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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: Biol Blood Marrow Transplant. 2010 Dec 19;17(5):608–619. doi: 10.1016/j.bbmt.2010.12.697

Effects of Sub-lethal Irradiation on Patterns of Engraftment after Murine Bone Marrow Transplantation

Jacob Andrade 1, Shundi Ge 2, Goar Symbatyan 3, Michael S Rosol 3, Arthur J Olch 4, Gay M Crooks 2
PMCID: PMC3086732  NIHMSID: NIHMS272790  PMID: 21176787

Abstract

Attempts to reduce the toxicity of hematopoietic stem cell transplantation have led to the use of a variety of immunosuppressive yet non-myeloablative preparative regimens that often include low dose irradiation. To determine the effects of low dose irradiation on the dynamics of donor cell engraftment after bone marrow transplantation (BMT), we coupled standard end-point flow cytometric analysis with in vivo longitudinal bioluminescence imaging performed throughout the early (<10 days) and late (days 10–90) post transplant period. To exclude the contribution of irradiation on reducing immunological rejection, severely immune deficient mice were chosen as recipients of allogeneic bone marrow. Flow cytometric analysis showed that sub-lethal doses of total body irradiation (TBI) significantly increased long-term (14 weeks) donor chimerism in the bone marrow compared to non-irradiated recipients (p<0.05). Bioluminescence imaging demonstrated that the effect of TBI (p<0.001) on chimerism was seen only after the first 7 days post BMT. Flow cytometry analysis at Day 3 showed no increase in the number of donor cells in irradiated BM, confirming that sub-lethal irradiation does not enhance marrow chimerism early after transplantation. Local irradiation also significantly increased late (but not early) donor chimerism in the irradiated limb. Intra-femoral injection of donor cells provided efficient early chimerism in the injected limb, but long-term systemic donor chimerism was highest with intravenous (IV) administration (p<0.05). Overall, the combination of TBI and IV administration of donor cells provided the highest levels of long-term donor chimerism in the marrow space. These findings suggest that the major effect of sub-lethal irradiation is to enhance long-term donor chimerism by inducing proliferative signals after the initial phase of homing.

Introduction

In classical preparative regimens for allogeneic marrow transplantation, patients receive full myeloablative conditioning and immune suppression to achieve complete hematopoietic reconstitution with donor cells. However, the toxicity associated with high dose radiation and chemotherapy excludes many patients from receiving this necessary treatment. High levels of bone marrow chimerism have also been achieved using non-myeloablative conditioning regimens that focus mostly on immune suppression (1–3). Several rodent models of non-myeloablative bone marrow transplantation have shown that engraftment can be enhanced by increasing the donor cell dose (4), changing the route of cell administration (5), or increasing immunological suppression to prevent rejection (6, 7).

Most of the clinical non-myeloablative preparative regimens include low dose irradiation (8). However, controversy exists over the mechanism by which low dose radiation acts to improve engraftment. Although lethal irradiation is believed to create “space” in the stem cell niche, the most primitive hematopoietic stem cells are relatively resistant to irradiation (9). Thus low dose irradiation is often considered to assist engraftment through lympho-ablation. An intriguing finding using a murine congenic transplant model showed that high dose irradiation of a local area of marrow increased engraftment both locally and also at distal sites, suggesting that at least high doses of irradiation enhance engraftment independent of immunological barriers (10).

The central goal addressed in this current paper was to assess the effect of sub-lethal irradiation provided as either TBI or as local irradiation, on the process of engraftment. Severely immune deficient mice (Non-Obese Diabetic Severe Combined Immune Deficient IL-2Rγnull, aka NSG) were used as recipients to exclude the contribution of irradiation on immunological rejection of allogeneic bone marrow. In addition to absent T and B cell development from the SCID mutation, the NOD mutation results in abnormal macrophage and dendritic cell function and the IL2Rγ null mutation results in NK cell deficiency. Thus severe defects exist in both the adaptive and innate immune systems of the NSG mouse (11).

Engraftment is a process that results from a combination of early homing and lodging of donor stem cells to the recipient endosteal niche, with subsequent proliferation and redistribution throughout the marrow spaces (12–14). Engraftment can be measured in vivo by quantifying the number of donor cells circulating in the recipients’ peripheral blood. However this method does not accurately determine the chimerism of donor HSCs in the recipient bone marrow and is limited by the number of repeated bleeds from a single animal. The standard quantitative measurement of donor chimerism in the marrow cavity uses flow cytometric analysis of cells isolated from the bone marrow of euthanized animals. Although very sensitive, this type of analysis is poorly suited for longitudinal studies of engraftment patterns. In this study, we combine end-point flow cytometry with a non-invasive bioluminescence imaging approach, developed in our laboratory (15), to study the effect of sub-lethal irradiation on the dynamics of early and late donor cell engraftment in immune deficient mice.

