Our study identifies the unexpected key role of host macrophages in modulating GVHD morbidity and mortality after allo-HCT. In this paper, we show that host macrophages persist in lymphoid organs for several days after allo-HCT and are critical to limit host tissue damage by donor alloreactive T cells. We also establish that pretransplant CSF-1 administration improves GVHD in transplanted animals through the expansion of the host macrophage pool.
These results came as a surprise because the current dogma suggests that host APCs, including DCs and macrophages, contribute to the induction of GVHD. This concept is based on experiments showing that the pretransplant conditioning regimen leads to the release of inflammatory cytokines by host macrophages (Hill et al., 1997
) and that the concomitant depletion of DC and macrophages improves GVHD (Zhang et al., 2002b
). In this study, we revisited the role of macrophages in GVHD by developing means to target host macrophages while sparing host DC before allo-HCT. To this end, we targeted CSF-1R to reduce macrophages, but not DC numbers, in lymphoid organs. CSF-1 is required for macrophage development, survival, and proliferation in vivo, and mice that lack CSF-1 or the CSF-1R also lack macrophages in lymphoid tissues (Cecchini et al., 1994
). We have shown in a series of studies (Helft et al., 2010
) that although CSF-1R controls the homeostasis of specific DC subsets in nonlymphoid tissues (Bogunovic et al., 2009
; Ginhoux et al., 2009
), it does not control the maintenance of lymphoid organ DC, and csf-1r−/−
mice have intact lymphoid organ DC populations (Ginhoux et al., 2006
). In this paper, we show that CSF-1R blockade before transplant eliminates macrophages, but not DC, in lymphoid organs and, unexpectedly, enhanced donor T cell expansion and exacerbated GVHD morbidity and mortality after allo-HCT.
To further establish the role of macrophages in GVHD, we administered low-dose Lip-Clod 10 d before transplant to deplete host macrophages, whereas host DC, which has a half-life in lymphoid tissues that does not exceed 3 d (Merad and Manz, 2009
), would have completely recovered at the time of transplant. Our results revealed that, in contrast to a previous study in which higher Lip-Clod doses administered 7 and 2 d before transplant led to the depletion of both DC and macrophages and improved GVHD (Zhang et al., 2002b
), low-dose Lip-Clod administered 10 d before transplant depleted host macrophages, but not DC, and aggravated GVHD.
Anti–CSF-1R mAb administration also reduced the number of circulating monocytes and affected the Gr-1low
monocyte subset more dramatically, suggesting that CSF-1R controls the differentiation of Gr-1high
monocytes in vivo. Because monocytes also limit T cell expansion after organ transplant (Garcia et al., 2010
), they could potentially also modulate GVHD outcome in mice treated with anti–CSF-1R mAb. The role of circulating monocytes in GVHD, however, appears to be unlikely, as circulating monocytes are sensitive to radiation and nearly absent from the host at the time of transplant. In addition, mice that received Lip-Clod 10 d before allo-HCT and have recovered the number of monocytes to normal levels (Tacke et al., 2006
) at the time of transplant developed more severe GVHD compared with control groups (unpublished data).
Our data also suggest that host macrophages improve GVHD by limiting the expansion of donor alloreactive T cells. The immunomodulatory role of macrophages has already been reported in several settings. In tumors, for example, macrophages modulate T cell function through several mechanisms that include, but are not limited to, the production of iNOS, arginase1, and IDO (Pollard, 2004
; Allavena et al., 2008
). Although these molecules have been shown to modulate GVHD after allo-HCT (Drobyski et al., 1994
; Krenger et al., 1996
; Blazar et al., 2003
; Banovic et al., 2005
; Jasperson et al., 2009
), blockade of IL-10, IDO, iNOS, or arginase 1 at least individually failed to interfere with macrophage ability to suppress donor T cell expansion in vitro. Blockade of TGF-β was able to partly restore donor T cell proliferation, suggesting its potential role in regulating recipient macrophage ability to control donor T cell expansion in vivo.
