Treg cells: Collection, processing, storage and clinical use

Pathology – Research and Practice 207 (2011) 209–215

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Pathology – Research and Practice journal homepage: www.elsevier.de/prp

Review

Treg cells: Collection, processing, storage and clinical use Nicola Daniele a , Maria Cristina Scerpa b , Fabiola Landi a , Maurizio Caniglia c , Massimino Jan Miele b , Franco Locatelli c,d , Giancarlo Isacchi a , Francesco Zinno a,∗ a

Immunohematology Section, Tor Vergata University and SIMT, IRCCS Bambino Gesù Pediatric Hospital, Rome, Italy SIMT, IRCCS Bambino Gesù Pediatric Hospital, Rome, Italy Pediatric Hematology and Oncology, IRCCS Bambino Gesù Pediatric Hospital, Rome, Italy d University of Pavia, Italy b c

a r t i c l e

i n f o

Article history: Received 29 November 2010 Received in revised form 17 January 2011 Accepted 2 February 2011 Keywords: Treg cells collection Treg cells storage FOXP3 GvHD Stem cells transplant

a b s t r a c t T regulatory cells are fundamental in the maintenance of immune homeostasis and self-tolerance. Experimental models suggest the existence of two functional types of Treg cells designated naturally occurring and induced. Interest in Treg cells increased with evidence from experimental mouse and human models demonstrating that the immunosuppressive potential of these cells can be utilized in the treatment of various pathological conditions. The existence of a subpopulation of suppressive T cells was the subject of significant controversy among immunologists for many years. T regulatory cells limit immune activation through a variety of direct and indirect interactions, many of which are yet to be determined. Fully understanding Treg cells biology will lead us to harnessing the capacity of these cells in order to develop strategies to prevent autoimmune disorders and tolerance to transplantation. Efficient isolation, expansion and cryopreservation strategies that comply with Good Manufacturing Practice (GMP) guidelines are prerequisites for the clinical application of human CD4+ CD25+ CD127low FOXP3+ regulatory T cells. © 2011 Elsevier GmbH. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . “Natural” Treg cells and “inducible” Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The “old” FOXP3 marker and the “new” CD127 marker for Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical application of the Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The ability of the Treg cells to prevent Graft-versus-host disease (GvHD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction between Treg cells and NK in transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory T cells in tumor immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory T cells in infection diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treg cells and their rule in autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GMP Isolation, Treg cells expansion and their cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction The immune system must be capable of mounting an effective immune response against foreign/microbial agents, but must not

∗ Corresponding author at: SIMT Ospedale Pediatrico Bambino Gesù, Piazza S. Onofrio, 4, 00165 Rome, Italy. Tel.: +39 0668592892; fax: +39 0668592167. E-mail addresses: [email protected], [email protected] (F. Zinno). 0344-0338/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2011.02.003

be self-reactive. Mechanisms at both central and peripheral levels exist to maintain tolerance against self-antigens [1]. Centrally, self-reactive clones are eradicated during thymocyte differentiation, and peripherally, various mechanisms exist to control the self-reactive clones that have escaped central tolerance. Among these, CD4+ CD25+ regulatory T cells (Treg ) play a critical role in the maintenance of peripheral immunological tolerance by limiting autoimmune process and inflammatory responses [1,2].

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Fig. 1. Title: Timeline This figure shows the major steps that have led to the discovery of Treg cells in human models [69–76,8].

