Natural antibodies do not inhibit xenogeneic transplantation of human PBL in lethally irradiated mice

Abstract: Attempts to transfer human peripheral blood lymphocytes (hu-PBL) into lethally irradiated mice resulted in limited engraftment in recipients lacking natural antibodies (nAb) and could not be achieved in immunologically normal mice. It has been proposed that nAb with antihuman specificity play a major role in the rejection of the hu-PBL graft. In the present study we demonstrate that, following intensification of the conditioning protocol (thymectomy, supralethal dose of TBI, and radioprotection with bone marrow for donors with severe combined immune deficiency (SCID), transplants of 50 to 70 × 10⁶ hu-PBL were successfully engrafted in BALB/c, CBA/J and C3H/HeJ mice—regardless of the initial high levels of nAb. The percentage of human CD45⁺ cells in peritoneal lavage was not statistically different from that obtained in congenitally immune-deficient corresponding strains (SCID and CBA/N) lacking natural antibodies. Significant differences in engraftment of hu-PBL, between different human donors, were related neither to the nAb content (r = 0.29) nor to the ABO(H) blood group. The transfer of serum with high level of nAb into SCID and CBA/N mice or incubation of hu-PBL in such a serum prior to implantation, did not impede the engraftment and did not decrease the production of human immunoglobulins. These data demonstrate that the presence of nAb in supralethally irradiated normal mice does not inhibit the engraftment of hu-PBL, emphasizing the role of cellular mediated mechanisms in xenograft rejection.     

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.     

Delayed type hypersensitivity-associated cytokines in islet xenotransplantation: limited efficacy of interleukin-2- and tumor necrosis factor-alpha-blockade in interferon-gamma receptor-deficient mice

Abstract: To investigate the role of interferon (IFN)- and tumor necrosis factor (TNF)- and their potential to replace each other in the process of fetal porcine islet-like cell cluster (ICC) xenograft rejection, mice with a targeted disruption of the IFN- receptor gene and wild-type controls were transplanted with fetal porcine ICCs under the kidney capsule and given post-transplant treatment with the TNF--inhibiting agent MDL 201,449A. Some of the MDL 201,449A-treated IFN- receptor-deficient mice received additional treatment with cyclosporinee (CsA). Evaluation of the xenografts was performed 7 days after transplantation (all groups), and in IFN- receptor-deficient mice treated with MDL 201 449 A, also 10 and 13 days after transplantation. On day 7 after transplantation, a few CD3⁺ cells were seen accumulated peripherally in the ICC xenograft. Moderate to abundant numbers of F4/80⁺ and Mac-1⁺ cells surrounded a few remaining ICCs present within the xenograft. Histochemical visualization of cyanide-resistant endogenous peroxidase activity for detection of eosinophils demonstrated only small numbers of eosinophils present within the xenograft by day 7 after transplantation. An increased amount of eosinophilic granulocytes was not found until day 10 after transplantation, i.e. at a time when ICC xenograft rejection has already been completed. However, two out of six IFN- receptor-deficient mice given post-transplant treatment with CsA and MDL 201,449A exhibited intact ICC xenografts with ICCs arranged in chords and duct-like structures on day 7 after transplantation. Taken together, findings in this study indicate that, in the pig-to-mouse model, IFN-, TNF-, and interleukin-2 seem to be of importance to fetal porcine ICC xenograft rejection. Nevertheless, in a majority of animals, other cytokines eventually substitute for the lack of IFN-, TNF- and interleukin-2.     

