Modification of T Cell Responses by Stem Cell Mobilization Requires Direct Signaling of the T Cell by G-CSF and IL-10

Kelli P. A. MacDonald, Laetitia Le Texier, Ping Zhang, Helen Morris, Rachel D. Kuns, Katie E. Lineburg, Lucie Leveque, Alistair L. Don, Kate A. Markey, Slavica Vuckovic, Frederik O. Bagger, Glen M. Boyle, Bruce R. Blazar and Geoffrey R. Hill
J Immunol April 1, 2014, 192 (7) 3180-3189; DOI: https://doi.org/10.4049/jimmunol.1302315

Abstract

The majority of allogeneic stem cell transplants are currently undertaken using G-CSF mobilized peripheral blood stem cells. G-CSF has diverse biological effects on a broad range of cells and IL-10 is a key regulator of many of these effects. Using mixed radiation chimeras in which the hematopoietic or nonhematopoietic compartments were wild-type, IL-10−/−, G-CSFR−/−, or combinations thereof we demonstrated that the attenuation of alloreactive T cell responses after G-CSF mobilization required direct signaling of the T cell by both G-CSF and IL-10. IL-10 was generated principally by radio-resistant tissue, and was not required to be produced by T cells. G-CSF mobilization significantly modulated the transcription profile of CD4+CD25+ regulatory T cells, promoted their expansion in the donor and recipient and their depletion significantly increased graft-versus-host disease (GVHD). In contrast, stem cell mobilization with the CXCR4 antagonist AMD3100 did not alter the donor T cell’s ability to induce acute GVHD. These studies provide an explanation for the effects of G-CSF on T cell function and demonstrate that IL-10 is required to license regulatory function but T cell production of IL-10 is not itself required for the attenuation GVHD. Although administration of CXCR4 antagonists is an efficient means of stem cell mobilization, this fails to evoke the immunomodulatory effects seen during G-CSF mobilization. These data provide a compelling rationale for considering the immunological benefits of G-CSF in selecting mobilization protocols for allogeneic stem cell transplantation.

This article is featured in In This Issue, p.2935

Introduction

Graft-versus-host disease (GVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation, with the resultant multiorgan damage and immune deficiency significantly impairing overall transplant survival. The use of recombinant human G-CSF–mobilized stem cell grafts has lead to rapid immune and hematopoietic reconstitution, and superior short term and long term disease free survival (1, 2). T cells from donors treated with G-CSF have a reduced capacity to induce GVHD on a per cell basis relative to those from control-treated donors (3). The mechanism by which G-CSF prevents GVHD has been suggested to be the result of Th2 and regulatory T cells (Treg) differentiation of naive donor T cells (3, 4). There is additional data suggesting G-CSF may also reduce GVHD through effects on dendritic cells, monocytes and NK cells (reviewed in Ref. 4). Indeed, the attenuation of T cell function by G-CSF is generally thought to be an indirect effect via effects on these innate cells.

We have previously demonstrated that pegylated G-CSF is superior to standard G-CSF for the prevention of GVHD, due to the enhanced generation of IL-10–producing Treg (5). Indeed, the induction of Th2 cells and Tregs by G-CSF are well described and dependent on the dose and type of G-CSF molecule used (3, 4, 6). In these studies, we investigated the greater unanswered question surrounding how T cell function is modified by G-CSF and whether these effects are recapitulated by mobilization with CXCR4 antagonists, which are also used for stem cell mobilization.

Materials and Methods

Mice

Female C57BL/6 (B6.WT, H-2b, CD45.2+), B6.Ptprca (H-2b, CD45.1+), and B6D2F1 (H-2b/d, CD45.2+) mice were purchased from the Animal Resources Centre (Perth, WA, Australia). B6.IL-10−/− (H-2b) and B6.G-CSFR−/− (H-2b) were supplied from the Australian National University and D. Link (Washington University, St. Louis, MO), respectively. B6.Foxp3-eGFP, B6.Foxp3-GFP-Luciferase-DTR (B6.Foxp3-luci), and CD40−/− mice were supplied by the QIMR Berghofer Medical Research Institute animal facility. The mice were used between 8 and 12 wk of age. Mice were housed in sterilized microisolator cages and received acidified autoclaved water (pH 2.5) after transplantation.

Stem cell mobilization

Recombinant human pegylated-G-CSF (Amgen, Thousand Oaks, CA) was diluted with normal saline (Baxter, Deerfield, IL) and given as a single dose s.c. at 12 μg/animal on day −6 prior to transplant (5). AMD3100 (Sigma-Aldrich, St. Louis, MO) was diluted with PBS and given s.c. at 200 μg/animal per dose (7). Donor mice received AMD3100 or saline on days −4, −3, −2, and −1 at 24-h intervals, and spleens were harvested 1 h after the last injection. All donor spleens were harvested on day 0. Where indicated, natural Treg (nTreg) were depleted in vivo by i.p. administration of anti-CD25 mAb (PC61) during G-CSF mobilization at days −3 and −1 pretransplant.

