Immunomodulatory Functions of BTLA and HVEM Govern Induction of Extrathymic Regulatory T Cells and Tolerance by Dendritic Cells.

Dendritic cells (DCs) initiate immunity and also antigen-specific tolerance mediated by extrathymic regulatory T (Treg) cells, yet it remains unclear how DCs regulate induction of such tolerance. Here, we report that efficient induction of Treg cells was instructed by BTLA+DEC205+CD8+CD11c+ DCs and the immunomodulatory functions of BTLA. In contrast, T cell activation in steady state by total CD11c+ DCs that include a majority of DCs that do not express BTLA did not induce Treg cells and had no lasting impact on subsequent immune responses. Engagement of HVEM, a receptor of BTLA, promoted Foxp3 expression in T cells through upregulation of CD5. In contrast, T cells activated in the absence of BTLA and HVEM-mediated functions remained CD5lo and therefore failed to resist the inhibition of Foxp3 expression in response to effector cell-differentiating cytokines. Thus, DCs require BTLA and CD5-dependent mechanisms to actively adjust tolerizing T cell responses under steady-state conditions.

CD5 instructs extrathymic regulatory T cell development in response to self and tolerizing antigens.

Self-reactive T cells can escape thymic deletion and therefore some of these potentially autoaggressive T cells need to convert into regulatory T (Treg) cells to help control responses against self. However, it remains unknown how peripheral self-reactive T cells are specifically instructed to become Treg cells. We report that CD5, whose expression is upregulated in T cells by self and tolerizing antigens in the thymus and periphery, governed extrathymic Treg cell development. CD5 modified effector cell-differentiating signals that inhibit Treg cell induction. Treg cell conversion of Cd5(-/-) and CD5(lo) T cells was inhibited by even small amounts of interleukin-4 (IL-4), IL-6, and interferon-γ (IFN-γ) produced by bystander lymphocytes, while CD5(hi) T cells resisted this inhibition of Treg cell induction. Our findings further revealed that CD5 promoted Treg cell induction by blocking mechanistic target of rapamycin (mTOR) activation. Therefore CD5 instructs extrathymic Treg cell development in response to self and tolerizing antigens.

TLR7 Signaling Regulates Th17 Cells and Autoimmunity: Novel Potential for Autoimmune Therapy.

Innate regulation through TLR signaling has been shown to be important for promoting T cell subset development and function. However, limited information is known about whether differential TLR signaling can selectively inhibit Th17 and/or Th1 cells, which are important for controlling excessive inflammation and autoimmune responses. In this article, we demonstrate that activation of TLR7 signaling in T cells can inhibit Th17 cell differentiation from naive T cells and IL-17 production in established Th17 cells. We further report that downregulation of STAT3 signaling is responsible for TLR7-mediated inhibition of Th17 cells due to induction of suppressor of cytokine signaling 3 and 5. TLR7-mediated suppression of Th17 cells does not require dendritic cell involvement. In addition, we show that TLR7 signaling can suppress Th1 cell development and function through a mechanism different from Th17 cell suppression. Importantly, our complementary in vivo studies demonstrate that treatment with the TLR7 ligand imiquimod can inhibit Th1 and Th17 cells, resulting in the prevention of, and an immunotherapeutic reduction in, experimental autoimmune encephalomyelitis. These studies identify a new strategy to manipulate Th17/Th1 cells through TLR7 signaling, with important implications for successful immunotherapy against autoimmune and inflammatory diseases.

Peripherally Induced Tolerance Depends on Peripheral Regulatory T Cells That Require Hopx To Inhibit Intrinsic IL-2 Expression.

Dendritic cells (DCs) can induce peripheral immune tolerance that prevents autoimmune responses. Ag presentation by peripheral DCs under steady-state conditions leads to a conversion of some peripheral CD4(+) T cells into regulatory T cells (Tregs) that require homeodomain-only protein (Hopx) to mediate T cell unresponsiveness. However, the roles of these peripheral Tregs (pTregs) in averting autoimmune responses, as well as immunological mechanisms of Hopx, remain unknown. We report that Hopx(+) pTregs converted by DCs from Hopx(-) T cells are indispensible to sustain tolerance that prevents autoimmune responses directed at self-Ags during experimental acute encephalomyelitis. Our studies further reveal that Hopx inhibits intrinsic IL-2 expression in pTregs after antigenic rechallenge. In the absence of Hopx, increased levels of IL-2 lead to death and decreased numbers of pTregs. Therefore, formation of Hopx(+) pTregs represents a crucial pathway of sustained tolerance induced by peripheral DCs, and the maintenance of such pTregs and tolerance requires functions of Hopx to block intrinsic IL-2 production in pTregs.

Landscape of Hopx expression in cells of the immune system.

Homeodomain only protein (Hopx) is a regulator of cell differentiation and function, and it has also emerged as a crucial marker of specific developmental and differentiation potentials. Hopx expression and functions have been identified in some stem cells, tumors, and in certain immune cells. However, expression of Hopx in immune cells remains insufficiently characterized. Here we report a comprehensive pattern of Hopx expression in multiple types of immune cells under steady state conditions. By utilizing single-cell RNA sequencing (scRNA-seq) and flow cytometric analysis, we characterize a constitutive expression of Hopx in specific subsets of CD4+ and CD8+ T cells and B cells, as well as natural killer (NK), NKT, and myeloid cells. In contrast, Hopx expression is not present in conventional dendritic cells and eosinophils. The utility of identifying expression of Hopx in immune cells may prove vital in delineating specific roles of Hopx under multiple immune conditions.

