Cord blood CD4+CD25+-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 750-758 ◽  
Author(s):  
Wayne R. Godfrey ◽  
Darrin J. Spoden ◽  
Ying G. Ge ◽  
Seth R. Baker ◽  
Baoling Liu ◽  
...  
Keyword(s):  
Cord Blood ◽  
Cell Lines ◽  
Cell Function ◽  
Interleukin 2 ◽  
Suppressor Cell ◽  
Treg Cells ◽  
T Regulatory Cell ◽  
Suppressor Activity ◽  
Chemokine Production ◽  
Suppressor Function

AbstractCD4+CD25+ T regulatory (Treg) cells have been shown to critically regulate self and allograft tolerance in mice. Studies of human Treg cells have been hindered by low numbers present in peripheral blood and difficult purification. We found that cord blood was a superior source for Treg-cell isolation and cell line generation compared with adult blood. Cord blood CD4+CD25+ cells were readily purified and generated cell lines that consistently exhibited potent suppressor activity, with more than 95% suppression of allogeneic mixed lymphocyte reactions (MLRs) (29 of 30 donors). Cultured Treg cells blocked cytokine accumulation in MLRs, with a less robust inhibition of chemokine production. These cell lines uniformly expressed CD25, CD62L, CCR7, CD27, and intracellular cytotoxic T-lymphocyte antigen-4 (CTLA4). FoxP3 protein, but not mRNA, was specifically expressed. Upon restimulation with anti-CD3/CD28 beads, the cultured Treg cells produced minimal cytokines (interleukin-2 [IL-2], interferon-γ [IFN-γ], and IL-10) and preferentially expressed tumor growth factor-β (TGF-β) latency associated protein. Cytokine production, however, was restored to normal levels by restimulation with phorbol myristate acetate (PMA)/ionomycin. Cord blood–derived cultured suppressor cell function was predominantly independent of IL-10 and TGF-β. These results demonstrate cord blood contains a significant number of Treg precursor cells capable of potent suppressor function after culture activation. Banked cord blood specimens may serve as a readily available source of Treg cells for immunotherapy.

In vitro–expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell–stimulated MLR cultures

Blood ◽  
2004 ◽  
Vol 104 (2) ◽  
pp. 453-461 ◽  
Author(s):  
Wayne R. Godfrey ◽  
Ying G. Ge ◽  
Darrin J. Spoden ◽  
Bruce L. Levine ◽  
Carl H. June ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Cell Lines ◽  
Cell Function ◽  
Interleukin 2 ◽  
Suppressor Cells ◽  
Suppressor Cell ◽  
Treg Cells ◽  
Cell Number ◽  
Model Systems

Abstract CD4+CD25+ T-regulatory (Treg) cells have been shown to critically regulate self- and allograft tolerance in several model systems. Studies of human Treg cells have been restricted by the small number present in peripheral blood and their naturally hypoproliferative state. To better characterize Treg suppressor cell function, we determined methods for the isolation and expansion of these cells. Stringent magnetic microbead-based purification was required for potent suppressor cell line generation. Culture stimulation with cell-sized Dynabeads coated with anti-CD3 and anti-CD28 monoclonal antibodies, CD4+ feeder cells, and interleukin 2, provided for marked expansion in cell number (100-fold), with retention and enhancement of suppressor function. The potent Treg cell lines suppressed proliferation in dendritic cell-driven allo-mixed lymphocyte reaction (MLR) cultures by more than 90%. The Treg-derived suppressor cells functioned early in allo-MLR because expression of activation antigens and accumulation of cytokines was nearly completely prevented. Importantly, cultured Treg cells also suppressed activated and matured dendritic cell-driven responses. These results demonstrate that short-term suppressor cell lines can be generated, and they can express a very potent suppressive activity. This approach will enable more detailed biologic studies of Treg cells and facilitate the evaluation of cultured Treg cells as a novel form of immunosuppressive therapy. (Blood. 2004;104:453-461)

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.