Human Treg cells were first characterized as CD4+ CD25+ T cells in 2001 by several groups based on the finding in 1995 that mouse Treg cells constitutively express CD25 [3–6]. Similarly, in 2003, FOXP3 was described as a master control gene for mouse Treg cell development and function, and subsequent studies have confirmed FOXP3 as a specific marker for human Treg cells (Fig. 1) [7–9]. “Natural” Treg cells and “inducible” Treg cells To date, two major types of CD4 Treg cells have been identified: “natural” Treg cells (nTreg ) that constitutively express CD25 and FOXP3, and so-called adaptative or “inducible” Treg cells (iTreg ). Natural Treg cells originate from the thymus as CD4+ cells expressing high levels of CD25 together with the transcription factor (and lineage marker) FOXP3. Natural Treg cells represent approximately 5–10% of the total CD4+ T cell population, and can be first seen at the single-positive stage of T lymphocyte development [10]. They are positively selected thymocytes with a relatively high avidity for self-antigens. The signal to develop into Treg cells is thought to come from interactions between the T cell receptor and the complex of MHC II with self peptide expressed on the thymic stroma [11]. Natural Treg cells are essentially cytokineindependent. Adaptative or inducible Treg cells originate from the thymus as single-positive CD4 cells. They differentiate into CD25 and FOXP3 expressing Treg cells (iTreg cells) following adequate antigenic stimulation in the presence of cognate antigen and specialized immunoregulatory cytokines such as TGF-␤, IL-10, and IL-4 [12]. The “old” FOXP3 marker and the “new” CD127 marker for Treg cells Human FOXP3 is a 47 kDa protein member of the forkhead/wingedhelix family of transcription factors. The members of

the fox family are both transcriptional repressors and activators, and have a forkhead (FKH) domain, which is critical for DNA binding and nuclear localization [13]. FOXP3 acts as a transcriptional repressor for promoters of key cytokine genes such as IL-2 and GM-CSF [14,15]. Recent studies suggest that FOXP3 physically and functionally interacts with transcription factors that play key roles in the expression of multiple cytokine genes such as nuclear factor of activated T cells (NFAT), acute myeloid leukemia 1/Runt-related transcription factor 1 (AML1/Runx1), and possibly nuclear factor␬B (NF-␬B) [16,17]. In general, transcription factors have protein domains that allow them to interact simultaneously with DNA, with other protein cofactors, and potentially with the basal transcription machinery. In the case of FOXP3, the proline-rich N-terminus is a transrepression domain required for suppression of NFAT-mediated gene transcription, a central domain that contains a C2H2 zinc finger, a leucine zipper involved in protein/protein interactions, and a C-terminal region that contains the forkhead DNA-binding domain and nuclear targeting sequences (Fig. 2) [18]. Mutations that affect any of these key functional domains may alter or abrogate the ability of the transcription factor to regulate gene expression. With regard to the other marker, Liu and colleagues demonstrated that CD127 expression is down-modulated on Treg cells, inversely correlating with the expression of Treg marker FoxP3. The study of Liu W. and coll. argues that FoxP3 interacts with the CD127 promoter and might contribute to reduced expression of CD127 in Treg cells [19]. CD127 is part of the heterodimeric IL-7 receptor that is composed of CD127 and common ␥ chain, which is shared by other cytokine receptors (IL-2R, IL-4R, IL-9R, IL-15R, and IL-21R). CD127 is expressed on thymocytes, T- and B-cell progenitors, mature T cells, monocytes, and some other lymphoid and myeloid cells. Several studies have shown that IL-7R plays an important role in the proliferation and differentiation of mature T cells, and in vitro

Fig. 2. Title: structural domains of FOXP3 Pro: proline rich region; ZnF: zinc finger domain; LZ: leucine zipper region; FHD: forkhead box; NFAT: nuclear factor of activated T-cells. NFAT help Fox-P3 to repress IL-2, activate CTLA-4, activate CD25 and suppress normal T-cells when expressed in them. AML/Runx-1 organizes and facilitates assembly of transcriptional activation complexes. FOXP3 acetylated by TIP60 enhances binding of FOXP3 to the IL-2 promoter.

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experiments show that the expression of CD127 is down-regulated following T cell activation [20,21].

