scholarly journals The High Affinity CXCR4 Inhibitor, BL-8040, Impairs the Infiltration, Migration, Viability, and Differentiation of Regulatory T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2804-2804
Author(s):  
Michal Abraham ◽  
Inbal Mishalian ◽  
Abi Vainstein ◽  
Liron Shemesh-Darvish ◽  
Ella Sorani ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Cancer Metastasis ◽  
Treg Cells ◽  
Pancreatic Tumors ◽  
Cxcr4 Antagonist ◽  
High Affinity ◽  
Tumor Tissues

Abstract Introduction: Regulatory T (Treg) cells, an immunosuppressive subset of CD4+ T cells characterized by the expression of the master transcription factor forkhead box protein P3 (FOXP3), are a component of the immune system with essential roles in maintaining self-tolerance. Treg cells which are indispensable for preventing autoimmunity, also suppress effective tumor immunity (Togashi et al. Nat Rev Clin Oncol 2019) Treg cells abundantly infiltrate into tumor tissues, present in the tumor microenvironment where they promote tumor development and progression by dampening antitumor immune responses. The abundantly infiltrate of Treg cells into tumor tissues is often associated with poor prognosis in cancer patients (Tanaka et al Eur. J. Immunol. 2019). The chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1/CXCL12) are critically involved in immune cell trafficking. CXCR4 overexpression, which has been identified in multiple cancer types, also supports cancer metastasis, recurrence and therapeutic resistance. More importantly, CXCR4 was shown to enhance tumor immune evasion by recruiting Treg (Santagata et al. Oncotarget. 2017) Objective: To study the effect of the high affinity CXCR4 antagonist, BL-8040, on the biology of Treg cells. To study how BL-8040 affects the ability of these cells to penetrate into the tumors, their migratory ability, their survival and also the differentiation of naive T cells towards Treg. Methods:C57BL/6 mice bearing LivMet pancreatic tumors and control mice were used for in-vivo study. In-vitro study was done with CD4 +CD25 hiFOXP3 + (Treg) cells which were isolated from fresh whole blood. CD4 +CD25 - cells were served as T conventional cells (Tconv). Differentiation of Treg cells was done from Naïve CD4+ T cells which were isolated from cord blood and stimulated with anti-CD3/CD28, TGF-b, IL-2 with or without BL-8040 for 6 days. Results: When mice bearing pancreatic cancer were treated with BL-8040, we found a significant reduction in the number of infiltrating Treg into the tumor. Following treatment with BL-8040 there was no alteration in the number of Treg in the blood neither in control mice nor in mice bearing tumors. To further understand the mechanism by which BL-8040 effected Treg cells we isolated Treg and Tconv cells and found that Treg cells express lower level of CXCR4, as compared to Tconv (Figure1). Further to, when we compared their motility, we found that Treg migration less efficiently towards CXCL12. Despite this, BL8040 more efficiently suppressed CXCL12 induced migration of Treg compared to Tconv. 100 nM of BL8040 was found to inhibits the migration of 82 % from the Treg compared to only 56.6% of Tconv cells (Figure 2). CXCR4 involves classical pathways of cell survival. In order to study the role of CXCR4 in the viability of Treg, we incubated Treg and Tconv cells in the presence of BL-8040 for 24 hr. We found that Treg cells are more sensitive to BL-8040 treatment with 19.2% of cell death compared to only 3.5% of Tconv cell death (Figure 3). Treg are one of the lineages of T helper (Th) cells which differentiated from naïve CD4 T cells. We found that BL-8040 inhibits the differentiation of naive CD4 T cells toward Treg. 10uM of BL-8040 shows a 5-fold inhibition in Treg differentiation from naïve CD4 T cells (Figure 4). Conclusions: In this work we show that the CXCR4 antagonist, BL-8040, can act as an immunomodulator by affecting the biology of regulatory T cells. BL8040 reduce the amount of infiltrating Treg into the tumors, impaired the migratory capacity of Treg toward CXCL12 and induces their cell death. Furthermore, BL-8040 was found to inhibit the differentiation of naïve CD4 T cells toward Treg. Taking all these together, BL-8040 may therefore improve the anticancer immune response, without impairing the activity of Tconv and thus can potentially serve as an effective immunomodulatory agent for cancer. Figure 1 Figure 1. Disclosures Abraham: Biokine Therapeutics: Current Employment. Vainstein: BioLineRx LTD: Current Employment. Shemesh-Darvish: BioLineRx LTD: Current Employment. Sorani: BioLineRx LTD: Current Employment. Eizenberg: Biokine Therapeutics Ltd: Current Employment. Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding.

