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,
*
Gene Therapy Program, and Departments of
Medicine and
Pediatrics, Louisiana State University Health Sciences Center, New Orleans, LA 70112; and
Bone Marrow Transplantation Program, Tulane University, New Orleans, LA 70118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Besides enrichment for HSC, donor grafts also contain most other blood-derived cell types. Here, specifically, the dynamic equilibrium between T cells and HSC in the donor graft has been proven to be pivotal for several transplantation outcome parameters. T cells are critical accessory cells needed for successful and accelerated donor cell engraftment (10, 11). Moreover, in allogeneic transplantations, donor T cells are largely responsible for the development of graft-vs-host disease (GVHD) and also for the therapeutically desired graft-vs-leukemia effect (GVL) (12). Surprisingly, despite a 10-fold increase in T cells in PBSC collections, PBSC transplants appear not to have an increased incidence or severity in GVHD. Some studies even suggest reduced GVHD with PBSC transplantations (5, 9, 10, 12, 13, 14). Other studies showed a significantly enhanced GVL effect after PBSC infusion compared with BM transplantation and a survival benefit (5, 15, 16, 17).
Because very little is known about the mechanism of HSC mobilization, the optimal mobilizing procedure has not yet been defined. Because the primitive human HSC has not yet been phenotypically identified, it is unknown whether PBSC obtained with current protocols will ultimately maintain lifelong hemopoietic repopulation (18, 19). The mobilization protocol determines the type and amount of mobilized precursor cells. HSC mobilization was originally described after chemotherapy and G-CSF administration (20, 21). Current procedures use combinations of chemotherapy regimens, with or without the administration of hemopoietic cytokines (G-CSF and/or GM-CSF; Refs. 1, 2, 3). Significant improvement in the mobilization of CD34+ cells was reported recently by using cytokine combinations with soluble stem cell factor (SCF) incorporated into G-CSF-mobilizing protocols (22, 23, 24). Moreover, use of mobilizing cytokines significantly affects and alters the composition of accessory cells contained within the PBSC collection. Accessory cells, specifically T cells and dendritic cells, facilitate engraftment and are capable of modulating the alloreactive potential (12, 13, 25). In summary, use of PBSC has favorably impacted the outcomes in BM transplantation. Because mobilizing cytokines have led to significant gains, the investigation of novel and optimized mobilization procedures continues to be a major goal of transplantation research.
We have reported previously that overexpression of mouse (m)IL-17 induces granulopoiesis in mice (26). IL-17 is a novel cytokine that is expressed in activated T cells (27, 28, 29). Many of IL-17’s biological effects may be due to its ability to stimulate the release of secondary cytokines, several of which are known HSC mobilizers (IL-8, G-CSF, and SCF; Refs. 22, 23, 26, 28, 30). In support of this, we found that IL-17-mediated granulopoiesis was attributable in part to a direct effect of mIL-17 on G-CSF release and the induction of the transmembrane form of SCF (31). Although both G-CSF and SCF are known stem cell-mobilizing compounds, it does not predict mobilizing activity for IL-17. IL-17 releases a panel of other cytokines and substances with unknown effects on the hemopoietic compartment. These may interfere or antagonize HSC mobilization. IL-17 could be expressed without toxicity in mice at sustained high levels, suggesting the potential suitability as a therapeutic agent in man (26, 31). Given the importance of T cells as critical accessory cells in stem cell grafts, we decided to investigate whether IL-17 mobilizes PBSC. To test the hypothesis that IL-17 can effectively mobilize PBSC, we conducted proof-of-principle experiments with a previously described murine model in which mIL-17 was overexpressed with recombinant adenovirus (Ad) technology (Ad-mIL-17), which permits sustained in vivo production of IL-17 (26, 31).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice ages 6–8 wk were purchased from the National Cancer Institute breeding facility (Frederick, MD). All mice were maintained under specific pathogen-free conditions in the vivarium of Louisiana State University Health Sciences Center (New Orleans, LA). Autoclaved food and water was provided ad libitum. Drinking water was acidified (pH 2.5) and supplemented with polymyxin (106 U/L; Sigma, St. Louis, MO).
Donor mice
Splenectomy was performed under sterile conditions with immediate closure of the incision by removable surgical clamps and application of local antibiotic ointment. Mice were used for additional experiments after a resting period of at least 4 wk. A total of 3 x 109 PFU of either Ad-mIL-17 or AdCMVLuc as control were administered via i.v. injection through the internal jugular vein.