Clinical application of the Treg cells The ability of the Treg cells to prevent Graft-versus-host disease (GvHD) Despite decades of improvement in care and recipient quality of life, allogeneic stem cell transplantation continues to produce significant, long-term complications. Specifically, the process of allogeneic HSCT can initiate a cascade of autoimmune events known as Graft-versus-host disease (GvHD). GvHD is a major cause of morbidity and mortality after allogeneic HSCT and donor lymphocyte infusion [22]. The main risk factor for development of GvHD is human leukocyte antigen (HLA) disparity of donor T cells with reactivity against recipient histocompatibility antigens [22]. The number of mismatched HLA class I antigens correlates with instance of GvHD and poor engraftment kinetics [23]. Clinically, GvHD is divided into acute and chronic forms. The acute or fulminant form of the disease is normally observed within the first 100 days post-transplant, and is a major challenge to transplants owing to associated morbidity and mortality [24]. The chronic form of GvHD normally occurs after 100 days posttransplants. The appearance of moderate to severe cases of chronic GvHD adversely influences long-term survival [25]. Intravenously administered corticosteroids, such as prednisone, are the standard of care in acute GvHD and chronic GvHD [26]. Nonresponders are offered second-line therapy, with different combinations of immunosuppressive agents. The outcome for such patients is poor, the quality of life unsatisfactory, and overall survival is usually low (1 year 30% in the largest trials) [27,28]. To overcome these limitations, several novel strategies are currently under investigation, including the use of Treg cells in HSC transplantation. Although initial studies trying to correlate Treg cells numbers and the risk of GvHD brought controversial results, several authors have now reported a lower cumulative incidence of GvHD in patients receiving a PBSCT containing higher Treg cells numbers [28–32]. CD4+ CD25+ Treg cells are actively involved in immune-mediate mechanisms after allogeneic stem cell transplantation. This was demonstrated in studies where the co-transplantation of large numbers of donor-type CD4+ CD25+ Treg cells effectively suppressed GvHD induced by conventional (CD25−) alloreactive T cells [33,34]. The protective effect of adoptively transferred Treg cells in the Graft versus Host Disease perhaps is the joined result of direct suppression of effector T cells in lymphoid organs as well as down-modulation of APC function with respect to presentation of alloantigens [35]. To date, Treg cells have been adoptively transferred into hematopoietic stem cell transplant (HSCT) recipients in Italy, Germany, and USA (Table 1) [36]. In one of such trials, the group led by M. Martelli reported that transfer of freshly isolated CD4+ CD25+ T cells (consisting of CD25high 25.6% ± 11.2 and FOXP3+ cells 64% ± 1, mean ± SD) 3 days prior to transplantation of haploidentical CD34+ stem cells favors immune reconstitution (Blood, ASH Annual Meeting Abstracts, Nov 2009; 114: 4). No GvHD was observed in 17 of 20 valuable patients. Two patients developed grade I cutaneous self-limited untreated GvHD and one developed grade III GvHD (this patient has received the lowest Treg cells doses). This study suggest that in the setting of haploidentical stem cell transplanta-

