Flt3 ligand expands CD4 FoxP3 regulatory T ... - Wiley Online Library

Eur. J. Immunol. 2013. 43: 533–539

Clinical immunology

DOI: 10.1002/eji.201242603

SHORT COMMUNICATION

Flt3 ligand expands CD4+FoxP3+ regulatory T cells in human subjects Oliver Klein∗1 , Lisa M. Ebert∗1 , Damien Zanker∗1 , Katherine Woods1 , Bee Shin Tan1 , Jitka Fucikova1,2 , Andreas Behren1 , Ian D. Davis1 , Eugene Maraskovsky3 , Weisan Chen∗1 and Jonathan Cebon∗1 1

Ludwig Institute for Cancer Research, Melbourne Centre for Clinical Sciences, Heidelberg, Victoria, Australia 2 Department of Immunology, Charles University, University Hospital Motol, Prague, Czech Republic 3 CSL Limited, Parkville, Victoria, Australia CD4+ CD25+ FoxP3+ naturally occurring regulatory T (Treg) cells play a crucial role in the maintenance of immune tolerance and in preventing autoimmune pathology. Interventions that expand Treg cells are highly desirable, as they may offer novel treatment options in a variety of autoimmune and transplantation settings. Paralleling previous preclinical studies, we demonstrate here that administration of the hematopoietic growth factor Flt3L to human subjects increases the frequency and absolute number of Treg cells, and reduces the ratio of CD8+ T cells to Treg cells in the peripheral blood. The increase in Treg cells was due to enhanced Treg-cell proliferation rather than release of Treg cells from the thymus. Further studies revealed that Flt3L-induced proliferation of Treg cells was an indirect effect that occurred via the interaction of Treg cells with the Flt3L-expanded pool of CD1c+ myeloid dendritic cells. On the basis of these findings, Flt3L may represent a promising agent for promoting immune tolerance in a variety of clinical settings.

Keywords: Flt3 ligand

r

Immune tolerance

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Regulatory T (Treg) cells

Introduction FoxP3-expressing Treg cells play a major role in maintaining peripheral tolerance and limiting self-harming effects of immune responses [1]. There is significant interest in exploiting the suppressive activity of Treg cells clinically for the treatment of conditions such as autoimmune disease, graft-versus-host disease, and allograft rejection. Current attempts are mainly focused on isolating Treg cells from blood, expanding them ex vivo, and re-infusing them into patients [2]. However, this approach is limited by insufficient expansion and loss of suppressive capacity in some of the

Correspondence: Dr. Oliver Klein e-mail: [email protected]

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

expanded cells [3]. It also requires the necessary logistics to produce cell products and will be very costly. Strategies to manipulate Treg cells in vivo could overcome these hurdles. Although various approaches in mice have produced increases in Treg-cell frequencies [4, 5], initial attempts in humans using a CD28 superagonist antibody led to a disastrous outcome [6]. Flt3L is a hematopoietic growth factor important for the development of myeloid and plasmacytoid DCs. Accordingly, injection of Flt3L into mice leads to dramatic increases in these DC subsets in secondary lymphoid organs and blood [7]. Similarly, daily subcutaneous injections of Flt3L in humans result in several-fold

∗

These authors contributed equally to this work.

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Figure 1. Flt3L expands Treg cells in human peripheral blood. (A) Changes in the absolute numbers of leukocyte populations before (D0) and after (D15) daily Flt3L treatment in 26 patients are shown as mean + SEM. (B) The proportion of CD4+ T cells with a Treg-cell phenotype (CD25+ FoxP3+ ) was determined before and after Flt3L treatment. (C) Representative flow cytometry staining for Treg cells before and after Flt3L treatment, gated on CD4+ T cells. (D) The absolute numbers of Treg cells per mL of blood before and after treatment in 22 patients are shown. (E) Timecourse showing the percentage of Treg cells (as a proportion of total CD4+ T cells) during and following treatment with Flt3L. (F) The ratio of CD8+ T cells to Treg cells was determined before and after treatment by dividing the percentage of lymphocytes with a CD8+ phenotype by the percentage of lymphocytes with a Treg-cell phenotype. Data shown are representative of one experiment performed. Statistical significance determined by paired, two-tailed t-test.

