Proliferation Of Foxp3 Research Articles

Involvement of Tumor Necrosis Factor Receptor Type II in FoxP3 Stability and as a Marker of Treg Cells Specifically Expanded by Anti-Tumor Necrosis Factor Treatments in Rheumatoid Arthritis.

To study the involvement of Treg cells expressing tumor necrosis factor receptor type II (TNFRII) in exerting control of inflammation in experimental models and in the response to anti-TNF treatments in patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA). The role of TNFRII in Treg cells was explored using a multilevel translational approach. Treg cell stability was evaluated by analyzing the methylation status of the Foxp3 locus using bisulfite sequencing. Two models of inflammation (imiquimod-induced skin inflammation and delayed-type hypersensitivity arthritis [DTHA]) were induced in TNFRII-/- mice, with or without transfer of purified CD4+CD25+ cells from wild-type (WT) mice. In patients with RA and those with SpA, the evolution of the TNFRII+ Treg cell population before and after targeted treatment was monitored. Foxp3 gene methylation in Treg cells was greater in TNFRII-/- mice than in WT mice (50% versus 36.7%). In cultured Treg cells, TNF enhanced the expression, maintenance, and proliferation of Foxp3 through TNFRII signaling. Imiquimod-induced skin inflammation and DTHA were aggravated in TNFRII-/- mice (P < 0.05 for mice with skin inflammation and P < 0.0001 for mice with ankle swelling during DTHA compared to WT mice). Adoptive transfer of WT mouse Treg cells into TNFRII-/- mice prevented aggravation of arthritis. In patients with RA receiving anti-TNF treatments, but not those receiving tocilizumab, the frequency of TNFRII+ Treg cells was increased at 3 months of treatment compared to baseline (mean ± SEM 65.2 ± 3.1% versus 49.1 ± 5.5%; P < 0.01). In contrast, in anti-TNF-treated patients with SpA, the frequency of TNFRII+ Treg cells was not modified. TNFRII expression identifies a subset of Treg cells that are characterized by stable expression of Foxp3 via gene hypomethylation, and adoptive transfer of TNFRII-expressing Treg cells ameliorates inflammation in experimental models. Expansion and activation of TNFRII+ Treg cells may be one of the mechanisms by which anti-TNF agents control inflammation in RA, but not in SpA.

Early reduction of regulatory T cells is associated with acute rejection in liver transplantation under tacrolimus-based immunosuppression with basiliximab induction.

Regulatory T (Treg) cells are important in preventing acute rejection (AR) in solid organ transplantation, but the clinical relevance of the different kinetics early after liver transplantation (LT) in acute rejectors and non-rejectors is unclear. We analyzed peripheral blood samples of 128 LT recipients receiving basiliximab induction plus tacrolimus immunosuppression. Samples were obtained at pretransplant, D7, and D30 after LT. Frequency and phenotype of Tregs were analyzed by flow cytometry. The predictive value of Treg frequency at D7 was assessed for suspected acute rejection (SAR) and was validated for biopsy-proven AR (BPAR). We found that the frequencies of total and activated Tregs at D7 were significantly lower in recipients with SAR and BPAR. Treg was more reduced in BPARs by in vitro tacrolimus treatment in the presence of basiliximab. Moreover, an early reduction of Treg frequency in rejectors was associated with a greater increase in Treg apoptosis and further attenuated IL-2 signaling. D7 Treg frequency was an independent risk factor for SAR, which was also validated for BPAR. In conclusion, first-week peripheral blood Treg frequency correlates with AR after LT under tacrolimus-based immunosuppression, which needs to be proven in larger, geographically and clinically diverse populations.

Inhibition of the PI3K-Akt-mTOR signaling pathway in T lymphocytes in patients with active tuberculosis

To investigate PI3K-Akt-mTOR signaling pathway changes and the proliferation of FoxP3+Treg cells in patients with active tuberculosis. We isolated PBMCs and CD4+CD25+FoxP3+Treg cells from peripheral blood collected from patients with active tuberculosis and healthy controls. We compared the proportion and MFI of PI3K-Akt-mTOR pathway components and PTEN by flow cytometry using specific cell-surface and intracellular markers. Moreover, we detected the specific secretory proteins ESAT-6 and Ag85B, cytokines IL-10, TGF-β1 and IL-35 in serum by ELISA. Compared with healthy controls, the proportions of CD3+Akt+, CD3+p-Akt+, CD3+mTOR+, CD3+p-mTOR+ and CD3+PTEN+ cells, in the T lymphocyte population of patients with active tuberculosis, were decreased (p<0.05), while CD3+FoxP3+ cells were increased (p=0.013). Similarly, for CD4+CD25+FoxP3+Treg cells, the proportions of Akt+ cells, p-Akt+ cells, mTOR+ cells, p-mTOR+ cells and PTEN+ cells were decreased (p<0.05) in patients with active tuberculosis. Compared with healthy controls, the levels of ESAT-6 and Ag85B were higher in patients with active tuberculosis (p<0.001). Levels of IL-10 and TGF-β1 were higher (p<0.001), whereas the level of IL-35 was lower (p<0.001). The PI3K-Akt-mTOR signaling pathway in T lymphocytes and CD4+CD25+FoxP3+Treg cells was inhibited, which could explain why M.tuberculosis can induce FoxP3+Treg cell to expand.

