The Macrophage-depleting Agent Clodronate Promotes Durable Hematopoietic Chimerism and Donor-specific Skin Allograft Tolerance in Mice

Hematopoietic chimerism is known to promote donor-specific organ allograft tolerance; however, clinical translation has been impeded by the requirement for toxic immunosuppression and large doses of donor bone marrow (BM) cells. Here, we investigated in mice whether durable chimerism might be enhanced by pre-treatment of the recipient with liposomal clodronate, a macrophage depleting agent, with the goal of vacating BM niches for preferential reoccupation by donor hematopoietic stem cells (HSC). We found that liposomal clodronate pretreatment of C57BL/6 mice permitted establishment of durable hematopoietic chimerism when the mice were given a low dose of donor BM cells and transient immunosuppression. Moreover, clodronate pre-treatment increased durable donor-specific BALB/c skin allograft tolerance. These results provide proof-of-principle that clodronate is effective at sparing the number of donor BM cells required to achieve durable hematopoietic chimerism and donor-specific skin allograft tolerance and justify further development of a tolerance protocol based on this principle.

the number of "unoccupied" BM niches remains unclear, despite the fact that donor-derived HSC engraftment can be enhanced when recipient HSCs are mobilized 18 . Moreover, whether pharmacologic enhancement of donor HSC engraftment would be sufficient to induce durable mixed chimerism and donor-specific tolerance has never been addressed.
Previously, we have attempted unsuccessfully to enhance mixed chimerism induction and donor-specific tolerance by mobilizing recipient HSCs with AMD3100 and/or G-CSF before allogeneic BM infusion 9 . In the present study, we have attempted to mobilize recipient HSCs from BM niches by systemic depletion of recipient macrophages, and have found the approach effective in inducing durable mixed chimerism and robust donor-specific tolerance to skin allografts in recipient animals.

Methods
Mice. Male C57BL/6, BALB/c and CBA/Ca mice 8 to 12 weeks old (body weight range: 26 ~ 28 gram) were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA) and were maintained in our specific pathogen-free animal facility. All care and handling of animals was carried out in accordance with guidelines provided in the Guide for the Care and Use of Laboratory Animals published by the U.S. Department of Health and Human Services. All studies were reviewed and approved by the Animal Care and Use Committee of the NIAID, NIH.
Bone marrow and skin transplantation. Allogeneic BM transplantation was modified from a protocol described elsewhere 19 . Briefly, BALB/c BM cells were obtained by flushing long bones, and were suspended in complete medium containing Ammonium-Chloride-Potassium lysing buffer to remove red blood cells. Unseparated BM cells were injected i.v. into C57BL/6 recipient mice via tail vein. The number of cells injected was either 2 × 10 7 or 5 × 10 7 , which are respectively below and above the threshold for establishing hematopoietic chimerism in our system without BM conditioning using radiation or cytotoxic agents. The immunosuppression protocol used in all experiments involved three consecutive 1 mg injections (days 0, 2 and 4 post-BM transplantation) of each of the following monoclonal antibodies: rat anti-mouse CD4 (CD4 cell non-depleting; YTS 177), anti-CD40 ligand (MR1) and anti-mouse CD8 (CD8 cell depleting; YTS 169), all from BioXcell (West Lebanon, NH). In addition, two doses of 12 mg/kg of rapamycin (LC Laboratories, Woburn, MA) were administered i.p. at days 6 and 30 post-transplantation 8 .
Where indicated, skin transplantation was performed as previously described by grafting full thickness tail skin measuring 1 × 1 cm on the two lateral flanks, one for the BALB/c donor type and the other for the CBA/Ca third party 8 . Grafts were observed daily after the removal of the bandage at day 7 post-transplantation and were considered rejected when complete loss of viable donor epithelium had occurred.
