Decreased regulatory T‐cells and CD4+/CD8+ ratio correlate with disease onset and progression in patients with generalized vitiligo

The aim of present study was to evaluate CD4⁺/CD8⁺ ratio and CD4⁺CD25^hiFoxP3⁺ Tregs in GV patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4⁺/CD8⁺ ratio and Tregs in 82 patients and 50 controls. CD8⁺ T‐cell counts were significantly higher in GV patients as compared with controls (p = 0.003). Active GV patients showed higher CD8⁺ T‐cell counts compared with stable GV patients (p = 0.001). The CD4⁺/CD8⁺ ratio decreased significantly in patients as compared with controls (p = 0.001). Moreover, the ratio in active GV patients significantly lowered as compared with stable GV patients (p = 0.002). Significant decrease in Treg cell percentage and counts in GV patients was observed compared with controls (p = 0.009, p = 0.008) with significant reduction in FoxP3 expression (p = 0.024). Treg cell percentage and counts were significantly decreased in active GV patients compared with stable GV patients (p = 0.007, p = 0.002). Our results suggest that an imbalance of CD4⁺/CD8⁺ ratio and natural Tregs in frequency and function might be involved in the T‐cell mediated pathogenesis of GV and its progression.     

MicroRNA‐126 regulates the induction and function of CD4+ Foxp3+ regulatory T cells through PI3K/AKT pathway

Recent evidence showed that limited activation of PI3K/Akt pathway was critical for induction and function sustainment of CD4⁺Foxp3⁺ regulatory T cells (Tregs). However, the underlying mechanism remains largely unknown. In this study, we reported that miR‐126 was expressed in mouse and human Tregs. Further study showed that silencing of miR‐126 using miR‐126 antisense oligonucleotides (ASO) could significantly reduce the induction of Tregs in vitro. Furthermore, miR‐126 silencing could obviously reduce the expression of Foxp3 on Tregs, which was accompanied by decreased expression of CTLA‐4 and GITR, as well as IL‐10 and TGF‐β, and impair its suppressive function. Mechanistic evidence showed that silencing of miR‐126 enhanced the expression of its target p85β and subsequently altered the activation of PI3K/Akt pathway, which was ultimately responsible for reduced induction and suppressive function of Tregs. Finally, we further revealed that miR‐126 silencing could impair the suppressive function of Tregs in vivo and endow effectively antitumour effect of CD8⁺T cells in adoptive cell transfer assay using a murine breast cancer model. Therefore, our study showed that miR‐126 could act as fine‐tuner in regulation of PI3K‐Akt pathway transduction in the induction and sustained suppressive function of Tregs and provided a novel insight into the development of therapeutic strategies for promoting T‐cell immunity by regulating Tregs through targeting specific miRNAs.     

Age-Related CD4+CD25+Foxp3+ Regulatory T-Cell Responses During Plasmodium berghei ANKA Infection in Mice Susceptible or Resistant to Cerebral Malaria

Different functions have been attributed to CD4⁺CD25⁺Foxp3⁺ regulatory T-cells (Tregs) during malaria infection. Herein, we describe the disparity in Treg response and pro- and anti-inflammatory cytokines during infection with Plasmodium berghei ANKA between young (3-week-old) and middle-aged (8-month-old) C57BL/6 mice. Young mice were susceptible to cerebral malaria (CM), while the middle-aged mice were resistant to CM and succumbed to hyperparasitemia and severe anemia. The levels of pro-inflammatory cytokines, such as TNF-α, in young CM-susceptible mice were markedly higher than in middle-aged CM-resistant mice. An increased absolute number of Tregs 3-5 days post-inoculation, co-occurring with elevated IL-10 levels, was observed in middle-aged CM-resistant mice but not in young CM-susceptible mice. Our findings suggest that Treg proliferation might be associated with the suppression of excessive pro-inflammatory Th1 response during early malaria infection, leading to resistance to CM in the middle-aged mice, possibly in an IL-10-dependent manner.     