Cord Blood CD4+CD25+ Derived T Regulatory Cell Lines Express FoxP3 Protein and Manifest Potent Suppressor Function.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2124-2124 ◽  
Author(s):  
Wayne R. Godfrey ◽  
Darrin J. Spoden ◽  
Ying G. Ge ◽  
Seth R. Baker ◽  
Baoling Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Cell Lines ◽  
Cell Biology ◽  
Suppressor Cells ◽  
Treg Cells ◽  
Cell Contact ◽  
Tgf Beta ◽  
Foxp3 Mrna ◽  
Chemokine Production

Abstract CD4+CD25+ T regulatory (Treg) cells have been shown to critically regulate self and more recently allograft tolerance in mice. Studies of human Treg have been hindered by the presence of CD25-dim conventional T cells that copurify during Treg isolation. We compared adult and cord blood sources for Treg, with the hypothesis that cord blood should lack significant numbers of memory cells or environmentally reactive T cells. We found that cord blood was a superior source for Treg isolation compared to adult blood. CD4+CD25+ cells were readily purified, and generated cell lines that consistently exhibited potent suppressor activity, with >95% suppression of allogeneic MLR (29/30 donors). The cells could markedly suppress an allo-MLR at a 1/16-1/32 suppressor/responder cell ratio. The cultured Treg cells blocked cytokine accumulation in MLR, with a less robust inhibition of chemokine production. Cultured Treg uniformly expressed CD25, CD62L, CCR7, CD27, and intracellular CTLA4. Upon re-stimulation with anti-CD3/CD28 beads, the cultured Treg produced minimal cytokines (IL2, IFN-gamma, and IL10), and preferentially expressed TGF-beta latency associated protein on the cell surface, while conventional CD4+CD25− derived cell lines did not. Cytokine production however, could be largely restored by stimulation with PMA/ionomycin. Abundant FoxP3 mRNA was detected in fresh, cultured, and anti-CD3/CD28 bead restimulated CD4+CD25+ cells (approximately 2–4 fold less than cyclophillin A). Low amounts of FoxP3 mRNA were present in fresh CD25− cells, and they increased 32 fold on culture. However, FoxP3 protein, as assessed by western blot, was specifically expressed in the CD25+ derived cell lines, and was not detected in the CD25− derived cell lines. Restimulation with anti-CD3/CD28 beads led to increased expression of FoxP3 protein in CD25+ derived cell lines, but not in CD25− derived cell lines. Cord blood derived cultured suppressor cells were not cytolytic in chromium release assays, and mediated suppression of alloreactivity that was cell contact dependent and predominantly independent of IL10 and TGF-beta. Antibodies to GITR, GITR-L, OX40, OX40-L, CTLA4, and PD1 did not significantly affect suppression of the culture activated Treg cells. These results demonstrate virtually pure populations of potent suppressor cells can be cultured from cord blood, and these cell lines form an ideal model system for the evaluation of suppressor cell biology and mechanisms of action.

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 Essential role for interleukin-2 for CD4+CD25+ T regulatory cell development during the neonatal period

2005 ◽  
Vol 201 (5) ◽  
pp. 769-777 ◽  
Author(s):  
Allison L. Bayer ◽  
Aixin Yu ◽  
Dennis Adeegbe ◽  
Thomas R. Malek
Keyword(s):  
Treg Cell ◽  
Interleukin 2 ◽  
Treg Cells ◽  
Cell Development ◽  
Wild Type ◽  
T Regulatory Cell ◽  
Neonatal Mice ◽  
Early Developmental ◽  
Normal Neonates

Although many aspects of CD4+CD25+ T regulatory (Treg) cell development remain largely unknown, signaling through the IL-2R represents one feature for the production of Treg cells. Therefore, the present study was undertaken to further define early developmental steps in the production of Treg cells, including a more precise view on the role of interleukin (IL)-2 in this process. After adoptive transfer of wild-type Treg cells into neonatal IL-2Rβ−/− mice, only a small fraction of donor Treg cells selectively seeded the lymph node (LN). These donor Treg cells underwent rapid and extensive IL-2–dependent proliferation, followed by subsequent trafficking to the spleen. Thus, IL-2 is essential for Treg cell proliferation in neonatal LN. The number and distribution of Treg cells in the periphery of normal neonatal mice closely paralleled that seen for IL-2Rβ−/− mice that received Treg cells. However, for normal neonates, blockade of IL-2 decreased Treg cells in both the thymus and LN. Therefore, two steps of Treg cell development depend upon IL-2 in neonatal mice, thymus production, and subsequent expansion in the LN.

