Intrahepatic regulatory T cells are phenotypically distinct from their peripheral counterparts in chronic HBV patients

Peripheral blood CD4+CD25+ regulatory T cells (Treg) prevent the development of strong HBV-specific T cell responses in vitro. In this study, we examined the phenotype of FoxP3+ regulatory T cells in the liver of patients with a chronic HBV infection. We showed that the liver contained a population of CD4+FoxP3+ cells that did not express CD25, while these cells were absent from peripheral blood. Interestingly, intrahepatic CD25-FoxP3+CD4+ T cells demonstrated lower expression of HLA-DR and CTLA-4 as compared to their CD25+ counterparts. Patients with a high viral load have a higher proportion of regulatory T cells in the liver, but not in blood, compared to patients with a low viral load. In conclusion, the intrahepatic Treg are phenotypically distinct from peripheral blood Treg. Our data suggest that the higher proportion of intrahepatic Treg observed in patients with a high viral load may explain the lack of control of viral replication.     

Effects of intrauterine exposure to maternal-derived HBeAg on T cell immunity in cord blood

Immature immune system and immune tolerance induced by exposure to HBeAg in utero and/or shortly after infection in newborns were reportedly the causes of chronic HBV infection. To investigate the effect of maternal-derived HBeAg on neonatal T cell immunity, we analysed and compared T cell phenotypes and functions among neonates born to HBsAg⁺/HBeAg⁺ mothers (HBeAg⁺ neonates), HBsAg⁺/HBeAg⁻ mothers (HBeAg⁻ neonates) and healthy control mothers (HC neonates), using flow cytometry. The results showed that neonatal T cell phenotypes were similar regardless of HBeAg exposure. Upon anti-CD3 and anti-CD28 stimulation in HBeAg⁺ neonates, CD4⁺ T cell production of IFN-γ (P < .05) was significantly enhanced, while CD8⁺ T cells secreted significantly more IL-2 compared with those in HBeAg⁻ and HC groups (P < .05). Moreover, similar levels of IFN-γ and IL-10 were observed in the culture supernatant after stimulation with rHBsAg, rHBcAg or rHBeAg among HBeAg⁺, HBeAg⁻ and HC neonates, whereas HBeAg⁺ neonates produced more TNF-α than HBeAg⁻ neonates upon stimulation with rHBcAg. In conclusion, the results indicated that the HBsAg⁺/HBeAg⁺ maternal environment did not influence the phenotypes of cord blood T cells but boosted neonatal non-specific Th1-type cytokine production.     

Circulating Tregs correlate with viral load reduction in chronic HBV-treated patients with tenofovir disoproxil fumarate

Limited response to current hepatitis B virus (HBV) drugs is possibly due to inadequate host cytotoxic cellular responses. Circulating Tregs have been shown to be associated with chronicity of HBV infection, but their profile during antiviral therapy has not been studied. We analyzed the frequency and effect of Tregs on cellular immune responses against HBV in 35 chronic hepatitis B eAg−ve and eAg+ve patients treated with tenofovir 300 mg/day. Frequency of Tregs and their modulatory role in cytokine-secreting cells were determined after stimulation with HBsAg or HBcAg in the absence or presence of Tregs and after blockage of PD-1/PDL-1 in peripheral blood mononuclear cells (PBMCs). Prior to therapy, eAg−ve patients had lower HBV DNA levels, reduced CD8 T cells, increased Tregs, and T cells expressing PD1. After 12 weeks of therapy, >2 log HBV viral reduction was observed in both groups, along with an increase frequencies of CD8 T cells in eAg−ve patients and increased expression of chemokine receptors/Toll-like receptors in both groups. PD-1 expression on CD8 cells in PBMCs was decreased in both groups during therapy but not on Tregs. In eAg–ve group, sustained increase of Tregs was observed till week 12, which declined at week 24. In both groups, after 24 weeks, depletion of CD4⁺CD25⁺ Tregs from PBMCs enhanced HBV-specific T cell responses, and blockage of PD-1/PDL1 pathway did enhance pro-inflammatory cytokine production in eAg+ve patients but not in eAg−ve. We conclude that Tregs induced by HBV replication in vivo are expanded in eAg−ve patients more. Reduction in HBV DNA by tenofovir partially restored adaptive immune responses and also reduced the Tregs. Blockage of PD-1/PDL1, enhanced cytokine production in eAg+ve patients but not in eAg−ve, suggests that distinctly different immunologic mechanisms are involved in eAg+ve and eAg−ve patients.     

