells. Viral infection induces numerous cytokines that influence T cells, including type I interferons (IFN-I), interleukin-2 (IL-2), IL-6, IL-7, IL-10, IL-15, and IL-21 (8). We activated Chat-GFP P14 T cell receptor (TCR) transgenic T cells in vitro with the GP33 peptide in the presence or absence of these cytokines. The only condition that resulted in Chat induction in P14 cells in vitro was IL-21 with peptide stimulation (Fig. 1D and fig. S2A). We evaluated the contribution of IL-21 signaling to Chat induction in vivo by infecting IL-21 receptor-deficient (Il21r^−/−) mice (9) expressing the Chat-GFP reporter with LCMV-Cl13. We observed a decrease in the fraction of both CD4⁺ and CD8⁺ T cells expressing Chat-GFP in Il21r^−/− mice (Fig. 1E). Mice heterozygous for Il21r (Il21r^+/−) showed a mixed phenotype. The expression of Chat-GFP in B cell populations was not reduced in Il21r^−/− animals (Fig. 1E). Chat-GFP⁺ cells in Il21r^−/− mice also demonstrated a lower mean fluorescence intensity (MFI) for the reporter molecule, suggesting reduced expression (Fig. 1F and fig. S2B).

IL-21 is critical for antiviral immunity (10–12). Thus, we investigated the role of IL-21-induced Chat in T cells (T-Chat) by using Chat^flox mice (13) crossed with CD4-cre mice (14) to generate Chat^flox/flox CD4-cre⁻ (Chat^WT) and Chat^flox/flox CD4-cre⁺ (T-Chat^KO) animals. Cre-driven recombination occurs at the double-positive stage in the thymus (14), resulting in deletion of Chat in both CD4⁺ and CD8⁺ T cells (fig. S3A) and a subsequent failure to produce ACh (fig. S3B). Notably, the loss of Chat specifically within T cells resulted in a failure to control LCMV-Cl13 in a subset of the animals (Fig. 2A), revealing that Chat expression in T cells is required during chronic infection. This failure to control LCMV-Cl13 corresponded with the attrition of virus-specific CD8⁺ T cells over time (Fig. 2B), poor cytokine production (Fig. 2C), and increased expression of inhibitory receptors (Fig. 2, D and E). There was no difference in the number of antiviral T cells in LCMV-Arm-infected T-Chat^KO mice (fig. S4), which has also been reported for Il21^−/− animals (15). Although we observed high Chat expression in T_FH and GC B cells (fig. S1), there were no deficits in either antiviral CD4⁺ T cell numbers or in the anti-LCMV antibody response in T-Chat^KO mice (fig. S5).

Loss of IL-21 signaling results in decreased T cell infiltration of tissues in bone marrow chimeras (16, 17). Thus, we evaluated tissue infiltration by Il21r^−/− T cells during LCMV-Cl13 infection using intravascular staining (18). We found a reduction in virus-specific T cells that had migrated into infected livers of Il21r^−/− mice (Fig. 3A). Chat-expressing T cells reduce blood pressure by producing ACh (3, 6), which may facilitate T cell entry into tissues by slowing blood flow. Consequently, we also found a reduction in virus-specific CD8⁺ T cells in both the liver and salivary gland of T-Chat^KO mice after LCMV-Cl13 infection (Fig. 3, B and C). No difference in the number of circulating virus-specific cells was found in either Il21r^−/− or T-Chat^KO mice (fig. S6, A to C). We observed a similar trend in the liver for virus-specific CD4⁺ T cells (fig. S6D). Poor migration into tissues could affect viral control, as fewer migrated cytotoxic T lymphocytes (CTLs) would result in the poor elimination of infected cells. In vivo CTL activity in the liver was impaired for two epitopes examined 8 days postinfection in T-Chat^KO mice (Fig. 3D), despite equivalent expression of granzyme B and degranulation (Fig. 3E) by noncirculating CTLs. Furthermore, this diminution in CTL activity was observed only for the GP33 epitope in the spleen (Fig. 3D), suggesting that the poor CTL activity in the liver was not due to intrinsic defects in the cells but rather their impaired infiltration of tissues.

