Transient Expression Of FOXP3 Research Articles

TCR-induced FOXP3 expression by CD8+ T cells impairs their anti-tumor activity

Adoptive cell transfer therapy using CD8+ T lymphocytes showed promising results eradicating metastatic malignancies. However, several regulatory mechanisms limit its efficacy. We studied the role of the expression of the transcription factor FOXP3 on CD8+ T cell function and anti-tumor immunity. Here we show that suboptimal T cell receptor stimulation of CD8+ T cells upregulates FOXP3 in vitro. Similarly, CD8 T cells transferred into tumor-bearing mice upregulate FOXP3 in vivo. Cell-intrinsic loss of FOXP3 by CD8+ T cells resulted in improved functionality after TCR stimulation and better antitumor responses in vivo. Inhibition of the FOXP3/NFAT interaction likewise improved CD8+ T cell functionality. Transcriptomic analysis of cells after TCR stimulation revealed an enrichment of genes implicated in the response to IFN-γ, IFN-α, inflammatory response, IL-6/JAK/STAT, G2M checkpoint and IL-2/STAT signaling in FOXP3-deficient CD8+ T cells with respect to FOXP3-wt CD8+ T cells. Our results suggest that transient expression of FOXP3 by CD8+ T cells in the tumor microenvironment restrains their anti-tumor activity, with clear implications for improving T cell responses during immunotherapy.

Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function.

BackgroundThere is circumstantial evidence that IFNy+ Treg might have clinical relevance in transplantation. IFNy+ Treg express IFNy receptors and are induced by IFNy. In the present study we investigated in kidney transplant recipients with good long-term stable graft function the absolute cell counts of IFNy+ Treg subsets and whether their expression of Foxp3 is stable or transient.MethodHelios expression determined by eight-color-fluorescence flow cytometry and methylation status of the Foxp3 Treg specific demethylation region (TSDR) served as indicators for stability of Foxp3 expression. Methylation status was investigated in enriched IFNy+ and IFNy- Treg preparations originating from peripheral blood using high resolution melt analysis. A total of 136 transplant recipients and 52 healthy controls were studied.ResultsProportions of IFNy+ Treg were similar in patients and healthy controls (0.05% and 0.04% of all CD4+ lymphocytes; p = n.s.). Patients also had similar absolute counts of IFNy producing Helios+ and Helios- Treg (p = n.s.). Most of the IFNy+ and IFNy- Treg in transplant recipients had a methylated Foxp3 TSDR, however, there was a sizeable proportion of IFNy+ and IFNy- Treg with demethylated Foxp3 TSDR. Male and female patients showed more frequently methylated IFNy+ and IFNy- Treg than male and female controls (all p<0.05).ConclusionsKidney transplant recipients with good long-term stable graft function have similar levels of IFNy+ Treg as healthy controls. IFNy+ and IFNy- Treg subsets in patients consist of cells with stable and cells with transient Foxp3 expression; however, patients showed more frequently methylated IFNy+ and IFNy- Treg than controls. The data show increased levels of Treg subsets with stable as well as transient Foxp3 expression in patients with stable allograft acceptance compared to healthy controls.

TCR usage, gene expression and function of two distinct FOXP3(+)Treg subsets within CD4(+)CD25(hi) T cells identified by expression of CD39 and CD45RO.

FOXP3+ regulatory T (Treg) cells are indispensable for immune homeostasis, but their study in humans is complicated by heterogeneity within Treg, the difficulty in purifying Tregs using surface marker expression (e.g. CD25) and the transient expression of FOXP3 by activated effector cells. Here, we report that expression of CD39 and CD45RO distinguishes three sub-populations within human CD4(+)CD25(hi) T cells. Initial phenotypic and functional analysis demonstrated that CD4(+)CD25(hi)CD39(+)CD45RO(+) cells had properties consistent with effector Treg, CD4(+)CD25(hi)CD39(-)CD45RO(-) cells were naïve Treg and CD4(+)CD25(hi)CD39(-)CD45RO(+) cells were predominantly non-Treg with effector T-cell function. Differences in these two newly identified Treg subsets were corroborated by studies of gene expression and TCR analysis. To apply this approach, we studied these two newly identified Treg subsets in ankylosing spondylitis, and showed impairment in both effector and naïve Treg. This work highlights the importance of discriminating Treg subsets to enable proper comparisons of immune regulatory capacity in healthy individuals and those with inflammatory disease.