The effect of icariin on autoimmune premature ovarian insufficiency via modulation of Nrf2/HO-1/Sirt1 pathway in mice

Primary ovarian insufficiency (POI) is a common gynecological disease. Autoimmunity is a common cause of POI. Icariin (ICA) plays a therapeutic role in many autoimmune diseases. This study aims to investigate the effect of ICA on autoimmune POI mice and its effect on immune regulation. Sixty-three female BALB/c mice were randomized into three groups (control, POI, POI + ICA). POI and POI + ICA group were hypodermically injected with zona pellucida three peptides (pZP3) to induce autoimmune POI. Then the POI + ICA group was gavaged with ICA. A vaginal smear was to observe estrous cycles, hematoxylin-eosin staining was to count follicles. Enzyme-linked immunosorbent analysis determined serum FSH, LH, AMH, and anti-zona pellucida antibody (AZPAb) levels. In addition, flow cytometry detected the expression of Th1 cells and Treg cells, and Western blot was used to detect the expression of Nuclear factor E2 related factor 2(Nrf2), heme oxygenase-1 (HO-1), and Sirtuin-1 (Sirt1) proteins. pZP3 treatment decreased serum AMH levels and increased FSH, LH, and AZPAb levels. Additionally, decreases in the number of healthy follicles at all stages and an increase in the number of atretic follicles. Abnormal ovarian structure and an arrested estrous cycle were also noted. However, ICA rescued POI through up-regulating Nrf2, HO-1, and Sirt1 expressions and up-regulating Treg expressions. ICA treatment improved the structure of the injured ovarian and its function in autoimmune POI mice. The mechanism is achieved by increasing the expression of Nrf2/HO-1/Sirt1 pathway in the ovary and increasing Treg cells' expression.     

Altered frequency of CD8+CD11c+ T cells and expression of immunosuppressive molecules in lymphoid organs of mouse model of colorectal cancer

CD11c is a member of the β2-integrin family typically used to define myeloid dendritic cells (DCs). Recent reports identify CD11c-expressing CD8⁺ T cells as a new subset of CD8⁺ regulatory T cells (Treg). Evidence exists that CD11c⁺CD8⁺ T cells may exert their effector or regulatory functions under different conditions. To date, no studies have addressed the frequency of CD11c⁺ T cells in cancer. Limited evidence exists in terms of expression of immune-checkpoint receptors, programmed cell death protein 1 (PD-1) and T-lymphocyte-associated antigen 4 (CTLA-4), as well as forkhead box P3 (Foxp3) in mouse lymphoid organs. Here, we have assessed CD11c⁺CD8⁺ and CD11c⁺CD4⁺ T cells, Foxp3, PD-1, and CTLA-4 expressing CD4⁺ T cells and CD8⁺ T cells in different tissues from three groups of male BALB/c mice—young, mature, and those with colorectal cancer (CRC). Analysis of CD3⁺CD11c⁺ T cells in the bone marrow (BM), spleen, and lymph nodes (LN) in each group showed a higher percentage of CD3⁺CD11c⁺ T cells in the BM from all groups and in the lymphoid organs of the cancer group compared with the young and mature groups. CD4^low and CD4^high cell fractions in mice BM have different expression patterns for Foxp3 and CTLA-4. We have observed a higher frequency of CD8⁺PD-1⁺ T cells in the BM, spleen, and LN of CRC mice compared with normal mice. T-cell exhaustion is associated with inhibitory receptor PD-1. According to the regulatory roles of CD11c expression in CD8⁺ T cells, we have proposed that the elevated percentage of CD11c, Foxp3, CTLA-4, and PD-1 expressing T cells were associated with immune response dysregulation in CRC.     

Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4⁺ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2^−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2^−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2^−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.     

Use of Tregs as a cell‐based therapy via CD39 for benign prostate hyperplasia with inflammation