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tion, the infusion of freshly purified Treg cells prior to transplant provides long-term protection from GvHD and robust immune reconstitution [37]. In another clinical trial, led by Matthias Edinger from Regensburg, Germany, freshly isolated bead-selected donor Treg cells were infused into HSCT recipients. So far, nine patients have been included in the study with no side effects [36]. Interaction between Treg cells and NK in transplantation In an original article, Yu J. and colleagues studied a Treg subpopulation developed in the thymus and spleen of AlloNKs + Chemo-treated mice on day 23 after Haplo-HSCT. These Treg cells showed potent inhibitory effects on the conventional haploidentical mixed lymphocyte reaction in vitro [38]. Furthermore, it should be noted that donor conventional DCs are the major population in presenting alloantigens after BM transplantation, and maintainable immaturity of CD11chi DCs promotes induction of peripheral tolerance after Allo-HSCT by impairing differentiation of Th1 cells and increasing Treg cells in recipients (Markey et al.) [39]. According to these data, Yu J. and colleagues observed that the donor-derived immature DCs persisted for at least 1 week until newly amplified CD4+ CD25+ CD127− Treg subset was detected in the thymus and spleen. The functional test highlighted that the immature CD11c+ DCs isolated from the thymus of Allo-NKs + Chemo-treated mice were capable of inducing the amplification of functional CD4+ CD25+ CD127− Treg cells from C57Bl/6 CD3+ T cells. These results indicated that Allo-NKs pretreatment contributes to developing immuno tolerance to haploidentical donor antigens by assembling donor-derived immature DCs and amplifying recipient-derived Treg cells in the thymus. In his work, Yu suggests that the recipient-derived Treg population is the most important trigger for the induction of HvG tolerance [38]. In contrast to Yu, Kohrt demonstrated, in studies on mice, that donor CD4+ CD25+ Treg cells are required for protection against GvHD after TLI/ATS conditioning. In particular, Kohrt observed, 6 days after transplantation, an important influence of host Natural Killer T cells and host IL-4 on the accumulation of donor Treg cells in the TLI/ATS-conditioned host spleen [40]. Regulatory T cells in tumor immunity The role of naturally occurring CD4+ CD25+ Treg cells in tumor immunity has been pioneering the subject of an important experiment in which administration of cell-depleting anti-CD25 monoclonal antibody before tumor inoculation eradicated syngeneic tumors [41]. Treg cells as regulatory elements have the ability to actively suppress immune responses and represent a predominant tolerance-inducing modality. The vast majority of the studies on Treg cells in cancer are performed on patients with solid malignancies. In particular, a large number of CD4+ CD25+ Treg cells are present in tumors and draining lymph nodes in tumor-bearing rodents and also patients with cancers in head and neck, lung, liver, gastrointestinal tracts, pancreas ovary or breast [42]. In the 1980s, Robert North and colleagues formulated the concept that Treg cells depletion was an interesting approach for increasing immune reactivity against cancer [43]. To date, different strategies aimed to deplete Treg cells or to functionally inactivate Treg cells are currently under development or in clinical evaluation. In particular, CD25 expression remains the principal target for Treg depleting strategies [44]. Several studies in murine models and cancer patients depleted Treg cells based on CD25 expression using different modalities.

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Table 1 Clinical trials in the Oncohematology field. No.

Title

Sponsors

Recruitment

Study results

Conditions

Phases

1

T-regulatory cell infusion post umbilical cord blood transplant in patients with advanced hematologic cancer Amplifying Graft-versus-tumor effect by donor regulatory T-cell depletion before donor lymphocytes infusion

Masonic Cancer Center, University of Minnesota

Suspended

No results available

Phase I

Assistance Publique - Hôpitaux de Paris Université Paris XII Université Paris VI Masonic Cancer Center, University of Minnesota

Completed

No results available

Leukemia/lymphoma/multiple myeloma and plasma cell neoplasm/myelodysplastic syndromes Hematological malignancy/relapse

Not yet recruiting

No results available

Hematologic malignancy

Phase I Phase II

Masonic Cancer Center, University of Minnesota

Terminated

No results available

Graft Versus host disease/leukemia/lymphoma/multiple myeloma

Phase I

Dana-Farber Cancer Institute Miltenyi Biotec GmbH

Recruiting

No results available

Hematologic malignancies

Phase I

Baylor College of Medicine

Recruiting

No results available

Leukemia/Hodgkin lymphoma/non Hodgkin lymphoma/myelodysplastic disease/myeloproliferative disorders Leukemia/lymphoma/multiple myeloma and myelodysplastic syndromes Chronic lymphocytic B-leukemia/B-CLL

Not reported

2

4

5

6

T-regulatory cell and CD3 depleted double umbilical cord blood transplantation in hematologic malignancies Umbilical cord blood T-regulatory cell infusion followed by donor umbilical cord blood transplant in treating patients with high-risk leukemia or other hematologic diseases Infusion of donor lymphocytes depleted of CD25+ regulatory T-cells in patients with relapsed hematologic malignancies T-Reg cell kinetics for patients receiving stem cell transplant

