Epigenetic and transcriptional analysis supports human regulatory T cell commitment at the CD4+CD8+ thymocyte stage

Vanhanen, Reetta; Leskinen, Katarzyna; Mattila, Ilkka; Saavalainen, Päivi; Arstila, T. Petteri

doi:10.1016/j.cellimm.2019.104026

Cited by 17 publications

(11 citation statements)

References 45 publications

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“…Most of them (76.68% ± 2.03%) exhibited a CD4 + CD8 + double-positive (DP) phenotype, while 12.57% ± 1.23% were CD4 + single-positive (SP) cells, and 6.98% ± 1.26% were CD8 + SP cells ( Supplementary Figure 1B ). Because it has been shown that CD4 + CD8 + DP thymic Treg cells significantly contribute to the Treg pool in the human thymus ( 30 – 32 ), we decided to directly isolate CD25 + thymocytes (2.36% ± 0.34%) without previous depletion of CD8 + cells. The average frequency of FOXP3 + cells on isolated CD25 + thymocytes was 67.08% ± 2.22% (representative plot in Supplementary Figure 1C ).…”

Section: Resultsmentioning

confidence: 99%

A Novel GMP Protocol to Produce High-Quality Treg Cells From the Pediatric Thymic Tissue to Be Employed as Cellular Therapy

et al. 2022

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Due to their suppressive capacity, the adoptive transfer of regulatory T cells (Treg) has acquired a growing interest in controlling exacerbated inflammatory responses. Limited Treg recovery and reduced quality remain the main obstacles in most current protocols where differentiated Treg are obtained from adult peripheral blood. An alternate Treg source is umbilical cord blood, a promising source of Treg cells due to the higher frequency of naïve Treg and lower frequency of memory T cells present in the fetus’ blood. However, the Treg number isolated from cord blood remains limiting. Human thymuses routinely discarded during pediatric cardiac surgeries to access the retrosternal operative field has been recently proposed as a novel source of Treg for cellular therapy. This strategy overcomes the main limitations of current Treg sources, allowing the obtention of very high numbers of undifferentiated Treg. We have developed a novel good manufacturing practice (GMP) protocol to obtain large Treg amounts, with very high purity and suppressive capacity, from the pediatric thymus (named hereafter thyTreg). The total amount of thyTreg obtained at the end of the procedure, after a short-term culture of 7 days, reach an average of 1,757 x106 (range 50 x 106 – 13,649 x 106) cells from a single thymus. The thyTreg product obtained with our protocol shows very high viability (mean 93.25%; range 83.35% – 97.97%), very high purity (mean 92.89%; range 70.10% – 98.41% of CD25+FOXP3+ cells), stability under proinflammatory conditions and a very high suppressive capacity (inhibiting in more than 75% the proliferation of activated CD4+ and CD8+ T cells in vitro at a thyTreg:responder cells ratio of 1:1). Our thyTreg product has been approved by the Spanish Drug Agency (AEMPS) to be administered as cell therapy. We are recruiting patients in the first-in-human phase I/II clinical trial worldwide that evaluates the safety, feasibility, and efficacy of autologous thyTreg administration in children undergoing heart transplantation (NCT04924491). The high quality and amount of thyTreg and the differential features of the final product obtained with our protocol allow preparing hundreds of doses from a single thymus with improved therapeutic properties, which can be cryopreserved and could open the possibility of an “off-the-shelf” allogeneic use in another individual.

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Section: Resultsmentioning

confidence: 99%

A Novel GMP Protocol to Produce High-Quality Treg Cells From the Pediatric Thymic Tissue to Be Employed as Cellular Therapy

et al. 2022

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“…It is highly interesting which epigenetic changes are exerted by the particular stimuli. It is widely known that the sustained suppressive phenotype of Tregs requires progressive demethylation in Tregs-specific signature genes (23). The majority of the studies focus on FoxP3 gene and its regulation, because FoxP3 is a master regulator that provides Tregs function and ensures phenotype maintenance.…”

