Identity of mysterious CD4<Superscript>+</Superscript>CD25<Superscript>-</Superscript>Foxp3<Superscript>+</Superscript>cells in systemic lupus erythematosus

Various abnormalities in CD4⁺CD25⁺ regulatory T cells (Tregs) in systemic lupus erythematosus (SLE) include increased Foxp3⁺ cells that are CD25 negative. Barring methodological technical factors, these cells could be atypical Tregs or activated non-Treg CD4⁺ cells that express Foxp3. Two groups have reached opposite conclusions that could possibly reflect the subjects studied. One group studied untreated new-onset SLE and suggested that these T cells were mostly CD25^-Foxp3⁺ non-Tregs. The other group studied patients with long-standing disease and suggested that these cells are mostly dysfunctional Tregs. A third group reported increased Foxp3⁺CD4⁺CD25^dim rather than CD25^- cells in active SLE and these were also non-Tregs. Thus, it is likely that not all Foxp3⁺ T cells in SLE have protective suppressive activity.     

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.     

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.     

Increased tumor-infiltrating CD8+Foxp3+ T lymphocytes are associated with tumor progression in human gastric cancer

Background

CD8⁺Foxp3⁺ T lymphocytes have been detected in tumors. However, the distribution, phenotypic features, and regulation of these cells in gastric cancer remain unknown.

Methods

The levels of CD8⁺Foxp3⁺ T lymphocytes in the peripheral blood, tumor-draining lymph nodes, non-tumor tissues, and tumor tissues of patients with gastric cancer were detected by flow cytometry. Foxp3 induction in CD8⁺Foxp3^? T cells was investigated in vitro. The suppressive function of CD8⁺Foxp3⁺ T lymphocytes was analyzed by their effect on CD4⁺ T-cell proliferation and IFN-γ production. The percentages of CD8⁺Foxp3⁺ T lymphocytes were evaluated for the association with tumor stage.

Results

The frequency of CD8⁺Foxp3⁺ T lymphocytes in tumor tissues was significantly higher than that in non-tumor tissues, and similar results were also observed in tumor-draining lymph nodes compared with peripheral blood. Most intratumoral CD8⁺Foxp3⁺ T lymphocytes were activated effector cells (CD45RA^?CD27^?). TGF-β1 levels were positively correlated with the frequency of CD8⁺Foxp3⁺ T lymphocytes in tumor tissues, and in vitro TGF-β1 could induce the generation of CD8⁺Foxp3⁺ T lymphocytes in a dose-dependent manner. Furthermore, intratumoral CD8⁺Foxp3⁺ T lymphocytes suppressed the proliferation and IFN-γ production of CD4⁺ T cells. Finally, intratumoral CD8⁺Foxp3⁺ T lymphocytes were significantly increased with tumor progression in terms of tumor-node-metastasis (TNM) stage.

Conclusions

Our data have shown that increased intratumoral CD8⁺Foxp3⁺ T lymphocytes are associated with tumor stage and potentially influence CD4⁺ T-cell functions, which may provide insights for developing novel immunotherapy protocols against gastric cancer.     

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.     

Relationship between the anti-inflammatory properties of salmeterol/fluticasone and the expression of CD4+CD25+Foxp3+ regulatory T cells in COPD

Background

Salmeterol and fluticasone combination (SFC) has anti-inflammatory effects and improves clinical symptoms in patients with chronic obstructive pulmonary disease (COPD). However, the anti-inflammatory mechanism of SFC remains unclear. In this study, we investigated the inflammatory responses of COPD, as well as the relationship of the inflammatory factors with the levels of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Foxp3⁺Tregs) after SFC therapy.

Methods

Twenty-one patients with moderate or severe COPD received treatment with 50/500 μg of SFC twice a day for 12 weeks. Before and after treatment, the patients were evaluated using the Modified Medical Research Council (MMRC) dyspnea scale and by conducting a 6-min walk test. The number of neutrophils, monocytes and lymphocytes in induced sputum were counted. Levels of cytokines, including pre-inflammatory IL-8, TNF-α, IL-17A and cytokine IL-10, in the sputum supernatant and peripheral blood were measured by ELISA. The proportion of Foxp3⁺Tregs in the total CD4⁺ T cell of the peripheral blood was determined by flow cytometry. The relationship between IL-17A levels and the percentage of Foxp3⁺Tregs was analyzed by statistical analysis.

Results

After treatment with SFC, the forced expiratory volume in 1 s as a percentage of predicted values (FEV1%) and the 6-min walk distance in the COPD patients significantly increased, while dyspnea scores decreased. The total number of cells, neutrophils, and the percentage of neutrophils in induced sputum reduced notably, while the proportion of monocytes was significantly increased. Levels of the inflammatory cytokines IL-8, TNF-α, and IL-17A in the sputum supernatant and in the blood were markedly lowered, while IL-10 levels were unchanged. The proportion of Foxp3⁺Tregs in the total CD4⁺T cell population in the peripheral blood was drastically higher than that before treatment. The level of IL-17A was negatively correlated with the proportion of Foxp3⁺Tregs in CD4⁺T cells.

