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ORIGINAL RESEARCH ARTICLE

Alloimmune Injury and Rejection of Human Skin Grafts on Human Peripheral Blood Lymphocyte–Reconstituted Non-Obese Diabetic Severe Combined Immunodeficient ß₂-Microglobulin-Null Mice

Nicole A. Turgeon^*, Scott J. Banuelos^*, Leonard D. Shultz, Bonnie L. Lyons, Neal Iwakoshi^*, Dale L. Greiner^*, John P. Mordes^*, Aldo A. Rossini^* and Michael C. Appel^*,1

^* University of Massachusetts Medical School, Worcester, Massachussetts 01605 and
The Jackson Laboratory, Bar Harbor, Maine 04609

	Abstract

Top Abstract Introduction Methods and Materials Results Discussion References

 
Small animal models with the capacity to support engraftment of a functional human immune system are needed to facilitate studies of human alloimmunity. In the present investigation, non-obese diabetic (NOD) severe combined immunodeficient (scid) ß₂-microglobulin-null (B2m^null) mice engrafted with human peripheral blood lymphocytes (hu-PBL-NOD-scid B2m^null mice) were used as in vivo models for studying human skin allograft rejection. Hu-PBL-NOD-scid B2m^null mice were established by injection of human spleen cells or PBLs and transplanted with full-thickness allogeneic human skin. Human cell engraftment was enhanced by injection of anti-mouse CD122 antibody. The respective contributions of human CD4⁺ and CD8⁺ cells in allograft rejection were determined using depleting antibodies. Human skin grafts on unmanipulated NOD-scid B2m^null mice uniformly survived but on chimeric hu-PBL-NOD-scid B2m^null mice exhibited severe immune-mediated injury that often progressed to complete rejection. The alloaggressive hu-PBLs did not require prior in vitro sensitization to elicit targeted effector cell activity. Extensive mononuclear cell infiltration directed towards human-origin endothelium was associated with thrombosis and fibrin necrosis. No evidence of graft-versus-host disease was detected. Either CD4⁺ or CD8⁺ T cells may mediate injury and alloimmune rejection of human skin grafts on hu-PBL-NOD-scid B2m^null mice. It is proposed that Hu-PBL-NOD-scid B2m^null mice engrafted with human skin will provide a useful model for analysis of interventions designed to modulate human allograft rejection.

Key Words: hu-PBL-scid • skin allograft • graft rejection • transplantation

	Introduction

Top Abstract Introduction Methods and Materials Results Discussion References

 
A major goal of transplantation immunology is to achieve allograft survival in the absence of chronic immunosuppression (1–3). The realization of this goal would lead to durable graft survival without the toxicity and complications of long-term immunosuppression. As knowledge of immune system activation and tolerance induction advances, a large number of immune-based therapeutic reagents are being developed for clinical investigation (2, 4–6). Study of these reagents invariably originates using small animal models, primarily mice and rats, that ultimately provide the underlying mechanistic rationale for developing human-targeted therapy. Translation of rodent results to humans is not always successful, however, and many approaches effective in small animal models fail in larger animal species and in humans (2, 5, 7). Importantly, reagents directed towards human immune cells and molecules, particularly anti-human lymphocyte antibodies like alemtuzumab (Campath-1H, Millennium Pharmaceuticals, Cambridge, MA) (8), often cannot be tested directly in rodent models due to species specificity of the reagent.

Because testing investigational therapeutics in humans is often constrained by technical and ethical considerations, most experimental reagents are evaluated in nonhuman primates prior to initiation of phase I clinical trials. However, high cost, limited primate availability, and genetic heterogeneity hamper such studies, in particular the rapid screening of new reagents. Consequently, expensive small-scale studies are often marred by unacceptable variability, necessitating additional experimentation to achieve statistical validation. In addition, human immune-based therapeutics must have a high level of cross-reactivity with the nonhuman primate used in the analysis.

These limitations have generated a need for standardized small animal models that will facilitate the in vivo study of human immune responses without putting patients at risk and that are not restricted by limited animal availability and prohibitive cost. Our laboratories have developed mouse models that support human lymphocyte engraftment for in vivo studies of human immune responses and efficacy analyses of human-specific therapeutics (9, 10). We have based our approach on early observations that human fetal hematopoietic tissues (11) and adult peripheral blood mononuclear cells (PBLs) (12) can engraft in C.B-17 mice homozygous for the severe combined immunodeficient Prkdc^scid (scid) mutation (9). Unfortunately, low levels of engraftment and an inconsistent ability to generate functional human immune responses have compromised the utility of this model. The latter issue has remained problematic despite numerous reports suggesting that primary and secondary immune responses can be elicited in hu-PBL-scid mice (13–15).

