Histone deacetylase inhibition facilitates GM-CSF-mediated expansion of myeloid-derived suppressor cells in vitro and in vivo

HDAC inhibition augments hematopoietic stem and progenitor cell expansion following GM-CSF administration, and skews bone marrow cell development toward myeloid-derived suppressor cells.

Abstract

Chromatin-modifying HDACi exhibit anti-inflammatory properties that reflect their ability to suppress DC function and enhance regulatory T cells. The influence of HDACi on MDSCs, an emerging regulatory leukocyte population that potently inhibits T cell proliferation, has not been examined. Exposure of GM-CSF-stimulated murine BM cells to HDACi led to a robust expansion of monocytic MDSC (CD11b⁺Ly6C⁺F4/80^intCD115⁺), which suppressed allogeneic T cell proliferation in a NOS- and HO-1-dependent manner with similar potency to control MDSCs. The increased yield of MDSCs correlated with blocked differentiation of BM cells and an overall increase in HSPCs (Lin^–Sca-1⁺c-Kit⁺). In vivo, TSA enhanced the mobilization of splenic HSPCs following GM-CSF administration and increased the number of CD11b⁺Gr1⁺ cells in BM and spleen. Increased numbers of Gr1⁺ cells, which suppressed T cell proliferation, were recovered from spleens of TSA-treated mice. Overall, HDACi enhance MDSC expansion in vitro and in vivo, suggesting that acetylation regulates myeloid cell differentiation. These findings establish a clinically applicable approach to augment this rare and potent suppressive immune cell population and support a novel mechanism underlying the anti-inflammatory action of HDACi.

Introduction

Histone acetylation classically modulates gene expression, whereby acetyl groups bound to lysine residues of histone proteins relax DNA binding, permitting gene accessibility and transcription. HDACi increase the extent of histone acetylation by inhibiting removal of acetyl groups from histones, resulting in tighter DNA binding and reduction in gene expression [1]. Histone acetylation offers a precise regulatory mechanism, where only a small proportion of genes is regulated by HDAC inhibition [2]. However, new evidence demonstrates nonhistone protein acetylation also to be an important post-translational modification, which suggests that acetylation is a more global regulatory mechanism than appreciated originally [3].

TSA is a naturally occurring antifungal metabolite produced by Streptomyces, which potently inhibits HDAC activity. TSA and other HDACi are well-known antineoplastic agents that modulate gene expression, leading to cell cycle arrest, differentiation, or apoptosis [4]. The HDACi SAHA (Vorinostat) is U.S. Food and Drug Administration-approved for the treatment of cutaneous T cell lymphoma. Recently, HDACi have been shown to suppress inflammatory disease [5] and to inhibit experimental GVHD [6], systemic lupus erythematosus [7], and colitis [8]. One mechanism that correlates with these anti-inflammatory effects of HDACi is the ability of these agents to target DC and other myeloid lineage APC functions [2, 5, 9–11].

DCs are professional BM-derived APCs with an unparalleled ability to stimulate naïve and memory T cells and regulate their function [12–14]. It has been demonstrated recently that HDACi reduce the stimulatory capacity of these potentially potent APCs [2, 5, 9–11]. Specifically, HDACi inhibit DC differentiation [9, 15] and reduce the expression of MHC gene products, costimulatory molecules, and proinflammatory cytokines by these cells, rendering them less immunostimulatory [9]. In addition, HDACi increase DC expression of IDO [10, 11], reduce DC production of Th1 cell-attracting chemokines, and selectively inhibit the induction of Th1 responses as a result of reduced bioactive IL-12 secretion [2]. Although studies of the anti-inflammatory activity of HDACi have focused on conventional mDCs, their influence on MDSCs, which have emerged recently as important regulators of immune reactivity [16], has not been investigated.

MDSCs are a rare, heterogenous population of incompletely differentiated, immature myeloid and MP cells. They expand from BM progenitors under inflammatory conditions, particularly in cancer [16], and in response to GM-CSF ex vivo [17] and in vivo [18]. Murine MDSCs coexpress CD11b and Gr1 and comprise 20–30% and 2–4%, respectively, of normal murine BM and splenocyte populations [16]. MDSCs potently inhibit T cell proliferation and are therefore regarded as important regulators of immune reactivity [16]. They have emerged as potential therapeutic agents based on their ability to suppress GVHD [17] and to mediate experimental transplant tolerance [19]. Given the ability of HDACi to impair DC differentiation [9, 15], we hypothesized that TSA might increase the generation of MDSCs from GM-CSF-stimulated BM cells by preventing the differentiation of BM cells into mature myeloid cells.

