Plerixafor Compared with G-CSF Alloreactivity of T Cells Mobilized

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Differences in the Phenotype, Cytokine Gene Expression Profiles, and In Vivo Alloreactivity of T Cells Mobilized with Plerixafor Compared with G-CSF This information is current as of September 23, 2021. Andreas Lundqvist, Aleah L. Smith, Yoshiyuki Takahashi, Susan Wong, Erkut Bahceci, Lisa Cook, Catalina Ramos, Abdul Tawab, J. Philip McCoy, Jr., Elizabeth J. Read, Hanh M. Khuu, Charles D. Bolan, Jungnam Joo, Nancy Geller, Susan F. Leitman, Gary Calandra, Cynthia Dunbar, Roger Kurlander and Richard W. Childs Downloaded from J Immunol 2013; 191:6241-6249; Prepublished online 15 November 2013; doi: 10.4049/jimmunol.1301148

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Differences in the Phenotype, Cytokine Gene Expression Proﬁles, and In Vivo Alloreactivity of T Cells Mobilized with Plerixafor Compared with G-CSF

Andreas Lundqvist,*,† Aleah L. Smith,* Yoshiyuki Takahashi,‡ Susan Wong,* Erkut Bahceci,x Lisa Cook,* Catalina Ramos,* Abdul Tawab,{ J. Philip McCoy, Jr.,* Elizabeth J. Read,‖ Hanh M. Khuu,‖ Charles D. Bolan,‖ Jungnam Joo,‖ Nancy Geller,‖ Susan F. Leitman,‖ Gary Calandra,# Cynthia Dunbar,* Roger Kurlander,{ and Richard W. Childs*

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34 cells into circulation. Recently, plerixafor has been used Downloaded from as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression + levels in CD4 T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In http://www.jimmunol.org/ contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8+ T cells and altered expression levels of 16 cytokine-associated genes in CD3+ T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC- matched, minor histocompatibility–mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell–mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF. The Journal of by guest on September 23, 2021 Immunology, 2013, 191: 6241–6249.