Recipient mice
Lethal 
irradiation of recipients was performed with a cobalt
source in two fractions of 550 rad each 4 h apart (Gammacell 1000;
Atomic Energy of Canada, Ottawa, Canada). For Y chromosome engraftment
studies, male donors and female recipients were used. Secondary
recipients were female mice that received identical ablative radiation
treatment. Blood was collected via cardiac puncture or retroorbitally
and transferred into heparinized tubes. Organs were harvested under
sterile conditions. BM cells from both femurs were flushed into medium
(DMEM-15% FBS; Life Technologies, Rockville, MD).
Donor cell preparation
Mice were sacrificed at different time points after vector infusion (Ad-mIL-17 or AdCMVLuc) and blood collected via cardiac puncture in sterile, heparinized tubes. After RBC lysis, cells were washed and resuspended in PBS with 2% FBS. Nucleated cell counts were performed with a hemocytometer and trypan blue staining to account for viability of cells. Smears from peripheral blood were prepared by standard techniques and stained with a modified Wright-Giemsa stain (Diff-Quick; Baxter, Deerfield, IL). Donor cells were normalized for their content in mononuclear cells and reinfused into recipient mice via intrajugular vein injection.
Vector constructs
The Ad vector construction for mIL-17 and luciferase encoding recombinant viruses (Ad-mIL-17 and AdCMVLuc) has been described in detail by our group (26). Briefly, viruses were propagated on 911 cells with endotoxin-free conditions and CsCl density purification as described previously (32, 33). mIL-17 production was measured in 911 cell supernatants by a standard bioassay as described below (28, 34). Virus preps were screened for replication-competent Ad by propagation on A549 cells. This assay has a sensitivity of 1 contaminant/108 PFU. All viral preps had a PFU:particle ratio of <100:1. All lots of rAd contained <1 endotoxin U/ml as measured by the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).
Colony-forming assays
Methylcellulose cultures for CFU-granulocyte macrophage (GM), and CFU with high proliferative potential (CFU-HPP) were prepared with MethoCult GF 3434, obrained from Stem Cell Technologies (Vancouver, Canada), as described previously (26). Briefly, spleen, peripheral blood (1 x 105), and BM (1 x 104) cells were suspended in 1 ml of methylcellulose medium and plated in 35-mm tissue culture dishes (Fisher, Pittsburgh, PA). The dishes were placed in a dedicated incubator and maintained there at 37°C under a 5% CO2 atmosphere and saturated humidified conditions. Analysis for colony formation was conducted at day 10 with an inverted microscope with the definition a colony as an accumulation of at least 50 cells. CFU-HPP is defined as an accumulation of at least 200 cells.
Flow cytometry
To determine relative differences between stem cell frequencies in peripheral blood of AdIL-17- and AdCMVLuc-treated animals, flow cytometry was performed to measure Lin-/lowc-kit+Sca1+ cells. The method was followed as previously described by Okada et al. and Ikuta and Weissman (35, 36). Briefly, 5 x 106 peripheral blood white blood cells were labeled with a mixture of anti-lineage markers (anti-B220, anti-GR-1, anti-Mac-1, anti-CD4, anti-CD8, and TER119 for erythroid lineage; The Jackson Laboratory, Bar Harbor, ME). The dullest 5% of the Lin-/low cells were gated and analyzed for the dual expression of c-kit (anti-CD117) and Sca1 (BD PharMingen, San Diego, CA).
Quantitative real-time PCR (kinetic PCR)
To determine sex-mismatched chimerism in mice, a Y chromosome-sensitive and -specific quantitative kinetic PCR procedure was developed by our group, which is specific for the genes zfy1 and the zfy2. The procedure and its validation has been described in detail by our group.4 Briefly, samples consisted of 5 x 105 mononuclear cells from peripheral blood or BM. DNA was extracted from cell pellets with a blood DNA extraction kit (Qiagen, Chatsworth, CA). Samples and standard DNA (4 µl) in duplicate aliquots were pipetted into black-sided, clear-bottom 96-well plates (Corning Costar, Cambridge, MA), briefly agitated, and immediately analyzed on a FL-600 microplate fluorescence reader (Biotek Instruments, Winooski, VT). The mean values of duplicate readings were used for further calculations and generation of standard curves. The DNA concentration of each sample was determined from the standard curve. To generate male/female DNA standards, limiting dilution of male cells was performed that were subsequently admixed to female cells.