TNFR25 Expression on CD4+CD25+ T Cells: Down Modulation of Regulatory Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3165-3165
Author(s):  
Vadim Deyev ◽  
Melinda Roskos ◽  
Robert B. Levy ◽  
Eckhard R. Podack
Keyword(s):  
T Cells ◽  
Cell Function ◽  
Ex Vivo ◽  
Cell Activity ◽  
Treg Cells ◽  
Regulatory Activity ◽  
T Regulatory Cell ◽  
Tnf Receptor Family ◽  
Receptor Family

Abstract TNFR25 (“DR3”) is a member of the TNF receptor family that is expressed by activated CD4+ and CD8+ T cells. To determine if activated CD4+CD25+ T cells also expressed this TNFR family molecule, B6 CD4+CD25+ T cells were stimulated with anti-CD3/CD28 coated beads (kind gift of Dr. B. Blazar, U. Minn.) and expanded for 3–4 days. TNFR25 expression was readily detected on CD4+CD25+ FoxP3+ T cells. Since other members of the TNF receptor family (GITR, OX40, 4–1BB) are known to influence T regulatory cell function, we investigated whether TNFR25 signaling can regulate CD4+CD25+ T cell activity. TNFR25 triggering in B6-wt T regulatory CD4+CD25+ cells with the recombinant TNFR25 ligand TL1A or agonistic anti-TNFR25 antibody (4C12) resulted in reduction of their ability to suppress anti-CD3 induced ex-vivo proliferation of CD4+CD25− cells. 4C12 mediated TNFR25 signaling also reduced B6-wt Treg mediated inhibition of peptide induced proliferation of OVA-specific B6 CD8+ (OT-I) cells. To further investigate a role for TNFR25 in Treg cell regulation, TNFR25 (full length) transgenic mice were generated and bred onto the BL/6 background. CD4+CD25+ cells from these TNFR25 tg mice were found to possess diminished T regulatory activity in vitro as determined by their diminished inability to regulate proliferation by B6-wt CD4+ and OT-I CD8+ T cells. To assess their in vivo regulatory activity, B6-wt and B6 TNFR25 tg Treg cells were examined for their ability to inhibit graft vs. host disease (GVHD) following allogeneic MHC class I/II mismatched BMT. In contrast to B6-wt Treg cells, TNFR25 tg Treg cells exhibited significantly diminished ability to regulate the onset of GVHD in vivo as assessed by weight loss and clinical symptoms. Using agonistic antibody, stimulation of TNFR25 on transgenic Treg cells was also found to effectively remove the ex-vivo regulatory activity expressed by this population. To exclude any possible direct co-stimulatory effects of 4C12 antibody on the responding proliferating cells, CD4+CD25−T cells from TNFR25 dominant negative transgenic mice were employed. 4C12 mab again abolished Treg cell inhibitory activity. The effect of TNFR25 agonists on T reg cell activity in vivo is being further investigated in both mouse models of GVHD and IBD diseases. Initial observations administering 4C12 post-allogeneic BMT together with B6-wt Treg cells indicate a reduction in their ability to regulate GVHD. In total, these studies identify TNFR25 as a new potential target for augmenting CD4+ and CD8+ responses by concomitant direct co-stimulation of effecter cells and inhibition of T regulatory cell function.

scholarly journals Cloned natural suppressor cell lines derived from the spleens of neonatal mice.

1985 ◽  
Vol 162 (1) ◽  
pp. 297-310 ◽  
Author(s):  
R B Schwadron ◽  
D M Gandour ◽  
S Strober
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Suppressor Cells ◽  
Suppressor Cell ◽  
Cytolytic Activity ◽  
Target Cell Line

