AU4169800A

AU4169800A - Methods for improving graft acceptance in a recipient by administration of a cytokine profile altering agent

Info

Publication number: AU4169800A
Application number: AU41698/00A
Authority: AU
Inventors: Albert Edge
Original assignee: Diacrin Inc
Current assignee: Diacrin Inc
Priority date: 1999-03-05
Filing date: 2000-03-02
Publication date: 2000-09-21
Also published as: WO2000051630A3; WO2000051630A2; EP1158999A2; CA2364279A1

Description

WO 00/51630 PCT/USOO/05647 METHODS FOR IMPROVING GRAFT ACCEPTANCE IN A RECIPIENT BY ADMINISTRATION OF A CYTOKINE PROFILE ALTERING AGENT Background of the Invention 5 The process of T cell maturation in the thymus is critical to the development of central immune tolerance to most self antigens. While autoreactive T cells are eliminated by clonal deletion through negative selection at the CD4+/CD8+ "double positive" T cell stage (Kappler et al. 1987. Cell. 49:273), not all self antigens are seen in the thymus. Peripheral mechanisms of tolerance have been described that are thought to 1o contribute to preventing autoimmunity to peripheral antigens (Miller, et al. 1994. Curr Opin Immunol. 6:892). Once a CD4+ or CD8+ T cell has reached the periphery, it can be inactivated by several mechanisms including deletion by apoptosis and inactivation by anergy. The ability of the immune system to develop tolerance to self peripheral antigens provides a 15 possible mechanism by which to promote acceptance of transplanted tissues (Dallman, et al. 1993. Immunol Rev. 133:18). For instance, cells that express Fas ligand have been found to be protected against activated T cells by inducing apoptosis and cell death (Griffith, et al., 1995. Science. 270:1189; Griffith, et al., Immunity, 1996. 5:7; French, et al., 1996. J. Cell Biol. 133:335). In fact, several tissues that express Fas ligand have 20 been identified as "immune privileged" and tissue from these organs has been successfully transplanted. Based on this finding, cells have been made to express heterologous Fas ligand by transfection or by making transgenic animals. These Fas ligand-expressing cells have been demonstrated to have increased survival upon transplantation. (Fandrich et al. 1998. Transplant. Proc. 30:2360) 25 Another way peripheral tolerance has been induced has been by blocking either of the two crucial interactions between a T cell and an antigen presenting cell. Two types of interactions between a T cell and APC are required for a naive T cell to become activated and develop effector function. The T cell receptor must first recognize and bind (signal one) to the antigen peptide-MHC molecule complex on the APC (Schwartz,. 30 1992. Cell, 71:1065; Lenschow, D.J. 1993. Curr. Opin. Immunology, 5:747). The second interaction (signal two) is required between the CD28 molecule on T cells and WO 00/51630 PCTIUSOO/05647 -2 the newly expressed costimulatory B7.1 or B7.2 molecules on the APC (Schwartz, R.H. 1992, Cell, 71:1065; Lenschow, D.J. 1993. Curr. Opin. Immunology, 5:747; Bluestone, J.A. 1995. Immunity, 2:555-559). In the absence of either signal one or signal two, responding T cells become anergized. Blocking either T cell receptor s signaling (e.g., using anti-CD3 antibody) or costimulatory signals (e.g., using a soluble form of a costimulatory molecule such as CTLA4Ig) have been shown to promote graft acceptance (Schaub et al. 1998. J. Am Soc. Nephrol. 9:891). Inducing tolerance to xenotransplants presents a particular set of problems. The mechanisms of xenograft rejection appear to differ from those associated with io alloreativity (Auchincloss, H. 1988. Transplantation. 46:1; Hardy, MA. 1982. Transplantation. 33:237; Homan et al. 1981. Transplantation 31:164; Croy et al. 1985. Transplantation. 37:84; Newlands, E.S. 1975. Transplantation. 20:13; Terasaka etl al. 1981. Immunology 36:699; Widmer et al. 1976. J. Exp. Med. 144:305; Peck et al. 1976. Transplant Review. 29:189). Xenograft rejection is thought to involve primarily is humoral immunity. The humoral immune response to a xenograft can develop in several ways, both of which ultimately lead to rejection of the transplant. Depending upon the species combination of the donor and the recipient, the recipient may have preexisting anti-donor antibodies that fix complement and lead to hyperacute rejection. In cases where there is no preexisting antibody, the xenograft recipient generates anti-graft 20 antibodies over several days post transplantation that lead to rejection. (Bach. 1997. Nature Medicine. 3:196). When hyperacute rejection is prevented, cellular immune responses have also been shown to lead to xenograft rejection (Fryer et al. 1994. Transplantation Immunology 2:87). The cellular response to xenografts can develop in several ways. 25 Recognition of an antigen from a foreign species can involve direct recognition by the T cell of the foreign MHC-peptide complexes on the graft or indirect recognition of the foreign antigens as processed peptides in association with recipient antigen presenting cells (APC) (Auchincloss, H.,Transplant. Reviews, 1990. 4:1; Auchincloss, H., et al., Proc. Natl. Acad. Sci., 1993. 90: p. 3373). In order for direct recognition of xenogeneic 3 o antigens to occur, the match between the xenogeneic MHC and the self MHC must be sufficient to allow direct recognition of the target cells by the recipient T cell receptor WO 00/51630 PCT/USOO/05647 -3 (Auchincloss, H. 1990. Reviews. 4:14; Moses, R.D. et al. 1990. . Exp. Med. 172:567). Regardless of the mechanism by which cellular immunity develops to a xenotransplant, cellular infiltrates have been identified in xenografts that survive antibody-mediated rejection. s The profile of cytokines that are produced during an immune response largely determines whether a cellular or humoral immune response develops. For example, T lymphocytes produce an array of cytokines. These cytokines are not produced constitutively by T cells, but are induced following receptor-mediated T cell activation. Given the lack of availability of human organs for transplantation, methods for io reducing the immune response of a transplanted subject which leads to rejection of donor tissue would be of great benefit. Promoting acceptance of an allograft or a xenograft without affecting immune responses to other, unrelated antigens would be highly desirable and would represent a major advance over the use of general immunosuppressants. 15 Summary of the Invention The instant invention is based, at least in part, on the discovery that biasing the cytokine response of a graft recipient to one dominated by Th2 cytokines promotes graft acceptance. 20 Applicants demonstrate that biasing the cytokine secretion profile of human responder T cells towards a Th2 phenotype leads to an inhibition of the human anti porcine immune response, thus providing a method for achieving acceptance of xenografts. This enhanced Th2 response leads to long-term graft acceptance even though previous work has demonstrated that the presence of Th2 cytokines correlates 25 with rejection of xenografts (Morris et al. 1995. J Immunol. 154:2470; Wren et al. 1993. Transplantation. 56:905; and Medbury. 1997. Transplantation. 64:1307). This discovery provides methods for promoting the acceptance of grafts. Accordingly, in one aspect the invention provides methods for promoting xenograft acceptance in a subject by administering an isolated xenogeneic cell and a 30 cytokine profile altering agent to the subject in order to promote acceptance of the xenogeneic cell in the subject.

WO 00/51630 PCT/USOO/05647 -4 In one embodiment, the cytokine profile altering agent is a cytokine selected from the group consisting of IL-4 and IL- 10. In another embodiment, the cytokine profile altering agent is a cytokine fusion protein selected from the group consisting of IL-41g and IL- 10Ig. In another embodiment, the cytokine profile altering agent is an 5 antibody. In a preferred embodiment, the antibody is selected from the group consisting of: an antibody which binds to IL-2, IFN-gamma, and IL-12. In one embodiment, the cell is a xenogeneic cell, e.g., a porcine cell. In one embodiment, the cell is selected from the group consisting of: a fetal cell, a stem cell, and a progenitor cell. In another embodiment, the xenogeneic cell is obtained from a pig 1o which is predetermined to be free from at least one organism selected from the group consisting of: zoonotic, cross-placental and organotropic organisms. In one embodiment, the cell is selected from the group consisting of a pancreatic islet cell, a kidney cell, a cardiac cell, a muscle cell, a liver cell, a lung cell, an endothelial cell, a central nervous system cell, a peripheral nervous system cell, an 15 epithelial cell, an eye cell, a skin cell, an ear cell, and a hair follicle cell. In another aspect, the invention pertains to a method for promoting xenograft acceptance by i) modifying a xenogeneic cell to express an exogenous polypeptide comprising a cytokine profile altering agent (e.g., by introducing a heterologous gene encoding a cytokine such as 11-4 or IL- 10) to produce a modified xenogeneic cell; ii) 20 administering the modified xenogeneic cell to a subject in order to improve xenograft acceptance in the subject. In one embodiment, the method further includes administering xenogeneic lymphoid cells to the subject. In a preferred embodiment, the xenogeneic lymphoid cells are administered intravenously. In one embodiment, a lymphoid cell is administered 25 into the portal vasculature of the subject. In one embodiment, a xenogeneic cell is administered intravenously. In another embodiment, the method further comprises administering an immunosuppressive agent to the subject. In a preferred embodiment an immunosuppressive agent is selected from the group consisting of methylprednisolone, 30 cyclosporin A, and FK506.

WO 00/51630 PCT/US00/05647 -5 In another embodiment, the method further comprises introducing a soluble form of a costimulatory molecule to the subject. In another aspect, the invention pertains to a method for promoting acceptance of a xenogeneic cell or tissue in a human subject comprising: i) administering an isolated s xenogeneic cell to the subject wherein the cell or tissue bears a surface antigen capable of causing an immune response against the cell or tissue in the subject, said antigen being modified, masked, or eliminated to decrease the immune response; and ii) administering a cytokine profile altering agent to the subject such that acceptance of the xenogeneic cell is promoted in the subject. 10 In one embodiment a surface antigen on the transplanted cell is an MHC class I antigen or an MHC class II antigen. In one embodiment, the masking agent is a non lytic anti-MHC class I antibody fragment or an anti-MHC class II antibody or fragment thereof. In one embodiment, the anti-MHC class I antibody fragment is an anti-MHC class I F(ab')2 fragment. 15 In another aspect, the invention pertains to a method for promoting Th2 cytokine production in a subject comprising: i) administering a xenogeneic cell or tissue to the subject wherein the cell or tissue bears a surface antigen capable of causing an immune response against the cell or tissue in the subject, said antigen being modified, masked, or eliminated to decrease the immune response, such that upon introduction of the 20 composition into the subject lysis of the cell or tissue is prevented; and ii) determining that Th2 cytokine production is promoted in the subject. Brief Description of the Figures Figure 1A shows that PT85 inhibits primary human anti-porcine responses. 25 Human peripheral blood mononuclear cells (PBMC) were incubated with MHC-class I+/Il- porcine smooth muscle cells (SMC) or embryonic brain cells (EBC) for 7 days in the presence or absence of antibody (Ab), and cells were pulsed with 3 H-thymidine for last 20 hours of incubation. The anti-human MHC class I reactive W6/32 mAb is not reactive with pig MHC class I. F(ab')2 fragment of normal mouse serum (mIg) was used 3o as control. The graph represents the mean and standard deviation (SD) of triplicate wells.

