Regulatory Innate Lymphoid Cells Control Innate Intestinal Inflammation.

An emerging family of innate lymphoid cells (termed ILCs) has an essential role in the initiation and regulation of inflammation. However, it is still unclear how ILCs are regulated in the duration of intestinal inflammation. Here, we identify a regulatory subpopulation of ILCs (called ILCregs) that exists in the gut and harbors a unique gene identity that is distinct from that of ILCs or regulatory T cells (Tregs). During inflammatory stimulation, ILCregs can be induced in the intestine and suppress the activation of ILC1s and ILC3s via secretion of IL-10, leading to protection against innate intestinal inflammation. Moreover, TGF-ß1 is induced by ILCregs during the innate intestinal inflammation, and autocrine TGF-ß1 sustains the maintenance and expansion of ILCregs. Therefore, ILCregs play an inhibitory role in the innate immune response, favoring the resolution of intestinal inflammation.

Regulatory T Cell-Derived TGF-ß1 Controls Multiple Checkpoints Governing Allergy and Autoimmunity.

The mechanisms by which regulatory T (Treg) cells differentially control allergic and autoimmune responses remain unclear. We show that Treg cells in food allergy (FA) had decreased expression of transforming growth factor beta 1 (TGF-ß1) because of interleukin-4 (IL-4)- and signal transducer and activator of transciription-6 (STAT6)-dependent inhibition of Tgfb1 transcription. These changes were modeled by Treg cell-specific Tgfb1 monoallelic inactivation, which induced allergic dysregulation by impairing microbiota-dependent retinoic acid receptor-related orphan receptor gamma t (ROR-γt)+ Treg cell differentiation. This dysregulation was rescued by treatment with Clostridiales species, which upregulated Tgfb1 expression in Treg cells. Biallelic deficiency precipitated fatal autoimmunity with intense autoantibody production and dysregulated T follicular helper and B cell responses. These results identify a privileged role of Treg cell-derived TGF-ß1 in regulating allergy and autoimmunity at distinct checkpoints in a Tgfb1 gene dose- and microbiota-dependent manner.

Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-ß.

Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8(+) tissue-resident memory T cells (TRM cells) require active transforming growth factor-ß1 (TGF-ß) for epidermal residence. Here we found that integrins αvß6 and αvß8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-ß. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvß6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-ß, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-ß by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication.

TGF-ß1 impairs IgA class switch recombination and production in porcine Peyer's patches B cells.

Secretory IgA is crucial for preventing the invasion of entero-pathogens via intestinal mucosa. While it is well-established that Transforming growth factor ß1 (TGF-ß1) regulates IgA production in human and mouse B cells, our previous investigation revealed different functions of TGF-ß1 in IgA generation in pigs compared with humans and mice, with the underlying mechanism remaining elusive. In this study, IgM+ B cells from porcine Peyer's patches (PPs) were isolated and stimulated with recombinant porcine TGF-ß1 to evaluate the effect of TGF-ß1 on pigs. The results showed that antibody production from B cells of PPs was impaired by TGF-ß1 ex vivo. Furthermore, TGF-ß1 treatment led to a decrease in the expression of germ-line transcript αand postswitch transcript α. Moreover, we observed that TGF-ß1 predominantly inhibited the phosphorylation of p38-mitogen-activated protein kinases (MAPK), confirming the involvement of the p38-MAPK pathway in porcine IgA generation and IgA class switch recombination. The application of p38-MAPK inhibitor resulted in decreased B-cell differentiation levels. Collectively, this study demonstrates that exogenous TGF-ß1 restrains the production and class switch recombination of IgA antibodies by inhibiting p38-MAPK signaling in porcine PPs B cells, which may constitute a component of TGF-ß1-mediated inhibition of B-cell activation.

The HIV-1 envelope protein gp120 impairs B cell proliferation by inducing TGF-ß1 production and FcRL4 expression.

The humoral immune response after acute infection with HIV-1 is delayed and ineffective. The HIV-1 envelope protein gp120 binds to and signals through integrin α4ß7 on T cells. We found that gp120 also bound to and signaled through α4ß7 on naive B cells, which resulted in an abortive proliferative response. In primary B cells, signaling by gp120 through α4ß7 resulted in increased expression of the immunosuppressive cytokine TGF-ß1 and FcRL4, an inhibitory receptor expressed on B cells. Coculture of B cells with HIV-1-infected autologous CD4(+) T cells also increased the expression of FcRL4 by B cells. Our findings indicated that in addition to mediating chronic activation of the immune system, viral proteins contributed directly to HIV-1-associated B cell dysfunction. Our studies identify a mechanism whereby the virus may subvert the early HIV-1-specific humoral immune response.

