Low-Dose Administration of Recombinant IL-2 Following T Cell Depleted Bone Marrow Transplantaion Induces Expansion of CD4+CD25+ FOXP3+ Regulatory T Cells in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2136-2136
Author(s):  
Emmanuel Zorn ◽  
Despina Lista ◽  
Haesook Kim ◽  
Roberto Bellucci ◽  
Christine Canning ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Low Dose ◽  
Cellular Therapy ◽  
Chronic Gvhd ◽  
Foxp3 Expression ◽  
P Value ◽  
Adoptive Cellular Therapy

Abstract Regulatory T cells (Treg) play a key role in controlling immune responses following allogeneic hematopoietic stem cell transplantation (HSCT). In murine models, infusion of purified CD4+CD25+ Treg at the time of transplant is sufficient to prevent acute GVHD. In humans, development of acute as well as chronic GVHD has been associated with reduced numbers of Treg following allogeneic HSCT, suggesting that defective reconstitution of this functional cell type can contribute to exacerbation of alloimmune responses. Based on these results, adoptive cellular therapy using purified and in vitro expanded populations of Treg has been proposed as a therapeutic strategy to correct chronic GVHD. Treg are mainly characterized by the constitutive expression of the IL-2 receptor ? chain, CD25 and proliferate in response to IL-2 in vitro. In vivo, the effects of IL-2 on Treg populations are unknown. To examine this question we quantified changes in Treg in 9 patients with CML who previously received low dose IL-2 following allogeneic HSCT. Patients enrolled in this protocol received a daily intravenous infusion of 2 X 105 U IL-2/m2 for 3 months, starting 3 months after CD6 depleted allogeneic bone marrow transplantation (BMT). No patient developed GVHD following IL-2 administration and overall toxicity was minimal. The predominant immunologic effect of IL-2 reported in the initial study was a marked increase in NK cell populations characterized as CD3-CD16+CD56+ as well as total CD56+ cells. In this retrospective analysis we investigated populations of CD4+CD25+ T cells before and 1 to 2 months after the beginning IL-2 treatment. Using RNA extracted from patient PBMC we also assessed the level of expression of the specific transcription factor FOXP3 by quantitative PCR as an alternate marker of Treg in vivo. As initially reported, all 9 patients showed a marked increase in CD3-CD56+ cells 1 to 2 months post IL-2 administration. In contrast, the percent of CD3+ T cells were either unchanged or slightly decreased. The percent of CD4+CD25+ cells within the CD3+ T cell population increased during IL-2 treatment (median: 5.8 pre IL-2 vs 7.6 post IL-2, p-value=0.02). Likewise, FOXP3 expression in the CD3+ population showed 5 to 19 fold increase in 8 of 9 patients during this period (median: 3817 AU pre IL-2 vs. 18924 AU post IL-2, p-value=0.055). These results indicate that administration of low dose IL-2 can augment Treg cells in vivo as reflected by increased ratio of CD4+CD25+/CD3+ T cells as well as higher levels of FOXP3 expression. These studies suggest that prolonged treatment with low dose IL-2 can effectively expand CD4+CD25+ Treg in vivo. This represents a novel strategy for expanding regulatory T cells in vivo and may be useful alone or in conjunction with adoptive cellular therapy with Treg.

scholarly journals IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo

Blood ◽  
2006 ◽  
Vol 108 (5) ◽  
pp. 1571-1579 ◽  
Author(s):  
Emmanuel Zorn ◽  
Erik A. Nelson ◽  
Mehrdad Mohseni ◽  
Fabrice Porcheray ◽  
Haesook Kim ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Nk Cells ◽  
Low Dose ◽  
Fold Increase ◽  
Foxp3 Expression ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3+ regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2′-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.

Functional effects of chimeric antigen receptor co-receptor signaling domains in human regulatory T cells

2020 ◽  
Vol 12 (557) ◽  
pp. eaaz3866 ◽  
Author(s):  
Nicholas A. J. Dawson ◽  
Isaac Rosado-Sánchez ◽  
German E. Novakovsky ◽  
Vivian C. W. Fung ◽  
Qing Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cellular Therapy ◽  
In Vitro Assays ◽  
In Vivo Model ◽  
Receptor Signaling ◽  
Signaling Domain ◽  
Signaling Domains

Antigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptors (CARs) are a potent immunosuppressive cellular therapy in multiple disease models and could overcome shortcomings of polyclonal Treg therapy. CAR therapy was initially developed with conventional T cells, which have different signaling requirements than do Tregs. To date, most of the CAR Treg studies used second-generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3ζ, but it was not known if this CAR design was optimal for Tregs. Using a human leukocyte antigen–A2–specific CAR platform and human Tregs, we compared 10 CARs with different co-receptor signaling domains and systematically tested their function and CAR-stimulated gene expression profile. Tregs expressing a CAR encoding CD28wt were markedly superior to all other CARs tested in an in vivo model of graft-versus-host disease. In vitro assays revealed stable expression of Helios and an ability to suppress CD80 expression on dendritic cells as key in vitro predictors of in vivo function. This comprehensive study of CAR signaling domain variants in Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy.

Human CIK Maintain Their In Vitro and In Vivo Anti-Tumor Ability after Cryopreservation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1062-1062
Author(s):  
Jeanette Baker ◽  
Kevin Sheehan ◽  
Gina Monterola ◽  
Nancy Staines ◽  
Robert S. Negrin
Keyword(s):  
T Cells ◽  
Cytotoxic Activity ◽  
Cellular Therapy ◽  
Cell Activity ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Adoptive Cellular Therapy ◽  
Cik Cells

Abstract Adoptive cellular therapy holds promise for improving the outcome of hematopoietic cell transplantation (HCT). At present, donor lymphocyte infusion post-HCT is efficacious for only a limited number of diseases, yet can induce significant graft versus host disease (GVHD). To improve the outcome of this approach, it would be beneficial to identify populations of T cells that retain graft versus tumor (GVT) effects with reduced propensity for GVHD. We have previously described studies of murine expanded Cytokine Induced Killer (CIK) cells which are ex vivo activated and expanded T cells that express both T and NK markers. CIK cells mediate cytotoxicity both in vivo and in vitro in a non- MHC restricted NKG2D dependent manner. Human CIK cells were expanded from PBMC from 9 healthy donors, cultured with IFNg, CD3 and IL-2 and maintained in AastromRepliCell® biochambers for 21–28 days. We aimed to determine whether cryopreservation of the CIK affects viability, cytotoxicity and phenotype. Cells were cryopreserved immediately after harvest at 10x106/ml and stored in liquid nitrogen vapor phase. CIK viability was not compromised with cryopreservation and cells thawed at 1, 2, 4, 8, 10 and 28 weeks after freezing were 96% viable (range 95%–99%). Immediately upon thawing, CIK cells showed diminished cytotoxicity against the B cell lymphoma cell lines DB and SUDHL4 with 6–10% killing at the 40:1 E:T ratio. However, thawed CIK cells regained their pre-freeze cytotoxic activity against these targets within 5 hours of being placed in reactivation medium containing IL-2 at 300 IU/ml. Reactivation of the CIK cells was extended up to 48 hours but showed no further increase in cytotoxicity beyond that attained at 5 hours; nor did increasing the IL-2 concentration to 1500 IU/ml in the reactivation medium improve CIK cell activity over the same time course. Cell viability declined during reactivation, decreasing from an average 96% upon thawing to 60% over 48 hours. Thawed CIK cells placed in reactivation medium maintained their cytotoxic activity up to 14 days in vitro. The cytotoxicity of reactivated CIK cells was assessed in vivo using SCID mice inoculated IP with 1x106 human ovarian cancer UCI-101 cells expressing the firefly luciferase gene. The mice were treated weekly with 2x107 cryopreserved and thawed human CIK cells that were re-cultured for 5 hours before injection. Following each administration of CIK cells, there was a reduction of tumor signal. Weekly treatments resulted in a better survival outcome for the mice receiving CIK cells as compared to PBS control mice. This study demonstrates that human CIK cells may be reactivated after cryopreservation and regain their cytotoxic potential. These finding have important implications for the application of these cells as adoptive cellular therapy.

scholarly journals CD8α+ plasmacytoid precursor DCs induce antigen-specific regulatory T cells that enhance HSC engraftment in vivo

Blood ◽  
2011 ◽  
Vol 117 (8) ◽  
pp. 2494-2505 ◽  
Author(s):  
Yiming Huang ◽  
Larry D. Bozulic ◽  
Thomas Miller ◽  
Hong Xu ◽  
Lala-Rukh Hussain ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Foxp3 Expression ◽  
Cell Receptor ◽  
Third Party ◽  
Antigen Specificity ◽  
Clinical Potential