Stem cell transplantation

Mice were transplanted as described previously (5, 8, 9). Briefly, on day −1, B6D2F1 mice received 1100 cGy total body irradiation ([137Cs] source at 108 cGy/min), split into two doses separated by 3 h to minimize gastrointestinal toxicity. B6 (107) donor splenocytes were typically T cell depleted and added to purified T cell fractions (3 × 106/per animal; see below), then injected i.v. on day 0. Transplanted mice were monitored daily and those with GVHD clinical scores of 6 were sacrificed and the date of death registered as the next day in accordance with institutional animal ethics committee guidelines. The GVHD induced in this study is to MHC and is severe in nature in the absence of G-CSF mobilization with early death consistent with the original publications from the Ferrara group (3, 10). Importantly, the transplant recipients all received the same mobilized T cell–depleted splenocyte stem cell source such that differential survival reflects differential T cell function. Previous studies have demonstrated similar neutrophil recovery after transplantation in animals receiving unstimulated versus G-CSF–stimulated allogeneic and syngeneic spleen between days 8 and 12, the time of maximal mortality (5). Target organ GVHD in recipients of allogeneic T cell replete but not T cell–depleted grafts also has been noted at this time (8). Thus, differential survival across groups should reflect GVHD and early deaths because of engraftment failure should be equivalent across groups. Mixed chimeric mice were generated by transplanting 5 × 106 B6.WT, B6.IL-10−/−, or B6.G-CSFR−/− T cell–depleted bone marrow (BM) cells into irradiated (1000 cGy) B6.WT or B6.IL-10−/− recipients, which were then allowed to reconstitute over 4 mo before use as allograft recipients. In some experiments, a combination of both B6.WT and B6.IL-10−/− or B6.WT and B6.G-CSFR−/− BM cells were transplanted in 1:1 or 4:1 ratios (5 × 106 cell total) as described in figures. The degree of systemic GVHD was assessed by scoring as previously described (maximum index = 10) (11).

Cell preparation

T cells were purified using magnetic bead depletion of non-T cell splenocytes. Briefly, following red cell lysis, splenocytes were incubated with purified mAb (CD19, B220, Gr-1, CD11b, and Ter119). After incubation with Abs, cells were incubated with goat anti-rat IgG BioMag beads (Qiagen Pty, Chadstone Centre, VIC, Australia) for 20 min on ice, and then placed on a magnet. Subsequent CD3+ T cell purities were >90% and 2–3 × 106 T cells were added to T cell–depleted grafts per animal. For total T cell depletion, splenocytes were incubated with hybridoma supernatants containing anti-CD4 (RL172), anti-CD8 (TIB211) and anti-Thy1.2 (HO-13-4) mAbs followed by incubation with rabbit complement (Cederlane Laboratories, Hornby, ON, Canada) as described previously (8). Resulting cell suspensions contained <1% contamination of viable CD3+ T cells. Donor T cells in mixed chimeric donors were sorted by FACS based on CD45.1 and CD45.2 staining by Moflo (DakoCytomation) to >98% purity.

FACS analysis

The following mAbs were purchased from BioLegend (San Diego, CA): FITC-conjugated CD45.2 (104) and IgG2a isotype control; PE-conjugated CD3 (2C11), CD25 (7D4), CD44 (IM7), CD45.1 (A20), CD54 (3E2), L-selectin (MEL-14), CD103 (M290), CD127 (5B/199), CCR6 (29-2L17), CXCR5 (2G8), GARP (YG1C86); IgG2b isotype control; allophycocyanin-conjugated CCR4 (CD794), CCR7 (4B12), GITR (YGITR 765), and biotinylated CD45.1 and IgG2a isotype control. Streptavidin-PE-Cy5 was from DakoCytomation (Carpinteria, CA). Purified mAb against CD3 (KT3), CD19 (HB305), Gr1 (RB6-8C5), Thy1.2 (HO-13-4), and Ter119 were produced “in-house.”

nTreg suppression assays

For in vitro suppression assays, CFSE-labeled, FACS-purified CD4+CD25− (B6, CD45.1+) were seeded at 5 × 104/well in 96-well round-bottom plates, with CD11c+ MACS bead (Miltenyi Biotec) purified dendritic cells (DC) (B6, CD45.2+) at 5 × 103/well, in the absence or presence of Treg (B6.Foxp3-GFP, CD45.2+) at 5 × 104/well and supplemented with anti-CD3 (2C11, 1 μg/ml). Cells were harvested for CFSE dilution analysis after 72 h of culture. For in vivo suppression assays, on day −1, recipient B6D2F1 mice received 1100 cGy total body irradiation ([137Cs] source at 108 cGy/min), split into 2 doses separated by 3 h. On day 0, recipients were transplanted with 5 × 106 BM cells (B6, CD45.2+) with or without 0.7 × 106 nTreg from saline or G-CSF–treated B6.Foxp3-eGFP donors. On day 2, recipients were transplanted with 0.7 × 106 CellTrace violet proliferation dye (Invitrogen)–labeled BioMag-purified total T cells from anti-CD25 mAb (PC61)–treated (250 μg i.p. on days −3 and −1) B6.Ptprca (CD45.1+). After 72 h, spleens were harvested, and violet dye dilution was assessed by FACS in the CD4+ and CD8+ CD45.1+ T cell compartments (12).