Materials and Methods

Animals

Transgenic donor mice (Caliber Life Sciences) and NOD/SCID/IL-2γc−/− recipients (NSG) mice (Jackson Laboratories) were kept in separate pathogen-free facilities under approved protocols of the CHLA Institutional Animal Care and Use Committee.

Bone Marrow Transplantation

Mononuclear bone marrow cells from FVB mice (H-2Kq) ubiquitously expressing luciferase under the β-actin promoter were transplanted into 8–10 week old NSG recipients (H-2Kd). Recipients were sub-lethally irradiated with a dose of 2.7Gy to the whole body (TBI) or right hind limb (RHL IR) before transplantation. Whole bone marrow cells were injected directly into the right or left femur (intra-femoral) or intravenously (IV) through the tail vein using a 28-gauge needle.

Irradiation

Total body irradiation was administered using a MK-1-68A Cesium-137 Gamma animal irradiator (J.L. Shepherd and Associates) or a Clinac 2100C 6 MV x-ray Linear Accelerator (Varian Medical Systems, Inc). Local irradiation to the right hind limb was accomplished by placing the limbs of anesthetized animals within a single radiation field encompassing the entire hind limb, including the femoral head but not the pelvis. A 1.5 cm thick layer of artificial tissue was used to buildup the dose to the mid-plane of the leg to 2.7Gy in a single dose. The radiation dose 0.5 cm outside the field border falls to less than 10% of the central dose.

Flow Cytometric Analysis of Multi-lineage Engraftment

Mice were euthanized at 3 days or 10–14 weeks after bone marrow transplantation by inhalation of a mixture of 75% CO2/25% O2. Hematopoietic cells were harvested from the left and right femurs, spleen and peripheral blood of each animal. Single cell suspensions were stained in 1% fetal bovine serum in phosphate buffered saline. Flow cytometry events were acquired by a FACSCalibur (BDBioscience) and analyzed using FlowJo software (Tree Star, Inc). Cells were initially gated using the forward and side scatter distribution profile of hematopoietic cells in order to eliminate any contribution from debris, prior to analysis of donor chimerism by monoclonal antibodies against mouse H-2Kq (BD Pharmingen). Contribution of each hematopoietic lineage was determined using CD11b (monocyte), Ly-6c/g (granulocyte), CD3ε (T cell), B220 (B cell) and NK1.1 (NK cell) (BD Pharmingen).

Bioluminescence Imaging

Mice were imaged serially beginning 24 hours after transplantation (day 1) as follows: a dose of 125 mg/Kg of luciferin (Promega Corporation) diluted in phosphate buffered saline was introduced by intraperitoneal injection 8 minutes before imaging. Mice were anesthetized with 2–3% isoflurane with oxygen in an induction chamber then placed into a Xenogen IVIS 100 imaging system (Caliber Life Sciences), and maintained under anesthesia during image acquisition. Acquisition time began at 3 minutes each on the ventral and dorsal surface of each animal and decreased to 1 minute after 1 week, and 1 second after 1 month because of camera photon saturation. Data was analyzed using the Living Image 2.5 software included with the imagining system. With the software, regions of interest where drawn around the whole body and the right and left hind limb of each mouse to determine the donor cell bioluminescence measured in photons/second/cm2/sr. Negative control mice were injected with saline instead of cells at the time of initial injection. During imaging, negative controls were used to determine threshold for detection of luciferase specific bioluminescence.

Statistical Analysis

Statistical analysis was performed with Prism Software (GraphPad Software, Inc.). Data are presented as mean ± standard error of the mean (SEM). Before statistical analysis, data was transformed logarithmically in order to use parametric tests because of the low sample size in each group. The unpaired t test was used for comparison of 2 groups of flow cytometry data. One-Way ANOVA using Bonferroni’s multiple comparison post-tests was used for flow cytometry data with 3 or more groups. Two-Way ANOVA using Bonferroni’s multiple comparison post-test was used to analyze longitudinal bioluminescence data at each time point. Early chimerism was analyzed by combining repeated measurements for each animal within each group for the first 10 days after transplant, followed by a paired T-test (hind limb bioluminescence) or unpaired T-test (whole body bioluminescence). Late chimerism was analyzed by combining repeated measurements for each animal within each group after 10 days post-transplant, followed by a paired T-test (hind limb bioluminescence) or unpaired T-test (whole body bioluminescence).

Results

Sub-lethal total body irradiation increases long-term donor chimerism

To evaluate the effect of sub-lethal irradiation on donor cell engraftment, severely immune deficient mice were transplanted with a range of doses of wild type donor bone marrow cells in the presence or absence of sub-lethal (2.7Gy) total body irradiation. Immune deficient mice were used as recipients to eliminate any contribution of immune rejection from the process of engraftment. 14 weeks after bone marrow transplantation, recipient mice were euthanized and cells were harvested from bone marrow, spleen and peripheral blood to assess donor cell chimerism by flow cytometry based on expression of the donor MHC class I type H-2Kq in recipient tissue (Figure 1).