7

Allogeneic bone marrow transplantation using less intensive therapy

Masonic Cancer Center, Minnesota

Recruiting

No results available

8

Lymphocytic B-leukemia (B-CLL) w/human IL-2 gene modified & human CD40 ligand-expressing autologous tumor cells

Completed

No results available

9

T-cell recovery in patients with leukemia, advanced lymphoma, myelodysplastic syndrome, or myeloproliferative disorder receiving alemtuzumab and undergoing donor stem cell transplant

Baylor College of Medicine/The Methodist Hospital System/Center for Cell and Gene Therapy, Baylor College of Medicine Baylor College of Medicine

Recruiting

No results available

10

Haploidentical natural killer cells to treat refractory or relapsed acute myelogenous leukemia (AML) Bendamustine combined with alemtuzumab in pretreated chronic lymphocytic leukemia (CLL)

Masonic Cancer Center, University of Minnesota Arbeitsgemeinschaft medikamentoese Tumortherapie/Mundipharma K.K. Abramson Cancer Center of the University of Pennsylvania

Recruiting

11

12

Genetically engineered lymphocyte therapy in treating patients with B-cell leukemia or lymphoma that is resistant or refractory to chemotherapy

Phase II

Phase I

Not reported

No results available

Chronic myeloproliferative disorders/leukemia/lymphoma/myelodysplastic syndromes/myelodysplastic/myeloproliferative neoplasms Leukemia, myelogenous, acute

Recruiting

No results available

Leukemia, lymphocytic, chronic, B-cell

Phase I Phase II

Recruiting

No results available

Hematopoietic/lymphoid cancer/adult acute lymphoblastic leukemia in remission

Not reported

Phase II

These studies are updated in January 2011 on the basis of information provided by National Institutes of Health (Bethesda, MD, USA) and published on the website of the Clinical Trials, a service of U.S. NIH (http://clinicaltrials.gov).

N. Daniele et al. / Pathology – Research and Practice 207 (2011) 209–215

3

Phase I Phase II

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Regulatory T cells in infection diseases In autoimmune diseases, Treg cells play an important role in controlling the immune response to self. In microbial infection, a balance must be set between an excessive immune response to the infection agent, resulting in immunopathological damage, and mounting an insufficient response allowing pathogen persistence. The importance of achieving this balance is highlighted by a murine model in which mice are infected with Helicobacter pylori. Adoptive transfer of lymph node cells, depleted of CD4+ CD25+ Treg cells, to T cell-deficient mice infected with H. pylori drives a more robust immune response to the bacterium than non-depleted lymph node cells but results in enhanced gastric inflammation causing damage to the host [45]. In vitro evidence leads us to think that natural Treg cells have a similar role in humans infected with H. pylori [46]. According to these results, Scholzen A. and colleagues suggest that infection with Plasmodium parasites can cause severe disease due to a lack of protective immune response to clear parassitemia, or to the host’s inability to control excessive inflammation resulting in immunophatology. Increases in CD4+ CD25+ Foxp3+ Treg cells have been observed in patients infected with Plasmodium falciparum [47]. Treg cells and their rule in autoimmunity Actually, Treg therapy is involved in the treatment of disorders with qualitative or quantitative impairment of Treg cells, including patients with rheumatoid arthritis, multiple sclerosis, and autoimmune polyglandular syndrome type II [48–50]. In addition, considerable preclinical evidence has accumulated to indicate that ex vivo expanded Treg cells have the capability to treat inflammatory bowel disease, systemic lupus erythematosus and to prevent type I diabetes [51–53]. Particularly, the prevention of type I diabetes is important since this disease is reaching epidemic proportions with millions of people requiring daily insulin injections to maintain normal blood sugar levels [54]. Diabetes mellitus type 1 (type 1 diabetes, IDDM, or juvenile diabetes) is a form of diabetes mellitus that results from autoimmune destruction of insulin-producing beta cells of the pancreas. Pancreas and islet transplants have been used to treat type 1 diabetes; however, islet transplants are currently still at the experimental trial stage [55]. In type 1 diabetes, the treatment with Treg cells, associated with drugs to eliminate Treg -resistant effector T cells, will lead to the induction of long-term tolerance and preservation of endogenous or transplanted ␤-cell mass without the need for long-term immunosuppression [56]. GMP Isolation, Treg cells expansion and their cryopreservation Although the existence of Treg cells is indisputable, using them for therapeutic purposes has not been straightforward, in fact the local microenvironment in which Treg cells reside can have a considerable influence on their functional status [57]. In addition, one of the obstacles in the implementation of clinical protocols using Treg cells is their low frequency in the peripheral blood leading to the need for ex vivo multiplication of the cells prior to their use in vivo [58]. Prior to their ex vivo expansion, Treg cells have to be isolated. The CliniMACS system (Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, 51429 Bergisch Gladbach, Germany) provides a relatively versatile method for GMP cell isolation, and this system has been developed to permit the automated separation of cells on a clinicalscale level in a closed and sterile system. Magnetic selection or