In vivo luminescence imaging

Recipients were injected with d-luciferin (0.5 mg) s.c. Five minutes later, anesthetized animals were imaged using the Xenogen imaging system (Xenogen IVIS 100; Caliper Life Sciences) to determine Treg expansion. Data were analyzed with Living Image Version 4 software (Xenogen).

Real-time PCR

Total RNA was extracted and prepared from sort-purified cell populations and IL-10R mRNA levels quantified as previously described (13). All measurements were normalized against the expression of the housekeeping gene, β2-microglobulin.

RNA microarray Treg trascriptome profiling

Splenic CD3+CD4+GFP+ Treg from saline (n = 4)- or G-CSF (n = 4)–treated B6.Foxp3-eGFP mice were sort purified (FACSAria; BD Biosciences Pharmingen) and mRNA extracted using a Picopure kit (Life Technologies) as per the manufacturer’s instructions. Biotinylated cRNA was prepared with the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX). Illumina MouseWG-6 v2.0 arrays were hybridized, washed and scanned with iScan according to Illumina standard processes and processed from raw images with Beadarray package for R and Bioconductor (14). Probes were filtered for quality, reannotated (15), and gene set enrichment analysis was performed using CAMERA for R (16).

Statistical analysis

Survival curves were plotted using Kaplan–Meier estimates and compared by log-rank analysis. A p value < 0.05 was considered statistically significant. Data are presented as mean ± SEM.

Results

The immunomodulatory properties of G-CSF on donor T cell function is a result of effects on both hematopoietic and nonhematopoietic tissue

G-CSF is increasingly recognized to mediate unexpected and diverse effects on nonhematopoietic tissue. To study which cells contribute to the effects of stem cell mobilization with G-CSF, we generated B6 chimeras in which nonhematopoietic tissue was wild-type (WT) or G-CSFR deficient (G-CSFR−/−) in conjunction with hematopoiesis that was either WT or G-CSFR−/− as illustrated in Fig. 1A. Of note, comparison of splenic T cells from naive WT and G-CSFR−/− mice demonstrated no difference in the number or frequency of naive or memory populations within the splenic CD4+ or CD8+ T cell compartments based on CD44 and L-selectin expression. The frequency and number of nTreg were also equivalent. In addition, TCR ligation with CD3 mAb induced similar frequencies of IFN-γ and TNF-producing cells within the CD4 and CD8 T cells (Supplemental Fig. 1), indicating that there is no intrinsic defect in T cell development or Th1/Tc1 cytokine production in the absence of G-CSFR signaling at steady state. The chimeras were then left 4 mo to reconstitute at which time >95% of hematopoietic tissue was of donor origin (17). Reconstituted chimeras were treated with G-CSF and donor T cells were purified and added to T cell–depleted spleen from naive B6.WT animals. The combined grafts were then transplanted into lethally irradiated B6D2F1 animals. The recipients of grafts that included T cells from mobilized donors in which only the hematopoietic compartment was WT had delayed GVHD mortality (Fig. 1B). In contrast, GVHD mortality was rapid in recipients of donor T cells where the hematopoietic compartment was deficient of the G-CSFR, irrespective of the G-CSFR expression status of the nonhematopoietic compartment, confirming that the majority of the protective effects of G-CSF were via direct effects on hematopoietic cells. However, when hematopoiesis was WT, the ability of G-CSF to signal through nonhematopoietic tissue provided additional protection, suggesting the presence of a second indirect mechanism.

FIGURE 1.
FIGURE 1.

G-CSF modulates the function of T cells through both hematopoietic and nonhematopoietic compartments. (A) BM chimeras were generated as outlined by transplanting T cell–depleted marrow from B6.WT or B6.G-CSFR–/– animals into B6.WT or B6.G-CSFR–/– recipients following 1000-cGy irradiation and allowing 4 mo for full reconstitution. These combinations of chimeras were then treated with G-CSF and donor T cells purified to >90% and transplanted with WT T cell–depleted spleen as a stem cell source into lethally irradiated (1100 cGy) B6D2F1 recipients. (B) Survival by Kaplan–Meier analysis. **p < 0.002 for recipients of T cells from B6.G-CSFR–/– → B6.WT and B6.G-CSFR–/– → B6.G-CSFR–/– chimeras versus B6.WT → B6.G-CSFR–/– and B6.WT → B6.WT chimeras. *p < 0.05 for recipients of T cells from B6.WT → B6.G-CSFR–/– versus B6.WT → B6.WT chimeras. Data pooled from two replicate experiments. Eighty-eight percent of recipients of T cell–depleted grafts alone (n = 8) survived the period of observation without evidence of GVHD (data not shown). (C) B6.WT donors were mobilized with G-CSF or AMD3100 or were untreated. T cells were purified from each cohort of donors and added to T cell–depleted splenocytes. Survival by Kaplan–Meier analysis; ***p < 0.0001 for recipients of T cells from G-CSF–mobilized donors versus AMD3100 or no mobilization (n = 11–16, combined from two experiments).