Abstract PR08: Antitumor activity and immune correlates of PEGylated human IL-10 (AM0010) alone or in combination with anti-PD-1

Abstract Background: The success of and the durability of immune therapy of cancer is thought to depend on the activation and expansion of tumor reactive and infiltrating CD8+ T cells. The response to immune checkpoint blockade, depends on a pre-existing, CD8 T cell-rich tumor microenvironment. IL-10 stimulates the antigen mediated cytotoxicity, survival and proliferation of intra-tumoral CD8+ T cells and simultaneously dampens chronic inflammation. T cell receptor mediated activation of CD8 T cells induces the expression of IL-10 receptors on these cells. IL-10 activates with STAT3 an essential survival and proliferation signal in antigen activated CD8 T cells. This also provides a mechanistic rationale for combining AM0010 and anti-PD1 in the clinic. To evaluate the clinical activity, tolerability and anti-tumor activity of AM0010 alone or in combination with chemotherapy or immune checkpoint inhibitors a multi-basket phase 1 study was conducted. Additional disease specific expansion cohorts for the combination of AM0010 with FOLFOX in pancreatic cancer or with nivolumab in RCC or NSCLC are currently evaluated Results: Tolerability and anti-tumor activity of AM0010 alone or in combination with chemotherapy or immune checkpoint inhibitors was established in this multi-basket phase 1 study. In monotherapy, objective responses were observed in pts with uveal melanoma, cutaneous T cell lymphoma and in 4 of 15 pts with RCC. Patients with advanced melanoma, RCC or NSCLC were also treated with AM0010 (daily SC) in combination with anti-PD-1 immune checkpoint blockade. Tumor responses were monitored following irRC. Immune responses were measured by analysis of serum cytokines, activation of blood derived T cells, peripheral T cell clonality and immunohistochemistry of tumor infiltrating CD8 T cells. In 19 pts, AMO010 10 μg/kg (n = 13) or 20 μg/kg (n = 6) in combination with anti-PD1 - pembrolizumab (2mg/kg) was well tolerated (observation period 10-15 months). Immune related TrAE occured in the frequency and severity as expected from pembrolizumab montherapy. The combination of AM0010 with pembrolizumab achieved objective responses (PR/CR) in 4 of 8 RCC pts, 2 of 5 NSCLC pts and 2 of 6 melanoma pts. 2 additional melanoma pts had tumor increase followed by decrease (pseudoprogression). Independent of the combination with either chemotherapy or anti-PD-1, AM0010 increased Th1 cytokines (IL-18, IFNγ, IL-7) in a dose dependent fashion. FasL and lymphotoxin beta - products of cytotoxic T cells - were also increased in the serum of AM0010 treated patients. In contrast, mediators of chronic inflammation, such as IL-23 and IL-17 and the immune suppressive cytokine TGFbeta were reduced in the serum of patients. AM0010 increased the number and proliferation of PD1+ activated CD8 T cells while decreasing the proliferation of FoxP3+ Tregs and TGFβ in the blood. AM0010 induced de-novo oligoclonal expansion of T cell clones in the blood of patients without affecting total lymphocyte counts. This clonal expansion appeared enhanced and accelerated in patients treated with a AM0010 anti PD-1 combination, but was also seen in patients which received a AM0010 - chemotherapy combination. AM0010 also increased the number of tumor infiltrating Phospho-STAT3+ CD8 T cells in tumors and the number of Granzyme+ PD1+ CD8+ T cells in tumor biopsies of treated patients. Conclusion: AM0010 alone or in combination with anti-PD1 is well-tolerated. The clinical activity and the observed CD8 T cell activation encourages the phase 2/3 studies of AM0010 in combination with anti-PD1 planned for later in 2016. Trial registration: www.clinicaltrials.gov NCT02009449. Citation Format: Aung Naing, Jeffrey R. Infante, Kyriakos P. Papadopoulos, Deborah J. Wong, Karen A. Autio, Gerald S. Falchook, Manish Patel, Shubham Pant, Amita Patnaik, Melinda Whiteside, Johanna C. Bendell, John Mumm, Ivan H. Chan, Gail L. Brown, Peter VanVlasselaer, J. R. Hecht, David S. Hong, Nizar M. Tannir, Martin Oft. Antitumor activity and immune correlates of PEGylated human IL-10 (AM0010) alone or in combination with anti-PD-1 [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR08.

