Elizabeth Nowak

IL-9 as a mediator of Th17-driven inflammatory disease

We report that like other T cells cultured in the presence of transforming growth factor (TGF) β, Th17 cells also produce interleukin (IL) 9. Th17 cells generated in vitro with IL-6 and TGF-β as well as purified ex vivo Th17 cells both produced IL-9. To determine if IL-9 has functional consequences in Th17-mediated inflammatory disease, we evaluated the role of IL-9 in the development and progression of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. The data show that IL-9 neutralization and IL-9 receptor deficiency attenuates disease, and this correlates with decreases in Th17 cells and IL-6–producing macrophages in the central nervous system, as well as mast cell numbers in the regional lymph nodes. Collectively, these data implicate IL-9 as a Th17-derived cytokine that can contribute to inflammatory disease.

Cellular sources and immune functions of interleukin-9

Interleukin-9 (IL-9) has attracted renewed interest owing to the identification of its expression by multiple T helper (TH) cell subsets, including TH2 cells, TH9 cells, TH17 cells and regulatory T (TReg) cells. Here, we provide a broad overview of the conditions that are required for cells to produce IL-9 and describe the cellular targets and nature of the immune responses that are induced by IL-9.

Retinoic Acid Can Directly Promote TGF-β-Mediated Foxp3+ Treg Cell Conversion of Naive T Cells

The article by Hill et al. (2008), published in the November 14, 2008 issue of Immunity, describes a mechanism by which retinoic acid (RA) enhances TGF-β-induced Foxp3 expression. The authors propose that RA does not act directly on naive T cells during activation in culture but rather indirectly via negative regulation of an accompanying population of effector or memory CD4+ CD44hi cells. They reasoned that the increased generation of Foxp3+ cells in response to RA in culture, as described previously (Coombes et al., 2007; Elias et al., 2008; Mucida et al., 2007; Sun et al., 2007; Xiao et al., 2008), represented the lifting by RA of inhibition imparted by accompanying CD4+CD44hi T cells, rather than by direct or indirect effects of RA on the Foxp3 expression of the primed naive T cells themselves. In order to assess the effects of RA on naive T cells in the absence of accompanying CD4+CD44hi T cells, we sorted (CD4+CD25−CD44low CD62L+) GFP− T cells (more than 99.7% purity) from Foxp3-eGFP reporter mice (Figure S1A available online) by flow cytometry. After 4 days of stimulation with anti-CD3 and anti-CD28, we stained CD4 cells with 7AAD to exclude dead cells; additionally, forward and side scatter (area versus width) was used to exclude doublets, and we evaluated Foxp3 expression via GFP staining. Addition of RA enhanced Foxp3 induction more than 50% by use of 1 or 10 ng/ml doses of TGF-β (Figure S1B). Because the sorting efficiency is not 100%, it is possible that extremely low numbers of “accompanying” memory or effector cells could still influence these results. To exclude this possibility, we used FACS-sorted CD4+CD25−CD44lo CD62L+ T cells, isolated from B7-1 and B7-2 double-deficient mice (Cd80−/− Cd86−/−), which even before sorting already contain less than 5% of memory or effector CD44hi cells (data not shown). RA also greatly enhanced Foxp3 induced by TGF-β in CD4+CD25−CD44lo CD62L+ naive T cells isolated from Cd80−/− Cd86−/− mice (Figure S1C). Moreover, we showed previously that RA is able to counterbalance the inhibitory effects of costimulation on TGF-β-mediated Foxp3 induction, with either CD4+CD25− or CD4+Foxp3− T cells (Benson et al., 2007). To confirm these results, we used OTII TCR transgenic CD4+CD25−CD44lo CD62L+ cells sorted by flow cytometry and tested the effects of RA by using increasing doses of anti-CD28 stimulation. We found that RA markedly enhanced TGF-β-mediated Foxp3 induction on pure naive CD4+ T cells that were stimulated with anti-CD3 and various doses of anti-CD28 (Figure S1D). The enhanced Foxp3 expression mediated by RA is more pronounced on naive monoclonal OTII TCR transgenic T cells as compared to polyclonal T cells, consistent with a lesser frequency of “contaminating” memory T cells. Finally, because we showed previously that RA-mediated enhanced expression of Foxp3 is greatly reduced in the absence of IL-2 (Mucida et al., 2007), we investigated the effects of RA on naive T cells with various doses of exogenous IL-2. Although IL-2-deficient mice develop inflammatory disorders, Il2−/−Cd80−/−Cd86−/− mice are healthy and, more importantly, they do not contain T regulatory cells. At steady state, ~99% of all CD4+ T cells isolated from Il2−/−Cd80−/−Cd86−/− mice are naive (data not shown). The CD4+ T cells were further sorted by flow cytometry so that highly purified naive CD4+CD25−CD44lo CD62L+ cells (more than 99.9% purity) were obtained. The sorted naive CD4+ Il2−/−Cd80−/−Cd86−/− T cells were tested for TGF-β-induced Foxp3 expression in the presence of increasing doses of IL-2 and anti-CD3 and anti-CD28 coated beads, with or without RA. The data showed that 1 nM RA distinctly enhanced TGF-β (1 ng/ml)-mediated Foxp3 induction in pure naive CD4+ T cells at all doses of IL-2 examined (Figures S1E and S1F). Strikingly, although the expression of Foxp3 was much reduced, RA enhanced TGF-β-mediated Foxp3 induction not only in the absence of memory or effector T cells but also in the absence of IL-2. These data demonstrate that RA mediates enhanced TGF-β-induced Foxp3 expression upon activation of pure naive T cells in the absence of accompanying CD4+CD44hi T cells. In addition, we confirmed, as Hill et al. (2008) proposed, that RA also efficiently counteracts inhibitory effects of CD44hi T cells on Foxp3 induction (data not shown), which indicates that RA is able to enhance Foxp3 expression both, via effects directly on the primed naive T cells as well as indirectly via inhibitory effects on accompanying CD4+CD44hi T cells. There is no doubt that the new findings by Hill et al. (2008) add an important new pathway by which RA can enhance Foxp3 induction, which had been suggested previously (Elias et al., 2008; Mucida et al., 2007; Xiao et al., 2008).Nevertheless, published data, together with the data presented here, disagree with the central statement proposed by Hill et al. (2008) that the enhanced expression of TGF-β driven Foxp3 mediated by RA is an indirect effect that requires suppression of accompanying CD4+CD44hi T cells rather than via direct or indirect effects on the primed T cells themselves. Under physiological conditions, naive T cells may be exposed to cytokines and effector or memory cells, and hence it is likely that during priming of naive T cells, both mechanisms of RA-mediated enhanced TGF-β driven Foxp3 expression in the primed T cells will synergize in vivo. Therefore, elucidating and understanding both processes by which RA affects naive and already differentiated T cells is important and may lead to the identification of possible targets for therapeutic interventions to treat various inflammatory and autoimmune diseases.