increase in the frequencies of myeloid and plasmacytoid DCs, and monocytes in the peripheral blood [8, 9]. It was recently demonstrated that peripheral Treg-cell homeostasis in mice is highly dependent on interactions with DCs, such that increases in DC number lead to increases in Treg-cell number [10]. Moreover, administration of Flt3L to mice induces significant expansion of Treg cells, as a secondary effect of DC expansion [11, 12]. Importantly, this increase in Treg cells was associated with amelioration of autoimmunity and graft-versushost disease. We demonstrate here that administration of Flt3L to humans expands Treg cells via a similar mechanism to that discovered in mice, suggesting that Flt3L may represent a novel treatment option in autoimmune and transplantation settings.

ies [8, 9]. In addition, we observed for the first time a significant increase in the percentage of Treg cells, as a proportion of CD4+ T cells, following Flt3L administration (mean 68% increase; p < 0.0001; n = 22; Fig. 1B-C). As shown in Figure 1D, the increase in Treg-cell frequency translated to an increase in Tregcell absolute number, with a greater than twofold increase in the mean number of Treg cells per mL of blood at day 15 (p < 0.05; n = 22). Several patients had samples available from additional time-points; time-course studies in these patients revealed that Treg-cell frequency was maintained or increased even further out to day 22, after Flt3L administration had ceased (Fig. 1E). We also assessed the ratio of CD8+ T cells to Treg cells, and detected a significant decrease after Flt3L treatment (Fig. 1F), which may favor the development of an immune-tolerant environment.

Results and discussion

Flt3L-expanded human Treg cells are suppressive

Flt3L increases the frequency and absolute number of human Treg cells

We next aimed to determine the suppressive activity of Treg cells after Flt3L treatment. FoxP3 and CD25 can both be transiently induced on activated effector T (Teff) cells, and it was therefore important to confirm that the increased number of CD4+ CD25+ FoxP3+ T cells after Flt3L treatment was due to an increase in genuine Treg cells. To this end, Treg cells were sorted from PBMCs

Daily subcutaneous injection of human subjects with Flt3L for 14 days led to large increases in monocytes, myeloid DCs, and plasmacytoid DCs in the blood (Fig. 1A), similar to previous stud C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Clinical immunology

T cells, the Treg cells from both time-points were equally suppressive on a per-cell basis (Fig. 2C), thereby confirming that Flt3Linduced Treg cells are functional.

Expansion is due to increased proliferation of pre-existing Treg cells

Figure 2. Flt3L-expanded Treg cells are suppressive. (A) Analysis of Treg-cell frequency in PBMC samples used for suppression assays, using the CD4+ CD25+ CD127 low/− phenotype for Treg-cell identification and sorting. Note that cells were rested in culture for 48 h prior to sorting. (B) Treg cells were assessed for FoxP3 expression immediately after sorting. A representative result from ten sorted samples is shown. (C) Treg cells were isolated from PBMCs collected before (Day 0) or after (Day 15) Flt3L treatment (n = 5), and tested in a suppression assay at a 2:1 responder:Treg-cell ratio. Data shown are representative of three experiments performed. Statistical significance determined by unpaired t-test.

collected before and 15 days after treatment, according to a CD4+ CD25+ CD127− phenotype, previously shown to be identical to CD4+ FoxP3+ Treg cells [13, 14]. Analysis of cells prior to sorting confirmed that, as expected, Treg cells identified using this phenotype were increased at day 15 compared with that on day 0 for all patients tested (Fig. 2A). Furthermore, analysis of Treg cells after sorting confirmed that the majority of sorted cells expressed FoxP3 (mean 87.1%; range 79–94% FoxP3+ ; Fig. 2B). When tested for their ability to suppress the proliferation of healthy donor CD8+


