Hyperlipidemia Alters Regulatory T Cell Function and Promotes Resistance to Tolerance Induction Through Costimulatory Molecule Blockade.

Recent work from our laboratory has shown that hyperlipidemia promotes accelerated rejection of vascularized cardiac allografts in mice by inducing anti-donor Th17 reactivity and production of IL-17. Here, we show that hyperlipidemia also affects FoxP3(+) regulatory T cells (Tregs). Hyperlipidemia promotes the development of Tregs that express low levels of CD25. Hyperlipidemia also promotes a decrease in central Tregs and an increase in effector Tregs that appears to account for the increase in the frequency of CD25(low) Tregs. Alterations in Treg subsets also appear to lead to alterations in Treg function. The ability of FoxP3(+) , CD25(high) , CD4(+) Tregs from hyperlipidemic mice to inhibit proliferation of effector T cells stimulated with anti-CD3 and CD28 was reduced when compared with Tregs from control mice. Regulatory T cells isolated from hyperlipidemic recipients exhibit increased activation of Akt, and a reduction in Bim levels that permits the expansion of FoxP3(+) CD25(low) CD4(+) T cells. Hyperlipidemic mice were also resistant to tolerance induction using costimulatory molecule blockade consisting of anti-CD154 and CTLA4Ig, a strategy that requires Tregs. Together, our data suggest that hyperlipidemia profoundly affects Treg subsets and function as well as the ability to induce tolerance.

MicroRNA-494 promotes cyclosporine-induced nephrotoxicity and epithelial to mesenchymal transition by inhibiting PTEN.

A major complication associated with cyclosporine (CsA) treatment is nephrotoxicity. In this study, we examined whether microRNAs play a role in cyclosporine-induced nephrotoxicity. Treatment of mice with CsA resulted in nephrotoxicity that was associated with an early increase in expression of microRNA mmu-miR-494 (miR-494). Similarly, tubular epithelial cell epithelial-mesenchymal transition (EMT) induced by CsA toxicity resulted in the upregulation of microRNA-494 and a decrease in PTEN levels in vitro. miR-494 directly targeted Pten and negatively regulated its expression. Preventing Pten targeting by miR-494 was sufficient to prevent CsA induced EMT. Knockdown of miR-494 prevented the downregulation of PTEN in tubular epithelial cells following CsA treatment and also prevented CsA induced EMT. Thus, miR-494 plays a major role in promoting CsA induced nephrotoxicity through its ability to target Pten thereby contributing to EMT. We suggest that manipulating miR-494 expression may represent a novel approach to preventing EMT associated with CsA induced nephrotoxicity.

Tolerance to MHC class II disparate allografts through genetic modification of bone marrow.

Induction of molecular chimerism through genetic modification of bone marrow is a powerful tool for the induction of tolerance. Here, we demonstrate for the first time that expression of an allogeneic MHC class II gene in autologous bone marrow cells, resulting in a state of molecular chimerism, induces tolerance to MHC class II mismatched skin grafts, a stringent test of transplant tolerance. Reconstitution of recipients with syngeneic bone marrow transduced with retrovirus encoding H-2I-A(b) (I-A(b)) resulted the long-term expression of the retroviral gene product on the surface of MHC class II-expressing bone marrow-derived cell types. Mechanistically, tolerance was maintained by the presence of regulatory T cells, which prevented proliferation and cytokine production by alloreactive host T cells. Thus, the introduction of MHC class II genes into bone marrow-derived cells through genetic engineering results in tolerance. These results have the potential to extend the clinical applicability of molecular chimerism for tolerance induction.

Persistent molecular microchimerism induces long-term tolerance towards a clinically relevant respiratory allergen.

