Co‐expression of LAG3 and TIM3 identifies a potent Treg population that suppresses macrophage functions in colorectal cancer patients

Regulatory T (Treg) cells are critical suppressors of inflammation and are thought to exert mainly deleterious effects in cancers. In colorectal cancer (CRC), Foxp3⁺ Treg accumulation in tumors was associated with poor prognosis. Hence, we examined the circulating Treg cells in CRC patients. Compared to controls, CRC patients presented mild upregulations in CD4⁺CD25^+/hi T cells and in the more canonical CD4⁺CD25^+/hiFoxp3⁺ Treg cells in peripheral blood mononuclear cells. Both of these Treg populations could be roughly divided into lymphocyte activation gene 3 negative T cell immunoglobulin and mucin‐domain containing‐3 negative (LAG3^?TIM3^?) and LAG3⁺TIM3⁺ subsets. In CRC patients, the LAG3⁺TIM3⁺ subset represented approximately half of CD4⁺CD25^+/hi T cells and greater than 60% of CD4⁺CD25^+/hiFoxp3⁺ Treg cells, which was significantly more frequent than in healthy controls. Compared to the LAG3^?TIM3^? CD4⁺CD25^+/hi T cells, the LAG3⁺TIM3⁺ CD4⁺CD25^+/hi T cells presented considerably higher transforming growth factor‐β and slightly higher interleukin (IL)‐10 secretion, together with higher cytotoxic T‐lymphocyte associated protein 4 and Foxp3 expression levels. Notably, macrophages following incubation with LAG3^?TIM3^? CD4⁺CD25^+/hi T cells and LAG3⁺TIM3⁺ CD4⁺CD25^+/hi T cells displayed different characteristics. Macrophages incubated with LAG3⁺TIM3⁺ CD4⁺CD25^+/hi T cells presented lower expression of major histocompatibility complex class II, CD80, CD86, and tumor necrosis factor‐α but higher expression of IL‐10, than macrophages incubated with LAG3^?TIM3^? CD4⁺CD25^+/hi T cells. Together, our investigations demonstrated that CRC patients presented an enrichment of circulating Treg cells, in which the LAG3⁺TIM3⁺ subset exhibited more potent expression of inhibitory molecules, and furthermore, the LAG3⁺TIM3⁺ Treg cells could suppress the proinflammatory activation of macrophages more potently than the LAG3^?TIM3^? Treg cells.     

Regulatory T cells ameliorate acetaminophen-induced immune-mediated liver injury

The contribution of innate immune cells to acetaminophen (APAP)-induced liver injury has been extensively investigated. However, the roles of T cell populations among adaptive immune cells in APAP-induced liver injury remain to be elucidated. Herein, we found that distinct CD4⁺ T cell subsets but not CD8⁺ T cells modulated APAP-induced liver injury in mice. After APAP challenge, more CD62L^lowCD44^hiCD4⁺ T cells appeared in the liver, accompanied by increased IFN-γ. The removal of CD4⁺ T cells by either antibody depletion or genetic deficiency markedly compromised pro-inflammatory cytokine levels and ameliorated liver injury. Meanwhile, we also found that the frequency and absolute number of Treg cells also increased. Treg cell depletion increased hepatic CD62L^lowCD44^hiCD4⁺ T cells, augmented pro-inflammatory cytokines, and exacerbated liver injury, while adoptive transfer of Treg cells ameliorated APAP-induced liver injury. Furthermore, the recruitment of Treg cells into the liver through specific expression of CXCL10 in the liver could ameliorate APAP-induced liver injury. Our investigation suggests that Th1 and Treg subsets are involved in regulating APAP-induced liver injury. Thus, modulating the Th1/Treg balance may be an effective strategy to prevent and/or treat APAP-induced liver injury.     