Alternative methods of stem cell mobilization do not attenuate donor T cell function

To investigate whether the modulation of donor T cell alloreactivity by G-CSF was a result of the stem cell mobilization process per se or a specific effect of the G-CSF molecule itself, we mobilized donors with AMD3100, a selective antagonist of the CXCR4 receptor, as described previously (7). Following AMD3100 mobilization, T cells were purified and compared with those from donors mobilized with G-CSF for their capacity to induce GVHD. Only T cells from animals mobilized with G-CSF had an attenuated capacity to induce GVHD because there was no protection afforded by AMD3100 mobilization (Fig. 1C).

Modulation of donor T cell function by G-CSF requires direct signaling of the T cell

We next investigated whether the protective effects of G-CSF signaling within the hematopoietic compartment was via direct or indirect effects on the T cell. To answer this question definitively in vivo, we generated mixed BM chimeras where hematopoiesis was predominantly WT, but where a small (20%) fraction of cells was also G-CSFR−/− (Fig. 2A). In this way, the reconstituted WT hematopoietic compartment can respond normally and expand in response to G-CSF so that any indirect downstream protective effects could be imparted on the G-CSFR−/− T cells. In contrast, if the effects of G-CSF on T cells required direct signaling, the G-CSFR−/− T cells would not be modulated and would thus induce fulminant GVHD. Surprisingly, the protective effects of G-CSF imparted during stem cell mobilization was entirely dependent on direct signaling through the T cell because WT but not G-CSFR−/− donor T cells were modulated by G-CSF, despite the presence of predominantly normal (WT) hematopoiesis (Fig. 2B).

FIGURE 2.
FIGURE 2.

The modulation of T cell function by G-CSF requires direct signaling of the T cell. (A) BM chimeras were generated as outlined by transplanting T cell–depleted marrow from B6.WT (CD45.1+) and B6.G-CSFR−/− (CD45.2+) animals into B6.WT (CD45.1+) recipients following 1000 cGy and allowing 4 mo for full reconstitution. These chimeras were then treated with G-CSF and B6.WT (CD45.1+) or B6.G-CSFR−/− (CD45.2+) T cells purified to >98% by magnetic bead depletion and FACS and transplanted with WT T cell–depleted spleen as a stem cell source into lethally irradiated (1100 cGy) B6D2F1 recipients. Unfractionated T cells from control-treated chimeras were >80% WT. (B) Survival by Kaplan–Meier analysis. One hundred percent of recipients of T cell–depleted grafts alone (n = 13) survived the period of observation without evidence of GVHD (data not shown). n values in GVHD groups are shown in the figure. ***p < 0.0001 for recipients of B6.WT T cells versus B6.G-CSFR−/− T cells from G-CSF–treated chimeras. Data pooled from three replicate experiments.

Donor T cell–derived IL-10 is not required for the promotion of regulatory function by stem cell mobilization with G-CSF

IL-10 is an important immunomodulatory cytokine known to be induced by stem cell mobilization (18), and we have previously shown that donor T cells from IL-10–deficient mice treated with G-CSF have an enhanced capacity to induce GVHD as compared with T cells from G-CSF–treated WT mice (5, 19). We therefore next generated chimeras in which hematopoietic tissue was WT, IL-10−/−, or a mixture of WT + IL-10−/− (Fig. 3A) to delineate whether donor T cells were required to secrete this cytokine following G-CSF mobilization to exert regulatory function. B6.WT + B6.IL-10−/− mixed chimeras were generated to permit the purification of donor T cells that could be modified by IL-10 generated from hematopoietic cells following G-CSF administration but unable to produce IL-10 themselves. The transplantation of grafts containing purified T cells from these chimeras demonstrated that IL-10 production was not required for T cells signaled by G-CSF to have attenuated ability to induce GVHD (Fig. 3B). Furthermore, G-CSF–modulated T cells were able to regulate the GVHD induced by naive T cells, regardless of their ability to secrete IL-10 (Fig. 3C).

FIGURE 3.
FIGURE 3.

IL-10 generation from donor T cells is not required for the protection following G-CSF signaling. (A) BM chimeras were generated as outlined by transplanting T cell–depleted marrow from B6.WT (CD45.1+) and/or B6.IL-10−/− (CD45.2+) animals into B6.WT (CD45.1+) recipients following 1000 cGy and allowing 4 mo for full reconstitution. These combinations of chimeras were then treated with G-CSF or control diluent and T cells purified to >90%. In mixed chimeras where both B6.WT (CD45.1+) and B6.IL-10−/− (CD45.2+) marrow was cotransplanted, B6.WT (CD45.1+) and B6.IL-10−/− (CD45.2+) T cells were purified to >98% by FACS and transplanted with WT T cell–depleted spleen as a stem cell source (n = 6/group) (B) or T cell replete spleen (n = 8–12/group) (C), to include GVHD-inducing naive T cells, into lethally irradiated B6D2F1 recipients. Survival by Kaplan–Meier analysis. *p < 0.02 for recipients of T cells from any G-CSF–treated chimera versus control-treated chimeras. ***p < 0.001 for recipients receiving B6.IL-10−/− T cells from G-CSF–treated WT + IL-10−/− BM mixed chimeras versus recipients of control grafts only.

