Dual targeting: Combining costimulation blockade and bortezomib to permit kidney transplantation in sensitized recipients.

Previous evidence suggests that a homeostatic germinal center (GC) response may limit bortezomib desensitization therapy. We evaluated the combination of costimulation blockade with bortezomib in a sensitized non-human primate kidney transplant model. Sensitized animals were treated with bortezomib, belatacept, and anti-CD40 mAb twice weekly for a month (n = 6) and compared to control animals (n = 7). Desensitization therapy-mediated DSA reductions approached statistical significance (P = .07) and significantly diminished bone marrow PCs, lymph node follicular helper T cells, and memory B cell proliferation. Graft survival was prolonged in the desensitization group (P = .073). All control animals (n = 6) experienced graft loss due to antibody-mediated rejection (AMR) after kidney transplantation, compared to one desensitized animal (1/5). Overall, histological AMR scores were significantly lower in the treatment group (n = 5) compared to control (P = .020). However, CMV disease was common in the desensitized group (3/5). Desensitized animals were sacrificed after long-term follow-up with functioning grafts. Dual targeting of both plasma cells and upstream GC responses successfully prolongs graft survival in a sensitized NHP model despite significant infectious complications and drug toxicity. Further work is planned to dissect underlying mechanisms, and explore safety concerns.

Microencapsulated adult porcine islets transplanted intraperitoneally in streptozotocin-diabetic non-human primates.

BACKGROUND: Xenogeneic donors would provide an unlimited source of islets for the treatment of type 1 diabetes (T1D). The goal of this study was to assess the function of microencapsulated adult porcine islets (APIs) transplanted ip in streptozotocin (STZ)-diabetic non-human primates (NHPs) given targeted immunosuppression. METHODS: APIs were encapsulated in: (a) single barium-gelled alginate capsules or (b) double alginate capsules with an inner, islet-containing compartment and a durable, biocompatible outer alginate layer. Immunosuppressed, streptozotocin-diabetic NHPs were transplanted ip with encapsulated APIs, and graft function was monitored by measuring blood glucose, %HbA1c, and porcine C-peptide. At graft failure, explanted capsules were assessed for biocompatibility and durability plus islet viability and functionality. Host immune responses were evaluated by phenotyping peritoneal cell populations, quantitation of peritoneal cytokines and chemokines, and measurement of anti-porcine IgG and IgM plus anti-Gal IgG. RESULTS: NHP recipients had reduced hyperglycemia, decreased exogenous insulin requirements, and lower percent hemoglobin A1c (%HbA1c) levels. Porcine C-peptide was detected in plasma of all recipients, but these levels diminished with time. However, relatively high levels of porcine C-peptide were detected locally in the peritoneal graft site of some recipients at sacrifice. IV glucose tolerance tests demonstrated metabolic function, but the grafts eventually failed in all diabetic NHPs regardless of the type of encapsulation or the host immunosuppression regimen. Explanted microcapsules were intact, "clean," and free-floating without evidence of fibrosis at graft failure, and some reversed diabetes when re-implanted ip in diabetic immunoincompetent mice. Histology of explanted capsules showed scant evidence of a host cellular response, and viable islets could be found. Flow cytometric analyses of peritoneal cells and peripheral blood showed similarly minimal evidence of a host immune response. Preformed anti-porcine IgG and IgM antibodies were present in recipient plasma, but these levels did not rise post-transplant. Peritoneal graft site cytokine or chemokine levels were equivalent to normal controls, with the exception of minimal elevation observed for IL-6 or IL-1ß, GRO-α, I-309, IP-10, and MCP-1. However, we found central necrosis in many of the encapsulated islets after graft failure, and explanted islets expressed endogenous markers of hypoxia (HIF-1α, osteopontin, and GLUT-1), suggesting a role for non-immunologic factors, likely hypoxia, in graft failure. CONCLUSIONS: With donor xenoislet microencapsulation and host immunosuppression, APIs corrected hyperglycemia after ip transplantation in STZ-diabetic NHPs in the short term. The islet xenografts lost efficacy gradually, but at graft failure, some viable islets remained, substantial porcine C-peptide was detected in the peritoneal graft site, and there was very little evidence of a host immune response. We postulate that chronic effects of non-immunologic factors, such as in vivo hypoxic and hyperglycemic conditions, damaged the encapsulated islet xenografts. To achieve long-term function, new approaches must be developed to prevent this damage, for example, by increasing the oxygen supply to microencapsulated islets in the ip space.