Figure 1. Sub-lethal irradiation increases long-term donor chimerism in bone marrow.

Figure 1

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Donor chimerism is shown as percentage of H-2Kq positive donor cells (mean ± SEM) detected by flow cytometry in (A) both femurs, (B) spleen and (C) peripheral blood of recipient mice 14 weeks after BMT. Recipients received either no irradiation or a 2.7Gy dose of TBI before intravenous injection of 1×106, 1×105, or 1×104 donor cells from whole bone marrow. Different colors within each bar reflect the proportion of each lineage detected by flow cytometry for markers of macrophages/granulocytes (red), B cells (yellow), T Cells (green), NK cells (purple), and “other” i.e. unclassified (gray) cells within the H-2Kq positive population. Analysis of Variance with Bonferroni's Adjustment Test (*P < 0.05) performed on logarithmic transformation of the data (in those groups with at least 2 mice) combined from 3 experiments as follows: No irradiation (N=2, N=1, N=2 animals each) vs Irradiation (N=3, N=2, N=1 animals each). (Abbreviations: TBI - total body irradiation; Macs/Grans – Macrophages/Granulocytes; FACS – Fluorescence-activated cell sorting).

Sub-lethal irradiation significantly increased donor chimerism in the bone marrow compared to non-irradiated recipients (69±5% compared to 6±3% in mice transplanted with 1×106 donor cells, p<0.05) 14 weeks after BMT (Figure 1A). As expected, in irradiated animals, levels of bone marrow chimerism correlated with cell dose. However, in non-irradiated animals, bone marrow chimerism was at equivalent low levels at all the cell doses tested.

Reconstitution of all the hematopoietic lineages from the donor cells was examined by flow cytometry using antibodies against common mouse lineage markers for macrophage, granulocytes, B cells, T cells, and NK cells. Donor chimerism in the bone marrow was predominantly from myeloid lineages (Figure 1A) and irradiation predominantly affected myeloid engraftment. BMT with wild type donor bone marrow, restored the predominantly lymphoid distribution of lineages in the NSG spleen, all of which were, as expected, of donor origin (Figure 1B). Based on the levels of donor chimerism and the lineage make-up of the chimerism, the donor cells found circulating in the peripheral blood presumably reflected contributions from engraftment of donor cells in the marrow and the subsequent donor-derived lymphopoiesis in the immune-deficient host thymus and spleen (Figure 1C). Thus, in the severely immune deficient host, sub-lethal irradiation significantly increased long-term bone marrow chimerism predominantly of the myeloid lineage.

Longitudinal analysis of donor cell engraftment in recipients with or without total body sub-lethal irradiation using bioluminescence Imaging

To analyze the effects of sub-lethal irradiation on the rate of engraftment, transgenic mice that express the firefly luciferase gene in all hematopoietic cells were used as bone marrow donors, thus permitting the use of bioluminescence imaging to quantify relative levels of engraftment longitudinally at various time points after bone marrow transplantation. Consistent with the flow cytometric data in Figure 1, sub-lethal irradiation increased long-term stable chimerism at all cell doses tested (Figure 2A, B, C) (1×106 p<0.001, 1×105 p<0.01, 1×104 p<0.05). In contrast, early levels of donor chimerism (defined as day 1–10 post transplant) rose rapidly, reaching similar levels irrespective of whether irradiation was given (Figure 2A, B, C); only after the first week was donor chimerism significantly increased by irradiation. Irradiated and non-irradiated recipients transplanted at all cell doses had significantly higher bioluminescence than background levels in negative control (non-transplanted) mice (Figure 2D,E).

Figure 2. Longitudinal analysis with bioluminescence imaging reveals a differential effect of sub-lethal irradiation on early versus late chimerism.