or adults with malignancies, mobilized peripheral blood quate numbers of peripheral blood stem cells (PBSCs) from progenitor cells (PBPC) are the preferred source of he- healthy donors, 5–10% of patients will mobilize poorly, necessi- F matopoietic stem cells (HSCs) for autologous transplants tating multiple large-volume apheresis or bone marrow harvesting and matched sibling allogeneic transplants because PBPCs engraft (2). Although G-CSF is generally well tolerated in healthy donors, faster than bone marrow–derived progenitor cells and are techni- it frequently causes bone pain, headache, myalgia, and in rare cally easier to collect (1). The administration of G-CSF results in circumstances, life-threatening adverse effects such as stroke, myo- successful mobilization of PBPCs in most healthy donors. Al- cardial infarction, and splenic rupture (3). The number of lym- though most regimens using G-CSF succeed in collecting ade- phocytes contained in G-CSF mobilized PBPC grafts is ∼10–15 times higher than BM grafts (4, 5). Despite containing substan- tially higher numbers of T cells, several prospective, randomized *Hematology Branch, National Heart, Lung and Blood Institute, National Institutes trials have shown that the incidence of acute graft-versus-host of Health, Bethesda, MD 20892; †Department of Oncology-Pathology, Karolinska Institutet, Stockholm S-17176, Sweden; ‡Nagoya University, Graduate School of disease (GVHD) is not higher in recipients of G-CSF mobilized x Medicine, Nagoya 466-8550, Japan; Section of Medical Oncology, Yale University PBPC allografts compared with BM allografts (4, 6–10). Although School of Medicine, New Haven, CT 06520; {Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892; ‖Department of a number of mechanisms have been postulated to account for this Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD phenomenon, alterations in cytokine-related genes in T cells mo- # 20892; and Sanofi Oncology, Cambridge, MA 02139 bilized with G-CSF including a polarization toward a type 2 cy- Received for publication May 13, 2013. Accepted for publication October 7, 2013. tokine (Th2) response is thought to play a pivotal role in reducing This work was supported by the intramural research program of the National Insti- the ability of T cells to trigger acute GVHD (11–14). The distinct tutes of Health, National Heart, Lung, and Blood Institute, Hematology Branch and cytokine production profile of Th2 cells has also been shown in the Swedish and European Research Councils (A.L.). animal models to contribute to the pathogenesis of chronic GVHD, Address correspondence and reprint requests to Dr. Richard Childs, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, whichhasbeenshowninrandomizedtrialstooccurmorecom- 10CRC, 3-5330, Bethesda, MD 20892. E-mail address: [email protected] monly with G-CSF mobilized transplants compared with bone mar- The online version of this article contains supplemental material. row transplants. Abbreviations used in this article: a-HCT, allogeneic hematopoietic cell transplanta- Plerixafor (Mozobil) is a bicyclam compound that inhibits the tion; GVHD, graft-versus-host disease; KLS, c-Kit+/Lin-/sca-1+; NHLBI, National interaction of stromal cell–derived factor-1 (SDF-1) to its cognate Heart, Lung and Blood Institute; PBPC, peripheral blood progenitor cell; PBSC, + peripheral blood stem cell; SDF-1, stromal cell–derived factor-1; Treg, regulatory receptor CXCR4 (15, 16). CXCR4 is present on CD34 hemato- T cell. poietic progenitor cells, and its interaction with SDF-1 plays www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301148 6242 EFFECT OF PLERIXAFOR MOBILIZATION ON LYMPHOCYTES a pivotal role in the homing of CD34+ cells in the bone marrow whole blood was incubated for 30 min at 4˚C with Abs. Each experiment (17, 18). Inhibition of CXCR4–SDF-1 binding by plerixafor re- was performed using the appropriate isotype controls. After incubation, leases CD34+ cells into the circulation, allowing them to be col- erythrocytes in these samples were lysed (Whole Blood Lysing Reagent; Coulter, Fullerton, CA), washed twice, and then fixed with paraformal- lected easily by apheresis (19). The addition of plerixafor to G-CSF dehyde. All samples were analyzed by using a CYAN MLE cytometer (Dako- has a potent synergistic effect on the mobilization PBPCs in pa- Cytomation, Ft. Collins, CO). Summit software (Dako-Cytomation) was used tients undergoing autologous transplantation. Plerixafor is approved for data acquisition and analysis. Mixtures of six fluorochrome-labeled mAbs in the United States in combination with G-CSF for hematopoietic were used in the same tube to assess the phenotype in different cell populations. CD3-PE-Cy7, CD4-allophycocyanin-Cy7, CD8-PerCP, CD16-allo- stem cell mobilization and collection for subsequent autologous phycocyanin-Cy7, CD56-PE-Cy5, CD19-allophycocyanin, and CD34-FITC transplantation in non-Hodgkin’s lymphoma and multiple myeloma were used to gate T cell, B cell, NK cell, and hematopoietic progenitor cell patients. populations. CD57-FITC, CD27-FITC, CD62L-FITC, HLA-DR–FITC, Plerixafor given as a single agent can also mobilize substantial CD45RA-FITC, pan-TCRgd-FITC, CD38-FITC, CD69-FITC, CXCR4-PE, numbers of hematopoietic progenitor cells into the circulation. A CD25-PE, CCR7-PE, CD71-PE, CD45RO-PE, and pan-TCRab-PE were used to assess the phenotype of CD4+ T cells and CD8+ T cells. All the phase one, dose-escalating study in healthy donors demonstrated mAbs were purchased from BD Biosciences (San Diego, CA) except for that large numbers of CD34+ cells were rapidly mobilized in pan-TCRgd-FITC and pan-TCRab-PE (Coulter, Fullerton, CA). healthy donors after a single s.c. injection of plerixafor with the Assessment of Th1, Th2, and Th3 cytokine gene expression peak CD34+ mobilizing effects occurring 6–9 h after 240 mg/kg levels in donors mobilized with plerixafor versus G-CSF plerixafor in the absence of any dose-limiting toxicities (20). Importantly, side effects were mild and transient, and did not Gene expression analysis was performed on CD3+ and CD4+ T cells include bone pain, fever, and myalgias commonly observed after obtained from the blood or apheresis collections of healthy subjects at Downloaded from baseline and after mobilization with 240 mg/kg plerixafor or G-CSF alone G-CSF administration. Based on these data, investigators have (10 mg/kg/day 3 5 d). Fresh samples were Ficolled (LSM; MP Bio- recently begun to explore the use of single-agent plerixafor to medicals), and CD3+ cells were isolated on fresh and thawed samples mobilize PBPCs for allogeneic transplantation, although its use in following manufacturers’ instructions using Human PanT Cell Isolation + this setting remains investigational. The first published study to Kit II (Miltenyi). CD4 T cell fractions were positively selected following manufacturers’ instructions using CD4 Microbeads (Miltenyi). Cryopre- use plerixafor alone to mobilize PBPCs for allogeneic transplan- 3 + served specimens were thawed in a 37˚C water bath, washed with 1 RPMI tation showed successful mobilization of CD34 cells in healthy 1640 and 10% heat inactivated FBS, and then were treated with DNase http://www.jimmunol.org/ stem cell donors and sustained trilineage hematopoietic reconsti- (Roche) for 30 min at 37˚C with the CD3+ and CD4+ T cell fractions being tution in 14 transplant recipients of plerixafor mobilized allografts isolated using the same procedure outlined earlier. RNA was isolated from CD3+ and CD4+ T cells using the RNeasy kit (21, 22). Leukapheresis products mobilized with plerixafor con- + + (Qiagen) according to the manufacturer’s instructions. Isolated CD3 RNA tained a higher number of CD3 T cells compared with G-CSF, was sent to SuperArray Bioscience Corporation where it was reverse tran- and in a limited analysis performed on four donors did not appear scribed and analyzed in quantitative real-time PCRs using the Syber Green to alter the phenotype of mobilized T cells. Because of its potential RT2 Profiler PCR Array Human Th1-Th2-Th3 plates. Baseline and post- use as a single mobilization agent in this setting, we performed a plerixafor or post–G-CSF samples were compared for each healthy subject. For assessment of Foxp3 gene expression levels from mobilized donors, detailed analysis of the phenotype, cytokine gene expression pro- RNA (1 mg) was obtained from purified CD4+ T cells. RNA was DNase- files, and immunological properties of T cells mobilized with pler- treated before reverse transcription using SuperScriptIII Reverse Tran- by guest on September 23, 2021 ixafor in healthy donors. Furthermore, we used an MHC-matched scriptase (Invitrogen). Five microliters of cDNA was added to a final mouse model of allogeneic PBSC transplantation to assess whether volume of 25 ml containing 13 Quantitech Multiplex PCR Kit (Qiagen), the phenotypic and genotypic differences observed in T cells mo- 0.4 mM of each forward and reverse primers for actin and Foxp3, and 0.2 mM of each probe. Actin and Foxp3 primer and probe sequences were bilized with plerixafor compared with G-CSF would impact trans- previously published (24, 25). All samples were run in triplicates on a plant outcome. Peltier Thermal Cycler PTC-200 (MJ Research) with a Chromo4 Contin- uous Fluorescence Detector 4. Thermal cycling conditions consisted of 95˚C Materials and Methods for 10 min followed by 94˚C for 1 min and 60˚C for 1 min (45 cycles). The Study design expression of Foxp3 was normalized to the actin levels within each individual sample, and the fold change from baseline was calculated for each Eleven healthy subjects had PBMCs collected at baseline and then were healthy subject. mobilized with 5 daily doses of s.c. G-CSF (10 mg/kg daily; National Heart, Lung and Blood Institute [NHLBI] protocols 04-H-0078, 99-H-0050, and Mixed lymphocyte reaction 99-H-0064) to analyze G-CSF mobilized cells. Two hours after the fifth The alloreactivity of mobilized cells collected at baseline and after pler- dose of G-CSF, subjects had PBMCs collected from the blood followed by ixafor mobilization were tested in an MLR by [3H]thymidine uptake and a 15- to 25-l apheresis procedure to collect mobilized cells. Twenty healthy CFSE dilution assays. Pooled and irradiated PBMCs from three healthy subjects underwent plerixafor mobilization on NHLBI protocols 04-H- volunteers were used as stimulators. For the thymidine uptake MLR, 5 3 0078 and 06-H-0179 to analyze plerixafor-mobilized cells. PBMCs were 105 responder mononuclear cells in 100 ml culture media were coincubated collected at baseline and then 6 h after a single s.c. injection of plerixafor with equal numbers of irradiated (50 Gy) stimulator cells in 96-well round- at a dose of 240 mg/kg (n = 12). Eight of these subjects also underwent a bottom plates. The culture media consisted of RPMI 1640 (Life Tech- 15- to 25-l apheresis beginning 6 h after plerixafor administration. Freshly nologies Life Technologies) supplemented with 5% human AB serum, collected apheresis products were analyzed by flow cytometry for cellular L-glutamine, gentamicin, and Hepes buffer. After 5 d, the wells were content. Cells were frozen in 10% DMSO for subsequent phenotypic, pulsed with [3H]thymidine (Amersham Pharmacia Biotech) at 1 mCi/well. genotypic, and in vitro and in vivo functional studies. Leukapheresis pro- After an additional 18-h incubation, the cells were harvested and thymi- cedures were performed using the model CS-3000 Plus (Baxter Healthcare, dine incorporation into DNA was quantified using a beta counter (Beckman Deerfield, IL) continuous-flow cell separator. Complete blood counts were Coulter). The results are reported as cpm. For the CFSE dilution assay assayed using an electronic counter, and CD34, CD3, and other cell counts MLR, responder cells were incubated in prewarmed working concen- were enumerated by flow cytometry, immediately before and after apher- trations of CFSE (Molecular Probes, Eugene, OR) in Dulbecco’s PBS esis, as previously described (23). (0.6 mM) for 15 min at 37˚C. The cells were then washed and resuspended Phenotypic analysis of cells in fresh, serum-free RPMI 1640, then were incubated with equal numbers of irradiated (50 Gy) stimulator cells for 5 d, and thereafter harvested and Six-color flow cytometry was used to analyze mononuclear cells obtained stained with propidium iodide, CD3-PerCP, and CD25-allophycocyanin from apheresis products or from peripheral blood after mobilization, as well mAbs (BD Pharmingen). Four-color flow cytometry was performed on a as from premobilization blood and blood obtained immediately before FACSCalibur dual-laser cytometer (Becton Dickinson, Mountain View, initiating apheresis. Peripheral blood was drawn by venipuncture into tubes CA). The precursor frequency of alloreactive T cells was estimated using containing heparin and stained within 8 h of collection. A 100-ml aliquot of FlowJo analysis software (Tree Star, Ashland, OR). The Journal of Immunology 6243

