Lihui Qin

Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immunosuppression with TGF-beta 1 or vIL-10.

The application of gene transfer techniques to organ transplantation offers the potential for modulation of immunity directly within an allograft without systemic side effects. Expression vectors and promoter elements are important determinants of gene transfer and expression. In this study, various vectors (naked plasmid DNA, retroviral vector, herpes simplex viral vector, and adenoviral vector) with various promoters (RSV-LTR, SV40, MuLV-LTR, HCMVie1) were directly compared to demonstrate the successful gene transfer and expression of beta-galactosidase in murine myoblasts in vitro and within murine heterotopic, nonvascularized cardiac isografts or allografts in vivo. Expression of transferred genes was not toxic to cells and strength of expression varied according to the type of vector. Plasmid DNA was expressed in myocytes, retroviral vector was expressed in the graft infiltrating cells, and herpes simplex and adenoviral vectors were expressed in both myocytes and graft-infiltrating cells. Preliminary studies evaluated the ability of these vectors to deliver immunologically important signals. Allografts injected with pSVTGF-beta 1, a plasmid-encoding transforming growth factor beta 1 (TGF-beta 1) under the control of the SV40 promoter, showed significant prolongation of graft survival of 26.3 +/- 2.5 days compared with 12.6 +/- 1.1 days for untreated allografts, and 12.5 +/- 1.5 days for the allografts injected with control plasmid (P < 0.05). Allografts injected with MFG-vIL-10, a retroviral vector encoding viral interleukin-10 under the control of the MuLV-LTR, showed prolongation of graft survival of 36.7 +/- 1.3 days versus 12.6 +/- 1.1 days for the untreated allograft, and 13.5 +/- 2.0 days for the allografts injected with control retroviral vector (P < 0.001). Both vectors were transcriptionally active in vivo and did not appear to have toxic effects. Gene therapy for transplantation can induce transient expression of immunologically relevant molecules within allografts that impede immune activation while avoiding the systemic toxicity of conventional immunosuppression.

Suppressor of Cytokine Signaling 1 Inhibits IL-10-Mediated Immune Responses

IL-10 has proved to be a key cytokine in regulating inflammatory responses by controlling the production and function of various other cytokines. The suppressor of cytokine signaling (SOCS) gene products are a family of cytoplasmic molecules that are essential mediators for negatively regulating cytokine signaling. It has been previously shown that IL-10 induced SOCS3 expression and that forced constitutive expression of SOCS3 inhibits IL-10/STAT3 activation and LPS-induced macrophage activation. In this report, we show that, in addition to SOCS3 expression, IL-10 induces SOCS1 up-regulation in all cell lines tested, including Ba/F3 pro-B cells, MC/9 mast cells, M1 leukemia cells, U3A human fibroblasts, and primary mouse CD4+ T cells. Induction of SOCS molecules is dependent on STAT3 activation by IL-10R1. Cell lines constitutively overexpressing SOCS proteins demonstrated that SOCS1 and SOCS3, but not SOCS2, are able to partially inhibit IL-10-mediated STAT3 activation and proliferative responses. Pretreatment of M1 cells with IFN-γ resulted in SOCS1 induction and a reduction of IL-10-mediated STAT3 activation and cell growth inhibition. IL-10-induced SOCS is associated with the inhibition of IFN-γ signaling in various cell types, and this inhibition is independent of C-terminal serine residues of the IL-10R, previously shown to be required for other anti-inflammatory responses. Thus, the present results show that both SOCS1 and SOCS3 are induced by IL-10 and may be important inhibitors of both IL-10 and IFN-γ signaling. IL-10-induced SOCS1 may directly inhibit IL-10 IFN-γ signaling, while inhibition of other proinflammatory cytokine responses may use additional IL-10R1-mediated mechanisms.

CTLA4Ig prolongs allograft survival while suppressing cell-mediated immunity.

T cell activation is the result of antigen-specific interactions with the TCR/CD3 complex and costimulation via other T cell surface receptors. Prevention of costimulation can result in clonal anergy. CTLA4Ig is a fusion protein that binds with high-affinity to the B7/BB1 ligand and blocks the interaction of this ligand with CD28 and CTLA4. We explored the immunosuppressive effects of CTLA4Ig in a murine nonvascularized heterotopic cardiac transplant model and in a model of cell mediated immunity. CTLA4Ig administered in vivo for two days at the time of transplantation resulted in significant prolongation of allograft survival (55 +/- 2.0 vs. 12.2 +/- 0.5 days for control, P < 0.03). Administration at later times or to previously primed animals produced no prolongation of graft survival. CTLA4Ig administered during in vivo immunization to the hapten TNP suppressed the contact sensitivity response and inhibited the subsequent in vitro generation of secondary TNP-specific CTL. CTLA4Ig administered in vivo had no effect on subsequent primary alloantigen-specific CTL or MLR responses--however, when added to culture the fusion protein inhibited the MLR response by 80%, but not the alloantigen-specific CTL response. CTLA4Ig inhibited CD4+ and CD8+ proliferative and cytokine responses to alloantigen. Flow cytometry showed no changes in distribution of subpopulations of T cells. These results confirm the immunosuppressive activity of CTLA4Ig in vivo in an allograft model and show that both CD4+ and CD8+ T cells are suppressed by CTLA4Ig. The most efficacious time of administration is during priming of the immune response at the time of antigen presentation.