Alloantigen specific T regulatory cells in transplant tolerance

CD4⁺CD25⁺Foxp3⁺T cells are regulatory/suppressor cells (Treg) that include non-antigen(Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naïve CD4⁺CD25⁺Treg develop into specific Tregs is unknown. We have studied DA rats tolerant to fully allogeneic PVG cardiac grafts that survived with out immunosuppression for over 100 days and identified the cellular basis of alloantigen specific tolerance. Key observations from our studies will be reviewed including how CD4⁺CD25⁺Tregs were first identified and the cytokine dependence of CD4⁺T cells that transfer alloantigen specific transplant tolerance which died in culture unless stimulated with both cytokine rich ConA supernatant and specific donor alloantigen. Both the tolerant CD4⁺CD25⁺ and CD4⁺CD25⁻ T cell populations are required to transfer tolerance, yet alone the CD4⁺CD25⁻ T cell effect rejection. Tolerance transfer occurs with a low ratio of CD4⁺CD25⁺T cells (< 1:10), whereas to induce tolerance with naive CD4⁺CD25⁺T cells requires both a ratio of > 1:1 and is not alloantigen specific.Recent findings on how naïve CD4⁺CD25⁺T cells developed into two separated pathways of alloantigen specific Tregs, by culturing them with alloAg with either IL-2 or IL-4 and donor alloantigen are described. IL-2 enhances IFN-γR and IL-5 mRNA while IL-4 induced a reciprocal profile with de novo IL-5Rα and increased IFN-γ mRNA expression. Both IL-2 and IL-4 alloactivated CD4⁺CD25⁺Tregs within 3–4 days of culture can induce alloantigen specific tolerance at ratios of 1:10. Long term, CD4⁺CD25⁺T cells from tolerant hosts given IL-2 cultured cells have increased IL-5 and IFN-γR mRNA; whereas hosts given IL-4 cultured cells had enhanced IL-5Rα mRNA expression and IL-5 enhanced their proliferation to donor but not third party alloAg.These findings suggest that Th1 and Th2 responses activate two pathways of alloantigen specific Tregs that can mediate transplant tolerance but are dependent upon cytokines produced by ongoing Th1 and/or Th2 immune responses.     

Downregulation of regulatory T cell function in patients with delayed fracture healing

Bone fracture healing is a multistage regenerative process that requires the collaboration of various cell types, with approximately 5%‐10% of fractures not healing properly. Accumulating evidence suggests that dysregulations in the immune system are associated with defective healing. In a cohort of 30 bone fracture patients between 50 and 62 years of age, 8 patients displayed delayed healing. Compared to the 22 normal healing patients, these 8 delayed healing patients presented significantly lower frequencies of CD4⁺CD25^hiFoxp3⁺ canonical regulatory T cells immediately following bone fracture and early on during the healing process. The CD4⁺CD25^+/hi T cells from delayed healing patients also presented reduced capacity to express transforming growth factor beta (TGF‐β), and presented reduced surface expression levels of inhibitory molecules, including CTLA‐4 and Lag‐3, compared to CD4⁺CD25^+/hi T cells from normal healing patients. Moreover, CD4⁺CD25^+/hi T cells from delayed healing patients were less potent in the suppression of CD4⁺CD25^? autologous conventional T cell proliferation, and presented reduced expansion capacity in response to interleukin (IL)‐2 stimulation. Overall, our results demonstrated multiple reductions in regulatory T cell function in delayed healing patients that could produce long‐lasting consequences in the bone fracture healing process.     

Follicular regulatory T cells infiltrated the ovarian carcinoma and resulted in CD8 T cell dysfunction dependent on IL-10 pathway