MHC-matched murine transplantation model (SPSS). Analysis of the real-time PCR arrays used a two-sample paired t 2 test to identify genes that were upregulated or downregulated after treat- Animals. Eight- to 12-wk-old BALB/c (H-2d, Ly9.1+, 7/4 ), B10.d2 (H-2d, 2 ment. The corresponding p values were evaluated based on the permutation Ly9.1 ,7/4+), and C57BL/6 (H-2b) mice were purchased from The technique. Because the number of distinct permutations is limited when the Jackson Laboratory (Bar Harbor, ME) and housed in microisolator cages sample size is small, the null statistics of nonsignificant genes was pooled at NHLBI pathogen-free facilities. Transplanted animals were provided to generate the permutation-based null distribution (27). The false dis- acidified water containing neomycin. All experiments were approved by covery rate was estimated using the q value function (28) given in R the NHLBI animal care and use committee (protocol no. HB-0112). language. A Student t test, Fisher’s exact test, or log-rank test was used to Mobilization. B10.d2 donor mice received either G-CSF (Amgen, Thousand assess the differences between mouse transplant groups. A p value ,0.05 3 Oaks, CA) given as s.c. injections daily 5d(10mg/mouse/day) or plerixafor was considered to be significant. (Genzyme, Cambridge, MA) given as a single s.c. injection (100 mg/mouse) to mobilize PBSCs. Mobilized PBSCs were collected from spleens harvested 6 h after the last injection for both groups. Spleens were also harvested from Results B10.d2 donor mice that received saline (HBSS) injections as controls. Mobilization with plerixafor in healthy subjects Flow cytometry. The phenotype of mobilized cells obtained from spleens (6 h after last injection) was evaluated after staining with fluorochrome-conjugated Apheresis products were collected from eight healthy subjects anti-CD3, CD4, CD8, CD19, DX5, Gr1, sca-1, c-Kit, CD25, and CD62L (BD mobilized with a single 240 mg/kg injection of plerixafor. Relative Pharmingen). Intracellular staining of Foxp3 was performed according to to the weight of the subjects mobilized, apheresis collections after manufacturer’s instructions (eBioscience, San Diego, CA). Regulatory T cells plerixafor mobilization (median 19.6 l apheresed; range 15–22 l) (Tregs) were determined by positive staining for CD4 and Foxp3. To measure contained a median 81 3 106 CD19+ B cells/kg, a median 274 3 lymphoid and myeloid chimerism, we stained peripheral blood collected after 6 + 6 + transplantation with Ly9.1-FITC, CD3-PerCP, and CD19-allophycocyanin 10 CD3 T cells/kg, and a median 1.6 3 10 CD34 cells/kg + (BD Pharmingen) and 7/4-PE (Cedarlane, Hornby, ON). For all flow cyto- (Table I). Plerixafor preferentially mobilized CD34 cells followed Downloaded from metric staining assays, cells were incubated with rat anti-mouse CD16/CD32 by monocytes and lymphocytes (Fig. 1A). Within the lymphocyte (BD Pharmingen) for 10 min at room temperature before incubation with compartment, B cells were preferentially mobilized followed by specific Abs. Cells were acquired on FACSCalibur (BD Biosciences) and + analyzed using Summit Software v3.3 (Dako Cytomation, Glostrup, Den- T cells and NK cells. Among CD19 B cells, CD20, k,andl mark) or FCS express software (DeNovo, Thornhill, ON). expression did not change from baseline, although the percentage Proliferation and suppression assays. CD4+ T cells were selected using of B cells expressing CD27 declined significantly in seven of eight