Absence of signaling into CD4⁺ cells via C3aR and C5aR enables autoinductive TGF-ß1 signaling and induction of Foxp3⁺ regulatory T cells.

Signaling through the G protein-coupled receptors for the complement fragments C3a and C5a (C3aR and C5aR, respectively) by dendritic cells and CD4(+) cells provides costimulatory and survival signals to effector T cells. Here we found that when signals from C3aR and C5aR were not transduced into CD4(+) cells, signaling via the kinases PI(3)Kγ, Akt and mTOR ceased, activation of the kinase PKA increased, autoinductive signaling by transforming growth factor-ß1 (TGF-ß1) initiated and CD4(+) T cells became Foxp3(+) induced regulatory T cells (iT(reg) cells). Endogenous TGF-ß1 suppressed signaling through C3aR and C5aR by preventing the production of C3a and C5a and upregulating C5L2, an alternative receptor for C5a. The absence of signaling via C3aR and C5aR resulted in lower expression of costimulatory molecules and interleukin 6 (IL-6) and more production of IL-10. The resulting iT(reg) cells exerted robust suppression, had enhanced stability and suppressed ongoing autoimmune disease. Antagonism of C3aR and C5aR can also induce functional human iT(reg) cells.

Induction and molecular signature of pathogenic TH17 cells.

Interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are often present at the sites of tissue inflammation in autoimmune diseases, which has led to the conclusion that T(H)17 cells are main drivers of autoimmune tissue injury. However, not all T(H)17 cells are pathogenic; in fact, T(H)17 cells generated with transforming growth factor-ß1 (TGF-ß1) and IL-6 produce IL-17 but do not readily induce autoimmune disease without further exposure to IL-23. Here we found that the production of TGF-ß3 by developing T(H)17 cells was dependent on IL-23, which together with IL-6 induced very pathogenic T(H)17 cells. Moreover, TGF-ß3-induced T(H)17 cells were functionally and molecularly distinct from TGF-ß1-induced T(H)17 cells and had a molecular signature that defined pathogenic effector T(H)17 cells in autoimmune disease.

Pulmonary Epithelial Cell-Derived Cytokine TGF-ß1 Is a Critical Cofactor for Enhanced Innate Lymphoid Cell Function.

Epithelial cells orchestrate pulmonary homeostasis and pathogen defense and play a crucial role in the initiation of allergic immune responses. Maintaining the balance between homeostasis and inappropriate immune activation and associated pathology is particularly complex at mucosal sites that are exposed to billions of potentially antigenic particles daily. We demonstrated that epithelial cell-derived cytokine TGF-ß had a central role in the generation of the pulmonary immune response. Mice that specifically lacked epithelial cell-derived TGF-ß1 displayed a reduction in type 2 innate lymphoid cells (ILCs), resulting in suppression of interleukin-13 and hallmark features of the allergic response including airway hyperreactivity. ILCs in the airway lumen were primed to respond to TGF-ß by expressing the receptor TGF-ßRII and ILC chemoactivity was enhanced by TGF-ß. These data demonstrate that resident epithelial cells instruct immune cells, highlighting the central role of the local environmental niche in defining the nature and magnitude of immune reactions.

Analysis of LAP+ and GARP+ Treg subsets in peripheral blood of patients with neuromyelitis optica spectrum disorders.