CD4+ T cells, without CD8+ or B lymphocytes, can reject xenogeneic skin grafts

Abstract: Previous experiments have shown that rejection of xenogeneic skin grafts by mice is particularly dependent on CD4⁺ T cells. There are two possible explantations for this finding: either 1) "help" provided by CD4⁺ T cells is essential for CD8⁺ T cell-, B cell-, or NK cell-mediated effector mechanisms of rejection, or 2) CD4⁺ cells are themselves responsible for rejection, perhaps by some nonspecific effector mechanism. To examine these two hypotheses, we transplanted pig skin onto SCID mice and then reconstituted the mice with selected subpopulations of lymphocytes. Mice that did not received CD4⁺ T cells were unable to reject their xenografts, whereas those receiving CD4⁺ cells could do so in the absence of CD8⁺ cells or B cells and even when additionally depleted of NK cells by treatment with anti-Asialo GM1 antibody. Additional experiments were performed both in vivo and vitro to confirm the absence in test mice of CD4⁺ or CD8⁺ and B lymphocytes, respectively. These results suggest that CD4⁺ T cells are not only necessary for rejection of xenogeneic skin grafts by mice, but that they can do so without CD8⁺ cells or B cells, and probably without NK cells. Since CD4⁺ cells in mice have been shown to recognize xenogeneic antigens indirectly, this suggests that a nonspecific effector mechanism may be involved in the rejection of xenografts. In these experiments allogeneic skin grafts behave quite differently as they could not be rejected by this mechanism.     

Alloreactive (CD4-Independent) CD8+ T Cells Jeopardize Long-Term Survival of Intrahepatic Islet Allografts

Despite success of early islet allograft engraftment and survival in humans, late islet allograft loss has emerged as an important clinical problem. CD8⁺ T cells that are independent of CD4⁺ T cell help can damage allograft tissues and are resistant to conventional immunosuppressive therapies. Previous work demonstrates that islet allografts do not primarily initiate rejection by the (CD4-independent) CD8-dependent pathway. This study was performed to determine if activation of alloreactive CD4-independent, CD8⁺ T cells, by exogenous stimuli, can precipitate late loss of islet allografts. Recipients were induced to accept intrahepatic islet allografts (islet 'acceptors') by short-term immunotherapy with donor-specific transfusion (DST) and anti-CD154 mAb. Following the establishment of stable long-term islet allograft function for 60–90 days, recipients were challenged with donor-matched hepatocellular allografts, which are known to activate (CD4-independent) CD8⁺ T cells. Allogeneic islets engrafted long-term were vulnerable to damage when challenged locally with donor-matched hepatocytes. Islet allograft loss was due to allo specific immune damage, which was CD8- but not CD4-dependent. Selection of specific immunotherapy to suppress both CD4- and CD8-dependent immune pathways at the time of transplant protects islet allografts from both early and late immune damage.     

Selective rejection of porcine islet xenografts by macrophages

Abstract:  Background: Our previous study has shown that porcine antigen‐primed and CD4+ T cell‐activated macrophages are capable of recognition and rejection of porcine xenografts after adoptive transfer. However, whether this is an absolute xenograft specific rejection remains to be confirmed. Methods:  Mouse islet allografts and neonatal porcine islet cell cluster (NICC) xenografts were admixed and transplanted under the left kidney capsule, and NICC xenografts alone were transplanted under the right kidney capsule of strepotozotocin‐induced diabetic NOD‐SCID mice. After achievement of normoglycemia, the NOD‐SCID recipients were transferred with macrophages purified from NICC transplant NOD‐SCID mice reconstituted with CD4+ T cells. Five weeks after macrophage transfer the left kidney with the admixed grafts were removed. Graft survival and function following macrophage transfer was assessed by blood glucose measurement and immunohistochemistry. Results and conclusions:  Adoptive transfer with activated macrophages did not affect the normalized blood glucose levels in NOD‐SCID recipients of admixed grafts until left nephrectomy 5 weeks post‐macrophage transfer. Insulin‐positive and porcine C‐peptide‐negative mouse islets were detected in the admixed grafts. The surviving mouse islets in the admixed grafts were surrounded but not infiltrated by macrophages. The nephrectomized recipients demonstrated sustained hyperglycemia and completely destroyed NICC xenografts in their remaining right kidneys 8 weeks after macrophage transfer. Taken together, these data provide direct evidence of porcine islet xenograft specific rejection by activated macrophages.     

The allogeneic response to cultured human skin equivalent in the hu-PBL-SCID mouse model of skin rejection.

