Inhibition of T Cell Alloreactivity by Gr-1⁺Mac-1⁺ Myeloid Suppressor Cells.

Graft-versus-host-disease (GVHD) remains the major complication of allogeneic stem cell transplantation (SCT). While pharmacological approaches have long been the mainstay of GVHD prevention, recent strategies have focused on cellular approaches, specifically the use of regulatory cell populations to prevent or mitigate the severity of GVHD. Gr-1⁺Mac-1⁺ myeloid-derived cells, termed myeloid suppressor cells (MSCs), are a regulatory cell population that has been shown to play an important role in the suppression of tumor and anti-infectious immunity. In these studies, we examined whether these cells were detectable in GVHD recipients and played any role in the suppression of alloreactive T cell responses in vitro and in vivo. Lethally irradiated Balb/c (H-2^d) mice were transplanted with MHC-incompatible C57BL/6 (H-2^b) bone marrow and spleen cells to induce GVHD. Immature myeloid cells with a Gr-1⁺Mac-1⁺ phenotype were found to constitute 60% of all splenocytes at day 10 post-SCT thereafter declining to 30% by day 21. MSCs isolated from the spleens of mice with GVHD expressed high levels of class I and II, but low levels of the costimulatory molecules CD80, CD86, CD40 and were negative for CD11c. Highly purified Gr-1⁺ Mac-1⁺ cells obtained from the spleens of GVHD mice 10 or 21 days post-SCT potently suppressed naive T cell proliferation in a standard MLC by 70% at a responder to suppressor ratio of 1:1. Notably, MSCs could also be generated in vitro from normal B6 BM after 6 days in culture with G-CSF and were equally potent at suppressing T cell alloreactivity. Suppression was partially mediated by nitric oxide (NO) as addition of the NO inhibitor L-NMA reversed 50% of the inhibitory effect. To determine whether MSCs exerted a suppressive effect in vivo, lethally irradiated Balb mice were transplanted with B6 BM plus naïve spleen cells with or without MSCs obtained from the spleens of GVHD animals 10 or 21 days post-BMT. The adoptive transfer of MSCs failed to protect mice from GVHD when assessed by overall survival, serial weight measurements or histological analysis. As an alternative approach to augment GVHD protection, G-CSF was administered for 14 days beginning at the time of BMT to enhance the in vivo survival of MSCs. G-CSF administration in both irradiated and non-irradiated MHC-incompatible GVHD mouse models, however, also had no protective effect. We reasoned that a possible explanation for the lack of an effect in vivo was that MSCs did not appropriately localize to nodal sites where GVHD is initiated. To examine this question, B6-green fluorescent protein (GFP) mice were used as donor animals to permit the detection and trafficking of MSCs in vivo. Adoptive transfer of B6 GFP-MSCs from the spleens of GVHD mice 10 days after SCT demonstrated that these cells could be detected in the secondary lymphoid organs of recipient mice 20 hours after BMT, but were completely absent by day 3, supporting the premise that the lack of protection was attributable, at least in part, to the inability of these cells to persist at nodal sites where T cell priming against host alloantigens was occurring. These results indicate that MSCs, which are present in the spleen of GVHD recipients, show potent T cell suppressive capacity in vitro, but fail to mediate a similar effect in vivo. Lack of protection appears to be due to the inability of these cells to localize to all critical secondary lymphoid sites underscoring the importance that the capability of regulatory cells to migrate to and persist at appropriate tissue sites plays in the design of cellular strategies to prevent GVHD.