WO 00/51630 PCT/USOO/05647 -6 Figure lB shows the inhibition of secondary human anti-porcine response. Cells from day 7 primary stimulation were re-stimulated with porcine SMC or EBC for 3 days, and cells were pulsed with 3 H-thymidine for the last 20 hours of incubation. No antibody was present in the secondary stimulation. 5 Figure 2A shows that PT-85 inhibits primary human anti-porcine responses. Tissue cultures were setup as described in Figure legend 1A. Thymidine incorporation was converted to percentage of the no antibody control values. Responder cells alone gave less than 10% of the full response. Stimulatory cells alone was less than 1% of the 1o full response. The graph represents a summary of 12 experiments with PBMC from 8 individuals. Figure 2B shows inhibition of secondary human anti-porcine responses. Tissue cultures were setup as described in figure legend 1B. Thymidine incorporation was 15 converted to percentage of a control (the no Ab group for SMC or mIgG group for EBC). Data represent a summary of 8 experiments with PBMC from 6 individuals for SMC as stimulators, and 2 experiments with PBMC from 2 individuals for EBC as stimulators. 20 Figure 3 shows inhibition of CD8+ T cells in primary human anti-porcine response. Human PBMC, CD8+ T cells or CD4+ T cells were incubated with porcine SMC for 7 days in the presence of absence of antibody (Ab), and cells were pulsed with 3 H-thymidine for the last 8 hours of incubation. 9-3 and 74-11-10 Ab are reactive with porcine MHC class I (see Table 2). 10.14 is reactive with porcine CD44. 25 Figure 4 shows that PT-85 inhibits IL-2 in the primary PBMC response against porcine SMC. Tissue cultures were set up as Figure 1A. Supernatants were harvested on the days indicated and used for ELISA. The results show IL-2 levels in the culture supernatants using PBMC from 4 individuals. 30 WO 00/51630 PCT/USOO/05647 -7 Figure 5 shows that PT-85 inhibits IFN-gamma in primary PBMC responses against porcine SMC. Tissue cultures were set up as described for Figure 1A. Supernatants were harvested on days as indicted and used for ELISA. The results show IFN-gamma levels in the culture supernatants using PBMC from 4 individuals. 5 Figure 6 shows sustained IL- 10 production in primary PBMC responses against porcine SMC. Tissue cultures were set up as Figure IA. Supernatants were harvested on days as indicted and used for ELISA. The results show IL- 10 levels in the culture supernatants using PBMC from 4 individuals. 10 Figure 7 shows inhibition of IFN-gamma during secondary response against porcine SMC. Tissue cultures were set up as Figure 1B. Supernatants were harvested at 48 hours and used for ELISA. The results show IFN-gamma levels in the culture supernatants using PBMC from 4 individuals. 15 Figure 8 shows enhanced IL- 10 production during secondary anti-porcine SMC response. Tissue cultures were set up as described in Figure lB. Supernatants were harvested at 48 hours and used for ELISA. The results show IFN-gamma levels in the culture supernatants using PBMC from 4 individuals. 20 Figure 9 shows enhanced IL- 10 production during secondary stimulation with anti-CD3 or anti-CD3/CD28 mABs. Cells from day 7 primary stimulation were re stimulated with immobilized anti-CD3 in the presence or absence of anti-CD28. Culture supernatants were harvested at 48 hours and used for ELISA. The results show IL-10 25 levels in culture supernatants using PBMC from 4 individuals. Figure 10 shows enhanced IL-4 production during secondary stimulation with anti-CD28 mABs. Cells from day 7 primary stimulation were re-stimulated with immobilized anti-CD3 in the presence or absence of anti-CD28. Culture supernatants 30 were harvested at 48 hours and used for ELISA. The results show IL-4 levels in culture supernatants using PBMC from 4 individuals.

WO 00/51630 PCTIUSOO/05647 Detailed Description of the Invention The instant invention is based, at least in part, on the discovery that biasing the cytokine response of a subject to a transplanted cell to one dominated by Th2 cytokines inhibits the immune response to the cell and improves graft acceptance. 5 As used herein, the following terms and phrases shall be defined as follows: As used herein the term "cell" includes an allogeneic cell or a xenogeneic cell. As used herein the phrase "xenogeneic cell" includes any cell which is derived from a different species from the recipient of the cell. As used herein the term "isolated" refers to a cell which has been separated from io its natural environment, e.g., which has been removed from the donor and which is no longer part of an intact organ. Isolated cells of the invention are cells which have been separated from the vascular tissue of the organ from which they originated. For example, the isolated cell can be in the form of a piece of tissue, e.g., an intact sheet of cells, e.g., a monolayer of cells. The term "intact sheet", as used herein, refers to a layer is of cells which remain adherent to one another after the cells are harvested. In a preferred embodiment, the cell is dissociated from the neighboring cells with which it was in contact in the donor. The isolated cells of the invention can be administered in the form of a cell suspension. More than one of the isolated cells of the invention constitute a population of 20 cells. The term population also includes cells which result from the proliferation of the isolated cells of the invention. A population of cells can be obtained from the same or different source(s), e.g., can originate from different types of donor tissue and can, therefore, comprise different cell types. The isolated cells can also be derived from different donor animals, e.g., the isolated cells can represent a pooled population of cells 25 derived from a number of different animals. The phrase "T helper cells" (Th cells) as used herein includes the meaning that this term is ordinarily given in the art. Prototypical Th cells bear the CD4 cell surface marker and recognize antigen in the context of MHC class II molecules. The phrase "cytotoxic T cells" (TC cells) as used herein includes the meaning that this term is 30 ordinarily given in the art. Prototypical TC cells bear the CD8 cell surface marker and recognize antigen in the context of MHC class I molecules.

WO 00/51630 PCTIUSOO/05647 -9 The term "Thl" refers to T helper cells of the type 1 phenotype which primarily secrete Th1 cytokines. As used herein the phrase "Th1 cytokine" refers to a cytokine preferentially produced by a prototypical T helper 1 type cell. "Th1 cytokines" are cytokines which primarily promote cellular immune responses, e.g., delayed type 5 hypersensitivity and cellular cytotoxicity. Th1 cytokines include, for example IL-2, IFN-gamma and/or lymphotoxin. In addition, the term Th1 cytokines as used herein includes those cytokines which may not be made by ThI cells, but which enhance the development of Th1 type cells and, thus, amplify ThI cytokine secretion. Thl -type cytokines need not be exclusively produced by Th1 cells. For example, IL-12 is made io primarily by monocytes and macrophages and has been found to drive T cell responses to the Th1 phenotype (e.g., U.S. patent 5,853,697). The term "Th2" refers to T helper cells of the type 2 phenotype which primarily secrete Th2 cytokines. As used herein the phrase "Th2 cytokine" refers to a cytokine which is preferentially produced by a prototypical T helper 2 type cell. Th2-type is cytokines may not be exclusively produced by Th2 cells. "Th2 cytokines" are cytokines which primarily promote B cell differentiation and/or activation and humoral immune responses. Th2 cytokines include, for example IL-4, IL-5, IL-6, IL-10, and/or IL-13. Th1 and Th2-type cytokines may be produced by cell types other than T helper cells, e.g., by monocytes, macrophages, nonB-nonT cells, cytotoxic T cells or by natural 20 killer cells. For example, Tc cells have been shown to be divisible into two groups based on their cytokine production just as Th cells are. In addition, the Thl/Th2 dichotomy may not be absolute, e.g., some T cells may produce both Th1 and Th2 cytokines. As used herein the phrase "cytokine profile altering agent" includes both Th2 25 cytokine promoting agents and Thl cytokine inhibitory agents. A cytokine profile altering agent biases T cell cytokine production to a Th2 phenotype. A "Th2 phenotype" can be demonstrated by augmenting levels of Th2 cytokines in the presence of the agent when compared with levels produced in the absence of the agent. A Th2 phenotype can also be demonstrated by the production of qualitatively more types of Th2 cytokines 30 (e.g., IL-4, IL-5, IL-10, or IL-13), i.e., a greater number of Th2 cytokines being produced in the presence of the agent than are produced in the absence of the agent. The WO 00/51630 PCT/USOO/05647 - 10 phrase "Cytokine profile altering agent" includes agents that lead to the production of decreased levels of Th1 cytokines and/or which lead to the production of fewer different Thl cytokines. In one embodiment a cytokine profile altering agent promotes Th2 cytokine production. In another embodiment, a cytokine profile altering agent inhibits 5 Th1 cytokine production. In another embodiment a cytokine profile altering agent both promotes Th2 cytokine production and inhibits Th1 cytokine production. Preferably, a cytokine profile altering agent promotes an increase in the ratio of Th2/Th 1 cytokines. As used herein, the term "fetal" includes cells which are derived from a donor during the fetal development of the donor. Depending upon the cell isolated, such fetal 1o cells can be terminally differentiated or can be capable of differentiation into more than one cell type, e.g., can be undifferentiated "precursor cells". As used herein the term "stem cell" includes an undifferentiated cell which is capable of proliferation and results in additional stem cells having the ability to differentiate into progenitor cells under appropriate conditions. The term "progenitor cell" as used herein refers to 15 undifferentiated cells derived from stem cells and which, under appropriate conditions, differentiate into terminally differentiated cells. The term "precursor cell" also includes totipotent cells (e.g., cells form early stage embryos which are unrestricted in their developmental capabilities). Such precursor cells can be used as sources of the cells, i.e., the cells for use in the invention can be derived from such precursor cells, e.g., in 20 vitro or in vivo. As used herein, the term "heterologous nucleic acid molecule" includes a nucleic acid molecule, preferably DNA, that does not occur naturally as part of the genome in which it is present or which is found in a location or locations in the genome that differs from that in which it occurs in nature. thus, heterologous DNA is not naturally 25 occurring in that position or is not endogenous to the cell into which it is introduced, but ratherhas been obtained from another cell. Generally, although not necessarily, such DNA encodes proteins that are not normally produced by the cell in which it is expressed. Heterologous DNA can be from the same species or, from a different species. In particularly preferred embodiments, it is mammalian, e.g., human. 30 Heterologous DNA may also be referred to as foreign DNA. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which is WO 00/51630 PCT/USOO/05647 - 11 expressed is herein encompassed by the term heterologous DNA. Examples of heterologous DNA include, but are not limited to, DNA that encodes proteins which provide a benefit to the recipient, e.g., which the recipient is incapable of synthesizing. The terms "heterologous protein", "heterologous polypeptide," "recombinant 5 protein", and "exogenous protein" are used interchangeably throughout the specification and refer to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. That is, the polypeptide is expressed from a heterologous nucleic acid. 10 As used herein the phrase "soluble form of a costimulatory molecule" includes forms of costimulatory molecules which are not expressed on the surface of a cell and which inhibit a costimulatory signal in a T cell. As used herein, the phrase "costimulatory molecule" collectively refers to costimulatory molecules on APCs (e.g., the B7 molecules B7-1 and B7-2) and their cognate ligands on T cells (e.g., CD28 and 15 CTLA4). Cells for Transplantation Any type of cell, tissue or whole organ can be used in practicing the instant methods. Preferably the cell for transplantation is an isolated cell. In preferred 20 embodiment, the cells are xenogeneic cells. Xenogeneic cells for use in the instant methods can be obtained from any donor which is of a different species than the recipient subject. In a preferred embodiment, the recipient subject is a human subject and the donor cells are porcine cells. In one embodiment, a xenogeneic cell is obtained from a species to which the 2s recipient subject does not have preexisting, natural antibodies. In such a situation, the species combination is said to be concordant. In another embodiment, a xenogeneic cell is obtained from a species to which the recipient subject has preexisting, natural antibodies. In such a situation, the species combination is said to be discordant.

WO 00/51630 PCT/USOO/05647 - 12 In one embodiment, a xenogeneic cell of the invention is capable of direct presentation of antigen to the T cells of a recipient subject. For example, the xenogeneic cell has class I MHC molecules which are similar enough to those of the recipient that the recipient T cells can directly recognize the MHC antigen on the surface of the 5 xenogeneic cell. In another embodiment, a xenogeneic cell of the invention has MHC class I molecules which are different from the class I molecules of the recipient such that recipient T cells cannot directly recognize antigen on the surface of the xenogeneic cell. In such a species combination the recipient T cells recognize xenoantigen indirectly after 1o processing and presentation by recipient antigen presenting cells. In another embodiment, a xenogeneic cell of the invention has costimulatory molecules that are not recognized by the costimulatory molecule counter receptors (e.g., CD28 and/or CTLA4) on recipient T cells. In a preferred embodiment, the combination of donor cell and recipient subject is is one in which antigen presentation is primarily direct, but in which indirect presentation may also occur. In one embodiment, graft acceptance is improved in a setting where the host response to the graft in the absence of the cytokine profile altering agent is predominantly a CD8+ cellular immune response. In one embodiment, graft acceptance is improved in a setting where the host response to the graft in the absence of the 20 cytokine profile altering agent is predominantly a CD4+ cellular immune response. In one embodiment, graft acceptance is improved in a setting where the host response to the graft in the absence of the cytokine profile altering agent is predominantly a humoral cellular immune response. The cells for use in the instant methods can be obtained from the donor organism 25 at any point in the donor's life e.g., the cell can be a fetal cell, a juvenile cell, or an adult cell. The cells of the invention can also be obtained at any stage of differentiation, for example, the cells can be stem cells or progenitor cells or can be differentiated. Undifferentiated cells of the invention can either be transplanted as undifferentiated cells and be allowed to differentiate in vivo or can be induced to differentiate in vitro prior to 30 transplantation using methods that are known in the art to cause differentiation into the cell type of interest.