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depletion of labeled target cells, for example from leukapheresis product, can be performed to isolate a desired cell population, or for the specific removal of a given cell population, based on the recognition of a surface antigen by highly specific antibodies. MACS Technology (Miltenyi Biotec GmbH, Germany) is based on the use of MACS MicroBeads (Miltenyi Biotec GmbH, Germany) – nanosized superparamagnetic particles coupled to specific antibodies – patented MACS Columns (Miltenyi Biotec GmbH, Germany), and MACS Separators (Miltenyi Biotec GmbH, Germany) strong permanent magnets. MACS MicroBeads are approximately 50 nm in diameter and are composed of an iron dextran matrix, which has an excellent safety profile. Cell separation with MACS Technology takes place within MACS Columns when placed in a MACS Separator. Magnetically labeled cells are retained within the column. The MACS Column matrix provides a magnetic field strong enough to retain target cells that are labeled with even minimal amounts of MACS MicroBeads. Unlabeled cells pass through and can be collected; labeled cells are released and collected after removal of the magnetic field. Two pioneering studies reported that CliniMACS GMP Treg cells isolation strategies, based on CD25pos enrichment, typically result in 40 –60% pure Treg cells, with moderate suppressive activity [59,60]. Peters et al. suggest that it is highly recommendable to exclude cytotoxic CD8pos T cells from CliniMACS isolated Treg populations. In their preliminary studies, this group found that Treg cells, isolated by CD25pos enrichment, following CD8pos depletion, contained a small contamination of B cells (1–3%), which could increase to up to 10% after T cell expansion. Peters et al. were able to prevent the contamination by B cells by including a CD19pos B cell depletion step in the isolation strategy [61]. Regarding the Treg cells expansion ex vivo, the most commonly used expansion protocol at present is based on stimulation by antiCD3/anti-CD28 beads in the presence of high doses of recombinant IL-2. This protocol results in the efficient expansion of polyclonal Treg cells, generating sufficient numbers of cells for cellular therapy [62]. In some cases, this protocol is supplemented with rapamycin. Gao et al. showed that rapamycin selectively blocks the proliferation of Teff cells while it expands human and mice Treg cells ex vivo [63]. Moreover, it has recently been shown that rapamycin is able not only to increase the number of Treg cells, but also to increase their donor-specific suppressive capability in vivo [64]. In any case, clinical implementation of Treg cells based therapy will be highly facilitated if Treg cells can be stored prior to infusion, as this will allow a more flexible timing of Treg cells therapy and/or therapeutic schemes with multiple Treg cells treatments over time, as suggested by Peters et al. [61]. In his work, Eyad Elkord found a dramatic reduction in Treg cells proportion following freezing. In fresh PBMC samples, from 6 healthy volunteers, the mean frequency of FoxP3+ cells as a proportion of CD4+ T cells was 5.58% (CI = 3.39–7.77). This frequency was dropped to 3.57% (CI = 1.94–5.22) following 3-week freezing, which is significantly lower than their level in fresh samples (paired T test, P = 0.0016). The author suggests that Treg cells are labile to cryopreservation conditions as significant proportions of these cells were reduced following freezing [65]. In contrast to the data reported by Elkord, Van Hemelen et al. did not detect substantial differences in the expression of FoxP3, or the marker CD25 within the CD4+ T cell population between freshly isolated and reconstituted cryopreserved PBMCs. Van Hemelen suggests that these differences are probably due to a different approach to cryopreservation between his group and Elkord et colleagues. In particular, in contrast to the ‘freezing medium’ used by Elkord and colleagues, consisting of 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO), Van Hemelen laboratory’s