Miltenyi immunomagnetic beads from the spleens of mobilized animals and donors, consistent with plerixafor preferentially mobilizing naive http://www.jimmunol.org/ then cocultured (50,000/well) with MHC-mismatched irradiated (50 Gy) Con type B cells; the median percentage of CD27+CD19+ B cells was A blasts (range 8000–500,000 cells) from C57BL/6 mice in 96-well plates in DMEM supplemented with 10% FCS, 2-ME (Invitrogen, Life Technologies, 35.1% at baseline and 19% after plerixafor mobilization (p = Carlsbad, CA). After a 72- to 96-h coculture, cells were pulsed with 1 mCi/ 0.011). The total WBC count and the absolute numbers of blood well [3H]thymidine for 16 h and then were harvested onto solid filters and neutrophils, monocytes, lymphocytes, and CD34+ cells increased measured in a microbeta scintillation counter (Perkin Elmer, Wellesley, MA). significantly from baseline after plerixafor administration (Fig. Con A activated T cell blasts were prepared by culturing C57BL/6 spleno- 3 6 1B–F). A detailed phenotypic analysis using six-color flow cytom- cytes at 5 10 cells/ml in DMEM supplemented with 10% FCS, 2-ME, and + + 5 mg/ml Con A (Sigma Aldrich, St. Louis, MO) for 48–96 h. etry of CD4 and CD8 lymphocyte subsets at baseline and 6 h after Transplantation. After mobilization, a single suspension of donor cells was a single injection of plerixafor or 2 h after the fifth dose of G-CSF prepared by homogenizing the spleen and filtering through a 70-mm nylon is shown in Table II. No significant change from baseline was by guest on September 23, 2021 mesh filter. Red cells were ACK lysed for 5 min at room temperature observed after mobilization with plerixafor in the percentage of (Biosource, Camarillo, CA). BALB/c recipient mice were conditioned with CD4+ and CD8+ T cells expressing the majority of surface markers 950 cGy total body irradiation (Shepherd Mark I 68A Cs 137; JL Shepherd and Associates, San Fernando, CA), then 3–6 h later were injected with analyzed including CD45RA, CD45RO, CD34, CD56, CD57, 15 3 106 mobilized splenocytes i.v. via tail vein injection. This GVHD CD27, CD71, and CD62L. Although the phenotype also did not model has been shown to be dependent on transferred CD4+ T cells. change after G-CSF mobilization in most CD4+ and CD8+ T cell Therefore, some experiments were performed with an identical number of populations, there was a significant decline in the percentage of CD4+ T cells. In such experiments, recipients were injected with 2 3 106 2 CD4 and CD8 T cells that expressed CD62L and in CD8 T cells CD4+ and 13 3 106 CD4 splenocytes i.v. via tail vein injection. Onset of GVHD symptoms including alopecia, hunched posture, loss of that expressed CD27 (Table II). activity, and weight loss was assessed twice weekly and was graded using a previously described GVHD scoring system (26). In brief, a total of 9 Impact of plerixafor on cytokine gene expression profiles in points represents the highest score: alopecia 0–4; weight loss 0–2 (,5% = T cells . 0; 5–15% = 1; 15% = 2); posture 0–2; and ear or eye irritation 0–1. All To examine whether plerixafor mobilization altered the cytokine mice were kept alive for up to 100 d, and no mice died because of GVHD. polarization of T cells, we analyzed cytokine gene expression Statistical analysis profiles using a Th1-Th2-Th3 RT-PCR plate in CD3+ T cells col- Differences between groups (baseline versus plerixafor) were tested (when lected from subjects mobilized with a single injection of plerixafor applicable) by paired samples t test using SPSS 10 statistical software versus CD3+ cells collected from subjects mobilized with five daily

