Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways.

Phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4(+) and CD8(+) lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform-specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4(+) and CD8(+) counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4(+) and CD8(+) lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity.

Transgenic expression of human CD47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation.

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable chimerism levels were observed in the peripheral blood of these recipients. In contrast, transplantation of WT progenitor cells resulted in little or no bone marrow engraftment and no detectable peripheral chimerism. These results demonstrate a substantial protective effect of hCD47 expression on engraftment and persistence of porcine cells in this model, presumably by modulation of macrophage phagocytosis.

Expansion of polyreactive B cells cross-reactive to HLA and self in the blood of a patient with kidney graft rejection.

Antibody rejection is often accompanied by nondonor HLA specific antibodies (NDSA) and self-reactive antibodies that develop alongside donor-specific antibodies (DSA). To determine the source of these antibodies, we immortalized 107 B-cell clones from a kidney transplant recipient with humoral rejection. Two of these clones reacted to HLA class I or MICA. Both clones were also reactive to self-antigens and a lysate of a kidney cell line, hence revealing a pattern of polyreactivity. Monoclonality was verified by the identification of a single rearranged immunoglobulin heavy chain variable region (VH) sequence for each clone. By tracking their unique CDR3 sequence, we found that one such polyreactive clone was highly expanded in the patient blood, representing ~0.2% of circulating B cells. The VH sequence of this clone showed evidence of somatic mutations that were consistent with its memory phenotype and its expansion. Lastly, the reactivity of the expanded polyreactive B-cell clone was found in the patient serum at time of rejection. In conclusion, we provide here proof of principle at the clonal level that human antibodies can cross-react to HLA and self. Our findings strongly suggest that polyreactive antibodies contribute to DSA, NDSA as well as autoantibodies, in transplant recipients.

Phosphorus fate and transport in soil columns loaded intermittently with influent of high phosphorus concentrations.

In this study, several columns of different lengths were filled with composite soils sampled from the field at corresponding depths and then loaded intermittently with influent of a high phosphorus concentration to evaluate phosphorus fate and transport in soil. The results indicate that the height of the mass transfer zone, solvent pore velocity, and soil's life expectancy for phosphorus removal increased with depth, while the retained phosphorus per kilogram of soil and the linear adsorption equilibrium coefficient, R, decreased with depth. An equation was developed to link liquid-phase phosphorus with solvent traveling time and soil depth. The results of X-ray diffraction and washout tests indicate that calcium-phosphorus precipitation and/or crystal growth occurred in the columns. The new protocol is useful for evaluation of phosphorus fate and transport in other subsurface systems, because it allows flexible adjustments in hydraulic loadings, feed solution, and sampling schemes.

Immortalized bone-marrow derived pig endothelial cells.

Primary cultures of porcine endothelial cells (EC) can only be maintained for a limited number of passages. To facilitate studies of xenogeneic human anti-pig immune responses in vitro, pig microvascular bone-marrow (BM) and macrovascular aortic EC were obtained from our herd of partially inbred miniature swine, homozygous for the major histocompatibility locus, and immortalized with a modified SV40 large T vector. The resulting BM-derived (2A2) and aortic (PEDSV.15) immortalized EC lines showed unlimited growth and EC phenotype as indicated by expression of von Willebrand Factor (vWF) and low density lipoprotein (LDL) receptors as well as by formation of typical cobblestone monolayers. Ultrastructural studies revealed morphological similarities in primary and immortalized EC. Flow cytometry analysis demonstrated constitutive SLA class I expression by all lines whereas SLA class II was only expressed after stimulation with porcine IFNgamma. Furthermore, pig CD34 mRNA was detected by Northern blot analysis in primary and immortalized aortic EC but not in 2A2. Both EC lines expressed a number of myeloid markers, adhesion molecules and xenoantigens, the latter being determined by binding of human natural antibodies. Gene transfer into the porcine EC lines was successfully performed by electroporation or calcium-phosphate transfection, as well as by adenoviral infection. Finally, the functional similarity between primary and immortalized EC was demonstrated in adhesion and cytotoxicity assays. Together, these results suggest that 2A2 and PEDSV. 15 represent valuable tools to study both human cellular and humoral immune responses in vitro against pig EC derived from microvascular and large vessels.