The establishment and characterization of cloned natural suppressor (NS) cell lines derived from the spleen of neonatal BALB/c mice are described. Cloned NS cells suppress the mixed leukocyte reaction (MLR) between normal adult responder and stimulator spleen cells with a 50-fold greater efficiency than fresh neonatal cells. Suppressive activity of both cells did not depend on the haplotype of the responder or stimulator cells, and was radioresistant. Cloned NS cells did not inhibit the uptake of [3H]thymidine by HT-2 cells proliferating in response to interleukin 2 (IL-2), nor the in vitro secretion of IL-1 by macrophages in response to lipopolysaccharide. Several experiments indicated that absorption of IL-2 could not explain the suppression of the MLR by the NS cells in the range of cell numbers tested. The results suggest that NS cells may suppress the MLR by interfering with early stages of T cell activation. The cell surface of a cloned NS cell line was examined using immunofluorescence staining, and was strongly positive for the Thy-1.2, Ly-5, and asialo-GM1 antigens. However, Lyt-1, Lyt-2, surface Ig, IE, MAC-1, and Fc and C3 receptor markers were not detected. In addition, NS cells showed no cytolytic activity against the YAC-1 target cell line. On the basis of these findings, cloned NS cells do not appear to be mature T cells, B cells, macrophages, or NK cells. The development of cloned NS cells may be useful in determining the identity and mechanism of action of nonspecific suppressor cells in the neonatal spleen, and their role in neonatal tolerance and maternal-fetal relationships.

T Regulatory Cells Obtained from Cord Blood Are Greatly Expandable and Exert Potent Suppressive Function.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4862-4862
Author(s):  
Giovanni F. Torelli ◽  
Roberta Maggio ◽  
Nadia Peragine ◽  
Barbarella Lucarelli ◽  
Maria S. De Propris ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
T Regulatory Cells ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Treg Cells ◽  
Suppressor Activity ◽  
Suppressive Function ◽  
Proliferative Effect ◽  
Expansion Capacity

Abstract CD4+CD25+ T-regulatory (Treg) cells have been shown to regulate self and allograft tolerance and to suppress allogeneic immune response. Studies in mouse models of bone marrow (BM) transplantation have shown that the infusion of culture-expanded Tregs can be effective in preventing and suppressing graft-versus-host disease (GVHD), while still allowing a graft-versus-leukemia effect (GVL). Cord blood (CB) represents the most recent source of hematopietic stem cell transplantation (HSCT) used in the unrelated transplant setting. Comparative studies have shown that unrelated CB transplant is an acceptable alternative to unrelated BM transplantation, characterized by a lower risk of transplant-related mortality due to GVHD. CB has been reported to contain Tregs, but minimal data are available on these cells. Aim of this study was to compare the suppressive functions of Tregs obtained from CB units with those obtained from the peripheral blood (PB) of patients who have undergone an allogeneic BM transplantation and of normal donors. Treg cells were purified from mononuclear cells obtained from CB units or PB using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with anti-CD3 and anti-CD28 MoAbs plus IL-2. To assess the suppressive functions of Treg cells, expanded CD4+CD25+ Tregs from CB units or PB were seeded with naïve autologous effector CD4+CD25- T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The immunophenotypic analysis of Tregs from CB units - in terms of expression of surface CD4, CD25, CD62L, cytoplasmic CTLA-4 and Foxp3 - before and after expansion, did not show any specific peculiarity. CB units (n = 6) contained a proportion of Tregs comparable to those contained in the PB of allotransplanted patients (n = 8) or in the PB of normal donors (n = 8) (mean % ± SD, CB 0.28% ± 0.41; PB patients 0.4% ± 0.4; PB donors CB 0.45% ± 0.29). On the contrary, Tregs from CB units and from the PB of allotransplanted patients showed a higher expansion capacity when compared to Tregs from the PB of normal donors (mean fold increase ± SD, CB units 11.5 ± 9.0; PB patients 9.6 ± 5.0; PB donors CB 5.2 ± 2.7). Preliminary data also show that Tregs expanded from CB units (n = 2) exert a higher suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC if compared to Tregs expanded from the PB of allotransplanted patients (n = 2) (mean fold reduction ± SD, CB 5.25 ± 3.7; PB patients 3.8 ± 3.3). These results demonstrate that Treg cells contained in CB units present an expansion capacity superior to that of Tregs from PB of normal donors; moreover, these expanded cells seem to exert a potent suppressive function of the proliferative effect in mixed lymphocyte reaction culture assays. These data offer further insights in the understanding of the biology of CB transplantation and may indicate a possible clinical use of expanded Tregs for the prevention and management of GVHD in the setting of CB transplantation.

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