BACKGROUND: Engineered tissues have been proposed for the treatment of a variety of conditions including the partial or complete replacement of human organs. To determine the basis for the rejection of these tissues, we analyzed the immune response to allogeneic human skin equivalent (HSE, also called Apligraf) in the humanized SCID mouse (hu-PBL-SCID). METHODS: Two models of hu-PBL-SCID were used for these studies. In one model, human skin or HSE was transplanted onto humanized mice so that graft survival could be analyzed. In the other model, skin grafts were allowed to heal on naive mice before humanization. This model was used to analyze the immunologic response to the vascularized skin allograft. Humanization was performed by adoptive transfer of human PBL into SCID mice by i.p. injection. RESULTS: Both human foreskin and HSE successfully engrafted onto naive SCID mice and remained stable for more than 6 months. In contrast, human foreskin was rejected by 21 days posttransplant in hu-PBL-SCID, whereas HSE consistently engrafted for more than 28 days. Treatment of HSE grafts with interferon-y for 5 days to induce maximal MHC class II molecule expression before grafting failed to induce rejection. HSE also engrafted onto hu-PBL-SCID mice that were exposed to alloantigen by prior injection with interferon-gamma-treated keratinocytes identical to those used to generate the HSE. In addition, we determined that humanization of SCID mice following engraftment and vascularization of human foreskin resulted in marked CD3+ T cell infiltrates and a lymphocyte-induced vasculitis. In contrast, the response in vascularized HSE was associated with minimal CD3+ T cell infiltration in the absence of vasculitis or morphological features of rejection. CONCLUSION: These results support the use of HSE and other allogeneic engineered tissues in humans provided that such tissues are limited in their antigen presenting capabilities. In addition, our findings suggest a critical function for the donor endothelial cell in rejection.     

CD8 T Cells Can Reject Major Histocompatibility Complex Class I-Deficient Skin Allografts

Following transplantation, recipient T cells can recognize and respond to donor antigens expressed directly on donor cells, and can respond to donor-derived peptides that have been processed and presented in the context of recipient MHC through the indirect pathway. Indirectly primed CD4⁺ T cells have been well studied in transplantation, but little information is available regarding whether indirectly primed CD8⁺ T cells participate in rejection. To address this, we placed MHC class I-deficient D^bK^b knockout skin grafts onto allogeneic H-2 ^k SCID recipients followed by adoptive transfer of purified H-2 ^k CD8⁺ T cells. The MHC class I-deficient grafts were rejected and only CD8⁺ T cells were detectable in the recipient lymphoid organs and in the skin grafts. Immunohistochemical analysis showed that CD8⁺ T cells were found in close proximity to vascular endothelial cells and to recipient infiltrating macrophages, suggesting specific interactions. The data demonstrate that cross-primed polyclonal CD8⁺ T cells can function as active participants in the effector phase of rejection. The findings confirm and extend previous studies using a monoclonal TCR transgenic T cell and shed light on mechanisms of acute and chronic graft injury that are potentially relevant to human transplant recipients.     

An Anti-CD103 Immunotoxin Promotes Long-Term Survival of Pancreatic Islet Allografts

Previous studies using knockout mice document a key role for the integrin CD103 in promoting organ allograft rejection and graft-versus-host disease. However, a determination of whether blockade of the CD103 pathway represents a viable therapeutic strategy for intervention in these processes has proven problematic due to the lack of reagents that efficiently deplete CD103⁺ cells from wild type hosts. To circumvent this problem, we conjugated the nondepleting anti-CD103 monoclonal antibody, M290, to the toxin, saporin, to produce an immunotoxin (M290-SAP) that efficiently depletes CD103⁺ cells in vivo . Herein, we show that M290-SAP dramatically reduces the frequency and absolute numbers of CD103-expressing leukocytes in the blood, spleen, mesenteric lymph nodes and intestinal epithelium of treated mice. We further demonstrate that M290-SAP promotes indefinite islet allograft survival in a fully MHC mismatched mouse model. The prolonged islet allograft survival resulting from M290-SAP treatment was associated with multiple effects in the host immune system including not only depletion of CD103-expressing leukocytes, but also an increase in CD4⁺CD25⁺FoxP3⁺ T regulatory cells and a predominance of effector-memory CD8 T cells. Regardless of the underlying mechanisms, these data document that depletion of CD103-expressing cells represents a viable strategy for therapeutic intervention in allograft rejection.     