Amphiregulin promotes the immunosuppressive activity of intrahepatic CD4+ regulatory T cells to impair CD8+ T-cell immunity against hepatitis B virus infection

Hepatitis B virus (HBV) infection causes liver diseases and hepatocellular carcinoma. Immunotolerance in HBV-infected patients is one of the factors that incur failure of HBV clearance and persistent HBV amplification. However, the mechanisms underlying immunotolerance after HBV infection are yet to be thoroughly understood. Using a novel HBV mouse model, we found for the first time that epidermal growth factor receptor (EGFR) is up-regulated on intrahepatic regulatory T (Treg) cells in HBV-infected mouse livers. The EGFR-positive Treg cells are more immunosuppressive than EGFR-negative Treg cells, demonstrated by higher expression of immunosuppressive cytokines and robust inhibition of CD8⁺ T-cell proliferation in vitro. Furthermore, EGFR-positive Treg cells potently restrain CD8⁺ T-cell-mediated anti-viral activity, leading to higher HBV burden in hepatocytes. Amphiregulin, a cytokine of the EGF family, is significantly up-regulated in HBV-infected livers, but the cellular sources of amphiregulin are still elusive. Amphiregulin promotes the immunosuppressive activity of EGFR-positive Treg cells in vitro, so as to profoundly inhibit production of anti-viral components in CD8⁺ T cells. Taken together, our discovery elucidated a novel mechanism contributing to immunotolerance and viral amplification after HBV infection. Our study may provide new clues for developing therapeutic strategies against HBV infection.     

PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients

Programmed death-1 (PD-1) is demonstrated to have an increased expression on antigen-specific T cells during chronic virus infections, and the blockage of PD-1/PD-ligand (PD-L1) pathway could restore the function of exhausted T cells. We measured the PD-1 expression levels on HBV-specific CD8 T cells and investigated the role of PD-1/PD-L1 pathway in T-cell responses of patients with different HBV infection statuses. Compared to the patients with convalescent acute hepatitis B, PD-1 expression on total CD8 T cells from chronic hepatitis B (CHB) patients was significantly upregulated, especially on the HBV pentamer-positive CD8 T cells. And PD-L1, but not PD-L2, was also significantly upregulated on PBMC from CHB patients. In CHB patients, HBV-specific T cells and cellular proliferation could be observed under the recombinant HBV-Ag stimulation in vitro, and blockade of PD-1 pathway significantly enhanced the IFN-gamma production and cellular proliferation of PBMC. Furthermore, PD-1 expression level on HBV-pentamers positive CD8 T cells was positively associated with plasma viral load in CHB patients. Thus, PD-1 upregulation on HBV-specific CD8 T cells is engaged in the dysfunction of T cells and high viraemia in CHB patients, and the antiviral T-cell responses could be improved by the blockade of this inhibitory PD-1/PD-L1 pathway.     

Systemic Immunomodulatory Effects of Combinatorial Treatment of Thalidomide and Dexamethasone on T Cells and Other Immune Cells

PurposeIn organ transplantation, the need for immune modulation rather than immune suppression has been emphasized. In this study, we investigated whether combinatorial treatments of with thalidomide (TM) and dexamethasone (DX) might be new approaches to induce systemic immunomodulation on T cells and other immune cells that regulate the expression of co-inhibitory molecules.Materials and MethodsNaïve splenic T cells from C57BL/6 mice were sort-purified and cultured in vitro for CD4⁺ T cell proliferation and regulatory T cell (Treg) conversion in the presence of TM or/and DX. Expression of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1) in proliferated and converted T cells was quantified by flow cytometry. We also quantified in vivo expression of CTLA-4 and PD-1 on splenic CD4⁺ T cells and other immune cells isolated from TM- or/and DX-treated mice. Mixed lymphocytes reactions (MLR) were performed to evaluate the capacity of immune cells in carrying out immune responses.ResultsCTLA-4 expressions in effector T cells in vivo and in Tregs in vivo/vitro significantly increased upon TM/DX combinatorial treatment. Corresponding to increased CTLA-4 expression in T cells, the expression of ligand molecules for CTLA-4 significantly increased in splenic dendritic cells in TM/DX-treated groups. In addition, MLR results demonstrated that splenocytes isolated from TM/DX-treated mice significantly suppressed the proliferation of T cells isolated from other strains.ConclusionBased on these results, we suggest that TM/DX combinatorial treatments might be efficient immunomodulatory methods for regulating T cell immunity.     