B7 family checkpoint regulators in immune regulation and disease

Fine-tuning the immune response and maintaining tolerance to self-antigens involves a complex network of co-stimulatory and co-inhibitory molecules. The recent FDA approval of ipilimumab, a monoclonal antibody blocking cytotoxic T lymphocyte antigen (CTLA)-4, demonstrates the impact of checkpoint regulators in disease. This is reinforced by ongoing clinical trials targeting not only CTLA-4, but also the programmed death (PD)-1 and B7-H4 pathways in various disease states. Recently, two new B7 family inhibitory ligands, V-domain Ig suppressor of T cell activation (VISTA) and B7-H6 were identified. Here, we review recent understanding of B7 family members and their concerted regulation of the immune response to either self or foreign pathogens. We also discuss clinical developments in targeting these pathways in different disease settings, and introduce VISTA as a putative therapeutic target.

A retinoic acid–dependent checkpoint in the development of CD4+ T cell–mediated immunity

Immune cell activation induces concurrent temporal and spatial retinoic acid signaling, and CD4+ T cell–specific loss of RA signals reduces effector function, migration, and polarity.

Tryptophan hydroxylase-1 regulates immune tolerance and inflammation

Tryptophan hydroxylase deficiency in mast cells breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation.

Transplantation survival is maintained by granzyme B+ regulatory cells and adaptive regulatory T cells.