BACKGROUND: Development of antigen-specific preventive strategies is a challenging goal in IgE-mediated allergy. We have recently shown in proof-of-concept experiments that allergy can be successfully prevented by induction of durable tolerance via molecular chimerism. Transplantation of syngeneic hematopoietic stem cells genetically modified to express the clinically relevant grass pollen allergen Phl p 5 into myeloablated recipients led to high levels of chimerism (i.e. macrochimerism) and completely abrogated Phl p 5-specific immunity despite repeated immunizations with Phl p 5. OBJECTIVE: It was unclear, however, whether microchimerism (drastically lower levels of chimerism) would be sufficient as well which would allow development of minimally toxic tolerance protocols. METHODS: Bone marrow cells were transduced with recombinant viruses integrating Phl p 5 to be expressed in a membrane-anchored fashion. The syngeneic modified cells were transplanted into non-myeloablated recipients that were subsequently immunized repeatedly with Phl p 5 and Bet v 1 (control). Molecular chimerism was monitored using flow cytometry and PCR. T cell, B-cell and effector-cell tolerance were assessed by allergen-specific proliferation assays, isotype levels in sera and RBL assays. RESULTS: Here we demonstrate that transplantation of Phl p 5-expressing bone marrow cells into recipients having received non-myeloablative irradiation resulted in chimerism persisting for the length of follow-up. Chimerism levels, however, declined from transient macrochimerism levels to persistent levels of microchimerism (followed for 11 months). Notably, these chimerism levels were sufficient to induce B-cell tolerance as no Phl p 5-specific IgE and other high affinity isotypes were detectable in sera of chimeric mice. Furthermore, T-cell and effector-cell tolerance were achieved. CONCLUSIONS AND CLINICAL RELEVANCE: Low levels of persistent molecular chimerism are sufficient to induce long-term tolerance in IgE-mediated allergy. These results suggest that it will be possible to develop minimally toxic conditioning regimens sufficient for low level engraftment of genetically modified bone marrow.

Critical role of proinflammatory cytokine IL-6 in allograft rejection and tolerance.

The proinflammatory cytokine IL-6 plays an important role in controlling T-cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL-6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild-type or IL-6-deficient C57BL/6 mice. We observed that production of IL-6 and IFN-γ was upregulated during allograft rejection in untreated wild-type mice. In IL-6-deficient mice, IFN-γ production was greater than that observed in wild-type controls, suggesting that IL-6 production affects Th1/Th2 balance during allograft rejection. CD28-B7 blockade by CTLA4-Ig inhibited IFN-γ production in C57BL/6 recipients, but had no effect on the production of IL-6. Although wild-type C57BL/6 recipients treated with CTLA4-Ig rejected fully MHC-mismatched BALB/c heart transplants, treatment of IL-6-deficient mice with CTLA4-Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL-6 production may represent an important part of costimulation blockade-based strategies to promote allograft acceptance and tolerance.

Homeostatic expansion permits T cells to re-enter the thymus and deliver antigen in a tolerogenic fashion.

We previously have shown that delivery of alloantigen on T cells can be used to induce tolerance through central deletion. Here, we analyzed the requirements for tolerance induced by T cells. Adoptively transferred allogeneic T cells undergo extensive homeostatic proliferation in the periphery of lethally irradiated hosts receiving a syngeneic bone marrow transplant, and acquire a memory-like cell surface phenotype. Analysis of the kinetics of thymic re-entry of transferred T cells revealed that T cells undergo homeostatic proliferation in the periphery prior to re-entry into the thymus. Prevention of homeostatic proliferation results in a failure of transferred T cells to re-enter the thymus. In the absence of homeostatic proliferation, adoptively transferred T cells were unable to induce tolerance. These date suggest that homeostatic proliferation of T cells resulting in an activated cell surface phenotype is required for thymic re-entry and is mechanistically linked to the ability of T cells to induce tolerance.

Persistence of antigen is required to maintain transplantation tolerance induced by genetic modification of bone marrow stem cells.

Genetic modification of hematopoietic stem cells (HSCs) resulting in a state of molecular chimerism can be used to induce donor-specific tolerance to allografts. However, the requirements for maintaining tolerance in molecular chimeras remain unknown. Here, we examined whether long-term expression of a retrovirally encoded alloantigen in hematopoietic cells is required to maintain donor-specific tolerance in molecular chimeras. To this end, mice were reconstituted with syngeneic bone marrow transduced with retroviruses carrying the gene encoding the allogeneic MHC class I molecule Kb. Following induction of molecular chimerism, mice were depleted of cells expressing Kb by administration of the anti-Kb monoclonal antibody Y-3. Mice that were effectively depleted of cells expressing the retrovirally encoded MHC class I antigen rejected Kb disparate skin allografts. In contrast, control molecular chimeras accepted Kb disparate skin allografts indefinitely. These data suggest maintenance of tolerance in molecular chimeras requires long-term expression of retrovirally transduced alloantigen on the progeny of retrovirally transduced HSCs.