WO 00/51630 PCTIUSOO/05647 - 13 The cells described herein can also be grown as a cell culture prior to transplantation, i.e., as a population of cells which grow in vitro, in a medium suitable to support the growth and/or differentiation of the cells. Media which can be used to support the growth of specific cell types is known in the art as are appropriate 5 differentiation factors and/or conditions. Cells can also be derived from any part of a donor, e.g., the cells can be derived from blood or bone marrow, or can be isolated from discrete differentiated organs. For example, in preferred embodiments, a cell is selected from the group consisting of: a pancreatic islet cell, a kidney cell, a cardiac cell, a muscle cell, a liver cell, a lung cell, an 10 endothelial cell, a central nervous system cell, a peripheral nervous system cell, an epithelial cell, an eye cell, a skin cell, an ear cell, and a hair follicle cell. In a preferred embodiment a cell for use in the claimed methods is a brain cell. In other preferred embodiments, a population of cells for transplantation is substantially free of endothelial cells, e.g., is separated from any vascular tissue that was associated with the organ from is which the cells were derived in the host. Preferably the cells of the invention are transplanted as isolated cells, not as whole organs. Cells can be separated from other cells and tissues which surrounds them using a variety of methods which are known in the art. For example, cells can be subjected to mild protease treatment, gentle trituration, or more vigorous mechanical 20 means of separation. The isolated cells of the invention can be transplanted as a population of cells. In preferred embodiments, populations of cells for administration comprise substantially pure populations of cells. Preferably such populations comprise cells of one type. Cells for transplantation can be purified using methods known in the art, e.g., 25 using antibodies (e.g., by panning or by cell sorting) or by limiting dilution cloning. For example, in one embodiment, a population of cells is at least about 80% pure. In another embodiment, a population of cells is at least about 85% pure. In yet another embodiment, a population of cells is at least about 90% pure. More preferably a population of cells is at least about 90% to 95% or about 98% to 99% pure. The purity 30 of a population of cells can be determined by methods known in the art. For example, markers which specifically detect a particular cell type, can be used to detect the WO 00/51630 PCT/USO0/05647 - 14 presence of the cell type and the percentage of the population of cells which is comprised of that cell type e.g., by staining for that marker. In a preferred embodiment, xenogeneic cells are obtained from donor animals which are inbred to minimize genetic variability. For example, inbred miniature swine 5 are preferred donors for cells. Pathogen-Free Cells In one embodiment, the cells of the invention are cells determined to be free from at least one organism which originates in the donor from which the cells are obtained, 10 which is capable of infecting the donor cells, and which transmits infection or disease to a recipient subject. Preferably the cell is determined to be free from at least two organisms. Xenogeneic cells with these characteristics can be obtained by screening the donor to determine if it is essentially free from organisms or substances which are capable of transmitting infection or disease to a xenogeneic recipient, e.g., a human is recipient, of the cells prior to isolating the cells. In preferred embodiments, the cells are porcine cells which are obtained from a swine which predetermined to be essentially free from pathogens which detrimentally affect humans. For example, the pathogens from which the swine are free can include, but are not limited to, one or more of pathogens from the following categories of pathogens: zoonotic, cross-placental, and organotropic 20 organisms. As used herein, "zoonotic" refers to organisms which can be transferred from pigs to man under natural conditions; "cross-placental" refers to organisms capable of crossing the placenta from mother to fetus; "organotropic" refers to organisms which selectively infect cells of a particular organ. Within each of these categories, the organism can be a parasite, bacterium, mycoplasma, and/or virus. For example, donor 25 animals, e.g., swine, can be free from parasites such as zoonotic parasites (e.g., toxoplasma), cross-placental parasites (e.g., eperythozoon suis or toxoplasma), neurotropic parasites (e.g., toxoplasma), ocular-infecting parasites, and/or mycoplasma, such as M. hypopneumonia. Additionally, the swine can be free from bacteria such as zoonotic bacteria (e.g., brucella, listeria, mycobacterium TB, leptospirillum), cross 30 placental bacteria (e.g., brucella, listeria, leptospirillum), neurotropic bacteria (e.g., listeria), and/or ocular-infecting bacteria. Specific examples of bacteria from which the WO 00/51630 PCT/USOO/05647 - 15 swine can be free include brucella, clostridium, hemophilus suis, listeria, mycobacterium TB, leptospirillum, salmonella and hemophilus suis. Additionally, the swine can be free from viruses such as zoonotic viruses, viruses that can cross the placenta in pregnant sows, neurotropic viruses, and ocular-infecting viruses. Zoonotic viruses include, for 5 example, a virus in the rabies virus group, a herpes-like virus which causes pseudorabies, encephalomyocarditis virus, swine influenza Type A, transmissible gastroenteritus virus, parainfluenza virus 3 and vesicular stomatitis virus. Cross placental viruses include, for example, viruses that cause porcine respiratory reproductive syndrome, a virus in the rabies virus group, a herpes-like virus which io causes pseudorabies, parvovirus, a virus that causes swine vesicular disease, teschen (porcine polio virus), hemmaglutinating encephalomyocarditis, cytomegalovirus, suipoxvirus, and swine influenza type A. Organotropic viruses include, e.g., neurotropic viruses, such as viruses in the rabies virus group, a herpes-like virus which causes pseudorabies, parvovirus, encephalomyocarditis virus, a virus which causes swine is vesicular disease, porcine poliovirus (teschen), a virus which causes hemmaglutinating encephalomyocarditis, adenovirus, parainfluenza 3 virus. Specific examples of viruses from which the swine are free include: a virus which causes (or results in) porcine respiratory reproductive syndrome, a virus in the rabies virus group, a herpes-like virus which causes pseudorabies, parvovirus, encephalomyocarditis virus, a virus which 20 causes swine vesicular disease, porcine poliovirus (teschen), a virus which causes hemmaglutinating encephalomyocarditis, cytomegalovirus, suipoxvirus, swine influenza type A, adenovirus, transmissible gastroenteritus virus, a virus which causes bovine viral diarrhea, parainfluenza virus 3, and vesicular stomatitis virus. In one embodiment, the pigs from which the xenogeneic cells are isolated are 25 essentially free from at least one of the organisms selected from the group consisting of: Toxoplasma, eperythrozoon, brucella, listeria, mycobacterium TB, leptospirillum, hemophilus suis, M. hypopneumonia, a virus which causes porcine respiratory reproductive syndrome, a virus which causes rabies, a virus which causes pseudorabies, parvovirus, encephalomyocarditis virus, a virus which causes swine vesicular disease, 30 porcine polio virus (teschen), a virus which causes hemagglutinating encephalomyocarditis, suipoxvirus, swine influenza type A, adenovirus, transmissible WO 00/51630 PCT/US00/05647 - 16 gastroenteritis virus, a virus which causes bovine viral diarrhea, and vesicular stomatitis virus. The phrase "essentially free or free from organisms or substances which are capable of transmitting infection or disease to a xenogeneic recipient" (also referred to herein as "essentially pathogen-free" or "pathogen free") when referring to a donor from 5 which cells are isolated or to the cells themselves means that donor does not contain organisms or substances in an amount which transmits infection or disease to a xenogeneic recipient, e.g. a human. In order to determine whether a donor animal, e.g., a pig harbors infectious agents which are capable of infecting and transmitting disease to a recipient any art io recognized method of detecting infectious agents can be used. (See e.g., Fishman. Xenotransplantation. 1:47. 1994 for exemplary screening methods and methods of maintaining donor animals free of infectious agents). For example, in one embodiment, nucleic acid molecules can be extracted from donor tissue using standard procedures. Under the appropriate conditions, is oligonucleotide primers capable of specifically amplifying nucleic acid molecules of an infectious agent can be used to amplify pathogen nucleic acid molecules, e.g., by polymerase chain reaction. The resulting samples can be screened to determine whether or not such sequences were present and amplified in the sample. In another embodiment, oligonucleitides capable of specifically hybridizing to infectious agent 20 nucleic acid molecules can be used as probes to detect the presence of infectious agents in a sample taken from the donor. In another embodiment, samples from the donor can be concentrated and examined, e.g., visually examined for signs of pathogens, e.g., protozoan cysts of trophozoites, helminth eggs and larvae. Protozoan cyst identification can be confirmed, 25 when required, by trichrome staining. In another embodiment, cells from a donor can be cocultured with cells which are capable of being infected by the pathogen which is being tested for and the ability of the donor cells to infect the test cells can be determined. For example, test cells can be observed for viral cytopathic effects: WO 00/51630 PCT/USOO/05647 - 17 Hemadsorbing viruses and hemaglutination testing can be detected using standard methods. A hemagglutination test is a test that detects viruses with the property to agglutinate erythrocytes, such as swine influenza type A, parainfluenza, and encephalomyocarditus viruses, in the test article. Hsuing, G.D. (1982) Diagnostic s Virology (Yale University Press, New Haven, CT);. Stites, Daniel P and Terr, Abba I, (1991), Basic and Clinical Immunology (Appleton & Lange, East Norwalk, CT). Fluorescent antibodies can also be used to stain cell suspensions of porcine cells to detect infectious agents, e.g., viruses. In addition, samples taken from donors can be cultured to determine whether 1o bacteria are present in the samples. If signs of bacterial growth are observed when the samples are initially screened for bacterial growth, a Gram stain is prepared and viewed microscopically at 1 00x oil immersion for the presence of any bacteria or fungi. Positive cultures can then be subcultured onto both chocolate agar plates with Iso Vitlex and onto BMB plates. The chocolate plate is incubated at 35-37*C in 5% C0 2 for 24 hours and 15 the BMB anaerobically at 35-37 0 C for 48 hours. Any yeast or fungi seen by gram stain is subcultured onto a Sabaroud dextrose/Emmons plate. The Vitek automated system can be to identify bacteria and yeast. Fungi can be identified via their macroscopic and microscopic characteristic. 20 Modified Cells The cells of the invention can be modified prior to use in transplantation. Preferably the cells to be modified are xenogeneic cells. In one embodiment, the cells of the invention are altered prior to administration to the recipient. In an unaltered state, the cells have one or more antigen on the cell surface which stimulates an immune 25 response against the cell when the cell is administered to a subject (also referred to herein as recipient or recipient subject). By altering the antigen, the normal immunological recognition of the cell, e.g., by the immune system cells of the recipient is disrupted. An "abnormal" immunological recognition of this altered form of the antigen can further augment graft survival. For example, in the case of masking MHC 30 class I molecules, as shown in the appended Examples, alteration of an antigen on the cell prior to introducing the cell into a subject provides another method by which the WO 00/51630 PCT/USOO/05647 - 18 production of Th2 cytokines is promoted. Accordingly such alteration can be used, e.g., in conjunction with a cytokine profile altering agent to further bias the T cell cytokine profile of the recipient towards a Th2 phenotype. As used herein, the term "altered" encompasses changes that are made to at least 5 one cell antigen(s) which reduces the immunogenicity of the antigen to thereby interfere with immunological recognition of the antigen(s) by the recipient's immune system. Cells can be altered, e.g., by modifying, masking, or eliminating the antigen such that such that upon introduction of the composition into the recipient, lysis of said cell is prevented. 10 Antigens that can be altered according to the current invention include antigens on a xenogeneic cell, e.g., a porcine cell, which can interact with an immune cell in a xenogeneic recipient subject and thereby stimulate a specific immune response against the xenogeneic cell in the recipient. The interaction between the antigen and the immune cell may be an indirect interaction or a direct interaction between the antigen 15 and a molecule present on the surface of the immune cell. As used herein, the term "immune cell" is intended to include a cell which plays a role in specific immunity (e.g., is involved in an immune response) or plays a role in natural immunity. Examples of immune cells include all distinct classes of lymphocytes (T lymphocytes, such as helper T cells and cytotoxic T cells, B lymphocytes, and natural killer cells), monocytes, 20 macrophages, other antigen presenting cells, dendritic cells, and leukocytes (e.g., neutrophils, eosinophils, and basophils). In a preferred embodiment, the antigen is one which interacts with a T lymphocyte in the recipient (e.g., the antigen normally binds to a receptor on the surface of a T lymphocyte). For example, in one embodiment the antigen is altered by masking the cell, e.g., 25 hiding an epitope of the antigen, for example using an antibody that binds to the antigen. In another embodiment, the antigen is altered by modifying the antigen, e.g., by expressing a form of the antigen which comprises an alteration in amino acid sequence such that an epitope of the antigen which would normally be recognized by the recipient's immune system is no longer so recognized. For example, a mutant form of a 30 cell surface molecule can be expressed by the cell using standard techniques, e.g., by using site directed mutagenesis. In yet another embodiment, an antigen is altered WO 00/51630 PCT/USOO/05647 - 19 eliminating the expression of the antigen from the surface of the cell. This can be accomplished, e.g., using enzymatic treatment, or by making a knock-out donor animal that no longer expresses the antigen to be altered. In one embodiment, the antigen to be altered on the cell is an MHC class I 5 antigen. Alternatively, an adhesion molecule on the cell surface, such as NCAM-1 or ICAM-1, can be altered. An antigen which stimulates a cellular immune response against the cell, such as an MHC class I antigen, can be altered prior to transplantation by contacting the cell with a molecule which binds to the antigen. A preferred molecule for binding to the antigen is an antibody, or fragment thereof (e.g., an anti-MHC class I lo antibody, or fragment thereof, an anti-ICAM- 1 antibody or fragment thereof, an anti LFA-3 antibody or fragment thereof, or an anti-p 2 microglobulin antibody or fragment thereof). A preferred antibody fragment is an F(ab') 2 fragment. Polyclonal or, more preferably, monoclonal antibodies can be used. Other molecules which can be used to alter an antigen (e.g., an MHC class I antigen) include peptides and small organic is molecules which bind to the antigen. Furthermore, two or more different epitomes on the same or different antigens on the cell surface can be altered. A particularly preferred monoclonal antibody for alteration of MHC class I antigens on xenogeneic cells is PT85 (commercially available from Veterinary Medicine Research Development, Pullman WA). PT85 can be used alone to alter MHC class I antigens or, if each antibody is 20 specific for a different epitope, PT85 can be used in combination with another antibody known to bind MHC class I antigens to alter the antigens on the cell surface. Suitable methods for altering a surface antigen on a cell for transplantation are described in greater detail in Faustman and Coe 1991. Science 252:1700-1702 and PCT publication WO 92/04033. Methods for altering multiple epitopes on a surface antigen on a cell for 25 transplantation are described in greater detail in PCT publication WO 95/26741, published on October 12, 1995, the contents of which are incorporated herein by reference. In another embodiment, the xenogeneic cells of the present invention can be altered to inhibit natural antibody-mediated hyperacute rejection of the cells. For 30 example, the cells of the invention may, in unaltered form, express an epitope on their surface which stimulates hyperacute rejection of the by natural antibodies in a recipient WO 00/51630 PCTIUSOO/05647 -20 subject. Such an epitope can be modified, reduced or substantially eliminated. This treatment of the cell inhibits subsequent binding of the epitope by natural antibodies in a recipient, thereby inhibiting hyperacute rejection. In a preferred embodiment, the epitope is a carbohydrate, preferably galactosyl (a&l,3)galactose (Gal (cal,3) Gal). 5 Epitopes on the surface of the xenogeneic cells, in one embodiment of the invention, are removed from the surface of a cell, such as by enzymatic or chemical treatment of the cell. For example, Gal (cl,3)Gal epitopes can be cleaved from a xenogeneic cell surface by treatment of the cell with an alpha-galactosidase. In another embodiment, formation of the epitope on the cell surface is inhibited. This can be 1o accomplished by inhibiting the activity of an enzyme which forms the epitope. For example, formation of Gal (al,3)Gal epitopes on the surface of a xenogeneic cell can be interfered with by inhibiting the activity of an alpha-1,3-galactosyltransferase within the cell, such as by introducing into the cell a nucleic acid which is antisense to a coding or regulatory region of an alpha-1,3-galactosyltransferase gene or by treating the cell with a 15 chemical inhibitor of the enzyme. In yet another embodiment, epitopes on the surface of a cell are altered by binding a molecule to the epitope, thereby inhibiting its subsequent recognition by natural antibodies in a recipient. For example, lectins, antibodies or antibody fragments can be bound to an epitope to inhibit its subsequent recognition by natural antibodies. Methods for altering epitopes on xenogeneic cell surfaces which 20 stimulate hyperacute rejection of the cells by natural antibodies in a recipient subject are described in greater detail in PCT Publication WO 95/33828, published on December 14, 1995, the contents of which are incorporated herein by reference. Cells Expressing Heterologous Proteins 25 In one embodiment, a cell of the invention is modified to express a heterologous gene product. As used herein, the term "modified" to express a heterologous gene product is intended to mean that the cell is treated in a manner that results in the production of a heterologous gene product by the cell. Preferably, the cell does not express the gene product prior to modification. Alternatively, modification of the cell 30 may result in an increased production of a gene product already expressed by the cell or WO 00/51630 PCT/USOO/05647 - 21 result in production of a gene product (e.g., an antisense RNA molecule) which decreases production of another, undesirable gene product normally expressed by the cell. In a preferred embodiment, a cell is modified to express a gene product by 5 introducing genetic material, such as a nucleic acid molecule (e.g., RNA or, more preferably, DNA) into the cell. The nucleic acid molecule introduced into the cell encodes a gene product to be expressed by the cell. The term "gene product" as used herein is intended to include proteins, peptides and functional RNA molecules. Generally, the gene product encoded by the nucleic acid molecule is the desired gene lo product to be supplied to a subject. Alternatively, the encoded gene product is one which induces the expression of the desired gene product by the cell (e.g., the introduced genetic material encodes a transcription factor which induces the transcription of the gene product to be supplied to the subject). A nucleic acid molecule introduced into a cell is in a form suitable for expression is in the cell of the gene product encoded by the nucleic acid. Accordingly, the nucleic acid molecule includes coding and regulatory sequences required for transcription of a gene (or portion thereof) and, when the gene product is a protein or peptide, translation of the gene product encoded by the gene. Regulatory sequences which can be included in the nucleic acid molecule include promoters, enhancers and polyadenylation signals, 20 as well as sequences necessary for transport of an encoded protein or peptide, for example N-terminal signal sequences for transport of proteins or peptides to the surface of the cell or for secretion. Nucleotide sequences which regulate expression of a gene product (e.g., promoter and enhancer sequences) are selected based upon the type of cell in which the 25 gene product is to be expressed and the desired level of expression of the gene product. For example, a promoter known to confer cell-type specific expression of a gene linked to the promoter can be used. A promoter specific for myoblast gene expression can be linked to a gene of interest to confer muscle-specific expression of that gene product. Muscle-specific regulatory elements which are known in the art include upstream 30 regions from the dystrophin gene (Klamut et al., 1989. Mol. Cell. Biol. 9:2396), the creatine kinase gene (Buskin and Hauschka, 1989. Mol. Cell Biol. 9:2627) and the WO 00/51630 PCT/USOO/05647 - 22 troponin gene (Mar and Ordahl, 1988. Proc. Natl. Acad Sci. USA. 85:6404). Regulatory elements specific for other cell types are known in the art (e.g., the albumin enhancer for liver-specific expression; insulin regulatory elements for pancreatic islet cell-specific expression; various neural cell-specific regulatory elements, including 5 neural dystrophin, neural enolase and A4 amyloid promoters). Alternatively, a regulatory element which can direct constitutive expression of a gene in a variety of different cell types, such as a viral regulatory element, can be used. Examples of viral promoters commonly used to drive gene expression include those derived from polyoma virus, adenovirus 2, cytomegalovirus and Simian Virus 40, and retroviral LTRs. 10 Alternatively, a regulatory element which provides inducible expression of a gene linked thereto can be used. The use of an inducible regulatory element (e.g., an inducible promoter) allows for modulation of the production of the gene product in the cell. Examples of potentially useful inducible regulatory systems for use in eukaryotic cells include hormone- regulated elements (e.g., see Mader, S. and White, J.H. 1993. Proc. 15 Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see, e.g. Spencer, D.M. et al. 1993. Science 262:1019-1024) and ionizing radiation-regulated elements (e.g., see Manome, Y. et al. 1993. Biochemistry 32:10607-10613; Datta, R. et al. 1992. Proc. Natl. Acad. Sci. USA 89:10149-10153). Additional tissue-specific or inducible regulatory systems which may be developed can also be used in accordance 20 with the invention. There are a number of techniques known in the art for introducing genetic material into a cell that can be applied to modify a cell of the invention. In one embodiment, the nucleic acid is in the form of a naked nucleic acid molecule. In this situation, the nucleic acid molecule introduced into a cell to be modified consists only of 25 the nucleic acid encoding the gene product and the necessary regulatory elements. Alternatively, the nucleic acid encoding the gene product (including the necessary regulatory elements) is contained within a plasmid vector. Examples of plasmid expression vectors include CDM8 (Seed, B., 1987. Nature 329:840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6:187-195). In another embodiment, the nucleic acid 30 molecule to be introduced into a cell is contained within a viral vector. In this situation, the nucleic acid encoding the gene product is inserted into the viral genome (or a partial WO 00/51630 PCT/USOO/05647 - 23 viral genome). The regulatory elements directing the expression of the gene product can be included with the nucleic acid inserted into the viral genome (i.e, linked to the gene inserted into the viral genome) or can be provided by the viral genome itself. Examples of methods which can be used to introduce naked nucleic acid into cells and viral s mediated transfer of nucleic acid into cells are described separately in the subsections below. A. Introduction of Naked Nucleic Acid into Cells 1. Transfection mediated by CaPO 4 : Naked DNA can be introduced into cells by forming a precipitate containing the DNA and calcium phosphate. For example, a io HEPES-buffered saline solution can be mixed with a solution containing calcium chloride and DNA to form a precipitate and the precipitate is then incubated with cells. A glycerol or dimethyl sulfoxide shock step can be added to increase the amount of DNA taken up by certain cells. CaPO 4 -mediated transfection can be used to stably (or transiently) transfect cells and is only applicable to in vitro modification of cells. 15 Protocols for CaPO 4 - mediated transfection can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, 1989., Section 9.1 and in Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, 1989., Sections 16.32-16.40 or other standard laboratory manuals. 20 2. Transfection mediated by DEAE-dextran: Naked DNA can be introduced into cells by forming a mixture of the DNA and DEAE-dextran and incubating the mixture with the cells. A dimethylsulfoxide or chloroquine shock step can be added to increase the amount of DNA uptake. DEAE-dextran transfection is only applicable to in vitro 25 modification of cells and can be used to introduce DNA transiently into cells but is not preferred for creating stably transfected cells. Thus, this method can be used for short term production of a gene product but is not a method of choice for long-term production of a gene product. Protocols for DEAE-dextran-mediated transfection can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing 30 Associates, 1989., Section 9.2 and in Molecular Cloning: A Laboratory Manual, 2nd WO 00/51630 PCTIUSOO/05647 - 24 Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, 1989., Sections 16.4 1 16.46 or other standard laboratory manuals. 3. Electroporation: Naked DNA can also be introduced into cells by incubating the 5 cells and the DNA together in an appropriate buffer and subjecting the cells to a high voltage electric pulse. The efficiency with which DNA is introduced into cells by electroporation is influenced by the strength of the applied field, the length of the electric pulse, the temperature, the conformation and concentration of the DNA and the ionic composition of the media. Electroporation can be used to stably (or transiently) transfect 10 a wide variety of cell types and is only applicable to in vitro modification of cells. Protocols for electroporating cells can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, 1989., Section 9.3 and in Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, 1989., Sections 16.54-16.55 or other standard 15 laboratory manuals. 4. Liposome-mediated transfection ("lipofection'): Naked DNA can be introduced into cells by mixing the DNA with a liposome suspension containing cationic lipids. The DNA/liposome complex is then incubated with cells. Liposome mediated transfection 20 can be used to stably (or transiently) transfect cells in culture in vitro. Protocols can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, 1989., Section 9.4 and other standard laboratory manuals. Additionally, gene delivery in vivo has been accomplished using liposomes. See for example Nicolau et al. 1987. Meth. Enz. 149:157-176; Wang and Huang 1987. Proc. 25 Natl. Acad Sci. USA 84:7851-7855; Brigham et al. 1989. Am. J. Med Sci. 298:278; and Gould-Fogerite et al. 1989. Gene 84:429-438. 5. Direct Injection: Naked DNA can be introduced into cells by directly injecting the DNA into the cells. For an in vitro culture of cells, DNA can be introduced by 30 microinjection. Since each cell is microinjected individually, this approach is very labor intensive when modifying large numbers of cells. However, a situation wherein WO 00/51630 PCT/USOO/05647 - 25 microinjection is a method of choice is in the production of transgenic animals (discussed in greater detail below). In this situation, the DNA is stably introduced into a fertilized oocyte which is then allowed to develop into an animal. The resultant animal contains cells carrying the DNA introduced into the oocyte. Direct injection has also 5 been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. 1991. Nature 332: 815-818; Wolff et al. 1990. Science 247:1465-1468). A delivery apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo can be used. Such an apparatus is commercially available (e.g., from BioRad). 1o 6. Receptor-Mediated DNA Uptake: Naked DNA can also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C.H. 1988. J. Bio. Chem. 263:14621; Wilson et al. 1992. J Biol. Chem. 267:963-967; and U.S. Patent No. 5,166,320). Binding of the DNA-ligand complex to the receptor facilitates uptake of the 1 DNA by receptor-mediated endocytosis. Receptors to which a DNA-ligand complex have targeted include the transferrin receptor and the asialoglycoprotein receptor. A DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. 1991. Proc. NatL. 20 Acad Sci. USA 88:8850; Cristiano et al. 1993. Proc. Nati. Acad. Sci. USA 90:2122 2126). Receptor-mediated DNA uptake can be used to introduce DNA into cells either in vitro or in vivo and, additionally, has the added feature that DNA can be selectively targeted to a particular cell type by use of a ligand which binds to a receptor selectively expressed on a target cell of interest. 25 Generally, when naked DNA is introduced into cells in culture (e.g., by one of the transfection techniques described above) only a small fraction of cells (about 1 out of 105) typically integrate the transfected DNA into their genomes (i.e., the DNA is maintained in the cell episomally). Thus, in order to identify cells which have taken up exogenous DNA, it is advantageous to transfect nucleic acid encoding a selectable 30 marker into the cell along with the nucleic acid(s) of interest. Preferred selectable markers include those which confer resistance to drugs such as G418, hygromycin and WO 00/51630 PCTUSOO/05647 - 26 methotrexate. Selectable markers may be introduced on the same plasmid as the gene(s) of interest or may be introduced on a separate plasmid. An alternative method for generating a cell that is modified to express a gene product involving introducing naked DNA into cells is to create a transgenic animal 5 which contains cells modified to express the gene product of interest. A transgenic animal is an animal having cells that contain a transgene, wherein the transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA molecule which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the io mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. Thus, a transgenic animal expressing a gene product of interest in one or more cell types within the animal can be created, for example, by introducing a nucleic acid molecule encoding the gene product (typically linked to appropriate regulatory elements, such as a tissue-specific enhancer) into the is male pronuclei of a fertilized oocyte, e.g., by microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Methods for generating transgenic animals, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009 and Hogan, B. et al., (1986) A Laboratory Manual, Cold Spring Harbor, New York, Cold Spring Harbor 20 Laboratory. A transgenic founder animal can be used to breed more animals carrying the transgene. Cells of the transgenic animal which express a gene product of interest can then be used to deliver the gene product to a subject in accordance with the invention. Alternatively, an animal containing a gene which has been modified by 25 homologous recombination can be constructed to express a gene product of interest. For example, an endogenous gene carried in the genome of the animal can be altered by homologous recombination (for instance, all or a portion of a gene could be replaced by the human homologue of the gene to "humanize" the gene product encoded by the gene) or an endogenous gene can be "knocked out" (i.e., inactivated by mutation). For 3o example, an endogenous gene in a cell can be knocked out to prevent production of that gene product and then nucleic acid encoding a different (preferred) gene product is WO 00/51630 PCTUSOO/05647 - 27 introduced into the cell. To create an animal with homologously recombined nucleic acid, a vector is prepared which contains the DNA which is to replace or interrupt the endogenous DNA flanked by DNA homologous to the endogenous DNA (see for example Thomas, K.R. and Capecchi, M. R. 1987. Cell 51:503). The vector is 5 introduced into an embryonal stem cell line (e.g., by electroporation) and cells which have homologously recombined the DNA are selected (see for example Li, E. et al. 1992. Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see for example Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. io (IRL, Oxford, 1987. pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA. Cells of the animal containing the homologously recombined DNA which express a gene 15 product of interest can then be used to deliver the gene product to a subject in accordance with the invention. B. Viral-Mediated Gene Transfer A preferred approach for introducing nucleic acid encoding a gene product into a 20 cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the gene product. Infection of cells with a viral vector has the advantage that a large proportion of cells receive the nucleic acid, which can obviate the need for selection of cells which have received the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which 25 have taken up viral vector nucleic acid and viral vector systems can be used either in vitro or in vivo. 1. Retroviruses: Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. 1990. Blood 76:27 1). A 3o recombinant retrovirus can be constructed having a nucleic acid encoding a gene product of interest inserted into the retroviral genome. Additionally, portions of the retroviral WO 00/51630 PCT/USOO/05647 - 28 genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can s be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, 1989, Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include y Crip, yCre, y2 and yAm. Retroviruses have been used to introduce a variety of genes io into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. 1985. Science 230:1395-1398; Danos and Mulligan 1988. Proc. Nati. Acad. Sci. USA 85:6460-6464; Wilson et al. 1988. Proc. NatL. Acad Sci. USA 85:3014 3018; Armentano et al. 1990. Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. i5 1991. Proc. Natl. Acad Sci. USA 88:8039-8043; Ferry et al. 1991. Proc. Natl. A cad. Sci. USA 88:8377-8381; Chowdhury et al. 1991. Science 254:1802-1805; van Beusechem et al. 1992. Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. 1992. Human Gene Therapy 3:641-647; Dai et al. 1992. Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. 1993. J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 20 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell. 25 2. Adenoviruses: The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. 1988. BioTechniques 6:616; Rosenfeld et al. 1991. Science 252:431-434; and Rosenfeld et al. 30 1992. Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known WO 00/51630 PCTIUSOO/05647 - 29 to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. 1992. cited supra), endothelial cells (Lemarchand et al. 1992. Proc. Nati. Acad. Sci. USA 89:6482 5 6486), hepatocytes (Herz and Gerard 1993. Proc. Natl. Acad Sci. USA 90:2812-2816) and muscle cells (Quantin et al. 1992. Proc. Natl. Acad Sci. USA 89:2581-2584). Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations io where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj Ahmand and Graham 1986. J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral El and E3 genes but retain is as much as 80 % of the adenoviral genetic material. 3. Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see 20 Muzyczka et al. Curr. Topics in Micro. and Immunol. 1992. 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. 1992. Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. 1989. J. Virol. 63:3822-3828; and McLaughlin et al. 1989. J Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV 25 can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. 1985. Mol. Cell. Biol. 5:3251 3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. 1984. Proc. Natl. Acad Sci. USA 81:6466-6470; Tratschin et al. 1985. Mol. Cell. Biol. 30 4:2072-2081; Wondisford et al. 1988. Mol. Endocrinol. 2:32-39; Tratschin et al. 1984. J. Virol. 51:611-619; and Flotte et al. 1993. J Biol. Chem. 268:3781-3790).