Because G-CSF treatment of IL-10–deficient mice failed to elicit the suppressive phenotype that occurs in WT mice, but T cell–derived IL-10 was not required for attenuation of GVHD, we considered the possibility that host nonhematopoietic cells, stimulated by G-CSF, may provide a source of IL-10 required for modulation of the T cell compartment. Therefore, we next used donor chimeras in which nonhematopoietic tissue was IL-10−/− or WT (Fig. 4A). When donor T cells were purified from these donors following G-CSF administration, T cells from donors in which nonhematopoietic tissue was WT had a significantly attenuated capacity to induce GVHD (Fig. 4B). In contrast, IL-10 derived only from hematopoietic tissue (in mice in which only the nonhematopoietic tissue is IL-10 deficient) had only a modest effect on donor T cell function following G-CSF administration. Importantly, these data demonstrate that the previously seen modulation of donor T cell function following direct signaling by G-CSF was largely lost in the absence of IL-10 generation from nonhematopoietic cells, and modification of donor T cell function by G-CSF requires signaling by IL-10 derived from nonhematopoietic tissue (in response to G-CSF) in conjunction with direct signaling by G-CSF. In contrast, the donor T cell does not need to secrete IL-10 itself at anytime in this process.

FIGURE 4.
FIGURE 4.

Modulation of T cell function by G-CSF requires the induction of IL-10 from nonhematopoietic cells. (A) BM chimeras were generated as outlined by transplanting T cell–depleted BM from B6.WT or B6.IL-10−/− animals into B6.WT or B6.IL-10−/− recipients following 1000 cGy and allowing 4 mo for full reconstitution. These combinations of chimeras were then treated with G-CSF and donor T cells purified to >90% and (B) transplanted along with WT T cell–depleted spleen as a stem cell source into lethally irradiated (1100 cGy) B6D2F1 recipients. Survival by Kaplan–Meier analysis. Data pooled from two replicate experiments. Eighty-six percent of recipients of T cell–depleted grafts alone (n = 7) survived the period of observation without evidence of GVHD (data not shown). n values in GVHD groups are shown in the figure. ***p < 0.0001 for recipients of T cells from G-CSF–treated B6.WT → B6.WT and B6.IL-10−/− → B6.WT chimeras versus all other chimeras. *p < 0.02 for recipients of T cells from G-CSF–treated B6.WT → B6.IL-10−/− versus B6.IL-10−/− → B6.IL-10−/− chimeras.

G-CSF modifies nTreg function

Because IL-10 is an important modifier of donor T cell function during stem cell mobilization, we next sought to confirm that the IL-10R was indeed expressed by T cells. As shown in Fig. 5A, the IL-10R was expressed by all T cell subsets as determined by real-time PCR, with the highest levels seen on the CD4+CD25+ nTreg subset. Because the functional relevance of the nTreg in GVHD following G-CSF mobilization is unknown we depleted >90% of these cells within the donor graft prior to transplantation by administration of the ant-CD25 (PC61) Ab or removal of Foxp3+ nTreg from B6.Foxp3.eGFP donors by FACS eGFP exclusion (Fig. 5B). As shown in Fig. 5C and 5D, G-CSF–mobilized grafts depleted of nTreg had a significantly enhanced propensity to induce GVHD. The same population from control-treated grafts played no role in preventing GVHD in the absence of prior treatment with G-CSF (data not shown). As expected, the number of CD4+ Treg in the spleens and lymph nodes at D7 posttransplant was significantly reduced in the recipients of Foxp3.eGFP Treg–depleted grafts (Fig. 5E), although a small but significantly reduced number of CD4+-induced Treg had emerged. However, the recently described Foxp3+CD8+-induced Treg population, which emerges early after transplant (20), was similar in recipients of nTreg replete and depleted grafts. Importantly, the depletion of Treg did not negate all the protective effects of G-CSF, consistent with the established effects of G-CSF on myeloid cells and Th2 differentiation in non-Treg subsets (4).

FIGURE 5.
FIGURE 5.