We demonstrate for the first time the ability of HDACi to enhance the generation of functional MDSCs in vitro and in vivo. TSA inhibited the differentiation of BM cells stimulated with GM-CSF in vitro and augmented HSPCs in BM cell cultures correlating with increased numbers of MDSCs. The related HDACi SAHA also increased phenotypic MDSCs in GM-CSF-exposed BM cultures. TSA administration following GM-CSF delivery enhanced the mobilization of splenic HSPCs and augmented the expansion of BM and splenic CD11b⁺Gr1⁺ cells in vivo. Greater numbers of splenic Gr1⁺ cells, with potent ability to suppress allogeneic T cell proliferation, were recovered from mice given TSA and GM-CSF than from those given GM-CSF alone. Taken together, these novel findings demonstrate the ability of HDACi to enhance the expansion of MDSCs in vitro and in vivo, correlating with their ability to expand HSPCs and impede BM cell differentiation.

MATERIALS AND METHODS

Animals

Experiments used 8- to 12-week-old male B6 (H2K^b) and BALB/c (H2K^d) mice from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in the specific pathogen-free facility of the University of Pittsburgh School of Medicine (Pittsburgh, PA, USA), and studies were conducted under an institutional Animal Care and Use Committee-approved protocol.

Cell culture and purification

BM cells were differentiated for 7 days, as described [20] in rmGM-CSF (1000 U/ml; Schering-Plough, Kenilworth, NJ, USA), alone or with rmIL-4 (1000 U/ml; R&D Systems, Minneapolis, MN, USA). On Day 7, nonadherent MDSCs were purified by labeling with PE-conjugated anti-Gr1 mAb and anti-PE immunomagnetic bead purification (Miltenyi Biotec, Auburn, CA, USA) [21]. mDCs were isolated as described [20]. TSA (0.1–10 nM; Sigma-Aldrich, St. Louis, MO, USA) or SAHA (10–500 nM; Selleck, Houston, TX, USA) was added to cultures on Days 2, 4, and 6.

Flow cytometry

Phenotypes were analyzed as described [20, 22] using fluorochrome-conjugated mAb and streptavidin (eBioscience, San Diego, CA, USA, or BD Biosciences, San Jose, CA, USA). HSPC and DC progenitors were analyzed on Day 4 of BM cell culture [23]. A Lin mAb cocktail consisting of anti-CD19, -B220, -CD3, -CD4, -CD8, -NK1.1, -Ter119, -CD11b, and -CD11c was used to set the Lin^– gate. Anti-CX₃CR1 was purchased from Abcam (Cambridge, MA, USA). Appropriately conjugated species- and isotype-matched IgG served as controls. Adjusted overall cell population frequency was calculated by multiplying the frequency of the indicated population by the number of events for the gate, divided by the total number of events recorded in the live cell singlet gate.

MDSC suppression assay

MDSC suppressor function was ascertained as described [24] with minor modifications. Isolated BM-derived Gr1⁺ MDSCs (B6) were rested overnight and tested for suppressor activity in allogeneic MLRs, where 1 × 10⁴ γ-irradiated DCs (B6; 20 Gy) stimulated CD3⁺ BALB/c T cell responders (4×10⁵) for 72 h in the presence of 0–3 × 10⁴ MDSCs in 96-well, round-bottom plates. MDSCs were rested for 16 h to allow LPS stimulation of DCs, which were also isolated on Day 7 and tested as stimulators (data not shown). norNOHA (500 μM; Calbiochem, Gibbstown, NJ, USA), L-NMMA (0.5 mM; Sigma-Aldrich), or SnPP (0.15 mM; Enzo Life Sciences, Farmingdale, NY, USA) was added where indicated. Alternatively, B6 splenic Gr1⁺ MDSCs (2×10⁵), isolated from treated mice, were tested for their ability to suppress proliferation of CD3⁺ BALB/c T cell (2×10⁵) responders stimulated with γ-irradiated B6 mDCs (5×10⁴).