WO 00/51630 PCT/USOO/05647 - 30 The efficacy of a particular expression vector system and method of introducing nucleic acid into a cell can be assessed by standard approaches routinely used in the art. For example, DNA introduced into a cell can be detected by a filter hybridization technique (e.g., Southern blotting) and RNA produced by transcription of introduced 5 DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR). The gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product, such as an enzymatic assay. If the gene product of interest to be lo expressed by a cell is not readily assayable, an expression system can first be optimized using a reporter gene linked to the regulatory elements and vector to be used. The reporter gene encodes a gene product which is easily detectable and, thus, can be used to evaluate the efficacy of the system. Standard reporter genes used in the art include genes encoding p-gammalactosidase, chloramphenicol acetyl transferase, luciferase and human 15 growth hormone. When the method used to introduce nucleic acid into a population of cells results in modification of a large proportion of the cells and efficient expression of the gene product by the cells (e.g., as is often the case when using a viral expression vector), the modified population of cells may be used without further isolation or subcloning of 20 individual cells within the population. That is, there may be sufficient production of the gene product by the population of cells such that no further cell isolation is needed. Alternatively, it may be desirable to grow a homogenous population of identically modified cells from a single modified cell to isolate cells which efficiently express the gene product. Such a population of uniform cells can be prepared by isolating a single 25 modified cell by limiting dilution cloning followed by expanding the single cell in culture into a clonal population of cells by standard techniques. C. Other Methods for Modifying a Cell to Express a Gene Product As an alternative to introducing a nucleic acid molecule into a cell to modify the 30 cell to express a gene product, a cell can be modified by inducing or increasing the level of expression of the gene product by a cell. For example, a cell may be capable of WO 00/51630 PCT/USOO/05647 -31 expressing a particular gene product but fails to do so without additional treatment of the cell. Similarly, the cell may express insufficient amounts of the gene product for the desired purpose. Thus, an agent which stimulates expression of a gene product can be used to induce or increase expression of a gene product by the cell. For example, cells 5 can be contacted with an agent in vitro in a culture medium. The agent which stimulates expression of a gene product may function, for instance, by increasing transcription of the gene encoding the product, by increasing the rate of translation or stability (e.g., a post transcriptional modification such as a poly A tail) of an mRNA encoding the product or by increasing stability, transport or localization of the gene product. io Examples of agents which can be used to induce expression of a gene product include cytokines and growth factors. Another type of agent which can be used to induce or increase expression of a gene product by a cell is a transcription factor which upregulates transcription of the gene encoding the product. A transcription factor which upregulates the expression of a is gene encoding a gene product of interest can be provided to a cell, for example, by introducing into the cell a nucleic acid molecule encoding the transcription factor. Thus, this approach represents an alternative type of nucleic acid molecule which can be introduced into the cell (for example by one of the previously discussed methods). In this case, the introduced nucleic acid does not directly encode the gene product of 20 interest but rather causes production of the gene product by the cell indirectly by inducing expression of the gene product. In yet another method, a cell is modified to express a gene product by coupling the gene product to the cell, preferably to the surface of the cell. For example, a protein can be obtained by purifying the cell from a biological source or expressing the protein 25 recombinantly using standard recombinant DNA technology. The isolated protein can then be coupled to the cell. The terms "coupled" or "coupling" refer to a chemical, enzymatic or other means (e.g., by binding to an antibody on the surface of the cell or genetic engineering of linkages) by which a gene product can be linked to a cell such that the gene product is in a form suitable for delivering the gene product to a subject. 3o For example, a protein can be chemically crosslinked to a cell surface using commercially available crosslinking reagents (Pierce, Rockford IL). Other approaches WO 00/51630 PCT/USOO/05647 - 32 to coupling a gene product to a cell include the use of a bispecific antibody which binds both the gene product and a cell-surface molecule on the cell or modification of the gene product to include a lipophilic tail (e.g., by inositol phosphate linkage) which can insert into a cell membrane. 5 Cytokine Profile Altering Agents Depending on the signals delivered to the T cells involved in the initial target antigen recognition, a CD4+ T cell response can be modulated toward a Thl or Th2 profile (Kuchroo et al. 1995. Cell. 80:707). In addition, certain cytokines, once io produced, can alter the profile of cytokines that are subsequently produced. A cytokine profile altering agent alters the profile of cytokine production from any cell type that produces cyokines. Preferably, such an agent alters the profile of cytokines produced by T cells. Production of cytokines after activation is short lived, occurring over intervals of 15 a few hours to a few days. Detailed understanding of T cell heterogeneity and an appreciation that T helper subset 1 (Thl ) and T helper subset 2 (Th2) subpopulations of T helper cells regulate either cell-mediated (DTH) or antibody-mediated immune responses is based on the work of Mosmann and colleagues who identified a cytokine production profile for each T cell subset (Mosmann, T.R. 1986. JImmunol. 136:2348; 20 Cher, D.J. and T.R. Mosmann, 1987 JImmunol, 138: p. 3688; Stout, R.D. and K. Bottomly. 1989. JImmunol. 142:760). This includes the preferential secretion of IL-2, IFN-gamma, and IL-15 for ThI cells and preferential secretion of IL-4, IL-5, IL-10, IL 13, and IL-16 for Th2 cells. Both subsets produce IL-3, granulocyte-macrophage CSF, and TNF. These profiles are frequently mutually exclusive due to cross regulation 25 between the Th1 and Th2 cells (Constant, S.L. and K. Bottomly. 1997 Annu Rev Immunol. 15:297). Th1 cytokines are primarily involved in promoting cellular responses. The major function of IL-2 is the activation of T cells and NK cells. IL-2 is secreted by helper T lymphocytes 4-12 hours following stimulation by antigen. The subsequent binding of 30 IL-2 to its receptor results in proliferation of the antigen activated T cells, enhanced secretion of lymphokines, and heightened expression of membrane receptors for the WO 00/51630 PCT/USOO/05647 -33 other growth factors. IL-2 principally enhances expansion of the Th1 subset of T cells. Moreover, IL-2 has been demonstrated to influence B cells and monocytes. In particular, activation of monocytes by IL-2 induces IL-1 secretion, enhances monocyte-mediated cytotoxicity, promotes proliferation of macrophage precursors, and increases s phagocytosis. Like IL-2, IFN-gamma augments the induction of the Th1 cells (Swain, S.L. 1991. Immunol Rev. 123:115) and is capable of regulating specific immune effector mechanisms by direct actions on helper T cells, cytotoxic T cells, and B cells. Conversely, IFN-gamma impedes the induction, proliferation, and effector functions of io the Th2 cell subset. IFN-gamma up regulates the expression of class I and class II MHC antigens on a variety of cell types. The consequence of this up-regulated class II MHC antigen expression is an augmented and accelerated immune response. Moreover, IFN gamma can induce the de novo expression of class II MHC antigens on epithelial, endothelial, and connective tissue cells, allowing these cells to become active in antigen is presentation and induction of specific T-cell immunity. In addition, IFN-gamma is a potent activator of macrophage and monocytes. Moreover, IFN-gamma augments cytotoxic immune responses by directly activating NK cells and CD8+ T cells. Many of the in vitro properties of IFN-gamma have been confirmed by in vivo animal model experiments. 20 In addition to IL-2 and IFN-gamma, IL- 12 has been identified as an important cytokine in the initiation of cell mediated Th1 response. Unlike IFN-gamma, the presence of IL- 12 during priming directly augments the Thl cells, but has no effect on Th2 cells. Th2 cytokines are important in the development of humoral immune responses 25 e.g., through their affects on B cells. In addition, Th2 cytokines have been found to positively amplify Th2 responses. For example, IL-4 and IL-10, as well as several other cytokines, have an important role in initiating the Th2 response (Swain, S.L., et al. 1990. JImmunol, 145:3796). In fact, the inclusion of anti-IL-4 antibody during priming of an immune response completely abrogates the generation of Th2 cells, suggesting that the 30 presence of IL-4, even if endogenously derived, is essential for Th2 differentiation. Although IL- 10 has also been reported to promote the development of Th2 cells, its WO 00/51630 PCT/USOO/05647 -34 major effect may be in suppressing ThI cells (Bromberg, J.S. 1995. Curr Opin Immunol. 7:639; de Vries, J.E. 1995. Ann Med. 27:537). In addition to these subsets of CD4+ T cells, studies have identified CD8+ T cells as Thl -like (TCl ) or Th2-like (TC2) in their cytokine production profiles (Sad, S., 5 R. et. al. 1995. Immunity. 2:271). Many of the CD8+ T cells that have been examined produce a pattern of cytokines very similar to that of Th1 clones, with the exception that they tend to make little, if any, IL-2 (Fong, T.A. and T.R. Mosmann, 1990. JImmunol, 144:1744. However, there are several reports (in mice) of CD8+ T cell populations with more Th2-like patterns, with production of IL-4 or IL-5 (Seder, R.A., et al 1992. J io Immunol. 148:1652;.Taguchi, T., et al. 1990. JImmunol, 1990. 145:68). Similarly, the cutaneous lesions in lepromatous leprosy in humans are characterized by large numbers of infiltrating CD8+ T cells that produce IL-4 and IL- 10 (Yamamura, M., et al., 1991. Science. 254:277-9 (published erratum appears in Science 1992Jan 3;255(5040):12). Th2-like CD8+ T cell clones have been established from such patients; these clones can is suppress the response of Thl-like cells, in part, through the activities of IL-4 (Salgame, P., et al. 1991. Science. 254:279. The cytokine profile of a recipient of a graft can be biased by the administration of a cytokine profile altering agent such that Th2 cytokine production after exposure to the agent is enhanced when compared with Th2 cytokine production in the absence of 20 the agent and/or Th 1 cytokine production is diminished upon exposure to cytokine profile altering agent. A cytokine profile altering agent is not a general immunosuppressant, e.g., cyclosporin Preferably a cytokine profile altering agent is a protein or polypeptide. A cytokine profile altering agent can lead to an increase the production of Th2 cytokines overall (or an increased ratio of Th2 to Th1 cytokines 25 overall), or can specifically lead to an increase in the production of one or more Th2 cytokines (or an increase in the ratio of a specific Th2 cytokine to Th 1 cytokines. Preferably the cytokine profile altering agent leads to decrease in the production of IL-2 and/or IFN-8. In another preferred embodiment the cytokine profile altering agent leads to an increase in the production of IL-4 and/or IL-10. Examples of cytokine profile 30 altering agents are provided below: WO 00/51630 PCT/USOO/05647 - 35 Cytokines And Cytokine Fusion Proteins In one embodiment, the cytokine profile altering agent is a cytokine which is administered to the transplant recipient. For example, cytokines such as IL-4 and IL- 10 have been found to bias T cell responses towards a Th2 phenotype. These cytokine 5 genes have been cloned and their sequences are available in the art (e.g., Viera et al. 1991. Proc. Nat!. Acad Sci 88:1172 (for IL-10) and Yokota et al. 1986. Proc. Nat!. Acad Sci. 83:5894 or Arai et al. 1989. J. Immunol. 142:274 (for IL-4). Any naturally occurring cytokine that biases a T cell response towards a Th2 phenotype can be administered to transplant recipient. The cytokine or cytokines to be administered can io be derived from any source, but are preferably human. In addition to naturally occurring cytokines, modified forms of cytokines can also be used, e.g., mutated forms that contain conservative substitutions, as long as they retain the ability to bind to their cognate cytokine receptor and bias a T cell response towards a Th2 phenotype. For example, a cytokine can be modified to reduce immunogenicity in the subject or to promote is increased half life. Exemplary methods of modifying amino acid sequences are known in the art. In one embodiment the cytokine that alters a T cell cytokine secretion profile is a fusion protein comprising the active portion of the cytokine (i.e., the portion that is capable of bias's a T cell cytokine secretion profile to a Th2 phenotype) and a second 20 non-cytokine protein. In a preferred embodiment, the second non-cytokine protein is a peptide derived from immunoglobulin molecule. Such a fusion protein, e.g., IL-41g or IL-10Ig, can be made using methods known in the art (see e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2:221; Linsley WO 93/00431 and U.S. Patent 5,770,197). 25 In another embodiment, cytokine analogs that mimic the function of cytokines can be used to bias an immune response towards a Th2 phenotype (e.g., U.S. patent 5,837,293). Antibodies In another embodiment, the cytokine profile altering agent is an antibody that 3o biases a response towards a Th2 phenotype can be administered to a transplant recipient. For example, antibodies to cytokines that reduce Th1 cytokine secretion can be WO 00/51630 PCT/USOO/05647 - 36 administered. For example, antibodies to IL-12, IL-2, and/or IFN-gamma can be administered. Antibodies for use in the claimed methods can be obtained commercially or can be made by immunization with the agent of interest. For example, IL-12 has been s cloned and sequenced (e.g., Wolf et al. 1991. J. Immunol. 146:3074) as have IL-2 (Taniguchi et al. 1983. Nature 302:305; Maeda et al. 1983. Biochem. Biophys. Res. Commun. 115:1040; Devos et al. 1983. Nucleic Acids Res. 11:4307) and antibodies can be generated against one or more of these cytokines and tested for their ability to bias an immune response towards a Th2 profile. 10 Antibodies can be from any source. However, to reduce the immunogenicity of the immunoglobulins themselves, antibodies are preferably of human origin or, if generated in other species, are "humanized" for administration to humans as described in the art. (e.g., Cancer Research. 1990. 50:1495; Brown et al. 1991. Proc. Natl. Acad. Sci. USA 88:2663; Kettleborough et al. 1991. Protein Engineering 4:773; US patent 15 5,853,697). Fragments of antibodies which maintain their ability to bias an immune response towards a Th2 profile are also included in the term "antibody." Methods of making such antibodies and fragments are known in the art (e.g., Harlow and Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring 20 Harbor, N.Y.) and can readily be screened e.g., for their ability to bind to the antigen to which they were raised using standard methods, e.g., using in an ELISA assay. The antibodies can be administered orally or parenterally in a pharmaceutically acceptable carrier to human subjects. suitable carriers for use in the present invention include, but are limited to, pyrogen-free saline. For parenteral administration a sterile 25 solution or suspension is prepared in a vehicle, e.g., saline, that may contain additives, such as ethyl oleate or isopropyl myristate, and can be injected, e.g., into subcutaneous or intramuscular tissues. Alternatively, the antibodies may be microencapsulated with either a natural or synthetic polymer into microparticles which produce a sustained release of antibody (Eldrige et al. 1989. Cur. Topics in Microbiol. and Immunol. 30 146:59; Oka et al. 1990. Vaccine. 8:573).