scholarly journals Evaluation of IL-17 Producing Memory Regulatory and Effector T Cells Expressing CD26 Molecule in Patients with Psoriasis

2018 ◽  
pp. 453-463
Author(s):  
Behnaz Esmaeili ◽  
Parvin Mansouri ◽  
Alipasha Meysamie ◽  
Maryam Izad
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Memory T Cells ◽  
Treg Cells ◽  
Effector Memory ◽  
Forkhead Box P3 ◽  
Forkhead Box ◽  
Significant Difference ◽  
Cytokine Levels

Memory regulatory T cells (Tregs) has been demonstrated to produce IL-17 in Psoriasis. Forkhead box P3 (Foxp3) has been demonstrated not to be reliable marker to evaluate Treg cells. Effector CD4+T cells also express Foxp3 after activation. Human T helper-17 cells (Th-17) express high level of surface CD26, while regulatory T cells are CD26 negative or low and this phenotype is stable even after activation of Treg cells. In this study, we aimed to analyze IL-17 producing Treg cells using CD26.      Memory T cells were isolated from 10 patients with psoriasis and 10 controls. Ex vivo stimulated IL-17 producing regulatory (Forkhead Box P3 (Foxp3)+CD25+CD26-/low) and effector (Foxp3+CD25+CD26hi) memory T cells were analyzed by flow cytometry. IL-23, IL-6, TNFα, TGFβ and IL-17 cytokine levels were also evaluated. No significant difference in IL-17+memory regulatory T cells was seen between patients and controls (p=0.19). A significant decrease in the percentage of IL-17 producing CD26hi effector memory T cells was observed in patients (p=0.04). However, the percentage of these cells was not different between patients with mild or severe form of psoriasis compared to controls (p=0.13). We could not find any significant difference regarding IL-23, IL-6, TNFα, TGFβ and IL-17 cytokine levels in plasma and cell culture supernatant samples between patients and controls.  Taken together, our results showed a reduced IL-17 producing effector memory CD26hi T cells in patients with psoriasis compared to controls. However, IL-17 producing memory regulatory CD4+T cells of patients showed no significant difference from that of controls

Reciprocal Interactions Between Conventional CD4 T Cells and Regulatory T Cells Alter the Properties of Regulatory T Cells and Promote the Development of IL-17 Producing Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 709-709
Author(s):  
Lequn Li ◽  
Jin Sub Kim ◽  
Vassiliki A Boussiotis
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Culture Conditions ◽  
Th2 Cells ◽  
Reciprocal Interactions ◽  
Ifn Γ ◽  
Th1 And Th2