To explore the mechanism behind Treg-cell expansion, we first investigated whether Flt3L had any direct effect on Treg cells. Although initial reports found no evidence for expression of Flt3 at the mRNA or protein level in mature lymphoid cells [15], low level Flt3 expression in T cells has been recently demonstrated [16]. We could also detect marginal expression of FLT3-mRNA in Treg cells in some donors using RT-PCR (Fig. 3A). However, culturing CFSE-labeled Treg cells with various concentrations of Flt3L failed to induce proliferation, excluding any cell-intrinsic effect of Flt3L on Treg cells (Fig. 3B). Indirect mechanisms to explain the Treg-cell increase after Flt3L administration include increased thymic output of Treg cells, the induction of a regulatory phenotype in conventional T cells, or increased proliferation of pre-existing Treg cells. Previous studies in mice have demonstrated that Flt3L can, under certain circumstances, lead to increased thymopoiesis through expansion of lymphoid progenitor cells [17]. To address this possibility, we assessed Treg cells for expression of the memory markers CD45RA and CD45RO, together with CD31, a marker of human CD4+ T cells that have recently emigrated from the thymus [18] (Fig. 3C). The percentage of CD31+ na¨ıve (CD45RA+ ) cells in the Treg population was low, consistent with the low thymic output in adults, and there was no significant increase between day 0 and 15, ruling out increased thymopoiesis as a major mechanism for the Flt3L-induced increase in Treg-cell numbers. This was supported by an unchanged ratio of CD45RA+ na¨ıve Treg cells to CD45RO+ memory Treg cells. To address the role of peripheral conversion of conventional T cells to Treg cells, we performed staining for the Helios transcription factor that has been reported to be expressed only by thymus-derived Treg cells [19], although this association is somewhat controversial [20]. As shown in Figure 3D, the proportion of Treg cells expressing Helios was unchanged after Flt3L administration, suggesting that peripheral conversion may not be a major mechanism by which Flt3L increases the pool of circulating Treg cells, although a more reliable marker of thymic-derived Treg cells is required to confirm this result. Finally, to determine whether the Flt3L-induced increase in Treg-cell frequency was due to enhanced proliferation, we performed intracellular staining for Ki67 (Fig. 3E). Prior to treatment, proliferating (Ki67+ ) cells were already more frequent within the Treg-cell population compared with the Teff-cell population, in keeping with the previously reported higher basal proliferation rate of Treg cells [21]. After 15 days of Flt3L treatment, there was a nearly twofold increase in the proportion of proliferating (Ki67+ ) Treg cells compared with the pretreatment measurement, without

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Figure 3. Expansion of Treg cells is driven by increased proliferation of pre-existing Treg cells. (A) FLT3 and β-ACTIN transcripts were detected using RT-PCR. Lanes 1, 3, and 5 show Treg cells purified from three donors, while lanes 2, 4, and 6 show Treg-cell-depleted T cells from the same donors. Lane 7 = human bone marrow. Actin was used as a loading control. (B) CFSE-labeled Treg cells were cultured alone, or with the indicated concentration of Flt3L, or with anti-CD3/CD28 beads for 5 days and then assessed for CFSE dye dilution. Data are shown as mean + SEM of three samples, and are pooled from two experiments performed. (C) The proportion of Treg cells expressing CD45RA, CD45RO, and CD31 was assessed by flow cytometry. Treg cells (CD4+ CD25+ FoxP3+ ) were gated, and the percentage of cells in the indicated populations determined as a proportion of total Treg cells. Data are shown as mean + SEM of n = 3 samples, and are representative of two experiments performed. (D) The proportion of total FoxP3+ CD4+ T cells that also expressed Helios was determined by flow cytometry for three patients, shown as mean + SEM. (E) Actively proliferating cells were identified by staining for Ki67. Gated CD4+ T cells were further gated into effector (FoxP3– ) and Treg (CD25+ FoxP3+ ) subsets, and assessed for intracellular staining with Ki-67 antibody. Top panel shows pooled results for three patients, while bottom panels show representative staining of the Treg-cell subset (solid lines = Ki-67; dotted lines = isotype control). Data are shown as mean + SEM. (F) Treg cells were isolated from healthy donor PBMCs, labeled with CFSE, and incubated alone or with CD14+ monocytes (mono), CD1c+ myeloid DCs (mDC), or plasmacytoid DCs (pDC) from the same donor. After 5 days, the proportion of divided (CFSE-low) cells was determined by flow cytometry and shown as mean + SEM of four samples, representative of three experiments performed. (G) CD1c+ DCs were analyzed on day 0 and 15 of Flt3L treatment for the expression of CD80, CD86, and HLA-DR by flow cytometry. Data are shown as mean + SEM of four samples, and are representative of three experiments performed. *p < 0.05; ns, not significant, unpaired t-test. D0 = pretreatment; D15 = day 15 of Flt3L treatment.

any increased proliferation in the CD4+ Teff cell compartment. Thus, the altered balance between Treg cells and Teff cells following Flt3L treatment appears to be primarily due to increased proliferation of pre-existing Treg cells.