Abstract CD8-positive/T-cell receptor–negative (CD8+/TCR−) graft facilitating cells (FCs) are a novel cell population in bone marrow that potently enhance engraftment of hemopoietic stem cells (HSCs). Previously, we showed that the CD11c+/B220+/CD11b− plasmacytoid-precursor dendritic cell (p-preDC) FC subpopulation plays a critical but nonredundant role in facilitation. In the present study, we investigated the mechanism of FC function. We report that FCs induce antigen-specific CD4+/CD25+/FoxP3+ regulatory T cells (Tregs) in vivo. The majority of chimeric Tregs were recipient derived. Chimeric Tregs harvested at ≥ 4 weeks after transplantation significantly enhanced engraftment of donor- and recipient-derived HSCs, but not third-party HSCs, in conditioned secondary recipients, demonstrating antigen specificity. Although Tregs were present 2 and 3 weeks after transplantation, they did not enhance engraftment. In contrast, week 5 and greater Tregs potently enhanced engraftment. The function of chimeric Tregs was directly correlated with the development of FoxP3 expression. Chimeric Tregs also induced significantly stronger suppression of T-cell proliferation to donor antigen in vitro. Removal of p-preDC FCs resulted in impaired engraftment of allogeneic HSCs and failure to produce chimeric Tregs, suggesting that the CD8α+ p-preDC subpopulation is critical in the mechanism of facilitation. These data suggest that FCs induce the production of antigen-specific Tregs in vivo, which potently enhance engraftment of allogeneic HSCs. FCs hold clinical potential because of their ability to remain tolerogenic in vivo.

scholarly journals Smad3 binding to the foxp3 enhancer is dispensable for the development of regulatory T cells with the exception of the gut

2012 ◽  
Vol 209 (9) ◽  
pp. 1529-1535 ◽  
Author(s):  
Susan M. Schlenner ◽  
Benno Weigmann ◽  
Qingguo Ruan ◽  
Youhai Chen ◽  
Harald von Boehmer
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Transcription Initiation ◽  
Foxp3 Expression ◽  
Cell Development ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Smad3 Binding

Regulatory T cells (T reg cells) are essential for the prevention of autoimmunity throughout life. T reg cell development occurs intrathymically but a subset of T reg cells can also differentiate from naive T cells in the periphery. In vitro, Smad signaling facilitates conversion of naive T cells into T reg cells but results in unstable Foxp3 expression. The TGF-β–Smad response element in the foxp3 locus is located in the CNS1 region in close proximity to binding sites for transcription factors implicated in TCR and retinoic acid signaling. From in vitro experiments it was previously postulated that foxp3 transcription represents a hierarchical process of transcription factor binding in which Smad3 would play a central role in transcription initiation. However, in vitro conditions generate T reg cells that differ from T reg cells encountered in vivo. To address the relevance of Smad3 binding to the CNS1 enhancer in vivo, we generated mice that exclusively lack the Smad binding site (foxp3CNS1mut). We show that binding of Smad3 to the foxp3 enhancer is dispensable for T reg cell development in newborn and adult mice with the exception of the gut.

scholarly journals DNA Methyltransferase Inhibition Promotes Th1 Polarization in Human CD4+CD25high FOXP3+ Regulatory T Cells but Does Not Affect Their Suppressive Capacity

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Sija Landman ◽  
Marjan Cruijsen ◽  
Paulo C. M. Urbano ◽  
Gerwin Huls ◽  
Piet E. J. van Erp ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Dna Methyltransferase ◽  
Hematological Malignancies ◽  
Foxp3 Expression ◽  
Significant Inhibition ◽  
Naturally Occurring ◽  
Suppressive Capacity

Regulatory T cells (Treg) can show plasticity whereby FOXP3 expression, the master transcription factor for Treg suppressor function, is lost and proinflammatory cytokines are produced. Optimal FOXP3 expression strongly depends on hypomethylation of the FOXP3 gene. 5-Azacytidine (Aza) and its derivative 5-aza-2′-deoxycytidine (DAC) are DNA methyltransferase inhibitors (DNMTi) that are therapeutically used in hematological malignancies, which might be an attractive strategy to promote Treg stability. Previous in vitro research primarily focused on Treg induction by DAC from naïve conventional CD4+ T cells (Tconv). Here, we examined the in vitro effect of DAC on the stability and function of FACS-sorted human naturally occurring CD4+CD25high FOXP3+ Treg. We found that in vitro activation of Treg in the presence of DAC led to a significant inhibition of Treg proliferation, but not of Tconv. Although Treg activation in the presence of DAC led to increased IFNγ expression and induction of a Thelper-1 phenotype, the Treg maintained their suppressive capacity. DAC also induced a trend towards increased IL-10 expression. In vivo studies in patients with hematological malignancies that were treated with 5-azacytidine (Vidaza) supported the in vitro findings. In conclusion, despite its potential to increase IFNγ expression, DAC does preserve the suppressor phenotype of naturally occurring Treg.