INTRODUCTION: Neuromyelitis optica spectrum disorder (NMOSD) is a group of antibody-mediated inflammatory demyelinating central nervous system diseases. T lymphocytes participate in NMOSD pathogenesis, with regulatory T cells (Treg) being the core in maintaining immune homeostasis. Studies have revealed that different Treg subsets play different roles in autoimmune diseases. The distribution of LAP+ or GARP+ Treg subsets in NMOSD may help us deeply understand their immune mechanism. METHODS: This study reviewed 22 NMOSD patients and 20 normal controls. Flow cytometric analysis was utilized to detect subsets of Treg cells expressing Foxp3, Helios, LAP, or GARP in peripheral blood. ELISA was used to detect plasma TGF-ß1 and IL-10. In addition, changes in the proportion of Treg cell subsets before and after glucocorticoid treatment in 10 patients were analyzed. RESULTS: Compared with healthy controls, LAP and GARP expressions were significantly downregulated in the peripheral blood of NMOSD patients. TGF-ß1 expression in NMOSD patients was lower and was positively correlated with the ratio of CD4+GARP+ Treg cells. After treatment with glucocorticoid, LAP and GARP expressions in the peripheral blood of NMOSD patients were upregulated. CONCLUSIONS: The proportion of Treg cells expressing LAP and GARP is downregulated, implying that Treg cells with the best inhibitory function are insufficient to maintain autoimmune homeostasis in NMOSD patients. Upregulation of Treg cells expressing LAP and GARP in NMOSD patients may be one of the mechanisms of glucocorticoid treatment.

TGF-ß1 - A truly transforming growth factor in fibrosis and immunity.

'Jack of all trades, master of everything' is a fair label for transforming growth factor ß1 (TGF-ß) - a cytokine that controls our life at many levels. In the adult organism, TGF-ß1 is critical for the development and maturation of immune cells, maintains immune tolerance and homeostasis, and regulates various aspects of immune responses. Following acute tissue damages, TGF-ß1 becomes a master regulator of the healing process with impacts on about every cell type involved. Divergence from the tight control of TGF-ß1 actions, for instance caused by chronic injury, severe trauma, or infection can tip the balance from regulated physiological to excessive pathological repair. This condition of fibrosis is characterized by accumulation and stiffening of collagenous scar tissue which impairs organ functions to the point of failure. Fibrosis and dysregulated immune responses are also a feature of cancer, in which tumor cells escape immune control partly by manipulating TGF-ß1 regulation and where immune cells are excluded from the tumor by fibrotic matrix created during the stroma 'healing' response. Despite the obvious potential of TGF-ß-signalling therapies, globally targeting TGF-ß1 receptor, downstream pathways, or the active growth factor have proven to be extremely difficult if not impossible in systemic treatment regimes. However, TGF-ß1 binding to cell receptors requires prior activation from latent complexes that are extracellularly presented on the surface of immune cells or within the extracellular matrix. These different locations have led to some divergence in the field which is often either seen from the perspective of an immunologists or a fibrosis/matrix researcher. Despite these human boundaries, there is considerable overlap between immune and tissue repair cells with respect to latent TGF-ß1 presentation and activation. Moreover, the mechanisms and proteins employed by different cells and spatiotemporal control of latent TGF-ß1 activation provide specificity that is amenable to drug development. This review aims at synthesizing the knowledge on TGF-ß1 extracellular activation in the immune system and in fibrosis to further stimulate cross talk between the two research communities in solving the TGF-ß conundrum.

The S1P(1)-mTOR axis directs the reciprocal differentiation of T(H)1 and T(reg) cells.

Naive CD4(+) T cells differentiate into diverse effector and regulatory lineages to orchestrate immunity and tolerance. Here we found that the differentiation of proinflammatory T helper type 1 (T(H)1) cells and anti-inflammatory Foxp3(+) regulatory T cells (T(reg) cells) was reciprocally regulated by S1P(1), a receptor for the bioactive lipid sphingosine 1-phosphate (S1P). S1P(1) inhibited the generation of extrathymic and natural T(reg) cells while driving T(H)1 development in a reciprocal manner and disrupted immune homeostasis. S1P(1) signaled through the kinase mTOR and antagonized the function of transforming growth factor-ß mainly by attenuating sustained activity of the signal transducer Smad3. S1P(1) function was dependent on endogenous sphingosine kinase activity. Notably, two seemingly unrelated immunosuppressants, FTY720 and rapamycin, targeted the same S1P(1) and mTOR pathway to regulate the dichotomy between T(H)1 cells and T(reg) cells. Our studies establish an S1P(1)-mTOR axis that controls T cell lineage specification.

Optimization strategies for expression of a novel bifunctional anti-PD-L1/TGFBR2-ECD fusion protein.

The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-ß1 receptor Ⅱ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-ß bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a "QKS" site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors. To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-ß1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone. In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.

Tuning Macrophage Phenotype to Mitigate Skeletal Muscle Fibrosis.