Humoral Compensation after Bortezomib Treatment of Allosensitized Recipients.

The efficacy of bortezomib monotherapy in desensitizing kidney transplant candidates with preformed donor-specific antibodies remains unclear. We evaluated the effect of bortezomib on preformed antibodies and upstream components of the B cell response in a primate model sensitized by fully mismatched allogeneic skin transplants to provide mechanistic insights regarding the use of bortezomib as a means of desensitization. Bortezomib treatment given intravenously twice weekly for 1 month (1.3 mg/m2 per dose) clearly reduced the numbers of antibody-producing cells and CD38+CD19+CD20- plasma cells in the bone marrow (P<0.05), but donor-specific alloantibody levels did not decrease. We observed a rapid but transient induction of circulating IgG+ B cells and an increased number of proliferating B cells in the lymph nodes after 1 month of treatment. Notably, bortezomib treatment induced germinal center B cell and follicular helper T cell expansion in the lymph nodes. These data suggest that bortezomib-induced plasma cell depletion triggers humoral compensation.

The role of B cells in solid organ transplantation.

The role of antibodies in chronic injury to organ transplants has been suggested for many years, but recently emphasized by new data. We have observed that when immunosuppressive potency decreases either by intentional weaning of maintenance agents or due to homeostatic repopulation after immune cell depletion, the threshold of B cell activation may be lowered. In human transplant recipients the result may be donor-specific antibody, C4d+ injury, and chronic rejection. This scenario has precise parallels in a rhesus monkey renal allograft model in which T cells are depleted with CD3 immunotoxin, or in a CD52-T cell transgenic mouse model using alemtuzumab to deplete T cells. Such animal models may be useful for the testing of therapeutic strategies to prevent DSA. We agree with others who suggest that weaning of immunosuppression may place transplant recipients at risk of chronic antibody-mediated rejection, and that strategies to prevent this scenario are needed if we are to improve long-term graft and patient outcomes in transplantation. We believe that animal models will play a crucial role in defining the pathophysiology of antibody-mediated rejection and in developing effective therapies to prevent graft injury. Two such animal models are described herein.

Bcl-xL protein protects from C/EBP homologous protein (CHOP)-dependent apoptosis during plasma cell differentiation.

Although it is known that the unfolded protein response (UPR) plays a significant role in the process of plasma cell differentiation, the contribution of the individual sensors of the UPR to this process remains unclear. In this study we examine the death signals and compensatory survival signals activated during B cell activation and the first stages of plasma cell differentiation. During in vitro differentiation of both primary murine B cells and the Bcl1 cell line, we demonstrate that in addition to activation of the physiological UPR, changes in the expression of several Bcl-2 proteins occur, which are consistent with a lowering of the apoptotic threshold of the cell. Specifically, we observed decreased expression of Bcl-2 and Mcl-1 and increased expression of the proapoptotic protein Bim. However, these changes were countered by Bcl-xL induction, which is necessary to protect differentiating cells both from ER stress-induced death by tunicamycin and from the death signals inherent in differentiation. Consistent with differentiating cells becoming dependent on Bcl-xL for survival, the addition of ABT-737 resulted in apoptosis in differentiating cells through the inhibition of sequestration of Bim. Confirming this result, differentiation in the context of RNAi-mediated Bcl-xL knockdown also induced apoptosis. This cell death is C/EBP homologous protein (CHOP)-dependent, connecting these events to the UPR. Thus plasma cell differentiation proceeds through a Bcl-xL-dependent intermediate.

Murine gammaherpesvirus 68 reactivation from B cells requires IRF4 but not XBP-1.