T follicular regulatory cells infiltrate the human airways during the onset of acute respiratory distress syndrome and regulate the development of B regulatory cells

T follicular regulatory (Tfr) cell is a CXCR5⁺Foxp3⁺ subset of T regulatory (Treg) cell with critical roles in regulating germinal center responses and modulating the immune environment in the lymph nodes. Studies have shown that the proportion of Tfr cells may increase during acute inflammation. In this study, we investigated the role of Tfr cells in acute respiratory distress syndrome (ARDS). We found that Tfr cells were significantly enriched in peripheral blood and in mini-bronchoalveolar lavage (BAL) during the onset of ARDS. Notably, Tfr cells represented the majority of Treg cells in the mini-BAL samples. Tfr cells also showed CTLA-4, IL-10, and TGF-β expression, but compared to the non-Tfr Treg cells, the CTLA-4 and IL-10 expression by Tfr cells were slightly reduced. Both Tfr cells and non-Tfr Treg cells suppressed the proliferation of autologous CD4⁺CD25^? T cells; however, the Tfr cells displayed slightly reduced suppression capacity. Subsequently, B cells were co-incubated with autologous Tfr cells or non-Tfr Treg cells. Interestingly, we found that the frequency of IL-10⁺ Breg cells was significantly higher following incubation with Tfr cells than with non-Tfr Treg cells, which suggested that Tfr cells were more potent at inducing IL-10⁺ Breg cells. Together, these results demonstrated that Tfr cells were a similar but distinctive subset of Treg cells. Given that Tfr cells were strongly enriched in ARDS patients, especially in the lung infiltrates, they may exert critical ameliorating effects in ARDS.     

Accumulation of CD5<Superscript>+</Superscript>CD19<Superscript>+</Superscript> B lymphocytes expressing PD-1 and PD-1L in hypertrophied pharyngeal tonsils

Programmed death-1 (PD-1) is one of the most important inhibitory co-receptors expressed predominantly on activated T and B lymphocytes whose expression could be sustained by permanent antigenic stimulation accompanying chronic or recurrent tonsillitis. The expression of PD-1 and PD-1L was analyzed using flow cytometry on hypertrophied tonsils collected from 57 children. We observed high expression of PD-1 and PD-1L on certain lymphocytes subpopulations of hypertrophied tonsils; among T cells, the expression of PD-1 on protein level was higher on CD4⁺ cells (70.3 %) than on CD8⁺ cells (35 %). Interestingly, a limited expression of PD-1 was observed on CD19⁺ B lymphocytes (6.5 %), while CD5⁺CD19⁺ B cells overexpressed PD-1 (52.5 %). Moreover, the expression of PD-1L was also higher on CD5⁺CD19⁺ B cells (16.5 %) than on CD19⁺ B cells (3.5 %) and on CD4⁺ T cells (20 %) than on CD8⁺ T cells (10 %). PD-1 and PD-1L expressions correlated only on CD5⁺CD19⁺ cells. In conclusion, high expression of PD-1 and PD-1L on T and B cells could represent hallmark of immune system adaptation to chronic antigenic exposition in patients with tonsillitis.     

LncRNA XLOC_003810 promotes T cell activation and inhibits PD-1/PD-L1 expression in patients with myasthenia gravis-related thymoma

This study aimed to investigate the effect of long non-coding RNA XLOC_003810 on the activation of CD4⁺ T cells and expression of PD-1/PD-L1 in patients with myasthenia gravis-related thymoma (MG-T). Thymus specimens and thymic mononuclear cells were obtained from MG and MG-T patients or cardiac surgery patients undergoing thoracotomy who were selected as negative controls (NC). XLOC_003810 expression was examined using quantitative real-time PCR (qRT-PCR). Frequency of CD4⁺ T cells and proportion of CD4⁺PD-1⁺ T cells and CD14⁺PD-L1⁺ monocytes were quantified by flow cytometry. The release of inflammatory cytokines was measured by qRT-PCR and enzyme-linked immunosorbent assay. Compared with the NC group, expression of XLOC_003810, frequency of CD4⁺ T cells and the production of inflammatory cytokines were increased in patients with MG and MG-T. XLOC_003810 overexpression significantly increased the frequency of CD4⁺ T cells, facilitated the production of inflammatory cytokines and decreased the proportion of CD4⁺PD-1⁺ T cells and CD14⁺PD-L1⁺ monocytes in the thymic mononuclear cells. In contrast, XLOC_003810 knockdown exerted the opposite effect. Together, XLOC_003810 promotes T cell activation and inhibits PD-1/PD-L1 pathway in patients with MG-T.     