Conclusion

SFC can reduce the levels of inflammatory factors and improve symptoms of COPD. The levels of inflammatory factors are associated with the variation of Foxp3⁺Tregs in COPD.

Trial registration

This study was registered with http://www.chictr.org (Chinese Clinical Trial Register) as follows: ChiCTR-TNC-10001270     

CD4+ T Cells Expressing Latency-Associated Peptide and Foxp3 Are an Activated Subgroup of Regulatory T Cells Enriched in Patients with Colorectal Cancer

Latency-associated peptide (LAP) - expressing regulatory T cells (Tregs) are important for immunological self-tolerance and immune homeostasis. In order to investigate the role of LAP in human CD4⁺Foxp3⁺ Tregs, we designed a cross-sectional study that involved 42 colorectal cancer (CRC) patients. The phenotypes, cytokine-release patterns, and suppressive ability of Tregs isolated from peripheral blood and tumor tissues were analyzed. We found that the population of LAP-positive CD4⁺Foxp3⁺ Tregs significantly increased in peripheral blood and cancer tissues of CRC patients as compared to that in the peripheral blood and tissues of healthy subjects. Both LAP⁺ and LAP⁻ Tregs had a similar effector/memory phenotype. However, LAP⁺ Tregs expressed more effector molecules, including tumor necrosis factor receptor II, granzyme B, perforin, Ki67, and CCR5, than their LAP⁻ negative counterparts. The in vitro immunosuppressive activity of LAP⁺ Tregs, exerted via a transforming growth factor-β–mediated mechanism, was more potent than that of LAP⁻ Tregs. Furthermore, the enrichment of LAP⁺ Treg population in peripheral blood mononuclear cells (PBMCs) of CRC patients correlated with cancer metastases. In conclusion, we found that LAP⁺ Foxp3⁺ CD4⁺ Treg cells represented an activated subgroup of Tregs having more potent regulatory activity in CRC patients. The increased frequency of LAP⁺ Tregs in PBMCs of CRC patients suggests their potential role in controlling immune response to cancer and presents LAP as a marker of tumor-specific Tregs in CRC patients.     

Gamma irradiation alters the phenotype and function of CD4CD25 regulatory T cells

To examine the effects of gamma irradiation on Tregs, changes in phenotype and suppression function in Tregs treated with or without gamma ray were analyzed. Purified CD4⁺CD25⁺ regulatory T cells were irradiated at different dosages with a ¹³⁷Cs source gamma ray at 4.8 Gy/min. After culture, the phenotype and function changes were determined by flow cytometry and [³H]-thymidine incorporation, respectively. A dose-dependent reduction of Tregs proliferation in response to gamma irradiation was noted, which paralleled the apoptosis induction of Tregs. Gamma irradiation downregulated the Tregs expression of CD45RO, CD62L, FOXP3, membrane TGF-β, but upregulated Bax and GITR. High dose gamma irradiation (30 Gy) significantly abolished the suppression of Tregs on CD4⁺CD25⁻ T cells proliferation. Thus Tregs not only influences the phenotype but also alters their suppressive capacities. Our findings suggest that radiotherapy may be an important strategy to alter the immunologic balance of Tregs and effector cells in cancer therapy.     

Identification of Tribbles-1 as a Novel Binding Partner of Foxp3 in Regulatory T Cells

In a previous study, we identified TRIB1, a serine-threonine kinase-like molecule, as a biomarker of chronic antibody-mediated rejection of human kidneys when measured in peripheral blood mononuclear cells. Here, we focused our analysis on a specific subset of peripheral blood mononuclear cells that play a dominant role in regulating immune responses in health and disease, so-called CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs). We isolated both human and murine Treg and non-Treg counterparts and analyzed TRIB1 and Foxp3 mRNA expression by quantitative PCR on the freshly isolated cells or following 24 h of activation. Physical interaction between the human TRIB1 and Foxp3 proteins was analyzed in live cell lines by protein complementation assay using both flow cytometry and microscopy and confirmed in primary freshly isolated human CD4⁺CD25^hiCD127⁻ Tregs by co-immunoprecipitation. Both TRIB1 and Foxp3 were expressed at significantly higher levels in Tregs than in their CD4⁺CD25⁻ counterparts (p < 0.001). Moreover, TRIB1 and Foxp3 mRNA levels correlated tightly in Tregs (Spearman r = 1.0; p < 0.001, n = 7), but not in CD4⁺CD25⁻ T cells. The protein complementation assay revealed a direct physical interaction between TRIB1 and Foxp3 in live cells. This interaction was impaired upon deletion of the TRIB1 N-terminal but not the C-terminal domain, suggesting an interaction in the nucleus. This direct interaction within the nucleus was confirmed in primary human Tregs by co-immunoprecipitation. These data show a direct relationship between TRIB1 and Foxp3 in terms of their expression and physical interaction and highlight Tribbles-1 as a novel binding partner of Foxp3 in Tregs.     