Our approach to overcoming these limitations is based on our finding that genetic modification of the mouse recipient improves the engraftment and function of the human immune system. We backcrossed the scid mutation onto the non-obese diabetic (NOD)/Lt strain, which expresses multiple defects in innate immunity, and the resulting NOD/LtSz-Prkdc^scid/Prkdc^scid mice (hereafter referred to as NOD-scid mice) display much improved engraftment of human cells (9). NOD immunodeficient mice are now the strain of choice for most human xenograft studies (9, 10). More recently, we backcrossed the ß₂-microglobulin-null (B2m^null) allele onto the NOD-scid strain and documented that NOD-scid B2m^null mice support even higher levels of human hematopoietic cell engraftment (16). ß₂-Microglobulin is a light chain for three disparate groups of molecules. These include class I major histocompatibility complex (MHC) molecules, the protective Fc receptor (FcRn), and the HFE iron-binding molecule that is required for appropriate iron uptake and storage. ß₂-Microglobulin is required for the transport of MHC class I molecules to the cell surface. Thus B2m^null mice lack MHC class I expression and have severely depressed natural killer (NK) cell activity. The FcRn molecule normally protects IgG from catabolism. Its absence in NOD-scid B2m^null mice results in shortened IgG half-life in the circulation as we have previously reported (16). Although the basis for the improved xenochimerism in NOD-scid B2m^null remains unclear, we have speculated that residual NK activity, which is lower than that detected in other scid strains, may play a key role (16, 17).

We now report the use of the hu-PBL-NOD-scid B2m^null mouse to establish a model system for human skin allograft rejection. We demonstrate that engrafted human immune cells can reproducibly injure and in some cases ultimately reject full-thickness human skin allografts. Our data suggest that human lymphocytes can initiate allograft rejection in hu-PBL-NOD-scid B2m^null mice and that CD4⁺ or CD8⁺ cells can mediate the final destructive effector cell activity.

	Methods and Materials

Top Abstract Introduction Methods and Materials Results Discussion References

 
Animals.
NOD/LtSz-Prkdc^scid/Prkdc^scid B2m^null (NOD-scid B2m^null) male and female mice 5–9 weeks old were obtained from colonies maintained by L.D.S. at The Jackson Laboratory (Bar Harbor, ME). All animals were certified to be free of Sendai virus, pneumonia virus of mice, murine hepatitis virus, minute virus of mice, ectromelia, lactate dehydrogenase–elevating virus, GD7 virus, Reo-3 virus, mouse adenovirus, lymphocytic choriomeningitis virus, polyoma, Mycoplasma pulmonis infection, and Encephalitozoon cuniculi infection. The mice were housed under specific pathogen-free conditions in microisolator cages and given ad libitum access to autoclaved food. All immunodeficient mice were maintained on an alternate schedule of autoclaved water and a suspension of sulfamethoxazole-trimethoprim (Goldline Laboratories, Ft. Lauderdale, FL) (16). Animals were maintained in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996) and the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.

Antibodies.
Directly conjugated anti-human CD3, CD4, CD8, CD20, CD33, CD45, and CD64 fluorochrome-conjugated monoclonal antibodies (mAbs) and anti-mouse CD45 fluorochrome-conjugated antibody used for flow cytometry were obtained from BD PharMingen (La Jolla, CA). Anti-mouse CD122 (interleukin-2 [IL-2] receptor ß-chain, clone TM-ß1, rat IgG2b) (18), the gift of T. Tanaka (Osaka University Medical Center), anti-human CD4 (clone OKT4, ATCC, Manassas, VA), and anti-human CD8 (clone OKT8, ATCC) were produced as ascites. Fibrin debris was removed from the collected ascites by filtration through nylon wool, and aliquots were frozen until used. Concentrations of mAb were quantified using anti-mouse IgG radial immunodiffusion plates (The Binding Site, San Diego, CA) (17) or by enzyme-linked immunosorbent assay for rat Ig. For in vivo depletion studies, the mAbs were diluted to a concentration of 1 mg/ml in phosphate-buffered saline (PBS) immediately prior to injection.

Skin Transplantation.
Discarded full-thickness human foreskin, obtained with internal review board approval, was prepared and transplanted as described (19). Skin grafts were subjected to visual and tactile inspection three times weekly for evidence of rejection or graft injury. Graft injury was classified according to visible evidence of erythema, thrombosis, and epithelial sloughing, affecting greater than 30% of the graft during the 36-day observation period.