Lipid-mediated gene transfer of viral IL-10 prolongs vascularized cardiac allograft survival by inhibiting donor-specific cellular and humoral immune responses.

The gene encoding the immunosuppressive cytokine viral interleukin-10 (vIL-10) was introduced into BALB/c (H-2d) vascularized cardiac allografts by perfusing the graft vasculature with DNA–liposome complexes, utilizing the experimental cationic lipid γAP DLRIE/DOPE and a plasmid encoding vIL-10 under the control of the HCMVie promoter. The DNA to lipid ratio and DNA dose were critical factors in obtaining optimal biologic effects. Gene transfer of vIL-10 with a 3:1 DNA to lipid weight ratio using 375 μg DNA significantly prolonged allograft survival in MHC-mismatched C57BL/6 (H-2b) recipients (16.00 days) compared with both unmodified allografts (8.14 days) and vIL-10 antisense controls (8.28 days). Enhanced graft survival was specific to vIL-10 expression since treatment with antisense plasmid or anti-vIL-10 monoclonal antibody (mAb) abrogated the effect. Prolonged survival was associated with a novel histology characterized by a moderate mono- nuclear infiltrate, edema, and diffuse fibrillar/collagen deposition in the interstitium. Despite these morphologic changes, myocytes remained viable and vessels were patent. Limiting dilution analysis revealed transient infiltration of IL-2 secreting, donor-reactive, helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) in vIL-10 expressing grafts on day 7, that decreased significantly by day 14. Similarly, vIL-10 gene transfer inhibited the accumulation of donor-specific HTL and CTL in the spleen, compared with antisense controls. Prolonged survival was also associated with a marked decrease in IgM and IgG alloantibody production, with little to no IgG isotype switching. These results show that viral IL-10 gene transfer inhibits graft rejection in a clinically relevant model by inhibiting donor-specific cellular and humoral immune responses.

Differential IL-10R1 Expression Plays a Critical Role in IL-10-Mediated Immune Regulation

In this study, we characterized the differential receptor-binding specificity, affinity, and Janus kinase-STAT activation of cellular IL-10 (cIL-10) compared with viral IL-10 (vIL-10). Only cells expressing IL-10R1 bind human IL-10 or vIL-10. IL-10R2 does not bind to cIL-10 or vIL-10 alone and its presence does not enhance the receptor-binding affinity of cIL-10 or vIL-10, but it is essential for both cIL-10- and vIL-10-mediated signal transduction and immune regulation. Responses initiated by cIL-10 and vIL-10 were compared in B cell and mast cell lines, and demonstrated that the inability of vIL-10 to stimulate immune responses, as compared with human IL-10, is due to failure to initiate signaling. Absent signal transduction is due to low level expression of cell surface IL-10R1, since overexpressing IL-10R1 allows vIL-10 to initiate cIL-10-like signals and subsequent biological responses. These results are similar in primary cells, since splenocytes respond to both cIL-10 and vIL-10, while thymocytes respond only to cIL-10 and have very low mouse IL-10R1 but not mouse IL-10R2 expression. These data demonstrate that IL-10R1 expression plays a critical role in determining whether cells respond to IL-10. Modulation of cell surface IL-10R1 density might be an important mechanism for determining whether IL-10 leads to immunostimulation or immunosuppression in vivo.

Gene Transfer for Transplantation Prolongation of Allograft Survival with Transforming Growth Factor-β1