Tolerance to solid organ transplants through transfer of MHC class II genes.

Donor/recipient MHC class II matching permits survival of experimental allografts without permanent immunosuppression, but is not clinically applicable due to the extensive polymorphism of this locus. As an alternative, we have tested a gene therapy approach in a preclinical animal model to determine whether expression of allogeneic class II transgenes (Tg's) in recipient bone marrow cells would allow survival of subsequent Tg-matched renal allografts. Somatic matching between donor kidney class II and the recipient Tg's, in combination with a short treatment of cyclosporine A, prolonged graft survival with DR and promoted tolerance with DQ. Class II Tg expression in the lymphoid lineage and the graft itself were sequentially implicated in this tolerance induction. These results demonstrate the potential of MHC class II gene transfer to permit tolerance to solid organ allografts.

Regulated expression of an MHC class II gene from a promoter-inducible retrovirus.

Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine transplantation model by retrovirus-mediated transfer of allogeneic MHC class II genes into bone marrow cells (BMCs) of recipient animals. Graft survival correlated with transient expression of the somatic transgene (Tg) in the induction phase of tolerance. With the aim of investigating the effects of timing and threshold levels of Tg expression on induction of hyporesponsiveness to the grafted tissues, two recombinant retrovirus constructs containing the tetracycline binary regulatory system were used to achieve conditional expression of either the green fluorescent protein (tetGFP) as a control, or the porcine MHC class II DRbeta chain (tetDRB). Effective downregulation of GFP gene transcription was demonstrated in transduced murine fibroblasts after doxycycline treatment, leading to a > 90% reduction of GFP fluorescence. Similar diminution of the DRB gene transcription was achieved in transduced pig endothelial cells (ECs). Drug-dependent downregulation of DRBc gene expression in SLAd pig ECs coincided with complete inhibition of allogeneic activation of anti-class IIc-primed SLAd T cells. These in vitro results suggest that the binary tetracycline retrovirus system may also be adequate to regulate MHC class II Tg expression in vivo.

Dendritic cells enriched from swine thymus co-express CD1, CD2 and major histocompatibility complex class II and actively stimulate alloreactive T lymphocytes.

Initial characterization and partial purification of thymic dendritic cells (DC) from miniature swine were carried out with the ultimate goal of using these cells to induce transplantation tolerance in this preclinical animal model. Immunohistochemical analysis of swine thymic tissue sections has shown DC to be large cells located in the medullary and the cortico-medullary regions as evidenced by the presence of surrounding Hassal bodies. These cells exhibit membrane processes and express the CD1, granulocyte/macrophage (G/M), and major histocompatibility complex (MHC) class II surface antigens, as well as the S100 cytosolic and nuclear markers found in humans to be specific for DC. Dendritic cells were purified from thymi following collagenase treatment, Percoll gradient centrifugation, and adhesion steps to plastic. Cells similar in morphology and phenotype to those described in tissue sections were detected in the lighter density layers of the gradient and represented 0.02% of the starting cell number. Removal of plastic nonadherent cells showed enrichment levels similar to those reported for murine and human DC. Two-colour flow cytometric analysis of purified pig DC identified these cells as MHC class IIhi, CD1+, CD2+, and G/M+. The dendritic nature of these cells was confirmed by their potent ability to stimulate alloreactive T lymphocytes. Modification of porcine thymic DC by transfer of allogeneic MHC genes and reinjection into the DC donor should permit testing of the role of this DC subset in the induction of transplantation tolerance.

MHC class II alpha/beta heterodimeric cell surface molecules expressed from a single proviral genome.

Transplantation tolerance to renal allografts can be induced in large animal preclinical models if the donor and recipient have identical major histocompatibility complex (MHC) class II loci. Such class II matching is, however, not clinically achievable owing to the extreme diversity of class II sequences. With the ultimate goal of creating a somatic class II match in the bone marrow of an allograft recipient, the aim of the study is to develop a double-copy retrovirus construct to express both chains of the MHC class II DQ glycoprotein on a single transduced cell. Analysis of the expression patterns of the retroviral DQ transgenes in both virus producer and transduced fibroblasts revealed correct transcription and stable surface expression of the DQ heterodimers. In addition, we demonstrate that both the DQA and DQB sequences are functional within the same proviral copy, a prerequisite for efficient induction of transplantation tolerance following transduction of bone marrow precursor cells. The DQ double-copy retrovirus vector showed efficient expression of the transferred class II cDNA in murine colony-forming units for the granulocyte-monocyte lineage (CFU-GM), indicating that it is suitable for gene therapy of multimeric proteins in hematopoietic cells.