Immunoblot

Immunoblotting was performed as described [20] on lysates from >1 × 10⁶ BM cells or isolated Gr1⁺ cells using primary mAb to acetyl-histone H4 (Lys8; Cell Signaling Technology, Beverly, MA, USA), arginase-1 (BD PharMingen, San Diego, CA, USA), HO-1 (Enzo Life Sciences), iNOS (Abcam), β-actin (Cell Signaling Technology), and GAPDH (Novus Biologicals, Littleton, CO, USA).

Hydrodynamic plasmid transfection of mice and TSA administration

B6 mice were injected with mGM-CSF-pcDNA3 (50 μg; Dr. Joyce Solheim, University of Nebraska Medical Center, Omaha, NE, USA) or eGFP-pcDNA3 (50 μg; Addgene, Cambridge, MA, USA) expression vectors as described [25]. TSA administration (1 mg/kg/day, i.p.) was initiated 1 day later and continued through Day 5. Spleens and femoral BM were harvested for analysis on Day 6.

Statistical analyses

Results are expressed as means ± 1 sd. Significant differences between means were determined using a one-tailed Student's t test and GraphPad Prism (GraphPad Software, La Jolla, CA, USA), and P < 0.05 was considered significant.

Online Supplemental material

Supplemental material includes Supplemental Fig. 1, demonstrating the dose-dependent effects of TSA and SAHA on BM cell cultures and the phenotype of MDSCs generated in SAHA cultures. Supplemental Fig. 2 displays the gating strategy used to identify HSPCs and MPs in BM cell culture. Supplemental Fig. 3 shows additional comparisons of the suppressive capacity of MDSCs generated from cultures containing IL-4, in addition to GM-CSF. Supplemental Fig. 4 demonstrates enhanced differentiation of CD11c⁺ DCs from GM-CSF- and IL-4-stimulated BM cell cultures exposed to TSA. The DCs isolated from these cultures demonstrated a dose-dependent reduction in cell surface molecules with increasing concentrations of TSA.

RESULTS

TSA enhances BM cell proliferation in response to GM-CSF and especially GM-CSF + IL-4 stimulation and skews myeloid lineage differentiation

We first examined the influence of TSA on murine BM cell cultures stimulated with GM-CSF or GM-CSF + IL-4. Under both conditions, addition of TSA (10 nM) led to a significant increase in total cells recovered on Day 7 (Fig. 1A). Although TSA modestly increased the proliferation of BM cells stimulated with GM-CSF alone, addition of TSA to GM-CSF in the presence of IL-4 enhanced cell proliferation three- to fourfold (Fig. 1A) and in a dose-dependent manner (Supplemental Fig. 1A). We next investigated the identity of the BM-derived cells expanded in TSA-treated cultures. Growth in GM-CSF alone led to a higher frequency of mDCs (CD11b⁺CD11c⁺) than growth in GM-CSF + IL-4 (Fig. 1B). Under both conditions, 10 nM TSA reduced the incidence of CD11b⁺CD11c⁺ DCs (Fig. 1B), consistent with impairment of DC differentiation by HDACi [9, 15]. As reported previously [26], pDCs (B220⁺CD11c⁺) were not generated to a significant degree in GM-CSF alone or GM-CSF + IL-4, and this was unchanged in the presence of TSA (Fig. 1C).

Figure 1. TSA enhances the expansion of BM-derived cells stimulated with GM-CSF ± IL-4 and favors the development of CD11b⁺Gr1⁺ cells in GM-CSF-stimulated BM cell cultures.