Benign prostatic hyperplasia (BPH) occurs most commonly among older men, often accompanied by chronic tissue inflammation. Although its aetiology remains unclear, autoimmune dysregulation may contribute to BPH. Regulatory T cells (Tregs) prevent autoimmune responses and maintain immune homeostasis. In this study, we aimed to investigate Tregs frequency, phenotype, and function in BPH patients and to evaluate adoptive transfer Tregs for immunotherapy in mice with BPH via CD39. Prostate specimens and peripheral blood from BPH patients were used to investigate Treg subsets, phenotype and Treg‐associated cytokine production. Sorted CD39^+/? Tregs from healthy mice were adoptively transferred into mice before or after testosterone propionate administration. The Tregs percentage in peripheral blood from BPH patients was attenuated, exhibiting low Foxp3 and CD39 expression with low levels of serum IL‐10, IL‐35 and TGF‐β. Immunohistochemistry revealed Foxp3+ cells were significantly diminished in BPH prostate with severe inflammatory. Although the Tregs subset was comprised of more effector/memory Tregs, CD39 was still down‐regulated on effector/memory Tregs in BPH patients. Before or after testosterone propionate administration, no alterations of BPH symptoms were observed due to CD39‐ Tregs in mice, however, CD39⁺Tregs existed more potency than Tregs to regulate prostatic hyperplasia and inhibit inflammation by decreasing IL‐1β and PSA secretion, and increasing IL‐10 and TGF‐β secretion. Furthermore, adoptive transfer with functional Tregs not only improved prostate hyperplasia but also regulated muscle cell proliferation in bladder. Adoptive transfer with Tregs may provide a novel method for the prevention and treatment of BPH clinically.     

4-1BB Signaling in Conventional T Cells Drives IL-2 Production That Overcomes CD4+CD25+FoxP3+ T Regulatory Cell Suppression

Costimulation with the recombinant SA-4-1BBL agonist of 4-1BB receptor on conventional CD4⁺ T cells (Tconvs) overcomes the suppression mediated by naturally occurring CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs). The mechanistic basis of this observation has remained largely unknown. Herein we show that Tconvs, but not Tregs, are the direct target of SA-4-1BBL-mediated evasion of Treg suppression. IL-2 produced by Tconvs in response to 4-1BB signaling is both necessary and sufficient for overcoming Treg suppression. Supernatant from Tconvs stimulated with SA-4-1BBL contains high levels of IL-2 and overcomes Treg suppression in ex vivo Tconv:Treg cocultures. Removal of IL-2 from such supernatant restores Treg suppression and repletion of Tconv:Treg cocultures with exogenous recombinant IL-2 overcomes suppression. This study establishes 4-1BB signaling as a key circuit that regulates physical and functional equilibrium between Tregs and Tconvs with important implications for immunotherapy for indications where a fine balance between Tregs and Teffs plays a decisive role.     

Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4+CD25+ regulatory T cells

Naturally occurring CD4⁺CD25⁺ regulatory T cells (Tregs) are required to limit immune‐induced pathology and to maintain homeostasis during the early‐phase of sepsis. This study aimed to investigate the role of interleukin (IL)‐38, a newly described member of the IL‐1 cytokine family, in mediated immune response of CD4⁺CD25⁺ Tregs in sepsis. Here, we provide evidence that expressions of IL‐38 and its receptor were detected in murine CD4⁺CD25⁺ Tregs. Stimulation of CD4⁺CD25⁺ Tregs with LPS markedly up‐regulated the expression of IL‐38. Treatment with rmIL‐38 dramatically enhanced the immunosuppressive activity of CD4⁺CD25⁺ Tregs after LPS stimulation and in septic mice induced by CLP, resulting in amplification of helper T cell (Th) 2 response and reduction in the proliferation of effector T cells. These effects were robustly abrogated when anti–IL‐38 antibody was administered. Administration of rmIL‐38 improved the survival rate of CLP mice. In addition, CD4⁺CD25⁺ Tregs depletion before the onset of sepsis obviously abolished IL‐38–mediated protective response. These findings suggest that IL‐38 enhances the immunosuppressive activity of CD4⁺CD25⁺ Tregs, which might contribute to the improvement of host immune function and prognosis in the setting of sepsis.     

Effect of HIF1α on Foxp3 expression in CD4+CD25− T lymphocytes

The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4⁺CD25^? T lymphocytes. CD4⁺CD25^? T lymphocytes were sorted from PBMC using a CD4⁺CD25⁺ regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF‐lenti) and those containing empty expression vector (control‐lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF‐lenti) and those containing control vector (sicontrol‐lenti) were also generated. The sorted CD4⁺CD25^? T lymphocytes were infected with HIF‐lenti, control‐lenti, siHIF‐lenti, and sicontrol‐lenti, respectively. Approximately 72 hr after transduction, real‐time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4⁺CD25^? T lymphocytes cultured under 21% O₂, 5% CO₂ (normoxia) and 1% O₂, 5% CO₂ (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4⁺CD25^? T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4⁺ T lymphocytes which may promote CD4⁺ T lymphocytes to convert to Treg.