UNLABELLED: Gammaherpesviruses display tropism for B cells and, like all known herpesviruses, exhibit distinct lytic and latent life cycles. One well-established observation among members of the gammaherpesvirus family is the link between viral reactivation from latently infected B cells and plasma cell differentiation. Importantly, a number of studies have identified a potential role for a CREB/ATF family member, X-box binding protein 1 (XBP-1), in trans-activating the immediate early BZLF-1 or BRLF1/gene 50 promoters of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), respectively. XBP-1 is required for the unfolded protein response and has been identified as a critical transcription factor in plasma cells. Here, we demonstrate that XBP-1 is capable of trans-activating the murine gammaherpesvirus 68 (MHV68) RTA promoter in vitro, consistent with previous observations for EBV and KSHV. However, we show that in vivo there does not appear to be a requirement for XBP-1 expression in B cells for virus reactivation. The MHV68 M2 gene product under some experimental conditions plays an important role in virus reactivation from B cells. M2 has been shown to drive B cell differentiation to plasma cells, as well as interleukin-10 (IL-10) production, both of which are dependent on M2 induction of interferon regulatory factor 4 (IRF4) expression. IRF4 is required for plasma cell differentiation, and consistent with a role for plasma cells in MHV68 reactivation from B cells, we show that IRF4 expression in B cells is required for efficient reactivation of MHV68 from splenocytes. Thus, the latter analyses are consistent with previous studies linking plasma cell differentiation to MHV68 reactivation from B cells. The apparent independence of MHV68 reactivation from XBP-1 expression in plasma cells may reflect redundancy among CREB/ATF family members or the involvement of other plasma cell-specific transcription factors. Regardless, these findings underscore the importance of in vivo studies in assessing the relevance of observations made in tissue culture models. IMPORTANCE: All known herpesviruses establish a chronic infection of their respective host, persisting for the life of the individual. A critical feature of these viruses is their ability to reactivate from a quiescent form of infection (latency) and generate progeny virus. In the case of gammaherpesviruses, which are associated with the development of lymphoproliferative disorders, including lymphomas, reactivation from latently infected B lymphocytes occurs upon terminal differentiation of these cells to plasma cells-the cell type that produces antibodies. A number of studies have linked a plasma cell transcription factor, XBP-1, to the induction of gammaherpesvirus reactivation, and we show here that indeed in tissue culture models this cellular transcription factor can trigger expression of the murine gammaherpesvirus gene involved in driving virus reactivation. However, surprisingly, when we examined the role of XBP-1 in the setting of infection of mice-using mice that lack a functional XBP-1 gene in B cells-we failed to observe a role for XBP-1 in virus reactivation. However, we show that another cellular factor essential for plasma cell differentiation, IRF4, is critical for virus reactivation. Thus, these studies point out the importance of studies in animal models to validate findings from studies carried out in cell lines passaged in vitro.

Viral-induced CD28 loss evokes costimulation independent alloimmunity.

BACKGROUND: Belatacept, a B7-specific fusion protein, blocks CD28-B7 costimulation and prevents kidney allograft rejection. However, it is ineffective in a sizable minority of patients. Although T-cell receptor and CD28 engagement are known to initiate T-cell activation, many human antigen-experienced T-cells lose CD28, and can be activated independent of CD28 signals. We posit that these cells are central drivers of costimulation blockade resistant rejection (CoBRR) and propose that CoBRR might relate to an accumulation of CD28(-) T-cells resulting from viral antigen exposure. MATERIALS AND METHODS: We infected C57BL/6 mice with polyomavirus (a BK virus analog), murine cytomegalovirus (a human cytomegalovirus analog), and gammaherpesvirus (HV68; an Epstein-Barr virus analog) and assessed for CD28 expression relative to mock infection controls. We then used mixed lymphocyte reaction (MLR) assays to assess the alloreactive response of these mice against major histocompatibility complex-mismatched cells. RESULTS: We demonstrated that infection with polyomavirus, murine CMV, and HV68 can induce CD28 downregulation in mice. We showed that these analogs of clinically relevant human viruses enable lymphocytes from infected mice to launch an anamnestic, costimulation blockade resistant, alloreactive response against major histocompatibility complex-mismatched cells without prior alloantigen exposure. Further analysis revealed that gammherpesvirus-induced oligoclonal T-cell expansion is required for the increased alloreactivity. CONCLUSIONS: Virus exposure results in reduced T-cell expression of CD28, the target of costimulation blockade therapy. These viruses also contribute to increased alloreactivity. Thus, CD28 downregulation after viral infection may play a seminal role in driving CoBRR.