Interplay of phosphorylation kinetics and methylation dynamics leads to complex behavior of Foxp3 and PTEN in computational model of T cell differentiation (IRM6P.709)

Abstract We have recently published a mathematical model that demonstrates that differential activation of signaling pathways downstream of TCR by high and low concentrations of antigen can be modeled by varying the duration of antigen stimulation, which results in a mixed population of regulatory (Treg), helper (Th) and inactive cells, a prediction we confirmed experimentally. Analysis of the dynamics of this scenario demonstrated an important role for a feedback loop involving Foxp3, PTEN, and Akt-mTOR signaling, which affects the timing of transient Foxp3 expression and PTEN activity, and the ratio of different phenotypes in the mixed population. We hypothesized that the correlation in the dynamics of Foxp3 and PTEN, which we previously modeled as a direct link between Foxp3 and PTEN, stems from the regulation of both Foxp3 and PTEN expression by the transcription factor Foxo1. We updated the model to test this hypothesis, and preliminary results suggest that the interplay of rapid modulation of Foxo1 activity by Akt with the slower kinetics of methylation/demethylation at the Foxp3 promoter mediated by mTOR can produce dramatic changes in the resulting distribution of phenotypes when the timing of these events is varied. To calibrate this timing in the model, we are currently conducting experiments that will help us determine the time scale for Foxo1 inactivation by Akt and for the effect of Foxo1 on Foxp3 and PTEN expression.

Regulatory T Cells Stay on Course

Regulatory T (Treg) cells are thought to be susceptible to conversion into effector T cells. In this issue of Immunity, Miyao et al. (2012) provide evidence that the apparent plasticity of murine Foxp3(+) Treg cells is largely restricted to a defined subset of poorly committed Foxp3(+) cells.

Plasticity of Foxp3+ T Cells Reflects Promiscuous Foxp3 Expression in Conventional T Cells but Not Reprogramming of Regulatory T Cells

The emerging notion of environment-induced reprogramming of Foxp3(+) regulatory T (Treg) cells into helper T (Th) cells remains controversial. By genetic fate mapping or adoptive transfers, we have identified a minor population of nonregulatory Foxp3(+) T cells exhibiting promiscuous and transient Foxp3 expression, which gave rise to Foxp3(-) ("exFoxp3") Th cells and selectively accumulated in inflammatory cytokine milieus or in lymphopenic environments including those in early ontogeny. In contrast, Treg cells did not undergo reprogramming under those conditions irrespective of their thymic or peripheral origins. Moreover, although a few Treg cells transiently lose Foxp3 expression, such "latent" Treg cells retained their memory and robustly re-expressed Foxp3 and suppressive function upon activation. This study establishes that Treg cells constitute a stable cell lineage, whose committed state in a changing environment is ensured by DNA demethylation of the Foxp3 locus irrespectively of ongoing Foxp3 expression.

Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with rheumatoid arthritis

In this study, we investigated the hypothesis that regulatory T cells (T(reg)) are involved in the immunomodulatory effects of statins on rheumatoid arthritis (RA) patients. The 12-week study cohort consisted of 55 RA patients and 42 control subjects allocated to either a group treated with atorvastatin (AT) (20 mg/day) or a non-AT group. T(reg) numbers, suppressive function, serum inflammatory markers, and disease activity were evaluated before and after the therapy. Furthermore, the effects of AT on the frequency and suppressive function of T(reg) were determined in vitro. Our data revealed that the suppressive function of T(reg) from RA patients significantly decreased compared with that of control subjects. AT significantly reduced erythrosedimentation, C-reactive protein, and disease activity. Concomitantly, T(reg) numbers and suppressive functions were significantly improved by AT. Consistent with the in vivo experiments, AT promoted the generation of T(reg) from primary T cells and enhanced preexisting T(reg) function in vitro. Moreover, we showed that PI3K-Akt-mTOR and ERK signal pathways were involved in the induction of T(reg) by AT. In conclusion, AT significantly increased T(reg) numbers and restored their suppressive function in the RA patients, and this may be relevant in the modulation of uncontrolled inflammation in this disorder.