Myeloid cells are critical to the development of fibrosis following muscle injury; however, the mechanism of their role in fibrosis formation remains unclear. In this study, we demonstrate that myeloid cell-derived TGF-ß1 signaling is increased in a profibrotic ischemia reperfusion and cardiotoxin muscle injury model. We found that myeloid-specific deletion of Tgfb1 abrogates the fibrotic response in this injury model and reduces fibro/adipogenic progenitor cell proliferation while simultaneously enhancing muscle regeneration, which is abrogated by adaptive transfer of normal macrophages. Similarly, a murine TGFBRII-Fc ligand trap administered after injury significantly reduced muscle fibrosis and improved muscle regeneration. This study ultimately demonstrates that infiltrating myeloid cell TGF-ß1 is responsible for the development of traumatic muscle fibrosis, and its blockade offers a promising therapeutic target for preventing muscle fibrosis after ischemic injury.

Airway macrophage-intrinsic TGF-ß1 regulates pulmonary immunity during early-life allergen exposure.

BACKGROUND: Early life represents a major risk window for asthma development. However, the mechanisms controlling the threshold for establishment of allergic airway inflammation in early life are incompletely understood. Airway macrophages (AMs) regulate pulmonary allergic responses and undergo TGF-ß-dependent postnatal development, but the role of AM maturation factors such as TGF-ß in controlling the threshold for pathogenic immune responses to inhaled allergens remains unclear. OBJECTIVE: Our aim was to test the hypothesis that AM-derived TGF-ß1 regulates pathogenic immunity to inhaled allergen in early life. METHODS: Conditional knockout (Tgfb1ΔCD11c) mice, with TGF-ß1 deficiency in AMs and other CD11c+ cells, were analyzed throughout early life and following neonatal house dust mite (HDM) inhalation. The roles of specific chemokine receptors were determined by using in vivo blocking antibodies. RESULTS: AM-intrinsic TGF-ß1 was redundant for initial population of the neonatal lung with AMs, but AMs from Tgfb1ΔCD11c mice failed to adopt a mature homeostatic AM phenotype in the first weeks of life. Evidence of constitutive TGF-ß1 signaling was also observed in pediatric human AMs. TGF-ß1-deficient AMs expressed enhanced levels of monocyte-attractant chemokines, and accordingly, Tgfb1ΔCD11c mice exposed to HDM throughout early life accumulated CCR2-dependent inflammatory CD11c+ mononuclear phagocytes into the airway niche that expressed the proallergic chemokine CCL8. Tgfb1ΔCD11c mice displayed augmented TH2, group 2 innate lymphoid cell, and airway remodeling responses to HDM, which were ameliorated by blockade of the CCL8 receptor CCR8. CONCLUSION: Our results highlight a causal relationship between AM maturity, chemokines, and pathogenic immunity to environmental stimuli in early life and identify TGF-ß1 as a key regulator of this.

Structure-based discovery and redesign of TGF-ß1 Elbow epitope recognition by its type-II receptor in hypertrophic scarring biotherapy.

Transforming growth factor-ß1 (TGF-ß1) signaling pathway has been implicated in the fibroblast activation of hypertrophic scarring (HS). Previously, we proposed a new biotherapeutic strategy to combat HS by disrupting the intermolecular interaction of TGF-ß1 with its cognate type-II receptor (TßR-II). Here, we further demonstrate that the binding site of TGF-ß1 to TßR-II is not overlapped with the conformational wrist epitope and linear knuckle epitope that are traditionally recognized as the functional binding sites of bone morphogenetic protein-2 (BMP-2) to its type-II receptor (BMPR-II), which can thus be regarded as a new functional site we called elbow epitope. Structural, energetic, and dynamic investigations reveal that the elbow epitope consists of two sequentially discontinuous, spatially vicinal segments Loop30-34 and Turn90-95 ; they cannot work effectively to independently interact with TßR-II. Rational redesign of the epitope is performed using an integrated in silio-in vitro method based on crystal and modeled structure data. In the procedure, the two epitope segments are split from the interface of TGF-ß1-TßR-II complex and then connected with each other in a head-to-tail manner by adding a flexible poly-(Gly)n linker between them, thus resulting in a series of combined peptides. We found that the peptide affinity reaches maximum at n = 2, which shares a consistent binding mode with the elbow epitope at native complex interface. The linker of either too long (n > 2) or too short (n < 2) cannot properly place the gap space between the two segments, thus impairing the binding compatibility of designed peptides with TßR-II active site.