     

非霍奇金淋巴瘤患者外周血中CD4+CD25+ 调节性T 细胞 亚群变化初探

目的:检测非霍奇金淋巴瘤(non-Hodgkin’s lymphoma,NHL)患者外周血中CD4+CD25+调节性T细胞(CD4+CD25+regulatoryT cell,Treg)的改变,探讨Treg与NHL的相关性。方法:病例组(n=60)为本院收治的初诊NHL患者,对照组(n=60)为本院健康体检者,用流式细胞技术联合标记CD4、CD25检测对照组及病例组化疗前、化疗后的外周血中CD4+CD25+调节性T细胞的分布特点。结果:(1)病例组化疗前外周血中CD4+细胞比例显著低于对照组(P<0.05),CD4+CD25+调节性T细胞比例显著高于对照组(P<0.05);(2)病例组化疗后,CD4+细胞比例明显高于化疗前(P<0.05),CD4+CD25+调节性T细胞比例明显低于化疗前(P<0.05);(3)病例组化疗后CD4+细胞比例与对照组无显著差异(P>0.05),而CD4+CD25+调节性T细胞比例显著高于对照组(P<0.05)。结论:非霍奇金淋巴瘤患者外周血中CD4+CD25+调节性T细胞比例升高,存在机体免疫抑制,化疗可降低CD4+CD25+调节性T细胞比例。     

Aging is associated with increased regulatory T‐cell function

Regulatory T‐cell (Treg, CD4⁺CD25⁺) dysfunction is suspected to play a key role in immune senescence and contributes to increased susceptibility to diseases with age by suppressing T‐cell responses. FoxP3 is a master regulator of Treg function, and its expression is under control of several epigenetically labile promoters and enhancers. Demethylation of CpG sites within these regions is associated with increased FoxP3 expression and development of a suppressive phenotype. We examined differences in FoxP3 expression between young (3–4 months) and aged (18–20 months) C57BL/6 mice. DNA from CD4⁺ T cells is hypomethylated in aged mice, which also exhibit increased Treg numbers and FoxP3 expression. Additionally, Treg from aged mice also have greater ability to suppress effector T‐cell (Teff) proliferation in vitro than Tregs from young mice. Tregs from aged mice exhibit greater redox remodeling–mediated suppression of Teff proliferation during coculture with DCs by decreasing extracellular cysteine availability to a greater extent than Tregs from young mice, creating an adverse environment for Teff proliferation. Tregs from aged mice produce higher IL‐10 levels and suppress CD86 expression on DCs more strongly than Tregs from young mice, suggesting decreased T‐cell activity. Taken together, these results reveal a potential mechanism of higher Treg‐mediated activity that may contribute to increased immune suppression with age.     

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease

The adoptive transfer of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.     

Indirubin Increases CD4+CD25+Foxp3+ Regulatory T Cells to Prevent Immune Thrombocytopenia in Mice

Indirubin, a traditional Chinese medicine, is used to treat autoimmune diseases in clinics. However, the effects of indirubin on the immunosuppressive CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) have not been addressed. Thus, we aimed to investigate the effects of indirubin on CD4⁺CD25⁺Treg cells in immune thrombocytopenia (ITP) CBA mice, which were established by immunization with Wistar rat platelets. 50 mg/kg indirubin treatment daily for 4 weeks significantly decreased anti-platelet antibody production and prevented the decrease of platelets caused by immunization in ITP mice. Consistently, indirubin significantly enhanced the percentage and cell number of CD4⁺CD25⁺Foxp3⁺Treg cells in the peripheral blood, spleen and lymph nodes. We also observed a significant increase of the frequency and cell number of CD4⁺CD25⁺Foxp3⁺Treg cells in the thymus upon indirubin treatment. Furthermore, CD4⁺CD25⁺Treg cells from indirubin-treated mice showed similar immunosuppression on T effector cells as compared to those from control mice. Altogether, indirubin ameliorates ITP by enhancing CD4⁺CD25⁺Foxp3⁺Treg cell level with preserving immunosuppressive function.     