Figure 2

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Whole body bioluminescence was measured in recipient mice after intravenous BMT with luciferase expressing transgenic donor cells, beginning 24 hours after BMT and serially through the course of 96 days. Recipients received either no irradiation (NIR) or 2.7Gy TBI before intravenous injection of 1×106, 1×105, or 1×104 donor whole bone marrow cells. (A) Logarithmic graph of mean whole body bioluminescence, measured in photons/second/cm2/sr, and SEM plotted against days after BMT of TBI (N=6) and NIR (N=3) recipients showing only those that received 1×106 donor cells. (B) Logarithmic plot of mean whole body bioluminescence and SEM of TBI (N=2) and NIR (N=1) recipients showing only those that received 1×105 donor cells. (C) Logarithmic plot of mean whole body bioluminescence and SEM of TBI (N=1) and NIR (N=2) recipients showing only those that received 1×104 donor cells. Repeated measurements of bioluminescence was combined for the first 10 days (Early) or greater than 10 days (Late) before using the unpaired T-test for statistical significance comparing TBI versus NIR recipient at each cell dose (A–C). P -value for significance using the unpaired T-test is printed in each figure, while NS indicates no statistical significance. TBI produced significantly higher late (but not early) chimerism at each cell dose (A–C). (D) Logarithmic plot of mean whole body bioluminescence against days after BMT for all recipient groups. SEM omitted for clarity. Data from control (non-transplanted) mice are shown in black, mice receiving no irradiation (NIR) in blue, and those receiving TBI in red. Within each group a solid (1×106 cells), dashed (1×105 cells) or dotted (1×104 cells) line denotes the transplanted cell dose. Two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-test for statistical significance (*P > 0.05, **P > 0.01) was used to compare each recipient group versus control (N=5) at each time point. (E) Representative images of donor cell bioluminescence in TBI, NIR, and control recipient mice at 1, 4, 8, 14, and 96 days after BMT. TBI and NIR recipients received a dose of 1×106 donor cells at BMT. In the right column the images from each mouse at day 96 are scaled to decrease signal intensity and more clearly demonstrate the anatomic distribution of chimerism in the mouse receiving TBI. All statistical analysis was performed on logarithmic transformation of the data combined from 3 experiments. (Abbreviations: TBI – Total Body Irradiation; NIR – no irradiation; NS – not statistically significant).

Donor chimerism during the early post transplant period

The similar levels of bioluminescence measured in the early post transplant period for irradiated and non-irradiated recipients suggested that equivalent numbers of total donor cells were present in the recipient regardless of irradiation. As the limited sensitivity of bioluminescence imaging may not detect differences in low levels of engraftment, flow cytometry analysis was used to further analyze the effects of irradiation on donor chimerism during the early stages of engraftment. Again, immune deficient mice received either no irradiation or 2.7Gy of total body irradiation before receiving 1 × 106 wild type bone marrow cells intravenously from luciferase transgenic mice. Recipients were sacrificed 3 and 7 days after bone marrow transplantation to assess total cell counts isolated from the right femur using a hemacytometer. In addition to bone marrow, cells from the spleen were harvested from recipients 3 days after BMT to assess donor cell chimerism by flow cytometric analysis of H-2Kq positive donor cells (Figure 3).

Figure 3. Donor chimerism in the early post transplant period.

Figure 3

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Recipients received either no irradiation or a 2.7 Gy dose of TBI before intravenous injection of 1×106 donor whole bone marrow cells. A) The total number of viable cells (mean ± SEM) isolated from the right femur of sub-lethally irradiated and non-irradiated mice was counted using a hemocytometer at 3 and 7 days after BMT. B) Donor chimerism was determined as the percentage (mean ± SEM) of H-2Kq positive donor cells detected by flow cytometry 3 days after BMT. The total number of donor cells (mean ± SEM) was calculated by multiplying the total cell count by the percentage of donor chimerism in (C) femur and (D) spleen of recipient mice 3 days after BMT. Data was analyzed using the unpaired test for statistical significance comparing non-irradiated to irradiated recipients for donor cell count and chimerism. Statistic analysis was performed on data from 5 mice per group from a single experiment. (Abbreviations: TBI – total body irradiation; BMT- bone marrow transplant; N.S. – not statistically significant).

Sub-lethal total body irradiation significantly decreased (p<0.001) the total number of cells (host plus donor) found in the femur of recipients 3 and 7 days after BMT (Figure 3-A). Although the percentage of donor cells was increased in the femurs of irradiated (2.8±0.2%) compared to non-irradiated (0.8±0.1%) recipients (Figure 3-B) 3 days after BMT, when the total cellularity was taken into account, the marrow of irradiated animals was found in fact to contain a lower number of donor cells than non-irradiated bone marrow (1.3±0.5×104in irradiated vs 8.2±1.5×104 in non-irradiated femurs, p=0.002) (Figure 3-C). In contrast in the spleen, the total number of donor cells found in irradiated (6±2×104) and non-irradiated (7±1×104) recipients was not significantly different (Figure 3-D).

Intra-femoral injection produces lower long-term donor chimerism in bone marrow than intravenous administration of donor cells in sub-lethally irradiated immune deficient recipients

Several studies using xenogeneic transplant models have reported that purified human stem and progenitor populations engraft immune deficient mice more efficiently if injected directly into the bone marrow, presumably by avoiding the problem of inefficient homing to marrow through the intravenous route (16, 17). We therefore tested if intra-femoral injection of whole allogeneic bone marrow would also provide higher levels of engraftment than intravenous infusion in the sub-lethally irradiated setting. Surprisingly, intra-femoral injection did not increase long-term chimerism based on FACS analysis of harvested tissues 10 weeks after transplantation (Figure 4). In fact, intravenous administration of donor cells produced significantly higher (p<0.05) systemic marrow chimerism (69±3%) than seen in animals given donor cells by direct intra-femoral injection (40±10%) (Figure 4A). Again, high levels of donor chimerism were seen in the spleen irrespective of the route of administration (Figure 4B).