Establishment of Hu-PBL-NOD-scid B2m^null Mice.
Human xenochimeric mice were prepared using human spleen cells or human PBLs (both termed hu-PBL-NOD-scid B2m^null mice). Single-cell suspensions of spleen cells from human cadaveric donors were prepared as described (17). Briefly, spleen tissues were dissociated into single-cell suspensions and subjected to red blood cell lysis and three washes with RPMI-1640. Nucleated cell counts and viability were assayed by trypan blue exclusion using a hemocytometer and cell viability was greater than 85% in all cases. PBLs were prepared by Ficoll-Hypaque density gradient centrifugation. Using a dosage regimen described by Murray et al. (20), mice were injected intraperitoneally with 100–300 x 10⁶ human cells coincident with skin grafting or 4 weeks after transplantation of skin grafts. Engraftment was facilitated by intraperitoneal injection of 1.0 mg rat anti-mouse IL-2 receptor ß-chain (CD122) mAb (clone TM-ß1) 12–24 hr prior to human cell engraftment. TM-ß1 rat anti-mouse CD122 mAb recognizes the ß-chain of the mouse IL-2R, is expressed on subpopulations of CD8⁺ T cells and most NK1.1⁺ cells, and has been shown to both attenuate NK activity and enhance human PBL xenochimerism in immunodeficient mice (13, 17, 21).

For analysis of the effects of in vivo anti-human CD4 and CD8 antibody treatments on human lymphocyte engraftment and the ability of human T cells to reject human skin allografts, NOD-scid B2m^null mice were injected intraperitoneally with anti-human CD4 or anti-human CD8 mAb (17). In these experiments 1 mg of anti-human CD8 mAb was injected immediately prior to cell transfer. However, depletion monotherapy against CD4⁺ cells was delayed for 2 weeks based on our previous observation that human PBLs fail to engraft in NOD-scid mice if anti-CD4 mAb therapy is initiated at the time of lymphocyte injection (17, 22). One milligram of anti-human CD4 mAb was administered 2 weeks after human spleen cell injection.

Flow Cytometry.
Red blood cells were removed from single-cell suspensions of spleens recovered from hu-PBL-NOD-scid B2m^null mice by hypotonic lysis, and the leukocytes were suspended in PBS plus 5% fetal bovine serum and prepared for two-color flow cytometry analysis as described (17). Multiparameter flow cytometry analysis used fluorescein isothiocyanate and phycoerythrin conjugated anti-human CD3, CD4, CD8, CD20, CD33, CD45, or CD64 mAb plus anti-mouse CD45 (Ly5). Following labeling, cells were fixed in PBS containing 1% paraformaldehyde and 0.02% NaN₃ and at least 10,000 events were acquired on Becton Dickinson FACS equipment (Becton Dickinson, San Jose, CA). The data were analyzed using WinList software (Verity Software House, Inc., Topsham, ME). For all analyses, mouse CD45⁺ cells were concurrently analyzed and were excluded from calculated percentages of human cells. Matching isotype antibodies were used as negative controls and the reported values have been corrected using these isotype control values.

Histology and Immunohistochemistry.
For immunohistochemistry, human skin grafts including adjacent margins of host mouse skin were recovered 30–36 days after skin transplantation, fixed in 10% buffered formalin, and embedded in paraffin. Sections (4 µm) were cut and stained with hematoxylin and eosin or used for immunohistochemistry. Immunohistochemical staining was performed with mAb specific for human CD45 (Fisher Scientific, Savannah, GA) or the UEA-1 lectin (Vector, Inc., Burlingame, CA) using a standard avidin-biotin complex methodology as described (23). Controls included omission of primary antibodies and the use of species-matched nonbinding control antibodies. The sections were developed using 3-diaminobenzidine and counterstained with hematoxylin for visualization by light microscopy. To monitor animals for evidence of graft-versus-host disease (GVHD), ear, tongue, intestine, liver, and pancreas were recovered from host animals for conventional histological examination and stained with hematoxylin and eosin. A qualified pathologist who was unaware of the treatment of the tissue donor evaluated all tissues.

	Results

Top Abstract Introduction Methods and Materials Results Discussion References

 
Establishment of Human Skin Grafts on NOD-scid B2m^null Mice.
Human full-thickness foreskin successfully grafted on 13 of 13 NOD-scid B2m^null mice. Within 2 weeks, skin graft sites were uniformly healed in all animals and representative histological features are shown in Figure 1A–D. The graft integrity remained intact throughout the 36-day experimental observation period without any grossly visible evidence of rejection. Microscopic evaluation of these grafts failed to identify any inflammatory lesions and revealed normal epidermis and dermis architecture and keratinocyte maturation (Fig. 1A–D). The majority of blood vessels in the dermis of the human skin graft were of human origin as demonstrated by binding the human endothelium-specific UEA-lectin (Fig. 1D) (23, 24). Erythrocytes were observed within these vessels without any evidence of thrombosis or exudation.

