Isolation of Treg cells and Treg cell suppression/death assay

2007 ◽  
Author(s):  
Pushpa Pandiyan
Keyword(s):  
Treg Cell ◽  
Treg Cells ◽  
Cell Suppression

Combination of Cord Blood T Regulatory Cells with Ruxolitinib Decreases Side Effects and Improves Survival in Xenogenic Graft Vs. Host Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 2-2
Author(s):  
Ke Zeng ◽  
Meixian Huang ◽  
Li Li ◽  
Mi-Ae Lyu ◽  
Hongbing Ma ◽  
...  
Keyword(s):  
Side Effects ◽  
Cord Blood ◽  
Treg Cell ◽  
Inflammatory Cytokines ◽  
Cell Function ◽  
Treg Cells ◽  
Host Disease ◽  
Suppressor Function ◽  
Cell Suppression ◽  
Suppression Assay

Background: Recent approval of ruxolitinib (rux) for steroid refractory graft versus host disease (GvHD) has revolutionized the field and provided tremendous choice for the patients. However, the side effects including thrombocytopenia leads to dug discontinuation and intolerance. We have previously shown that adoptive therapy with cord blood (CB) derived T regulatory (Treg) cells can prevent and treat GvHD. We hypothesized that the addition of CB Treg therapy to rux based therapy can augment overall efficacy. Methods: CellTrace Violet suppression assay was performed to evaluate the suppressor function of CB Treg cells in the presence or absence of rux. Xenogenic GvHD mouse model was utilized where the NSG mice underwent sublethal irradiation on day -1 followed by injection of 1x107 donor peripheral blood (PB) mononuclear cells (MNCs) on day 0. Oral rux at 1 mg daily was fed continuously to the mice in the presence or absence of 1x107 CB Treg cells, tagged with CellTrace Violet dye, administered on days +4, +7, +11, +18. Mice were followed every other day for weight, GvHD score and survival. Serial blood draws were performed to analyze for cell compartment and cytokine assays. Results: We examine whether the addition of rux impacts the suppressor function of CB Treg cells. Varying concentration of rux including 0.01, 0.05, 0.1 and 0.5 µM were added at 24, 48 and 96 hours of the cell suppression assay. The addition of rux 0.05 µM at 48 hours of the cell suppression culture led to a restoration of poor cell function (Figure A). Lowest GVHD score was reported in the rux+CB Treg combination arms at day +14 when compared to rux alone or CB Treg alone arm which (Figure B) translated into a superior survival in the rux +CB Treg arm (Figure C). Furthermore, an increase in the hemoglobin level (Figure D) and the platelet count (Figure E) was demonstrated in the rux+CB Treg arm. Addition of rux led to longer persistence of the injected CB Treg cells on day 14 (data not shown) which correlated with the increase in the plasma level of the pro-Treg cell signaling markers including IL-7 (Figure F) and IL-15 (Figure G). A decrease in the IL-4 production supported the increased Treg cell function (Figure H). A synergistic suppression of inflammatory cytokines including IL-17 (Figure I) and IL1A (Figure J) was evident in the rux+CB Treg arm. Conclusion: The combination of CB Tregs with ruxolitinib leads to improved overall survival, decreased inflammatory cytokines and improved hematologic parameters. Such combination should be explored in a clinical setting Figure Disclosures Sadeghi: Cellenkos Inc.: Current Employment. Parmar:Cellenkos Inc.: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals Efficient IL-2R signaling differentially affects the stability, function, and composition of the regulatory T-cell pool

2021 ◽  
Author(s):  
Marc Permanyer ◽  
Berislav Bošnjak ◽  
Silke Glage ◽  
Michaela Friedrichsen ◽  
Stefan Floess ◽  
...  
Keyword(s):  
T Cell ◽  
Treg Cell ◽  
Cell Function ◽  
Interleukin 2 ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Receptor Expression ◽  
Spontaneous Mutant ◽  
Cell Pool ◽  
And Function

AbstractSignaling via interleukin-2 receptor (IL-2R) is a requisite for regulatory T (Treg) cell identity and function. However, it is not completely understood to what degree IL-2R signaling is required for Treg cell homeostasis, lineage stability and function in both resting and inflammatory conditions. Here, we characterized a spontaneous mutant mouse strain endowed with a hypomorphic Tyr129His variant of CD25, the α-chain of IL-2R, which resulted in diminished receptor expression and reduced IL-2R signaling. Under noninflammatory conditions, Cd25Y129H mice harbored substantially lower numbers of peripheral Treg cells with stable Foxp3 expression that prevented the development of spontaneous autoimmune disease. In contrast, Cd25Y129H Treg cells failed to efficiently induce immune suppression and lost lineage commitment in a T-cell transfer colitis model, indicating that unimpaired IL-2R signaling is critical for Treg cell function in inflammatory environments. Moreover, single-cell RNA sequencing of Treg cells revealed that impaired IL-2R signaling profoundly affected the balance of central and effector Treg cell subsets. Thus, partial loss of IL-2R signaling differentially interferes with the maintenance, heterogeneity, and suppressive function of the Treg cell pool.