Table I. Cellular content of plerixafor mobilized apheresis products

Apheresis Total Total Total CD34+ Age (y) Sex Weight (kg) Volume (l) T Cells/kg (106) B Cells/kg (106) Cells/kg (106) 40 M 85 20 254 42 1.78 35 M 75 15 293 42 4.25 49 F 64 18.6 263 41 0.92 45 M 85 20 332 115 0.86 56 F 71 22 285 100 1.61 18 F 58 22 512 172 5.4 32 M 68 19 53 118 1.42 35 M 130 20 133 62 1.58 Mean 19.6 265 87 2.2 6244 EFFECT OF PLERIXAFOR MOBILIZATION ON LYMPHOCYTES

T cells had a statistically nonsigniﬁcant decrease in precursor frequency against third-party allogeneic stimulators as compared with T cells at baseline (Fig. 3B). A similar decline in alloreactivity against third-party allogeneic cells was observed in mononuclear cells after G-CSF mobilization as assessed by the 5-d 3H MLR assay (Fig. 3C) or CFSE (Fig. 3D). Impact of plerixafor on Foxp3 gene expression in CD4+ T cells In murine transplant studies, Tregs transplanted with allogeneic PBPCs appear to reduce the risk for GVHD. In humans undergoing allogeneic transplantation, an inverse correlation between GVHD and Foxp3 mRNA expression has been reported (29). Therefore, we analyzed FOXP3 expression by RT-PCR in CD4 cells isolated from healthy subjects after mobilization with plerixafor versus G-CSF. Compared with baseline, no statistically signiﬁcant changes in Foxp3 gene expression were observed in CD4+ T cells mobilized with either plerixafor or G-CSF (Fig. 4).

Mobilization of murine lymphocyte subsets after plerixafor Downloaded from versus G-CSF administration Mobilized PBSCs were collected from spleens of mice harvested 6 h after the last injection of G-CSF versus plerixafor. Compared with HBSS (saline) controls, there was a trend toward an increase in the absolute number of monocytes (p = 0.09) and lymphocytes

(p = 0.09) in the blood after plerixafor mobilization (Fig. 5A). http://www.jimmunol.org/ The absolute number of circulating lymphocytes in the blood after G-CSF mobilization was similar to plerixafor mobilization (p = 0.68), although there was a trend toward a higher number of monocytes being mobilized with plerixafor compared with G-CSF (p = 0.07). Both mobilizing agents increased absolute circulating granulocyte numbers, but granulocyte numbers in the blood after G-CSF mobilization were significantly higher compared with saline controls (p = 0.02) and plerixafor mobilized mice (p = 0.007). Similarly, absolute numbers of c-Kit+/Lin-/sca-1+ (KLS) cells were by guest on September 23, 2021 highest in spleens of G-CSF–treated mice. Spleens of G-CSF–treated mice contained a 3.6-fold and a 1.7-fold higher number of KLS cells compared with HBSS controls (p = 0.002) and plerixafor-treated mice (p = 0.07), respectively. Spleens from plerixafor-treated mice contained 2.2-fold higher numbers of KLS cells compared with HBSS controls (p =0.02). Compared with saline controls, spleen sizes appeared grossly similar after plerixafor mobilization and were larger after G-CSF mobilization (Supplemental Fig. 1A). There was an ∼2-fold in- FIGURE 1. Mobilization of blood mononuclear cells after a single dose A crease in the number of cells contained within the spleen after of plerixafor in healthy subjects. ( ) Fold change in leukocyte subsets. , Changes in absolute cell numbers of leukocytes (B), lymphocytes (C), mobilization with G-CSF compared with saline controls (p neutrophils (D), monocytes (E), and CD34-positive cells (F) after a single 0.001). Splenocyte cell numbers after plerixafor also increased sig- dose of plerixafor. Blood samples were collected before the start of mo- nificantly (p = 0.04) compared with saline controls, although this bilization and 6 h after a single injection of 240 mg/kg plerixafor imme- increase was smaller than that observed with G-CSF (Fig. 5A). The diately before apheresis. Each symbol represents an individual subject. higher number of splenocytes observed in G-CSF mobilized spleens *p , 0.01, **p , 0.001. was mostly due to an increase in the number of splenic granulocytes. To identify cellular phenotypic differences that might impact doses of G-CSF alone. None of the 84 cytokine genes examined GVHD and graft-versus-leukemia, we harvested and stained mo- was significantly altered from baseline in CD3+ cells mobilized bilized splenocytes with cell-specific differentiation markers. The with plerixafor. In contrast, 16 of 84 (19%) cytokine-related genes percentages of splenocytes expressing T cell, NK cell, B cell, and were significantly altered from baseline in CD3+ T cells after G-CSF granulocyte markers after plerixafor mobilization were similar to mobilization (Fig. 2). saline control–injected animals (Table III). In contrast, animals mobilized with G-CSF had a significantly higher percentage of Impact of plerixafor on T cell alloreactivity splenic granulocytes, a significantly lower percentage of CD4+ The impact of plerixafor mobilization on T cell alloreactivity was T cells and B cells, and a similar number of CD8+ T cells com- assessed by two different MLR methods. Compared with baseline, pared with plerixafor-injected animals. There was no difference in plerixafor mobilized mononuclear cells had slightly decreased al- the percentage of regulatory CD4+/Foxp3+ T cells mobilized be- loreactivity against third-party cells using a standard 5-d 3H MLR tween any of the groups (Table III). Furthermore, CD4+ T cells (Fig. 3A). Alloreactivity assessed using a CFSE MLR prolifera- mobilized with plerixafor had similar proliferation against MHC- tion assay gated on CD3+ T cells showed plerixafor mobilized mismatched cells as was observed with CD4+ T cells mobilized The Journal of Immunology 6245

Table II. Effect of plerixafor or G-CSF mobilization on circulating T cell subsets