View larger version (155K): [in this window] [in a new window]   Figure 1. Histology of human skin allografts and mouse tissues in hu-PBL-NOD-scid B2mnull mice. Histological analysis of full-thickness human foreskin grafts on untreated NOD-scid-ß2mnull mice or mice injected with human spleen cells within 24 hr of skin transplantation. All tissues were recovered 36 days after skin grafting for analysis, and representative histological data are shown. In untreated NOD-scid B2mnull mice, fully healed and vascularized skin grafts are apparent (A–D). Human skin grafts on mice injected with anti-CD122 and engrafted with allogeneic human spleen cells at levels >1% exhibit extensive human mononuclear cell infiltration and evidence of graft injury (E–L). A destructive mononuclear cell inflammation within the dermis and vascular thrombosis are shown in panels E–H. In some grafted hu-PBL-NOD-scid ß2mnull mice, complete graft rejection characterized by epithelial sloughing and vasculature necrosis was seen (I–L). Normal morphology without mononuclear cell inflammation was observed in host mouse tissues (tongue, ear, intestine) that typically hallmark evidence of GVHD (M–O). Original magnification x100 (A, E, I, M, N, O) and x250 (B, C, D, F, G, H, J, K, L).

View larger version (20K): [in this window] [in a new window]   Figure 2. Human cell engraftment and skin allograft injury in hu-PBL-NOD-scid-B2mnull mice. (A) Human-origin cells in the spleens of NOD-scid B2mnull mice were determined 36 days after human cell injection and placement of full-thickness human foreskin grafts. The percentage of animals engrafted defined as >1% human CD45+ cells in the spleen of hu-PBL-NOD-scid-B2mnull mice is shown. Mice were given anti-CD122 mAb (n = 11), anti-CD122 mAb plus anti-human CD4 (n = 6), or anti-CD122 mAb plus anti-human CD8 mAb (n = 5). (B) Percentage of hu-PBL-NOD-scid B2mnull mice displaying evidence of human skin graft injury (affecting >30% of entire graft) or complete rejection in each experimental group.

Histological examination of the human skin grafts revealed the presence of intense human CD45⁺ mononuclear cell infiltrates diffusely interspersed throughout the dermis (Fig. 1F and J). UEA-1⁺ human vascular endothelial cells detected were markedly reduced in the infiltrated grafts and surviving UEA-1⁺ microvessels were thrombosed and necrotic (Fig. 1H and L). Epidermal damage varied from localized inflammation and mononuclear infiltration subjacent to the basal layer of keratinocytes, to selected cases progressing to a complete erosion of the epithelium and sloughing of the entire skin allograft. Representative examples of these severe graft rejection lesions are shown in Figure 1I–L.

Absence of Graft-Versus-Host Disease.
The infiltration and rejection of human skin grafts could be due to allogeneic rejection by the engrafted human spleen cells or to a nonspecific GVHD that induced bystander destruction of the human allograft. To discriminate between these possibilities, we examined tissues that are commonly infiltrated with mononuclear cells during GVHD. We observed no human (or mouse) mononuclear cell infiltration into tongue, skin, or intestine (Fig. 1M–O). Little to no human mononuclear cell infiltration was observed in nonhuman mouse tissues, even in mouse skin adjacent to the human skin allograft undergoing rejection (data not shown).

Engraftment and Rejection of Human Skin Allografts in Hu-PBL-NOD-scid B2m^null Mice Given Anti-Human CD4 or Anti-Human CD8 mAb.
We next determined the effect of depletion monotherapy with anti-human CD4 or anti-human CD8 mAb on the engraftment and function of human spleen cells injected into hu-PBL-NOD-scid B2m^null mice. Previously, we demonstrated that anti-human CD4 mAb treatment initiated at the time of human PBL injection prevents engraftment of all human lymphocytes (17, 22). Therefore, injection of anti-human CD4 mAb was delayed until 2 weeks after human spleen cell injection to permit human lymphoid cell engraftment. Surprisingly, this delay in monotherapy administration only produced incomplete CD4⁺ cell depletion. We observed that the overall frequency of human CD45⁺ cell–engrafted mice was unaltered by administration of anti-human CD8 mAb (five of five, 100%) at the time of human cell injection or by delayed administration of anti-human CD4 (six of six, 100%; Fig. 2A). The percentage of human CD3⁺, CD4⁺, and CD8⁺ cells of the total spleen cells of human skin–grafted mice is shown in Figure 3A. Human CD20⁺ (B cells), CD33⁺ (myeloid precursor), and CD64⁺ (macrophage/monocyte) were not detected (<1%) in the spleens of recipients. The short-term survival of these hematopoietic cells following human PBL transfer was consistent with an earlier report (17).