We tested whether Chat expression in T cells functions in a cell-intrinsic manner by transplanting Chat^WT or T-Chat^KO TCR transgenic P14 T cells into congenic recipient Chat^WT or T-Chat^KO mice and infecting them with LCMV-Cl13. We then quantified migration capacity of the P14 cells (Fig. 3F). Briefly, we examined the total noncirculating D^b(GP33)⁺ in the spleen and organs and determined what percentage of these D^b(GP33)⁺ cells were donor P14 cells. This percentage in each organ was then compared against the percentage in the spleen of that individual animal to determine whether the P14 cells had migrated in a superior (ratio > 1), inferior (ratio < 1), or equivalent (ratio = 1) manner compared with the endogenous D^b(GP33)⁺ T cells (Fig. 3F). In control mice (WT P14→WT recipients and KO P14→KO recipients), the frequency of P14 cells was similar in the spleen and organs, resulting in a ratio of ~1 (Fig. 3G and fig. S6G). However, in T-Chat^KO mice receiving Chat^WT P14 cells, we observed a greater frequency of Chat^WT P14 cells in the liver and kidney than would be predicted by their rate of occurence in the spleen (Fig. 3G and fig. S6G). Thus, Chat^WT cells were more efficient at seeding these peripheral organs than T-Chat^KO cells in the same animal. When T-Chat^KO P14 cells were transplanted into a Chat^WT recipient, they migrated just as well as endogenous Chat^WT cells, indicating that the observed differences were not due to an intrinsic failure of T-Chat^KO cells to adhere or sense chemokines. We postulated that this migratory advantage of Chat^WT cells in a T-Chat^KO host was due to local changes in the vasculature induced by the presence of Chat⁺ T cells and would still be present in Chat^WT recipients of T-Chat^KO P14 cells.

Vasodilation is critical for immune responses and is one of the hallmarks of inflammation facilitating the entry of immune cells into infected tissues. Not only do Il21r^−/− mice exhibit smaller arterial connections in the brain (19), T-Chat^KO mice exhibit higher blood pressure than Chat^WT littermates (6), indicating that they also have smaller arteries. ACh signaling has long been known to induce vasodilation (20). We posited that Chat⁺ T cells induced by infection are the primary mediators of vasodilation via the release of ACh and that loss of Chat in T cells would consequently abrogate infection-driven vasodilation. Upon imaging the liver arterial vasculature of naïve and LCMV-Cl13-infected Chat^WT and T-Chat^KO mice (movies S1 to S4), we found that infection-induced vasodilation in the liver was completely abrogated in T-Chat^KO mice, in contrast to their Chat^WT counterparts (Fig. 4, A and B), resulting in fewer detectable terminal branches (fig. S7A) and smaller mean vessel diameter at equivalent branch depths (fig. S7B).

The blood vessel phenotype in T-Chat^KO mice was reversed with short-term treatment with the vasodilator minoxidil (Fig. 4C, fig. S7, and movie S5). Thus, these differences were not developmental but reflective of poor vasodilation in the absence of T-Chat. Furthermore, treatment of wild-type mice with the vasoconstrictor L-NAME was sufficient to recapitulate the vascular phenotype observed in T-Chat^KO mice (Fig. 4C, fig. S7, and movie S6). Minoxidil treatment was sufficient to restore viral control in T-Chat^KO mice and also augmented viral control in Chat^WT animals (Fig. 4D). Moreover, viral titers were significantly higher in wild-type mice treated with L-NAME on days 6 through 12 postinfection when compared with phosphate-buffered saline (PBS)-treated controls (Fig. 4E). Thus, vasodilation mediated by Chat-expressing T cells is critical for appropriate viral control.

IL-21 supports antiviral immunity beyond Chat induction and vasomodulation (21). Indeed, treatment with minoxidil was not sufficient to fully rescue Il21r^−/− mice, although this treatment did reduce viral titers compared with vehicle-treated Il21r^−/− mice (Fig. 4F). Efficient migration of effector T cells into tissues is critical for the control of viral infections (22) and is also of great interest for immunotherapy directed at tumors (23). In addition to its other reported roles during infection, IL-21 signaling enhances the efficacy of expanded tumor-infiltrating lymphocytes to combat cancer (24, 25). Here, we report that IL-21, a cytokine critical for control of chronic infection (10–12), drives the expression of Chat in T cells to facilitate their migration into infected tissues. These findings underscore the role for IL-21 during the host response to infection and establish a cholinergic mechanism for regulating cellular migration into tissues.