The transcription factor XBP-1 is essential for the development and survival of dendritic cells.

Dendritic cells (DCs) play a critical role in the initiation, maintenance, and resolution of an immune response. DC survival is tightly controlled by extracellular stimuli such as cytokines and Toll-like receptor (TLR) signaling, but the intracellular events that translate such extracellular stimuli into life or death for the DC remain poorly understood. The endoplasmic reticulum (ER) stress, or unfolded protein response (UPR), is a signaling pathway that is activated when unfolded proteins accumulate in the ER. The most conserved arm of the UPR involves IRE1alpha, an ER transmembrane kinase and endoribonuclease that activates the transcription factor XBP-1 to maintain ER homeostasis and prevent activation of cell death pathways caused by sustained ER stress. We report that XBP-1 is essential for DC development and survival. Lymphoid chimeras lacking XBP-1 possessed decreased numbers of both conventional and plasmacytoid DCs with reduced survival both at baseline and in response to TLR signaling. Overexpression of XBP-1 in hematopoietic progenitors rescued and enhanced DC development. Remarkably, in contrast to other cell types we have examined, the XBP-1 pathway was constitutively activated in immature DCs.

Partial tolerance of autoreactive B and T cells to erythrocyte-specific self-antigens in mice.

BACKGROUND: Breakdown of humoral tolerance to RBC antigens may lead to autoimmune hemolytic anemia, a severe and sometimes fatal disease. The underlying mechanisms behind the breakdown of humoral tolerance to RBC antigens are poorly understood. DESIGN AND METHODS: In order to study the pathogenesis of autoimmune hemolytic anemia, we developed a murine model with RBC-specific expression of a model antigen carrying epitopes from hen egg lysozyme and ovalbumin. RESULTS: Humoral tolerance was observed; this was not broken even by strong immunogenic stimulation (lysozyme or ovalbumin with adjuvant). Autoreactive CD4(+) T cells were detected by tetramer enrichment assays, but failed to activate or expand despite repeat stimulation, indicating a nonresponsive population rather than deletion. Adoptive transfer of autoreactive CD4(+) T cells (OT-II mice) led to autoantibody (anti-lysozyme) production by B cells in multiple anatomic compartments, including the bone marrow. CONCLUSIONS: These data demonstrate that B cells autoreactive to RBC antigens survive in healthy mice with normal immune systems. Furthermore, autoreactive B cells are not centrally tolerized and are receptive to T-cell help. As the autoreactive T cells are present but non-responsive, these data indicate that factors that reverse T-cell non-responsiveness may be central to the pathogenesis of autoimmune hemolytic anemia.

XBP-1 specifically promotes IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in primary B cells.

Differentiation of B cells into plasma cells requires X-box binding protein-1 (XBP-1). In the absence of XBP-1, B cells develop normally, but very little immunoglobulin is secreted. XBP-1 controls the expression of a large set of genes whose products participate in expansion of the endoplasmic reticulum (ER) and in protein trafficking. We define a new role for XBP-1 in exerting selective translational control over high and sustained levels of immunoglobulin M (IgM) synthesis. XBP-1(-/-) and XBP-1(+/+) primary B cells synthesize IgM at comparable levels at the onset of stimulation with lipopolysaccharide or CpG. However, later there is a profound depression in synthesis of IgM in XBP-1(-/-) B cells, notwithstanding similar levels of micromRNA. In marked contrast, lack of XBP-1 does not affect synthesis and trafficking of other glycoproteins, or of immunoglobulin light chains. Contrary to expectation, degradation of proteins from the ER, using TCRalpha or US11-mediated degradation of class I major histocompatibility complex molecules as substrates, is normal in XBP-1(-/-) B cells. Furthermore, degradation of membrane mu was unaffected by enforced expression of XBP-1. We conclude that in primary B cells, the XBP-1 pathway promotes synthesis and secretion of IgM, but does not seem to be involved in the degradation of ER proteins, including that of mu chains themselves.

Gammaherpesvirus-driven plasma cell differentiation regulates virus reactivation from latently infected B lymphocytes.