In vivo macrophage depletion protocol. Our protocol of in vivo macrophage depletion was modified from that described previously 17 . Mice were retro-orbitally injected with 200 or 300 μL of a clodronate-loaded liposome suspension (Encapsula NanoSciences, Brentwood, TN) every other day for four doses during the week prior to BM transplantation and beginning immunosuppression. Control mice were injected with 300 μL PBS-loaded liposomes using the same schedule. Some mice received intra-nasal administration of liposomal clodronate to deplete lung macrophages; in that case, a total of 150 μL liposomal clodronate was administered on days one and two before BM transplantation 20 .
Statistics. Data were analyzed using unpaired parametric t-tests (two-tailed) with Prism 6 (GraphPad Software) and are presented as the mean ± SEM of summary data (the data were approximately normally distributed).

Results
Clodronate pretreatment induces durable mixed hematopoietic chimerism associated with robust, donor-specific skin allograft tolerance. The use of liposomal clodronate in mice, especially frequent and large intravenous doses, is known to be toxic and even fatal in a subset of animals 22 . We confirmed this in a preliminary experiment, in which we tested 100, 200 and 300 μL doses of liposomal clodronate given IV every other day for a total of four doses (n = 5 in each group). We observed one, two and two deaths per group, respectively (overall mortality rate of 33.3%), occurring between days 2 and 14 after the first injection; no deaths were observed in a group receiving control liposomes (n = 5) (data not shown). Since the majority of animals survived, we decided this was sufficient to test the concept that this macrophage-depleting agent could enhance mixed chimerism and induce donor-specific tolerance. Consistent with our preliminary results, we found an overall mortality rate of 31.8% across all the experiments detailed below (14 deaths in 44 mice receiving IV liposomal clodronate). Stratified by the doses studied, we found that 5 out of 15 mice (33.3%) receiving 200 μL of liposomal clodronate died, and 9 out of 29 mice (31.03%) receiving 300 μL of liposomal clodronate died, whereas no deaths were observed in mice receiving intranasal liposomal clodronate (n = 6), i.v. control liposomes (n = 12) or no liposomes (n = 13) ( Table 1). With one exception, all mice receiving liposomal clodronate that survived this early phase of high mortality survived until the end of the study (> 180 days after clodronate administration) and were not distinguishable from control mice with regard to weight and overt behavior. The outlier mouse had received 200 μl of liposomal clodronate and died on day 62 after the first injection.
Using the same dosing schedule specified above, we found that injection of 300 μl of liposomal clodronate resulted in a 50% reduction of CD11b + F4/80 + macrophages in the BM compared to control liposome-injected animals on the day after the last injection (p < 0.001, n = 2 experiments), consistent with previously published studies 17 . No effect was observed for liposomal clodronate on the absolute levels of neutrophils, B cells or T cells in the blood (data not shown).
On the day after the last liposomal clodronate or control liposome injection of C57BL/6 recipient mice, we infused allogeneic BALB/c donor BM to test whether mixed chimerism induction could be enhanced under transient immunosuppression conditions (administration on days 0, 2 and 4 post-BMT of depleting anti-CD8 mAb and non-depleting anti-CD4 mAb and CD40L mAb; rapamicin on day 6 and day 30 post-BMT) (Fig. 1). Infusion of 20 million donor BM cells into recipient mice receiving control liposomes induced only a background level of mixed chimerism in peripheral blood, ranging from 0.05 ± 0.02% to 0.14 ± 0.07%, both at 30 and 90 days after transplantation. In contrast, in mice receiving 50 million BM cells and the same immunosuppression schedule,  without any clodronate pretreatment, durable mixed chimerism was established (0.86 ± 0.21% and 2.01 ± 0.66% at 30 and 90 days, respectively) (Supplemental Fig. 1A). These benchmarks are consistent with our previous report 9 . C57BL/6 mice receiving 20 million BALB/c donor BM cells (i.e., the dose we demonstrated to be below a threshold required to establish hematopoietic chimerism under transient immunosuppression) showed clear evidence of durable mixed hematopoietic chimerism when they were pre-treated with four every other day injections of 200 μl of liposomal clodronate before receiving the transplant, in contrast to mice receiving the same doses of control liposomes and BM cells (Supplemental Fig. 1A).