Inhibition of CD26 peptidase activity significantly improves engraftment of retrovirally transduced hematopoietic progenitors.

It has previously been shown that inhibition of CD26 (DPPIV/dipeptidylpeptidase IV) peptidase activity improves homing of hematopoietic stem cells (HSCs) to the bone marrow and increases engraftment efficiency. Here, we demonstrate that treatment of retrovirally transduced mouse bone marrow cells with the tri-peptide Diprotin A (Ile-Pro-Ile), a specific inhibitor of CD26, significantly enhances engraftment of retrovirally transduced HSCs. Treatment of transduced bone marrow cells with Diprotin A permitted long-term expression of a retrovirally encoded MHC class I gene on multiple hematopoietic cell lineages after transplantation of a suboptimal number of transduced cells. Secondary transfer experiments revealed that expression of the transduced MHC class I gene resulted from engraftment of transduced HSCs. Expression of the allogeneic MHC class I antigen on bone marrow-derived cells following transplantation of Diprotin A-treated cells was sufficient to induce transplantation tolerance. Therefore, inhibition of CD26 activity significantly enhances engraftment of limited numbers of genetically modified HSCs, resulting in physiologically relevant levels of gene transfer.

Gene therapy progress and prospects: gene therapy in organ transplantation.

One major complication facing organ transplant recipients is the requirement for life-long systemic immunosuppression to prevent rejection, which is associated with an increased incidence of malignancy and susceptibility to opportunistic infections. Gene therapy has the potential to eliminate problems associated with immunosuppression by allowing the production of immunomodulatory proteins in the donor grafts resulting in local rather than systemic immunosuppression. Alternatively, gene therapy approaches could eliminate the requirement for general immunosuppression by allowing the induction of donor-specific tolerance. Gene therapy interventions may also be able to prevent graft damage owing to nonimmune-mediated graft loss or injury and prevent chronic rejection. This review will focus on recent progress in preventing transplant rejection by gene therapy.

Induction of molecular chimerism by gene therapy prevents antibody-mediated heart transplant rejection.

In order for xenotransplantation to become a clinical reality, and fulfill its promise of overcoming shortages of human organs and tissues, rejection mediated by the host's immune system must first be overcome. In primates, preformed natural antibodies that bind the carbohydrate antigen Galalpha1-3Galbeta1-4GIcNAc-R (alphaGal), which is synthesized by UDP galactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha(1-3)galactosyltransferase (E.C. 2.4.1.151) or simply alphaGT, mediate rigorous rejection of transplanted pig organs and tissues. In alphaGT knockout mice (GT0 mice), which like humans contain in their serum antibodies that bind alphaGal, expression of a retrovirally transduced alphaGT in bone marrow-derived cells is sufficient to prevent production of alphaGal-reactive antibodies. Here, we demonstrate that reconstitution of lethally irradiated GT0 mice with alphaGT-transduced bone marrow cells from GT0 littermates prevents antibody-mediated rejection of cardiac transplants from wild-type mice. These data suggest that gene therapy can be used to induce immunological tolerance to defined antigens and thereby overcome transplant rejection.

Defining the requirements for peptide recognition in gene therapy-induced T cell tolerance.