The Ratios of CD8+ T Cells to CD4+CD25+ FOXP3+ and FOXP3- T Cells Correlate with Poor Clinical Outcome in Human Serous Ovarian Cancer

Ovarian cancer is an immune reactive malignancy with a complex immune suppressive network that blunts successful immune eradication. This suppressive microenvironment may be mediated by recruitment or induction of CD4⁺ regulatory T cells (Tregs). Our study sought to investigate the association of tumor-infiltrating CD4⁺CD25⁺FOXP3⁺ Tregs, and other immune factors, with clinical outcome in serous ovarian cancer patients. We performed immunofluorescence and quantification of intraepithelial tumor-infiltrating triple positive Tregs (CD4⁺CD25⁺FOXP3⁺), as well as CD4⁺CD25⁺FOXP3^-, CD3⁺ and CD8⁺ T cells in tumor specimens from 52 patients with high stage serous ovarian carcinoma. Thirty-one of the patients had good survival (i.e. > 60 months) and 21 had poor survival of < 18 months. Total cell counts as well as cell ratios were compared among these two outcome groups. The total numbers of CD4⁺CD25⁺FOXP3⁺ Tregs, CD4⁺CD25⁺FOXP3^-, CD3⁺ and CD8⁺ cells were not significantly different between the groups. However, higher ratios of CD8⁺/CD4⁺CD25⁺FOXP3⁺ Treg, CD8⁺/CD4⁺ and CD8/CD4⁺CD25⁺FOXP3^- cells were seen in the good outcome group when compared to the patients with poor outcome. These data show for the first time that the ratios of CD8⁺ to both CD4⁺CD25⁺FOXP3⁺ Tregs and CD4⁺CD25⁺FOXP3^- T cells are associated with disease outcome in ovarian cancer. The association being apparent in ratios rather than absolute count of T cells suggests that the effector/suppressor ratio may be a more important indicator of outcome than individual cell count. Thus, immunotherapy strategies that modify the ratio of CD4⁺CD25⁺FOXP3⁺ Tregs or CD4⁺CD25⁺FOXP3^- T cells to CD8⁺ effector cells may be useful in improving outcomes in ovarian cancer.     

Glucose metabolism characteristics and TLR8-mediated metabolic control of CD4+ Treg cells in ovarian cancer cells microenvironment

Immunotherapy is expected to become the most promising new treatment for ovarian cancer owing to its immunogenicity. However, immunosuppression in the tumor microenvironment is a major obstacle to the efficacy of tumor therapy. Studies have found different metabolism ways of regulatory T cells (Tregs) in the cancer environment may be related to the immunosuppression and Toll-like receptor 8 (TLR8) can reverse the suppression function of Tregs. But it is still unclear that if the TLR8-mediated function reversal is associated with the change of glucose metabolism of Tregs. It was found that the positive expression rates of Glut1, HIF-1α, and Ki67 in CD4⁺ Treg cells of OC were significantly higher than that in benign ovarian tumor and HC, and also significantly higher than that in CD4⁺ Teffs of OC. What’s more, compared with CD4⁺ Teff group, CD4⁺ Tregs highly expressed seven genes and three proteins related to glucose metabolism and had higher levels of glucose uptake and glycolysis. After activating TLR8 signal of CD4⁺ Tregs, the proliferation level of naive CD4⁺ T cells was higher than that of the control group. At the same time, the expression levels of eight genes and five proteins related to glucose metabolism in CD4⁺ Treg cells with TLR8 activated were decreased and levels of glucose uptake and glycolysis were also lower. Furthermore, TLR8 signaling also downregulated the mTOR pathway in CD4⁺ Tregs. CD4⁺ Tregs pretreated with 2-deoxy-d-Glucose (2-DG) and galloflavin also attenuated the inhibition of Teffs proliferation. Although CD4⁺ Tregs pretreated with 2-DG and galloflavin before activating TLR8 signal had no significant difference compared with the group only treated with inhibitors, which suggested TLR8-mediated reversal of CD4⁺ Treg cells inhibitory function in ovarian cancer cells co-cultured microenvironment had a causal relationship with glucose metabolism.Subject terms: Glycobiology, Tumour immunology     

A tolerogenic semimature dendritic cells induce effector T-cell hyporesponsiveness by activation of antigen-specific CD4+CD25+ T regulatory cells that promotes skin allograft survival in mice