Combination of pegylated interferon-alpha and nucleos(t)ide analogue treatment enhances the activity of natural killer cells in nucleos(t)ide analogue experienced chronic hepatitis B patients

A combination of pegylated interferon-alpha (peg-IFN-α) and nucleos(t)ides analogue (NA) therapy can effectively reduce hepatitis B surface antigen (HBsAg), especially in NA-experienced chronic hepatitis B (CHB) patients. However, the immune mechanism of this therapy is unclear. Forty NA-experienced CHB patients were enrolled into this study. The frequencies of peripheral blood natural killer (NK) cells, dendritic cells (DCs), CD4⁺ T cells, CD8⁺ T cells, T helper (Th) cells, regulatory T cells (T_reg), B cells and follicular T helper (Tfh) cells were evaluated by flow cytometry. Seven of the 40 patients converted to peg-IFN-α combined with NA treatment, while the other 33 continued to NA therapy. The decrease in HBsAg was more pronounced in the combination treatment group, and only patients receiving combination treatment achieved HBsAg loss. The frequency and absolute number of CD56^bright NK cells in the combination treatment group increased significantly compared with the NA treatment group, whereas the CD56^dim NK cells were decreased. In the NA treatment group, the proportions of CD4⁺ T_N, CD8⁺ T_N, CD19⁺ B and cytotoxic T lymphocyte antigen-4 (CTLA-4)⁺CD4⁺ T cells were increased, while the proportions of CD4⁺ T_EM, CD8⁺ T_EM, CD25⁺CD4⁺ T_reg, CD25^highCD4⁺ T_reg, CD127^lowCD25⁺ T_reg, programmed cell death 1 (PD-1)⁺CD4⁺ T, PD-1⁺CD8⁺ T, CTLA-4⁺CD8⁺ T, CCR4⁺CD25⁺ T_reg and CCR4⁺CD25^high T_reg cells were decreased after therapy. For NA-experienced CHB patients who achieved low HBsAg levels, combination treatment is more likely to result in HBsAg decline and HBsAg clearance by increasing the activity of CD56^brightNK cells.     

Intrahepatic Expression of Programmed Death-1 and its Ligands in Patients with HBV-Related Acute-on-Chronic Liver Failure

Hepatitis B virus (HBV) infection is a major public health problem, and HBV-related acute-on-chronic liver failure (HBV-ACLF) has an extremely poor prognosis due to a lack of understanding of pathogenesis as well as a lack of effective treatments. Signals from the inhibitory receptor programmed death-1 (PD-1) have been demonstrated to be involved in regulating the pathogenesis of infectious diseases. However, the expression of PD-1 and its ligands in HBV-ACLF patients has yet to be evaluated. In this study, the expression of PD-1 and its ligands, PD-L1 and PD-L2, in liver biopsies from HBV-ACLF as well as chronic hepatitis B (CHB) patients were analyzed by immunohistochemistry. The results showed that all three molecules were observed in the HBV-ACLF samples and their levels were significantly higher than they were in CHB. Immunofluorescence double-staining showed that PD-1 was found on CD3⁺, CD8⁺ T cells, CD56⁺ NK cells, CD68⁺ macrophages, CK-18⁺ epithelial cells, and CD16⁺ monocytes. The PD-L1 expression was observed on all cell types detected and the PD-L2 was chiefly on CK-18⁺ epithelial cells and CD31⁺ endothelial cells. Interestingly, high levels of virus-induced procoagulant molecule fibrinogen-like protein 2 (FGL2) were observed in liver sections from HBV-ACLF, and PD-L1 and PD-L2 expression was also observed on FGL2⁺ cells in these patients. Our combined results suggest that the expression of PD-L1 and PD-L2 may be biomarkers to identify and diagnose ACLF, and a clear understanding of their functional roles should further elucidate the pathogenesis of this disease.     