Amplification with the sense primer 5'-TGG AGA GCC ACA AGC TAA CCA-3' and antisense primer 5'-TCC CAG CAT GAG AAA GAT TCT TC-3', which were designed with Primer Express Software (Integrated DNA Technologies, Coralville, IA), generated an 81-nt amplicon. A labeled custom-made probe was designed to recognize a 33-nt sequence on the antisense strand of the amplicon (5'-6-carboxyfluorescein-CAG-TCA-TCA-CAT-TCG-GTG-GTC-TTT-TCT-GTC-TTA-TAMRA-3'; PerkinElmer Applied Biosystems, Foster City, CA). The final buffer concentration per sample was calculated to be 50 mM potassium chloride, 10 mM Tris, 0.1 mM EDTA, (equivalent of 1x TaqMan buffer A), 0.05 mM passive reference (Rox plus proprietary components), 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.4 mM dUTP, 7 mM MgCl2, 300 nM sense primer, 900 nM antisense primer 100 nM TaqMan probe, 0.5 µl AmpErase Uracil N-glycosylase (1 U/µl), and 1.25 µl of AmpliTaq Gold DNA Polymerase (5 U/µl). Samples were amplified by using an ABI 5700 sequence detection system (PerkinElmer). After the initial 2-min 50°C incubation step, two additional heating/cooling cycles (95°C for 1.5 min and 55°C for 2.0 min) were added immediately before AmpliTaq Gold DNA activation with a 6-min incubation at 95°C. This was followed with 45 amplification cycles (94°C for 15 s, 58°C for 20 s, and 60°C for 35 s).
For each reaction series, cycle threshold (Ct) readings from known male/female standard mixtures provided the values used to generate standard curves for each reaction series. The Ct number was computed for each sample using the Sequence Detection Software (PerkinElmer). The Ct baseline was preset at 10 SD above the equipment background. The standard curves for zfy1/zfy2 kinetic PCR were generated by plotting the mean of triplicate Ct values vs the log of the Y copy number and calculation of a regression line. Only those reactions where the standard curves had a slope value of -3.3 ± 0.1 and an r coefficient >0.990 were accepted for further analysis. The copy number for the unknown samples was determined by applying the mean Ct value of triplicates to the standard curve which was concurrently obtained, using the software provided by the manufacturer (PerkinElmer). The Y chromosome copy number was adjusted based on the total amount of DNA present in each reaction.
Data and statistical analysis
For all samples, statistical significance of differences was performed with Student’s t test, which was provided within a statistical software program (Sigma Plot; SPSS Science, Chicago, IL). Values of p < 0.05 were assumed for statistical significance. Survival analysis was performed by log-rank nonparametric testing and expressed as Kaplan Meier curve.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the effect of Ad-mIL-17 on peripheral blood in
splenectomized mice, morphologic analysis of white blood cells was
performed over a 10-day period. Within 24 h, a statistically
significant increase in white blood cells was observed that reached a
maximum on day 7 (4-fold). This increase was predominantly attributable
to an increase in the absolute neutrophil count, which increased by up
to 13-fold on day 7. This neutrophil expansion was sustained over the
entire 10-day observation period (Fig. 1
A). Monocytes increased by
10-fold with a maximum on day 3 (p < 0.05).
Lymphocytes also increased with the maximum observed on day 3 at
2.6-fold (p < 0.05) (n = 8
mice/group; Fig. 1B). To determine the optimal time of PBSC
mobilization after Ad-mIL-17 administration, peripheral blood was
analyzed over a 10-day time course. Hemopoietic precursor frequency in
peripheral blood was analyzed with CFU assays. Precursors of
granulocytes, erythrocytes, macrophages, and monocytes
(CFU-GM/granulocyte-erythrocyte-macrophage-monocyte (GEMM)), as well as
precursors with HPP (CFU-HPP), increased significantly in the Ad-mIL-17
group over the control AdCMVLuc group with maxima on days 3 and 7 (Fig. 2, A and B). To
determine HSC frequency in peripheral blood, morphologic phenotyping
for the previously described murine HSC population
Lin-/lowc-kit+Sca1+
was performed (35, 36). This cell population increased
significantly in the Ad-mIL-17 group with a maximum on day 3 (1.4% vs
0.03%; Fig. 2C).