Co-stimulatory molecules in islet xenotransplantation: CTLA4Ig treatment in CD40 ligand-deficient mice

Previous work has demonstrated that short-term systemic administration of cytotoxic T lymphocyte antigen-4 (CTLA-4) Ig blocks human pancreatic islet xenograft rejection in mice and induces long-term, donor-specific tolerance, whereas studies on pig pancreatic islet rejection in mice have failed to demonstrate a role for CTLA4Ig in preventing rejection. Treatment with anti-CD40 ligand (L) monoclonal antibodies alone is somewhat effective in prolonging the survival of islet xenografts, but ineffective when applied to skin xenografts. However, simultaneous blockade of the CD28 and CD40 co-stimulatory pathways prolongs the survival of pig skin on recipient mice. To evaluate the role of CD28 and CD40 co-stimulatory pathways in pig islet-like cell cluster (ICC) xenograft rejection in mice, CD40L-deficient mice transplanted with fetal porcine ICCs were given posttransplant treatment with human (h) CTLA4Ig or a human IgG1 chimeric mAb (hL6). Xenografts were evaluated 6 or 12 days after transplantation. Fetal porcine ICC xenografts were protected from rejection in hCTLA4Ig-treated CD40L-deficient mice, whereas xenograft rejection persisted in untreated CD40L-deficient mice. Simultaneous blockade of the CD28 and CD40 co-stimulatory pathways is mandatory to inhibit ICC xenograft rejection in the pig-to-mouse model, because the CD28 and CD40 co-stimulatory pathways seem capable of efficiently substituting for one another.     

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft‐infiltrating Th17 cells and activated B cells

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig‐to‐humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45⁺ cells comprising activated human B cells, CD4⁺CD44⁺IL‐17⁺ Th17 cells, and CD68⁺ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long‐term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI‐SP), and peri‐transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI‐SP treatment in controlling human immune cells in promoting long‐term islet xenograft survival.     

Type I Interferons Are Not Critical for Skin Allograft Rejection or the Generation of Donor-Specific CD8+ Memory T Cells

Type I interferons (IFN-I) link innate to adaptive immunity in microbial infection, autoimmune disease and tumor immunity. It is not known whether IFN-I have an equally central role in alloimmunity. Here we tested this possibility by studying skin allograft survival and donor-specific CD8⁺ T-cell responses in mice that lack the IFN-I receptor (IFN-IR^−/−). We found that IFN-IR^−/− mice reject fully allogeneic wild-type skin grafts at the same rate as wild-type recipients. Similarly, allograft rejection was not delayed if IFN-IR^−/− male skin was transplanted to syngeneic IFN-IR^−/− female mice. Quantitation of the male (H-Y)-specific CD8⁺ T-cell response in these mice revealed normal generation of donor-specific CD8⁺ effector T cells but fourfold reduction in CD8⁺ memory T cells. Memory CD8⁺ T cells generated in the absence of IFN-IR had normal phenotype and recall function, assessed by ex vivo cytokine production and the ability of IFN-IR^−/− mice to mount second set rejection. Finally, these memory T cells were maintained at a constant number despite their inability to respond to IFN-1. Our findings indicate that IFN-I cytokines are not critical for acute allograft rejection or for the expansion and differentiation of donor-specific CD8⁺ T cells into long-lived, functional memory T cells.     

Aqueous Humor Alloreactive Cell Phenotypes, Cytokines and Chemokines in Corneal Allograft Rejection

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45⁺CD4⁺, CD45⁺CD8⁺ and CD45⁺CD14⁺ cells were found in aqueous during rejection; no CD45⁺ cells were seen in control samples. Higher proportions of CD45⁺ cells found in aqueous during rejection were CD14⁺, denoting monocyte/macrophage lineage, than were CD4⁺ or CD8⁺. Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1α and eotaxin. The role of CD14⁺ cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.     

Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin-10 in humanized mice

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ(-/-) mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.     

A critical role for human CD4+ T-cells in rejection of porcine islet cell xenografts

T-cell-mediated rejection is likely to present a significant barrier to porcine islet xenotransplantation. Little is known, however, about human anti-porcine islet rejection because no suitable model exists to study this process. To address this problem, we have developed an immunodeficient mouse model to study rejection of fetal porcine islet cell clusters (ICCs) by human lymphocytes. Transplantation of porcine ICCs into hyperglycemic recombinase activating gene-deficient (R-) mice restores normal blood glucose levels within 5 weeks. Adoptive transfer of in vitro-stimulated human peripheral blood mononuclear cells into R- mice before islet cell transplantation leads to acute cellular rejection of porcine ICCs. The first human cells observed to infiltrate rejecting grafts are CD4+ T-cells. Although CD8+ T-cells are observed within the grafts at later time points, CD4+ T-cells predominate until the graft is destroyed. Adoptive transfer of purified human CD4+ T-cells before ICC transplantation is sufficient to cause acute cellular rejection. These data demonstrate that human CD4+ T-cells play a critical role in porcine ICC xenograft rejection.     