The aggravating effect of CSF-1R blockade identified in this paper is consistent with a recently published study showing that an antibody to CSF-1R (M279 clone), distinct from the AFS98 clone used in our study, also aggravated GVHD outcome after allo-HCT in mice (MacDonald et al., 2010
). Although this study did not identify the cellular target that control GVHD aggravation upon injection of the M279 Ab clone, it is likely that it differs from the cells targeted by the AFS98 clone. In contrast to AFS98, injection of the M279 Ab clone did not deplete spleen red pulp and LN medullary macrophages and affected mainly LN subcapsular macrophages, perifollicular macrophages in the spleen, and tissue-resident macrophages. The different target cell populations between the clones might explain the differential fate of donor T cells observed in the two studies. Whereas we found that AFS treatment significantly increased the donor T cell pool as early as 18 h after transplant and did not affect donor T regulatory cell differentiation in vivo, the M279 Ab clone failed to modulate the numbers of donor allogeneic T cells but affected Th1 differentiation and reduced donor T regulatory cell expansion in recipient mice.
Our results also suggest that host macrophages limit the donor T cell pool directly through their ability to engulf and clear donor T cells. CD47 is an integrin-associated protein ubiquitously expressed on all hematopoietic cells (Matozaki et al., 2009
) and its receptor called SIRPα (signal regulatory protein α) is highly expressed on macrophages (unpublished data) and on a subset of DC (Ginhoux et al., 2009
). We found that naive T cells express lower CD47 levels compared with other cell types. We also found that whereas naive allogeneic CD47−/−
T cells are rapidly eliminated upon injection into lethally irradiated recipient animals compared with wild-type T cells, the survival of allogeneic CD47−/−
T cells is dramatically prolonged when recipient mice are depleted of their macrophage content before transplant. These results strongly support CD47 having a key role in controlling macrophage ability to modulate the donor allogeneic T cell pool and are consistent with prior studies showing that CD47 expression by donor hematopoietic cells determine their ability to survive in recipient animals (Fraser et al., 1995
; Blazar et al., 2001
; Abe et al., 2002
; Rozemuller et al., 2004
; Ide et al., 2007
; Takenaka et al., 2007
Further supporting the importance of host macrophages in regulating allo-HCT outcome, we found that pretransplant injections of the cytokine CSF-1 increased the host macrophage pool, limited the expansion of donor alloreactive T cells, and improved GVHD morbidity and mortality. Because the half-life of CSF-1 in the circulation does not exceed 10 min (Bartocci et al., 1987
), these results strongly suggest that CSF-1 pretransplant treatment improved the survival of mice after allo-HCT by promoting the survival of host macrophages and their ability to modulate donor T cell–mediated tissue damage. In contrast to a previous report showing that CSF-1 administration after allo-HCT interferes with donor cell engraftment in recipient mice injected with T cell–depleted allografts (Blazar et al., 1992
), pretransplant use of CSF-1 did not affect the engraftment of donor hematopoietic cells in our study (unpublished data). CSF-1 after transplant administration was also found to prevent fungal infection in patients after allo-HCT (Nemunaitis et al., 1993
), although CSF-1 posttransplant therapy to treat GVHD should be used with caution, as CSF-1 should also be able to prolong the survival of macrophages that infiltrate GVHD target tissue and promote tissue inflammation and fibrosis (Facon et al., 1995
; Namba et al., 2007
; Wynn and Barron, 2010
In conclusion, our study establishes for the first time the differential role of host APC in allo-HCT. In this paper, we show that although both host DCs and macrophages survive the conditioning regimen, they have opposite contribution to GVHD outcome. Host DCs prime donor T cells against host tissue antigens and initiate GVH reactions. In contrast, remaining host macrophages reduce the donor T cell pool through their ability to engulf alloreactive T cells and to modulate their proliferation and, consequently, limit the severity of GVHD. Our study also identifies pretransplant CSF-1 therapy as a novel clinical strategy for the modulation of GVHD severity in patients undergoing allo-HCT.