G-CSF modifies nTreg function. (A) Relative expression of IL-10R mRNA in sort-purified T cell populations as described in Materials and Methods. (B) Depletion of nTreg by anti-CD25 mAb (PC61) at days −3 and −1 or Foxp3-eGFP depletion by FACS as detailed. (C) Survival by Kaplan–Meier analysis of lethally irradiated B6D2F1 recipients receiving splenic grafts from G-CSF–treated donors that had been pretreated with anti-CD25 or control Ab. *p = 0.012 for recipients of grafts from G-CSF and control Ab–treated donors versus G-CSF and anti-CD25 Ab–treated donors (n = 24/group, combined from three experiments). (D) Survival by Kaplan–Meier analysis of lethally irradiated B6D2F1 recipients receiving splenic grafts from G-CSF–treated B6.Foxp3-eGFP donors that had or had not been depleted of eGFP+ cells by FACS. All recipients of TCD grafts survived (n = 7) without GVHD. *p = 0.017 for recipients of grafts from G-CSF B6.Foxp3-EGFP depleted versus nondepleted donors (n = 13–16/group, combined from two experiments). (E) At day 7 posttransplant, CD4+eGFP+ and CD8+eGFP+ Treg were enumerated in the spleen, mLN, and pLN from recipients of Foxp3-eGFP+ replete or depleted grafts from G-CSF–treated B6.Foxp3-GFP donors. Data are represented as mean ± SEM.

G-CSF mobilization promotes nTreg expansion and alters their mRNA transcriptome

Because nTreg were shown to be an important mediator of the protection afforded by G-CSF mobilization, we next compared the number, phenotype and function of nTreg from saline- versus G-CSF–treated donors. G-CSF treatment induced a small but significant increase in the absolute number of CD4+GFP+ nTreg compared with the saline-treated group but did not alter CD4+GFPneg effector cell number, resulting in a significant increase in the Treg:T effector cell ratio (Fig. 6A). However, phenotypic analysis of Treg-associated cell surface markers failed to identify any differentially expressed surface Ags on splenic CD4+GFP+ nTreg from saline- versus G-CSF–treated donors (Fig. 6B). Similarly, in both in vitro and in vivo Treg suppression assays, there was no measurable difference in the suppressive function of (CD4+GFP+) nTreg sort-purified from saline or G-CSF–treated B6.Foxp3.eGFP donors (Fig. 6C and 6D, respectively). We next examined the effect of G-CSF mobilization on Treg survival posttransplant. For these studies, we used B6.Foxp3-Luci mice (that express luciferase off of the Foxp3 promoter) as donors to facilitate tracking and quantification of Treg expansion post transplant. Bioluminescence imaging at days 3 and 8 posttransplant, and FACS analysis confirmed that the numbers of CD4+ Treg in recipients of grafts from G-CSF were significantly and specifically increased in comparison with recipients of saline-treated B6.Foxp3-Luci donors (Fig. 6E, 6F).

FIGURE 6.
FIGURE 6.

G-CSF enhances nTreg expansion pre- and posttransplant. (A) Comparison of absolute number of splenic CD3+CD4+GFP− and CD3+CD4+GFP+ T cell populations in saline or G-CSF–treated B6.Foxp3-eGFP mice (n = 11/group). (B) Phenotype of splenic CD3+CD4+GFP+ Treg from saline or G-CSF–treated B6.Foxp3-eGFP mice. (C) Following saline or G-CSF treatment, splenic CD4+Foxp3+ cells were sort purified from B6.Foxp3-eGFP mice and cultured with CFSE-labeled sort-purified CD4+CD25−CD45.1+ B6.WT T cells, CD45.2+ B6.WT DC, and 1 μg/ml CD3. CFSE dilution was examined in CD45.1+ cells after 72 h. FACS plots representative of one of two separate experiments. (D) B6D2F1 recipients were lethally irradiated and 24 h later transplanted with or without sort-purified CD4+Foxp3+ Treg (7 × 105) from saline or G-CSF–treated B6.Foxp3-eGFP. Violet dye–labeled unfractionated CD45.1+ CD3+ T cells (7 × 105) were injected 2 d later, and their proliferation was monitored in the spleen by violet dye dilution 3 d later. (E) Lethally irradiated B6D2F1 recipients were transplanted with grafts from saline of G-CSF–treated B6.Foxp3.Luci donors (luciferase, GFP, and the DT receptor–driven off of the Foxp3 promoter). Recipients were imaged at days 3 and 8 after transfer and bioluminescence quantified. (F) On day 8 following transplant, spleens were harvested and splenic CD4+ and CD8+ eGFP+ or eGFP−CD3+ T effector and Treg cells enumerated. Data are representative of one of two similar experiments and presented as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