Effects of ultraviolet B irradiation on fetal pig islet-like cell clusters

Abstract: Ultraviolet B (UV-B) irradiation of donor islets has previously been shown to result in the prolongation of their survival when transplanted into rodents. This study examined the in vitro and in vivo effects of UV-B irradiation on fetal pig islet-like cell clusters (ICCs), which like adult islets are being transplanted to reverse diabetes. Under control conditions, fetal pig ICCs were able to stimulate both human and pig peripheral blood mononuclear cells (PBMC) in mixed islet lymphocyte culture (MILC). Exposure of the ICCs to UV-B irradiation significantly reduced their ability to stimulate PBMC of both species in MILC when 600 J/m² but not lower doses (300 and 400 J/m²) of irradiation were applied. In contrast, all doses of UV-B irradiation were effective in inhibiting the ability of pig and human PBMC to stimulate human PBMC in a mixed lymphocytes culture (MLC). This demonstrates that UV-B irradiation is effective in reducing xeno immunogenicity of pig antigens. A toxic effect of all doses of UV-B irradiation on ICCs was demonstrated in vitro with a reduction in ³H-thymidine incorporation of 57, 71, 64, and 80% at 150, 300, 450, and 600 J/m², respectively. Toxicity of UV-B irradiation was also demonstrated when treated ICCs were transplanted beneath the renal capsule of SCID mice. The insulin content of the ICCs, 6 weeks after transplantation, was significantly reduced in the 600 J/m² group ( P <0.05). ICCs treated with UV-B irradiation (300 J/m²) in vitro and then transplanted beneath the renal capsule of BALB/c mice were rejected within 2 weeks as were untreated ICCs. Injection of cyclosporine (12.5 mg/kg/day) into these mice did not alter the results. It is concluded that UV-B irradiation is toxic to fetal pig ICCs and, in low dose, unable to prevent their rejection when transplanted into mice.     

Pig islet xenograft rejection in a mouse model with an established human immune system

Abstract: Background: Xenotransplantation from pigs provides a potential solution to the severe shortage of human pancreata, but strong immunological rejection prevents its clinical application. A better understanding of the human immune response to pig islets would help develop effective strategies for preventing graft rejection. Methods: We assessed pig islet rejection by human immune cells in humanized mice with a functional human immune system. Humanized mice were prepared by transplantation of human fetal thymus/liver tissues and CD34⁺ fetal liver cells into immunodeficient mice. Islet xenograft survival/rejection was determined by histological analysis of the grafts and measurement of porcine C‐peptide in the sera of the recipients. Results: In untreated humanized mice, adult pig islets were completely rejected by 4 weeks. These mice showed no detectable porcine C‐peptide in the sera, and severe intra‐graft infiltration by human T cells, macrophages, and B cells, as well as deposition of human antibodies. Pig islet rejection was prevented by human T‐cell depletion prior to islet xenotransplantation. Islet xenografts harvested from T‐cell‐depleted humanized mice were functional, and showed no human cell infiltration or antibody deposition. Conclusions: Pig islet rejection in humanized mice is largely T‐cell‐dependent, which is consistent with previous observations in non‐human primates. These humanized mice provide a useful model for the study of human xenoimmune responses in vivo.     