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‘freezing medium’ contains 50% FCS, 40% HBSS, and 10% DMSO. In Van Hemelen’s protocol, after the isolation, the PBMCs are first resuspended in FCS/HBSS medium alone, whereafter the DMSO is gently added to the suspension, instead of immediately dissolving the PBMCs with the freezing medium already containing DMSO. To reconstitute the frozen samples, Van Hemelen et al. gradually dilute the contents of the rapidly thawed cryovials with 10 ml of 37 ◦ C culture media (RPMI-1640 (Lonza/Bio-Whittaker) supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 ␮M ␤mercaptoethanol [66]. Song Y.C., in his paper, highlights that this gradual dilution is fundamental to avoid osmotic stress, reducing cell damage [67]. According to Van Hemelen, we suppose that cryopreservation protocol rather than the process of cryopreservation per se may influence the survival of Treg cells. Moreover, Peters et al. studied the feasibility of CliniMACS Treg cells cryopreservation in liquid nitrogen, and their results indicate that Treg cells can survive cryopreservation, as thawed populations showed 70–80% cell viability. In addition, Peters et al. noted decreased suppressive activity of thawed Treg populations, which could be restored by Treg cells expansion. An alternative approach would be to expand Treg cells prior to cryopreservation [61]. Anyway, to date, as Kivling suggests, the knowledge of how cryopreservation can alter the suppressive function of Treg cells is still limited probably due to the insufficient information on how Treg cells act suppressively. We should also consider that the different subpopulations of Treg cells act in diverging directions [68]. Conclusions and future directions The interest in Treg cells and enthusiasm for their potential therapeutic application have intensified over the recent years. Thus Treg research is very active, and new papers emerge almost daily. Understanding the exact mechanisms by which Treg cells exert their influence is an area of intense research and will likely define parameters that can be manipulated therapeutically to intervene in several disease processes. To date, there are 53 ongoing clinical trials with the application of Treg cellular therapy. Regarding the future, it would be necessary to improve cryopreservation protocols for these cells. Furthermore, we must consider that each cell is unique and there is no single cryopreservation protocol. It is important to consider that there is a knowledge gap in the field of cryopreservation: we do not understand the mechanism of action for DMSO and why certain cell types survive and others do not. For the future, molecular mechanisms of damage, during Treg cells cryopreservation, must be elucidated and alternatives to DMSO are needed for Treg cells cryopreservation. Progress in understanding Treg cells biology and the optimization of protocols of Treg cells manipulation will promote the translation of research-based knowledge to the clinic. Conflict of interest All authors declared no conflict of competing interests. Acknowledgements The work was supported by Associazione “Davide Ciavattini” ONLUS. In addition, we appreciate the help from Dr. Grenga Lucia for comments. References [1] S.F. Ziegler, FOXP3: not just for regulatory T cells anymore, Eur. J. Immunol. 37 (1) (2007) 21–23.

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