View larger version (19K): [in this window] [in a new window]   Figure 3. Engraftment of human lymphocytes in the spleen of NOD-scid B2mnull mice. The percentages of total human lymphocytes (CD45+), T lymphocytes (CD3+), and T-lymphocyte subsets (CD4+ and CD8+) in the spleen of hu-PBL-NOD-scid B2mnull mice 36 days after human spleen cell injection are shown. Hu-PBL-NOD-scid B2mnull mice were treated with anti-mouse CD122 mAb (A, n = 11), anti-mouse CD122 mAb plus anti-human CD4 mAb (B, n = 6), or anti-mouse CD122 mAb plus anti-human CD8 mAb (C, n = 5). Error bars represent the mean value ± SEM.

Surprisingly, no differences were encountered in the frequency of skin allograft injury or rejection in hu-PBL-NOD-scid B2m^null mice treated only with anti-CD122 as compared with mice additionally treated with either anti-human CD4 or anti-human CD8 mAb. Evidence of graft injury was apparent in six of six (100%) and five of five (100%) of recipient mice treated with anti-human CD4 or CD8 mAb, respectively (Fig. 2B). The severity of graft rejection lesions was comparable between hu-PBL-NOD-scid B2m^null mice given supplemental depleting monotherapy and those given skin allografts and anti-CD122 alone (data not shown).

Human PBLs Mediate Graft Injury in Hu-PBL-NOD-scid B2m^null Mice with Established Human Skin Allografts.
We next tested the ability of human PBLs to mediate skin allograft injury in NOD-scid B2m^null mice with established human skin grafts. Recipient mice (n = 3) were given full-thickness human skin, and the skin grafts were permitted to heal in for 4 weeks (Fig. 4A and B). Animals were then treated with anti-mouse CD122 mAb 12 hr prior to an intraperitoneal injection with 100 x 10⁶ human PBLs. Recipient mice were xenochimeric as demonstrated by a mean human CD45⁺ percentage of 31.9% (range, 15.6%–58.2%) in the recipient spleen 13 days later. Skin allograft injury affecting 30%–60% of the total graft was observed in all three mice. Histological analysis of these skin allografts revealed abnormalities indistinguishable from those described above (Fig. 4C and D).

View larger version (147K): [in this window] [in a new window]   Figure 4. Skin allograft injury in NOD-scid B2mnull mice engrafted with human PBLs. Human skin xenografts were allowed to fully heal in NOD-scid B2mnull mice (A and B). Three mice with fully healed skin xenografts were then given 100 x 106 human PBLs (C and D). In contrast to control animals (A and B), destructive mononuclear cell inflammation is uniformly apparent at 13 days within the dermis, resulting in necrosis of the supporting vasculature (C and D). Extensive sloughing of the epidermis is also seen (C). Higher magnification shows typical vasculitis and fibrin necrosis of a microvessel in the dermis (D) that is absent in controls (B). Original magnification panels A and C = x100, panels B and D = x250. Hematoxylin and eosin staining.

	Discussion

Top Abstract Introduction Methods and Materials Results Discussion References

In the hu-PBL-NOD-scid B2m^null mice given human skin allografts, immune injury was evident even in the nearly complete absence of human CD8⁺ cells. The immune response in hu-PBL-NOD-scid B2m^null mice given human skin grafts is characterized by extensive mononuclear cell infiltration of the graft that appears to be targeted to human-origin endothelium and produces vascular occlusion with coagulation necrosis. Complete sloughing (rejection) of the graft was observed in some cases. The destructive infiltration into the allografts was not due to collateral xenoreactive GVHD, as no evidence of mononuclear infiltration was observed in host tissues typically affected during GVHD. Finally, nonspecific inflammatory cytokines related to granulation tissue in the graft bed did not appear to play a role in the observed graft injury, because an acute vasculitic reaction was observed in animals bearing healed-in skin grafts given human PBLs. These data validate the hu-PBL-NOD-scid B2m^null mouse grafted with human skin as a model for the study of human allograft rejection.