(A) B6 mouse BM cells were grown in GM-CSF ± IL-4, with or without 10 nM TSA. Addition of TSA to cultures led to enhanced expansion of total cells in GM-CSF and especially, GM-CSF + IL-4. Absolute cell number is the cell count/20 ml culture, starting with 3–4 × 10⁶ BM cells. Means + 1 sd for n = 3 separate experiments are shown. *P < 0.05 when compared with untreated control, determined by unpaired Student's t test. Cell samples were analyzed by flow cytometry for phenotypic (B) mDC (CD11b⁺CD11c⁺), (C) pDC (B220⁺CD11c⁺), or (D) putative MDSCs (CD11b⁺Gr1⁺). (B) TSA reduced the percentage of mDC by one-half when added to GM-CSF cultures and by approximately one-third when added to GM-CSF + IL-4-stimulated cultures. (C) Cultures stimulated with GM-CSF ± IL-4 produced few pDCs, and this was unchanged by TSA treatment. (D) TSA led to increased generation of CD11b⁺Gr1⁺ cells in GM-CSF-stimulated cultures, and this effect was reversed by addition of IL-4. (E) CD11b⁺CD11c^– cells were gated and analyzed for Gr1 expression. The majority of these cells expressed Gr1 in the absence of IL-4, whereas most cells were Gr1^– in the presence of IL-4, regardless of exposure to TSA. (F) Cells from GM-CSF-stimulated cultures were analyzed further for expression of Ly6C and -G (Gr1 epitopes), F4/80, and CD115. TSA-exposed cultures showed a threefold increase in CD11b⁺Ly6C⁺ cells, which were mostly F4/80^intCD115⁺. Data are representative of n = 2 or more independent experiments.

There is evidence that GM-CSF expands murine MDSCs from BM cells in vitro [17, 27] and in vivo [18]. We found that TSA had contrasting effects on the generation of CD11b⁺Gr1⁺-presumptive MDSCs, depending on whether the BM cells were differentiated in GM-CSF alone or GM-CSF + IL-4 (Fig. 1D). Specifically, addition of TSA to cultures stimulated with GM-CSF alone led to an increase in the incidence of CD11b⁺Gr1⁺ cells, from 30% to 70% (Fig. 1D). Similarly, addition of SAHA, a clinically used HDACi, to GM-CSF-stimulated BM cell cultures led to a dose-dependent increase in total cells (Supplemental Fig. 1B) and the frequency of CD11b⁺Gr1⁺ cells (Supplemental Fig. 1C and D). By contrast, in GM-CSF + IL-4-stimulated cultures, TSA reduced the incidence of CD11b⁺Gr1⁺ cells but enhanced the incidence of CD11b⁺Gr1^– cells (from 55% to 80%; Fig. 1D). Within the CD11b⁺CD11c^– gate, TSA enhanced the proportion of CD11b⁺Gr1⁺ cells in GM-CSF-stimulated cultures and alternatively, CD11b⁺Gr1^– cells when GM-CSF + IL-4 was used (Fig. 1E). Therefore, TSA differentially alters BM cell myeloid lineage commitment when stimulated with GM-CSF, depending on the presence of IL-4.

MDSCs are divided into granulocytic and monocytic subsets, depending on their expression of the Gr1 epitopes, Ly6G and Ly6C, respectively, and further classified according to their expression of CD115 (M-CSFR) and F4/80 [16]. Cells isolated from GM-CSF-stimulated BM cultures were assessed for their expression of these surface markers. Addition of TSA to these cultures increased the frequency of CD11b⁺Ly6C⁺ cells, which were predominantly F4/80^intCD115⁺, approximately threefold (Fig. 1F). SAHA demonstrated a comparable effect on GM-CSF-mediated BM cell differentiation, favoring CD11b⁺Ly6C⁺F4/80^intCD115⁺ cells (Supplemental Fig. 1E).

TSA expands HSPCs correlating with increased growth of myeloid cells

As TSA could induce differing myeloid lineage cell expansion, depending on the presence of IL-4 (Fig. 1), we considered that its effect might be to enhance the proliferation of an upstream MP with divergent differentiation directed by appropriate hematopoietic growth factors. To ascertain the influence of TSA on MPs, BM cells cultured in GM-CSF ± IL-4 were harvested on Day 4 following 2 days of exposure to TSA (10 nM) and assessed for myeloid lineage progenitors, as demonstrated in Supplemental Fig. 2. TSA significantly increased the frequency of HSPCs (Lin^–Sca-1⁺c-Kit⁺), irrespective of the presence of IL-4 (Fig. 2A and B) but exerted no significant enhancing effect on the frequency of MPs, MDPs or CDPs. However, cultures containing exogenous IL-4 exhibited an increased frequency of MPs compared with those stimulated with GM-CSF alone (Fig. 2A). In agreement with the increased frequency of HSPCs in cultures exposed to TSA, the absolute number of HSPCs in these cultures was increased approximately threefold, irrespective of the presence of IL-4 (Fig. 2C). BM cells exposed to GM-CSF and TSA overnight demonstrated enhanced acetylation of histone H4 (Fig. 2D). These findings indicate that the increased number of myeloid lineage cells induced by TSA (Fig. 1) correlated with early expansion of HSPCs, with the fate of the HSPC determined by specific exogenous growth factor addition to the cultures.