Strategies to Induce Marked Prolongation of Secondary Skin Allograft Survival in Alloantigen-Primed Mice

Alloreactive memory T cells mediate accelerated rejection. We investigated the effect of polyclonal anti-T-cell antibody (ALS) and rapamycin (RAPA) on skin allograft survival in naïve or alloantigen-primed mice. ALS prolonged graft survival in both naïve and alloantigen-primed mice. T-cell depletion by ALS was associated with increased CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ memory T cells. Addition of RAPA to ALS extended graft survival in naïve mice, but had no effect on secondary allograft survival in alloantigen-primed mice. In adoptive transfer experiments, RAPA inhibited alloantigen-stimulated proliferation and allograft rejection by naïve T cells. In contrast, alloantigen-primed memory T cells, particularly CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ T cells, were resistant to RAPA in response to alloantigen and mediated accelerated rejection in the presence of RAPA. Resistance to RAPA by alloantigen-primed mice was overcome by the use of high-dose ALS, which achieved marked prolongation of secondary skin allograft survival (>100 days). Inhibition of CD122⁺ T cells and/or OX40/OX40L costimulation blockade, combined with low-dose ALS and RAPA, was also effective. These results demonstrate that tolerance may be achieved in allosensitized individuals by T-cell depletion- and RAPA-based strategies employing high-dose ALS or targeting CD122⁺CD8⁺ T cells and/or the OX40/OX40L costimulatory pathway.     

Characterization of human killer cell reactivity against porcine target cells: differential modulation by cytokines

Abstract: The cytotoxic cell response to porcine cells by human lymphocytes, and the modulation of cytolytic cellular activity by human cytokines were investigated. Human peripheral blood mononuclear cells (PBMC) and purified lymphocyte subsets were co-cultured with fresh irradiated porcine stimulator cells and examined for the development of lytic activity and for their proliferative response. Porcine target cells included a new cell line, MS-PBMC-J2 (designated J2; SLA-DR⁺MHC class I⁺CD2⁺CD3^–CD8⁺CD16⁺CD45⁺), aortic and microvascular endothelial cells. Initial results showed that natural killer (NK) cells were fivefold more efficient in killing porcine target cells compared with T cells. IL-12 augmented the killing of porcine target cells by human NK cells beyond that induced by stimulation with cells alone. In contrast, IL-2 and IL-15 often induced substantial human NK cell mediated killing of porcine target cells, including endothelial cells in the case of IL-2 where such targets were examined, even in the absence of stimulator cells. Finally, neither IL-18 nor IL-8 had any effect beyond background on NK cell mediated killing of porcine target cells. These findings show that cytokines that would be produced in a xenograft setting clearly modulate the ability of human cytolytic cells to kill porcine targets. In addition, fresh unstimulated human NK cells lysed J2 and porcine aortic endothelial cells, but not porcine microvascular endothelial cells, suggesting the possibility of rapid attack of xenografts by NK cells, and differential susceptibility of endothelial cells from different vascular structures to this attack.     

Ex Vivo-Expanded Natural CD4+CD25+ Regulatory T Cells Synergize With Host T-Cell Depletion to Promote Long-Term Survival of Allografts

Foxp3⁺CD4⁺CD25⁺ natural regulatory T (nT_reg) cells have been shown in immunodeficient mice to suppress allograft rejection after adoptive cotransfer. We hypothesized that immunotherapy using ex vivo -expanded nT_reg could suppress allograft rejection in wild-type mice. Donor alloantigen (alloAg) specificity of naive splenic nT_reg was enriched in vitro by culturing with anti-CD3/CD28-coated Dynabeads plus bone marrow-derived dendritic cells (BM-DC) in the presence of interleukin (IL)-2 or IL-2 plus transforming growth factor (TGF)-β. On average, 96.2% fresh CD4⁺CD25⁺ nT_reg were intracellular Foxp3⁺. By d+20 in culture, 6.4% nT_reg were Foxp3⁺ following expansion with IL-2 alone, and 14.4% or 19.7% nT_reg were Foxp3⁺ when expanded with IL-2 plus 0.5 or 2.5 ng/mL TGF-β, respectively. In vitro , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg exerted stronger donor alloAg-specific suppression than cells with IL-2 alone in mixed lymphocyte reaction (MLR) assays. In vivo , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg expressed high-level Foxp3 following infusion, effectively overcame acute rejection and induced long-term survival of donor but not third-party heart allografts in peritransplant host T-cell-depleted mice. Long-term surviving allografts were noted to possess Foxp3⁺ graft-infiltrating cells of exogenous and endogenous origins. In conjunction with transient host T-cell depletion, therapeutic use of ex vivo -expanded nT_reg may be a practical means of preventing acute allograft rejection.