The Significance of mTOR Inhibitor, Everolimus in TGF-β-Induced Regulatory T cells from Cord Blood

Abstract Abstract 2180 Background: Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Cord blood (CB) is rich in naïve T cells and is a promising source of inducible Tregs (iTregs), since it was reported that stable iTregs may be derived exclusively from naïve T cells. However, the standard method for iTregs has not yet been established. Here we studied the impact of mTOR inhibitors, rapamycin (Rap) and everolimus (Eve), on ex vivo expansion of iTregs from CB-CD4+ T cells. Methods: CB-CD4+ T cell were isolated using anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured in a flask coated with anti-CD3/CD28 MAbs and supplemented with IL-2 and TGF-β in the presence or absence of Rap or Eve. After two weeks of culture, the total number of CD4+ T cells was calculated, and the incidence of CD25+Foxp3+ cell population among those was estimated by FACS. Results and Discussions: Both Rap and Eve significantly increased the incidence of CD25+Foxp3+ cell population in CD4+ T cells. However, Rap apparently inhibited their growth and did not increase the absolute number of CD25+Foxp3+ cells in comparison to the control. On the other hand, Eve contributed to efficient expansion of iTregs at the concentration between 1 and 50ng/ml without no significant inhibition of their growth. Expansion of CD4+ T cells with TGF-β and Eve yielded 71.5 ±23.5% purity of CD25+Foxp3+ cells which also expressed CTLA-4 as well as the memory phenotype, while the purity obtained with TGF-β only was 47.4±30.0% and that without TGF-β/Eve was 7.3±4.5%. Thus, an average of 2.95±2.8 x107 iTregs were obtained from the initial input of 5×104 CD4+ T cells. The resulting iTregs with TGF-β, TGF-β/Rap and TGF-β/Eve inhibited the proliferation of CFSE-labeled T cells stimulated with allogeneic dendritic cells. The precise mechanism for Foxp3 induction by mTOR inhibitors still remains to be elucidated. Furthermore, we found that expression of CD26 (DPP-IV) was significantly down-regulated in CD4+ T cells expanded with TGF-β and profoundly with TGF-β/Eve, while CD127 was negative after culture in all the conditions. Mean fluorescence intensity of CD26 indicated 67.5 in CD4+ T cells without TGF-β, 1.58 with TGF-β, 0.18 with TGF-β/Rap and 0.12 with TGF-β/Eve, respectively. Accordingly, CD26 negativity may be an indicator of iTregs together with Foxp3. Conclusion: mTOR inhibitor, Eve, is an efficient co-inducer of iTregs and applicable to ex vivo expansion of iTregs in a clinical setting. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Unique Role of mTOR Inhibitor, Everolimus in Inducible Regulatory T Cells

Blood ◽

10.1182/blood.v120.21.4349.4349 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4349-4349

Author(s):

Tokiko Nagamura-Inoue ◽

Yuki Yamamoto ◽

Seiichiro Kobayashi ◽

Kazuo Ogami ◽

Kiyoko Izawa ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cd4 T Cells ◽

Mtor Inhibitor ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Foxp3 Expression ◽