Abstract Abstract 709 The differentiation and functional specialization of effector T cells allows for effective immune response to diverse insults. However, tight regulation of effector T cell responses is required for effective control of infections and avoidance of autoimmunity. Naïve CD4 T cells can differentiate into IFN-γ-secreting type I (Th1) cells and IL-4-secreting type II (Th2) cells. Recently, the Th1/Th2 paradigm of T helper (Th) cells differentiation has been expanded following the discovery of a third subset of effector Th cells that produce IL-17 (Th17). Regulatory T (Treg) cells have a remarkable ability to prevent naïve T cell differentiation into Th1 and Th2 cells and to suppress immune responses driven by Th1 and Th2 effector cells. The role of Treg cells in regulating IL-17 production remains undetermined. Some studies suggest that Treg cells may promote differentiation of naïve T cells into Th17 cells in the context of inflammatory cytokine milieu. The aim of our present study was to determine the role of Treg cells and conventional CD4+ T cells (Tconv) in the differentiation of IL-17 producing cells in the absence of exogenous cytokines and insults. Naïve Tconv cells stimulated with anti-CD3 mAb in the presence of antigen presenting cells (APCs) secreted significant amounts of IFN-γ and IL-4 but no detectable levels of IL-17, whereas Treg cells were incapable of producing any of these cytokines under the same culture conditions. Production of IFN-γ and IL-4 was significantly reduced by addition of Treg cells in the cultures of Tconv cells with anti-CD3 mAb and APC. In contrast, production of IL-17 was considerably enhanced in these co-culture conditions and the level of IL-17 displayed a positive correlation with the number of Treg cells added in the culture. To evaluate whether TCR-mediated stimulation of both Treg and Tconv cells was required for IL-17 production, we used Tconv cells and Treg cells from two different TCR transgenic mouse strains in H-2b background, 2D2 (MOG35-55-specific) and OT-II (OVA323-339-specific), respectively, and co-cultured them in the presence of APCs (H-2b). Production of IL-17 was not observed when either MOG peptide or OVA peptide alone was added in the cultures. In contrast, addition of both MOG and OVA resulted in production of IL-17, suggesting that simultaneous activation of Tconv and Treg cells was essential for induction of IL-17. To determine the source of IL-17 during co-culture of Treg and Tconv cells, we purified Treg cells from C57/B6 mice and co-cultured them with Tconv cells from the B6 congenic mouse strain B6.PL, which carry the Thy1a (Thy1.1) allele and can be easily recognized by flow cytomeric analysis using a Thy1.1-specific mAb. Detailed evaluation during co-culture revealed that a significant proportion of Thy1.1- T cells (the source of Treg) gradually downregulated expression of Foxp3 while obtaining expression of IL-17. In contrast, there was no significant change in the expression of either Foxp3 or IL-17 in the Thy1.1+ population (the source of Tconv), suggesting that Treg was the main source of IL-17 when stimulated in the presence of antigen and activated Tconv cells. Several cytokines have been implicated in the induction of IL-17, in particular, TGF-β. For this reason, we investigated the potential involvement of TGF-β in this conversion process. Addition of TGF-β to Tconv cultured with APCs and anti-CD3 mAb in the absence of Treg cells resulted in upregulation of Foxp3 but not IL-17. In contrast, addition of TGF-β neutralizing antibody to Tconv cultured with APC and anti-CD3 mAb in the presence of Treg, suppressed IL-17 production. Moreover, assessment of TGF-β signaling in Tconv and Treg cells revealed a dramatically increased level of Smad3 phosphorylation in Treg compared to Tconv cells, indicating a reduced threshold of TGF-β mediated signaling in Treg cells. Taken together, our data indicate that reciprocal interactions of Treg and Tconv cells are required for conversion of Treg into IL-17 producing cells and that TGF-β-mediated signaling is required for this process. In addition, our results provide evidence that Treg may convert into proinflammatory effectors producing IL-17, under conditions that promote Tconv differentiation into Treg cells. These observations provide a new dimension to our understanding of Treg cells functions and may have important implications in therapeutic strategies using Treg cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Unique Chemotactic Response Profile and Specific Expression of Chemokine Receptors Ccr4 and Ccr8 by Cd4+Cd25+ Regulatory T Cells

2001 ◽  
Vol 194 (6) ◽  
pp. 847-854 ◽  
Author(s):  
Andrea Iellem ◽  
Margherita Mariani ◽  
Rosmarie Lang ◽  
Helios Recalde ◽  
Paola Panina-Bordignon ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Chemokine Receptors ◽  
Treg Cells ◽  
Chemotactic Response ◽  
Receptor Expression ◽  
T Cell Subsets ◽  
Response Profile

