Ex Vivo–Expanded but Not In Vitro–Induced Human Regulatory T Cells Are Candidates for Cell Therapy in Autoimmune Diseases Thanks to Stable Demethylation of the FOXP3 Regulatory T Cell–Specific Demethylated Region

Abstract Regulatory T cells (CD4+CD25hiCD127loFOXP3+ T cells [Tregs]) are a population of lymphocytes involved in the maintenance of self-tolerance. Abnormalities in function or number of Tregs are a feature of autoimmune diseases in humans. The ability to expand functional Tregs ex vivo makes them ideal candidates for autologous cell therapy to treat human autoimmune diseases and to induce tolerance to transplants. Current tests of Treg function typically take up to 120 hours, a kinetic disadvantage as clinical trials of Tregs will be critically dependent on the availability of rapid diagnostic tests before infusion into humans. Here we evaluate a 7-hour flow cytometric assay for assessing Treg function, using suppression of the activation markers CD69 and CD154 on responder T cells (CD4+CD25− [Tresp]), compared with traditional assays involving inhibition of CFSE dilution and cytokine production. In both freshly isolated and ex vivo expanded Tregs, we describe excellent correlation with gold standard suppressor cell assays. We propose that the kinetic advantage of the new assay may place it as the preferred rapid diagnostic test for the evaluation of Treg function in forthcoming clinical trials of cell therapy, enabling the translation of the large body of preclinical data into potentially useful treatments for human diseases.

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Regulatory T Cells Contribute to Resistance against Lyme Arthritis

Infection and Immunity ◽

10.1128/iai.00160-20 ◽

2020 ◽

Vol 88 (11) ◽

Author(s):

Emily M. Siebers ◽

Elizabeth S. Liedhegner ◽

Michael W. Lawlor ◽

Ronald F. Schell ◽

Dean T. Nardelli

Keyword(s):

T Cells ◽

T Cell ◽

Lyme Disease ◽

Regulatory T Cells ◽

Regulatory T Cell ◽

Interleukin 10 ◽

Lyme Arthritis ◽

Interleukin 17 ◽

Content Type

ABSTRACT The symptoms of Lyme disease are caused by inflammation induced by species of the Borrelia burgdorferi sensu lato complex. The various presentations of Lyme disease in the population suggest that differences exist in the intensity and regulation of the host response to the spirochete. Previous work has described correlations between the presence of regulatory T cells and recovery from Lyme arthritis. However, the effects of Foxp3-expressing CD4+ T cells existing prior to, and during, B. burgdorferi infection have not been well characterized. Here, we used C57BL/6 “depletion of regulatory T cell” mice to assess the effects these cells have on the arthritis-resistant phenotype characteristic of this mouse strain. We showed that depletion of regulatory T cells prior to infection with B. burgdorferi resulted in sustained swelling, as well as histopathological changes, of the tibiotarsal joints that were not observed in infected control mice. Additionally, in vitro stimulation of splenocytes from these regulatory T cell-depleted mice resulted in increases in gamma interferon and interleukin-17 production and decreases in interleukin-10 production that were not evident among splenocytes of infected mice in which Treg cells were not depleted. Depletion of regulatory T cells at various times after infection also induced rapid joint swelling. Collectively, these findings provide evidence that regulatory T cells existing at the time of, and possibly after, B. burgdorferi infection may play an important role in limiting the development of arthritis.

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Regulatory T-cell therapy in Crohn’s disease: challenges and advances

Gut ◽

10.1136/gutjnl-2019-319850 ◽

2020 ◽

Vol 69 (5) ◽

pp. 942-952 ◽

Cited By ~ 8

Author(s):

Jennie N Clough ◽

Omer S Omer ◽

Scott Tasker ◽

Graham M Lord ◽

Peter M Irving

Keyword(s):

T Cells ◽

Crohn’S Disease ◽

T Cell ◽

Crohn's Disease ◽

Regulatory T Cells ◽

Cell Therapy ◽

Regulatory T Cell ◽

Critical Role ◽

Significant Proportion ◽

T Cell Therapy

The prevalence of IBD is rising in the Western world. Despite an increasing repertoire of therapeutic targets, a significant proportion of patients suffer chronic morbidity. Studies in mice and humans have highlighted the critical role of regulatory T cells in immune homeostasis, with defects in number and suppressive function of regulatory T cells seen in patients with Crohn’s disease. We review the function of regulatory T cells and the pathways by which they exert immune tolerance in the intestinal mucosa. We explore the principles and challenges of manufacturing a cell therapy, and discuss clinical trial evidence to date for their safety and efficacy in human disease, with particular focus on the development of a regulatory T-cell therapy for Crohn’s disease.

