Isolation strategies of regulatory T cells for clinical trials: Phenotype, function, stability, and expansion capacity

Ukena, Sya N.; Höpting, Matthias; Velaga, Sarvari; Ivanyi, Philipp; Grosse, Jens; Baron, Udo; Ganser, Arnold; Franzke, Anke

doi:10.1016/j.exphem.2011.08.010

Cited by 47 publications

(36 citation statements)

References 33 publications

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“…While there is limited information on TSDR methylation status in human eTregs(9, 17, 28), no data are available for human iTreg protocols proposed for cell therapy. We hypothesized that TSDR methylation might be directly linked to Treg stability, i.e.…”

Section: Resultsmentioning

confidence: 99%

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Ex Vivo–Expanded but Not In Vitro–Induced Human Regulatory T Cells Are Candidates for Cell Therapy in Autoimmune Diseases Thanks to Stable Demethylation of the FOXP3 Regulatory T Cell–Specific Demethylated Region

Rossetti

Spreafico

Saidin

et al. 2015

The Journal of Immunology

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Treg1 cell therapy is a promising approach for transplant rejection and severe autoimmunity. Unfortunately, clinically meaningful Treg numbers can be obtained only upon in vitro culture. Functional stability of human expanded (e)Tregs2 and induced (i)Tregs3 has not been thoroughly addressed for all proposed protocols, hindering clinical translation. We undertook a systematic comparison of eTregs and iTregs to recommend the most suitable for clinical implementation, and then tested their effectiveness and feasibility in rheumatoid arthritis (RA)4. Regardless of the treatment, iTregs acquired suppressive function and FOXP3 expression, but lost them upon secondary restimulation in the absence of differentiation factors, which mimics in vivo reactivation. In contrast, eTregs expanded in the presence of Rapamycin (rapa)5 retained their regulatory properties and FOXP3 demethylation upon restimulation with no stabilizing agent. FOXP3 demethylation predicted Treg functional stability upon secondary TCR engagement. Rapa eTregs suppressed conventional T cell (Tconv)6 proliferation via both surface (CTLA-4) and secreted (IL-10, TGF-β and IL-35) mediators, similarly to ex vivo Tregs. Importantly, Treg expansion with rapa from RA patients produced functionally stable Tregs with yields comparable to healthy donors (HD)7. Moreover, rapa eTregs from RA patients were resistant to suppression reversal by the pro-inflammatory cytokine TNF-α, and were more efficient in suppressing synovial Tconv proliferation compared to their ex vivo counterparts, suggesting that rapa improves both Treg function and stability. In conclusion, our data indicate Treg expansion with rapa as the protocol of choice for clinical application in rheumatological settings, with assessment of FOXP3 demethylation as a necessary quality control step.

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Section: Resultsmentioning

confidence: 99%

“…For instance, TGF-β alone is sufficient to induce suppressive functions in mouse but not human T cells(14, 15). Furthermore, the proposed strategies to obtain stable eTregs in humans(8-10, 16, 17) were tested under diverse and non-comparable experimental conditions. The characterization is even more scattered for human iTregs(11).…”

Section: Introductionmentioning

confidence: 99%

Ex Vivo–Expanded but Not In Vitro–Induced Human Regulatory T Cells Are Candidates for Cell Therapy in Autoimmune Diseases Thanks to Stable Demethylation of the FOXP3 Regulatory T Cell–Specific Demethylated Region

Rossetti

Spreafico

Saidin

et al. 2015

The Journal of Immunology

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“…Cell surface marker CD127 could enrich human Treg Cells selectively for in vitro functional studies and has the potential in vivo therapy [11]. Therefore, the CD4+CD25+CD127-population has recently been suggested in preclinical studies to be most suitable for human adoptive transfer studies and represent accurately the level of human Treg cells [11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

High Level of CD4+CD25+CD127- Treg Cells in Donor Graft is Associated with a Low Risk of aGVHD after allo-HSCT for Children with Hematologic Malignancies