Semimature dendritic cells (smDCs) can induce autoimmune tolerance by activation of host antigen-specific CD4⁺CD25⁺ regulatory T (Treg) cells. We hypothesized that donor smDCs injected into recipients would induce effector T-cell hyporesponsiveness by activating CD4⁺CD25⁺Treg cells, and promote skin allograft survival. Myeloid smDCs were derived from C57BL/6J mice (donors) in vitro. BALB/c mice (recipients) were injected with smDCs to generate antigen-specific CD4⁺CD25⁺Treg cells in vivo. Allograft survival was prolonged when BALB/c recipients received either C57BL/6J smDCs prior to grafting or C57BL/6J smDC-derived CD4⁺CD25⁺Treg cells post-grafting, and skin flaps from these grafts showed the highest IL-10 production regardless of rapamycin treatments. Our findings confirm that smDCs constitute an independent subgroup of DCs that play a key role for inducing CD4⁺CD25⁺Treg cells to express high IL-10 levels, which induce hyporesponsiveness of effector T cells. Pre-treating recipients with donor smDCs may have potential for transplant tolerance induction.     

Characterization of age‐associated exhausted CD8+ T cells defined by increased expression of Tim‐3 and PD‐1

Aging is accompanied by altered T‐cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD‐1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T‐cell exhaustion and aging‐associated immune dysfunction. In this study, we demonstrate that T‐cell immunoglobulin mucin domain‐3 (Tim‐3), another exhaustion marker, is up‐regulated on aged T cells, especially CD8⁺ T cells. Tim‐3‐expressing cells also produced PD‐1, but Tim‐3⁺PD‐1⁺ CD8⁺ T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim‐3^?PD‐1⁺ cells. Tim‐3⁺PD‐1⁺ CD8⁺ T cells showed more evident properties associated with exhaustion than Tim‐3^?PD‐1⁺ CD8⁺ T cells: an exhaustion‐related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL‐10 and induced normal CD8⁺ T cells to produce IL‐10, which might contribute to immune dysregulation in aged mice. The generation of Tim‐3‐expressing CD8⁺ T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8⁺ T‐cell population with age‐associated exhaustion was distinguishable by its expression of Tim‐3. These results provide clues for understanding the alterations that occur in T‐cell populations with age and for improving dysfunctions related to the aging of the immune system.     

CD8+ T cells exhaustion induced by myeloid‐derived suppressor cells in myelodysplastic syndromes patients might be through TIM3/Gal‐9 pathway

CD8⁺ T cells play a central role in antitumour immunity, which often exhibit ‘exhaustion’ in the setting of malignancy and chronic viral infection due to T cell immunoglobulin and mucin domain 3 (TIM3) and myeloid‐derived suppressor cells (MDSCs). Our team previously found that overactive MDSCs and exhausted TIM3⁺CD8⁺ T cells were observed in myelodysplastic syndromes (MDS) patients. However, it is not obvious whether MDSCs suppress CD8⁺ T cells through TIM3/Gal‐9 pathway. Here, Gal‐9, as the ligand of TIM3, was overexpressed in MDSCs. The levels of Gal‐9 in bone marrow supernatants, serum and culture supernatants of MDSCs from MDS patients were elevated. CD8⁺ T cells from MDS or normal controls produced less perforin and granzyme B and exhibited increased early apoptosis after co‐culture with MDSCs from MDS. Meanwhile, the cytokines produced by CD8⁺ T cells could be partially restored by TIM3/Gal‐9 pathway inhibitors. Furthermore, CD8⁺ T cells produced less perforin and granzyme B after co‐culture with excess exogenous Gal‐9, and the function of CD8⁺ T cells was similarly restored by TIM3/Gal‐9 pathway inhibitors. Expression of Notch1, EOMES (associated with perforin and granzyme B secretion), p‐mTOR and p‐AKT (associated with cell proliferation) was decreased in CD8⁺ T cells from MDS after co‐culture with excess exogenous Gal‐9. These suggested that MDSCs might be the donor of Gal‐9, and TIM3/Gal‐9 pathway might be involved in CD8⁺ T cells exhaustion in MDS, and that TIM3/Gal‐9 pathway inhibitor might be the promising candidate for target therapy of MDS in the future.