Enhanced induction of CD4+CD25+FOXP3+ suppressive function in memory versus naïve CD4+CD25-FOXP3- T-cells is proportional to strength of activating stimulus. (50.2)

Abstract CD4+CD25+FOXP3+ regulatory T-cells (Tregs) play a vital role in suppressing autoimmune responses. We have previously shown that, in humans, all activated T-cells attain a state of transient FOXP3 expression that correlates with transient suppressive ability. In fact, peripheral generation of induced Tregs (iTregs) is likely the dominant source of regulatory cells in healthy adults. Through the use of a sensitive flow-based suppression assay, we observed that both memory and naïve CD4+CD25-FOXP3- T-cells developed regulatory ability following a variety of activating stimuli. This suppressive ability was not due to competition for nutrients or antigen presenting cells. Interestingly, memory T-cells displayed significantly greater FOXP3 induction compared to naïve counterparts, and this phenotype correlated to significantly enhanced suppressive function. Furthermore, acquisition of suppressive ability in both the memory and naïve iTreg compartments was proportional to the strength of activating stimulus. While blockade of activation using anti-IL-2, CTLA-4 Ig, anti-TGF-B, indomethacin or cyclosporin A did not affect iTreg generation, methotrexate drastically preempted the induction of regulatory ability. Moreover, irradiation of iTregs also abrogated regulatory function. These studies suggest that iTreg development and function may vary dependent on precursor origin and emphasize the importance of characterizing the iTreg compartment prior to therapeutic application.

Conversion of naïve T Cells into regulatory T Cells after tolerance induction in the murine liver

Introduction: Recently, we have demonstrated that sublethal doses of concanavalin A (ConA) induce tolerance towards ConA hepatitis in mice within one week. Tolerance is mediated by IL-10 producing regulatory T cells (Tregs). In vivo and in vitro data suggested that highly suppressive ConA-primed Foxp3+ Tregs might convert naïve CD4+ T cells into immunosuppressive Foxp3+ T cells. Hence, this study was intended to identify the relevance of “infectious tolerance“ as one mechanism of ConA tolerance. Methods: Splenic CD4+ T cells from DEREG (“depletion of regulatory T cells“) mice were co-cultivated with splenic Tregs from saline- or ConA-pretreated wt mice. Moreover, liver non-parenchymal cells (NPCs) or total splenocytes were isolated from DEREG mice. Cells were stimulated with anti-CD3 mAb for 72hrs. Foxp3 expression correlating with GFP expression and expression of surface markers with suppressive/regulatory function were quantified by FACS analysis. Results: Tregs from ConA- or saline-treated mice could not induce Foxp3 expression in the co-culture system. However, a transient FoxP3/GFP expression was detectable in cultures of whole splenocytes and liver NPCs, respectively, suggesting the requirement of APCs in Treg conversion. PD1, CCR5, and CXCR3 expression were upregulated on Tregs from ConA-treated mice, whereas CD45RB and CD62L were downregulated. Conclusion: Different migration patterns and surface expression profiles seem to characterize highly suppressive ConA-primed Tregs. Moreover, conversion of naïve T cells into Foxp3+ T cells by Tregs alone might be of minor importance for controlling ConA-induced inflammation; however, differentiation of IL-10 secreting FoxP3-negative Tr1 cells might be induced by Tregs in an infectious manner. Further studies will identify the mechanism of the transient FoxP3 expression upon ConA challenge in vivo and the potential of liver-specific APCs, e.g. LSECs or hepatocytes to convert naïve intrahepatic T cells into Tregs.

Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than OKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-β. Both humanized antibodies induced significantly lower production of IFN-γ and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-γ production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with OKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity.

Diabetes is not prevented by Foxp3-transduced CD4 +T cells under the IL-12Rβ2 promoter control

Our previous studies have shown that Foxp3 under the control of IFN-γ promoter (IγP-Foxp3) converts pathogenic CD4 +Th1 cells into regulatory T cells (Tregs), which control diabetes in non-obese diabetic (NOD) mice. Here, we tested the other hypothesis that transient expression of Foxp3 as controlled by the transient expression of IL-12Rβ2 during Th1 cell derivation is sufficient to convert cells to Tregs. Foxp3, under the control of IL-12Rβ2 promoter (Iβ2P), was lentivirally transduced into naïve CD4 +T cells from NOD mice. Iβ2P-Foxp3-transduced CD4 +T cells could not effectively suppress the incidence of diabetes when transferred into NOD mice. Furthermore, we found that Iβ2P-Foxp3-transduced CD4 +T cells, stimulated by a high dose of autoantigen, did not suppress CD4 +T cell activation, produce CD4 +Foxp3 +Tregs, and up-regulate CTLA4 expression. These results suggest that Iβ2P cannot mediate Foxp3 to convert pathogenic CD4 +Th1 cells into Tregs which control diabetes in NOD mice.

Is FOXP3 a bona fide marker for human regulatory T cells?

FOXP3 is a specific marker for naturally occurring regulatory T cells (nTreg). FOXP3 expression is correlated with development and function of nTreg. Recent evidence suggests that, upon activation, human effector T (Teff) cells and type 1 regulatory T (Tr1) cells can express FOXP3, albeit transiently. While the role of transient FOXP3 expression in Teff cells is poorly understood, it is becoming clear that it does not correlate with suppressor function. Furthermore, FOXP3-independent mechanisms, mediated by IL-10, contribute to the induction and suppressor functions of Tr1 cells.