To gain further insight into the modulatory effects of G-CSF on nTreg, we performed genome wide microarray mRNA expression analysis. Analysis of gene expression shows distinct expression profiles in highly purified nTreg from saline- and G-CSF–treated B6.Foxp3-eGFP mice (Supplemental Table I; GEO submission GSE54616, http://www.ncbi.nlm.nih.gov/geo/). Principal component analysis of the full set of 24291 probes sets remaining after specificity and quality filtering, show clear separation of these two populations (data not shown). Similarly, unsupervised hierarchical clustering demonstrated distinct gene expression programs with clear blocks of differentially expressed genes (Fig. 7A). Of note, G-CSF induced the upregulation of several genes or gene families implicated in enhanced nTreg survival. In this regard, STAT3 is an established inhibitor of nTreg stability (21), and the expression of protein inhibitor of activated STAT3 (Pias3) was significantly increased in nTreg from G-CSF–treated animals, which may then result in greater nTreg stability. In addition, a number of autophagy-related gene (Atg) family members (Atg2a, Atg16l1, and Atg4c) were also found to be upregulated in nTreg from G-CSF–treated donors. Autophagy is a cell survival mechanism which involves recycling of intracellular components to generate nutrients and energy in times of stress such as during stem cell mobilization and transplantation. Thus, the induction of this pathway may contribute to the observed higher Treg numbers by enhancing their survival. Furthermore, β-catenin stablilization has been shown to extend nTreg survival (22, 23), and in this study, gene set enrichment analysis revealed a significant downregulation of the β-catenin destruction complex in nTreg from G-CSF–treated compared with saline-treated mice. Interestingly, the gene most differentially expressed in response to G-CSF treatment was the AT-hook transcription factor AKNA (Fig. 7B, Supplemental Table I). AKNA is expressed by multiple lineages of immune cells including T and B cells and plays a crucial role in the regulation of inflammatory responses (24). In B cells, AKNA binds to the promoter region of both the CD40 and CD40L genes to coordinate maturation (25). Because G-CSF upregulated AKNA mRNA in nTreg, we next asked whether the putative downstream molecule CD40 also contributes to Treg development and survival in vivo. Intriguingly, examination of the T cell compartment in CD40−/− mice demonstrated a preferential decrease of CD4+Foxp3+ T cells relative to CD4+Foxp3neg T cells in both the thymus and spleen, consistent with a dominant role for CD40–CD40L interactions in the generation and maintenance of Treg (Fig. 7C).

FIGURE 7.
FIGURE 7.

G-CSF modulates the nTreg mRNA transcriptome. (A) RNA micorarray analysis of splenic CD4+GFP+ nTreg isolated from saline or G-CSF–treated B6.Foxp3-GFP mice. Heat map of 1500 most differentially expressed probe sets after hierarchical clustering with columns showing distinct clustering of nTreg from saline- or G-CSF–treated donors. (B) Relative expression of Akna mRNA by Treg from saline- and G-CSF–treated WT animals. Data was normalized using the quantile method. (C) Frequency and absolute numbers of T cell subsets in the thymus and spleen in WT and CD40−/− mice. Combined data from two experiments presented as mean ± SEM (n = 6). *p < 0.02, **p < 0.01, ****p < 0.0001.

Discussion

G-CSF mobilization is known to elicit broad immunomodulatory effects on multiple cell types including cells of both myeloid and lymphoid lineages. In this regard, G-CSF modulation of T cell cytokine production and differentiation is thought to be critical for the improved outcomes associated with patients receiving G-CSF–mobilized PBMCs compared with standard BM (24). Stem cell mobilization with G-CSF is known to profoundly influence T cell differentiation. These effects include the promotion of Th2 and Th17 differentiation at low and high doses (3, 26) without impairment in cytolytic responses and thus graft-versus-leukemia effects (25). However the actual mechanism by which G-CSF alters T cell function remains to be elucidated. The protective effects of G-CSF mediated through hematopoietic tissue may be via direct effects on T cells or as is the current paradigm, indirect effects, downstream of soluble products, including at least IL-10, generated by expanded myeloid and nonhematopoietic cells. The ability of G-CSF to directly stimulate T cells is controversial but at least one study has demonstrated the presence of G-CSFR on T cells when studied at a single cell level accompanied by an ability to respond to G-CSF in vitro, as determined by GATA3 expression (27). Nevertheless, the expression of the G-CSFR on T cells is extremely low at steady state, and to our knowledge, direct effects of G-CSF on T cells in vivo have never been studied.

In this study, we demonstrate that although G-CSF signaling within both the hematopoietic and non-hematopoietic compartment can modulate T cell function. These effects are maximal following direct signaling of the T cell by G-CSF in conjunction with IL-10 that is principally derived from nonhematopoietic tissue. Thus, although the T cell needs to be exposed to IL-10 in vivo during stem cell mobilization, it does not need to generate IL-10 itself thereafter. Systemic levels of IL-10 are increased after clinical stem cell mobilization with G-CSF (18), which we have also noted (data not shown). Previous reports have demonstrated that G-CSF administration can result in the expansion of a Tr1 population (as opposed to the nTreg studied here) (6, 18) which suppress in vitro via IL-10. While in vitro studies demonstrate that monocytes can be an important source of IL-10 after G-CSF (18, 19), our data confirm that in vivo, it is in fact radio-resistant cells that are the critical sources of this immunomodulatory cytokine. The cells involved are likely to be radio-resistant tissue macrophages, NK cells, or the stromal differentiated products thereof (e.g., fibroblasts, kupffer cells, microglia) or vascular endothelium that are known to express the G-CSFR (28).

From a clinical perspective, the modulatory effects of G-CSF are restricted to situations where stem cells are mobilized with G-CSF and are not a feature of stem cell mobilization per se. Thus alternative means of mobilization that involve the direct targeting of the CXCR4 axis do not alter T cell function and would be predicted to have a negative effect on allogeneic stem cell transplant outcome if used in isolation of G-CSF, an important potential issue now these agents are in routine practice.