Gammaherpesviruses chronically infect their host and are tightly associated with the development of lymphoproliferative diseases and lymphomas, as well as several other types of cancer. Mechanisms involved in maintaining chronic gammaherpesvirus infections are poorly understood and, in particular, little is known about the mechanisms involved in controlling gammaherpesvirus reactivation from latently infected B cells in vivo. Recent evidence has linked plasma cell differentiation with reactivation of the human gammaherpesviruses EBV and KSHV through induction of the immediate-early viral transcriptional activators by the plasma cell-specific transcription factor XBP-1s. We now extend those findings to document a role for a gammaherpesvirus gene product in regulating plasma cell differentiation and thus virus reactivation. We have previously shown that the murine gammaherpesvirus 68 (MHV68) gene product M2 is dispensable for virus replication in permissive cells, but plays a critical role in virus reactivation from latently infected B cells. Here we show that in mice infected with wild type MHV68, virus infected plasma cells (ca. 8% of virus infected splenocytes at the peak of viral latency) account for the majority of reactivation observed upon explant of splenocytes. In contrast, there is an absence of virus infected plasma cells at the peak of latency in mice infected with a M2 null MHV68. Furthermore, we show that the M2 protein can drive plasma cell differentiation in a B lymphoma cell line in the absence of any other MHV68 gene products. Thus, the role of M2 in MHV68 reactivation can be attributed to its ability to manipulate plasma cell differentiation, providing a novel viral strategy to regulate gammaherpesvirus reactivation from latently infected B cells. We postulate that M2 represents a new class of herpesvirus gene products (reactivation conditioners) that do not directly participate in virus replication, but rather facilitate virus reactivation by manipulating the cellular milieu to provide a reactivation competent environment.

Functional aspects of factor VIII expression after transplantation of genetically-modified hematopoietic stem cells for hemophilia A.

BACKGROUND: Major complications with respect to the development of gene therapy treatments for hemophilia A include low factor VIII (fVIII) expression and humoral immune responses resulting in inhibitory anti-fVIII antibodies. We previously achieved sustained curative fVIII activity levels in hemophilia A mice after nonmyeloablative transplantation of genetically-modified hematopoietic stem cells (HSCs) encoding a B-domain deleted porcine fVIII (BDDpfVIII) transgene with no evidence of an immune response. METHODS: Mouse HSCs were transduced using MSCV-based recombinant virus encoding BDDpfVIII and transplanted into hemophilia A mice. Transplanted mice were followed for donor cell engraftment, fVIII expression and activity, and generation of anti-fVIII immune response. RESULTS: We now show that: (i) the protein expressed by hematopoietic cells has a specific activity similar to that of purified protein; (ii) BDDpfVIII expressed from hematopoietic cells effectively induces thrombus formation, which is shown using a new method of in vivo analysis of fVIII function; (iii) naïve and pre-immunized mice receiving HSC gene therapy are nonresponsive to challenges with recombinant human fVIII; (iv) nonresponsiveness is not broken after stringent challenges with BDDpfVIII; and (v) T cells from these mice are unresponsive to BDDpfVIII presentation. Furthermore, stem cells isolated from donors with high titer anti-human fVIII antibodies show no defects in donor cell engraftment or the ability to express BDDpfVIII. CONCLUSIONS: These results demonstrate that HSC gene therapy can be an effective alternative treatment for individuals with hemophilia A and may benefit patients by inducing immunological nonresponsiveness to fVIII replacement products.

T Cell Repertoire Maturation Induced by Persistent and Latent Viral Infection Is Insufficient to Induce Costimulation Blockade Resistant Organ Allograft Rejection in Mice.