scholarly journals Signaling through C5a receptor and C3a receptor diminishes function of murine natural regulatory T cells

2013 ◽  
Vol 210 (2) ◽  
pp. 257-268 ◽  
Author(s):  
Wing-hong Kwan ◽  
William van der Touw ◽  
Estela Paz-Artal ◽  
Ming O. Li ◽  
Peter S. Heeger
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cell Function ◽  
Foxp3 Expression ◽  
C5a Receptor ◽  
Self Tolerance ◽  
T Cell Immune Responses

Thymus-derived (natural) CD4+ FoxP3+ regulatory T cells (nT reg cells) are required for immune homeostasis and self-tolerance, but must be stringently controlled to permit expansion of protective immunity. Previous findings linking signals transmitted through T cell–expressed C5a receptor (C5aR) and C3a receptor (C3aR) to activation, differentiation, and expansion of conventional CD4+CD25− T cells (T conv cells), raised the possibility that C3aR/C5aR signaling on nT reg cells could physiologically modulate nT reg cell function and thereby further impact the induced strength of T cell immune responses. In this study, we demonstrate that nT reg cells express C3aR and C5aR, and that signaling through these receptors inhibits nT reg cell function. Genetic and pharmacological blockade of C3aR/C5aR signal transduction in nT reg cells augments in vitro and in vivo suppression, abrogates autoimmune colitis, and prolongs allogeneic skin graft survival. Mechanisms involve C3a/C5a-induced phosphorylation of AKT and, as a consequence, phosphorylation of the transcription factor Foxo1, which results in lowered nT reg cell Foxp3 expression. The documentation that C3a/C3aR and C5a/C5aR modulate nT reg cell function via controlling Foxp3 expression suggests targeting this pathway could be exploited to manipulate pathogenic or protective T cell responses.

Therapeutic potential of low-dose IL-2 in a chronic GVHD patient by in vivo expansion of regulatory T cells

Cytokine ◽  
2016 ◽  
Vol 78 ◽  
pp. 22-26 ◽  
Author(s):  
Nayoun Kim ◽  
Young-Woo Jeon ◽  
Young-Sun Nam ◽  
Jung-Yeon Lim ◽  
Keon-Il Im ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Low Dose ◽  
Therapeutic Potential ◽  
Chronic Gvhd

Stability of Foxp3 Expression Is a Critical Factor In the Ability of Regulatory T Cells to Mitigate Graft Versus Host Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 731-731
Author(s):  
Amy Beres ◽  
Richard Komorowski ◽  
William R. Drobyski
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Critical Factor ◽  
Foxp3 Expression ◽  
Spleen Cells ◽  
Graft Versus Host