A significant component of the protective effects of G-CSF is mediated by nTreg that are disproportionately (to effector cells) numerically increased both during stem cell mobilization and early after transplantation. Intriguingly, G-CSF does not result in major changes in the phenotype or measurable suppressive qualities of nTreg in vitro however the intracellular transcriptome and suppressive effects in vivo are profoundly modified. In addition to the molecules associated with Treg survival mentioned in the results, G-CSF significantly upregulated molecules known to be involved in pathways that regulate T cell function including neuropilin-1 (29), Hdac4 (30), Bcl-6 (31), Jak1 (32), CD83 (33), Atg2a (34), Tgfb1 (35), chd4 (36), ccr5 (37). Similarly, DNAM-1 (38), pim1 (39), IER3 (40), and IL-10 were downregulated, the latter consistent with the finding in this study that nTreg do not need to make IL-10 themselves for effective function. Taken together, these data confirm profound transcriptional regulation by G-CSF, an effect that our data suggests may at least in part mediated by direct receptor ligation.

The G-CSF–induced increase in AKNA mRNA expression by Treg is particularly intriguing given the established role for this transcription factor in the control of inflammation (24). In addition, mice deficient in AKNA are growth retarded, develop systemic inflammation and die early in the neonatal period (24), a phenotype that bears a remarkable similarity to the scurfy phenotype that occurs as a result of Foxp3 or Treg deficiency (41). Treg are known to suppress autoimmune disease and the suppressive function and number of Treg are reduced during active disease. Although AKNA is reported to be expressed by CD4 T cells, no studies thus far have characterized the expression or function of AKNA in Treg. Notably, AKNA was reported to be diminished in CD4+ T cells from patients with the autoimmune disease Vogt-Koyanagi-Harada syndrome (42); however, the CD4 T cell population used for protein analysis in those studies contained both effector cells and Treg. Thus, it is tempting to speculate that disrupted AKNA expression in Treg may contribute to diminished tolerance in the settings of autoimmunity and transplantation. One of the reported functions of AKNA is to promote the expression of CD40 and CD40L, suggesting that signaling through this pathway may be important in Treg development and survival. In support of this, we demonstrate a significant impairment in Treg development and maintenance in CD40-deficient mice. Further investigations are under way to validate AKNA-dependent CD40–CD40L signaling as an important pathway in nTreg survival and to determine the mechanism by which CD40 signaling contributes to Treg biology.

The current widely accepted paradigm is that G-CSF modifies T cells during stem cell transplantation indirectly, via effects on DC (18, 43) and/or monocytes (19, 44). Although it is clear that these innate cells are modified by G-CSF and the populations themselves can influence adaptive immunity during allogeneic SCT, direct evidence for their role as the dominant pathway altering T cell responses is lacking. Instead, our data suggest that the direct effects of G-CSF on the T cell, in conjunction with the induction of IL-10 from nonhematopoietic tissue is a major mechanism by which this cytokine influences T cell function and subsequently modifies disease pathology. Although the expression of the G-CSFR on nonmyeloid cells has been reported by studies using mRNA or specific Abs, the validity of the latter has at least been called into question (45). The data in this paper report biological effects in vivo in relation to exposure of T cells to G-CSF that do, or do not express the G-CSFR and so are free of these technical limitations. It must be noted however, the expression of the G-CSFR by T cells is undoubtedly very low and any effects of G-CSF are presumably only seen when G-CSF levels are high enough to saturate all other cellular receptors. Nevertheless, this is now a common clinical scenario and the ability to modulate both innate and adaptive immune responses has obvious potential clinical implications. For patients receiving allogeneic stem cell products mobilized with G-CSF, these regulatory effects are obviously highly desirable in helping to separate GVHD and graft-versus-leukemia responses. Finally, the demonstration of the critical requirement for the activation of G-CSF and IL-10 signaling pathways for modulation of T cell function during G-CSF stem cell mobilization raises the question as to whether G-CSFR and/or IL-10/IL-10R polymorphisms might contribute to the striking individual variability in responsiveness to G-CSF therapy and GVHD outcomes. Thus, future studies examining the genetic polymorphisms in these genes in G-CSF peripheral blood stem cell donors could serve to identify polymorphisms associated with poor responders who might benefit from adjunct therapy with other mobilizing agents such as AMD3100. Importantly, these studies could also provide unique insight into why some patients receiving G-CSF stem cell grafts develop significant GVHD and others less so.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by grants from the National Health and Medical Research Council and Cancer Council Queensland. K.P.A.M. is a Cancer Council Queensland Senior Research Fellow. G.R.H. is a National Health and Medical Research Council Australian Fellow and Queensland Health Senior Clinical Research Fellow. K.A.M. is a National Health and Medical Research Council Clinical Training Fellow.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    B6
    C57BL/6
    BM
    bone marrow
    DC
    dendritic cell
    GVHD
    graft-versus-host disease
    nTreg
    natural regulatory T cell
    Treg
    regulatory T cell.

  • Received September 5, 2013.
  • Accepted January 30, 2014.

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