Skin allotransplantation with donor-type (BALB/c) and third-party (CBA/Ca) allografts was performed next in these chimeric mice to determine whether donor-specific skin allograft tolerance could be established in association with clodronate-dependent hematopoietic chimerism. Skin allotransplantation was first performed (primary allograft) 90 days after the BM transplant, a time-point when all the recipient mice had been off immunosuppression for at least two months. As expected, all third-party CBA/Ca primary allografts were rapidly rejected (fewer than 20 days) after skin transplantation in all groups tested. The BALB/c donor-type primary allografts were also rapidly rejected in mice receiving 20 million donor BM cells without liposomal clodronate pretreatment. In contrast, prolonged donor-specific primary skin allograft survival was observed in mice pretreated with liposomal clodronate. In particular, in liposomal clodronate-pretreated mice, rejection was markedly delayed in 3 out of 7 animals; in the other 4 mice, no evidence of rejection was observed for up to 90 days post-primary skin allotransplantation (Supplemental Fig. 1B), at which point the animals were sacrificed to evaluate immune organ chimerism (see below).
We next carried out a second independent set of experiments, this time increasing the dose of liposomal clodronate to 300 μL per injection. Mixed hematopoietic chimerism was also observed in these animals, 1.67 ± 0.22% and 1.87 ± 0.26% at 30 and 90 days, respectively ( Fig. 2A), which are similar to the levels of chimerism we had observed using 200 μL per injection of liposomal clodronate (Supplemental Fig. 1A). Again, animals receiving control liposomes had only background levels of chimerism, and animals receiving 50 million BM cells without liposomes had durable and high levels of chimerism, similar in magnitude to what was observed with clodronate-pretreated mice receiving a subthreshold dose of 20 million BM cells (Fig. 2A). Furthermore, only 2  (Fig. 2B,C). The other twelve donor-type primary BALB/c allografts survived for the full post-primary skin transplant observation period (90 days). Rejection of the donor-type BALB/c primary skin allografts in the two exceptions was markedly delayed (35 days post-primary skin transplantation) compared to the time of the primary third party CBA/Ca allograft rejection (which occurred in 100% of mice), as well as compared to the time of donor type primary allograft rejection in mice not receiving liposomal clodronate. This rate of donor-type BALB/c primary skin allograft survival was similar to the survival rate in C57BL/6 mice receiving 5 × 10 7 donor BM cells (cells) without liposomal clodronate pretreatment (Fig. 2B,C).
To test the robustness of the apparent donor-specific primary skin allograft tolerance in these 12 mice that had received 300 μL of liposomal clodronate per injection, donor-type and third party secondary skin allotransplantation was performed. Ten of the 12 mice tolerated both the primary and secondary donor-type BALB/c allografts for the full observation period after secondary transplantation (45 days), whereas all third party CBA/Ca secondary skin allografts were rejected in fewer than 20 days. Rejection of primary and secondary donor-type allografts in the other two mice occurred 20 and 30 days after the time of secondary skin transplantation (Fig. 2B,D).