WO 00/51630 PCT/USOO/05647 - 37 For administration to transplant recipients, antibodies can be administered at a dosage readily determined by one of ordinary skill in the art. For example, the antibodies can be administered in a single dosage, e.g., between 10 mg and 20 mg/kg of body weight. Alternatively, patients can be given a dosage of 10 mg to 20 mg/kg 5 weekly until colitis symptoms subside. For oral administration, 500 mg to 1000 can be given. For parenteral administration, 10 mg to 20 mg/kg of body weight can be administered as a single or as a weekly intravenous injection. The skilled clinician will consider the age, weight, and condition of the individual in determining a final dose. For administration of antibodies in particulate form, 500 mg to 100 mg can be io microencapsulated as described for slow release over a prolonged period. Administration of Cells and Agents A variety of methods can be used for administering cells to subjects. For example, cells can be directly administered to the site of an organ in the recipient. 15 Alternatively, the cells are administered systemically. In one embodiment, in order to achieve systemic administration, the cells are injected intraperitoneally, (Wilson, J. et al. 1991. Clin. Biotech. 3(1):21-25). In a preferred embodiment, in order to achieve systemic administration the cells are injected intravenously. For example, the cells can be injected into the portal vein (Kay, M. 1993. Cell Trans. 2:405-406; Tejera, J.L. et al. 20 1992. Transplan. Proc. 24(l):160-161; Wiederkehr, J.C. et al. 1990. Transplantation 50(3):466-476), or the mesenteric vein (Grossman, M. et al. 1994. Nature Gen. 6:335 341; Wilson, J.M. et al. 1990. Proc. Natl. Acad Sci. 87:8437-8441). Intrasplenic injection of the cells (Rhim, J.A. et al. 1994. Science 263:1149-1152; Kay, M.A. 1993. Cell Trans. 2:405-406; Wiederkehr, J.C. et al. 1990. Transplantation 50(3):466-476), or 25 infusion of the cells into the splenic artery can also be performed. In another embodiment, the cells for use in the claimed methods can be delivered to multiple sites in the recipient. For example, the cells can be delivered directly to an organ and can also be administered systemically.