Baseline After Plerixafor p* Baseline After G-CSF p* CD3+CD4+ T cells CD45RA+ 45.0 6 8.2 50.8 6 12.4 0.1 41.8 6 10.5 31.0 6 4.5 0.06 CD45RO+ 49.4 6 9.3 45.3 6 10.3 0.06 57.0 6 14.3 58.4 6 5.8 0.26 CD57+ 3.3 6 3.3 2.4 6 1.9 0.28 5.7 6 4.8 6.5 6 9.8 0.50 CD27+ 87.7 6 6.8 81.6 6 17.6 0.42 70.5 6 25.9 57.3 6 31.4 0.20 CD25+ 16.2 6 11.1 11.7 6 10.0 0.1 23.9 6 19.1 10.6 6 7.1 0.20 CD62L+ 34.1 6 32.4 27.4 6 32.7 0.1 67.8 6 15.2 29.5 6 22.9 0.03 CD71+ 1.4 6 0.9 1.8 6 1.5 0.55 0.6 6 0.4 1.2 6 0.8 0.31 HLA-DR+ 5.3 6 1.6 3.8 6 1.2 0.1 5.8 6 1.9 4.4 6 2.1 0.40 CD3+CD8+ T cells CD45RA+ 58.2 6 24.5 65.5 6 23.9 0.11 55.2 6 12.2 51.4 6 3.2 0.79 CD45RO+ 43.0 6 21.0 34.9 6 17.8 0.09 49.3 6 13.3 46.6 6 4.4 0.13 CD57+ 7.0 6 3.3 5.0 6 1.77 0.09 22.0 6 17.4 21.9 6 14.0 0.41 CD27+ 86.4 6 10.4 83.8 6 22.2 0.71 65.0 6 9.7 50.2 6 13.0 0.03 CD25+ 4.0 6 3.2 1.3 6 1.1 0.12 5.5 6 6.5 4.6 6 3.7 0.58 CD62L+ 32.0 6 33.7 29.6 6 37.1 0.39 47.1 6 14.1 16.4 6 12.4 0.01 CD71+ 0.7 6 0.4 0.7 6 0.36 0.88 0.5 6 0.3 0.8 6 0.9 0.65 HLA-DR+ 8.2 6 3.3 4.9 6 2.1 0.08 14.9 6 11.3 8.6 6 6.3 0.27 Downloaded from Values are reported as percent of total CD4+ or CD8+ T cells. *Comparison of baseline and postplerixafor or G-CSF on T cell subsets. The p values were calculated as paired t test. with G-CSF or CD4+ T cells obtained from saline controls (Fig. recipients of G-CSF (n = 6) and plerixafor mobilized splenocytes 5B). Similar to human T cells, a decline in CD62L expression was (n = 6). The incidence of skin GVHD in recipients of plerixafor

observed on murine T cells after mobilization with G-CSF (Sup- and saline mobilized splenocytes was comparable. In contrast, http://www.jimmunol.org/ plemental Fig. 1B). recipients of G-CSF mobilized splenocytes had a reduced incidence of skin GVHD compared with recipients of either plerixafor Comparison of transplant outcome in a murine MHC-matched or saline mobilized splenocytes (p = 0.02, Fisher test; p , 0.05, transplantation model using allografts mobilized with log-rank test; Fig. 5C). Furthermore, recipients of G-CSF mobi- plerixafor versus G-CSF lized splenocytes had signiﬁcantly less weight loss on day 15 post- BALB/c recipient mice were conditioned with total body irradi- transplantation compared with mice transplanted with HBSS ation and then were transplanted i.v. with 15 3 106 splenocytes after mobilization with G-CSF versus plerixafor. To compensate + for the higher CD4 T cell numbers in the spleens of plerixafor by guest on September 23, 2021 mobilized mice, we also conducted experiments in which transplant recipients had the lymphocyte dose adjusted to 2 3 106 CD4+ T cells combined with 13 3 106 CD42 splenocytes mobilized with either G-CSF or plerixafor. Lymphoid and myeloid chimerism assessed 30 d after transplantation by ﬂow cytometry was .90% donor in origin in BALB/c

FIGURE 2. Th1 and Th2 gene expression proﬁles in CD3+ T cells assessed by real-time PCR. Left panel, Heat map showing real-time PCR expression levels of 16 genes in CD3+ T cells that changed signiﬁcantly from baseline after G-CSF mobilization in 5 healthy subjects. All samples were normalized to the center of the mean of the pre–G-CSF samples, with red denoting upregulated expression and green denoting downregulated FIGURE 3. The impact of mobilization on alloreactivity. The allo- expression (10% false discovery rate). Right panel, Gene expression levels reactivity of PBMCs before and after mobilization with plerixafor (A, B)or of plerixafor did not change from baseline in the 84 measured Th1-Th2- G-CSF (C, D) against third-party PBMC stimulators was measured using Th3 genes, including the 16 genes that were altered with G-CSF treatment a[3H]thymidine uptake (A, C), or CFSE dilution assay gated on viable that are displayed. CD3+ T cells (B, D). 6246 EFFECT OF PLERIXAFOR MOBILIZATION ON LYMPHOCYTES