Multi-lineage chimerism was found in tolerant animals, with donor-derived B lymphocytes the major component. Mice in the low dose clodronate group (200 μL/injection) were sacrificed at 180 days
after BM infusion (i.e. 90 days after primary skin allograft transplantation) to check the status of mixed chimerism in immune organs (spleen, lymph node, BM) as well as in liver, lung and kidney. Only background levels of hematopoietic chimerism were observed in mice that had received 20 million donor BM cells without liposomal clodronate pretreatment (Supplemental Fig. 1C). In contrast, in the liposomal clodronate-pretreated mice receiving 20 million BM cells, 3-6% mixed hematopoietic chimerism was observed in immune organs of the mice still tolerant to donor-type skin allografts (Supplemental Fig. 1C, and data not shown). Mixed chimerism was also observed in the liver and lungs of the clodronate-pretreated mice; no chimerism was observed in the kidney (data not shown). On the other hand, in the few clodronate pretreated mice that had rejected their skin allografts, mixed chimerism did not rise above the background level (data not shown). In those mice receiving 50 million donor BM cells without clodronate pre-treatment, 2-4% mixed chimerism was also observed in immune organs of mice remaining tolerant to donor-type skin allografts (Supplemental Fig. 1C). The same pattern of mixed hematopoietic chimerism was observed in the same organs for the high dose liposomal clodronate-treated group of mice described above (300 μL/injection) (Fig. 3). It is important to note that since the high dose but not the low dose clodronate group of mice was subjected to secondary skin allotransplantation, mice in this group were sacrificed even later than the low dose group (at 225 days, instead of 180 days as for the low dose group, after BM infusion; i.e. 135 days after primary skin allograft transplantation) (Fig. 3). Lineage analysis of donor-derived cells in the BMs of recipient mice treated with either high or low dose clodronate revealed that CD19-positive B lymphocytes predominated, followed by CD11b + F4/80 + macrophages, CD4 + T lymphocytes, and CD49b + NK cells (Fig. 4). Histological analysis of chimeric recipient mice revealed the presence of donor MHC class I-expressing cells in situ in the BM, mainly in the peripheral sub-endosteal space (Fig. 5). Some of these donor-derived cells were found adjacent to cells expressing Nestin, a marker of mesenchymal stromal cells in BM niches (Fig. 5E). Both findings suggested that engraftment of the donor-derived cells had occurred in the recipient BM stem cell niche.
Donor-specific skin allograft tolerance is associated with specific Vβ T-cell deletion. One of the mechanisms by which mixed donor-recipient chimerism is thought to promote donor-specific tolerance is by central (intrathymic) and peripheral deletion of donor-reactive T cells from the recipient 23 . In our transplantation model, this can be reflected by the degree of deletion of specific Vβ T cells 8 . In particular, only the BALB/c MHC class II molecules present superantigen to TCR bearing Vβ 5 and Vβ 11 subdomains, leading to deletion of T cells expressing these Vβ molecules 8 . Thus, we tested whether specific Vβ T cell deletion was present in our model by comparing the percentages of Vβ 5 and Vβ 11 T cells in tolerant and non-tolerant skin allograft recipients as well as in naive animals; Vβ 6-expressing T cells, which do not recognize endogenous superantigens, served as control. The percentages of Vβ 6-expressing CD4 + and CD8 + T cells did not differ among the groups. In contrast, a significant reduction of Vβ 11-expressing CD4 + T cells was observed in the spleen and lymph node of mice tolerant to the skin allografts compared to the other two groups (Fig. 6). A similar decrease was also observed in the Vβ 5-expressing CD4 + T cells in the lymph node. No difference was observed for CD8 + T cells.

Systemic administration of liposomal clodronate and transient immunosuppression are critical for durable mixed chimerism induction.
A possible mechanism underlying our finding is that depletion of macrophages in the lungs decreased the passive entrapment of infused donor BM cells (first-pass effect), thus increasing the probability of BM niche engraftment. To test this, we performed BM infusion after administering liposomal clodronate via the intranasal route, a classical approach to selectively deplete lung macrophages 20 . In a control group of mice, BM infusion was performed after systemic macrophage depletion but in the absence of any immunosuppression. In contrast to mice receiving both systemic macrophage depletion and transient immunosuppression, no mixed chimerism was observed 30 days after BM infusion in either mice receiving intranasal clodronate or mice not receiving immunosuppression, suggesting the importance of systemic macrophage depletion and transient immunosuppression in the induction of mixed chimerism (Fig. 7).