Recombinant retrovirus vectors for the expression of MHC class II heterodimers.

Class II antigens are critical in determining the fate of vascularized allografts across major histocompatibility differences. We have recently developed a new approach to induce transplantation tolerance in miniature swine by creating MHC class II antigen "molecular chimerism" in bone marrow cells of potential recipients through retrovirus-mediated gene transfer. As part of this project, the ability of a recombinant double-expression vector (ZQ32N) to express MHC class II DQA and DQB was investigated. Flow cytometry analyses of ZQ32N transfected virus-producer cells demonstrated the cell surface expression of DQa/DQb heterodimers, thus suggesting a correct transcription, translation, and transport of the swine polypeptides to the cell surface. The analyses of RNA isolated from virus particles produced from ZQ32N transfected virus-producer cells indicated the DQ sequences to be correctly packaged. However, the DQ-negative cells transduced with the ZQ32N retrovirus did not show any DQ-retrovirus surface expression. Southern and Northern blot analyses of ZQ32N transfected and transduced cells strongly suggested DNA rearrangements and deletions which could account for transgene expression loss. An analysis of transduced cell genomes suggested DNA recombinations targeted to homologous sequences within the recombinant provirus. The implications of the sequence instability in designing vectors for gene therapy of organ transplantation are discussed.

Transfer of porcine MHC DRalpha into IEalpha-deficient murine bone marrow results in reduced IE-restricted Vbeta usage.

BACKGROUND: Allogeneic bone marrow transplantation has proven effective for inducing specific tolerance to subsequent solid organ allografts, although the clinical applicability of this approach is limited by the morbidity and mortality associated with this procedure. As an alternative, we are investigating the transfer of allogeneic MHC class II genes into recipient bone marrow cells (BMC), using the miniature swine as a model. METHODS: To understand the mechanism of tolerance induction achieved through class II gene transfer, BMC from C57BL/10 mice, which lack expression of the MHC class II DRalpha equivalent (H-2 IEalpha), were transduced with a retrovirus vector for swine DRalpha. RESULTS: Expression of the DRA-vector in bone marrow-derived cells was demonstrated by Northern analysis of colonies grown in vitro from transduced myeloid progenitors. Taking advantage of the fact that the introduced DRalpha chain was able to form heterodimers with endogenous IEbeta, surface expression of the transgene was demonstrated on splenocytes harvested 1, 17, and 28 weeks after bone marrow transplantation. Transgene expression was confirmed by reverse transcriptase-polymerase chain reaction in the thymus of those animals killed at weeks 17 and 28. Finally, the effects of bone marrow transduction on central tolerance induction was demonstrated by the progressive decrease of IE-reactive T-cell clones bearing Vbeta5 and Vbeta11 T cell receptors in the peripheral blood cells of engineered recipients. CONCLUSIONS: Our results support the notion that transplantation tolerance, induced by class II gene transfer into syngeneic BMC, results in part from durable deletional unresponsiveness of graft-specific alloreactive T cells.

Expression of an allogeneic MHC DRB transgene, through retroviral transduction of bone marrow, induces specific reduction of alloreactivity.