Section: Discussionmentioning

confidence: 99%

Antigenic Challenge Influences Epigenetic Changes in Antigen-Specific T Regulatory Cells

et al. 2021

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BackgroundHuman regulatory T cells (Tregs) are the fundamental component of the immune system imposing immune tolerance via control of effector T cells (Teffs). Ongoing attempts to improve Tregs function have led to the creation of a protocol that produces antigen-specific Tregs, when polyclonal Tregs are stimulated with monocytes loaded with antigens specific for type 1 diabetes. Nevertheless, the efficiency of the suppression exerted by the produced Tregs depended on the antigen with the best results when insulin β chain peptide 9-23 was used. Here, we examined epigenetic modifications, which could influence these functional differences.MethodsThe analysis was pefromed in the sorted specific (SPEC, proliferating) and unspecific (UNSPEC, non-proliferating) subsets of Tregs and Teffs generated by the stimulation with monocytes loaded with either whole insulin (INS) or insulin β chain peptide 9-23 (B:9-23) or polyclonal cells stimulated with anti-CD3/anti-CD28 beads (POLY). A relative expression of crucial Tregs genes was determined by qRT-PCR. The Treg-specific demethylated region (TSDR) in FoxP3 gene methylation levels were assessed by Quantitative Methylation Specific PCR (qMSP). ELISA was used to measure genomic DNA methylation and histone H3 post-translational modifications (PTMs).ResultsTregs SPECB:9-23 was the only subset expressing all assessed genes necessary for regulatory function with the highest level of expression among all analyzed conditions. The methylation of global DNA as well as TSDR were significantly lower in Tregs SPECB:9-23 than in Tregs SPECINS. When compared to Teffs, Tregs were characterized by a relatively lower level of PTMs but it varied in respective Tregs/Teffs pairs. Importantly, whenever the difference in PTM within Tregs/Teffs pair was significant, it was always low in one subset from the pair and high in the other. It was always low in Tregs SPECINS and high in Teffs SPECINS, while it was high in Tregs UNSPECINS and low in Teffs UNSPECINS. There were no differences in Tregs/Teffs SPECB:9-23 pair and the level of modifications was low in Tregs UNSPECB:9-23 and high in Teffs UNSPECB:9-23. The regions of PTMs in which differences were significant overlapped only partially between particular Tregs/Teffs pairs.ConclusionsWhole insulin and insulin β chain peptide 9-23 affected epigenetic changes in CD4+ T cells differently, when presented by monocytes. The peptide preferably favored specific Tregs, while whole insulin activated both Tregs and Teffs.

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“…Multiple studies have helped to elucidate the dynamic role of epigenetics in Treg cell development and maintenance [ 11 , 75 , 76 , 77 ]. One report showed that in humans, Treg commitment begins at the CD4 + CD8 + DP stage, and that Treg precursor cells have a fully demethylated FOXP3 enhancer, which leads to stable FOXP3 expression [ 78 ]. Another study showed that treatment with the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) promotes Treg cell signature gene expression, including FOXP3 , CD25, GITR , and CTLA-4 , in CD4 + CD25 hi Treg precursor cells and induces development of Treg cells [ 79 ].…”

Section: Epigenetic Changes During T Cell Differentiationmentioning

confidence: 99%

New Insights into Epigenetic Regulation of T Cell Differentiation

Dutta

Venkataganesh

Love

2021

Cells

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Immature CD4− CD8− thymocytes progress through several developmental steps in the thymus, ultimately emerging as mature CD4+ (helper) or CD8+ (cytotoxic) T cells. Activation of naïve CD4+ and CD8+ T cells in the presence of specific cytokines results in the induction of transcriptional programs that result in their differentiation into effector or memory cells and in the case of CD4+ T cells, the adoption of distinct T-helper fates. Previous studies have shown that histone modification and DNA methylation play important roles in each of these events. More recently, the roles of specific epigenetic regulators in T cell differentiation have been clarified. The identification of the epigenetic modifications and modifiers that control mature T cell differentiation and specification has also provided further insights into how dysregulation of these processes can lead to cancer or autoimmune diseases. In this review, we summarize recent findings that have provided new insights into epigenetic regulation of T cell differentiation in both mice and humans.

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Epigenetic and transcriptional analysis supports human regulatory T cell commitment at the CD4+CD8+ thymocyte stage

Cited by 17 publications

References 45 publications

A Novel GMP Protocol to Produce High-Quality Treg Cells From the Pediatric Thymic Tissue to Be Employed as Cellular Therapy

A Novel GMP Protocol to Produce High-Quality Treg Cells From the Pediatric Thymic Tissue to Be Employed as Cellular Therapy

Antigenic Challenge Influences Epigenetic Changes in Antigen-Specific T Regulatory Cells

New Insights into Epigenetic Regulation of T Cell Differentiation

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