scholarly journals Granzyme B is not required for regulatory T cell–mediated suppression of graft-versus-host disease

Blood ◽  
2010 ◽  
Vol 115 (9) ◽  
pp. 1669-1677 ◽  
Author(s):  
Sheng F. Cai ◽  
Xuefang Cao ◽  
Anjum Hassan ◽  
Todd A. Fehniger ◽  
Timothy J. Ley
Keyword(s):  
Immune Responses ◽  
Treg Cell ◽  
Graft Versus Host Disease ◽  
Granzyme B ◽  
Target Organ Damage ◽  
Organ Damage ◽  
Treg Cells ◽  
Host Disease ◽  
Graft Versus Host

Abstract Regulatory T (Treg) cells can suppress a wide variety of immune responses, including antitumor and alloimmune responses. The mechanisms by which Treg cells mediate their suppressive effects depend on the context of their activation. We previously reported that granzyme B is important for Treg cell–mediated suppression of antitumor immune responses. We therefore hypothesized that granzyme B may likewise be important for suppression of graft-versus-host disease (GVHD). We found that allogeneic mismatch induces the expression of granzyme B in mixed lymphocyte reactions and in a model of graft-versus-host disease (GVHD). However, wild-type and granzyme B–deficient Treg cells were equally able to suppress effector T (Teff) cell proliferation driven by multiple stimuli, including allogeneicantigen-presenting cells. Surprisingly, adoptive transfer of granzyme B–deficient Treg cells prevented GVHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, which demonstrated that granzyme B plays a nonredundant role in Treg cell–mediated suppression of antitumor responses. Taken together, these findings suggest that targeting specific Treg cell–suppressive mechanisms, such as granzyme B, may be therapeutically beneficial for segregating GVHD and graft-versus-tumor immune responses.

scholarly journals FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 4953-4960 ◽  
Author(s):  
Mojgan Ahmadzadeh ◽  
Aloisio Felipe-Silva ◽  
Bianca Heemskerk ◽  
Daniel J. Powell ◽  
John R. Wunderlich ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Metastatic Melanoma ◽  
Treg Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Production Capacity ◽  
Biological Marker ◽  
Foxp3 Expression ◽  
Treg Cells

Abstract Regulatory T (Treg) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and Treg cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate Treg cells from effector T cells. Similar to Treg cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3+CD4 T cells, unlike FOXP3− T cells, were unable to produce IL-2 and IFN-γ upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human Treg cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3+CD4 Treg cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3+CD4 Treg cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3- to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 Treg cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment.

517 Regulatory T cell functional identity is sustained by a glucose:lactate axis that is exploited in the tumor microenvironment

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A553-A553
Author(s):  
McLane Watson ◽  
Paolo Vignali ◽  
Steven Mullet ◽  
Abigail Overacre-Delgoffe ◽  
Ronal Peralta ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Treg Cell ◽  
High Glucose ◽  
Cell Function ◽  
Treg Cells ◽  
Effector T Cells ◽  
Flux Analysis ◽  
Major Barrier ◽  
Suppressive Function

BackgroundRegulatory T (Treg) cells are vital for preventing autoimmunity but are a major barrier to robust cancer immunity as the tumor microenvironment (TME) recruits and promotes their function. The deregulated cellular metabolism of tumor cells leads to a metabolite-depleted, hypoxic, and acidic TME. While the TME impairs the effector function of highly glycolytic tumor infiltrating CD8 T cells, Treg cell suppressive function is maintained. Further, studies of in vitro induced and ex vivo Treg cells reveal a distinct metabolic profile compared to effector T cells. Thus, it may be that the altered metabolic landscape of the TME and the increased activity of intratumoral Treg cells are linked.MethodsFlow cytometry, isotopic flux analysis, Foxp3 driven Cre-lox, glucose tracers, Seahorse extracellular flux analysis, RNA sequencing.ResultsHere we show Treg cells display heterogeneity in terms of their glucose metabolism and can engage an alternative metabolic pathway to maintain their high suppressive function and proliferation within the TME and other tissues. Tissue derived Treg cells (both at the steady state and under inflammatory conditions) show broad heterogeneity in their ability to take up glucose. However, glucose uptake correlates with poorer suppressive function and long-term functional stability, and culture of Treg cells in high glucose conditions decreased suppressive function. Treg cells under low glucose conditions upregulate genes associated with the uptake and metabolism of the glycolytic end-product lactic acid. Treg cells withstand high lactate conditions, and lactate treatment prevents the destabilizing effects of high glucose culture. Treg cells utilize lactate within the TCA cycle and generate phosphoenolpyruvate (PEP), a critical intermediate that can fuel intratumoral Treg cell proliferation in vivo. Using mice with a Treg cell-restricted deletion of lactate transporter Slc16a1 (MCT1) we show MCT1 is dispensable for peripheral Treg cell function but required intratumorally, resulting in slowed tumor growth and prolonged survival.ConclusionsThese data support a model in which Treg cells are metabolically flexible such that they can utilize ‘alternative’ metabolites present in the TME to maintain their suppressive identity. Further, our studies support the notion that tumors avoid immune destruction not only by depriving effector T cells of essential nutrients, but also by metabolically supporting regulatory T cells.