Adenosine A3 agonists reverse neuropathic pain via T cell-mediated production of IL-10.

The A3 adenosine receptor (A3AR) has emerged as a therapeutic target with A3AR agonists to tackle the global challenge of neuropathic pain, and investigation into its mode of action is essential for ongoing clinical development. Immune cell A3ARs, and their activation during pathology, modulate cytokine release. Thus, the use of immune cells as a cellular substrate for the pharmacological action of A3AR agonists is enticing, but unknown. The present study discovered that Rag-KO mice lacking T and B cells, as compared with WT mice, are insensitive to the anti-allodynic effects of A3AR agonists. Similar findings were observed in interleukin-10 and interleukin-10 receptor knockout mice. Adoptive transfer of CD4+ T cells from WT mice infiltrated the dorsal root ganglion (DRG) and restored A3AR agonist-mediated anti-allodynia in Rag-KO mice. CD4+ T cells from Adora3-KO or Il10-KO mice did not. Transfer of CD4+ T cells from WT mice, but not Il10-KO mice, into Il10-KO mice or Adora3-KO mice fully reinstated the anti-allodynic effects of A3AR activation. Notably, A3AR agonism reduced DRG neuron excitability when cocultured with CD4+ T cells in an IL-10-dependent manner. A3AR action on CD4+ T cells infiltrated in the DRG decreased phosphorylation of GluN2B-containing N-methyl-D-aspartate receptors at Tyr1472, a modification associated with regulating neuronal hypersensitivity. Our findings establish that activation of A3AR on CD4+ T cells to release IL-10 is required and sufficient evidence for the use of A3AR agonists as therapeutics.

A Two-Step Process of Effector Programming Governs CD4+ T Cell Fate Determination Induced by Antigenic Activation in the Steady State.

Various processes induce and maintain immune tolerance, but effector T cells still arise under minimal perturbations of homeostasis through unclear mechanisms. We report that, contrary to the model postulating primarily tolerogenic mechanisms initiated under homeostatic conditions, effector programming is an integral part of T cell fate determination induced by antigenic activation in the steady state. This effector programming depends on a two-step process starting with induction of effector precursors that express Hopx and are imprinted with multiple instructions for their subsequent terminal effector differentiation. Such molecular circuits advancing specific terminal effector differentiation upon re-stimulation include programmed expression of interferon-γ, whose production then promotes expression of T-bet in the precursors. We further show that effector programming coincides with regulatory conversion among T cells sharing the same antigen specificity. However, conventional type 2 dendritic cells (cDC2) and T cell functions of mammalian target of rapamycin complex 1 (mTORC1) increase effector precursor induction while decreasing the proportion of T cells that can become peripheral Foxp3+ regulatory T (pTreg) cells.

Effects of cinnamaldehyde on the glucose transport activity of GLUT1.

There is accumulating evidence that cinnamon extracts contain components that enhance insulin action. However, little is know about the effects of cinnamon on non-insulin stimulated glucose uptake. Therefore, the effects of cinnamaldehyde on the glucose transport activity of GLUT1 in L929 fibroblast cells were examined under both basal conditions and conditions where glucose uptake is activated by glucose deprivation. The data reveal that cinnamaldehyde has a dual action on the glucose transport activity of GLUT1. Under basal conditions it stimulates glucose uptake and reaches a 3.5 fold maximum stimulation at 2.0mM. However, cinnamaldehyde also inhibits the activation of glucose uptake by glucose deprivation in a dose dependent manner. Experiments with cinnamaldehyde analogs reveal that these activities are dependent on the α,ß-unsaturated aldehyde structural motif in cinnamaldehyde. The inhibitory, but not the stimulatory activity of cinnamaldehyde was maintained after a wash-recovery period. Pretreatment of cinnamaldehyde with thiol-containing compounds, such as ß-mercaptoethanol or cysteine, blocked the inhibitory activity of cinnamaldehyde. These results suggest that cinnamaldehyde inhibits the activation of GLUT1 by forming a covalent link to target cysteine residue/s. This dual activity of cinnamaldehyde on the transport activity of GLUT1 suggests that cinnamaldehyde is not a major contributor to the anti-diabetic properties of cinnamon.

Dual action of phenylarsine oxide on the glucose transport activity of GLUT1.

An early event in the toxic effects of organic arsenic compounds, such as phenylarsine oxide (PAO), is an inhibition of glucose uptake. Glucose uptake involving the glucose transporter, GLUT4 is inhibited by PAO indicating an importance of vicinal sulfhydryls in insulin-stimulated glucose uptake. However, the data on effects of PAO on GLUT1 are conflicting. This study investigated the effects of PAO on glucose uptake in L929 fibroblast cells, cells, which express only GLUT1. The data presented here reveal a dual effect of PAO. At low concentrations or short exposure times PAO stimulated glucose uptake reaching a peak activation of about 400% at 3 microM. At higher concentrations (40 microM), PAO clearly inhibited glucose uptake. At intermediate concentrations (10 microM), PAO had no effect under basal conditions but completely inhibited activation of glucose uptake by glucose deprivation and partially inhibited methylene blue-stimulated glucose uptake. PAO increased the specific binding of cytochalasin B to GLUT1 suggesting a direct interaction with the transporter. These data are most consistent with PAO interacting with multiple proteins that regulate the activity of this transporter, one of which may be GLUT1 itself. The identity of these proteins will require further investigation.