Abstract Abstract 731 Graft versus host disease (GVHD) is a proinflammatory T cell-mediated syndrome that is the major complication of allogeneic bone marrow transplantation (BMT). During the course of GVHD, there is a progressive loss of regulatory T cells (Tregs), leading to an imbalance between the effector and regulatory arms of the immune system. Tregs have been subdivided into two distinct subsets, termed natural and induced, which have overlapping yet unique characteristics. While the role of natural regulatory T cells (nTregs) in GVHD biology has been extensively examined, the role of induced regulatory T cells (iTregs) remains largely unknown. An attractive aspect of the latter cell population is that they can be differentiated in vitro from conventional T cells and expanded in large numbers making them a potential source for regulatory T cell therapy in vivo. To determine whether in vitro-expanded iTregs were able to suppress alloreactive donor T cell responses and to compare the efficacy of these cells relative to nTregs, studies were performed using an MHC-incompatible murine BMT model (B6[H−2b]−Balb/c[H−2d]). In initial studies, purified CD4+ Foxp3EGFP– T cells obtained from B6 Foxp3EGFP reporter mice were cultured with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and TGF-b. After three days in culture, approximately 60–70% of cells were Foxp3+, expressed GITR, CD25, and CD103, and were equally suppressive to nTregs in mixed lymphocyte cultures. To determine if iTregs were suppressive in vivo, lethally irradiated Balb/c mice were transplanted with either B6 BM alone, B6 BM and spleen cells, or B6 BM/spleen cells and in vitro-expanded iTregs. In contrast to in vitro results, adoptive transfer of iTregs failed to protect mice from lethal GVHD even when administered at high Treg: effector T cell ratios (5:1) and were much less effective than equivalent doses of nTregs at abrogating GVHD pathology. iTregs also had no additive effect when co-administered with nTregs. Notably, we observed that whereas transferred nTregs persisted for up to 60 days in transplanted animals, iTregs were undetectable after only 14 days in liver, lung, colon and spleen, indicating that reduced in vivo survival was a potential explanation for the lack of protection. Further examination, however, revealed that the inability to detect iTregs was primarily attributable to the loss of Foxp3 expression and the subsequent in vivo reversion of these cells to a proinflammatory phenotype characterized by the secretion of interferon-gamma. In prior studies (Chen et al, Blood, 2009), we demonstrated that blockade of IL-6 signaling augmented reconstitution of nTregs and reduced overall GVHD severity. To determine whether inhibition of IL-6 could stabilize Foxp3 expression and prevent phenotypic reversion of iTregs, lethally irradiated Balb/c recipients were transplanted with B6 BM and spleen cells along with in vitro-differentiated iTregs and then treated with either isotype control or anti-IL-6R-specific antibody. Analysis of cells obtained from spleen, liver, lung and colon revealed that blockade of IL-6 signaling did not prevent loss of Foxp3 expression or reversion of iTregs to a Th1 cytokine phenotype. While Tregs can be converted from conventional T cells in vitro, they can also be generated in vivo during inflammatory syndromes. We therefore examined whether in vivo induction of iTregs occurred during GVHD and the extent to which blockade of IL-6 signaling affected iTreg expansion and overall GVHD protection. To address this question, lethally irradiated Balb/c mice were transplanted with B6 Rag-1 BM cells and purified CD4+ Foxp3EGFP– T cells, and then treated with either anti-IL-6R or control antibody. We observed that in vivo conversion of Tregs was negligible in control animals (<1%), but that administration of anti-IL-6R antibody significantly increased the relative and absolute number of iTregs in GVHD target tissues with a commensurate reduction in overall pathological damage. Thus, blockade of IL-6 signaling was able to enhance reconstitution of iTregs in vivo, but had no discernible affect on adoptively transferred iTregs. In summary, these studies demonstrate that the stability of Foxp3 expression is a critical factor in the maintenance of transplantation tolerance and that instability of expression limits the utility of adoptively transferred iTregs as a source of cellular therapy for the abrogation of GVHD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Induced, but not natural, regulatory T cells retain phenotype and function following exposure to inflamed synovial fibroblasts

2020 ◽  
Vol 6 (44) ◽  
pp. eabb0606 ◽  
Author(s):  
Sujuan Yang ◽  
Ximei Zhang ◽  
Jingrong Chen ◽  
Junlong Dang ◽  
Rongzhen Liang ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Synovial Fibroblasts ◽  
Foxp3 Expression ◽  
Regulatory Function ◽  
Migration And Invasion ◽  
Effector Cells ◽  
T Effector Cells

Aberrant number and/or dysfunction of CD4+Foxp3+ Regulatory T cells (Tregs) are associated with the pathogenesis of rheumatoid arthritis (RA). A previous study has demonstrated that thymus-derived, natural Tregs (nTregs) prefer to accumulate in inflamed joints and transdifferentiate to TH17 cells under the stimulation of inflamed synovial fibroblasts (SFs). In this study, we made a head-to-head comparison of both Treg subsets and demonstrated that induced Tregs (iTregs), but not nTregs, retained Foxp3 expression and regulatory function on T effector cells (Teffs) after being primed with inflamed SFs. In addition, iTregs inhibited proliferation, inflammatory cytokine production, migration, and invasion ability of collagen-induced arthritis (CIA)–SFs in vitro and in vivo. Moreover, we noted that iTregs directly targeted inflamed SFs to treat autoimmune arthritis, while nTregs failed to do this. Thus, manipulation of the iTreg subset may have a greater potential for prevention or treatment of patients with RA.

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