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Sirolimus Inhibits Proliferation but Enhances Suppressive Activity of Rhesus Macaque Natural CD4 Regulatory T Cells

Blood ◽

10.1182/blood.v118.21.1008.1008 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1008-1008

Author(s):

Karnail Singh ◽

Natalia Kozyr ◽

Linda Stempora ◽

Allan D Kirk ◽

Christian P Larsen ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Ex Vivo ◽

Cd8 T Cell ◽

T Cell Proliferation ◽

Suppressive Activity ◽

Suppressive Capacity

Abstract Abstract 1008 Regulatory T cells (Tregs) have been shown to be potent inhibitors of autoimmunity, and to be capable of suppressing alloimmune responses that occur during both allograft rejection and graft-versus host disease. However, they have yet to gain widespread use clinically, due in part to the fact that it remains extremely costly and difficult to produce them in sufficient numbers and with sufficient suppressive capacity to significantly impact the alloimmune response. Here we have used our established non-human primate model to demonstrate that significant Treg expansion (up to 600-fold in 21 days) can be maintained, and suppressive capacity enhanced by exposing Treg cultures to a short burst of sirolimus at the end of the culture period. Using a highly sensitive and specific in vitro CFSE-MLR assay we show that Tregs significantly inhibit allo-proliferation of multiple T cell subpopulations including both CD4+ and CD8+ T cells (3.2 and 2.7-fold inhibition of proliferation, respectively), as well as their CD28+CD95+ and CD28-CD95+ subpopulations (2.2 and 2.1 and 1.9 and 2.7-fold inhibition of CD4+ and CD8+ subpopulation proliferation, respectively). Tregs were able to combine in vitro with the newly FDA-approved CTLA4-Ig analog belatacept to enhance the inhibition of alloproliferation that occurred with either agent alone (4.8-fold inhibition of CD8 T cell proliferation with Tregs + belatacept, compared to 3.0-fold or 1.9-fold inhibition of CD8 T cell proliferation with Tregs or belatacept alone, respectively). Importantly, we have found that the suppressive activity of ex-vivo expanded Tregs could be further enhanced by pulsing with sirolimus. Thus, while long-term culture of Tregs in the presence of sirolimus (1–1000 nM) profoundly inhibited Treg expansion (50–800 fold inhibition of expansion when cultured in the presence of 1–1000 nM sirolimus), a 48 hour pulse of sirolimus (100 nM) on days 20–21 of culture completely preserved Treg yields while doubling their suppressive function against CD8 proliferation when compared to unpulsed Tregs, p<0.01) A mechanistic evaluation of the increase potency observed with sirolimus pulsed Tregs (SPTs) has revealed several key differences that distinguish these cells from the less-potent unpulsed Tregs: SPTs were found to undergo fewer rounds of proliferation in an MLR when compared with unpulsed Tregs (14% proliferation in SPTs versus 37% proliferation in un-pulsed Tregs, p= 0.015), suggesting that the suppressive capability of Tregs may be inversely related to their proliferative capacity. SPTs were also shown to have significantly increased expression of CD25 (p=0.04) and total CTLA4 (p= 0.009) compared to unpulsed Tregs, implicating signaling through both of these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue by which to achieve enhanced Treg-based suppression of alloimmunity, in a manner that is amenable to large-scale ex-vivo expansion and to combinatorial therapy with novel, costimulation-blockade-based immunosuppression strategies. Disclosures: No relevant conflicts of interest to declare.