Expression of a retrovirally transduced MHC class I Ag, H-2K(b) (K(b)), in bone marrow-derived cells leads to specific prolongation of K(b) disparate skin grafts. To examine the extent to which peptides derived from K(b) contribute to the induction of tolerance, retroviruses carrying mutant K(b) genes designed to enter separate pathways of Ag presentation were constructed. Thymectomized and CD8 T cell-depleted mice that had been irradiated and reconstituted with bone marrow cells expressing a secreted form of K(b) showed prolongation of K(b) disparate skin graft survival. Skin graft prolongation was not observed when similar experiments were performed using mice that were not CD8 T cell depleted. This suggests that hyporesponsiveness can be induced in CD4 T cells, but not CD8 T cells by Ags presented via the exogenous pathway of Ag processing. Modest prolongation of skin allografts was observed in mice reconstituted with bone marrow cells transduced with retroviruses carrying a gene encoding a mutant K(b) molecule expressed only in the cytoplasm. Prolongation was also observed in similar experiments in mice that were thymectomized and CD4 T cell depleted following complete reconstitution, but not in mice that were reconstituted and then thymectomized and CD8 T cell depleted. Thus, hyporesponsiveness can be induced in a subset of CD8 T cells by recognition of peptides derived from K(b) through both the direct and indirect pathways of Ag recognition, while CD4 T cell hyporesponsiveness to MHC class I disparate grafts occurs only through the indirect pathway of Ag recognition.

Induction of B-cell tolerance by retroviral gene therapy.

The primary immunologic barrier to overcome before clinical xenotransplantation can be successful is rejection mediated by preformed natural antibodies in the host, directed toward a single carbohydrate epitope Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal) present on porcine tissue, encoded for by the enzyme glucosyltransferase UDP galactose:beta-D-galactosyl-1, 4-N-acetyl-D-glucosaminide alpha(1-3)galactosyltransferase (EC 2.4.1. 151) or simply alphaGT. Although we have shown previously that a gene therapy approach could be used to prevent production of natural antibodies specific for alphaGal, the ability to induce and maintain tolerance after rigorous antigen challenge would be required if similar approaches are to be used clinically. Here, we demonstrate in alphaGT knockout mice (GT(0) mice), which, like humans, contain in their serum antibodies that bind alphaGal, that the efficient transduction and expression of a retrovirally transduced alphaGT gene in bone marrow-derived cells induces stable long-term tolerance to the alphaGal epitope. GT(0) mice reconstituted with alphaGT-transduced bone marrow cells were unable to produce antibodies that bind alphaGal after extensive immunization with pig cells. Furthermore, using ELISPOT assays, we were unable to detect the presence of B cells that produce alphaGal reactive antibodies after immunization, suggesting that such B cells were eliminated from the immunologic repertoire after gene therapy. Interestingly, after tolerance to alphaGal is induced by gene therapy, the antiporcine non-alphaGal humoral response changes from a predominantly IgM to an IgG response. This suggests that once the natural antibody barrier is eliminated by the induction of tolerance, the antipig response changes to a typical T-cell-dependent response involving isotype switching. Thus, gene therapy approaches may be used to overcome immunologic responses leading to xenograft rejection, and similar gene therapy approaches could be used to overcome autoimmunity.

Human CD4+ T cells mediate rejection of porcine xenografts.

It has previously been demonstrated that xenograft rejection in rodents is dependent on CD4+ T cells. However, because of the lack of an appropriate in vivo model, little is known about the cellular basis of human T cell-mediated rejection of xenografts. In this study, we have evaluated the ability of human T cells to mediate rejection of porcine skin grafts in a novel in vivo experimental system using immunodeficient mice as recipients. Recombinase-activating gene-1-deficient mice (R-) lacking mature B and T cells were grafted with porcine skin and received human lymphocytes stimulated in vitro with irradiated porcine PBMC. Skin grafts on mice given either unseparated, activated human lymphocytes, or NK cell-depleted lymphocyte populations were rejected within 18 days after adoptive cell transfer. In contrast, skin grafts on mice given T cell-depleted human lymphocytes or saline showed no gross or histologic evidence of rejection up to 100 days after adoptive transfer. Purified CD4+ T cells were also able to mediate rejection of porcine skin grafts. These data suggest that human CD4+ T cells are sufficient to induce rejection of porcine xenografts. Thus, strategies directed toward CD4+ T cells may effectively prevent cellular rejection of porcine xenografts in humans.

Xenotransplantation--state of the art--update 1999.