Figure 2. Culture of BM cells in TSA leads to an increased frequency and absolute number of HSPCs.

B6 BM cells were harvested on Day 4 following 2 days of exposure to TSA (10 nM). (A) The relative frequency of HSPCs and specific progenitor cell populations was determined by flow cytometry. HSPCs were defined as Lin^–Sca-1⁺c-Kit⁺; MPs as Lin^–Sca-1^–c-Kit^hiFlt3⁺CX₃CR1^–; MDPs as Lin^–Sca-1^–c-Kit^hiFlt3⁺CX₃CR1⁺; and CDPs as Lin^–Sca-1^–c-Kit^loFlt3⁺CD115⁺CX₃CR1⁺. Data are means + 1 sd of n = 4 independent experiments. (B) Representative flow cytometry plots demonstrating the expansion of HSPCs by TSA. Plots shown are Lin^–-gated and representative of n = 4 independent experiments. (C) The absolute number of HSPCs and specific MP populations was determined/plate. Cell number was determined by multiplying the average total number of cells isolated/plate for each treatment by the frequency of each progenitor. Data are means + 1 sd of n = 3 independent experiments. *P < 0.05 when compared with untreated control, determined by unpaired Student's t test. (D) BM cells were stimulated with GM-CSF (1000 U/ml) for 2 h prior to treatment with TSA at the indicated concentrations overnight. Acetyl-histone H4 and β-actin were detected by immunoblot. Data are representative of n = 2 independent experiments.

Increased numbers of mDCs and functional MDSCs are generated in the presence of TSA

We next sought to verify the function of presumptive MDSCs recovered from TSA-treated GM-CSF- and GM-CSF + IL-4-stimulated cultures. A greater than twofold increase in the number of Gr1⁺ cells recovered by positive bead selection from GM-CSF alone-stimulated cultures was achieved in the presence of TSA (10 nM; Fig. 3A), verifying the increase in these cells determined by flow cytometry (Fig. 1). Cells isolated by Gr1-positive selection were primarily CD11b⁺Ly6C⁺F4/80^intCD115⁺ (Fig. 3B). The ability of the presumptive MDSCs to suppress alloreactive CD3⁺ T cell proliferation in MLRs was determined. CD11b⁺Gr1⁺ cells differentiated in TSA were as potent in suppressing allogeneic T cell responses on a per-cell basis as those from control cultures (Fig. 3C and Supplemental Fig. 3A). MDSCs from GM-CSF + IL-4-stimulated cultures [which yielded fewer cells (Fig. 3A)] were slightly more effective on a per-cell basis in suppressing CD3⁺ T cell proliferation than those generated in GM-CSF alone (Supplemental Fig. 3B and C).

Figure 3. Putative MDSCs expanded by TSA exhibit suppression of T cell proliferation.

(A) MDSCs were isolated by Gr1⁺ immunomagnetic bead selection from 7 days of cultures in GM-CSF ± IL-4, supplemented with TSA (10 nM). TSA increased the numbers of MDSCs from GM-CSF-stimulated cultures. Differentiation of BM cells in GM-CSF with TSA yielded approximately twice as many MDSCs as untreated cultures. Significantly more MDSCs were isolated from GM-CSF-stimulated cultures than those containing GM-CSF + IL-4. (B) The phenotype of MDSCs isolated by Gr1-positive selection was determined by flow cytometry. (C) MDSCs (B6) isolated from cultures described in A were tested as suppressors in allogeneic MLRs at graded ratios from 0 to 3 × 10⁴ MDSCs with 1 × 10⁴ DCs (B6) as stimulators and 4 × 10⁵ CD3⁺ T cells (BALB/c) as responders. Addition of TSA to GM-CSF-stimulated cultures did not alter the ability of MDSCs to suppress T cell proliferation. (D) Expression of arginase-1, iNOS, and HO-1 was detected by immunoblot of freshly isolated Gr1⁺ cells from GM-CSF-stimulated BM cell cultures or those containing TSA. Acetyl-histone H4 was also detected by immunoblot, and GAPDH was included as a loading control. (E) MLRs were set up as in C, with 3 × 10⁴ MDSCs. Inhibitors of arginase-1 (norNOHA), iNOS (L-NMMA), and HO-1 (SnPP) were added to cultures on Day 0. MDSCs from GM-CSF- and GM-CSF/TSA-stimulated cultures required iNOS and HO-1 to suppress T cell proliferation. (A) The bar graph shows the means + 1 sd of n = 2 independent experiments. (B) The dot plots are representative of n = 3 independent experiments. (C–E) The data are representative of n = 2 independent experiments. Error bars indicate sd of triplicate wells. Differences were considered significant compared with untreated control (*P<0.05) using a Student's t test.