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Restoring regulatory T-cell dysfunction in Alzheimer’s disease through ex vivo expansion

Brain Communications ◽

10.1093/braincomms/fcaa112 ◽

2020 ◽

Vol 2 (2) ◽

Author(s):

Alireza Faridar ◽

Aaron D Thome ◽

Weihua Zhao ◽

Jason R Thonhoff ◽

David R Beers ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Regulatory T Cell ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

T Cell Proliferation ◽

Inflammatory Macrophages

Abstract Inflammation is a significant component of Alzheimer’s disease pathology. While neuroprotective microglia are important for containment/clearance of Amyloid plaques and maintaining neuronal survival, Alzheimer inflammatory microglia may play a detrimental role by eliciting tau pathogenesis and accelerating neurotoxicity. Regulatory T cells have been shown to suppress microglia-mediated inflammation. However, the role of regulatory T cells in ameliorating the proinflammatory immune response in Alzheimer’s disease requires further investigation. Forty-six patients with Alzheimer disease, 42 with mild cognitive impairment and 41 healthy controls were studied. The phenotypes of peripheral regulatory T cells were assessed with multicolour flow cytometry. Regulatory T cells were co-cultured with responder T cells and proliferation was determined by 3H-thymidine incorporation. In separate experiments, regulatory T cells were added to induced pluripotent stem cell-derived pro-inflammatory macrophages and changes in interleukin-6/tumour necrosis-alpha transcripts and protein levels were measured. Freshly isolated regulatory T cells were expanded ex vivo in the presence of CD3/CD28 expander beads, interleukin-2 and rapamycin to promote their suppressive function. We found that the suppressive function of regulatory T cells on responder T-cell proliferation was compromised at the Alzheimer disease stage, compared with mild cognitive impairment and healthy controls. CD25 mean fluorescence intensity in regulatory T-cell population was also reduced in Alzheimer dementia patients. Regulatory T cells did not suppress pro-inflammatory macrophages at baseline. Following ex vivo expansion, regulatory T-cell suppression of responder T-cell proliferation and pro-inflammatory macrophage activation increased in both patients and controls. Expanded regulatory T cells exerted their immunoregulatory function on pro-inflammatory macrophages through a contact-mediated mechanism. In conclusion, regulatory T-cell immunophenotype and function are compromised in Alzheimer’s disease. Following ex vivo expansion, the immunomodulatory function of regulatory T cells is enhanced even at advanced stages of Alzheimer’s disease. Restoration of regulatory T-cell function could be explored as a means to modulate the inflammatory status of Alzheimer’s disease.

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Rabbit ATG but Not Horse ATG Promotes Expansion of Functional CD4+CD25highFoxP3 Regulatory T Cells In Vitro.

Blood ◽

10.1182/blood.v110.11.2312.2312 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2312-2312

Author(s):

Xingmin Feng ◽

Elena E. Solomou ◽

Keyvan Keyvanfar ◽

Thomas Herndon ◽

Jichun Chen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Autoimmune Diseases ◽