Organ transplantation is limited by the number of cadaveric human donor organs that become available. Xenotransplantation - the transplantation of organs and tissues between animal species - would supply an unlimited number of organs and offer many other advantages. The pig has been identified as the most suitable donor animal. Pig organs, when transplanted into humans or nonhuman primates, are, however, rejected hyperacutely within minutes by antibody-mediated complement activation. Human anti-pig antibodies have been identified as being directed against galactose alpha 1-3galactose (alpha Gal) epitopes on pig vascular endothelium. Methods have been successfully developed to prevent hyperacute rejection. These include (i) depletion or inhibition of recipient antibodies or complement and (ii) development of transgenic pigs that express a human complement-regulatory protein (e.g. hDAF). The persistence or return of anti-pig antibody, however, even following the use of hDAF pig organs, eventually leads to what has been variously termed "acute vascular rejection" or "delayed xenograft rejection", which is again believed to be largely antibody-dependent. Nevertheless, experimental pig-to-primate organ xenotransplantation now results in transplant function for days and weeks rather than minutes. Little is yet known of the nature of the acute cellular rejection response that is anticipated to follow, and of any subsequent chronic rejection that may develop. Tolerance to both the alpha Gal epitope and to swine leukocyte antigens (SLA) is being explored using gene therapy techniques and by the induction of hematopoietic cell chimerism. The development of genetically engineered pigs that do not express the alpha Gal epitope is also being pursued. Considerable progress has been made in recent years, but experimental results do not yet warrant the initiation of a clinical trial of organ xenotransplantation. However, trials are already underway of pig cell transplants in patients with diabetes and neurodegenerative conditions, such as Parkinson's disease.

Inhibition of xenoreactive natural antibody production by retroviral gene therapy.

The major barrier to transplantation across discordant species, such as from pig to human, is rejection mediated by xenoreactive natural antibodies (XNA) that bind the carbohydrate epitope Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal) on donor tissues. This epitope is synthesized by the enzyme glucosyltransferase uridine 5'-diphosphate galactose:beta-D-galactosyl-1, 4-N-acetyl-D-glucosaminide alpha(1-3)galactosyltransferase (E.C. 2.4.1.151), or simply alphaGT. When a functional alphaGT gene was introduced by retroviral gene transfer into bone marrow cells, alphaGal XNA production in a murine model ceased. Thus, genetic engineering of bone marrow may overcome humoral rejection of discordant xenografts and may be useful for inducing B cell tolerance.

Tolerization of anti-Galalpha1-3Gal natural antibody-forming B cells by induction of mixed chimerism.

Xenotransplantation could overcome the severe shortage of allogeneic organs, a major factor limiting organ transplantation. Unfortunately, transplantation of organs from pigs, the most suitable potential donor species, results in hyperacute rejection in primate recipients, due to the presence of anti-Galalpha1-3Gal (Gal) natural antibodies (NAbs) in their sera. We evaluated the ability to tolerize anti-Gal NAb-producing B cells in alpha1,3-galactosyltransferase knockout (GalT KO) mice using bone marrow transplantation (BMT) from GalT+/+ wild-type (WT) mice. Lasting mixed chimerism was achieved in KO mice by cotransplantation of GalT KO and WT marrow after lethal irradiation. The levels of anti-Gal NAb in sera of mixed chimeras were reduced markedly 2 wk after BMT, and became undetectable at later time points. Immunization with Gal+/+ xenogeneic cells failed to stimulate anti-Gal antibody production in mixed chimeras, whereas the production of non-Gal-specific antixenoantigen antibodies was stimulated. An absence of anti-Gal-producing B cells was demonstrated by enzyme-linked immunospot assays in mixed KO + WT --> KO chimeras. Thus, mixed chimerism efficiently induces anti-Gal-specific B cell tolerance in addition to T cell tolerance, providing a single approach to overcoming both the humoral and the cellular immune barriers to discordant xenotransplantation.

Long-term expression of the gene encoding green fluorescent protein in murine hematopoietic cells using retroviral gene transfer.