scholarly journals Bcl11b prevents fatal autoimmunity by promoting Treg cell program and constraining innate lineages in Treg cells

2019 ◽  
Vol 5 (8) ◽  
pp. eaaw0480 ◽  
Author(s):  
Theodore T. Drashansky ◽  
Eric Helm ◽  
Zhiguang Huo ◽  
Nina Curkovic ◽  
Preet Kumar ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Steady State ◽  
Treg Cell ◽  
Nk Cell ◽  
Treg Cells ◽  
Chromatin Accessibility ◽  
Peripheral Tolerance ◽  
Identity Control ◽  
And Function ◽  
Human And Mouse

Regulatory T (Treg) cells are essential for peripheral tolerance and rely on the transcription factor (TF) Foxp3 for their generation and function. Several other TFs are critical for the Treg cell program. We found that mice deficient in Bcl11b TF solely in Treg cells developed fatal autoimmunity, and Bcl11b-deficient Treg cells had severely altered function. Bcl11b KO Treg cells showed decreased functional marker levels in homeostatic conditions, inflammation, and tumors. Bcl11b controlled expression of essential Treg program genes at steady state and in inflammation. Bcl11b bound to genomic regulatory regions of Treg program genes in both human and mouse Treg cells, overlapping with Foxp3 binding; these genes showed altered chromatin accessibility in the absence of Bcl11b. Additionally, Bcl11b restrained myeloid and NK cell programs in Treg cells. Our study provides new mechanistic insights on the Treg cell program and identity control, with major implications for therapies in autoimmunity and cancer.

scholarly journals DNAM-1 regulates Foxp3 expression in regulatory T cells by interfering with TIGIT under inflammatory conditions

2021 ◽  
Vol 118 (21) ◽  
pp. e2021309118
Author(s):  
Kazuki Sato ◽  
Yumi Yamashita-Kanemaru ◽  
Fumie Abe ◽  
Rikito Murata ◽  
Yuho Nakamura-Shinya ◽  
...  
Keyword(s):  
Treg Cell ◽  
Intracellular Signaling ◽  
Cell Function ◽  
Inflammatory Diseases ◽  
Mammalian Target Of Rapamycin ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Cell Dysfunction ◽  
Inflammatory Conditions ◽  
Treg Cell Function

Regulatory T (Treg) cells that express forkhead box P3 (Foxp3) are pivotal for immune tolerance. Although inflammatory mediators cause Foxp3 instability and Treg cell dysfunction, their regulatory mechanisms remain incompletely understood. Here, we show that the transfer of Treg cells deficient in the activating immunoreceptor DNAM-1 ameliorated the development of graft-versus-host disease better than did wild-type Treg cells. We found that DNAM-1 competes with T cell immunoreceptor with Ig and ITIM domains (TIGIT) in binding to their common ligand CD155 and therefore regulates TIGIT signaling to down-regulate Treg cell function without DNAM-1–mediated intracellular signaling. DNAM-1 deficiency augments TIGIT signaling; this subsequently inhibits activation of the protein kinase B–mammalian target of rapamycin complex 1 pathway, resulting in the maintenance of Foxp3 expression and Treg cell function under inflammatory conditions. These findings demonstrate that DNAM-1 regulates Treg cell function via TIGIT signaling and thus, it is a potential molecular target for augmenting Treg function in inflammatory diseases.

scholarly journals Dysbiosis of Gut Microbiota and Its Relationship with the Regulatory T Cells in Patients with Aplastic Anemia 

