Abstract
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.
Highlights
hematopoietic stem cells (HSC) activity in the niche is tightly controlled; under certain circumstances, they can be induced to egress from the bone marrow (BM) and enter into the peripheral circulation, a process termed “mobilization”, which is still poorly understood[11,12]
C57BL/6 mice receiving 20 million BALB/c donor BM cells 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)
We have demonstrated that the macrophage-depleting agent clodronate promotes durable mixed hematopoietic chimerism and donor-specific skin allograft tolerance in mice
Summary
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). Leukocytes were purified from the blood, thymus, spleen, lymph node, BM, liver and lung from mice that had received BM transplantation, were washed after red blood cell lysis and incubated for 20 min with Fc blocking antibody (Clone 93, 1:50 BioLegend, San Diego, CA) followed by the relevant antibody combinations (1:50 dilution) and FACS staining buffer for 30 min at 4 °C. 3D tiled-images comprising the entire sternum fossae volume were collected for assessment of the number and distribution of donor and recipient cells. 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)
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