A high Treg/CD8 T cell ratio in ovarian carcinoma was negatively associated with the prognosis of the patients. The human follicular regulatory T (Tfr) cells are a newly characterized subset of Treg cells with features of both follicular helper T (Tfh) cells (CXCR5⁺) and canonical Treg cells (CD25⁺Foxp3⁺). The role of Tfr cells in ovarian cancer is yet unclear. We found that in peripheral blood, the ovarian cancer patients presented significantly higher levels of both CD4⁺CD25⁺CD127⁻CXCR5⁺ T cells and CD4⁺CD25⁺CD127⁻CXCR5⁺Foxp3⁺ T cells than the healthy controls. In resected tumor samples, Tfr cells represented a much greater percentage of lymphocytes than in peripheral blood. Interestingly, the circulating Tfr cells from ovarian cancer patients presented significantly higher TGFB1 and IL10 expression than their counterparts in healthy controls directly ex vivo, and significantly higher IL10 after stimulation. The tumor-infiltrating Tfr cells presented further upregulated expression of TGFB1 and IL10. In addition, the levels of TGFB1 and IL10 expression by Tfr cells negatively associated with the expression of IFNG in tumor-infiltrating CD8 T cells. In an in vitro CD8 T cell/Tfr cell coculture system, we found that Tfr cells could significantly suppress the activation of CD8 T cells, in a manner that was dependent on IL-10 and probably on TGF-β. Overall, our study found that Tfr cells could suppress CD8 T cells, and in ovarian cancer patients, the Tfr cells were increased in both frequency and function.     

Dysfunction of IL-10-producing type 1 regulatory T cells and CD4+CD25+ regulatory T cells in a mimic model of human multiple sclerosis in Cynomolgus monkeys

CD4⁺CD25⁺ Treg and IL-10⁺ Tr1 cells play a major role in controlling autoimmunity by suppressing self-reactive T cells. Dysfunction of Tregs appears to be a critical factor in the pathogenesis of autoimmune diseases. Multiple sclerosis (MS) is an inflammatory demyelinating disorder of CNS, where CD4⁺ T cells result in nervous tissue damage. The aim of this study was to investigate the protective role of Treg and Tr1 cells in a mimic model of human MS in Cynomolgus monkeys.This study indicated the suppressive capacity of Tregs from MS monkeys was impaired compared with naive controls. The population of CD4⁺CD25⁺ Tregs was decreased in acute stage of MS. However, they showed a restored function and percentage in remitting monkeys. In stable phase, CD4⁺CD25⁺ Tregs differentially expressed elevated level of CD62P cell adhesion molecule which contributes to the mechanism by which Treg cells inhibit CD4⁺ T cell responses. On the other hand, the percentage of CD4⁺IL-10⁺ Tr1 and suppressive function of Tr1 cells were found reduced in MS monkeys. IL-10 secretion was diminished almost 9-fold in active MS, and recovered in active MS. This deficit in IL-10 secretion was specific to CD3/CD46, but not to CD3/CD28 stimulation. The concentrations of IFN-γ secreted by CD3/CD46-activated T cells were also not affected.These results demonstrate that Tregs are dysfunctional in Cynomolgus monkey with MS. Loss of regulatory function appears to be an important factor in the pathogenesis of MS. Hence, to develop new approaches for induction of Tregs in vivo may be beneficial for the clinical treatment in autoimmune diseases.     

Thymol as a reciprocal regulator of T cell differentiation: Promotion of regulatory T cells and suppression of Th1/Th17 cells

Regulatory T cells (Tregs) are critical for maintaining immune response and enhancing their differentiation has therapeutic implications for autoimmune diseases. In this study, we investigated the effects of thymol a well-known monoterpene from Thyme on differentiation and function of Tregs. In vitro generation of Tregs from purified naïve CD4⁺CD25⁻ T cells in the presence of thymol was carried out. Suppressor activity of generated Tregs was examined by changes in the proliferation of CFSE-labeled conventional T cells. Thymol promotes differentiation of naïve CD4⁺CD25⁻ T cells to CD4⁺CD25⁺Foxp3⁺ Tregs [66.9–71.8% vs. control (47%)] and increased intensity of Foxp3 expression on Tregs (p < 0.01). In functional assay, an increased immune suppression by thymol-induced Tregs (≈2.5 times of untreated Tregs) was detected. For in vivo study, thymol was intraperitoneally administered to ovalbumin (Ova)-immunized mice. Flow cytometry assessment of spleens from thymol-treated Ova-immunized mice showed increased number of CD4⁺ Foxp3⁺ Tregs (>8%, p < 0.01(and decreased levels of CD4⁺T-bet⁺ Th1 and CD4⁺RORγt⁺ Th17 cells resulted in significant decreased Th1/Treg and Th17/Treg ratios. In ex vivo Ova challenge of splenocytes from thymol-treated Ova-immunized mice, similarly higher levels of CD4⁺ Foxp3⁺ Tregs, and also elevated TGF-β expression in CD4⁺Foxp3⁺ population (48.1% vs. 18.9% in untreated Ova-immunized group) and reduced IFN-γ-producing CD4⁺T-bet⁺ T cells and IL-17-producing CD4⁺RORγt⁺ T cells were detected. This led to marked decreased ratios of IFNγ/TGF-β and IL-17/TGF-β expressions. In conclusion, this study revealed thymol as a compound with enhancing effects on Treg differentiation and function, which may have potential benefits in treatment of immune-mediated diseases with Th1/Th17 over-activation.     