Induction Rate ◽

The Impact

Abstract Abstract 4349 Background: Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Unbalance between Tregs and effector T cells is involved in graft-versus-host disease (GvHD) and other autoimmune disorders. Adoptive use of inducible Tregs (iTregs) is a candidate immunosuppressive therapy, and major concern has been focused on sustained expression of Foxp3 in iTregs. We previously reported that iTregs can be efficiently expanded from cord blood (CB)-derived CD4+ T cells in the presence of IL2, TGFb and a mTOR inhibitor, Everolimus (Eve). However, the effect of Eve on in vitro induction of iTreg remains to be elucidated. Here we studied the impact of Eve on CB-CD4+ T cells. Methods: CD4+ T cells were prepared from CB with a purity of >95% and put into the flask coated with anti-CD3/CD28 MAb. For Treg induction, these cultures were supplemented with IL2, IL-2/TGFb, IL2/TGFb/Eve, or IL2/Eve and kept for two weeks. The resulting CD4+ T cells including variable proportion of iTregs were subjected to mixed lymphocyte reaction (MLR) along with CFSE-labeled autologous responder T cells and allogeneic dendritic cells (DCs) as stimulator. Results: The basal proportion of CD25+Foxp3+ cells in CB-CD4+ T cells was 0.60 ± 0.59%. After two weeks, the induction rate of CD25+Foxp3+CD4+ T cells was higher in the culture with IL2/TGFb/Eve than that with IL2/TGFb, but Eve itself could not significantly induce iTregs in the absence of TGFb (Figure1.). The iTreg ratio (CD25+Foxp3+ cells/total CD4+ T cells) was 79.3 ± 17.4% in the culture with IL2/TGFb/Eve, 53.1 ± 23.8% with IL2/TGFb, 35.5±18.6% with IL2/Eve and 22.7 ± 18.6% with IL2, respectively. There was no significant relationship between the dose of Eve and the iTreg ratio, but the highest ratio and induction rate of iTregs were observed at 10nM Eve. Thus, an average of 2.95 ± 2.8 ×107 iTregs was obtained from 5 ×104 CB-CD4+ T cells after two weeks of culture with IL2/TGFb/Eve. The iTreg-rich population cultured with IL2/TGFb/Eve and IL2/TGFb, but not IL2 alone, efficiently inhibited MLR triggered by allogeneic DCs (Figure 2.). These iTregs were also active in MLR using allogeneic responder T cells. Interestingly, IL2/Eve-treated CB-CD4+ T cells also inhibited MLR, irrespective of the low or moderate iTreg ratio. The inhibitory effect on MLR was much less observed by another mTOR inhibitor, rapamycin, rather than Eve (Figure2). Expression of CD26 on CD4+ T cells was inversely correlated to Foxp3 expression and significantly down-regulated by TGFb with or without Eve. Discussion: Treatment of CB-CD4+ T cells with IL2/TGFb/Eve results in the efficient ex vivo expansion of functional iTregs. Eve enhanced TGFb induction of Foxp3 expression, but did not induce Foxp3 expression by itself. mTOR is a complex of TORC1 and 2. Rapamycin is reported to inhibit TORC1, while Eve inhibits both of them, at general dose. In recent report, mTOR-deficient T cells (TORC1/2, not TORC1 alone) displayed normal activation and IL-2 production upon initial stimulation, but failed to differentiate into effecter T cells, instead, differentiated into Tregs. Although the direct mechanism to inhibit MLR by CB-CD4+ T cells treated with Eve remained to be elucidated, these results suggested the aberrant pathways of immunological inhibition. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CD26 Is Down-Regulated In Inducible Regulatory T Cells

Blood ◽

10.1182/blood.v116.21.4706.4706 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4706-4706

Author(s):

Tokiko Nagamura-Inoue ◽

Kazuo Ogami ◽

Seiichiro Kobayashi ◽

Naoyuki Takahashi ◽

Kazuaki Yokoyama ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cd4 T Cells ◽

Magnetic Beads ◽

Mtor Inhibitor ◽

Cultured Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Surface Markers ◽

Activated T Cells

Abstract Abstract 4706 Objectives: CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in allograft- and self-tolerance and thus have the potential for therapeutic application in immunological and allergic disorders. And several attempts of ex vivo expansion of Treg to enable the adaptive immunoregulatory therapy in humans. However the quality of the final products is hardly assessed because of no unique surface markers of the Treg to be distinguished from activated T cells. CD127 is useful surface markers for naïve Treg, but after induction culture with anti-CD28 and anti-CD3-MAbs, CD127 becomes negative and no more helpful to distinguish the iTreg in cultured T cells. Here we studied gene expressions and the influence factors in inducible Treg (iTreg) compared to those in activated T cells and report. Methods: CD4+ T cells from cord blood cells (CB) were isolated by anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured using a plastic plate coated with anti-CD28 and anti-CD3-MAbs in the medium containing recombinant human (rh) IL-2 and rhTGF-β. After 2-weeks of culture, Treg-rich populations were obtained in the culture with rhTGF-β. The gene expression array was performed between the cells cultured with and without TGF-β. To study the influence of mTOR inhibitor on Treg expansion, we added rapamycin to the culture system with TGF-β. CFSE-labeled responder T cells and autologous or allogeneic dendritic cells (DC) with or without expanded Treg-rich populations. Results: Mean fold induction of CD4+CD25+Foxp3+Treg/Treg % in the cultured cells derived from CB naïve CD4+ T cells was 7,403/7% of the cultured cells without TGF-β, 31,795/32% with TGF-β and 6,128/49% with TGF-β and rapamycin, respectively. Dipeptidyl peptidase IV (DPP-IV:CD26) is significantly down-regulated from high to intermediate (inter)/negative in the cultured cells with TGF-β compared to those without TGF-β, while CD127 indicated all negatice in the cultured cells. Mean of median intensity of CD26 indicated 64.2 in the cultured cells without TGF-β, 0.44 with TGF-β and 0.23 with TGF-β and rapamycin, respectively, while CD45RO became positive for all cultured cells from CD45RA+naive phenotype. Rapamycin augmented Foxp3 expression of the cultured cells with TGF-β, although expansion rate itself of iTreg was reduced. The resulting Treg-rich populations inhibited the proliferative response of CFSE-labeled CD4+ and CD8+ T cells to allogeneic DC. Discussions and conclusion: CD45RO+CD45RA-CD26high T cells is defined as memory phenotype, while iTreg cultured under the presence of TGF-β with or without Rapamycin indicated the unique population, CD45RO+CD26inter to negative. The mechanism of the down-regulation of CD26 remained to be solved. Our results indicated that CD26 may be useful marker to assess the final products of iTreg to distinguish from the activated T cells and mTOR inhibitor such as rapamycin will be helpful reagent to induce Treg and applicable to clinical use. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Suppressive Characteristics of Umbilical Cord Blood–derived Regulatory T Cells After Ex Vivo Expansion on Autologous and Allogeneic T Effectors and Various Lymphoblastic Cells