Myeloid DCs induce Treg-cell proliferation in vitro As Flt3L treatment led to large increases in myeloid DCs, plasmacytoid DCs, and monocytes in peripheral blood, we cocultured these antigen-presenting cell subsets with Treg cells to directly inves-


tigate whether any of these populations could induce Treg-cell proliferation. In mice, CD4+ CD8– lymphoid DCs are the only DC subset capable of inducing Treg-cell proliferation in the absence of antigen [22, 23]. Human CD1c+ myeloid DCs are seen as the human counterpart of this population [24], and accordingly, were the only cell type able to promote significant Treg-cell proliferation in our studies (Fig. 3F). Furthermore, flow cytometric analysis of CD1c+ DCs in patient samples revealed a significant increase in HLA-DR expression after Flt3L administration without any change in expression of the costimulatory molecules CD80 and CD86 (Fig. 3G). These data suggest that increased availability

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of MHC class II molecules on the expanded DC population contributed to the increased Treg-cell proliferation. This is in keeping with findings in an animal model where the TCR/MHC class II interaction was crucial for the Flt3L-mediated Treg-cell expansion [10].

Clinical immunology

Staining Kit (eBioscience, San Diego, CA), and Pacific Bluecoupled anti-human Helios antibody (Biolegend, Cambridge, UK). Anti-Ki67 from BD Biosciences was used to assess proliferation, as previously described [30].

Suppression assay

Concluding remarks Our results demonstrate that human Treg cells can be expanded in vivo by injection of Flt3L, in keeping with previous studies in mice [11,12]. This increase in Treg-cell frequency occurs indirectly, via expansion of myeloid DCs, which in turn induce Treg-cell proliferation. Our findings are supported by a recently described human DC deficiency syndrome that is associated with a significant reduction in circulating Treg-cell frequency [25], and by studies of normal human skin demonstrating that autologous Langerhans cells promote the proliferation of skin-resident Treg cells via an MHC class II-dependent mechanism [26]. The patients analyzed in this study were participants in a clinical trial designed to investigate potential immune-enhancing effects of Flt3L on endogenous and vaccine-induced anti-tumor immune responses in melanoma. We were unable to detect any significant induction or boosting of melanoma antigen-specific immune responses in our patients, which may be due to the Flt3Lmediated Treg-cell expansion described here [27]. On the other hand, agents that can shift the balance between Teff cells and Treg cells to promote an immune-tolerant environment may offer new treatment options for autoimmune conditions and the prevention of alloimmune responses in transplantation medicine [28,29]. Preclinical studies investigating Flt3L administration to mice [11, 12] demonstrated an increase in peripheral Treg-cell number of a very similar magnitude to that observed here (∼twofold). Importantly, this increase was sufficient to ameliorate colitis and prevent death from graft-versus-host disease. Our new data in humans suggest that Flt3L, which already has a well-established safety profile [8, 9], may have similar efficacy in the treatment of humanw disease.

Materials and methods

Frozen PBMC samples were thawed and rested in culture for 48 h. Live cells were collected using Lymphoprep, stained and sorted as follows: responder (CD8+ CD127+ CD25− ), Treg cells (CD4+ CD25+ CD127− ), and non-Treg cells (CD4+ CD25− CD127− ). CD8+ T cells were labeled with 5 μM CFSE and 2000 cells cultured with 1000 Treg cells in 96-well plates containing 1 μg/mL anti-CD3 (OKT3) antibody. The percentage of divided CD8+ T cells (CFSE-low) was determined after 4 days’ culture and used to calculate percent suppression as follows: 1 – (% divided test /% divided max) × 100, where ‘test’ was the suppressor population being tested, and ‘max’ was the non-Treg-cell control.