FIGURE 4. The impact of mobilization on CD4+ Foxp3 expression. Foxp3 expression after mobilization compared with baseline in CD4+ cells mobilized with plerixafor or G-CSF showed no fold-change in expression from baseline in healthy subjects mobilized with either plerixafor or G-CSF alone. Line represents the median change from baseline in 13 samples Downloaded from obtained after plerixafor mobilization and 7 samples obtained after G-CSF mobilization. mobilized splenocytes (p = 0.002) and mice transplanted with plerixafor mobilized splenocytes (p = 0.04). When the number of + 6 transplanted CD4 T cells was normalized to 2 3 10 cells across http://www.jimmunol.org/ all 3 groups, recipients of G-CSF mobilized splenocytes still had a significantly lower incidence (7/15 recipients developed GVHD within day +60; p , 0.05) of skin GVHD compared with plerixafor (16/17 recipients developed GVHD within day +60) or saline mobilized mice (16/17 recipients developed GVHD within day +60). Furthermore, total clinical GVHD scores were significantly lower (p , 0.05) in recipients of G-CSF mobilized splenocytes (1.6 6 0.3) compared with recipients of plerixafor (2.9 6 0.5) or saline mobilized splenocytes (2.7 6 0.4). by guest on September 23, 2021 Discussion Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. This study provides, to our knowledge, the first detailed analysis of both phenotypic and functional properties of lymphocytes mobilized with plerixafor. In this study, we show that alterations in T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. G-CSF and plerixafor mobilized a similar number of T cells into circulation. As reported by others, we observed the expression of CD62L on T cells to decline after G-CSF mobilization (30). In contrast, we observed no change in T cell phenotypic markers FIGURE 5. Animal transplant model. Donor B10.d2 mice were injected including CD62L expression after plerixafor mobilization. Nor- s.c. with either 10 mg G-CSF daily for 5 d, one injection of 100 mg pler- mally, CD62L2 T cells are effector memory cells; in contrast with ixafor, or saline control (HBSS). (A) Number of splenocytes and complete CD62L+ T cells, transplantation of CD62L2 T cells does not blood counts were obtained from G-CSF and plerixafor mobilized donors. B appear to cause GVHD in murine models (31). The impact of ( ) The alloreactivity of donor B10.d2 splenocytes in response to Con A blasts derived from C57BL/6 donors was assessed by MLR and showed no transplanting higher numbers of CD62L+ T cells using allogeneic significant differences between the three groups. (C) Incidence of skin grafts is unknown. Although animal models might suggest the GVHD in recipients receiving 15 3 106 mobilized total unfractionated incidence of GVHD could increase, it is important to consider that splenocytes (p , 0.05, G-CSF compared with HBSS controls). CD62L expression on CD3+ T cells alone may not reflect the true subtype of T cells. Studies have also shown that serum-soluble CD62L status were performed in our study, CD62L shedding after L-selectin levels are increased after G-CSF mobilization (32) and G-CSF treatment has previously been established to be a tempo- that some CD62L loss can be reversed with in vitro incubation rary phenomenon. Importantly, after mobilization with either pler- (30). Based on this observation, it is possible that the lower levels ixafor versus G-CSF, no differences in the percentage of T cells of CD62L found on G-CSF mobilized T cells occur as a conse- expressing CD45RA or CCR7, markers of naive (both) and central quence of shedding, rather than reflecting preferential mobiliza- memory (CCR7) cells, were observed. Although the expression of tion of an effector type T cell. Although no kinetic studies on CD27 on plerixafor mobilized T cells did not change, a significant The Journal of Immunology 6247

Table III. Phenotype of mobilized splenic lymphocytes our genotype study did not include an analysis that was specific for the CD4+ T cell subset. Recent data suggest reductions in Th17 HBSS G-CSF Plerixafor cells may be partially responsible for the avoidance of acute GVHD CD3 T cells 21.2 6 4.1 15.9 6 3.1* 20.3 6 3.5 in G-CSF mobilized allografts (38). However, using a Luminex CD4 T cells 12.1 6 2.6 8.9 6 1.4* 12.4 6 2.9 array analysis, we observed no changes in serum IL-17 levels CD8 T cells 6.9 6 1.9 5.2 6 2.1 6.4 6 1.6 after G-CSF mobilization. Instead, serum levels of G-CSF, IL- Treg cells 1.7 6 0.03 1.7 6 0.2 1.8 6 0.3 1Ra, IL-4, and MIP-1b increased significantly from baseline after NK cells 3.0 6 0.8 2.5 6 0.8 3.1 6 0.9 B cells 52.5 6 12.8 39.2 6 9.4** 51.5 6 8.6 G-CSF mobilization. In contrast with these findings, we observed Granulocytes 6.1 6 2.1 20.8 6 5.6* 7.5 6 2.8 no significant changes in any of these or other serum cytokines or chemokines measured after plerixafor mobilization (data not Values are represented as percentage 6 SD from 13–18 donors in each group. *p , 0.05 compared with HBSS and plerixafor mobilization, **p , 0.05 com- shown). pared with HBSS controls. Increasing data suggest Tregs play an important role in regulating GVHD after allogeneic hematopoietic cell transplantation decrease in CD27 expression was observed in G-CSF mobilized (a-HCT). Transplanting PBSC allografts that contain higher doses CD8+, but not CD4+, T cells. The functional consequence of of Tregs has been associated with a reduced incidence of GVHD 2 higher numbers of CD27 CD8 T cells using allogeneic grafts is (39–41). In preclinical murine models of a-HCT, injection of unknown. CD27 binds CD70 and is required for generation and CD4+CD25+ Tregs was shown to suppress the early expansion of long-term maintenance of T cell immunity, and was recently shown alloreactive donor T cells and their capacity to induce GVHD + to augment CD8 T cell activation (33, 34). One might therefore without abrogating graft-versus-tumor effector function (42).Fur- Downloaded from speculate that allografting of G-CSF mobilized CD8+ T cells thermore, data in humans have shown that supplementation of results in reduced GVHD. allografts with donor Tregs reduces the risk for acute GVHD after G-CSF mobilization has been shown to skew T cells toward a haploidentical a-HCT (43). Consistent with these observations, Th2-type phenotype characterized by increased expression of IL-4 a number of studies have recently reported that Treg numbers and IL-10, and decreased IFN-g production. Th2-type T cells are are decreased in patients who develop chronic GVHD (29, 44).