Journal of Immunotherapy ◽

10.1097/cji.0000000000000262 ◽

2019 ◽

Vol 42 (4) ◽

pp. 110-118 ◽

Cited By ~ 1

Author(s):

Thitinee Vanichapol ◽

Nutkridta Pongsakul ◽

Supanart Srisala ◽

Nopporn Apiwattanakul ◽

Somchai Chutipongtanate ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Lymphoblastic Cells

Download Full-text

Phenotypic and Epigenetic Adaptations of Cord Blood CD4+ T Cells to Maternal Obesity

Frontiers in Immunology ◽

10.3389/fimmu.2021.617592 ◽

2021 ◽

Vol 12 ◽

Author(s):

Suhas Sureshchandra ◽

Norma Mendoza ◽

Allen Jankeel ◽

Randall M. Wilson ◽

Nicole E. Marshall ◽

...

Keyword(s):

T Cells ◽

Cord Blood ◽

Single Cell ◽

Rna Sequencing ◽

Cd4 T Cells ◽

Maternal Obesity ◽

Ex Vivo ◽

Microbial Infection ◽

Single Cell Rna Sequencing ◽

The Impact

Pregravid obesity has been shown to disrupt the development of the offspring’s immune system and increase susceptibility to infection. While the mechanisms underlying the impact of maternal obesity on fetal myeloid cells are emerging, the consequences for T cells remain poorly defined. In this study, we collected umbilical cord blood samples from infants born to lean mothers and mothers with obesity and profiled CD4 T cells using flow cytometry and single cell RNA sequencing at resting and following ex vivo polyclonal stimulation. We report that maternal obesity is associated with higher frequencies of memory CD4 T cells suggestive of in vivo activation. Moreover, single cell RNA sequencing revealed expansion of an activated subset of memory T cells with maternal obesity. However, ex vivo stimulation of purified CD4 T cells resulted in poor cytokine responses, suggesting functional defects. These phenotypic and functional aberrations correlated with methylation and chromatin accessibility changes in loci associated with lymphocyte activation and T cell receptor signaling, suggesting a possible link between maternal obesogenic environment and fetal immune reprogramming. These observations offer a potential explanation for the increased susceptibility to microbial infection in babies born to mothers with obesity.

Download Full-text

Large Scale Selective Ex Vivo Expansion of CD4+CD25+FOXP3+ Regulatory T Cells from Peripheral Blood.

Blood ◽

10.1182/blood.v112.11.2344.2344 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2344-2344

Author(s):

Tokiko Nagamura-Inoue ◽

Kazuo Ogami ◽

Kazuaki Yokoyama ◽

Kiyoko Izawa ◽

Seiichiro Kobayashi ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Large Scale ◽