2021 ◽  
Author(s):  
Mengxiao Ren ◽  
Yongqin Ge ◽  
Jindan Qi ◽  
Shengli Xue ◽  
Miao Miao ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Aplastic Anemia ◽  
Treg Cell ◽  
Relative Abundance ◽  
Treg Cells ◽  
Cross Sectional Survey ◽  
Cross Sectional ◽  
Cell Counts ◽  
And Control ◽  
Healthy Participants

Abstract Background: The characteristics of gut microbiota (GM) and its relationship with the Regulatory T Cells (Treg) remains unclear in patients with aplastic anemia (AA). Methods: This study was a cross-sectional survey which included 12 AA patients consisted of 6 with severity aplastic anemia (SAA) and 6 with non-severity aplastic anemia (NSAA) and 6 healthy participants. The GM and its relationship with the Treg cells of AA patients were analyzed. Results: The results showed that the presence of compositional differences in the GM structure between the AA and Control groups. The bacterial communities were depleted of Clostridia class (e.g., Lachnospiraceae ND3007, Lachnospiraceae XPB1014, Lachnolostridium, Ruminococcaceae UCG 013 and Butyricicoccus genus) in AA group, especially in SAA group. Inversely, the relative abundance of Lactobacillus and Streptococcus genus from Bacilli class were increased significantly in patients with SAA. The relative abundance of Lachnospiraceae (r=0.663, p=0.029), Clostridiaceae 1 (r=0.619, p=0.042) and Clostridiales vadinBB60 group family (r=0.674, p=0.023) which from Clostridia class, were positively correlated with the Treg cell counts. Conclusion: We speculated that the decrease of some bacteria from Clostridia class may participate in the pathophysiological process of AA through reducing the Treg cell counts. Notwithstanding the low sample size, our data provided some clues that the treatment strategy of AA could start by adjusting the imbalance of GM, increasing Treg cell counts to improve the suppression of bone marrow hematopoiesis.

scholarly journals Transient Depletion of Foxp3+ Regulatory T Cells Selectively Promotes Aggressive β Cell Autoimmunity in Genetically Susceptible DEREG Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Deepika Watts ◽  
Marthe Janßen ◽  
Mangesh Jaykar ◽  
Francesco Palmucci ◽  
Marc Weigelt ◽  
...  
Keyword(s):  
T Cells ◽  
Treg Cell ◽  
Autoimmune Diabetes ◽  
Cell Activity ◽  
Spontaneous Diabetes ◽  
Nod Mice ◽  
Treg Cells ◽  
Β Cell ◽  
Cell Ablation ◽  
Tcr Repertoire

Type 1 diabetes (T1D) represents a hallmark of the fatal multiorgan autoimmune syndrome affecting humans with abrogated Foxp3+ regulatory T (Treg) cell function due to Foxp3 gene mutations, but whether the loss of Foxp3+ Treg cell activity is indeed sufficient to promote β cell autoimmunity requires further scrutiny. As opposed to human Treg cell deficiency, β cell autoimmunity has not been observed in non-autoimmune-prone mice with constitutive Foxp3 deficiency or after diphtheria toxin receptor (DTR)-mediated ablation of Foxp3+ Treg cells. In the spontaneous nonobese diabetic (NOD) mouse model of T1D, constitutive Foxp3 deficiency did not result in invasive insulitis and hyperglycemia, and previous studies on Foxp3+ Treg cell ablation focused on Foxp3DTR NOD mice, in which expression of a transgenic BDC2.5 T cell receptor (TCR) restricted the CD4+ TCR repertoire to a single diabetogenic specificity. Here we revisited the effect of acute Foxp3+ Treg cell ablation on β cell autoimmunity in NOD mice in the context of a polyclonal TCR repertoire. For this, we took advantage of the well-established DTR/GFP transgene of DEREG mice, which allows for specific ablation of Foxp3+ Treg cells without promoting catastrophic autoimmune diseases. We show that the transient loss of Foxp3+ Treg cells in prediabetic NOD.DEREG mice is sufficient to precipitate severe insulitis and persistent hyperglycemia within 5 days after DT administration. Importantly, DT-treated NOD.DEREG mice preserved many clinical features of spontaneous diabetes progression in the NOD model, including a prominent role of diabetogenic CD8+ T cells in terminal β cell destruction. Despite the severity of destructive β cell autoimmunity, anti-CD3 mAb therapy of DT-treated mice interfered with the progression to overt diabetes, indicating that the novel NOD.DEREG model can be exploited for preclinical studies on T1D under experimental conditions of synchronized, advanced β cell autoimmunity. Overall, our studies highlight the continuous requirement of Foxp3+ Treg cell activity for the control of genetically pre-installed autoimmune diabetes.

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