WO 00/51630 PCT/USOO/05647 - 38 In one embodiment, cells for transplantation can be bound to microcarrier beads such as collagen-coated dextran beads (Pharmacia, Uppsala, Sweden) (Wilson, J. et al. 1991. Clin. Biotech. 3(1):21-25) prior to transplantation. Cells can be administered in a pharmaceutically acceptable carrier or diluent as described herein. 5 Administration of the compositions and/or agents described herein can be in any pharmacological form that includes a therapeutically active amount of an agent and a pharmaceutically acceptable carrier. Administration of a therapeutically active amount of the subject agents and/or compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve treatment of the disorder in the case of the io transplant, and to inhibit rejection of the transplant in the case of an agent which alters a cytokine profile. A therapeutically active amount of an agent or composition may vary, for example, depending upon such factors as the disease state, age, sex or weight of the recipient, the type of cell transplanted, the site in the donor to which the transplanted 15 cells were administered, and the reason for administration. Such an amount can be readily determined by one of ordinary skill in the art. The optimal course of administration of the agents and/or compositions of the invention may also vary depending upon the subject to be treated. In certain embodiments a subject will be transplanted and treated with an agent that alters a 20 cytokine profile at the same time. In that instance, it may be desirable to administer the agent that alters a cytokine profile and the cell simultaneously, for example, in the form of a composition comprising both the cell and the agent. In other embodiments it will be desirable to transplant and administer the agent separately. In certain embodiments, staggered administration may be desirable to 25 achieve optimal alteration in a cytokine profile or optimal inhibition of the recipient immune response to the graft. For example, an agent which alters a cytokine profile can be administered alone prior to transplantation, or can be administered alone after transplantation. Cytokine profile altering agents can be administered systemically or locally using art recognized methods.

WO 00/51630 PCT/USOO/05647 - 39 A dosage regime may be adjusted to provide the optimum therapeutic response for each subject without undue experimentation. For example, the cytokine profile of the recipient can be measured (e.g., levels of cytokines can be measured in serum or by in situ staining or nucleic acid hybridization in the graft) to determine whether or not a 5 shift in cytokine profile has occurred in response to administration of the agent. Additionally or alternatively, graft rejection can be monitored in the recipient, e.g., by detecting the presence of cells or assaying for their continued function. Additionally or alternatively, cells (e.g. mixed or purified population of cells) can be removed from the recipient (e.g. , from periferal blood or from a biopsy specimen) and tested in vitro for 10 the cytokines made in response to graft antigens. Preferably, the cytokine production profile of the patient will be determined prior to transplantation and comparisions can be made to post transplantation cytokine production profiles. Alernatively, comparisons can be made to cytokine profiles of untransplanted, control individuals. Given the results of such assays, the skilled clinician will be capable of determining whether any of is these tests indicates that treatment with a cytokine profile altering agent should be modified. One skilled in the art will realize that dosages are best optimized by the practicing physician and methods for determining dosages are described, for example, in Remington's Pharmaceutical Sciences (Martin. Remington's Pharmaceutical Sciences, 20 Martin, latest edition, Mack Publishing Co., Easton, PA). To administer the subject agents or compositions by other than parenteral administration, it may be necessary to coat them with, or co-administer them with, a material to prevent its inactivation. An agent or composition of the present invention may be administered to an individual in an appropriate carrier or diluent, co 25 administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 7:27).