Magnetic Beads ◽

Ex Vivo ◽

Chronic Gvhd ◽

Ex Vivo Expansion ◽

Healthy Donors

Abstract CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in allograft- and self-tolerance and thus have the potential for therapeutic application in immunological and allergic disorders. However, the frequencies of Treg in peripheral blood are very low. Here we attempted the ex vivo expansion of Treg to enable the adaptive immunoregulatory therapy in humans. CD4+ T cells from peripheral blood of healthy donors or patients with chronic graft-versus-host disease (GVHD) were isolated by anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured using a plastic plate coated with anti-CD28 and anti-CD3-MAbs in the medium containing recombinant human (rh) IL-2 and rhTGF-b. After one week of culture, expanding cells were once detached from the plate and subjected to the fresh medium including rhIL-2 and rhTGF-b but not MAbs. After 2-weeks of culture, phenotypic and functional analyses were performed. Mixed lymphocyte reaction was done using CFSE-labeled responder T cells and autologous or allogeneic dendritic cells (DC) with or without expanded Treg-rich populations. Xenogeneic -GVHD in NOD-Scid mice was induced by the injection of human T cells expressing luciferace transgene, followed by in vivo bioluminescence imaging (BLI) analysis using a CCD camera. Our expansion procedure with TGF-b yielded 45-83% purity of Foxp3+CD25+CD4+Treg co-expressing CTLA-4, CD54 and GITR, while 8-42% purity without TGF-b(p<0.001). These cell populations also displayed CD45RO+CD45RA−CD26high+ memory phenotype. An average expansion rate of Treg was 62,200 fold (25,500–97,900) in healthy donors during the culture periods (n=5). Thus, we obtained an average of 4.7x108 Treg from the initial number of 5x105 CD4+ T cells in peripheral blood. Additionally, from peripheral CD4+ T cells in patients with chronic GVHD, Treg could be expanded equivalently to healthy donors. The resulting Treg-rich populations inhibited the proliferative response of CFSE-labeled T cells to autologous and allogeneic DC (Figure 1). The ex vivo expanded Treg-rich populations had the inhibitory effect on xeno-reactive T cells expressing luciferase transgene in a xenogeneic GVHD model (Figure 2). Our procedure has allowed efficient ex vivo expansion of Treg-rich populations from a small volume of peripheral blood, and will be applicable to clinical use. Figure 1. MLR inhibited by expanded Treg Figure 1. MLR inhibited by expanded Treg Figure 2. Xenogeneic GVHD diminished by expanded Treg. Figure 2. Xenogeneic GVHD diminished by expanded Treg.

Download Full-text

Significant Ex-Vivo Expansion of Cord Blood (CB) Natural Killer (NK) Cells and Concomitant Decrease in CB T-Cells by Genetically Reengineered K562 Cells (K562-mbIL15-41BBL)

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2008.12.135 ◽

2009 ◽

Vol 15 (2) ◽

pp. 44-45

Author(s):

J. Hochberg ◽

B. Mar ◽

J. Ayello ◽

N. Day ◽

C. van de Ven ◽

...

Keyword(s):

T Cells ◽

Cord Blood ◽

Nk Cells ◽

Natural Killer ◽

Ex Vivo ◽

K562 Cells ◽

Ex Vivo Expansion ◽

Concomitant Decrease

Download Full-text

Large Ex Vivo Expansion of Human Umbilical Cord Blood CD4+ and CD8+ T Cells

Journal of Hematotherapy ◽

10.1089/106161299320406 ◽

1999 ◽

Vol 8 (2) ◽

pp. 129-139 ◽

Cited By ~ 11

Author(s):

Danna Skea ◽

Nan-Hua Chang ◽

Robin Hedge ◽

Barbara Dabek ◽

Truman Wong ◽

...

Keyword(s):

T Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Cd8 T Cells ◽

Umbilical Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord

Download Full-text

Comparing the Effects of the mTOR Inhibitors Azithromycin and Rapamycin on In Vitro Expanded Regulatory T Cells

Cell Transplantation ◽

10.1177/0963689719872488 ◽

2019 ◽

Vol 28 (12) ◽

pp. 1603-1613 ◽

Cited By ~ 1

Author(s):

Marcus Bergström ◽

Malin Müller ◽

Marie Karlsson ◽

Hanne Scholz ◽

Nils Tore Vethe ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Mtor Inhibitors ◽

Allogeneic Transplantation ◽

Suppressive Effect ◽

And Function ◽

Treg Phenotype ◽

Suppression Assay

Adoptive transfer of autologous polyclonal regulatory T cells (Tregs) is a promising option for reducing graft rejection in allogeneic transplantation. To gain therapeutic levels of Tregs there is a need to expand obtained cells ex vivo, usually in the presence of the mTOR inhibitor Rapamycin due to its ability to suppress proliferation of non-Treg T cells, thus promoting a purer Treg yield. Azithromycin is a bacteriostatic macrolide with mTOR inhibitory activity that has been shown to exert immunomodulatory effects on several types of immune cells. In this study we investigated the effects of Azithromycin, compared with Rapamycin, on Treg phenotype, growth, and function when expanding bulk, naïve, and memory Tregs. Furthermore, the intracellular concentration of Rapamycin in CD4+ T cells as well as in the culture medium was measured for up to 48 h after supplemented. Treg phenotype was assessed by flow cytometry and Treg function was measured as inhibition of responder T-cell expansion in a suppression assay. The concentration of Rapamycin was quantified with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Azithromycin and Rapamycin both promoted a FoxP3-positive Treg phenotype in bulk Tregs, while Rapamycin also increased FoxP3 and FoxP3+Helios positivity in naïve and memory Tregs. Furthermore, Rapamycin inhibited the expansion of naïve Tregs, but also increased their suppressive effect. Rapamycin was quickly degraded in 37°C medium, yet was retained intracellularly. While both compounds may benefit expansion of FoxP3+ Tregs in vitro, further studies elucidating the effects of Azithromycin treatment on Tregs are needed to determine its potential use.

Download Full-text