BACKGROUND: Transfer of MHC class II genes, through allogeneic bone marrow (BM) transplantation, induced long-lasting acceptance of renal allografts in miniature swine. To adapt this approach to the clinic, we have now examined whether somatic transfer of allogeneic class II DR genes, into otherwise autologous bone marrow cells (BMC), can provide the matching required for inducing immune tolerance. METHODS: Autologous BMC were transduced ex vivo with recombinant retroviruses for allogeneic DRB followed by BM transplantation. The recipients were then challenged with kidney allografts solely matched to the DRB transgene. RESULTS: Five miniature swine received autologous BMC conditioned with growth factors and transduced with recombinant retrovirus vectors containing allogeneic (n=4) or syngeneic (n=1) class II DRB genes and a drug-resistance marker. Expression of retrovirus-derived products in BM-derived cells was demonstrated by the detection of drug-resistant colony-forming progenitors and the presence of DRB retrovirus transcripts in peripheral cells. Analysis of selective mixed lymphocyte reaction responses to DR or DQ antigens indicated decreased reactivity toward the transduced DR gene product. Among all of the animals receiving fully mismatched kidney allografts, but with DRB matched to the transduced DRB, the one with the highest gene transduction rate showed stable allograft function and essentially normal renal histology for 2.5 years. A control animal, which received a syngeneic DRB gene, rejected its kidney allograft in 120 days after an earlier rejection crisis. CONCLUSIONS: These studies demonstrate that allogeneic MHC gene transfer into BM provides a new strategy for inducing tolerance across MHC barriers.

HLA-Cw3 expression on porcine endothelial cells protects against xenogeneic cytotoxicity mediated by a subset of human NK cells.

There is increasing evidence that NK cells make an important contribution to human anti-porcine xenogeneic cytotoxicity. Most allogeneic as well as autologous normal cells are not susceptible to NK cell-mediated cytotoxicity because they express inhibitory molecules encoded within the MHC class I loci. The protective signal is delivered to NK cells through killer cell-inhibitory receptors expressing different MHC class I specificities. It has been proposed that xenogeneic target cells may be susceptible to NK cell-mediated lysis because their MHC class I molecules fail to be recognized by human killer cell-inhibitory receptors. To explore this hypothesis, we examined the effect of human MHC class I expression on porcine target cell lysis by human NK cells. An immortalized porcine bone marrow-derived endothelial cell line (2A2) was transfected with three different human MHC class I allelic genes (HLA-A2, -B27, or -Cw3). The cytotoxic activity of several GL183+ NK clones, which lysed untransfected porcine cells effectively, was substantially blocked by the presence of HLA-Cw3. In contrast, HLA-Cw3-positive cells were not protected against lysis by GL183- EB6+ NK clones. The expression of HLA-B27 or HLA-A2 molecules on pig target cells did not provide substantial protection from lysis by any of the NK clones tested. In addition to confirming the hypothetical basis of NK cell-mediated killing of xenogeneic targets, these results have practical implications as an approach to overcoming NK cell-mediated cytotoxicity, which may be an obstacle to pig-to-human xenotransplantation.

Molecular and cellular events implicated in local tolerance to kidney allografts in miniature swine.

Long-term tolerance to class I-mismatched renal allografts can be induced in miniature swine by treatment with a short course of cyclosporine (CsA). Kidney recipients treated with CsA and untreated control kidney recipients both demonstrated infiltration of the transplanted kidney by mononuclear cells, which reached a maximum between postoperative days 8 and 11. Recipients that did not receive the tolerizing regimen rejected their grafts between postoperative days 8 and 12 in this model. The kinetics of cytokine gene expression, including interleukin (IL)-1alpha, IL-1beta, IL-2, IL-6, IL-10, tumor necrosis factor, and interferon-gamma (IFN-gamma), within the grafted kidney of rejector and acceptor animals, were determined using Northern blot hybridization. A strong correlation between rejection and up-regulation of the IFN-gamma gene was observed, whereas animals with long-term tolerance showed low levels of IFN-gamma, but high levels of IL-10 gene transcription. None of the other cytokine genes demonstrated a reproducible pattern of expression that correlated with acceptance/rejection of allografts. Analysis of transcription patterns of cytokine genes in mononuclear cells purified from renal grafts confirmed the initial observations made on biopsies. The phenotype of graft-infiltrating cells (GIC) showed a dominance of CD8+ cells, with an average of 66% single-positive cells and 19% CD4/CD8 double-positive cells, compared with 30% and 14%, respectively, for peripheral cells. Predominance of CD8+ GIC was dictated neither by the MHC antigen disparity nor the rejector/acceptor status. These results, therefore, suggest that GIC represent a regulated combination of mononuclear cells producing local immune mediators that, in part, control the fate of allografts in this large animal model.