Chemokines dictate regional trafficking of functionally distinct T cell subsets. In rodents and humans, a unique subset of CD4+CD25+ cytotoxic T lymphocyte antigen (CTLA)-4+ regulatory T cells (Treg) has been proposed to control peripheral tolerance. However, the molecular basis of immune suppression and the trafficking properties of Treg cells are still unknown. Here, we determined the chemotactic response profile and chemokine receptor expression of human blood-borne CD4+CD25+ Treg cells. These Treg cells were found to vigorously respond to several inflammatory and lymphoid chemokines. Treg cells specifically express the chemokine receptors CCR4 and CCR8 and represent a major subset of circulating CD4+ T cells responding to the chemokines macrophage-derived chemokine (MDC)/CCL22, thymus and activation-regulated chemokine (TARC)/CCL17, I-309/CCL1, and to the virokine vMIP-I (ligands of CCR4 and CCR8). Blood-borne CD4+ T cells that migrate in response to CCL1 and CCL22 exhibit a reduced alloproliferative response, dependent on the increased frequency of Treg cells in the migrated population. Importantly, mature dendritic cells preferentially attract Treg cells among circulating CD4+ T cells, by secretion of CCR4 ligands CCL17 and CCL22. Overall, these results suggest that CCR4 and/or CCR8 may guide Treg cells to sites of antigen presentation in secondary lymphoid tissues and inflamed areas to attenuate T cell activation.

scholarly journals Shared TCR epitope cross-reactivity could permit dyads of Foxp3+ regulatory and IL-2-producing T cell precursors to escape thymic purge

2019 ◽  
Author(s):  
David Usharauli ◽  
Tirumalai Kamala
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Cd4 T Cells ◽  
Cross Reactivity ◽  
The Other ◽  
High Affinity ◽  
Mechanistic Basis ◽  
The One

The thymus-derived Foxp3+ regulatory T cells (Tregs) represent a unique population of CD4+ T cells responsible for maintaining dominant tolerance to auto-antigens, beneficial microbiota and potential irritants such as allergens on the one hand and efficient but balanced defense against pathogens on the other. How Tregs with high-affinity TCRs for thymically expressed epitopes survive thymic deletion or display such broad functionality is presently unclear. We recently introduced a novel framework dubbed SPIRAL (SPecific ImmunoRegulatory ALgorithm) which suggests that antigen cross-reactivity of thymic Treg repertoire could provide a mechanistic basis for its broad functionality. Here we further develop this model to propose how escape of high-affinity Tregs from thymic purge could be achieved in dyads with high-affinity natural IL-2-producing T cells (IL-2p T cells) sharing TCR epitope cross-reactivity. We believe this interpretation could reconcile contradictions related to Treg ontogeny in the thymus and their role in modulating antigen-specific immune responses.

scholarly journals Follicular regulatory T cells can access the germinal centre independently of CXCR5

2019 ◽  
Author(s):  
Ine Vanderleyden ◽  
Sigrid C. Fra-Bido ◽  
Silvia Innocentin ◽  
Hanneke Okkenhaug ◽  
Nicola Evans-Bailey ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Unique Feature ◽  
Treg Cells ◽  
Germinal Centre ◽  
Exact Role ◽  
Cell Homing ◽  
High Affinity ◽  
Reduced Frequency

SummaryThe germinal centre (GC) response is critical for generating high-affinity humoral immunity and immunological memory, which forms the basis of successful immunisation. Control of the GC response is thought to require follicular regulatory T (Tfr) cells, a subset of suppressive Foxp3+ Treg cells located within GCs. Relatively little is known about the exact role of Tfr cells within the GC, and the mechanism/s through which they exert their suppressive function. A unique feature of Tfr cells is their reported CXCR5-dependent localisation to the GC. Here we show that the lack of CXCR5 on Foxp3+ regulatory T cells resulted in a reduced frequency, but not an absence of, GC-localised Tfr cells. This demonstrates that additional, CXCR5-independent mechanisms facilitate Treg cell homing to the GC.