thought to be associated with a lower risk for causing acute GVHD Therefore, we measured the percentage of Tregs mobilized in http://www.jimmunol.org/ but may play a part in contributing to the increased incidence of mice and also measured Foxp3 gene expression in human CD4+ chronic GVHD, which occurs with the use of G-CSF mobilized T cells after plerixafor versus G-CSF administration. In mice, allografts compared with bone marrow transplants. We found that there was no significant difference in the percentage of regulatory T cells mobilized with plerixafor did not display any changes in CD4+/Foxp3+ T cells mobilized with either G-CSF or plerixafor. Th1- and Th2-type cytokines after nonspecific mitogen stimulation Furthermore, HBSS, G-CSF, or plerixafor mobilized murine Tregs (data not shown). However, similar to G-CSF mobilized T cells, did not differ in their suppressive activity of anti-CD3–stimulated we did observe a small reduction in responsiveness to allogeneic T cells in [3H]thymidine proliferation assays (data not shown). stimuli in vitro by [3H]thymidine uptake MLR in plerixafor mo- Furthermore, in humans, Foxp3 gene expression levels did not bilized T cells. Only minor changes in serum levels of IL-4, IL-10, change significantly from baseline and were similar in G-CSF and by guest on September 23, 2021 and IFN-g were found in mice receiving G-CSF compared with plerixafor mobilized CD4+ T cells. Based on these data, one HBSS-treated controls. However, we did observe significant de- would expect any impact that Tregs will have on GVHD would crease in serum levels of IL-8 in donors mobilized with G-CSF not differ significantly between allografts mobilized with pleri- (data not shown). Similar to this observation, investigators have xafor versus G-CSF. previously reported that IL-8 levels decline in patients with esoph- Because plerixafor does not alter the phenotype and cytokine ageal cancer after treatment with G-CSF (35). rIL-8 is known to polarization of T lymphocytes, one might postulate the T cell– directly suppress the spontaneous production of IL-4 by CD4+ mediated immune sequelae of allogeneic transplantation may T cells (36). Taken together, these data suggest that G-CSF–mediated differ when allografts are mobilized with plerixafor compared reductions in serum levels of IL-8 may lead to a shift toward a Th2 with G-CSF. To better interpret the clinical relevance of trans- phenotype in CD4+ cells potentially accounting for the reduced planting T cells with these differences, we explored a murine incidence of GVHD observed in this cohort. model of PBSC transplantation in which transplant recipients re- Remarkably, the cytokine gene expression profiles related to ceived a T cell replete plerixafor versus G-CSF mobilized allo- Th1/Th2/Th3 pathways differed significantly in T cells mobilized graft from MHC-matched, minor histocompatibility–mismatched with these two different agents. Real-time PCR assays showed donors. A single injection of plerixafor resulted in the rapid mo- T cells mobilized with plerixafor had cytokine gene expression bilization of KLS hematopoietic stem cells into the peripheral patterns that were similar to nonmobilized T cells. In contrast, blood of B10.d2 donor mice. The phenotype and MLR reactivity G-CSF altered a number of cytokine-related genes in mobilized to alloantigens of T cells mobilized into the spleen with plerixafor CD3+ T cells including both TH1 and TH2 cytokine-related was similar to T cells mobilized with G-CSF and nonmobilized genes, transcription factors, and genes regulating T cell activation. T cell splenocytes. However, the CD4+ T cell content was lower in Numerous prior studies have also reported that G-CSF induces G-CSF mobilized splenocytes, and recipients of G-CSF mobilized alterations in cytokine-related genes. G-CSF mobilized allografts cells displayed a lower incidence of GVHD compared with recip- contain more than a log higher T cell dose than bone marrow ients transplanted with plerixafor mobilized cells. Importantly, allografts but are not associated with an increased incidence of the cumulative incidence of GVHD was still lower in recipients of acute GVHD. This has led investigators to speculate that G-CSF– G-CSF mobilized grafts even when the transplanted number of induced alterations in cytokines inhibit T cells from causing acute CD4+ T cells was matched between cohorts. Of note, the incidence GVHD. Franzke et al. (37) showed G-CSF mobilization increased of GVHD did not differ between recipients of plerixafor mobilized gene expression of the Th2-related gene GATA3 in CD4+ T cells, splenocytes compared with recipients of nonmobilized splenocytes. with this effect being less apparent in nonsorted CD3+ cell frac- Transplanting large numbers of T cells that do not undergo changes tions. In our analysis, GATA3 levels in T cells did not change in their cytokine gene expression profiles in contrast with trans- significantly in CD3+ T cells after G-CSF mobilization, although planting T cells that undergo cytokine changes after G-CSF mo- 6248 EFFECT OF PLERIXAFOR MOBILIZATION ON LYMPHOCYTES bilization could potentially account for the higher incidence of bone-marrow stem-cell transplantation in haematological malignant diseases: a randomised trial. Lancet 355: 1231–1237. acute GVHD observed in this analysis with plerixafor mobilized 9. Blaise, D., M. Kuentz, C. Fortanier, J. H. Bourhis, N. Milpied, L. Sutton, allografts. Because CXCR4 is expressed on many different he- J. P. Jouet, M. Attal, P. Bordigoni, J. Y. Cahn, et al. 2000. 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