It has been difficult to demonstrate human allograft rejection in hu-PBL-scid mice. Various manipulations have been employed, including injection of human T cells from HLA-sensitized donors (25), irradiation of the recipient (26), or injection of large numbers of human splenic cells (27). Preactivation (priming) of human cells in vitro prior to injection and other manipulations have also been used in attempts to induce allograft or xenograft rejection in hu-PBL-scid mice (28–33). In many of these studies, severe GVHD ensued, making it difficult to distinguish between graft injuries resulting from allospecific versus nonspecific inflammation. A promising series of studies has been described for skin allograft damage in hu-PBL-scid mice (20, 23). The authors demonstrated morphological characteristics at the graft site (24, 34, 35) that closely resembled human allograft rejection (34). Damage to microvessels of human but not mouse-origin blood vessels was detected. Skin allograft damage was inhibited by immunosuppression using a combination of cyclosporine and rapamycin (20) or by administration of murine antibodies or fusion proteins that blocked human CD2/LFA-3 interactions (24). However, the allograft damage was restricted to the microvasculature and gross rejection (scabbing and epidermal sloughing) was not described. The murine host used in these studies was the C.B-17-scid mouse, and anti-mouse CD122 mAb was not used to facilitate human cell engraftment. Our studies using the NOD-scid B2m^null mouse with concurrent administration of anti-mouse CD122 mAb led to increased engraftment of human lymphocytes and, in some cases, full rejection of the skin allografts. Importantly, alloaggression by the human PBLs did not require in vitro sensitization or activation prior to injection. The role of NK cells in modulating human lymphocyte engraftment in immunodeficient mice has been suggested by the sequentially improved human cell engraftment in NOD-scid and NOD-scid B2m^null mice over that of C.B-17-scid mice (10, 16). In each recipient, human lymphocyte engraftment correlated inversely with levels of NK cell activity. Additionally, human CD4⁺ cell engraftment increased as NK cell activity decreased (16). Depletion monotherapy with anti-mouse CD122 mAb further attenuates mouse NK cell activity, resulting in enhanced human CD4⁺ cell engraftment (17). The data suggest that our combination of improved recipient strain characteristics with anti-mouse CD122 mAb optimizes the hu-PBL-SCID model for both human cell engraftment and allograft rejection.

Surprisingly, we observed that human CD8⁺ cells did not appear to be required for human skin allograft rejection in our model system. Although CD8⁺ cells are postulated to be important mediators of allograft rejection, it has been reported that skin allograft rejection occurs in mice genetically deficient in CD8⁺ cells (36, 37). In addition, mice genetically deficient in ß₂-microglobulin, a molecule required for MHC class I expression and CD8⁺ T-cell development, also exhibit allograft rejection, albeit it delayed (38). Conversely, we could not determine whether CD8⁺ cells alone could mediate skin allograft rejection in the absence of CD4⁺ cells. Our use of depleting anti-human CD4 mAb initiated 2 weeks after human cell injection did not fully ablate human CD4⁺ cells. This result was not expected based on our previous studies demonstrating that a similar dose of anti-human CD4 mAb given to NOD-scid mice at the time of human cell engraftment prevented the engraftment not only of human CD4⁺ cells but also of human CD8⁺ cells (17, 22). We are currently investigating whether the immunoglobulin half-life is shorter in NOD-scid B2m^null mice as compared with NOD-scid mice. The failure of anti-human CD4 mAb to deplete human CD4⁺ cells in hu-PBL-NOD-scid B2m^null mice may conceivably be due to their lack of a protective Fc receptor important for the IgG salvage pathway (16).

In summary, the data document the utility of the hu-PBL-NOD-scid B2m^null mouse to support human lymphocyte and skin engraftment. Engrafted lymphocytes are capable of allograft rejection as demonstrated by their ability to mediate injury, and in some cases, complete rejection of human skin grafts. Studies of human allograft injury in this model system should permit a controlled analysis of the efficacy of human-specific therapeutic reagents prior to translation to preclinical and clinical trials.

	Acknowledgments

 
The authors thank Linda Paquin, Stephanie Gibbons, and Jean Leif for technical assistance.

	Footnotes

 
This work was supported in part by Grants AI30389, AI10623, AI38757, AI24544, CS34196, 1PO1-DK32520, T32RR07068, and an institutional Diabetes Endocrinology Research Center (DERC) Grant DK52530 from the National Institutes of Health; by Grant 4-2002-431 from the Juvenile Diabetes Research Foundation (JDRF); and a grant from the Hood Foundation. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

¹ To whom requests for reprints should be addressed at Division of Diabetes, University of Massachusetts Medical School, 373 Plantation Street, Suite 218, Worcester, MA 01605. E-mail: michael.appel{at}umassmed.edu

	References

Top Abstract Introduction Methods and Materials Results Discussion References

 

