In this study, we analyzed the suitability of the NSG mouse as a model for human skin transplantation and allograft rejection. We confirm a previous report (12
) that a cellular infiltrate consisting of mononuclear cells and granulocytes of host origin invades the graft, leading to decreased graft integrity. Depletion of host Gr1+
cells depletes this cellular infiltrate, improves the healing process and preserves the human endothelium within the graft. Passenger human leukocytes within the graft survive, expand, and populate the peripheral tissues of the host. Healed-in human skin grafts were rejected following engraftment of unfractionated, CD4 or CD8 allogeneic human PBMC.
Numerous studies of human skin grafts on immunodeficient mice have been reported, and many have been based on the SCID.bg strain (12
). A comparison of SCID.bg with NSG as recipients of skin grafts documented that the grafts retained better integrity and human endothelium survival following transplantation on the SCID.bg strain than on the NSG strain (12
). However, human lymphohematopoietic cell engraftment is much better on the NSG strain (2
). We have shown that the NSG mouse is an excellent model for human islet alloimmunity (1
) in which preservation of graft endothelium is not an issue as most is destroyed during the islet isolation process (17
). To combine the superior lymphohematopoietic engraftment characteristics of the NSG mouse with the enhanced skin graft integrity of SCID.bg mice, we used anti-Gr1 mAb to prevent host Gr1+
cellular infiltration into the graft. This treatment decreased the cellular infiltrate leading to enhanced graft vasculature, morphology and preservation of graft endothelium.
The simplest interpretation of our data is that depletion of mouse granulocytes with anti-Gr1 mAb is responsible for improved graft outcome. However, a Gr1+
monocyte/macrophage population has been described (18
), and are one of the first cell populations to arrive at a wound site. They are postulated to be involved in the inflammatory response, wound healing, and tissue repair (19
). Two subsets of Gr1+
macrophages have been identified, a first wave of Gr1hi
macrophages consisting of macrophages that rapidly respond to and perpetuate the inflammatory process, and a second wave of Gr1lo
macrophages involved in tissue repair and healing (21
). Depletion of Gr1+
cells would be expected to deplete both granulocytes and the initial infiltration of Gr1hi
pro-inflammatory macrophages, reducing the inflammatory environment at the graft site. Alternatively, the Gr1lo
macrophages would be relatively resistant to depletion with anti-Gr1 mAb, and would promote tissue repair. Our observation that anti-Gr1 mAb enhances graft healing supports this model of sequential infiltration of different subsets of Gr1+
Although many models of human skin grafts on immunodeficient mice have been described (12
), the observation of human passenger leukocyte survival in the graft has not been reported. We observed that passenger leukocytes in human skin grafts on NSG mice survived and expanded following transplantation. Passenger leukocytes that reside within a graft have been shown to participate in allorejection, but paradoxically may also enhance graft acceptance through immunomodulation pathways (23
). In previous immunodeficient mouse models, host innate immunity, particularly host NK cells, prevent passenger leukocyte survival (24
). In NSG mice, NK cells are non-functional, and human tissues with inherent architecture, including passenger leukocytes, have been shown to engraft following primary human lung tumor transplantation (26
). Our data indicate that skin passenger leukocytes not only survive, but also migrate into host tissues. Higher levels of human CD45+
cells in the host tissues correlated with reduced graft pliability and increased transplant thickness, pigmentation, epidermal pealing and rough appearance (unpublished observations). We speculate that an increased pro-inflammatory environment at the graft site would increase a xenograft-graft-versus-host response of the passenger leukocytes, leading to their activation and migration into the host tissues. This interpretation is supported by our observation that blockade of TNF, a pro-inflammatory cytokine, reduces xenograft-versus-host disease in PBMC-engrafted NSG mice (27
). These observations also support our hypothesis that depletion of Gr1hi
macrophages would reduce the pro-inflammatory environment at the graft site, thus reducing the xenograft-versus-host response, and promote healing of the graft.
NSG mice exhibit enhanced engraftment with human lymphohematopoietic cells as compared with immunodeficient strains based on the BALB/c background (2
). We confirmed that PBMC-injected NSG mice treated with anti-Gr1 mAb and bearing healed-in human skin grafts engrafted at high levels. Interestingly, we also observed increased human allogeneic PBMC infiltration into the murine skin in these mice. We have previously observed infiltration of human PBMC into the skin of NSG mice (1
), but this infiltration was low and rarely led to hair loss. In contrast, this infiltration was dramatically increased in the presence of human skin grafts on NSG mice. This suggests a possible enhanced expansion of xenoreactive human T cells in the absence of host Gr1+
cells, or alternatively, the establishment of an allo-response against the allogeneic human leukocytes that have migrated from the graft into the murine tissues. We are currently investigating the role of xenograft response versus allogeneic response using NSG mice lacking MHC class I, which reduces xenoreactivity (27
) and human skin grafts depleted of passenger leukocytes.
The engraftment of human PBMC into NSG mice bearing allogeneic human skin grafts led to graft infiltration and rejection. We have previously used this model to demonstrate rejection of human islet allografts in NSG mice (1
). In that model, islets and PBMC were transplanted simultaneously. In the present study, PBMC were capable of rejecting a healed-in human graft. We observed an early human CD45+
cellular infiltrate that was associated with visual evidence of sloughing of the skin from the graft bed. Human vasculature was lost and human-origin epithelium and dermal cell populations were absent by ~4 weeks after PBMC injection. In addition our data show that either human CD4+
T cells, purified from PBMC, can mediate the rejection.
In summary, we have shown that human skin grafted onto NSG mice is infiltrated with a host myeloid cellular infiltrate that impairs graft healing. Depletion of host Gr1+ cells improves graft integrity, promotes survival of graft endothelium, and reveals the presence of graft passenger leukocytes that can populate the host peripheral tissues. These healed-in grafts can function as targets of allogeneic human PBMC, recapitulating the rejection of grafts in the clinic with respect to kinetics and morphology. These data suggest the NSG mouse will be a suitable model for investigations of wound healing and transplantation.