Figure 4. Direct injection of donor cells into the bone marrow cavity does not improve long-term hematopoietic chimerism in sub-lethally irradiated recipients.

Figure 4

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Donor chimerism is shown as %H-2Kq positive donor cells detected by flow cytometry in (A) Left and Right Femur, (B) Spleen and (C) Peripheral Blood of recipient mice 10 weeks after bone marrow transplantation (BMT). Recipients received 2.7Gy of ionizing radiation before receiving 1×106 donor bone marrow cells injected intravenously (IV, N=3) or directly into the right femur (intra-right femoral, IF, N=3). Shown are mean ± SEM for the total percentage of donor cells, with different hematopoietic lineages as described in Fig 1A. Analysis of variance with Bonferroni’s multiple comparison post-test was used to determine statistical significance comparing chimerism in the left versus right hind limb, within and between recipient groups. Left and right hind limb data was combined and analyzed using the unpaired test for statistical significance (*p<0.05) comparing systemic marrow engraftment in IV versus IF BMT. Statistics was performed on logarithmic transformation of data from one representative experiment. (Abbreviations: Macs/Grans – Macrophages/Granulocytes; FACS – Fluorescence-activated cell sorting).

Bioluminescence imaging demonstrated that intravenous infusion of donor marrow resulted in higher whole body signal levels compared with intra-femoral injection at almost all stages of engraftment, although by 10 weeks donor chimerism from intra-femoral administration had gradually increased to intravenous levels (Figure 5A,B). As whole body bioluminescence reflects donor cells not only in the marrow but also in spleen and peripheral blood, local measurements of signal intensity in each hind limb were then analyzed to compare dynamics of engraftment specifically in the marrow space with each route of administration. In animals given donor cell infusions intravenously, signal intensity was equivalent in each hind limb at all time points (Figure 5C) as would be expected with systemic administration. In animals in which marrow was injected directly into the right femur, early engraftment (up to 8 days) was higher in the injected limb than the non-injected limb, after which time signal intensity in both limbs increased to similar levels (Figure 5D). These findings suggest that mobilization of donor cells from the injected marrow and subsequent proliferation in distant sites delays equilibration of chimerism in all marrow niches. Thus, systemic chimerism increases more rapidly and efficiently when donor cells are given intravenously because although direct administration of donor cells into the marrow cavity produces early efficient engraftment in the local site of injection, re-distribution to distal sites is delayed.

Figure 5. Intra-femoral donor cell injection results in early local engraftment but delayed systemic engraftment relative to intravenous administration.

Figure 5

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Bioluminescence was measured serially in recipient mice after transplantation with transgenic donor marrow cells beginning 24 hours and ending at 65 days post BMT. Recipients received 2.7Gy TBI before intravenous (IV-BMT, N=6) or intra-right femoral (RF-BMT, N=5) injection of 1×106 donor cells. (A) Whole body bioluminescence (Mean ± SEM) for all mice given IV-BMT (red) or RF-BMT (green). (B) Representative images of donor cell bioluminescence in recipient mice at days 1, 4, 8, 14, and 65 shown from mice receiving IV-BMT (top) and RF-BMT (bottom). In the right column, all images are scaled to decrease signal intensity and more clearly demonstrate the anatomic distribution of engraftment at 65 days post BMT. (C) Bioluminescence signal (Mean ± SEM) gated on left (white) and right (solid red) hind limbs of IV-BMT recipients. (D) Bioluminescence signal (Mean ± SEM) gated on left (white) and right (solid green) hind limbs, after RF-BMT. In (A) repeated measurements of whole body bioluminescence were combined for the first 10 days (Early) or greater than 10 days (Late) before using the unpaired T-test for statistical significance (**P<0.01, NS = not significant) comparing IV-BMT versus RF-BMT. In (C) and (D) two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-test for statistical significance (*P > 0.05) was used to compare bioluminescence in the left compared to the right hind limb of IV-BMT (C) and IF-BMT (D) recipients at each time point. Statistics was performed on logarithmic transformation of the data combined from 3 experiments. (Abbreviations: IV-BMT – intravenous bone marrow transplant; RF-BMT – right femoral injection bone marrow transplant; NS – not statistically significant).

Sub-lethal total body irradiation produces higher long-term chimerism than local irradiation: Intravenous BMT

A recent report has indicated that local radiation increases long-term stable engraftment in both irradiated and non-irradiated sites when compared to no irradiation (10). However, these reports did not address the question of whether local irradiation can provide similar levels of chimerism to that seen with total body irradiation. We therefore directly compared engraftment patterns after local irradiation of the right hind limb to engraftment after total body irradiation. Following irradiation, recipients received luciferase-expressing donor bone marrow cells via intravenous injection into the tail vein and chimerism was assessed as before by FACS analysis of bone marrow 10 weeks post transplantation and longitudinally by bioluminescence imaging.