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C57BL/6 mice were lethally irradiated with 1100 rad before the
infusion of C57BL/6 mouse donor blood. Splenectomized donors had been
treated 3 days earlier with either 3 x 109
PFU Ad-mIL-17 or AdCMVLuc. Although no survivors were registered in
mice infused with AdCMVLuc-treated donor blood (up to 1 x
107 cells), survival was 85% in the group
infused with AdmIL-17-treated donor blood (1 x
106 cells). All mice in the AdCMVLuc donor blood
group had succumbed by day 17, but no further mortality was observed
after that time point in the Ad-mIL-17-mobilized blood recipients. A
total of 180 mice in four different experiments were used for these
survival studies. The data presented represent results from the
combined four experiments. (Fig. 3). All surviving mice became
long-term survivors maintaining normal hemopoiesis, with one cohort
followed for 1 year after treatment.
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To demonstrate that the Ad-mIL-17-mobilized donor PBSC had
engrafted and sustained functional hemopoiesis over a period of 3 mo,
individual male/female autotransplanted C57BL/6 mice were sequentially
analyzed by CBC and differential cell count at 4 and 12 wk after
transplantation (n = 6/group). Hemopoietic
reconstitution in Ad-mIL-17 PBSC autotransplanted C57BL/6 mice was
observed with cell numbers reaching levels close to nontransplanted
control animals at 4 wk and full hemopoietic restoration compared with
untransplanted animals at 3 mo (Fig. 4).
T and B lymphocyte and also NK cell lineages were present at normal
levels (data not shown). Male donor cells were detected in peripheral
blood of female recipients by Y chromosome-specific kinetic PCR at 1
and 3 mo in significant levels. Results from 10 individual mice are
depicted in Fig. 5.
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Mice autotransplanted with Ad-IL-17-mobilized donor PBSC were
evaluated at 1 year and results were compared with nontransplanted
littermates. White blood count (WBC) and differential count were
statistically not different (Fig. 4
). BM CFU (CFU-GEMM and CFU-HPP)
also were not different (data not shown). To demonstrate that the
Ad-IL-17-mobilized donor PBSC indeed had engrafted and repopulated mice
for prolonged time periods (>6 mo), four mice were analyzed for
presence of BM Y chromosome sequences at 1 year after transplantation.
Engraftment was significant and ranged between 45 and 75% (Fig. 6A). Second transplantation experiments were
conducted with BM from female recipients that had been transplanted 3
mo earlier with Ad-IL-17-mobilized male donor PBSC. One donor mouse
provided cells for three recipients. The secondary female recipients
were analyzed for presence of Y chromosome sequences at 4 mo after
transplantation. A total of 12 animals were analyzed. In all secondary
recipients, male-derived cells were detected at significant levels
(45–95%; Fig. 6B). The data suggest that Ad-mIL-17 can
mobilize PBSC with long-term repopulating ability.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To achieve optimal mobilization and avoid pooling of mobilized cells in the spleen, donor mice were splenectomized prior to vector administration. mIL-17 pharmacokinetics in this splenectomized model were very similar to our previously reported results where mIL-17 plasma levels rose to 40 U/ml of mIL-17 at 12 h after vector administration, 60 U/ml at 24 h, and to a peak level at 72 h (120 U/ml; Ref. 26). From there on, a slow linear decline was observed, with levels still at 70 U/ml/ml on day 10 (data not shown). Thus, stable mIL-17 levels within a 3-fold range could be achieved with the Ad-mIL-17 expression system throughout a 10-day period.
As previously reported by our group in unsplenectomized mice, Ad-mIL-17 treatment resulted in a substantial increase of the total WBC. This was caused predominantly by expansion of neutrophils (26, 31). However, in splenectomized animals, monocytes also increased significantly by 10-fold and lymphocytes by almost 3-fold. Previously, the increase in peripheral blood lymphocytes and monocytes was noted. However, it did not reach statistical significance. We conclude that in these previous experiments, both BM-derived cell types were siphoned and pooled within the spleen and therefore did not appear in the peripheral blood at significant levels.