Regulatory T-Cell Plasticity

During the past 2 decades, human and animals studies have clearly documented the crucial role of inflammation in the development and complications of atherosclerosis.1 Both innate immunity and adaptive immunity are involved in this process. The first evidence that suggested a role of adaptive immunity in atherosclerosis was the widespread detection of the major histocompatibility class II in human atherosclerotic plaques and the presence of a large amount of CD3+ lymphocytes in human and mouse atherosclerotic lesions. Most of the T cells in mouse and human atherosclerotic plaques are CD4+ T-helper (Th) cells expressing the αβ T-cell antigen receptor. Among CD4+ T cells, Th1 cells have been shown to exert proatherogenic effects, whereas regulatory T cells (Tregs) display atheroprotective properties. The role of Th2 and Th17 cells is still debated.1 Helper T-cell subsets are defined by the production of cytokines or the expression of characteristic lineage-defining transcription factors. Th1 cells are generated on priming in the presence of interleukin (IL)-12 that promotes the expression of the transcription factor T-bet and stimulates the production of the Th1 prototypical cytokine interferon-γ (IFN-γ).2 Tregs have a central role in the dominant control of immunologic tolerance and maintenance of immune homeostasis. They were first identified in mice and later in humans.3 The transcription factor FoxP3 is essential for the generation and the functions of Tregs.4 FoxP3 deficiency leads to a multiorgan autoimmune disease as can be observed in the scurfy mouse and in human immune dysregulation, polyendocrinopathy, enteropathy X–linked syndrome patients.5 Natural or thymic FoxP3+ Tregs acquire regulatory lineage commitment already on maturation in the thymus,3 whereas adaptive or peripheral FoxP3+ Tregs can be induced from mature CD4+ Th cells in the periphery under the influence of different stimulations, especially transforming …

Donor Specific Regulatory T-Cell Expansion Is Associated With the Maintenance of Allograft Tolerance Induced By Transient Mixed Chimerism.

Background: We have previously reported successful induction of solid organ allograft tolerance in nonhuman primates (NHP) via a mixed chimerism approach. Since these recipients achieved tolerance after only transient hematopoietic chimerism, peripheral mechanisms are considered to be operative in the maintenance of tolerance. In the current study, we evaluated the role of donor specific regulatory T cells (Treg) in NHP that achieved long-term allograft survival. Method:Nine cynomolgus monkeys that achieved long-term (>360 days) allograft survival without maintenance immunosuppression [kidney (n=6), kidney/heart (n=2), and lung (n=1)] were investigated. Three recipients [heart alone (n=2) and kidney (n=1)] that received the same conditioning regimen but rejected their allografts after discontinuation of immunosuppression were evaluated as controls. Anti-donor and anti-third party responses in these recipients were evaluated by ELISPOT (INF-g) and expansion of donor specific Tregs was evaluated by MLR against donor and third party cells with CFSE/Foxp3 staining. Expanded T cells were sorted and their suppressive function was evaluated by inhibition of T cell activation by CD3/CD28 beads. Results: Donor-specific hyporesponsiveness was observed with ELISPOT in long-term survivors, but substantial anti-donor responses in recipients with rejection. (Fig.1) Foxp3+ cells proliferated significantly more after MLR with donor PBMC than when stimulated with self or third party PBMC in 5/7 long-term survivors. (Fig.2 representative flow cytometric analysis).Figure: No Caption available.Sorted Treg dominant cells expanded after MLR with donor cells showed suppressive effects in T cell activation. In contrast, there was no proliferation of FoxP3+ cells observed after MLR with donor cells in recipients with rejection. Conclusion: These data suggested that Tregs play a critical role in the maintenance of long-term allograft tolerance following induction of transient mixed chimerism.