Zhang¹,

Zhu²,

Changying³

et al. 2013

J Cell Sci Ther

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Acute Graft-Versus-Host Disease (aGVHD) is the major problem for patient undergoing allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT). Previous study showed the significant role of CD4+CD25+ Treg cells in inhibiting aGVHD. This retrospective study of 50 children with hematological malignancies undergoing allo-HSCT investigated the influence of donor CD4+CD25+CD127-Treg cells on aGVHD. The proportion of Treg cells in graft is significantly higher in patient with grade 0-I aGVHD than in patients with grade II-IV aGVHD (3.08 ± 0.72% vs. 2.52 ± 0.86%, P=0.016). There was no significant difference on Treg cells proportion in graft between relapsed and non relapsed patients (3.20 ± 0.80% vs. 2.80 ± 0.81% P=0.549). CD4+CD25+CD127-Treg cells in donor graft can reduce the incidence of aGVHD after children received allo-HSCT without increasing the risk of relapse. Graft CD4+CD25+CD127-Treg cells level is a valuable biomarker to predict aGVHD.

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“…Mononucleated cells were isolated from fresh blood samples by Ficoll density gradient centrifugation. Peripheral blood mononuclear cells (PBMCs) were thawed, cultured overnight in RPMI, 10% FCS and 1% Pen/ Strep and subsequently analyzed by FACS using monoclonal antibodies against (Ukena et al, 2011c). Cells were gated on CD4 þ CD25 high Foxp3 þ and CD4 þ CD25 À and the resulting data were further analyzed with FlowJo software (TreeStar).…”

Section: Phenotypic Characterization Of Tregs and Cd4 þ T Effector Cellsmentioning

confidence: 99%

Reconstitution and Phenotype of Tregs in CMV Reactivating Patients Following Allogeneic Hematopoietic Stem Cell Transplantation

Velaga

Ukena

Höpting

et al. 2012

Immunological Investigations

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In experimental and clinical settings Tregs prevent graft-versus-host disease (GvHD) by inhibiting the proliferation and function of conventional T cells (Tconv). The suppressive potency of Tregs might also lead to the inhibition of protective antiviral T cell responses. As the control of CMV reactivation is important to improve the clinical outcome in allogeneic HSCT, we analyzed the Treg reconstitution in CMV reactivating patients with and without GvHD (n=47) in the first 6 months following transplantation. Most importantly, CMV reactivation does not correlate with the numerical reconstitution of CD4(+)CD25(high)CD127(-) Tregs. During CMV reactivation the proportion of Tregs within the CD4(+) T cell population decreased significantly independent of GvHD manifestation. A comprehensive FACS analysis was performed in order to characterize the phenotype of Tregs and Tconv cells in greater detail for activation, co-stimulation, proliferation, suppressive function and migratory capability. Interestingly, Tregs of patients with CMV reactivation showed a significantly higher CXCR3 expression. CD4(+) Tconv cells expressed significantly higher protein levels of the proliferation marker Ki67 correlating with a numerical increase of CD4(+) T cells. Our results indicate that Tregs are not inhibiting pathogen clearance by Tconv following HSCT, which is of high relevance for future Treg cell-based clinical trials in allogeneic HSCT.

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Isolation strategies of regulatory T cells for clinical trials: Phenotype, function, stability, and expansion capacity

Cited by 47 publications

References 33 publications

Ex Vivo–Expanded but Not In Vitro–Induced Human Regulatory T Cells Are Candidates for Cell Therapy in Autoimmune Diseases Thanks to Stable Demethylation of the FOXP3 Regulatory T Cell–Specific Demethylated Region

Ex Vivo–Expanded but Not In Vitro–Induced Human Regulatory T Cells Are Candidates for Cell Therapy in Autoimmune Diseases Thanks to Stable Demethylation of the FOXP3 Regulatory T Cell–Specific Demethylated Region

High Level of CD4+CD25+CD127- Treg Cells in Donor Graft is Associated with a Low Risk of aGVHD after allo-HSCT for Children with Hematologic Malignancies

Reconstitution and Phenotype of Tregs in CMV Reactivating Patients Following Allogeneic Hematopoietic Stem Cell Transplantation

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