Donor chimerism was analyzed at 10 weeks post transplantation by flow cytometry of cells isolated separately from the right (irradiated) and left (non-irradiated) hind limb. Using intravenous administration of donor marrow, total body irradiation provided significantly higher overall chimerism in marrow from both hind limbs compared to animals that received local irradiation of the right hind limb or no irradiation (No IR) (TBI 69±3% vs. local irradiation 7±2% and No IR 6±2%, p<0.001) (Figure 6A). As expected, in either non-irradiated or total body irradiated animals, there was no difference between levels of chimerism in the right hind limb and the left hind limb at 10 weeks post transplant. In animals irradiated locally at the right hind limb, and transplanted intravenously, there was a trend for higher chimerism in the irradiated limb (11±3%) compared to the non-irradiated left limb (4±1%) at 10 weeks post transplant, though these differences did not reach statistical significance.

Figure 6. Conditioning with total body irradiation results in higher levels of chimerism than with local marrow irradiation.

Figure 6

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Shown are the effects of local irradiation and TBI and route of donor cell administration on long-term donor chimerism as measured by FACS analysis at 10 weeks post BMT (A, C), and on the dynamics of engraftment as measured by longitudinal bioluminescence imaging (B, D, E, F, G). Animals were either given no irradiation (No IR, A, B: N=2), 2.7Gy total body irradiation (TBI; A, B: N=3), or 2.7Gy local irradiation to the right hind limb (RHL-IR), prior to either intravenous (IV) (A, B, E: N=3) or intra-femoral (C, D, F, G) administration of donor bone marrow. For animals given either no irradiation (C, D: N=4) or TBI (C, D: N=5), intra-femoral administration was into the right femur (RF-BMT). For animals given local irradiation to the RHL, donor cells were injected either into the irradiated right femur (RF-BMT; C, D, F: N=2), or into the non-irradiated left femur (LF-BMT; C, D, G: N=3). (A, C) % H-2Kq positive donor cells detected by flow cytometry from samples analyzed separately from the left and right femurs of recipient mice, 10 weeks after receiving BMT. Analysis of Variance with Bonferroni's Adjustment Test (***P< 0.001) was used to compare the left versus the right hind limb within each group and across each group. (B, D) Logarithmic plots of mean whole body bioluminescence versus days after IV (B) or intra-femoral (IF) BMT (D). Data for mice receiving No IR are shown in blue with solid circles, TBI are shown in red with solid circles, RHL IR are shown with empty black circles with either a red line for right femur injection (RF-BMT) or blue line for left femur injection (LF-BMT). Note: No IR and LF-BMT data overlap appearing as a single blue line in the graph in (D). Two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-test for statistical significance (***P > 0.001) was used to compare each recipient group at each time point. (E–G) To assess dynamics of engraftment in the marrow after local irradiation, bioluminescence signal was measured after gating on the left or right hind limb region. Schemas of experimental design are shown to the left of each set of data with (E) intravenous donor cell injection, (F) donor cell injection directly into the right irradiated femur (right) and (G) donor cell injection directly into the left (non-irradiated) femur. Repeated measurements of bioluminescence were combined for the first 10 days (Early) or greater than 10 days (Late) before using the paired T-test for statistical significance (*P<0.05, **P<0.01, or NS = not significant) comparing left or to right hind limb in E, F, and G. All statistics was performed on logarithmic transformation of the data combined from 2 experiments. (Abbreviations: Macs/Grans – Macrophages/Granulocytes; No IR – no irradiation; TBI – Total Body Irradiation; RHL IR – right hind limb irradiation; IV-BMT – intravenous bone marrow transplant; RF-BMT – right femoral injection bone marrow transplant; LF-BMT – left femoral injection bone marrow transplant; IR – irradiation; NS – not statistically significant).

Bioluminescence imaging of the same animals confirmed that, after the first week post transplant, systemic (whole body) chimerism, as measured by total bioluminescent signal of the whole animal, was significantly greater with TBI than with either local or no irradiation (Figure 6B). Consistent with previous data, the significant increase in chimerism seen with TBI was evident only after 7–10 days post transplant. Local irradiation conferred no increase in systemic chimerism compared to no irradiation at any time point. However, when imaging was restricted to each hind limb, a significantly higher signal was detected in the irradiated limb compared to the non-irradiated limb; again the effect was only seen after 7–10 days post transplant (Figure 6E). Thus local irradiation was significantly inferior to TBI in providing long-term chimerism after IV administration of BM.