1. Auchincloss H Jr, Sachs DH. Xenogeneic transplantation. Annu Rev Immunol 16:433–470, 1998.[Medline]
2. Gudmundsdottir H, Turka LA. Transplantation tolerance: Mechanisms and strategies? Semin Nephrol 20:209–216, 2000.[Medline]
3. Waldmann H. Transplantation tolerance: Where do we stand? Nat Med 5:1245–1248, 1999.[Medline]
4. Adler SH, Bensinger SJ, Turka LA. Stemming the tide of rejection. Nat Med 8:107–108, 2002.[Medline]
5. Carpenter C. Improving the success of organ transplantation (editorial). N Engl J Med 342:647–648, 2000.[Free Full Text]
6. Marmont AM. New horizons in the treatment of autoimmune diseases: Immunoablation and stem cell transplantation. Annu Rev Med 51:115–134, 2000.[Medline]
7. Rossini AA, Greiner DL, Mordes JP. Induction of immunological tolerance for transplantation. Physiol Rev 79:99–141, 1999.[Abstract/Free Full Text]
8. Gourishankar S, Turner P, Halloran P. New developments in immunosuppressive therapy in renal transplantation. Expert Opin Biol Ther 2:483–501, 2002.[Medline]
9. Greiner D, Hesselton RA, Shultz LD. SCID mouse models of human stem cell engraftment. Stem Cells 16:166–177, 1998.[Abstract/Free Full Text]
10. Greiner DL, Shultz LD. Use of NOD/LtSz-scid/scid mice in biomedical research. In: Leiter EH, Atkinson MA, Eds. NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer and Other Diseases. Austin, TX: R.G. Landes Co., pp173–203, 1998.
11. McCune JM, Namikawa R, Kaneshima H, Shultz LD, Lieberman M, Weissman IL. The SCID-hu mouse: Murine model for the analysis of human hematolymphoid differentiation and function. Science 241:1632–1639, 1988.[Abstract/Free Full Text]
12. Mosier DE, Gulizia RJ, Baird SM, Wilson DB. Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 335:256–259, 1988.[Medline]
13. Cao T, Leroux-Roels G. Antigen-specific T cell responses in human peripheral blood leucocyte (hu-PBL)-mouse chimera conditioned with radiation and an antibody directed against the mouse IL-2 receptor beta-chain. Clin Exp Immunol 122:117–123, 2000.[Medline]
14. Murphy WJ, Funakoshi S, Fanslow WC, Rager HC, Taub DD, Longo DL. CD40 stimulation promotes human secondary immunoglobulin responses in HuPBL-SCID chimeras. Clin Immunol 90:22–27, 1999.[Medline]
15. Nguyen H, Hay J, Mazzulli T, Gallinger S, Sandhu J, Teng Y, Hozumi N. Efficient generation of respiratory syncytial virus (RSV)-neutralizing human MoAbs via human peripheral blood lymphocyte (hu-PBL)-SCID mice and scFv phage display libraries. Clin Exp Immunol 122:85–93, 2000.[Medline]
16. Christianson SW, Greiner DL, Hesselton RA, Leif JH, Wagar EJ, Schweitzer IB, Rajan TV, Gott B, Roopenian DC, Shultz LD. Enhanced human CD4⁺ T cell engraftment in ß₂-microglobulin-deficient NOD-scid mice. J Immunol 158:3578–3586, 1997.[Abstract]
17. Wagar EJ, Cromwell MA, Shultz LD, Woda BA, Sullivan JL, Hesselton RM, Greiner DL. Regulation of human cell engraftment and development of EBV-related lymphoproliferative disorders in Hu-PBL-scid mice. J Immunol 165:518–527, 2000.[Abstract/Free Full Text]
18. Tanaka T, Tsudo M, Karasuyama H, Kitamura F, Kono T, Hatakeyama M, Taniguchi T, Miyasaka M. A novel monoclonal antibody against murine IL-2 receptor beta-chain: Characterization of receptor expression in normal lymphoid cells and EL-4 cells. J Immunol 147:2222–2228, 1991.[Abstract]
19. Turgeon N, Iwakoshi NN, Meyers W, Shultz L, Greiner DL, Mordes JP, Rossini AA. Analysis of human immune responses to human allografts in small animal models. Surg Forum 50:393–395, 1999.
20. Murray AG, Schechner JS, Epperson DE, Sultan P, McNiff JM, Hughes CC, Lorber MI, Askenase PW, Pober JS. Dermal microvascular injury in the human peripheral blood lymphocyte reconstituted-severe combined immunodeficient (HuPBL-SCID) mouse/skin allograft model is T cell mediated and inhibited by a combination of cyclosporine and rapamycin. Am J Pathol 153:627–638, 1998.[Abstract/Free Full Text]
21. Tournoy KG, Depraetere S, Pauwels RA, Leroux-Roels GG. Mouse strain and conditioning regimen determine survival and function of human leucocytes in immunodeficient mice. Clin Exp Immunol 119:231–239, 2000.[Medline]