Soluble PD-1 rescues the proliferative response of simian immunodeficiency virus-specific CD4 and CD8 T cells during chronic infection

Phenotypic and functional studies of the programmed death-1 (PD-1) molecule on CD4⁺ and CD8⁺ T cells were performed on peripheral blood mononuclear cells from uninfected and simian immunodeficiency virus (SIV)-infected rhesus macaques. These data demonstrated a rapid upregulation of PD-1 expression on tetramer-positive CD8⁺ T cells from MamuA.01⁺ SIV-infected macaques upon infection. Upregulation of PD-1 on total CD8⁺ T cells was not detectable. In contrast, CD4⁺ T-cell PD-1 expression was markedly higher in total CD4⁺ T cells during chronic, but not acute, infection and there was a correlation between the level of PD-1 expression on naive and central memory CD4⁺ T cells and the levels of viral loads. Such association was emphasized further by a marked decrease of PD-1 expression on tetramer-positive CD8 T cells as well as on CD4⁺ T cells on longitudinal samples collected before and after the initiation of antiretroviral therapy and downregulation of viral replication in vivo. Cloning of PD-1 and its two ligands from several non-human primate species demonstrated > 95% conservation for PD-1 and PD-L2 and only about 91% homology for PD-L1. Functional studies using soluble recombinant PD-1 protein or PD-1–immunoglobulin G fusion proteins induced marked increases in the SIV-specific proliferative responses of both CD4⁺ and CD8⁺ T cells from rhesus macaques. The results of these studies serve as a foundation for future in vivo trials of the use of rMamu-PD-1 to potentially enhance and/or restore antiviral immune responses in vivo.     

B7-H1 on myeloid-derived suppressor cells in immune suppression by a mouse model of ovarian cancer

Myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing hosts and are associated with immune suppression. Here, we described high level of expression of B7-H1 (CD274), PD-1 (CD279) and CTLA4 (CD152) by Gr-1⁺CD11b⁺ MDSCs obtained from both ascites and spleens of mice bearing the 1D8 ovarian carcinoma, whereas B7-DC (CD273), CD40 and CD86 were absent. In contrast, B7-H1, PD-1 and CTLA-4 expression was not detected on Gr-1⁺CD11b⁺ cells from naive mice. Expression of B7-H1 by Gr-1⁺CD11b⁺ cells from naive mice could be induced by co-culture with 1D8 ovarian carcinoma cells. Gr-1⁺CD11b⁺ cells derived from 1D8 tumor-bearing mice markedly suppressed antigen-specific immune responses, whereas Gr-1⁺CD11b⁺ cells from naive mice did not. siRNA-mediated knockdown of B7-H1 in Gr-1⁺CD11b⁺ cells of 1D8 tumor-bearing mice alleviated suppression of antigen-specific immune responses. Suppression of antigen-specific immune responses via B7-H1 on Gr-1⁺CD11b⁺ myeloid cells was mediated by CD4⁺CD25⁺ Foxp3⁺ T regulatory cells and required PD-1. Antibody blockade of either B7-H1 or PD-1 retarded the growth of 1D8 tumor in mice. This suggests that expression of B7-H1 on Gr-1⁺CD11b⁺ myeloid cells triggered by the 1D8 mouse model of ovarian carcinoma suppresses antigen-specific immunity via interaction with PD-1 on CD4⁺CD25⁺ Foxp3⁺ regulatory T cells.     

Function of T regulatory type 1 cells is down-regulated and is associated with the clinical presentation of coronary artery disease

T regulatory type 1 (Tr1) cells can promote tolerance and suppress inflammation. Atherosclerosis may be induced by the proinflammatory activation of cells in the vasculature and the immune system. Hence, we wondered whether defects in Tr1 function were a contributing factor to coronary artery disease (CAD). Data showed that the frequency of IL-10⁺ Tr1 cells was significantly lower in CAD patients than in controls. Compared to healthy controls, Tr1 cells from CAD patients presented lower CTLA-4 but higher PD-1 expression, in addition to lower IL-10 secretion. When co-incubated with Tconv cells, the CD4⁺CD49b⁺LAG-3⁺CD45RO⁺ Tr1 cells presented IL-10-dependent inhibitory effects, and those from CAD patients presented significantly lower suppression capacity than those from healthy controls. Interestingly, the characteristics of Tr1 cells were associated with clinical features of CAD patients. The frequency of Tr1 cells and the IL-10 and LAG-3 expression by Tr1 cells were negatively correlated with the BMI of the CAD patients. In addition, the Tr1 frequency and the LAG-3 and CTLA-4 expression on Tr1 cells were lower in CAD patients with higher numbers of narrowed vessels. Together, these results suggest that in CAD, Tr1 cells present multiple defects, which are associated with the clinical presentation of the disease.     

Age-related accumulation of T cells with markers of relatively stronger autoreactivity leads to functional erosion of T cells

Background

Although it is known that regulatory T cells (Tregs) can suppress the function of effector T cells, and may contribute to impaired immune response, the precise role of Tregs during the course of hepatitis B virus (HBV) infection remains to be elucidated. A newly identified subset of the CD4⁺Foxp3⁺ Tregs, the CD39⁺ Tregs, has been associated with viral infections and autoimmune diseases. Therefore, we hypothesized that this discrete Treg subset may contribute to the chronic infection of HBV.

