Circulating and tumor-infiltrating Foxp3⁺ regulatory T cell subset in Chinese patients with extranodal NK/T cell lymphoma

Foxp3⁺ regulatory T lymphocytes (Tregs) usually act as an immune suppressor and correlate with poorer survival in malignancies. This study aims to investigate the distribution and characterization of Foxp3⁺ subset in peripheral blood mononuclear cells (PBMCs) and tumor tissues from extranodal NK/T cell lymphoma (ENKTL). Our study showed the percentage of Foxp3⁺ subset from PBMC was significantly higher than that of healthy individuals (P<0.001). The Foxp3⁺ subset from PBMCs expressed CD45RO, CTLA4, GITR, CCR7, and had an IL-10^highIFNγ⁺TGFβ⁺IL-2^lowIL-17^low cytokine secreting phenotype. Interestingly, the existence of EBV antigen-specific CD8⁺Foxp3⁺ Tregs was discovered in ENKTL. Furthermore, the high density of Foxp3⁺ TILs was associated with improved progression-free survival (PFS) in ENKTL patients (P<0.05). Collectively, our study implicates that EBV antigens could induce antigen-specific CD8⁺Foxp3⁺ Tregs in ENKTL, and Foxp3⁺ TILs is an independent factor for PFS in ENKTL.     

Differential effects of IL-2 and IL-21 on expansion of the CD4+CD25+Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia

CD4⁺ CD25+ regulatory T cells are expanded in solid and hematological malignancies including Chronic Lymphocytic Leukemia (CLL). Several cytokines and co-stimulatory molecules are required for generation, survival and maintenance of their suppressive effect. We and others have shown direct cytotoxic effect of the novel common gamma chain cytokine interleukin- 21 (IL-21) on primary B cells from CLL patients. Since members of this family of cytokines are known to exhibit their effects on diverse immune cells, we have examined the effects of IL-21 on CLL patient derived regulatory T cell induction, expansion and the inhibitory effect on natural killer cells in vitro. We demonstrate here the expression of IL-21 receptor in CD4⁺CD25^High regulatory cells from CLL patients. In contrast to IL-2, the IL-21 cytokine failed to mediate expansion of regulatory T cells or induced expression of Foxp3 in CD4⁺CD25Intermediate or CD4+CD25^Dim/- T cells in whole blood derived from CLL patients. Interestingly, in contrast to their differential effects on expansion of the CD4⁺CD25⁺Foxp3⁺T cells, IL-2 and IL-21 exhibited a redundant role in T-reg mediated suppression of NK cell mediated antibody dependent cytotoxicity function. Given the infusion related toxicities and pro-survival effect of IL-2 in CLL, these studies provide a rationale to explore IL-21 as an alternate gamma chain cytokine in CLL therapy.     

IL-15 Augments TCR-Induced CD4+ T Cell Expansion In Vitro by Inhibiting the Suppressive Function of CD25High CD4+ T Cells

Due to its critical role in NK cell differentiation and CD8⁺ T cell homeostasis, the importance of IL-15 is more firmly established for cytolytic effectors of the immune system than for CD4⁺ T cells. The increased levels of IL-15 found in several CD4⁺ T cell-driven (auto-) immune diseases prompted us to examine how IL-15 influences murine CD4⁺ T cell responses to low dose TCR-stimulation in vitro. We show that IL-15 exerts growth factor activity on both CD4⁺ and CD8⁺ T cells in a TCR-dependent and Cyclosporin A-sensitive manner. In CD4⁺ T cells, IL-15 augmented initial IL-2-dependent expansion and once IL-15Rα was upregulated, IL-15 sustained the TCR-induced expression of IL-2/15Rβ, supporting proliferation independently of secreted IL-2. Moreover, IL-15 counteracts CD4⁺ T cell suppression by a gradually expanding CD25^HighCD4⁺ T cell subset that expresses Foxp3 and originates from CD4⁺CD25⁺ Tregs. These in vitro data suggest that IL-15 may dramatically strengthen the T cell response to suboptimal TCR-triggering by overcoming an activation threshold set by Treg that might create a risk for autoimmune pathology.     

Palmitate-derivatized human IL-2: a potential anticancer immunotherapeutic of low systemic toxicity

Purpose and experimental design

Recombinant human IL-2 (rhIL-2) is a potent cytokine and FDA-approved anticancer drug. However, its clinical use has been limited by severe toxicity, associated primarily with systemic administration with excess protein distributing freely throughout the body. We hypothesized that rhIL-2 in alternate forms permitting more restricted localization may exert stronger antitumor efficacy and less toxicity. Here, we have tested the utility of palmitate-derivatized rhIL-2. rhIL-2 was reacted with N-hydroxysuccinimide palmitate ester. The resultant lipidated rhIL-2 (pIL-2), when mixed with cells, could spontaneously transfer from solution to cell surfaces. Next, anticancer efficacy of pIL-2 was assessed in two modalities. For adoptive T cell therapy, antitumor cytotoxic T cells (CTLs) were protein transferred (“painted”) with pIL-2 and injected into mice bearing lymphoma. For in situ therapy, pIL-2 was injected intratumorally into mice bearing melanoma. Tumor growth and IL-2-associated toxicity were determined.