scholarly journals Defective IL-10 signaling in hyper-IgE syndrome results in impaired generation of tolerogenic dendritic cells and induced regulatory T cells

2011 ◽  
Vol 208 (2) ◽  
pp. 235-249 ◽  
Author(s):  
Masako Saito ◽  
Masayuki Nagasawa ◽  
Hidetoshi Takada ◽  
Toshiro Hara ◽  
Shigeru Tsuchiya ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Elevated Serum ◽  
Treg Cells ◽  
Th2 Cells ◽  
Hyper Ige Syndrome ◽  
And Function

Hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by recurrent staphylococcal infections and atopic dermatitis associated with elevated serum IgE levels. Although defective differentiation of IL-17–producing CD4+ T cells (Th17) partly accounts for the susceptibility to staphylococcal skin abscesses and pneumonia, the pathogenesis of atopic manifestations in HIES still remains an enigma. In this study, we examined the differentiation and function of Th1, Th2, regulatory T cells (Treg cells), and dendritic cells (DCs) in HIES patients carrying either STAT3 or TYK2 mutations. Although the in vitro differentiation of Th1 and Th2 cells and the number and function of Treg cells in the peripheral blood were normal in HIES patients with STAT3 mutations, primary and monocyte-derived DCs showed defective responses to IL-10 and thus failed to become tolerogenic. When treated with IL-10, patient DCs showed impaired up-regulation of inhibitory molecules on their surface, including PD-L1 and ILT-4, compared with control DCs. Moreover, IL-10–treated DCs from patients displayed impaired ability to induce the differentiation of naive CD4+ T cells to FOXP3+ induced Treg cells (iTreg cells). These results suggest that the defective generation of IL-10–induced tolerogenic DCs and iTreg cells may contribute to inflammatory changes in HIES.

scholarly journals Shared TCR epitope cross-reactivity could permit dyads of Foxp3+ regulatory and IL-2-producing T cell precursors to escape thymic purge

2019 ◽  
Author(s):  
David Usharauli ◽  
Tirumalai Kamala
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Cd4 T Cells ◽  
Cross Reactivity ◽  
The Other ◽  
High Affinity ◽  
Mechanistic Basis ◽  
The One

The thymus-derived Foxp3+ regulatory T cells (Tregs) represent a unique population of CD4+ T cells responsible for maintaining dominant tolerance to auto-antigens, beneficial microbiota and potential irritants such as allergens on the one hand and efficient but balanced defense against pathogens on the other. How Tregs with high-affinity TCRs for thymically expressed epitopes survive thymic deletion or display such broad functionality is presently unclear. We recently introduced a novel framework dubbed SPIRAL (SPecific ImmunoRegulatory ALgorithm) which suggests that antigen cross-reactivity of thymic Treg repertoire could provide a mechanistic basis for its broad functionality. Here we further develop this model to propose how escape of high-affinity Tregs from thymic purge could be achieved in dyads with high-affinity natural IL-2-producing T cells (IL-2p T cells) sharing TCR epitope cross-reactivity. We believe this interpretation could reconcile contradictions related to Treg ontogeny in the thymus and their role in modulating antigen-specific immune responses.

scholarly journals Regulatory T Cells Resist Virus Infection-Induced Apoptosis

2014 ◽  
Vol 89 (4) ◽  
pp. 2112-2120 ◽  
Author(s):  
Jenny W. Che ◽  
Anke R. M. Kraft ◽  
Liisa K. Selin ◽  
Raymond M. Welsh
Keyword(s):  
T Cells ◽  
Virus Infection ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Viral Infections ◽  
Treg Cells ◽  
Lymphocytic Choriomeningitis ◽  
Type I ◽  
Interleukin 7 ◽  
Induced Apoptosis