MDSCs suppress T cell proliferation by local depletion of l-arginine by arginase-1 and iNOS. NO produced by iNOS also has direct T cell-suppressive effects [16]. Likewise, the cytoprotective and immunoregulatory enzyme HO-1 is a critical mechanism for the T cell-suppressive capacity of MDSCs [28]. Cells isolated by Gr1-positive selection were immunoblotted to detect expression of arginase-1, iNOS, and HO-1 (Fig. 3D). Gr1⁺ cells isolated from TSA-exposed cultures had lower expression of iNOS and HO-1 and did not express arginase-1. As expected, these cells had higher levels of acetylated histone H4 compared with control. Arginase-1 and iNOS are inhibited by the addition of nor-NOHA and L-NMMA, respectively [17]. Additionally, HO-1 is selectively inhibited by SnPP. Addition of these inhibitors to MLRs containing MDSCs from GM-CSF-stimulated cultures demonstrated that iNOS and HO-1 are independently indispensable for the suppressive function of the MDSCs (Fig. 3E). The function of MDSCs expanded in the presence of TSA also required these enzymes. Thus, although Gr1⁺ cells isolated from TSA-exposed cultures displayed reduced levels of iNOS and HO-1, they still displayed potent T cell-suppressive function. Taken together, these results indicate that increased numbers of MDSCs can be recovered from BM cell cultures containing TSA and that the suppressive function and mechanism of these cells are similar to those from control cultures.

HDACi during the generation of mDCs from BM cells stimulated with GM-CSF + IL-4 led to increased numbers of a homogenous population of CD11c⁺ cells with reduced granularity compared with those from control cultures (Supplemental Fig. 4A and B). Consistent with previous reports [9, 10], exposure of BM cells stimulated with GM-CSF + IL-4 to increasing concentrations of TSA led to a dose-dependent reduction in several functionally important DC surface molecules, including CD40, CD80, CD86, MHC class II antigen (I-A^b), and the chemokine receptor CCR7 (Supplemental Fig. 4C and D).

TSA augments GM-CSF-mediated expansion of CD11b⁺Gr1⁺ BM cells in vivo

To extend our in vitro finding that TSA enhanced MDSC production from BM cells (Figs. 1 and 3), we tested its ability to promote expansion of these myeloid cells in vivo (Fig. 4). In mice given TSA, together with GM-CSF, a significant increase in the absolute number of BM cells was observed (Fig. 4A). TSA increased the absolute number and adjusted frequency of total BM CD11b⁺Gr1⁺ cells significantly compared with mice treated with GM-CSF alone (Fig. 4B–D). Conversely, B220⁺ cells, representing a nonmyeloid cell population, were reduced significantly (Fig. 4B and C). Of the two subsets of CD11b⁺Gr1⁺ cells, TSA exerted a greater effect on the Gr1^hi subset (Fig. 4B–D).

Figure 4. TSA enhances GM-CSF-mediated in vivo expansion of CD11b⁺Gr1⁺ cells in BM.