Peripheral blood of control virus-treated animals did not contain precursor cells (CFU) or HSC. However, both elements were observed at significant levels after Ad-mIL-17 treatment. A time course determined the optimal Ad-mIL-17 mobilizing effect at 72 h, where the maximum mobilization of primitive HSC (Lin-/lowc-kit+Sca1+) occurred. Colony-forming ability of peripheral blood was similar on days 3 and 7, with no statistical difference. Therefore, PBSC obtained at 72 h were used for subsequent rescue studies with lethally irradiated mice. Compared with animals infused with Ad-mIL-17-mobilized cells, control animals received excess cells (up to 3-fold white blood cells and 2-fold mononuclear cells) without the rescue of a single animal. Because Ad-mIL-17-mobilized PBSC protected mice from the immediate posttransplantation mortality, lineage committed precursor cells (CFU-GM) must have rapidly differentiated into mature and functional cells protecting the radiated host.
To determine donor cell contribution over time, the male Y chromosome sequences zfy1/zfy2 in female recipients were amplified by kinetic PCR. In peripheral blood, donor sequences were detected in all mice at 4 and 12 wk. In most animals, engraftment was in excess of 30% at both time points. Only two mice had a low percentage of male DNA (10%; mouse 1 and mouse 9) at 12 wk. One possible explanation is the recovery of endogenous hemopoiesis and loss of donor hemopoiesis. In this case, Ad-mIL-17-mobilized precursor cells would have secured survival during the radiation-induced pancytopenia period. However, the composition of peripheral blood, which consists of mature and functional cells, does not correspond stochiometrically to the HSC within the blood-forming organs. Rather, it is believed that the majority of functional blood cells are derived from few, sequentially activated proliferating stem cell clones (37). Should these activated clones be predominantly of host origin, the proportion of donor HSC in the blood-forming organs is not accurately represented by peripheral blood analysis. To eliminate this potential caveat, analysis directly from BM may provide more accurate results of donor/host HSC composition. However, it also is important to realize that BM support cells are more radio-resistant than hemopoietic precursor cells. Therefore, when donor marrow is procured from transplanted animals, it is quite likely that also host-derived stroma and connective tissue cells will be carried over for analysis. Therefore, even in the event of complete replacement of host hemopoiesis with donor HSC, some detection of host DNA is also expected, precluding the result of an entirely donor derived-BM. The BM of four mice was analyzed 1 year after Ad-mIL-17-mobilized PBSC transplantation with male donor DNA contributing 45–75% to total DNA. In the second transplantation series, the BM of each recipient was found to have male donor DNA ranging from 45 to 95%. Although individual variation was observed between mice, donor cells constituted a major portion of host hemopoiesis after a second transplantation. Taken together, the data support that Ad-mIL-17 can mobilize primitive PBSC that can durably reconstitute hemopoiesis in mice.
Given the critical role of T cells in engraftment, follow-up studies are needed to compare directly the engraftment kinetics of IL-17-mobilized PBSC to conventional mobilized PBSC. T cells have numerous roles in transplantation biology, part of which could be explained by mediating cytokines. The hypothesis that IL-17 may have a potential role in GVHD or GVL of allogeneic transplantation is intriguing and requires further investigation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul Schwarzenberger, Gene Therapy Program/Hematology-Oncology, Louisiana State University Health Sciences Center, 533 Bolivar Street, CSB Room 611, New Orleans, LA 70112. E-mail address: pschwa1{at}lsuhsc.edu
3 Abbreviations used in this paper: PBSC, peripheral blood stem cell; HSC, hemopoietic stem cells; BM, bone marrow; GVHD, graft-vs-host disease; GVL, graft-vs-leukemia effect; m, mouse; Ad, adenovirus; Ct, cycle threshold; GM, granulocyte-macrophage; HPP, high proliferative potential; GEMM, granulocyte-erythrocyte-macrophage-monocyte; SCF, stem cell factor.
4 E. P. Bryne, W. Huang, V. M. Wallace, M. K. Shean, L. Zhang, Q. Zhong, C. Theodossiou, H. Blakesley, J. K. Kolls, and P. Schwarzenberger. Quantitative real-time (kinetic) PCR: an accurate and simplified method for chimerism analysis in sex-matched murine transplantation. Submitted for publication.
Received for publication March 30, 2001. Accepted for publication June 13, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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