Sub-lethal total body irradiation produces higher long-term chimerism than local irradiation: intra-femoral BMT

To minimize any effect on homing, we next examined the effects of TBI versus local irradiation when donor cells were injected directly into the marrow cavity. For locally irradiated animals, donor cells were injected either into the irradiated (local) femoral cavity or into the non-irradiated (distal) femoral cavity. Again, donor chimerism at 10 weeks post transplant was analyzed separately by flow cytometry of cells isolated from the right and left hind limbs. In all experiments using intra-femoral BMT, TBI produced significantly higher long-term marrow chimerism than did local irradiation, either when local irradiation was applied to the injected or non-injected limb (40±10% with TBI vs. 7±3% with injection into the locally irradiated femur, p<0.05; or vs. 4±1% with injection into the non-irradiated femur, p<0.01) (Figure 6C). Using TBI, chimerism in injected and non-injected limbs was no different by 10 weeks post BMT, consistent with data in Figure 4. When donor cells were injected into the same (right) limb that was locally irradiated, mean chimerism of the right injected limb (11±5%) was higher than in the left non-injected (2±1%), non-irradiated limb at 10 weeks, though these differences did not reach statistical significance. When donor cells were injected into the distal (left) limb that had not been irradiated, the donor chimerism of both limbs was no different to that seen in non-irradiated animals. Thus, a small effect was seen with local irradiation only when infusion of bone marrow was directly into the irradiated site and not if marrow was administered distal to the irradiation.

Data from whole body bioluminescence imaging was consistent with endpoint analysis by FACS: donor chimerism after intra-femoral injection was again seen to be significantly greater with TBI than with local irradiation or no irradiation (NIR) (Figure 6D). Again, bioluminescence signal was the same in all animals for the first week after transplantation with the benefits of TBI appearing only after day 8 (p<0.001). No difference in overall chimerism was seen with local irradiation versus no irradiation (Figure 6D).

Bioluminescence imaging of locally irradiated mice reveals that the effects of irradiation on HSC engraftment are restricted to the irradiated site

As we had previously noted that splenic chimerism is high with or without irradiation, we hypothesized that splenic engraftment would contribute to the whole body bioluminescence signals seen in both non-irradiated and locally irradiated animals (Figure 6B, D) and may thus obscure engraftment differences specifically in the marrow space. We thus next analyzed separately the signals arising from the right and left hind limbs to assess whether local irradiation would change the dynamics of engraftment specifically in the marrow either in the setting of intravenously administered donor cells (Figure 6E) or in animals injected with donor cells into the irradiated (right) femur (Figure 6F) or in the non-irradiated (left) femur (Figure 6G). Using this approach, our interest was to examine if radiation affected not only local engraftment but also the efficiency of migration to marrow distal to the injection site.

As mentioned above, local irradiation had no effect on early donor chimerism in the marrow after intravenous infusion, as signals from irradiated and non-irradiated hind limbs were identical during the first week (Figure 6E). However, a significantly higher (p<0.05) signal was detected from the irradiated limb compared to the non-irradiated limb after 1 week (Figure 6E).

When bone marrow was injected locally, the signal was statistically higher in the injected limb during the first week after transplantation, irrespective of whether the limb injected was on the side that received irradiation (Figure 6F, p<0.05) or on the side contralateral to irradiation (Figure 6G, p<0.01). The higher initial donor chimerism in the injected sites was maintained into the later post transplant period. However, long-term donor chimerism was highest in the limbs that received donor cells injected into the irradiated sites. These findings lead us to conclude that the effects of local sub-lethal irradiation are limited to the irradiated microenvironment rather than induction of systemic factors that can influence distal sites of engraftment.

Discussion

The clinical use of non-myeloablative preparative regimens for hematopoietic stem cell transplantation continues to increase since the first observations that donor chimerism could be accomplished with significantly reduced toxicity to the recipient. Most of these regimens combine low dose irradiation, either as TBI or total lymphoid irradiation, with lympho-ablative drugs. It is not clear how the low dose irradiation improves engraftment but it has been thought that the main mechanism for this effect is by increasing host lympho-ablation and thus preventing immunological rejection. However, some experimental models suggest that sub-lethal irradiation improves stem cell engraftment through effects on the marrow microenvironment in a dose dependent manner (18). To examine the effects of low dose irradiation on non-immunological mechanisms of engraftment, we analyzed the kinetics of donor engraftment in a severely immune deficient mouse recipient. We show that sub-lethal total body irradiation does significantly increase marrow engraftment after allogeneic transplantation, independent of any effects on lympho-ablation.

Through the use of bioluminescence imaging, it was revealed that although long-term chimerism is increased by sublethal irradiation, this is not as a result of increased early engraftment. The increased percentage of donor cells in irradiated bone marrow early post transplant detected by flow cytometry was a reflection of the rapid loss of host cells after irradiation. In fact, irradiation reduced rather than increased the absolute number of donor cells in the host bone marrow. This data is consistent with previously published reports comparing homing and engraftment of donor cells in non-irradiated and lethally irradiated transplant recipients (17, 19, 20). Damage to stromal cells after irradiation has been shown to decrease retention of hematopoietic cells in the bone marrow leading to an increase in lodgment of donor cells in non-hematopoietic organs such as the liver, lung and skeletal muscle after transplant (20, 21). This may explain the equivalent whole body bioluminescence seen in our imaging studies of irradiated and non-irradiated recipients during the first week after transplantation.