Results

Initial characterization studies of healthy peripheral CD39⁺FoxP3⁺CD4⁺ T cells revealed that the majority were CD45RA^- Treg cells. Subsequent analysis of HBV-infected patients (38 asymptomatic HBV carriers (AsCs), 37 chronic active hepatitis B (CAH), 29 HBV-associated acute-on-chronic liver failure (ACLF)) and healthy individuals (25 controls) was conducted to assess association with HBV copy number and the liver injury marker alanine aminotransferase (ALT). A higher percentage of CD39⁺ Tregs was detected within the population of FoxP3⁺CD4⁺ T cells in peripheral blood of AsCs patients. Moreover, the percentage of CD39⁺ Tregs was significantly less in CAH and ACLF patients. The increased proportions of circulating CD39⁺ Tregs were positively correlated with serum viral load, but inversely correlated with serum ALT level.

Conclusion

These findings not only suggest that CD39⁺ Treg cells may be involved in HBV disease progression but also identify CD39⁺ Tregs as a dynamic immune regulatory cell population that may represent a new target of immunomodulatory therapeutic interventions.     

Aging and human CD4 regulatory T cells

Alterations in immunity that occur with aging likely contribute to the development of infection, malignancy and inflammatory diseases. Naturally occurring CD4⁺ regulatory T cells (Treg) expressing high levels of CD25 and forkhead box P3 (FOXP3) are essential for regulating immune responses. Here we investigated the effect of aging on the number, phenotypes and function of CD4⁺ Treg in humans. The frequency and phenotypic characteristics of CD4⁺, FOXP3⁺ T cells as well as their capacity to suppress inflammatory cytokine production and proliferation of CD4⁺, CD25⁻ T cells (target cells) were comparable in young (age ≤40) and elderly (age ≥65) individuals. However, when CD4⁺, FOXP3⁺ Treg and CD4⁺, CD25⁻ T cells were co-cultured at a ratio of 1:1, the production of anti-inflammatory cytokine IL-10 from CD4⁺, CD25⁻ T cells was more potently suppressed in the elderly than in the young. This finding was not due to changes in CTLA-4 expression or apoptosis of CD4⁺, FOXP3⁺ Treg and CD4⁺, CD25⁻ T cells. Taken together, our observations suggest that aging may affect the capacity of CD4⁺, FOXP3⁺ T cells in regulating IL-10 production from target CD4⁺ T cells in humans although their other cellular characteristics remain unchanged.     

Analysis of CD8+ Treg cells in patients with ovarian cancer: a possible mechanism for immune impairment

Regulatory T (Treg) cells may participate in mediating a suppressive microenvironment that blunts successful anti-tumor immunotherapy. Recent studies show that CD8⁺ Treg cells might impede effective immune responses to established tumors. However, there is limited research regarding CD8⁺ Treg cells in ovarian cancer (OC) patients. Here, we investigated CD8⁺ Treg cells in OC patients and their in vitro induction. The immunohistochemistry of tumor-infiltrating lymphocytes revealed a significant correlation between the intratumoral CD8⁺ T cells and the forkhead box p3 (Foxp3)⁺ cells in the intraepithelial and stromal areas of advanced OC tissues. We examined the expression of Treg markers in CD8⁺ T cells from the peripheral blood and fresh tumor tissues of OC patients using flow cytometry. Our results indicated an increase in the CD8⁺ Treg cell subsets of OC patients compared with those in patients with benign ovarian tumors and healthy controls, including an increased expression of CD25, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and Foxp3 and decreased CD28 expression. To demonstrate whether the tumor microenvironment could convert CD8⁺ effector T cells into suppressor cells, we used an in vitro transwell culturing system. Compared with the CD8⁺ T cells cultured alone, the CD8⁺ Treg cells induced in vitro by coculture with SK-OV-3/A2780 showed increased CTLA-4 and Foxp3 expression and decreased CD28 expression. In addition, the in vitro-induced CD8⁺ Treg cells inhibited naïve CD4⁺ T-cell proliferation, which was partially mediated through TGF-β1 and IFN-γ. Our study suggests that CD8⁺ Treg cells were increased in OC patients and could be induced in vitro, which may be the way that tumors limit antitumor immunity and evade immune surveillance.