22. Foy TM, McIlraith M, Masters SR, Dunn JJ, Rossini AA, Shultz LD, Hesselton RA, Wagar EJ, Lipsky PE, Noelle RJ, Greiner DL. Blockade of CD40-CD154 interferes with human T cell engraftment in scid mice. Cell Transplantation 7:25–35, 1998.[Medline]
23. Murray AG, Petzelbauer P, Hughes CC, Costa J, Askenase P, Pober JS. Human T-cell-mediated destruction of allogeneic dermal microvessels in a severe combined immunodeficient mouse. Proc Natl Acad Sci USA 91:9146–9150, 1994.[Abstract/Free Full Text]
24. Sultan P, Schechner JS, McNiff JM, Hochman PS, Hughes CC, Lorber MI, Askenase PW, Pober JS. Blockade of CD2-LFA-3 interactions protects human skin allografts in immunodeficient mouse/human chimeras. Nat Biotechnol 15:759–762, 1997.[Medline]
25. Kawamura T, Niguma T, Fechner JH Jr., Wolber R, Beeskau MA, Hullett DA, Sollinger HW, Burlingham WJ. Chronic human skin graft rejection in severe combined immunodeficient mice engrafted with human PBL from an HLA-presensitized donor. Transplantation 53:659–665, 1992.[Medline]
26. Huppes W, Hoffmann-Fezer G. Peripheral blood leukocyte grafts that induce human to mouse graft-vs.-host disease reject allogeneic human skin grafts. Am J Pathol 147:1708–1714, 1995.[Abstract]
27. Alegre ML, Peterson LJ, Jeyarajah DR, Weiser M, Bluestone JA, Thistlethwaite JR. Severe combined immunodeficient mice engrafted with human splenocytes have functional human T cells and reject human allografts. J Immunol 153:2738–2749, 1994.[Abstract]
28. Friedman T, Shimizu A, Smith RN, Colvin RB, Seebach JD, Sachs DH, Iacomini J. Human CD4⁺ T cells mediate rejection of porcine xenografts. J Immunol 162:5256–5262, 1999.[Abstract/Free Full Text]
29. Friedman T, Smith RN, Colvin RB, Iacomini J. A critical role for human CD4⁺ T-cells in rejection of porcine islet cell xenografts. Diabetes 48:2340–2348, 1999.[Abstract]
30. Platt JL. New directions for organ transplantation. Nature 392:11–17, 1998.[Medline]
31. Sawada T, DellaPelle PA, Seebach JD, Sachs DH, Colvin RB, Iacomini J. Human cell-mediated rejection of porcine xenografts in an immunodeficient mouse model. Transplantation 63:1331–1338, 1997.[Medline]
32. Shiroki R, Nazirrudin B, Hoshinaga K, Naide Y, Scharp DW, Mohanakumar T. Human peripheral blood lymphocyte-reconstituted, severe combined immunodeficient mice as a model for porcine islet xenograft rejection in humans. Artif Organs 20:878–882, 1996.[Medline]
33. Zeng Y, Peterson L, Levisetti M, Torres M, Montag A, Thistlethwaite JR, Jr. Immunomodulation of human islets results in prolonged in vivo islet graft survival. Transplant Proc 27:611–612, 1995.[Medline]
34. Dvorak HF, Mihm MC Jr., Dvorak AM, Barnes BA, Manseau EJ, Galli SJ. Rejection of first-set skin allografts in man. The microvasculature is the critical target of the immune response. J Exp Med 150:322–337, 1979.[Abstract/Free Full Text]
35. Lorber MI, Wilson JH, Robert ME, Schechner JS, Kirkiles N, Qian HY, Askenase PW, Tellides G, Pober JS. Human allogeneic vascular rejection after arterial transplantation and peripheral lymphoid reconstitution in severe combined immunodeficient mice. Transplantation 67:897–903, 1999.[Medline]
36. Iwakoshi NN, Mordes JP, Markees TG, Phillips NE, Greiner DL, Rossini AA. Treatment of allograft recipients with donor specific transfusion and anti-CD154 antibody leads to deletion of alloreactive CD8⁺ T cells and prolonged graft survival in a CTLA4-dependent manner. J Immunol 164:512–521, 2000.[Abstract/Free Full Text]
37. Krieger NR, Yin DP, Fathman CG. CD4⁺ but not CD8⁺ cells are essential for allorejection. J Exp Med 184:2013–2018, 1996.[Abstract/Free Full Text]
38. Grusby MJ, Auchincloss H Jr., Lee R, Johnson RS, Spencer JP, Zijlstra M, Jaenisch R, Papaioannou VE, Glimcher LH. Mice lacking major histocompatibility complex class I and class II molecules. Proc Natl Acad Sci USA 90:3913–3917, 1993.[Abstract/Free Full Text]

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