Detection of Flt3 by RT-PCR Treg cells were isolated from bulk PBMCs using the Treg isolation kit (Milteny Biotech). The Treg-cell-depleted fraction of CD4+ T cells was used as a source of non-Treg cells. RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA synthesized as previously described [31]. The FLT3 primers were designed and PCR performed as described [15].

Treg-cell proliferation Treg cells, monocytes, CD1c+ myeloid DCs, and plasmacytoid DCs were isolated from PBMCs of healthy donors using Treg-cell, myeloid, and plasmacytoid DC isolation kits and CD14 microbeads (Miltenyi Biotec, Auburn, CA). The Treg cells were stained with 1 μM CFSE, and 2 × 105 cells were incubated with the autologous DC populations in a 5:1 ratio for 5 days. Alternatively, CFSElabeled Treg cells were cultured with human Flt3L (R&D Systems, Minneapolis, MN), or anti-CD3/CD28 beads (Invitrogen) for 5 days.

Statistical analysis Clinical trial PBMCs were obtained from patients with resected stage II/III/IV melanoma participating in a clinical trial involving Flt3L administration [27]. Patient PBMCs were cryopreserved in 10% DMSO, and stored in liquid nitrogen. The trial was approved by the Austin Health Human Research Ethics Committee.

Pre- and posttreatment data were compared using a paired, twotailed t-test, calculated using Prism software (GraphPad Software, La Jolla, CA). Other analyses were performed using an unpaired t-test.

Flow cytometry Leukocyte populations were characterized using fluorochromecoupled antibodies to surface markers from BD Biosciences (Franklin Lakes, NJ), allophycocyanin-labeled anti-human FoxP3 C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Acknowledgments: This work was supported by a Grant-in-Aid to LME and JC from the Cancer Council of Victoria and Operational Infrastructure Support from the Victorian State Government. EM is

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an employee of CSL Limited and an Honorary Senior Research Fellow of the Ludwig Institute for Cancer Research. JF was supported by a grant from the Ministry of Education, Czech Republic. IDD was supported by a Victorian Cancer Agency Clinician Researcher Fellowship and is an honorary NHMRC Practitioner Fellow. JC is an NHMRC Practitioner Fellow.

Eur. J. Immunol. 2013. 43: 533–539

13 Liu, W., Putnam, A. L., Xu-Yu, Z., Szot, G. L., Lee, M. R., Zhu, S., Gottlieb, P. A. et al., CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J. Exp. Med. 2006. 203: 1701–1711. 14 Seddiki, N., Santner-Nanan, B., Martinson, J., Zaunders, J., Sasson, S., Landay, A., Solomon, M. et al., Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J. Exp. Med. 2006. 203: 1693–1700.

Conflict of interest: E.M. is employed by CSL Limited, but no reagent or potential product of CSL was used in this work. All other authors declare no financial or commercial conflict of interest.

15 Rosnet, O., Schiff, C., Pebusque, M. J., Marchetto, S., Tonnelle, C., Toiron, Y., Birg, F. et al., Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells. Blood 1993. 82: 1110–1119. 16 Astier, A. L., Beriou, G., Eisenhaure, T. M., Anderton, S. M., Hafler, D. A. and Hacohen, N., RNA interference screen in primary human T cells reveals FLT3 as a modulator of IL-10 levels. J. Immunol. 2010. 184: 685–

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28 Koreth, J., Matsuoka, K., Kim, H. T., McDonough, S. M., Bindra, B., Alyea,

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Abbreviation: Teff cell: effector T cell

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Full correspondence: Dr. Oliver Klein, Ludwig Institute for Cancer Research, Melbourne Centre for Clinical Sciences, 147-163 Studley Road, Heidelberg, VIC 3084, Australia Fax: +61-3-9457-6698 e-mail: [email protected] Current address: Lisa M. Ebert, Centre for Cancer Biology, SA Pathology, Adelaide, SA 5000, Australia

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Received: 12/4/2012 Revised: 25/9/2012 Accepted: 29/10/2012 Accepted article online: 2/11/2012

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