ABSTRACTRegulatory T (Treg) cells are important in the maintenance of self-tolerance, and the depletion of Treg cells correlates with autoimmune development. It has been shown that type I interferon (IFN) responses induced early in the infection of mice can drive memory (CD44hi) CD8 and CD4 T cells into apoptosis, and we questioned here whether the apoptosis of CD44-expressing Treg cells might be involved in the infection-associated autoimmune development. Instead, we found that Treg cells were much more resistant to apoptosis than CD44hi CD8 and CD4 T cells at days 2 to 3 after lymphocytic choriomeningitis virus infection, when type I IFN levels are high. The infection caused a downregulation of the interleukin-7 (IL-7) receptor, needed for survival of conventional T cells, while increasing on Treg cells the expression of the high-affinity IL-2 receptor, needed for STAT5-dependent survival of Treg cells. The stably maintained Treg cells early during infection may explain the relatively low incidence of autoimmune manifestations among infected patients.IMPORTANCEAutoimmune diseases are controlled in part by regulatory T cells (Treg) and are thought to sometimes be initiated by viral infections. We tested the hypothesis that Treg may die off at early stages of infection, when virus-induced factors kill other lymphocyte types. Instead, we found that Treg resisted this cell death, perhaps reducing the tendency of viral infections to cause immune dysfunction and induce autoimmunity.

Intratumoral CD4+CD25+ Regulatory T-Cell-Mediated Suppression of Infiltrating CD4+ T-Cells in B-Cell Non-Hodgkin Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3312-3312
Author(s):  
Zhi-Zhang Yang ◽  
Anne J. Novak ◽  
Mary J. Stenson ◽  
Thomas E. Witzig ◽  
Stephen M. Ansell
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Regulatory T Cells ◽  
B Cell ◽  
Tumor Immunity ◽  
Cd4 T Cells ◽  
The United States ◽  
Treg Cells ◽  
Autologous Tumor

Abstract Background: Non-Hodgkin lymphomas (NHL) are increasing in incidence and are now the fifth most common tumor diagnosed each year in the United States. Most NHLs are of B-cell origin but the tumor tissue is variably infiltrated with T-cells. Our group has shown in diffuse B-cell large cell lymphoma, that a high number of intratumoral CD4+ T-cells predicts a better overall survival. It has been shown that recently-characterized CD4+CD25+ regulatory T-cells (Treg cells) played an important role in the mediation of anti-tumor immunity. However, there is no data on the role of intratumoral Treg cells in suppression of autologous infiltrating CD4+ T-cells in B-cell NHL. Goal: To investigate the effect of intratumoral Treg cells on the proliferation of tumor-infiltrating CD4+CD25- T-cells, to determine the underlying mechanism of the T-cell suppression, and evaluate the role that malignant B-cells may play in the recruitment of Treg cells to the site of B-cell NHL. Results: We identified a subset of CD4+CD25+ T-cells over-represented in biopsy specimens of B-cell NHL (these cells comprise 17% of cells in lymphoma biopsies, compared 7% of peripheral blood mononuclear cells, 12% of cells in inflammatory tonsil and 6% of cells in tumor free lymph nodes; p-value =0.001). These CD4+CD25+ T-cells are memory-like T-cells (CD45RO+ and CD45RA−) and express high levels of CTLA-4 and Foxp3 when compared to autologous tumor-infiltrating CD4+CD25- T-cells. Importantly, these CD4+CD25+ T-cells displayed the ability to suppress the proliferation and cytokine (IFN-g and IL-4) production of tumor-infiltrating CD4+CD25- T-cells in response to PHA stimulation. Treatment with anti-B7-H1 antibody or PD-1 fusion protein enhanced the proliferation of infiltrating CD4+CD25- T-cells when co-cultured with intratumoral CD4+CD25+ T-cells. Our results suggest that interaction between B7-H1 and PD-1 accounts for about 30% of intratumoral Treg cell-mediated inhibition of autologous infiltrating CD4+CD25- T-cells in tumor sites of B-cell NHL. Lastly, we found that CCL22 secreted by lymphoma B-cells is involved in the chemotaxis and migration of intratumoral CD4+CD25+ T-cells which express chemokine receptor CCR4, but not CCR8. Conclusion: Our results suggest that tumor microenvironmental CD4+CD25+ regulatory T-cells are important regulators of tumor immunity and that these cells are recruited to the area of lymphoma involvement by the malignant B-cells.