Indirubin modulates CD4+ T‐cell homeostasis via PD1/PTEN/AKT signalling pathway in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by an immune mediated decrease in platelet number. Disturbance of CD4⁺ T‐cell homeostasis with simultaneous decrease of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) as well as unrestricted proliferation and activation of peripheral CD4⁺ effector T cells underpin the pathophysiology of ITP. Indirubin is an active ingredient of a traditional Chinese herb called Indigofera tinctoria L. which is clinically used for the treatment of ITP patients. Whether indirubin targets the Tregs/effector T cell‐axis to restore platelet number is unknown. In our in vitro studies, Indirubin could significantly enhance the number and function of Tregs and meanwhile dampen the activation of effector T cells in a dose‐dependent manner. Indirubin was observed to restore the expression of programmed cell‐death 1 (PD1) and phosphatase and tensin homolog (PTEN) on the CD4⁺ T cells of ITP patients, leading to the subsequent attenuation of the AKT/mTOR pathway. Furthermore, these observations were recapitulated in an active murine model of ITP with a prominent platelet response. Thus, our results identified a potentially novel mechanism of the therapeutic action of indirubin in the treatment of ITP through regulating the homeostasis of CD4⁺ T cells in a PD1/PTEN/AKT signalling pathway.     

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.     

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.     

Combination of rapamycin and IL-2 do not affect antigen presentation ability of rat B cell and could promote Tregs proliferation and inhibitory activity

Rapamycin (RPM), a powerful agent used clinically in transplant recipients, induces CD4⁺CD25⁺ regulatory T cells (Tregs) which play an important role in induction of immune tolerance. However, long-term use of RPM has negative side effects. In this report, we found that combination with the low dose RPM and high dose IL-2 did not affect antigen presentation of rat B cells to Tregs, and could efficiently promote Tregs proliferation and enhance their inhibitory activities in vitro. In addition, the combination of low dose RPM and high dose IL-2 enhanced mRNA expression of Foxp3, TGF-β1 and Pim-2 in Tregs but not in CD4⁺CD25⁻ T effector cells (Teffs). The Tregs inhibitory activity is positively associated with mRNA expressions of TGF-β1 and Pim-2 while unrelated to the Foxp3 mRNA expression. Our present study offers one approach to expand functional Tregs in vitro, which maybe used for clinical immune tolerance induction.     

Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones

Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4⁺ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4⁺Foxp3^- and CD4⁺Foxp3⁺ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4⁺ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.     

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.     

CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

The peripheral Foxp3⁺ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4⁺ T cells can be readily converted to Foxp3⁺ iTreg in vitro, and memory CD4⁺ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3⁺ T cells from various CD4⁺ T-cell subsets in human peripheral blood. Though naive CD4⁺ T cells were readily converted to Foxp3⁺ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3⁺ T cells did not express the memory marker CD45RO as do Foxp3⁺ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4⁺ T cells, defined as CD62L⁺ central memory T cells, could be induced by TGF-β to differentiate into Foxp3⁺ T cells. It is well known that Foxp3⁺ T cells derived from human CD4⁺CD25^- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4⁺CD62L⁺ central memory T cell-derived Foxp3⁺ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4⁺ T cell-derived Foxp3⁺ T cells. But further research showed that mouse CD4⁺ central memory T cells also could be induced to differentiate into Foxp3⁺ T cells, such Foxp3⁺ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4⁺CD62L⁺ central memory T cells as a novel potential source of iTreg.     

Immune checkpoint blockade impairs immunosuppressive mechanisms of regulatory T cells in B-cell lymphoma

In malignant disease, CD4⁺Foxp3⁺ regulatory T cells (Tregs) hamper antitumor immune responses and may provide a target for immunotherapy. Although immune checkpoint blockade (ICB) has become an established therapy for several cancer entities including lymphoma, its mechanisms have not been entirely uncovered. Using endogenously arising λ-MYC-transgenic mouse B-cell lymphomas, which can effectively be suppressed by either Treg ablation or ICB, we investigated which mechanisms are used by Tregs to suppress antitumor responses and how ICB affects these pathways. During tumor development, Tregs up-regulated Foxp3, CD25, CTLA-4 and IL-10, which correlated with enhanced immunosuppressive functions. Thus, in contrast to other tumors, Tregs did not become dysfunctional despite chronic stimulation in the tumor microenvironment and progressive up-regulation of PD-1. Immunosuppression was mediated by direct contacts between Tregs and effector T cells and by IL-10. When λ-MYC mice were treated with ICB antibodies, Tregs revealed a less profound up-regulation of Foxp3, CD25 and IL-10 and a decreased suppressive capacity. This may be due to the shift towards a pro-inflammatory milieu fostered by ICB. In summary, an ICB-induced interference with Treg-dependent immunosuppression may contribute to the success of ICB.