WO 00/51630 PCT/USOO/05647 - 40 The active agent or composition may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. 5 Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and lo suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In many cases, it will be is preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating active composition 20 or agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable 25 solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (e.g., agent or composition) plus any additional desired ingredient from a previously sterile-filtered solution thereof. When the active agent or composition is suitably protected, as described above, the protein may be orally administered, for example, with an inert diluent or an 3o assimilable edible carrier. As used herein "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic WO 00/51630 PCTIUS0O/05647 -41 and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the agent that alters a cytokine profile, it can be used in the therapeutic compositions of the invention. Supplementary active s compounds can also be incorporated into the compositions. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound io calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active agent or composition for the treatment is of individuals. In one embodiment of the invention, a cytokine profile altering agent is administered to a subject transiently, pre-transplantation, post-transplantation, or both. In another embodiment, the cytokine profile altering agent is administered chronically, i.e., over a prolonged period of time post-transplantation. Administration of the cytokine 20 profile altering agent to the subject can begin prior to transplantation of the cells into the subject. For example, initiation of agent administration can be a few days (e.g., one to fourteen days) before transplantation. Alternatively, agent administration can begin the day of transplantation or a few days after transplantation. Preferably, administration of the agent is continued for sufficient time such that a measurable change in cytokine 25 secretion profile occurs in the recipient or until graft acceptance is promoted in the recipient. Administration of the agent can continue chromically in order to maintain graft acceptance. Preferably donor cells will not be rejected by the recipient when administration of the agent ceases. For example, the agent can be administered for as short as three days or longer than three months following transplantation or over the 30 lifetime of the recipiant. Typically, the cytokine profile altering agent is administered for at least one week following transplantation. Induction of tolerance to the WO 00/51630 PCT/USOO/05647 - 42 transplanted cells in a subject can be measured by the lack of rejection of the transplanted cells after administration of the cytokine profile altering agent has ceased, e.g., by measuring continued graft function or presence of donor cells in the recipient. To accomplish these methods of administration, the cells and/or compositions of 5 the invention can be inserted into a delivery device which facilitates introduction of the cells and/or compositions into the subject. Such delivery devices include tubes, e.g., catheters, for infusing or injecting cells and fluids into the body of a recipient subject. In a preferred embodiment, the tubes additionally have a needle, e.g., a syringe, through which the cells of the invention can be introduced into the subject at a desired location. 10 The cells (and compositions containing the cells) of the invention can be inserted into such a delivery device, e.g., a syringe, e.g., syringe pump, in different forms. For example, the cells (and/or agent that alters a cytokine profile) can be suspended in a solution or embedded in a support matrix when contained in such a delivery device. As used herein, the term "solution" includes a pharmaceutically acceptable carrier or diluent 15 in which the cells of the invention remain viable. Pharmaceutically acceptable carriers and diluents include sterile saline and aqueous buffer solutions. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid to the extent that easy syringability exists. Preferably, the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of 20 microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Solutions of the invention can be prepared by incorporating cells and/or agent as described herein in a sterile pharmaceutically acceptable carrier or diluent and, as required, other ingredients. Support matrices in which the cells can be incorporated or embedded include 25 matrices which are recipient-compatible and which degrade into products which are not harmful to the recipient. Natural and/or synthetic biodegradable matrices are examples of such matrices. Natural biodegradable matrices include collagen matrices. Synthetic biodegradable matrices include synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid. These matrices can provide support and protection 30 for certain types of cells in vivo.

WO 00/51630 PCT/USOO/05647 - 43 Treatment Of Disorders Using Cells The subject cells can be transplanted into subjects having a disorder that would benefit by the transplantation of such a cell. To assess their therapeutic potential in humans, the cells of the invention can be introduced into animal transplantation models 5 of various disorders. The survival and/or function of transplanted cells can be monitored. For example, the ability of the cells to synthesize a protein which is not produced in active form in the recipient can be determined. This can be done, for example, by detecting the presence of the protein itself or an activity of the protein, e.g., an enzymatic activity. Additionally or alternatively, the cells themselves can be io detected, for example, by taking biopsy specimens and detecting the transplanted cells. Additional Treatments In certain embodiments, in addition to the administration of cytokine profile altering agents, additional agents can be administered to the subject to further inhibit the is immune response of the recipient subject and facilitate acceptance of the xenograft. Exemplary additional agents include: Soluble Forms Of Costimulatory Molecules In one embodiment, a soluble form of a costimulatory molecule can additionally 20 be administered to the transplant recipient. Soluble forms of costimulatory molecules have been found to block the transduction of a costimulatory signal in a T cell. In one embodiment, the soluble form of a costimulatory molecule is a soluble form of CTLA4. DNA sequences encoding the human and murine CTLA4 protein are known in the art, see e.g., Dariavich, et al. (1988) Eur. J. Immunol. 18(12), 1901-1905; Brunet, J.F., et al. 25 (1987) supra; Brunet, J.F. et al. (1988) Immunol. Rev. 103:21-36; and Freeman, G.J., et al. (1992) J. Immunol. 149, 3795-3801. In one embodiment, the soluble CTLA4 protein comprises the entire CTLA4 protein. In a preferred embodiment, a soluble CTLA4 protein comprises the extracellular domain of a CTLA4 protein. For example, a soluble, recombinant form of the extracellular domain of CTLA4 has been expressed in yeast 30 (Gerstmayer et al. 1997. FEBS Lett. 407:63). In other embodiments, the soluble WO 00/51630 PCT/USO0/05647 - 44 CTLA4 proteins comprise at least a portion of the extracellular domain of CTLA4 protein which retains the ability to bind to B7-1 and/or B7-2. In one embodiment the soluble CTLA4 protein or portion thereof is a fusion protein comprising at least a portion of CTLA4 which binds to B7-1 and/or B7-2 and at s least a portion of a second non-CTLA4 protein. In preferred embodiments, the CTLA4 fusion protein comprises a CTLA4 extracellular domain which is fused at the amino terminus to a signal peptide, e.g., from oncostatin M (see e.g., W093/0043 1). In a particularly preferred embodiment, a soluble form of CTLA4 is a fusion protein comprising the extracellular domain of CTLA4 fused to a portion of an lo immunoglobulin molecule. Such a fusion protein, CTLA4Ig, can be made using methods known in the art (see e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2:221; Linsley WO 93/00431 and U.S. Patent 5,770,197). In one embodiment, the soluble form of B7-1 or B7-2 or a combination of B7-1 and B7-2 can additionally be administered to the transplant recipient. DNA sequences 15 encoding B7 proteins are known in the art, see e.g., B7-2 (Freeman et al. 1993 Science. 262:909 or GenBank Accession numbers P42081 or A48754); B7-1 (Freeman et al. J. Exp. Med 1991. 174:625 or GenBank Accession numbers P33681 or A45803. In one embodiment, the soluble B7 protein comprises an entire B7 protein. In a preferred embodiment, a soluble B7 protein comprises the extracellular domain of a B7 protein. 20 For example, a soluble, recombinant form of the extracellular domain of CTLA4 has been expressed in yeast (Gerstmayer et al. 1997. FEBS Lett. 407:63). In other embodiments, the soluble B7 proteins comprise at least a portion of the extracellular domain of B7 protein which retains the ability to bind to CTLA4 and/or CD28. In one embodiment the soluble B7 protein or portion thereof is a fusion protein 25 comprising at least a portion of B7 which binds to CD28 and/or CTLA4 and at least a portion of a second non-B 7 protein. In preferred embodiments, the B7 fusion protein comprises a B7 extracellular domain which is fused at the amino terminus to a signal peptide, e.g., from oncostatin M (see e.g., W093/0043 1). In a particularly more preferred embodiment, a soluble form of B7 is a fusion 30 protein comprising the extracellular domain of B7 fused to a portion of an immunoglobulin molecule. Such a fusion protein, a B7Ig, can be made using methods WO 00/51630 PCT/US00/05647 -45 known in the art (see e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2:221; Linsley WO 93/00431, U.S. Patent 5,770,197, and U.S. Patent 5,580,756). Administration of Lymphocytes 5 In one embodiment, administration of a population of lymphocytes taken from the same donor or a related donor can accompany administration of a cytokine profile altering agent. In a preferred embodiment, the population of lymphocytes comprises T lymphocytes. In preferred embodiments, the population of lymphocytes is a xenogeneic population of lymphocytes. 10 In one embodiment, the lymphocytes are administered systemically. In a preferred embodiment, the lymphocytes are administered intravenously. In another embodiment, the lymphocytes are administered intraperitoneally. In yet another embodiment, the lymphocytes are administered intrasplenically. In a preferred embodiment, the lymphocytes are administered into the portal 1 vasculature of the subject (Morita et al. 1998. Proc. Natl. A cad. Sci. 95:6947; Hirakawa et al. 1993. Transplant. Proc. 25:346; Zhang et al. 1994. Eur. J. Immunol. 24:1558; Yu et al. 1994. surgery. 116:229). The administration of the cells of the invention into this vessel can be accompanied by a step which involves maintaining portal blood pressure. As used herein, a "maintaining portal blood pressure" of a subject refers to maintaining a 20 relatively stable blood pressure level in a subject during and after transplantation of the cells of the invention relative to the portal blood pressure level of the subject prior to transplantation of the cells, e.g., lymphocytes, of the invention. This may involve temporarily decreasing portal blood pressure so that the infusion of cells does not increase portal blood pressure. 25 In one embodiment, the portal blood pressure of a subject is decreased or maintained through the use of an transjugular intra-hepatic porto-systemic shunt (TIPS). See e.g., Rossle, M. and Ring, E.J. in Progress in Liver Disease (Saunders, 1994) Vol. XII:177-189; Ochs, A. et al. 1995. N. Engl. J Med 332(18):1192-1196; Rossle, M. et al. 1994. N. Engl. J. Med 330(3):165-171. The TIPS procedure includes the passage of a 30 tube e.g., needle, catheter, which acts as a shunt, from the jugular vein into a hepatic vein and then advancing the tube through the liver parenchyma into a portal vein branch.

WO 00/51630 PCTIUSOO/05647 - 46 This shunt allows blood flowing through the portal venous system to pass directly from the portal vein into the hepatic vein, thereby bypassing the liver parenchyma. Bypass of the liver parenchyma results in a decrease or at least a maintenance of the portal blood pressure in a subject after transplantation of the cells of the invention. The step of 5 decreasing or maintaining the portal venous blood pressure in a subject can be performed prior to, during, or after transplantation of the cells of the invention. Preferably, the cells of the invention are administered to the subject via the TIPS catheter into the portal vein, thereby eliminating the need for providing an additional route of administration for the cells. In a preferred embodiment, the step of decreasing or maintaining the portal 10 venous blood pressure in a subject is performed after transplantation of the cells of the invention. Typically, cells of the invention are transplanted and the portal blood pressure is decreased or maintained by performance of the following steps: 1) transjugular cannulation of the portal vein; 2) transplantation of cells into the liver via the portal vein, e.g., through the use of the cannula in the portal vein; and 3) placement 15 of the TIPS within the liver. Immunosuppressants In another embodiment, the subject method further comprises administration of an immunosuppressive drug or general immunosuppessant. A preferred agent for use in 20 inhibiting T cell activity in a recipient subject is an immunosuppressive drug. The term "immunosuppressive drug or agent" is intended to include pharmaceutical agents which inhibit or interfere with normal immune function. A preferred immunsuppressive drug is cyclosporin A. Other immunosuppressive drugs which can be used include FK506, RS-61443, and deoxyspergualin. In one embodiment, the immunosuppressive drug is 25 administered in conjunction with at least one other therapeutic agent. Additional therapeutic agents which can be administered include steroids (e.g., glucocorticoids such as prednisone, methyl prednisolone and dexamethasone) and chemotherapeutic agents (e.g., azathioprine and cyclosphosphamide). Suitable immunosuppressive drugs are commercially available (e.g., cyclosporin A is available from Sandoz, Corp., East 30 Hanover, NJ).