Tim3/Gal9 interactions between T cells and monocytes result in an immunosuppressive feedback loop that inhibits Th1 responses in osteosarcoma patients

The Tim3/Gal9 pathway is associated with immunosuppression and worse clinical outcome in multiple cancers. To illustrate the specific mechanism of Tim3/Gal9 interaction in osteosarcoma, we examined expression, function, and regulation of Tim3/Gal9 in various cells from osteosarcoma patients. Data showed that CD4⁺ T cells, CD8⁺ T cells, and monocytes from both peripheral blood and tumor of osteosarcoma patients contained high frequencies of Tim3⁺ cells, while the Gal9 expression was primarily found in regulatory T cells (Tregs) from osteosarcoma patients and was elevated compared to that in non-cancer controls. The Tim3⁺ CD4⁺ and CD8⁺ T cells presented lower proliferation capacity compared to their Tim3⁻ counterparts, which could be reverted by blocking Tim3 or Gal9. Interestingly, purified Tim3⁺ CD4⁺ T cells secreted more interferon gamma (IFNγ) than purified Tim3⁻ CD4⁺ T cells, but IFNγ production by Tim3⁺ CD4⁺ T cells was vulnerable to Gal9-mediated suppression. In monocytes, Tim3 expression was associated with high interleukin (IL)-10 and low IL-12 cytokine secretion profile. Exogenous recombinant Gal9, as well as CD4⁺ CD25⁺ Treg supernatant, further decreased IL-12 expression in monocytes. In CD4⁺ T cell-monocyte coculture experiments, Tim3⁺ monocytes inhibited IFNγ expression from total CD4⁺ T cells and the development of IFNγ response in naive CD4⁺ T cells. Blocking the Tim3/Gal9 pathway reverted these effects. Together, these results suggested that in osteosarcoma patients, Tim3 expression did not directly mediate immune suppression, but the interaction between Tim3⁺ T cells and monocytes, naive CD4⁺ T cells, and Gal9-expressing CD4⁺ CD25⁺ Tregs could resulting in progressive suppression of Th1 responses.     

Colorectal tumors are enriched with regulatory plasmablasts with capacity in suppressing T cell inflammation

Inflammation plays a critical role in the initiation of colorectal cancer but is also required to mediate antitumor immunity in established tumors. Therefore, identifying the cellular and molecular components in colorectal tumors is necessary for the understanding of tumor progression and the development of novel treatment strategies. In this study, we demonstrated that a specific subtype of regulatory B cells, the CD19^loCD27^hi plasmablasts, was enriched in the colorectal tumor microenvironment. This CD19^loCD27^hi plasmablast subset presented high interleukin 10 (IL-10) expression but not transforming growth factor-β (TGF-β) secretion. Phenotypically, the tumor-infiltrating IL-10⁺ CD19^loCD27^hi plasmablasts presented lower CD24, CD38, and IgA, and higher Tim-1 and IgG expression compared to the IL-10⁻ CD19^loCD27^hi plasmablasts. The tumor-infiltrating IL-10⁺ CD19^loCD27^hi plasmablasts were found to be gut-homing due to their higher expression of α4β7 while peripheral blood B cells did not show the same characteristic. When cocultured with autologous T cells, CD19^loCD27^hi plasmablasts demonstrated potent activity in suppressing interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) expression but did not promote Foxp3 expression. Overall, this study demonstrate that in colorectal cancer, CD19^loCD27^hi plasmablasts make up a large percentage in tumor-infiltrating lymphocytes and possess potent immunoregulatory functions, and thus could be utilized in future therapeutic strategies.     