Induction of Mouse Naive T Cells Transition into CD4+ CD2+ FoxP3+ Regulatory T Cells in a Modified Culture Medium As a Potential Armament in Graft-Versus-Host Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5426-5426
Author(s):  
Tzeon-Jye Chiou ◽  
Tan-Hwa Chu ◽  
Sin-Tak Chu ◽  
Woan-Fang Tzeng
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Activated T Cells ◽  
Allogeneic Hsct

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) has been used to treat some of hematological malignancies and inherited or acquired non-malignant disorders. Unfortunately, graft-versus-host disease (GVHD) occurred approximately 15% in transplant recipients and impacts on the outcome of allogeneic HSCT. At present, no effective modality could completely prevent the GVHD from allogeneic HSCT patients. CD4+ CD25+ FoxP3+ regulatory T cells (Tregs) have been shown to be important in maintaining immune homeostasis and preventing autoimmunity. However, 5% to 10% Tregs could be measured in human CD4+ T cells and few Tregs would convert to conventional activated T cells because of losing FoxP3 expression orn Tregs in suppression of T cell activation. It had been reported to correlate with the occurrence and severity of GVHD in some study. In order to study the potential use of CD4+ CD25+ FoxP3+ Tregs for the prevention of GVHD, we attempt to evaluate the better efficient method to increase the number of induced Treg cells (i Tregs) in donor and stabilize the FoxP3 ini Treg cells. Using mouse as a model, the splenocytes were prepared from mouse spleen. Before having biological function,i Treg cells need to stabilize the FoxP3 protein expression. Using retinoic acid (RA, 0.1-5ng/ml) as a stabilizer of the FoxP3 protein expression can keep thei Treg cells in stable. The endogenous regulatory T cells (n Treg) can inhibit T cell activation, thereby affecting T cells intoi Treg efficiency. We should remove the n Treg cells from the CD4+ T cells. Therefore, CD4+ T cells were isolated by negative selection, and then using the n Treg removing kit, we harvested the CD4+ CD62L+ naïve T cells fori Treg cell induction. For this purpose, naïve CD4+ cells were harvested, and then activated with anti-CD3/CD28 Dynabeads in the presence of IL-2, TGF-β1 and retinoic acid (RA) containing RPMI1640 medium. During the Tregs induction, the activated T cells were performed under low nutrient supplement (5% FBS) for three days then refreshed the cells into the full nutrient supplement (10% FBS) for another four days. The harvested cells were analyzed by flow cytometry method with fluorescence-conjugated CD-antibodies, including CD4, CD25, CD127, CD62L and FoxP3. Currently, the removal of n Treg cells could improve the efficiency of i Treg cell formation from 15% to 70-80% under this modified culture method (Fig.1). Further improvement of human peripheral blood regulatory T cell generation efficiency is our ongoing target. Our study showed that the combination of IL-2, TGF-β1 and RA in 3-day-nutrient-deprived medium could convert naïve CD4+ CD62L+ T cells to CD4+ CD25+ FoxP3+i Treg cells and stabilize FoxP3 expression in thei Treg cells efficiently. Further, we will develop thei Treg suppression assay to clarify the biological function ofi Tregs in vitro. GVHD mouse model will be established by using allogeneic HSCT to verify the function of i Tregs in vivo, too. Disclosures No relevant conflicts of interest to declare.

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