CD28:CD80/86 pathway costimulation blockade (CoB) with the CD80/86-specific fusion protein CTLA4-Ig prevents T cell-mediated allograft rejection in mice. However, in humans, transplantation with CoB has been hampered by CoB-resistant rejection (CoBRR). CoBRR has been attributed in part to pathogen-driven T cell repertoire maturation and resultant heterologous alloreactive memory. This has been demonstrated experimentally in mice. However, prior murine models have used viral pathogens, CoB regimens, graft types, and/or antigen systems atypically encountered clinically. We therefore sought to explore whether CoBRR would emerge in a model of virus-induced memory differentiation designed to more closely mimic clinical conditions. Specifically, we examined mouse homologs of clinically prevalent viruses including murine polyomavirus, cytomegalovirus, and gammaherpesvirus 68 in the presence of clinically relevant maintenance CoB regimens using a fully MHC-mismatched, vascularized allograft model. Infected mice developed a significant, sustained increase in effector memory T cells consistent with that seen in humans, but neither developed heterologous alloreactivity nor rejected primarily vascularized heterotopic heart transplants at an increased rate compared with uninfected mice. These results indicate that memory acquisition alone is insufficient to provoke CoBRR and suggest that knowledge of prior latent or persistent viral infection may have limited utility in anticipating heterologous CoB-resistant alloimmunity.

XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). We have investigated here the contribution of the UPR transcription factors XBP-1, ATF6alpha, and ATF6beta to UPR target gene expression. Gene profiling of cell lines lacking these factors yielded several XBP-1-dependent UPR target genes, all of which appear to act in the ER. These included the DnaJ/Hsp40-like genes, p58(IPK), ERdj4, and HEDJ, as well as EDEM, protein disulfide isomerase-P5, and ribosome-associated membrane protein 4 (RAMP4), whereas expression of BiP was only modestly dependent on XBP-1. Surprisingly, given previous reports that enforced expression of ATF6alpha induced a subset of UPR target genes, cells deficient in ATF6alpha, ATF6beta, or both had minimal defects in upregulating UPR target genes by gene profiling analysis, suggesting the presence of compensatory mechanism(s) for ATF6 in the UPR. Since cells lacking both XBP-1 and ATF6alpha had significantly impaired induction of select UPR target genes and ERSE reporter activation, XBP-1 and ATF6alpha may serve partially redundant functions. No UPR target genes that required ATF6beta were identified, nor, in contrast to XBP-1 and ATF6alpha, did the activity of the UPRE or ERSE promoters require ATF6beta, suggesting a minor role for it during the UPR. Collectively, these results suggest that the IRE1/XBP-1 pathway is required for efficient protein folding, maturation, and degradation in the ER and imply the existence of subsets of UPR target genes as defined by their dependence on XBP-1. Further, our observations suggest the existence of additional, as-yet-unknown, key regulators of the UPR.

Successful desensitization with proteasome inhibition and costimulation blockade in sensitized nonhuman primates.

The detrimental effects of donor-directed antibodies in sensitized transplant patients remain a difficult immunologic barrier to successful organ transplantation. Antibody removal is often followed by rebound. Proteasome inhibitors (PIs) deplete antibody-producing plasma cells (PCs) but have shown marginal benefit for desensitization. In an allosensitized nonhuman primate (NHP) model, we observed increased germinal center (GC) formation after PI monotherapy, suggesting a compensatory PC repopulation mediated via GC activation. Here we show that costimulation blockade (CoB) targets GC follicular helper T (Tfh) cells in allosensitized NHPs. Combined PI and CoB significantly reduces bone marrow PCs (CD19+CD20-CD38+), Tfh cells (CD4+ICOS+PD-1hi), and GC B cells (BCL-6+CD20+); controls the homeostatic GC response to PC depletion; and sustains alloantibody decline. Importantly, dual PC and CoB therapy prolongs rejection-free graft survival in major histocompatibility complex incompatible kidney transplantation without alloantibody rebound. Our study illustrates a translatable desensitization method and provides mechanistic insight into maintenance of alloantibody sensitization.

Alloimmune injury and rejection of human skin grafts on human peripheral blood lymphocyte-reconstituted non-obese diabetic severe combined immunodeficient beta2-microglobulin-null mice.

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) beta2-microglobulin-null (B2mnull) mice engrafted with human peripheral blood lymphocytes (hu-PBL-NOD-scid B2mnull mice) were used as in vivo models for studying human skin allograft rejection. Hu-PBL-NOD-scid B2mnull 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 B2mnull mice uniformly survived but on chimeric hu-PBL-NOD-scid B2mnull 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 B2mnull mice. It is proposed that Hu-PBL-NOD-scid B2mnull mice engrafted with human skin will provide a useful model for analysis of interventions designed to modulate human allograft rejection.