Enhancement of the antitumor effect of HER2-directed CAR-T cells through blocking epithelial-mesenchymal transition in tumor cells.

The efficacy of chimeric antigen receptor T (CAR-T) cell therapy in solid tumors is far from satisfactory. In this study, we investigated the influence of epithelial-mesenchymal transition (EMT) on the antitumor effect of CAR-T cells and explored the potential efficacy of combining CAR-T cells with inhibitors targeting EMT. We successfully induced EMT in tumor cells with TGF-ß1, and the antitumor effect of HER2-directed CAR-T cells was significantly suppressed by EMT. Upregulation of PD-L1 was observed in tumor cells undergoing EMT, and change in PD-L1 expression during the EMT process was dependent on the MEK/ERK and PI3K/Akt pathways. Inhibition of the TGF-ß1 pathway could block the EMT process in tumor cells and restore their susceptibility to HER2-directed CAR-T cells in vitro. In addition, targeting the TGF-ß1 pathway significantly enhanced the antitumor effect of HER2-directed CAR-T cells in vivo. Our findings suggest that blocking EMT could potently enhance the antitumor effect of CAR-T cells, which provides a promising approach to improving the therapeutic efficacy of CAR-T cell therapy in solid tumors.

IL-37 Attenuates Lung Fibrosis by Inducing Autophagy and Regulating TGF-ß1 Production in Mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive and destructive lung disease with a poor prognosis resulting in a high mortality rate. IL-37 is an anti-inflammatory cytokine that inhibits innate and adaptive immunity by downregulating proinflammatory mediators and pathways. However, the exact role of IL-37 in lung fibrosis is unclear. In this study, we found that the IL-37 protein was expressed in alveolar epithelial cells (AECs) and alveolar macrophages in healthy controls but significantly reduced in patients with IPF. IL-37 significantly inhibited oxidative stress-induced primary mouse AEC death in a dose-dependent manner, and knockdown of IL-37 significantly potentiated human lung cancer-derived AEC (A549 cells) death. IL-37 attenuated constitutive mRNA and protein expression of fibronectin and collagen I in primary human lung fibroblasts. IL-37 inhibited TGF-ß1-induced lung fibroblast proliferation and downregulated the TGF-ß1 signaling pathway. Moreover, IL-37 enhanced beclin-1-dependent autophagy and autophagy modulators in IPF fibroblasts. IL-37 significantly decreased inflammation and collagen deposition in bleomycin-exposed mouse lungs, which was reversed by treatment with the autophagy inhibitor 3-methyladenine. Our findings suggested that a decrease in IL-37 may be involved in the progression of IPF and that IL-37 inhibited TGF-ß1 signaling and enhancement of autophagy in IPF fibroblasts. Given its antifibrotic activity, IL-37 could be a therapeutic target in fibrotic lung diseases, including IPF.

Silicon, an important exposure marker in vivo in silicosis research.

PURPOSE: The degree of silicosis exposure is closely related to the progress of silicosis. At present, we use animal and human studies to explore whether silicon can be an important exposure marker in the development of silicosis. METHODS: Rats were randomly divided into 2 groups: (1) controls; and (2) silicosis. Rats in the silicosis group were killed at 4, 8, 12, 16, 24 h, 3, 7, 14, 21, and 28 days. Hematoxylin-eosin (HE) and immunohistochemistry (IHC) were performed to observe the histomorphology of lung tissue. The expression levels of CC16 and SP-D were detected using ELISA kits. In addition, we conducted a population study. Workers who have been selected to work in an iron mine for more than 1 year as research objects. The population was divided into four groups: silicosis exposure group (workers exposed to silica dust for more than 1 year in an iron mine were selected); patients group (silicosis patients); observation group (evidence of disease not meeting formal diagnostic criteria) and control group. Both the levels of trace silicon in the urine and blood of rats and human subjects were measured with ICP-MS. RESULTS: Serum levels of silicon were immediately increased in rats exposed to silicon dust. Similarly, our population study revealed that the silicon level in the silica exposure group and the observing group (exposed but no obvious symptoms) were significantly increased over that of the control group (P < 0.05). In subjects with extended exposure to silica, the serum and urine silicon level in exposed workers appeared to rapidly increase, reaching its peak in 1-5 years, followed by a gradual decline thereafter. Workers exposed to dust for less than 10 years were divided into subgroups by 2-year limit. The levels of serum silicon, urine silicon, TGF-ß1, and TNF-α were significantly higher than that of control group. CONCLUSION: Changes of the serum levels of silicon occurred earlier than the expression of cytokines such as TNF-α, TGF-ß1, CC16, and SP-D. The level of silicon in workers rapidly increased after exposure to silica, and the change occurred before the expression of TGF-ß1 and TNF-α. As a whole, the findings suggest that determining the level of silicon in vivo might be an effective exposure marker in the diagnosis and pathogenesis of silicosis.