B6 mice receiving murine GM-CSF plasmid or control eGFP plasmid were treated with TSA (1 mg/kg/day) or vehicle (DMSO) for 5 days. (A) Viable BM cells from one femur were enumerated using trypan blue exclusion. The absolute number (B) and adjusted frequency (C) of Gr1^hi and Gr1^lo subsets of BM CD11b⁺Gr1⁺ cells were identified by flow cytometry. (D) Representative flow cytometry plots of CD45⁺-gated cells (1×10⁴ events), demonstrating MDSC gates. (A–C) The data are plotted as means + 1 sd for n = 3 independent experiments with two to four animals/group. Significance values (*P<0.05) were determined by unpaired Student's t test for total CD11b⁺Gr1⁺ cells and B220⁺ cells.

TSA enhances GM-CSF-mediated expansion of splenic MDSCs in vivo, correlating with enhanced mobilization of peripheral HSPCs

In BM cell cultures, TSA enhanced the number and frequency of HSPCs (Fig. 2), correlating with an increase in myeloid cells (Figs. 1 and 3 and Supplemental Figs. 1 and 4). We ascertained whether this effect of TSA could also be seen in vivo following GM-CSF administration (Fig. 5). Although no significant effect was observed on HSPCs residing in the BM (Fig. 5A and B), TSA significantly enhanced the absolute number (Fig. 5C) and frequency (Fig. 5D and E) of HSPCs in the spleens of mice treated with GM-CSF.

Figure 5. TSA increases the mobilization of peripheral HSPCs following GM-CSF administration.

B6 mice given GM-CSF or eGFP plasmid were treated with TSA or vehicle (DMSO). HSPCs (Lin^–c-Kit⁺Sca-1⁺) were identified in BM cells (A and B) and splenocyte populations (C and D) by flow cytometry. The absolute number of HSPCs in the BM from one femur (A) and the spleen (C) was determined from the adjusted frequency of HSPCs in the BM (B) and spleen (D). Data are plotted as means + 1 sd of n = 5–9 from three independent experiments. Significance values (*P<0.05) were determined by unpaired Student's t test. (E) Representative flow cytometry plots of Lin^–-gated cells demonstrating splenic HSPCs.

Unlike BM cells (Fig. 4A), the absolute number of splenocytes was not increased significantly in mice given TSA in combination with GM-CSF (Fig. 6A). However, as observed in the BM (Fig. 4B–D), TSA significantly increased the absolute number and frequency of splenic CD11b⁺Gr1⁺ cells in animals given GM-CSF (Fig. 6B–D). CD11b⁺Gr1⁺ cells that expanded in the spleen were predominantly Gr1^lo. mDC expansion was not increased significantly by TSA in the spleens of mice given GM-CSF (data not shown), reflecting the need for IL-4 to allow expansion of mDCs by TSA (Supplemental Fig. 4A). As in the BM, B220⁺ cells were not increased significantly in the spleen (Fig. 6B and C); however, the trend in the increased absolute number reflected an increased absolute number of splenocytes in TSA-treated mice given GM-CSF (Fig. 6A). These splenocytes yielded a significantly increased number of Gr1⁺ cells (Fig. 6E), which were functionally intact suppressors of allogeneic T cell proliferation in MLRs (Fig. 6F). Thus, these data confirm in vivo our in vitro finding that TSA augments GM-CSF-mediated expansion of MDSCs correlating with an increased number of splenic HSPCs.

Figure 6. TSA enhances GM-CSF-mediated expansion of suppressive CD11b⁺Gr1⁺ cells in the spleen.

(A) Splenocytes were enumerated using trypan blue exclusion from mice receiving hydrodynamic infusion of GM-CSF or eGFP plasmid and treated with TSA or vehicle (DMSO). The absolute number (B) and adjusted frequency (C) of Gr1^hi and Gr1^lo subsets of splenic CD11b⁺Gr1⁺ cells were determined by flow cytometry. B220⁺ cells were included as a negative control. (D) Representative flow cytometry plots of CD45⁺-gated cells (5×10⁴ events), demonstrating CD11b⁺Gr1^hi and CD11b⁺Gr1^lo gates. (E) Gr1⁺ cells were isolated by positive bead selection from 50 × 10⁶ pooled splenocytes of each treatment group and live cells enumerated by trypan blue exclusion. (F) Isolated Gr1⁺ cells were tested as suppressors in MLRs using B6 DCs as stimulators and BALB/c CD3⁺ T cells as responders. Positive controls consisted of DC + T cells, in the absence of added Gr1⁺ cells. (A–C) The data are plotted as means + 1 sd for n = 4 independent experiments with two to four animals/group. Significance (*P<0.05) was determined by unpaired Student's t test for total CD11b⁺Gr1⁺ cells and B220⁺ cells. (E and F) The data are means + 1 sd of n = 2 independent experiments with two to four mice/group. Significance values (*P<0.05) were determined by unpaired Student's t test (E) and paired Student's t test (F).