Orally administered lactoperoxidase ameliorates dextran sulfate sodium-induced colitis in mice by up-regulating colonic interleukin-10 and maintaining peripheral regulatory T cells

We previously demonstrated orally administered bovine lactoperoxidase (LPO) ameliorated dextran sulfate sodium-induced colitis in mice. Here, we examine the mechanism of action of LPO. Three days after colitis induction, expression of interferon-γ mRNA in colonic tissue was significantly decreased in mice administered LPO; while mRNA expression of interleukin (IL)-10 and regulatory T cell (Treg) marker, Foxp3, were significantly increased. The proportion of CD4⁺CD25⁺ Tregs in peripheral CD4⁺ T cells was also significantly elevated when LPO was administered. Nine days after colitis induction, the severity of colitis symptoms, including body weight loss and colon shortening, was reduced and expression of IL-10 mRNA was increased in mice administered LPO. The proportion of CD4⁺CD25⁺ Tregs in peripheral leukocytes was also significantly elevated when LPO was administered. These results suggest LPO ameliorates colitis by up-regulating colonic anti-inflammatory cytokines and maintaining peripheral regulatory T cells.     

CD4+CD25+Foxp3+ T cells contribute to the antiasthmatic effects of Astragalus membranaceus extract in a rat model of asthma

Astragalus membranaceus (AM), a traditional Chinese medicinal herb, has been widely used for centuries to treat asthma in China. Previous studies demonstrated that AM had inhibitory effects on airway hyperresponsiveness, inflammation and airway remodeling in murine models of asthma. However, it remained unclear whether the beneficial effects of AM on asthma were associated with CD4⁺CD25⁺Foxp3⁺ Treg cells; this issue is the focus of the present work. An asthma model was established in Sprague–Dawley (SD) rats that were sensitized and challenged with ovalbumin. Bronchoalveolar lavage fluid (BALF) was assessed for inflammatory cell counts and cytokine levels. Airway hyperresponsiveness was detected by direct airway resistance analysis. Lung tissues were examined for cell infiltration, mucus hypersecretion and airway remodeling. CD4⁺CD25⁺Foxp3⁺ Treg cells in the BALF and Foxp3 mRNA expression in lung tissues were examined. The oral administration of AM significantly reduced airway hyperresponsiveness to aerosolized methacholine and inhibited eosinophil counts and reduced IL-4, IL-5 and IL-13 levels and increased INF-γ levels in the BALF. Histological studies showed that AM markedly decreased inflammatory infiltration, mucus secretion and collagen deposition in the lung tissues. Notably, AM significantly increased population of CD4⁺CD25⁺Foxp3⁺ Treg cells and promoted Foxp3⁺ mRNA expression in a rat model of asthma. Together, these results suggest that the antiasthmatic effects of AM are at least partially associated with CD4⁺CD25⁺Foxp3⁺ Tregs.     

LncRNA XLOC_003810 modulates thymic Th17/Treg balance in myasthenia gravis with thymoma

Imbalance of T helper 17 (Th17)/regulatory T (Treg) cells is involved in the pathogenesis of myasthenia gravis with thymoma (MG-T). Long non-coding RNAs (lncRNAs) are implicated in the regulation of Th17/Treg balance. This study was designed to explore the role of XLOC_003810, a novel lncRNA, in regulating the Th17/Treg balance in MG-T. The thymic CD4⁺ T cells were isolated from control subjects and MG-T patients. The Th17/Treg balance was evaluated by determining proportions of Th17 and Treg cells and expression of Th17- and Treg- associated molecules. Lentivirus-mediated silencing and overexpression of XLOC_003810 in CD4⁺ T cells were performed. The results showed that XLOC_003810 expression was higher in MG-T thymic CD4⁺ T cells than that in the control group. Furthermore, the ratio of Th17/Treg cells, proportion of Th17 cells and levels of Th17-associated molecules were significantly increased, whereas the proportion of Treg cells and levels of Treg-associated molecules were decreased in MG-T thymic CD4⁺ T cells. Importantly, the Th17/Treg imbalance in MG-T thymic CD4⁺ T cells was aggravated by XLOC_003810 overexpression, whereas it was attenuated by XLOC_003810 silencing. Collectively, XLOC_003810 modulates thymic Th17/Treg balance in MG-T patients, providing the scientific basis for the clinical targeted therapy of MG-T.     