ObjectiveThe authors tested the ability of plasmid gene transfer to express transforming growth factor-β1 (TGF-β1), prolong allograft survival, and evaluate promoter effects on gene expression. Summary Background DataDelivery of immunosuppressants directly to allografts using gene transfer and gene therapy approaches may inhibit immune activation while avoiding the systemic toxicity of conventional immunosuppression. Candidate genes include soluble cytokines, which could be expressed at low levels throughout the graft while inducing a local immunosuppressive effect. Transforming growth factor-β1 is a soluble cytokine that has pleiotropic immunosuppressive effects. MethodsCardiac grafts from syngeneic (CBA/J, H-2k) or allogeneic (C57BL/6, H-2b) donors were placed into CBA/J recipients. Purified plasmid DNA-encoding murine TGF-β1 or β-galactosidase (Lac Z) under the control of RSV, SV40, MMTV, or pancreatic elastase promoters was injected into grafts at surgery. The Lac Z expression was determined by histologic examination and TGF-β1 expression by graft survival. Cytotoxic T lymphocyte and flow cytometric analyses were performed to evaluate the immunosuppressive effects of TGF-β1 in vitro. ResultsPlasmid DNA-encoding TGF-β1 prolonged survival from 12.6 ± 1.1 days to 26.3 ± 2.5 days (p < 0.02, Students t test). The SV40 promoter was superior to the MMTV promoter in its ability to prolong survival. The effects of the plasmids were specific because Lac Z, antisense TGF-β1 inserts, or pancreatic elastase promoter did not prolong allograft survival. Histologic examination demonstrated Lac Z expression at least 14 days post-transplant in myocardial cells. Both RSV and SV40 promoters were effective in this respect, while a control null promoter was not. Toxicity testing showed that gene transfer of TGF-β1 did not alter survival or histology of syngeneic grafts. In addition, plasmids and purified TGF-β1 protein were not toxic to myoblasts in vitro. Recombinant TGF-β1 inhibited cytotoxic T lymphocyte generation and altered T cell surface receptor expression and subset expansion in vitro. ConclusionGene transfer/therapy with plasmid DNA encoding TGF-β1 in vivo achieves immunologic effects that prolong allograft survival. Multiple promoters effectively induce plasmid expression, which is achieved in cardiac myocytes for at least 2 weeks without toxicity or adverse systemic effects.

L-Selectin-Dependent Lymphoid Occupancy Is Required to Induce Alloantigen-Specific Tolerance

Maneuvers that interfere with signals 1, 2, 3, or Ag processing can result in indefinite allograft survival. However, they are not applicable to all tissues, strains, or species, suggesting that there are additional levels of immune regulation. We hypothesized that secondary lymphoid organs are important for interactions among lymphocytes, alloantigen, and immunosuppressants that lead to tolerance. To explore this, cardiac allografts were performed with a tolerogenic immunosuppressive regimen. Concurrent administration of anti-L-selectin (CD62L) Ab, which prevents lymph node homing, prevents indefinite allograft survival and tolerance. Anti-CD62L Ab is not costimulatory, and Fab and F(ab′)2 anti-CD62L have similar activities. Flow cytometry and histologic examination show that Ab shifts T cells away from lymph nodes and into spleen, peripheral blood, and graft. Tolerance is not induced in CD62L−/− mice, and adoptive transfer of CD62L−/−, but not CD62L+/+, T cells prevents tolerization in wild-type recipients. FTY720, an immunosuppressant that promotes chemokine-dependent, but CD62L-independent, lymph node homing, reverses the Ab effect. Blockade of other homing receptors also prevents tolerization. These results indicate that T lymphocytes use CD62L-dependent migration for alloantigen-specific tolerance, and suggest that lymph nodes or other lymphoid tissues are an important site for peripheral tolerization to alloantigen.

Blockade of multiple costimulatory receptors induces hyporesponsiveness: inhibition of CD2 plus CD28 pathways.

T-lymphocyte activation requires engagement of the T cell receptor with antigen-major histocompatibility complex, and simultaneous ligation of costimulatory pathways via the lymphocyte receptors CD2 and CD28/ CTLA4. Anti-CD2 monoclonal antibody (mAb) blocks the interaction of the antigen-presenting cell receptor CD48 with its ligand CD2, whereas CTLA4Ig binds with high affinity to the antigen-presenting cell ligands B7-1 and B7-2, blocking their interaction with CD28/CTLA4. We tested the immunosuppressive effects of simultaneously blocking both costimulatory pathways. Using donor C57BL/6J (H2b) hearts transplanted to CBA/J (H2k) recipients, anti-CD2 mAb plus CTLA4Ig administered at the time of transplantation prolonged cardiac allograft mean survival time to >120 days compared with untreated controls (12.2+/-0.5 days, P<0.01), anti-CD2 mAb alone (24.8+/-1.0 days, P<0.01), or CTLA4Ig alone (55.0+/-2.0 days, P<0.01). Retransplantation of these recipients with donor-specific and third-party grafts demonstrated that hyporesponsiveness and tolerance were achieved. In vitro stimulation of lymphocytes from tolerant recipients with donor-specific alloantigen resulted in normal cytotoxic T lymphocyte and mixed lymphocyte reaction responses, showing that clonal deletion or anergy did not occur, but that graft adaptation or suppression likely helped to maintain long-term graft survival. In vitro combinations of anti-CD2 mAb and CTLA4Ig suppressed the generation of allogeneic cytotoxic T lymphocytes (58%) and the mixed lymphocyte reaction (36%); CTLA4Ig was more effective in this regard and the two agents were not synergistic. Anti-CD2 mAb and CTLA4Ig suppressed mitogen-driven proliferation in differential fashions, suggesting that they affected independent signaling pathways. Anti-CD2 mAb and CTLA4Ig also inhibited interleukin (IL)-2, IL-4, and IL-2 receptor (CD25). These data indicate that anti-CD2 mAb plus CTLA4Ig induces hyporesponsiveness and tolerance. The mechanism is likely related to the initial disruption of independent pathways of T-lymphocyte activation leading to antigen-specific long-term graft survival.

