Ex vivo expansion of regulatory T cells from long-term Belatacept-treated kidney transplant patients restores their phenotype and suppressive function but not their FOXP3 TSDR demethylation status

Cortés-Hernández, Arimelek; Alvarez-Salazar, Evelyn Katy; Arteaga-Cruz, Saúl; Alberú-Gómez, Josefina; Soldevila, Gloria

doi:10.1016/j.cellimm.2020.104044

Cited by 11 publications

(12 citation statements)

References 63 publications

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“…Therefore, the increase in FOXP3 expression and suppressive function of the expanded allo Tregs did not correlate with the demethylation status of the Foxp3 locus. Such discordance has been previously reported by our group in both expanded iTregs (22) and nTregs (21). Moreover, another study showed that the hypomethylation of TSDR in Tregs from juvenile idiopathic arthritis was not associated with their FOXP3 levels (64).…”

Section: Discussionsupporting

confidence: 50%

“…On the other hand, the allo Tregs from both patients and healthy controls displayed a heterogeneous expression of Helios, a transcription factor that is required in Tregs to induce the expression of Treg-related genes (48), enhance suppressive function (49), and maintain a stable phenotype during inflammatory responses (50). Although some allo Tregs showed low expressions of Helios, we (21) and others (33) have described that expanded Tregs may retain their suppressive function despite a reduced Helios expression, suggesting that this marker may not necessarily define the functional status of Tregs.…”

Section: Discussionmentioning

confidence: 72%

“…However, the proportion of Helios + cells was slightly lower in cryopreserved expanded Tregs (Figure 8F) compared to that in recently thawed Tregs. In this context, we and others have previously reported the decrease of Helios after in vitro stimulation (21,33). After successful expansion of cryopreserved Tregs, we investigated the in vitro function of these cells.…”

Section: Allospecific Tregs Maintain Their Immunosuppressive Phenotype After Long-term Cryopreservationmentioning

confidence: 91%

“…Live proliferating CD4 + CD25 + CTV − Tregs (allospecific Tregs) (Figure S1B) were sorted using a MoFlo XDP cell sorter, collected in RPMI medium with 20% FBS, and cultured for 3 days in expansion medium supplemented with IL-2 (50 U/ml) plus 10% human AB serum. Then, the allospecific Tregs were polyclonally expanded using a modified protocol described previously (21,22). Briefly, the allospecific Tregs (2.5 × 10 4 cells/well) were stimulated with anti-CD3/anti-CD28 beads (bead/Treg ratio of 1:2) for 4 days in expansion medium with 10% human AB serum, IL-2 (300 U/ml), TGF-b (2.5 ng/ml), and rapamycin (100 nM).…”

Section: Isolation and Expansion Of Allospecific Tregsmentioning

confidence: 99%

“…However, the large-scale production of human allospecific Tregs for immunotherapy has remained a challenge due to the lack of optimized protocols to allow their purification and efficient expansion, preserving their functional and phenotypic stability (19). In addition, studies have shown that long-term expansion of Tregs results in the loss of FOXP3 and may convert to potentially inflammatory T cells (20,21). Recently, our group has applied a protocol that allows the generation of a large number of functionally stable allogeneic induced Tregs (iTregs) after long-term polyclonal expansion (22).…”

Section: Introductionmentioning

confidence: 99%

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Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines

et al. 2021

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The adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited due to their low frequency in peripheral blood and the scarce development of efficient protocols to ensure their purity, expansion, and stability. Here, we describe a new experimental protocol that allows the long-term expansion of highly purified allospecific natural Tregs (nTregs) from both healthy controls and chronic kidney disease (CKD) patients, which maintain their phenotype and suppressive function under inflammatory conditions. Firstly, we co-cultured CellTrace Violet (CTV)-labeled Tregs from CKD patients or healthy individuals with allogeneic monocyte-derived dendritic cells in the presence of interleukin 2 (IL-2) and retinoic acid. Then, proliferating CD4+CD25hiCTV− Tregs (allospecific) were sorted by fluorescence-activated cell sorting (FACS) and polyclonally expanded with anti-CD3/CD28-coated beads in the presence of transforming growth factor beta (TGF-β), IL-2, and rapamycin. After 4 weeks, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded alloTregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of inflammatory cytokines (IFN-γ, IL-4, IL-6, or TNF-α). Unexpectedly, the long-term expansion resulted in an increased methylation of the specific demethylated region of Foxp3. Interestingly, alloTregs from both normal individuals and CKD patients maintained their immunosuppressive phenotype and function after being expanded for two additional weeks under an inflammatory microenvironment. Finally, phenotypic and functional evaluation of cryopreserved alloTregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients.

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Section: Discussionsupporting

confidence: 50%

Section: Discussionmentioning

confidence: 72%

Section: Allospecific Tregs Maintain Their Immunosuppressive Phenotype After Long-term Cryopreservationmentioning

confidence: 91%

Section: Isolation and Expansion Of Allospecific Tregsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines

et al. 2021

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Regulatory T cell homeostasis: Requisite signals and implications for clinical development of biologics

Skartsis

Müller

Ferreira

2023

Clinical Immunology

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Benefit of Belatacept in Cord Blood-Derived Regulatory T Cell-Mediated Suppression of Alloimmune Response

Zhang

et al. 2021

Cell Transplant

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The role of Regulatory T cells (Tregs) in tolerance induction post-transplantation is well-established, but Tregs adoptive transfer alone without combined immunosuppressants have failed so far in achieving clinical outcomes. Here we applied a set of well-designed criteria to test the influence of commonly used immunosuppressants (belatacept, tacrolimus, and mycophenolate) on cord blood-derived Tregs (CB-Tregs). Our study shows that while none of these immunosuppressants modulated the stability and expression of homing molecules by CB-Tregs, belatacept met all other selective criteria, shown by its ability to enhance CB-Tregs-mediated in vitro suppression of the allogeneic response without affecting their viability, proliferation, mitochondrial metabolism and expression of functional markers. In contrast, treatment with tacrolimus or mycophenolate led to reduced expression of functional molecule GITR in CB-Tregs, impaired their viability, proliferation and mitochondrial metabolism. These findings indicate that belatacept could be considered as a candidate in Tregs-based clinical immunomodulation regimens to induce transplant tolerance.

show abstract

Ex vivo expansion of regulatory T cells from long-term Belatacept-treated kidney transplant patients restores their phenotype and suppressive function but not their FOXP3 TSDR demethylation status

Cited by 11 publications

References 63 publications

Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines

Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines

Regulatory T cell homeostasis: Requisite signals and implications for clinical development of biologics

Benefit of Belatacept in Cord Blood-Derived Regulatory T Cell-Mediated Suppression of Alloimmune Response

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