Mouse Macrophages Driven to a Novel State of Activation Prolong Allograft Survival in Unconditioned, Non-immunosuppressed Recipients

Paloma Riquelme¹, James Hutchinson¹, Stefan Tomiuk², Hans Schlitt¹, Edward Geissler¹

¹Department of Surgery and Experimental Surgery, University Hospital Regensburg, Regensburg; ²Miltenyi Biotec GmbH, Bergisch Gladbach; Germany

Human regulatory macrophages (M reg) have been extensively investigated as a potential cell-based immunosuppressive treatment. Using culture conditions analogous to those under which human M regs arise, we have now generated a mouse counterpart of the human cell. Identifying and accurately characterising the mouse M reg has provided a convenient model to study the mechanisms of M reg action in vivo and also to define an optimal immunosuppressive context for the delivery of M reg therapy. Mouse monocytes are driven to the M reg phenotype over a seven day culture period during which the cells are exposed to M-CSF and IFN-g. The resulting M regs adopt a characteristic morphology and express markers distinguishing them from monocytes, monocyte-derived DCs and M0-, M2a-, and M2c-polarised macrophages. To better assess the interrelation of M regs and these other cell types, whole genome expression profiling studies were undertaken. In clustering analyses, M regs were found to constitute a distinct subgroup most closely related to immune complex-stimulated macrophages. At a 1:1 ratio, M regs completely suppressed polyclonally-stimulated T cell proliferation: this effect was extinguished by inhibitors of inducible nitric oxide synthase. In a heterotopic heart transplant model, a single intravenous administration of 5x10⁶ donor-strain M regs prior to transplantation significantly prolonged allograft survival in unconditioned, non-immunosuppressed recipients using both the stringent C3H-to-BALB/c (32.6±4.5 vs. 8.7±0.2 days; p<0.001) and B6-to-BALB/c (31.1±12 vs 9.7±0.4 days; p=0.002) strain combinations. The graft-protective effects of M regs were specific to donor cells as recipient (9.6±0.4 days; p>001) and third party-derived M regs (11.0±0.6 days; p<0.001) were not effective. Co-treatment with M regs and 1 mg/kg/day rapamycin for 10 days posttransplant enhanced the effect of M regs (64.1±8.6 days) compared to M reg treatment alone (p=0.006) or rapamycin alone (p=0.02) and some recipients accepted grafts indefinitely. It is concluded that mouse M regs represent a novel, phenotypically distinct subset of macrophages which bear a close resemblence to human M regs in terms of their mode of derivation, marker phenotype and in vitro functions. The graft-protective effect of M reg treatment in mice is further enhanced by concurrent administration of rapamycin.