Bioluminescence imaging revealed that expansion of donor cells began after ~7 days following engraftment and continued for approximately 30 days after BMT. This late expansion was significantly increased with sub-lethal irradiation. These data are consistent with the hypothesis that irradiation induces changes in the recipient marrow microenvironment that stimulate donor cell expansion rather than facilitate homing. The finding that local irradiation enhanced long term chimerism of the irradiated limb but not of distal sites suggests that the effect of radiation is limited to the local irradiated microenvironment rather than due to the release of systemic factors. Of note, even in animals given intra-femoral transplants, TBI produced higher levels of overall marrow chimerism than did local irradiation, presumably because of the larger area of marrow space affected by TBI.

Intra-femoral transplantation has been reported to improve engraftment compared to intravenous transplantation in allogeneic (22) and xenogeneic BMT models (5, 23, 24). However, in our studies, we found that intravenous administration produced higher levels of marrow chimerism than did intra-femoral administration of donor cells. Several aspects of experimental design may explain the different findings in our studies compared to those of the previous reports. Almost all the data comparing intra-femoral with intravenous transplantation used small numbers of purified human stem and progenitor cells, transplanted into immune deficient mice (5, 24–26). It is likely that homing in the xenogeneic model is relatively inefficient and that direct administration into the marrow space provides an advantage over systemic infusion in the xenogeneic setting. In addition it would be expected that depletion of T cells and other mature cells from the more purified stem cell grafts used in other reports further reduce efficiency of homing and engraftment. In our studies, unfractionated allogeneic bone marrow was used rather than purified stem cells, thus potentially further facilitating engraftment in the intravenous transplants.

As whole body bioluminescence imaging reflects a composite of donor cells circulating from marrow to other lympho-hematopoietic organs, we also performed bioluminescence analysis of hind limbs separately to assess chimerism specifically in each marrow space. In mice given TBI, the signal from donor cells was higher at the injected site than in distal sites during the first week. However, after the first week, locally injected cells had mobilized to and proliferated in distant sites, eventually leading to long-term systemic chimerism. Thus, although locally injected limbs had high levels of chimerism initially, engraftment at distal sites was delayed compared to intravenously transplanted animals.

One interesting report found that animals receiving locally delivered sub-lethal irradiation to the mediastinum or hind limbs alone had increased donor chimerism in local and distal sites compared to animals that received no irradiation (10). The authors suggested that their data supports a model in which local irradiation first induces local proliferation of engrafted donor cells at irradiated sites, which subsequently equilibrate systemically to increase chimerism in non-irradiated sites. This previous report used only intravenous administration of donor cells, and thus did not address how cells would respond if injected directly into the irradiated or non-irradiated marrow spaces.

We found that regardless of injection route, recipients with 2.7Gy delivered as TBI had significantly higher long-term systemic engraftment than recipients receiving 2.7Gy of irradiation locally restricted to the right hind limb. However, again there was no difference in early whole body bioluminescence between TBI, locally irradiated and non-irradiated recipients. It should be noted that several differences in experimental design between our study and that of Ito et al. might account for the greater effect of local irradiation seen in the latter study (10). Ito et al used both a higher radiation dose (7Gy) and larger exposure area (27) than our study (mediastinal was estimated as 20% compared with 7.5% for a single limb). In addition, Ito et al. used a 200-fold higher cell dose compared to our studies.

In summary, the highest levels of long-term chimerism were seen with the combination of total body irradiation and intravenous administration of donor cells. The effect of sub-lethal irradiation was first detected at least a week after transplantation; during the first week, bioluminescence was unaffected by either systemic or local irradiation. Thus, sub-lethal irradiation increases long-term chimerism most likely through direct effects on the marrow microenvironment that induce proliferation of donor cells, an effect that is enhanced through both systemic administration of donor cells and by maximizing the area of irradiation.

Acknowledgements

The authors sincerely thank our colleagues Ann George and Judy Zhu; Lora Barsky, Jessica Scholes, and Ewa Zielinska from the Flow Cytometry Core; Gevorg Karapetyan from the Small Animal Imaging Core; The Animal Care Facility at the Saban Research Institute all at CHLA. We express gratitude for the insightful discussions with Drs. Andrew Cuddihy, Denis Evseenko, and Gautam Dravid. We thank Dr. David Gjertson at UCLA for advice on statistical analysis. Additionally, JA would like to express his deepest appreciation for the guidance of Dr. Brian E. Henderson from USC. This work was supported by NHLBI Award Number F31HL085926 (JA), 1P01AI072686 (GMC) and 2P01 - HL073104 (GMC).

Footnotes

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