P060 Impact of intestinal inflammation on Foxp3+ regulatory T cells

Background: Regulatory Foxp3+ T cells (Treg) maintain immune tolerance by controlling systemic autoimmune and allergic diseases but are also crucial for control of intestinal homeostasis and oral tolerance to dietary antigens and to the microbiota. Breakdown of intestinal homeostasis in chronic inflammatory bowel disease is associated with inability of Treg to control inflammation, suggesting that the inflamed intestinal microenvironment is deleterious for Treg. Methods: In this study we explored, using two models of colitis (DSS colitis and TNBS colitis), the impact of intestinal inflammation on the number, survival, phenotype and regulatory function of Treg and conversion from CD4+ T cells. Results: We found that intestinal inflammation increased the number Foxp3+ Treg in Mesenteric Lymph Nodes (MLN), although their frequency decreased due to the inflammatory infiltrate. Colitis was associated with increased proliferation of Foxp3+Ki67+ Treg in MLN and LP. MLN Foxp3+ Treg at the steady state comprised both Helios+Neuropilin+ and Helios Neuropilin , which are believed to represent natural (nTreg) versus induced (iTreg) Treg, respectively and intestinal inflammation decreased the frequency of nTreg. Ex vivo Treg suppression assay showed that MLN Treg from colitic mice maintain their suppressive function, when removed from the inflamed intestine. Adoptive transfer of naive CD4+ T cells (containing nTreg) in congenic recipient mice showed that colon inflammation exacerbated recruitment of CD4+ T cells into the lamina propria. However, in vivo conversion of CD4+CD25+ Foxp3 pre-Treg (sorted from Foxp3-egfp transgenic mice) into Foxp3+ Treg was impaired in colitic recipient mice. Finally, transfer of Foxp3-egfp+ cells in DSS mice showed a significant decrease of colitis severity. Conclusions: Taken together our data illustrate that Treg can be activated and recruited in the inflamed intestine but that intestinal inflammation impairs Treg conversion from CD4+ T cells.

P061 Identification of the mesocolic mesothelium as a novel source of fibroblasts – implications for Crohn's disease

Background: Regulatory Foxp3+ T cells (Treg) maintain immune tolerance by controlling systemic autoimmune and allergic diseases but are also crucial for control of intestinal homeostasis and oral tolerance to dietary antigens and to the microbiota. Breakdown of intestinal homeostasis in chronic inflammatory bowel disease is associated with inability of Treg to control inflammation, suggesting that the inflamed intestinal microenvironment is deleterious for Treg. Methods: In this study we explored, using two models of colitis (DSS colitis and TNBS colitis), the impact of intestinal inflammation on the number, survival, phenotype and regulatory function of Treg and conversion from CD4+ T cells. Results: We found that intestinal inflammation increased the number Foxp3+ Treg in Mesenteric Lymph Nodes (MLN), although their frequency decreased due to the inflammatory infiltrate. Colitis was associated with increased proliferation of Foxp3+Ki67+ Treg in MLN and LP. MLN Foxp3+ Treg at the steady state comprised both Helios+Neuropilin+ and Helios Neuropilin , which are believed to represent natural (nTreg) versus induced (iTreg) Treg, respectively and intestinal inflammation decreased the frequency of nTreg. Ex vivo Treg suppression assay showed that MLN Treg from colitic mice maintain their suppressive function, when removed from the inflamed intestine. Adoptive transfer of naive CD4+ T cells (containing nTreg) in congenic recipient mice showed that colon inflammation exacerbated recruitment of CD4+ T cells into the lamina propria. However, in vivo conversion of CD4+CD25+ Foxp3 pre-Treg (sorted from Foxp3-egfp transgenic mice) into Foxp3+ Treg was impaired in colitic recipient mice. Finally, transfer of Foxp3-egfp+ cells in DSS mice showed a significant decrease of colitis severity. Conclusions: Taken together our data illustrate that Treg can be activated and recruited in the inflamed intestine but that intestinal inflammation impairs Treg conversion from CD4+ T cells.

Low-Dose Antigen Promotes Induction of FOXP3 in Human CD4+ T Cells

Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.

An IL-2 Paradox: Blocking CD25 on T Cells Induces IL-2–Driven Activation of CD56bright NK Cells

Daclizumab (Dac), an Ab against the IL-2R alpha-chain, inhibits brain inflammation in patients with multiple sclerosis, while expanding CD56(bright) immunoregulatory NK cells in vivo. We hypothesized that this unexpected expansion is paradoxically IL-2 driven; caused by the increased availability of T cell-derived IL-2 for NK cell signaling. To this end, we performed ex vivo functional analyses of CD56(bright) NK cells and T cells from patients in clinical trials with Dac. We developed in vitro models to investigate mechanisms for ex vivo observations. We observed that Dac treatment caused decreased numbers and proliferation of FoxP3(+) T regulatory cells (Tregs), a model T cell population known to be dependent on IL-2 for proliferation and survival. As anticipated, Dac therapy inhibited IL-2 signaling in all T cells; however, we also observed functional adaptation of T cells to low IL-2 signal in vivo, characterized by the concomitant enhancement of IL-7 signaling on all T cells and parallel increase of CD127 expression by Tregs. In contrast, IL-2 signaling on CD56(bright) NK cells was not inhibited by Dac and their in vivo proliferation and cytotoxicity actually increased. Mechanistic studies indicated that the activation of CD56(bright) NK cells was likely IL-2 driven, as low doses of IL-2, but not IL-15, mimicked this activation in vitro. Our study provides insight into the role that IL-2 and CD25 play in functional regulation of two important immunoregulatory cell populations in humans: FoxP3(+) Tregs and CD56(bright) NK cells.