In Vitro Culture ◽

Mononuclear Cells ◽

Foxp3 Expression ◽

Therapeutic Effects ◽

Lymphocyte Depletion

Abstract CD4+CD25+ regulatory T cells (Treg) are believed to play important roles in suppressing immune responses and maintaining tolerance. Treg have the ability to prevent the development of autoimmune diseases, graft rejection and graft versus host disease (GVHD) in mice and perhaps also in humans. Immunosuppressive drugs such as rabbit ATG (rATG), horse ATG (hATG) and cyclosporine A (CsA) are widely used in conditioning for transplantation and for the treatment of autoimmune diseases and GVHD, but their effects on Treg remain to be fully elucidated. Lopez et al. (J Am Soc Nephrol. 2006;17:2844–2853.) first reported that in vitro culture of human peripheral blood mononuclear cells (PBMC) with rATG resulted in expansion of CD4+CD25+ T cells. In the current study, we show that in vitro culture of normal human PBMC with low dose rATG (10 μg/ml) resulted in marked expansion of CD4+CD25high T cells (rATG 8.00±0.95% versus untreated 0.99±0.11%, n=10, p<0.0001) and CD4+CD25high FoxP3 T cells (rATG 2.24±0.11% versus untreated 0.90±0.10%, n=10, p<0.0001). rATG exposure converted CD4+CD25− T cells into CD4+CD25+ T cells, which proliferated better in comparison to CD4+CD25− T cells. In immunoblots of protein extracted from PBMC treated with rATG, there was increased expression of FoxP3 and nuclear factor of activated T cells (NFAT1) in CD4+CD25− and CD4+CD25+ T cells; rATG-induced NFAT1 expression correlated with FoxP3 expression. Expanded Treg suppressed autologous T-cell proliferation after T-cell receptor (TCR) stimulation by 64% when cultured with autologous PBMC at a 1:1 ratio, consistent with functional activity. Culture supernatants of PBMC treated with rATG showed increased levels of IL-10, compared with supernatants of PBMC treated with hATG or CsA, but no differences in INF-γ, IL-2, and IL-4. Unexpectedly, hATG did not expand but rather decreased Treg [For CD4+CD25high T cells (n=10): hATG 0.67±0.10% versus untreated 0.99±0.11%, p=0.0386; For CD4+CD25high FoxP3 T cells (n=10): hATG 0.62±0.08% versus untreated 0.90±0.10%, p=0.0435]. Furthermore, rATG and hATG showed differences in binding to lymphocytes, they contained different amounts of CD3 and TCRαβ antibodies, and they induced different activation states (expression of glucocorticoid-induced tumor necrosis factor receptor, cytotoxic T lymphocyte-associated antigen-4, and CD62L) for CD4+ T cells. In vitro, Treg expansion mediated by rATG occurred at submitogenic concentrations (< 50 μg/ml) rather than at lymphocyte depletion levels (50–100 μg/ml). Our findings suggest that rATG expanded Treg by converting CD4+CD25− T cells into CD4+CD25+ T cells, probably through a mechanism of transcription regulation, and enhanced NFAT1 expression, in turn conferring on CD4+CD25− T cells FoxP3 expression and regulatory activity. The therapeutic effects of rATG in the treatment of autoimmune diseases and GVHD may occur due to not only lymphocyte depletion but also enhanced Treg cell number and function. Our observation may provide a useful method for expansion of Treg in cellular treatment in transplantation and autoimmune diseases.

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FcRγ-Dependent Facilitating Cells Are Direct Inducers of Regulatory T Cells.

Blood ◽

10.1182/blood.v106.11.65.65 ◽

2005 ◽

Vol 106 (11) ◽

pp. 65-65

Author(s):

Kendra N. Taylor ◽

Vivek R. Shinde Patil ◽

Meredith Chittenden ◽

Yolonda L. Colson

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Regulatory T Cell ◽

In Vivo Studies ◽

Pcr Analysis

Abstract Facilitating cells (FC) are CD8+/αβγδTCR− bone marrow-derived cells that promote allogeneic stem cell (SC) engraftment without graft vs. host disease (GVHD) and induce donor-specific transplantation tolerance. However, the mechanism of FC-mediated allogeneic SC engraftment is not known. We have previously demonstrated that allogeneic SC engraftment promoted by FC result in no clinical evidence of GVHD and is associated with increased number of CD4+25+ regulatory T cells post transplant compared to recipients of bone marrow T cells that develop severe GVHD. This is consistent with the hypothesis that FC facilitate allogeneic SC engraftment and induce tolerance through the induction of a regulatory T cell network. Here we report that CpG activation of toll-like receptor 9 (TLR9) on FC induce CD4+25− naïve T cell differentiation into CD4+25+ regulatory T cells. CpG stimulated and unstimulated CD8+αβγδTCR− FC isolated from bone marrow of C57/BL6 mice by flow cytometric cell sorting FC were co-cultured with splenic CD4+25− T cells for 5 days. CpG stimulated FC co-culture gave rise to CD4+25+ T cells as determined by mRNA expression of the CD4+25+ regulatory T cell marker, FoxP3. In contrast, unstimulated FC in co-culture did not induce regulatory T cells. Because FcRγ is the dominant ITAM receptor (immunoreceptor tyrosine-based activating motif), on FC and given that in vivo studies have demonstrated a requirement for FcRγ expression on FC, we hypothesized that FcRγ gene expression is increased within activated FC. FcRγ gene expression in CpG stimulated and unstimulated FC were compared by real-time PCR analysis. CpG-mediated TLR signaling within FC result in increased gene expression of FcRγ. Taken together, these studies demonstrate that CpG stimulated FC induce the generation of CD4+25+ regulatory T cells in vitro and CpG activation results in increased gene expression of FcRγ, suggesting a requirement for FcRγ signaling in FC-mediated induction of regulatory T cells. These findings provide the first mechanistic evidence that FC are direct inducers of regulatory T cells. Further characterization of cooperative TLR and FcRγ signaling pathways in FC will be critical to defining the mechanism of FC-mediated SC engraftment and the identification of potential therapeutic targets for the clinical induction of tolerance in the future.