DISCUSSION

Several recent studies have described the ex vivo expansion of early stem cell progenitors using HDACi [29–33]. Although others have shown [29, 31, 32] that TSA increases HSPC proliferation under conditions favoring stem cell renewal, our finding that TSA expands HSPCs in GM-CSF ± IL-4-stimulated BM cell cultures reveals that this effect is maintained even under strong differentiation signals from GM-CSF. A similar effect has been seen with human CD34⁺ cells stimulated with G-CSF in the presence of the HDACi valproic acid [30]. To our knowledge, the present study presents the first evidence that in vivo administration of HDACi enhances peripheral HSPCs, likely as a result of their increased mobilization from the BM, as recent evidence demonstrates that HDACi may reduce HSPC adherence to BM stromal cells [34].

Also, we found that addition of TSA to BM cell cultures stimulated with GM-CSF skews the differentiation of myeloid cells depending on the presence of IL-4. Exposure of BM cell cultures to TSA reduced the phenotypic differentiation of DC (Fig. 1B), favoring a population of CD11c^–CD11b⁺ cells. These observations are in agreement with previous reports that HDACi block DC differentiation [9, 15]. The nonhistone protein STAT3 represents an intriguing target to further explain the mechanism of HDACi enhancement of MDSC expansion. STAT3 has a known acetylation site at lysine 685, which is required for its dimerization and transcriptional regulation [35], and exposure to HDACi promotes STAT3 activity by this mechanism [11]. GM-CSF [18] and M-CSF [36] are among the soluble factors that induce MDSC expansion. Many of these factors activate STAT3, which is currently believed to be the most important transcription factor regulating MDSC expansion [16]. Furthermore, STAT3 is indispensable for the maintenance of embryonic stem cell pluripotency and self-renewal [37]. These findings make STAT3 a strong candidate for the molecular regulation of MDSC and potentially HSPC expansion in our model.

Importantly, our findings provide a novel platform for the expansion of MDSCs in vitro. Typically, MDSCs are expanded in vivo using GM-CSF or inflammatory stimuli, such as bacterial LPS. GM-CSF has been used to generate monocytic MDSCs in vitro from BM cells, and these MDSCs required iNOS for suppressive activity [27]. Our data support these findings, where GM-CSF-expanded, monocytic MDSCs required iNOS activity to suppress T cell proliferation, but they did not require arginase-1 activity (Fig. 3E). In addition, MDSCs isolated from BM cultures in our studies required HO-1 for their suppressive activity, similar to those isolated from endotoxin-exposed mice [28]. In contrast, Highfill et al. [17] used GM-CSF to expand monocytic CD11b⁺Ly6C⁺ MDSCs, but in their system, the MDSCs required arginase-1 for suppressive activity. This discrepancy may result from our use of higher concentrations of GM-CSF (1000 U/ml vs. 250 U/ml), a longer culture period (7 days vs. 4 days), or different selection strategy (Gr1 vs. CD11b). TSA-expanded MDSCs demonstrated a similar suppressive potency and mechanism of suppression as control MDSCs, suggesting that the ability of HDACi to block complete myeloid differentiation [9, 15] leads to a build-up of immature myeloid cells and MPs with an intact, suppressive function.

In vitro and in vivo methods to generate MDSCs represent valuable tools to explore the potential use of these potent regulatory cells for therapeutic purposes. Two groups [17, 38] have generated MDSCs in vitro, which suppressed GVHD, and others have adoptively transferred MDSCs to alleviate experimental inflammatory bowel disease [39] and promote skin allograft survival [40]. The use of HDACi to enhance GM-CSF-mediated expansion of MDSCs will allow for further interrogation of these cells to develop their therapeutic potential.

AUTHORSHIP

B.R.R. designed and performed research, collected and analyzed data, and wrote the paper. A.C. and S.N. performed research and collected and analyzed data. A.W.T. and H.R.T. designed research, analyzed data, and wrote the paper.