BACKGROUND: A major goal in retroviral-based gene therapy is to establish methods that allow for selection and tracking of transduced cell populations. Green fluorescent protein (GFP) may be useful for gene therapy applications because it is a naturally fluorescent protein that can be detected using conventional flow cytometers facilitating rapid analysis and purification of transduced cell populations. However, it is unknown whether GFP can be stably expressed in vivo, particularly in multiple bone marrow-derived cell lineages. METHODS: A murine retrovirus carrying the gene encoding GFP was used to infect murine bone marrow cells (BMCs). These studies were conducted to (1) directly determine whether GFP could be used as a marker of BMC transduction, (2) determine whether GFP is capable of being expressed in multiple bone marrow-derived hematopoietic cell lineages, and (3) determine whether GFP could be used to follow the fate of transduced cells in vivo. RESULTS: Infection of BMCs with retroviruses carrying the gene encoding GFP resulted in a fluorescent signal in viable transduced cells that was detectable by flow cytometry. Expression of GFP was detected in multiple bone marrow-derived cell lineages after transduction, including stem cell antigen-positive (Sca-1+), lineage marker-negative (Lin-) cells. Using GFP as a selectable marker, we were able to enrich for transduced cells by cell sorting. Mice reconstituted with enriched populations of GFP+ cells showed a significant increase in the percentage of cells expressing GFP in the periphery when compared with mice reconstituted with unenriched transduced bone marrow. CONCLUSIONS: These data indicate that GFP can be used to select for transduced BMCs in vitro, expressed in multiple bone marrow-derived cell lineages, used to select transduced cells, and follow the fate of transduced cells long-term in vivo.

The mechanism of specific prolongation of class I-mismatched skin grafts induced by retroviral gene therapy.

In the present study, we examine the mechanism of specific hyporesponsiveness to major histocompatibility complex (MHC) class I-mismatched skin allografts induced by retrovirus-mediated gene transfer of an allogeneic class I gene into syngeneic bone marrow (BM). Using appropriate congenic recombinant mouse strains, we have mapped MHC determinants that are capable of restoring rapid rejection of Kb-bearing skin grafts. Our results indicate that either a single class I or a single class II alloantigen expressed on skin in association with Kb is able to restore the rapid rejection of Kb-mismatched skin grafts. These data suggest that third-party alloantigens expressed on skin in association with Kb abrogate hyporesponsiveness by providing T cell help. Consistent with this interpretation, spleen cells from mice reconstituted with Kb-transduced BM were unable to elicit a significant anti-Kb cytotoxic T lymphocyte response in vitro unless interleukin-2 was added to the culture medium. Skin graft survival was also analyzed on B10. AKM mice thymectomized 3-4 weeks post-reconstitution with Kb-transduced BM. Thymectomy did not result in significantly prolonged survival of B10. MBR skin grafts compared to euthymic controls, suggesting that even early after reconstitution, intrathymic deletion of Kb-reactive T cells must have been incomplete. Taken together, these data suggest that prolongation of skin allograft survival in this model is controlled at the level of T cell help.

Adult bone marrow contains precursors for CD5+ B cells.

To determine directly whether B cell precursors of adult origin are capable of generating CD5+ B cells, we reconstituted neonatal C3H. SCID mice with adult C57BL/6 bone marrow and analyzed splenic B cells 10 months later. Surface staining and flow cytometry revealed that the B cells were of donor origin and that 30% were CD5+. This confirms that in vivo generated CD5+ B cells can be adult derived. After anti-IgM (but not lipopolysaccharide) stimulation in vitro, virtually all of the B cells from the bone marrow-reconstituted mice expressed surface CD5. Sequence analysis of expressed VHDJH genes from the CD5+ B cells present after anti-IgM stimulation revealed a high frequency of N nucleotide addition in CDR3 regions. The presence of N nucleotides indicates that these sequences were derived from CD5+ B cells of adult origin rather than from long-lived fetal precursor B cells present in either the adult bone marrow at the time of transfer or adult spleen. These experiments demonstrate conclusively that adult bone marrow contains precursors for CD5+ B cells and that unlike fetal liver-derived precursors these express terminal deoxynucleotidyl transferase.