CD4+ CD25+ regulatory T cells approach the clinic

In 1995, Sakaguchi et al. [1] (CIT) described that CD4 T cells constitutively expressing the interleukin-2 receptor alpha chain (CD25) are potent suppressors of autoreactive T-cell responses. In their experimental system, they showed that the adoptive transfer of CD4 CD25 T cells inhibited the autoimmunity that is regularly observed in mice after early post-natal thymectomy. These experiments suggested that the suppressive T-cell population is thymus-derived and exported from the thymus only after birth and that there is no other peripheral tolerance mechanism compensating for the lack of these ‘regulatory’ T cells (Treg). A few years later, the transcription factor Foxp3 was identified as a master regulator for the development and peripheral function of those thymus-derived Treg cells, and it was shown that loss of function mutations in the foxp3 gene results in a lack of this natural Treg cell compartment [2 6]. In mice and humans, such mutations cause severe autoimmunity (scurfy and IPEX, respectively). Interestingly, boys with IPEX syndrome (immune dysregulation, polyendocrinopathy, eteropathy, X-linked) suffer from severe colitis and develop unusual immune responses, for example against nutritional antigens. In experimental models of colitis, Mottet et al. [7] showed that adoptively transferred Treg cells not only inhibit colitis induction but, more importantly, even cure established disease. Thus the adoptive transfer of natural Treg cells seems a promising strategy for the treatment of chronic colitis. In this issue of Cytotherapy, Sumida et al. [8] present methods for the enrichment of natural Treg cells from leukapheresis products of patients with ulcerative colitis (UC). For this purpose, they have adopted a strategy described previously for the isolation of Treg cells from healthy donors in allogeneic stem cell transplantation [9,10]. This isolation protocol was developed for the prevention of graft-versus-host disease after allogeneic stem cell transplantation, where the co-transplantation of conventional and Treg cells at a 1:1 ratio is envisaged. Sumida et al. [8] have added a CD8-depletion step in addition to the CD19-depletion and repetitive CD25enrichment cycles to get rid of the 2 5% of CD8 T cells that otherwise contaminate a Treg-enriched cell product. Overall, they demonstrate an approximately 55% enrichment of natural Treg cells from leukapheresis products of colitis patients, based on the proportion of FOXP3 cells in the target cell fraction. However, the final cell product does not contain solely Treg cells but also recently activated CD4 T cells expressing CD25 and probably also some non-regulatory T cells that transiently express FOXP3 after stimulation. Whether such transient FOXP3 expression confers suppressive activity is a matter of debate and doubted by several investigators in the field. Nevertheless, exclusive surface markers for the unequivocal identification and separation of Treg cells from activated conventional T cells have not yet been identified and the protocol presented by Sumida et al. [8] is the best currently available approach for the enrichment of Treg cells under good manufacturing practice (GMP) conditions. Although additional depletion of CD127 cells or enrichment of naive Treg cells (CD45RA ) have been suggested to improve Treg cell purities [11 13], such strategies remain to be evaluated at large scales with GMP-compatible reagents. Consequently, as long as we lack a technology that permits the generation of pure Treg cell products, we have to define the necessary degree of purity for each potential clinical application. Although leukocyte depletion from the peripheral blood of patients with UC is not yet a standard treatment world-wide [14], it

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Induction of FOXP3 expression in naive human CD4+FOXP3− T cells by T-cell receptor stimulation is transforming growth factor-β–dependent but does not confer a regulatory phenotype

AbstractThymic-derived natural T-regulatory cells (nTregs) are important for the induction of self-tolerance and the control of autoimmunity. Murine CD4+CD25−Foxp3− cells can be induced to express Foxp3 after T-cell receptor (TCR) activation in the presence of transforming growth factor β (TGFβ) and are phenotypically similar to nTregs. Some studies have suggested that TCR stimulation of human CD4+CD25− cells results in the induction of transient expression of FOXP3, but that the induced cells lack a regulatory phenotype. We demonstrate here that TCR stimulation alone was insufficient to induce FOXP3 expression in the absence of TGFβ, whereas high levels of FOXP3 expression could be induced in the presence of TGFβ. Although FOXP3 expression was stable, the TGFβ-induced FOXP3+ T cells were neither anergic nor suppressive and produced high levels of effector cytokines. These results suggest that even high levels of FOXP3 expression are insufficient to define a human CD4+ T cell as a T-regulatory cell.