Direct and Indirect Effect of TGFß on Treg Transendothelial Recruitment in HCC Tissue Microenvironment.

The balance between anti-tumor and tumor-promoting immune cells, such as CD4+ Th1 and regulatory T cells (Tregs), respectively, is assumed to dictate the progression of hepatocellular carcinoma (HCC). The transforming growth factor beta (TGFß) markedly shapes the HCC microenvironment, regulating the activation state of multiple leukocyte subsets and driving the differentiation of cancer associated fibroblasts (CAFs). The fibrotic (desmoplastic) reaction in HCC tissue strongly depends on CAFs activity. In this study, we attempted to assess the role of TGFß on transendothelial migration of Th1-oriented and Treg-oriented CD4+ T cells via a direct or indirect, CAF-mediated mechanisms, respectively. We found that the blockage of TGFß receptor I-dependent signaling in Tregs resulted in impaired transendothelial migration (TEM) of these cells. Interestingly, the secretome of TGFß-treated CAFs inhibited the TEM of Tregs but not Th1 cells, in comparison to the secretome of untreated CAFs. In addition, we found a significant inverse correlation between alpha-SMA and FoxP3 (marker of Tregs) mRNA expression in a microarray analysis involving 78 HCCs, thus suggesting that TGFß-activated stromal cells may counteract the trafficking of Tregs into the tumor. The apparent dual behavior of TGFß as both pro- and anti-tumorigenic cytokines may add a further level of complexity to the mechanisms that regulate the interactions among cancerous, stromal, and immune cells within HCC, as well as other solid tumors, and contribute to better manipulation of the TGFß signaling as a therapeutic target in HCC patients.

Ionizing radiation modulates the phenotype and function of human CD4+ induced regulatory T cells.

BACKGROUND: The use of immunotherapy strategies for the treatment of advanced cancer is rapidly increasing. Most immunotherapies rely on induction of CD8+ tumor-specific cytotoxic T cells that are capable of directly killing cancer cells. Tumors, however, utilize a variety of mechanisms that can suppress anti-tumor immunity. CD4+ regulatory T cells can directly inhibit cytotoxic T cell activity and these cells can be recruited, or induced, by cancer cells allowing escape from immune attack. The use of ionizing radiation as a treatment for cancer has been shown to enhance anti-tumor immunity by several mechanisms including immunogenic tumor cell death and phenotypic modulation of tumor cells. Less is known about the impact of radiation directly on suppressive regulatory T cells. In this study we investigate the direct effect of radiation on human TREG viability, phenotype, and suppressive activity. RESULTS: Both natural and TGF-ß1-induced CD4+ TREG cells exhibited increased resistance to radiation (10 Gy) as compared to CD4+ conventional T cells. Treatment, however, decreased Foxp3 expression in natural and induced TREG cells and the reduction was more robust in induced TREGS. Radiation also modulated the expression of signature iTREG molecules, inducing increased expression of LAG-3 and decreased expression of CD25 and CTLA-4. Despite the disconcordant modulation of suppressive molecules, irradiated iTREGS exhibited a reduced capacity to suppress the proliferation of CD8+ T cells. CONCLUSIONS: Our findings demonstrate that while human TREG cells are more resistant to radiation-induced death, treatment causes downregulation of Foxp3 expression, as well as modulation in the expression of TREG signature molecules associated with suppressive activity. Functionally, irradiated TGF-ß1-induced TREGS were less effective at inhibiting CD8+ T cell proliferation. These data suggest that doses of radiotherapy in the hypofractionated range could be utilized to effectively target and reduce TREG activity, particularly when used in combination with cancer immunotherapies.