Transplantation tolerance induced by regulatory T cells: In vivo mechanisms and sites of action

The mechanisms by which CD4⁺CD25⁺Foxp3⁺ T (Treg) cells regulate effector T cells in a transplantation setting and their in vivo homeostasis still remain to be clarified. Using a mouse adoptive transfer model, we analyzed the in vivo expansion, trafficking, and effector function of alloreactive T cells and donor-specific Treg cells, in response to a full-thickness skin allograft. Fluorescent-labeled CD4⁺CD25⁻ and antigen-specific Treg cells were transferred alone or co-injected into syngeneic BALB/c-Nude recipients transplanted with skins from (C57BL/6×BALB/c) F1 donors. Treg cells divided in vivo, migrated and accumulated in the allograft draining lymph nodes as well as within the graft. The co-transfer of Treg cells did not modify the early activation and homing of CD4⁺CD25⁻ T cells in secondary lymphoid organs. However, in the presence of Treg cells, alloreactive CD4⁺CD25⁻ T cells produced significantly less IFN-γ and were present in reduced numbers in the secondary lymphoid organs. Furthermore, time-course studies showed that Treg cells were recruited into the allograft at a very early stage after transplantation and effectively prevented the infiltration of effector T cells. In conclusion, suppression of rejection requires the early recruitment to the site of antigenic challenge of donor-specific Treg cells, which then mainly regulate the effector arm of T cell alloresponses.     

Epigenetic Modification of the FoxP3 TSDR in HAM/TSP Decreases the Functional Suppression of Tregs

HTLV-1 is a human retrovirus that is associated with the neuroinflammatory disorder HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). In these patients, HTLV-1 is primarily found in the CD4⁺CD25⁺ T cell subset (Regulatory T cells:Tregs), which is responsible for peripheral immune tolerance and is known to be dysfunctional in HAM/TSP. Recent evidence suggests that FoxP3 expression and function is determined epigenetically through DNA demethylation in the Treg-specific demethylated region (TSDR). We analyzed the methylation of the TSDR in PBMCs, CD4⁺ T cells, and CD4⁺CD25⁺ T cells from normal healthy donors (NDs) and HAM/TSP patients. We demonstrated that there is decreased demethylation in analyzed PBMCs and CD4⁺CD25⁺ T cells from HAM/TSP patients as compared to NDs. Furthermore, decreased TSDR demethylation was associated with decreased functional suppression by Tregs. Additionally, increased HTLV-1 Tax expression in HAM/TSP PBMC culture correlated with a concomitant decline in FoxP3 TSDR demethylation. Overall, we suggest that HTLV-1 infection decreases Treg functional suppressive capacity in HAM/TSP through modification of FoxP3 TSDR demethylation and that dysregulated Treg function may contribute to HAM/TSP disease pathogenesis.     

Activation of the aryl hydrocarbon receptor reduces the number of precursor and effector T cells,but preserves thymic CD4CD25Foxp3 regulatory T cells

Aryl hydrocarbon receptor (AhR) activation suppresses immune responses, including allergic sensitization, by increasing the percentage of regulatory (Treg) cells. Furthermore, AhR activation is known to affect thymic precursor T cells. However, the effect of AhR activation on intrathymic CD4⁺CD25⁺Foxp3⁺ Treg cells is unknown. Therefore, we investigated the effect of AhR activation on the percentage and number of CD4⁺CD25⁺Foxp3⁺ Treg cells during allergic sensitization in relevant immunological organs.