Results

In the lymphoma model, painting of the antitumor CTLs with pIL-2 markedly increased their viability and titer. In the melanoma model, intratumoral injection of pIL-2, but not rhIL-2, increased the number of activated CD8⁺ T cells (IFN-γ⁺) in the spleen, reduced lung metastasis and prolonged the survival of treated mice. Moreover, while repeated intratumoral injection of rhIL-2 at an excessively high dose (10 injections of 10,000 IU/mouse) caused marked vascular leakage syndrome, the same regimen using pIL-2 caused no detectable toxicity.

Conclusions

Transferring spontaneously from solution to cell surfaces, pIL-2 may bypass the current limitations of rhIL-2 and, thus, serve as a more effective and tolerable anticancer drug.     

Differential Effects of IL-12 on Tregs and Non-Treg T Cells: Roles of IFN-γ, IL-2 and IL-2R

Complex interactions between effector T cells and Foxp3⁺ regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4⁺Foxp3⁻ T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

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.     

Priming of naive CD8+ T cells in the presence of IL-12 selectively enhances the survival of CD8<Superscript>+</Superscript>CD62L<Superscript>hi</Superscript> cells and results in superior anti-tumor activity in a tolerogenic murine model

During the antigen-dependant activation process several subsets CD8⁺ T cells appear with different phenotypic and functional characteristics. Recent studies indicate that the state of T cell differentiation radically affects their ability to effectively respond to tumor challenge, with early effector CD8⁺ T (CD62L^high) cells having better anti-tumor activity. Thus strategies aimed at optimizing the generation of such subpopulations could significantly enhance the effectiveness of adoptive cell therapy (ACT) for cancer. In this study, we show that priming of naïve CD8⁺ T cells in the presence of IL-12 selectively rescued early CD8⁺ CD62L^hi from activation induced cell death and resulted in the increased accumulation of this subset of CD8⁺ T cells. Furthermore, we demonstrated that IL-12 directly modulated the expression of CD62L on activated CD8⁺ T cells. When used for ACT, naïve CD8⁺ T cells primed in vitro in the presence of IL-12 showed superior anti-tumor activity toward B16 melanoma. Importantly, using the Pmel-1 model, priming pmel-1 cells in vitro with IL-12 reduced the state of functional tolerance associated with the non-mutated “self” tumor antigen gp100, as demonstrated by significant tumor responses in the absence of vaccination. Together, our results suggest that in vitro conditioning of naïve CD8⁺ T cells with IL-12 prior to ACT could significantly enhance their anti-tumor activity.     

Regulatory T cells induced by B cells: a novel subpopulation of regulatory T cells

Regulatory T cells play a crucial role in the homeostasis of the immune response. In addition to CD4⁺Foxp3⁺ regulatory T cells, several subsets of Foxp3^- regulatory T cells, such as T helper 3 (Th3) cells and type 1 regulatory T (Tr1) cells, have been described in mice and human. Accumulating evidence shows that naïve B cells contribute to tolerance and are able to promote regulatory T cell differentiation. Naïve B cells can convert CD4⁺CD25^- T cells into CD25⁺Foxp3^- regulatory T cells, named Treg-of-B cells by our group. Treg-of-B cells express LAG3, ICOS, GITR, OX40, PD1, and CTLA4 and secrete IL-10. Intriguingly, B-T cell-cell contact but not IL-10 is essential for Treg-of-B cells induction. Moreover, Treg-of-B cells possess both IL-10-dependent and IL-10-independent inhibitory functions. Treg-of-B cells exert suppressive activities in antigen-specific and non-antigen-specific manners in vitro and in vivo. Here, we review the phenotype and function of Foxp3⁺ regulatory T cells, Th3 cells, Tr1 cells, and Treg-of-B cells.     

Effects of cyclophosphamide and IL-2 on regulatory CD4+ T cell frequency and function in melanoma patients vaccinated with HLA-class I peptides: impact on the antigen-specific T cell response

The frequency and function of regulatory T cells (Tregs) were studied in stage II–III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4⁺ T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8⁺ T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8⁺ T cell effectors when used in the inflammatory environment of vaccination.     

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.     

In vitro inhibition of monkeypox virus production and spread by Interferon-β

Background

Despite inducing a sustained increase in CD4+ T cell counts, intermittent recombinant IL-2 (rIL-2) therapy did not confer a better clinical outcome in HIV-infected patients enrolled in large phase III clinical trials ESPRIT and SILCAAT. Several hypotheses were evoked to explain these discrepancies. Here, we investigated the impact of low and high doses of IL-2 in Rhesus macaques of Chinese origin infected with SIVmac251 in the absence of antiretroviral therapy (ART).

Results

We demonstrated that rIL-2 induced a dose dependent expansion of CD4+ and CD8+ T cells without affecting viral load. rIL-2 increased CD4 and CD8 Treg cells as defined by the expression of CD25^highFoxP3⁺CD127^low. We also showed that rIL-2 modulated spontaneous and Fas-mediated CD4⁺ and CD8⁺ T cell apoptosis. The higher dose exhibited a dramatic pro-apoptotic effect on both CD4⁺ and CD8⁺ T cell populations. Finally, all the animals treated with rIL-2 developed a wasting syndrome in the month following treatment simultaneously to a dramatic decrease of circulating effector T cells.

Conclusion

These data contribute to the understanding of the homeostatic and dosage effects of IL-2 in the context of SIV/HIV infection.     

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     

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.