Discussion
In the present study, we have demonstrated that the macrophage-depleting agent clodronate promotes durable mixed hematopoietic chimerism and donor-specific skin allograft tolerance in mice. It has been known since the 1940's that a state of mixed hematopoietic chimerism in allotransplant recipients relative to the genetic background of the donor may be associated with donor-specific tolerance 24 . Despite the clinical promise of   donor-specific tolerance and the reality that it can be achieved by establishing mixed hematopoietic chimerism under specific laboratory conditions and in rare experiments of nature, the ability to translate knowledge in this area to a safe and effective treatment for patients has been limited not only by the need for harsh patient conditioning but also by the requirement for clinically unachievable doses of BM cells needed to establish chimerism. Any protocol that might reduce the BM cell dose requirement would represent a significant advance in this field. We have previously described a relatively transient immunosuppressive regimen in mice that supports establishment of hematopoietic chimerism in the absence of radiation and cytotoxic agents, but this regimen still requires 50 million BM cells from the allogeneic donor or else transplantation of an intact limb 8,9 . Chimerism is thought to involve engraftment of donor hematopoietic stem cells in BM niches. The probability of donor HSC engraftment is thought to depend in part on the number of niche vacancies in the recipient, which may be increased by targeting specific niche components with specific drugs. The idea is to transiently create space in the niches at the time of BM transplantation to allow allogeneic HSCs to enter and engraft. In this regard, we have previously tested plerixafor, an antagonist of the HSC retention factor and chemokine receptor CXCR4, and found that it could enhance hematopoietic chimerism after BM transplantation, but only transiently and without promoting donor-specific allograft tolerance 9 .
Several studies have demonstrated that monocyte/macrophage depletion may promote mobilization of HSCs from BM to peripheral blood and spleen in mice 17,25,26 . The mechanism is thought to involve loss of direct support of niche cell HSC retention activity by local BM macrophages, although other potential mechanisms may operate. Importantly, in animals pretreated with clodronate durable mixed chimerism occurred after infusing only 20 million total donor BM cells under transient immunosuppression conditions, and was associated with robust, donor-specific skin allograft tolerance. This was evidenced by the selective rejection of third-party but not donor-type fresh skin allografts transplanted 90 & 180 days after donor BM infusion, time-points at which the mice were free of immunosuppression. Our results using liposomal clodronate suggest that niches are fully competent after macrophage depletion in supporting allogeneic HSC engraftment, self-renewal and differentiation, as evidenced by the durable multilineage mixed chimerism and specific donor type Vβ T cell depletion we observed in chimeric recipient animals.
A number of other mixed chimerism approaches have been investigated trying to transplant clinically realistic doses of BM without irradiation or cytotoxic drugs 24 . Among them, only infusion of in vitro-prepared, polyclonal recipient-type regulatory T cells (Tregs) leads to engraftment of clinically feasible doses of fully allogeneic BM without cytoreductive recipient conditioning 27 . In that study, although long-term, donor-specific skin allograft tolerance was achieved, the robustness of tolerance was not determined by second-set grafting of fresh skin. In any case, preparing Tregs ex vivo is complicated and expensive and may not scale as a community-based treatment option.
Our study has several important limitations. First, although it did deplete macrophages in the BM and in other organs, we do not know exactly how clodronate is working. Second, clodronate was toxic and resulted in ~30% mortality in recipient mice, mainly due to cardiac complications (infarction and hemorrhage). We took advantage of the surviving mice, which represented the majority, to perform this proof-of-principle study; however, a role for one or more of the toxic side-effects of clodronate at the doses we used cannot be excluded with regard to promoting chimerism. Third, our data indicate that immunosuppression, although transient, is still needed to induce a state of mixed hematopoietic chimerism and possibly to prevent graft-versus-host disease (GVHD).
In conclusion, we have developed proof-of-principle that pretreatment of recipient mice with the macrophage-depleting agent liposomal clodronate can enhance induction of mixed hematopoietic chimerism and promote robust donor-specific skin allograft tolerance. In order to be translated to the clinic, further optimization of the protocol will be necessary, at the level of both safety and efficacy, or instead by identifying another less toxic macrophage-depleting agent able to enhance chimerism and tolerance.