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Faculty Opinions recommendation of Activation of the aryl hydrocarbon receptor induces human type 1 regulatory T cell-like and Foxp3(+) regulatory T cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.4942963.4875063 ◽

2010 ◽

Author(s):

Rachel R Caspi ◽

Ru Zhou

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Aryl Hydrocarbon Receptor ◽

Regulatory T Cell ◽

Human Type ◽

Aryl Hydrocarbon

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Regulatory T Cells Fail to Suppress Fast Homeostatic Proliferation In Vitro

Life ◽

10.3390/life11030245 ◽

2021 ◽

Vol 11 (3) ◽

pp. 245

Author(s):

Daniil Shevyrev ◽

Valeriy Tereshchenko ◽

Elena Blinova ◽

Nadezda Knauer ◽

Ekaterina Pashkina ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Autoimmune Diseases ◽

Peripheral Blood ◽

Treg Cells ◽

Homeostatic Proliferation ◽

Suppressive Activity ◽

Healthy Donors

Homeostatic proliferation (HP) is a physiological process that reconstitutes the T cell pool after lymphopenia involving Interleukin-7 and 15 (IL-7 and IL-15), which are the key cytokines regulating the process. However, there is no evidence that these cytokines influence the function of regulatory T cells (Tregs). Since lymphopenia often accompanies autoimmune diseases, we decided to study the functional activity of Tregs stimulated by HP cytokines from patients with rheumatoid arthritis as compared with that of those from healthy donors. Since T cell receptor (TCR) signal strength determines the intensity of HP, we imitated slow HP using IL-7 or IL-15 and fast HP using a combination of IL-7 or IL-15 with anti-CD3 antibodies, cultivating Treg cells with peripheral blood mononuclear cells (PBMCs) at a 1:1 ratio. We used peripheral blood from 14 patients with rheumatoid arthritis and 18 healthy volunteers. We also used anti-CD3 and anti-CD3 + IL-2 stimulation as controls. The suppressive activity of Treg cells was evaluated in each case by the inhibition of the proliferation of CD4+ and CD8+ cells. The phenotype and proliferation of purified CD3+CD4+CD25+CD127lo cells were assessed by flow cytometry. The suppressive activity of the total pool of Tregs did not differ between the rheumatoid arthritis and healthy donors; however, it significantly decreased in conditions close to fast HP when the influence of HP cytokines was accompanied by anti-CD3 stimulation. The Treg proliferation caused by HP cytokines was lower in the rheumatoid arthritis (RA) patients than in the healthy individuals. The revealed decrease in Treg suppressive activity could impact the TCR landscape during lymphopenia and lead to the proliferation of potentially self-reactive T cell clones that are able to receive relatively strong TCR signals. This may be another explanation as to why lymphopenia is associated with the development of autoimmune diseases. The revealed decrease in Treg proliferation under IL-7 and IL-15 exposure can lead to a delay in Treg pool reconstitution in patients with rheumatoid arthritis in the case of lymphopenia.

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764 Expansion with IL-15 increases cytotoxicity of Vγ9Vδ2 T cells and is associated with higher levels of cytotoxic molecules and T-bet

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0764 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A812-A812

Author(s):

Pia Aehnlich ◽

Per Thor Straten ◽

Ana Micaela Carnaz Simoes ◽

Signe Skadborg ◽

Gitte Olofsson

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

Ex Vivo ◽

Adoptive Cell Therapy ◽

Hematological Cancers ◽

Cytotoxic Molecules ◽

Vγ9vδ2 T Cells ◽

T Cell Expansion

BackgroundAdoptive cell therapy (ACT) is an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle.MethodsIn this study, we explored the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro.ResultsWe could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1000U/ml interleukin (IL)-2 and (b) 100U/ml IL-2+100U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in phenotype, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells harbor increased amounts of perforin, granzyme B and granulysin in a resting state and release more upon activation. IL-2/IL-15-expanded Vγ9Vδ2 T cells also showed higher levels of transcription factor T-bet, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity.ConclusionsThese results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

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