Combination of Electroporation and DNA/Dendrimer Complexes Enhances Gene Transfer into Murine Cardiac Transplants

Electroporation is a new gene delivery method to increase gene transfer and expression in vivo. Starburst polyamidoamine dendrimers have been demonstrated to augment gene expression in vitro and in vivo. We hypothesized that the combination of electroporation and dendrimer could enhance the gene transfer and gene expression in cardiac transplants. After immersion in DNA/dendrimer complexes or intracoronary transfer of DNA/dendrimer complexes, both nonvascularized and vascularized syngeneic cardiac grafts, respectively, were subjected to serial electrical pulses before transplantation. β‐Galactosidase reporter gene expression in the graft was determined by X‐Gal staining. Gene expression was enhanced 10‐ to 45‐fold in grafts immersed in DNA/dendrimer complexes, or after intracoronary transfer of DNA/dendrimer complexes, and subjected to 20 square wave 25‐ms pulses with a strength of 200 V/cm. The combination of electroporation and DNA/dendrimer complexes may provide a novel approach to enhance gene transfer and gene expression ex vivo.

Anti-CD2 and anti-CD3 monoclonal antibodies synergize to prolong allograft survival with decreased side effects.

Anti-CD3 monoclonal antibody suppresses immunity and prolongs allograft survival; however, it induces T cell activation and overproduction of soluble factors that result in a deleterious cytokine syndrome. Anti-CD2 mAb also prolongs allograft survival, by suppression of mature and precursor CD4 and CD8 T cells and NK cells, without an associated cytokine release. Because of the close physical and functional association of CD2 and CD3 on the T cell surface, we tested whether alpha CD2 mAb in combination with alpha CD3 mAb could act synergistically to prolong allograft survival, and whether the combination would affect the alpha CD3-associated cytokine syndrome. C57BL/6 (H-2b) hearts were transplanted to CBA (H-2k) recipients in a heterotopic nonvascularized model. Recipients received alpha CD2 (12-15) or alpha CD3 (145-2C11) mAb i.v. alone or in combination. Lymphocytes from treated animals were also analyzed by fluorescent flow cytometry and stimulated in vitro and assessed for proliferation and lymphokine production. Anti-CD2 and alpha CD3 each prolong allograft survival (mean survival time 22.4 +/- 1.0 and 27.4 +/- 3.3 days, respectively vs. 14.0 +/- 0.6 for control mAb, P < 0.001 for both vs. control). Combinations of mAbs show a more complicated interaction. Very low doses (1 microgram) of alpha CD2 and alpha CD3, which have no effect when given alone, are synergistic (16.5 +/- 1.3 days, P < 0.02). A high dose of alpha CD2 (100 micrograms), which is immunosuppressive, is additive with a moderate dose of alpha CD3 (10 micrograms), which is immunostimulatory. The two mAbs are again synergistic when a high dose of alpha CD2 (100 micrograms) is combined with a high dose of alpha CD3 (1 mg) (> 51.5 +/- 23.0 days, P < 0.001). Furthermore, high-dose alpha CD2 administered 48 h prior to high-dose alpha CD3 was a more effective combination for prolonging allograft survival than both antibodies administered simultaneously (67.1 +/- 10 vs. 35.8 +/- 0.7 days, P < 0.05). Anti-CD2 also diminishes the alpha CD3-associated cytokine syndrome, and prior in vivo treatment with alpha CD2 decreases the subsequent in vitro proliferative response to alpha CD3 and the alpha CD3-stimulated production of IL-2 and IL-4. Flow cytometry demonstrates that in general these mAbs do not deplete but leave T cell populations intact with altered receptor expression. These results show that the combination of alpha CD2 and alpha CD3 mAbs prolongs cardiac allograft survival in a synergistic fashion while decreasing the side effects of alpha CD3 mAb alone.(ABSTRACT TRUNCATED AT 400 WORDS)