Granzyme B (GZB) has been implicated as an effector mechanism in regulatory T cells (Treg) suppression. In a model of Treg-dependent graft tolerance, it is shown that GZB- deficient mice are unable to establish long-term tolerance. Moreover, mice overexpressing the inhibitor of GZB, serine protease inhibitor 6, are also resistant to tolerization to alloantigen. Graft survival was shorter in bone marrow-mixed chimeras reconstituted with GZB-deficient Treg as compared with wild-type Treg. Whereas there was no difference in graft survival in mixed chimeras reconstituted with wild-type, perforin-deficient, or Fas ligand-deficient Treg. Finally, data also show that if alloreactive effectors cannot express FoxP3 and be induced to convert in the presence of competent Treg, then graft tolerance is lost. Our data are the first in vivo data to implicate GZB expression by Treg in sustaining long-lived graft survival.

Disruption of the immune-checkpoint VISTA gene imparts a proinflammatory phenotype with predisposition to the development of autoimmunity

Significance The discovery of V domain-containing Ig suppressor of T-cell activation (VISTA) as a novel immune-checkpoint regulator comes at an exciting time, as the field of cancer immunotherapy has made significant progress owing to the clinical success of targeting immune-checkpoint proteins such as cytotoxic T lymphocyte-associated antigen 4 and programmed death 1 and ligand. Recent studies also show the promise of monoclonal antibody-mediated VISTA targeting for enhancing antitumor immunity in murine tumor models. The current study demonstrates the spectrum of immune alterations upon genetic disruption of VISTA in mice in the context of self-tolerance as well as immune response against neoantigen. These results enhance the understanding of the immune-regulatory role of VISTA and form the foundation for designing future clinical applications that target VISTA in treating human diseases. V domain-containing Ig suppressor of T-cell activation (VISTA) is a negative checkpoint regulator that suppresses T cell-mediated immune responses. Previous studies using a VISTA-neutralizing monoclonal antibody show that VISTA blockade enhances T-cell activation. The current study describes a comprehensive characterization of mice in which the gene for VISTA has been deleted. Despite the apparent normal hematopoietic development in young mice, VISTA genetic deficiency leads to a gradual accumulation of spontaneously activated T cells, accompanied by the production of a spectrum of inflammatory cytokines and chemokines. Enhanced T-cell responsiveness was also observed upon immunization with neoantigen. Despite the presence of multiorgan chronic inflammation, aged VISTA-deficient mice did not develop systemic or organ-specific autoimmune disease. Interbreeding of the VISTA-deficient mice with 2D2 T-cell receptor transgenic mice, which are predisposed to the development of experimental autoimmune encephalomyelitis, drastically enhanced disease incidence and intensity. Disease development is correlated with the increase in the activation of encephalitogenic T cells in the periphery and enhanced infiltration into the CNS. Taken together, our data suggest that VISTA is a negative checkpoint regulator whose loss of function lowers the threshold for T-cell activation, allowing for an enhanced proinflammatory phenotype and an increase in the frequency and intensity of autoimmunity under susceptible conditions.

Augmentation of regulatory B cell activity in experimental allergic encephalomyelitis by glatiramer acetate

We recently showed that B cells reduce CNS inflammation in mice with experimental allergic encephalomyelitis (EAE). Here, we demonstrate that adoptively transferred CD5/CD19+ B cells protect against EAE severity. Furthermore, we show that glatiramer acetate (GA), a therapeutic for relapsing multiple sclerosis treatment, amplifies this effect. Transfer of GA-conditioned B cells leads to increased production of immunoregulatory cytokines and reduced CNS inflammation, as well as decreased expression of the chemokine receptor, CXCR5, and elevated BDNF expression in the CNS. Thus B cells can protect against EAE, and GA augments this effect in maintaining immune homeostasis and controlling EAE disease progression.

Interleukin-9 as a T helper type 17 cytokine.

The production of interleukin‐9 (IL‐9) by CD4 T cells has gathered renewed interest as the result of the observation that its expression is broader than originally thought. This includes the production of IL‐9 by a recently characterized subset of CD4 helper T (Th) cells that are termed Th9 as well as production by additional T‐cell subsets including Th17 cells. There is an incomplete understanding as to which IL‐9‐producing T‐cell subsets develop under physiological conditions. We describe the conditions used to generate IL‐9 in Th17 cells in vitro. We also summarize conditions where both IL‐9 and IL‐17 are found in vivo and propose that Th17 cells producing IL‐9 may co‐exist and interact with Th9 cells during conditions of autoimmunity, allergy and infection.