WO 00/51630 PCT/USOO/05647 - 47 An immunsuppressive drug is administered in a formulation which is compatible with the route of administration. Suitable routes of administration include intravenous injection (either as a single infusion, multiple infusions or as an intravenous drip over time), intraperitoneal injection, intramuscular injection and oral administration. For 5 intravenous injection, the drug can be dissolved in a physiologically acceptable carrier or diluent (e.g., a buffered saline solution) which is sterile and allows for syringability. Dispersions of drugs can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Convenient routes of administration and carriers for immunsuppressive drugs are known in the art. For example, cyclosporin A can be io administered intravenously in a saline solution, or orally, intraperitoneally or intramuscularly in olive oil or other suitable carrier or diluent. An immunosuppressive drug is administered to a recipient subject at a dosage sufficient to achieve the desired therapeutic effect (e.g., inhibition of rejection of transplanted cells). Dosage ranges for immunosuppressive drugs, and other agents ,s which can be coadministered therewith (e.g., steroids and chemotherapeutic agents), are known in the art (See e.g., Freed et al. New Engl. J. Med. 1992. 327:1549: Spencer et al. 1992. New Engl. J. Med 327:1541; Widner et al. 1992. New Engl. J. Med. 327:1556; Lindvall et al. 1992. Ann. Neurol. 31:155; and Lindvall et al. 1992. Arch. Neurol. 46:615). A preferred dosage range for immunosuppressive drugs, suitable for treatment 20 of humans, is about 1-30 mg/kg of body weight per day. A preferred dosage range for cyclosporin A is about 1-10 mg/kg of body weight per day, more preferably about 1-5 mg/kg of body weight per day. Dosages can be adjusted to maintain an optimal level of the immunosuppressive drug in the serum of the recipient subject. For example, dosages can be adjusted to maintain a preferred serum level for cyclosporin A in a human subject 25 of about 100-200 ng/ml. It is to be noted that dosage values may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted over time to provide the optimum therapeutic response according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage 30 ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

WO 00/51630 PCT/USOO/05647 - 48 In one embodiment of the invention, an immunsuppressive drug is administered to a subject transiently after transplantation of the subject. Administration of the drug to the subject can begin prior to transplantation of the cells into the subject. For example, initiation of drug administration can be a few days (e.g., one to fourteen days) before s transplantation. Alternatively, drug administration can begin the day of transplantation or a few days after transplantation. Preferably, administration of the drug is continued for sufficient time such that, in combination with the cyokine profile altering agent, aceptance of the graft is promoted. In one embodiment, donor specific tolerance to the graft is induced. Acceptance of the graft is indicated by the presence of the transplanted lo cells after administration of the immunosuppressive drug has ceased. Tolerance can be determined using standard methods, e.g., failure of host immune cells to respond to donor antigen (e.g., by proliferating or in a cellular cytotoxicity assay). Acceptance of transplanted tissue can be determined morphologically (e.g., with biopsies of liver) or by assessment of the functional activity of the graft. i5 Another type of agent which can be used to inhibit the anti-graft immune response in a subject is an antibody, or fragment or derivative thereof, e.g., which depletes or sequesters immune cells in a recipient. For example, in one embodiment, antibodies which are capable of depleting or sequestering T cells in vivo when administered to a subject can be given. Such antibodies are known in the art. Typically, 20 these antibodies bind to an antigen on the surface of a T cell. Polyclonal antisera can be used, for example anti-lymphocyte serum. Alternatively, one or more monoclonal antibodies can be used. Preferred T cell-depleting antibodies include monoclonal antibodies which bind to CD2, CD3, CD4 or CD8 on the surface of T cells. Antibodies which bind to these antigens are known in the art and are commercially available (e.g., 25 from American Type Culture Collection). A preferred monoclonal antibody for binding to CD3 on human T cells is OKT3 (ATCC CRL 8001). The binding of an antibody to surface antigens on a T cell can facilitate sequestration of T cells in a subject and/or destruction of T cells in a subject by endogenous mechanisms. Alternatively, a T cell depleting antibody which binds to an antigen on a T cell surface can be conjugated to a 30 toxin (e.g., ricin) or other cytotoxic molecule (e.g., a radioactive isotope) to facilitate destruction of T cells upon binding of the antibody to the T cells. See WO 95/26740, WO 00/51630 PCT/USOO/05647 -49 published on 12 October 1995, for further details concerning the generation of antibodies which can be used in the present invention. Another type of antibody which can be used to inhibit T cell activity in a recipient subject is an antibody which inhibits T cell proliferation. For example, an 5 antibody directed against a T cell growth factor, such as IL-2, or a T cell growth factor receptor, such as the IL-2 receptor, can inhibit proliferation of T cells (See e.g., DeSilva, D.R. et al. 1991. J. Immunol. 147:3261-3267). Accordingly, an anti-IL-2 or an anti-IL-2 receptor antibody can be administered to a recipient to inhibit rejection of a transplanted cell (see e.g. Wood et al. 1992. Neuroscience 49:410). Additionally, both an anti-IL-2 10 and an anti-IL-2 receptor antibody can be coadministered to inhibit T cell activity or can be administered with another antibody (e.g., which binds to a surface antigen on T cells). An antibody which depletes, sequesters or inhibits T cells within a recipient can be administered at a dose and for an appropriate time to inhibit rejection of cells upon transplantation when administered in conjunction with a cytokine profile altering agent. is Antibodies are preferably administered intravenously in a pharmaceutically acceptable carrier or diluent (e.g., a sterile saline solution). Antibody administration can begin prior to transplantation (e.g., one to five days prior to transplantation) and can continue on a daily basis after transplantation to achieve the desired effect (e.g., up to fourteen days after transplantation). A preferred dosage range for administration of an antibody to a 20 human subject is about 0.1-0.3 mg/kg of body weight per day. Alternatively, a single high dose of antibody (e.g., a bolus at a dosage of about 10 mg/kg of body weight) can be administered to a human subject on the day of introduction of the cells and/or agent that alters a cytokine secretion profile into the subject. The effectiveness of antibody treatment in depleting T cells from the peripheral blood can be determined by comparing 25 T cell counts in blood samples taken from the subject before and after antibody treatment. Dosage regimes can be adjusted over time to provide the optimum therapeutic response according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and are not intended to limit the scope or 30 practice of the claimed composition.

WO 00/51630 PCT/USOO/05647 - 50 Methods Of Determining The Level Of Cytokine Production The cytokine secretion profile of a recipient can be determined pre and/or post xenotransplantation. In one embodiment, cells of the recipient (e.g., peripheral blood mononuclear cells) are cocultured in vitro with cells from the same species as the donor. 5 In a preferred embodiment, the cells are from the same donor (e.g., are cells that were cryopreserved at the time of transplantation), or are from another donor that is genetically related to the donor of the transplanted cells. The donor cells can be irradiated or fixed prior to coculturing them with the recipient responder cells using standard methods. The lymphokines secreted by the recipient cells can be measured io from the in vitro coculture supernatants using standard techniques, e.g., as described in the appended examples and as known in the art. For example, changes in cytokine levels can be determined using a commercially available ELISA kit (R&D systems Quantikine kit, Minneapolis, Minn.) or by bioassay using a cytokine dependent cell line, e.g., CTLL-2 (ATCC, Rockville, MD). IFN-gamma can be measured using a kit available is from Endogen (Cambridge, MA). In another example, cytokine production can be measured using an ELISPOT assay (e.g., Williams et al. 1994. J. Infect. Disease. 170:946-954). The transcription of Cytokine genes can also be measured (e.g., using PCR, RNA protection assays, or northern blot analysis). Cytokine production can be measured in either primary or secondary cocultures, e.g., as is known in the art and is 20 described in Examples. As an alternative or in addition to measuring cyokine profiles of a transplant recipient in response to donor antigen, the polyclonal response of the recipient T cells can be measured. Polyclonal T cell stimulators are known in the art and include, e.g., anti-CD3 antibody, and phorbol myristate acetate and ionomycin. 25 In one embodiment, limiting dilution analysis can be performed to ThI and Th2 cells isolated and a determination of the precursor frequency of each type of cell can be calculated pre and post transplantation as an indication of the cytokine secretion profile of the recipient and whether or not the secretion profile has been altered towards a Th2 phenotype.

WO 00/51630 PCTIUS00/05647 -51 When more sensitive detection of cytokines is desired, techniques other than measuring cytokines in in vitro culture can be employed. For example, the cytokines produced by the recipient can also be measured in situ, e.g., by removing peripheral blood mononuclear cells or by taking a biopsy sample from the recipient and staining the 5 cells for cytokines. For direct staining with fluorescence-conjugated antibodies to cell surface antigens using standard methods (e.g., U.S. patent 5,767,097). For example, the cells can be treated with Brefeldin A and stained with PE-labeled anti-IL-2 antibody and then washed in permeabilization buffer prior to FACS analysis. Alternatively, nucleic acid molecules can be isolated from recipient cells, e.g., from recipient peripheral blood io lymphocytes or from infiltrates into a graft site. mRNA levels can be measured or reverse transcribed into cDNA for amplification using the polymerase chain reaction prior to quantitation. PCR can be performed using parameters which have been optimized for the detection of cytokines. Primers for human cytokines are commercially available, e.g., from Stratagene, La Jolla, CA. Alternatively, mRNA encoding a 15 particular cytokine can be measured by in situ hybridization. The sequences of the Th2 and/or Th1 cytokines to be detected are known in the art and can be found, for example, on GenBank or in the references set forth supra. In addition, primers for amplifying these sequences and/or oligonucleotide probes that specifically bind to Thl and Th2 cytokine sequences are known in the art and can e.g., 20 be chemically synthesized. In addition, many are commercially available. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Genetics; Molecular 25 Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al. (Cold Spring Harbor Laboratory Press 1989.); Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel, F. et al. (Wiley, NY 1995.); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984)); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1984)); the 3 0 treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, WO 00/51630 PCT/USOO/05647 - 52 London (1987)); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds. (1986)); and Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972)). This invention is further illustrated by the following examples which should not s be construed as limiting. The contents of all references and published patents and patent applications cited throughout the application are incorporated herein by reference. Examples io Example 1 Blocking the human anti-porcine T cell response with anti-MHC class I antibody not only inhibited proliferation and induced subsequent hyporesponsiveness, it also led to dramatic increase in the type 2/type 1 cytokine ratio. The human PBMC response to porcine MHC class I+/class II- aortic smooth muscle cells (SMC) produced high levels 15 of IL-2 and IFN-y. In the presence of PT85 F(ab') 2 antibody. most of the response was blocked and the production of both IL-2 and IFN-y were inhibited. In a secondary response to porcine SMC without further PT85 blocking, proliferation was reduced relative to control group and cytokine production of IL-2 and IFN-y were again reduced while the production of IL-4 and IL-10 became enhanced. In addition, secondary 20 stimulation using anti-CD3/anti-CD28 immobilized antibody pair on previously (PT85) blocked cells also inhibited production of IL-2 and IFN-y while increased the levels of IL-4 and IL-l 0. These findings demonstrate that PT85 not only blocked proliferation but induced a cytokine profile change from type 1 to type 2. In addition, RNA protection assay demonstrated a clear inhibition of IL-I5 message during anti-class I masking 25 conditions both in the primary and secondary cultures. Since activated monocytes and dendritic cells are the only known producers of the potent T cell growth factor IL- 15, our findings support a direct role in inhibiting not only the pathway of direct recognition but the indirect pathway which requires the presentation of donor antigen peptide on responder APC. These results are qualitatively similar to the altered peptide ligand or co 30 receptor deficient TCR signal that lead to conditions of unresponsiveness. Thus, a single WO 00/51630 PCT/US00/05647 - 53 treatment with PT85 F(ab')2 directed against the MHC class I molecule on donor cells biases the T cell cytokine secretion profile towards Th2 phenotype and induces T cell tolerance in vitro that can provide long-term graft survival in the porcine to human cell transplantation. 5 The following methods were used in the examples: Construction of chimeric MHC Class I: Total RNA was isolated from Balb/c fibroblast cell lines using RNAzolTM B following the manufacture's procedure (Tel-Test, Friendswood, TX). The first strands of cDNA from mRNA were synthesized using the io Advantage RT-for-PCR TM following the manufacture's procedure (Clontech, Palo Alto, CA). The cDNA of H2Dd was amplified by PCR using a 5' primer containing an Xho I linker tail (CGA TCT CGA GAT GGG GGC GAT GGC TCC GCG CAC) (SEQ ID NO: 1) and a 3' primer containing an Hind III linker tail (ATC GAA GCT TTC ACA CTT TAC AAT CTG GGA GAG) (SEQ ID NO:2) to facilitate cloning into pGEM-7Zf 15 (Promega, Madison, WI). The H2Dd clone was sequenced to assure reliable amplification. The PD1 gene was used as a template for the chimeric constructs. The MMP construct: H2Dd/pGEM-7Zf was linearized with Bsm I and partially digested with BsrD I in order to excise exon 4 portion of this gene. The exon 4 of pig PD1 corresponding to the a3 domain was amplified by PCR using primers that contained the 20 junctions of mouse exon 3/pig exon 4 and pig exon 4/mouse exon 5. The 5' primer also contains the Bsm I site for ligation to the mouse exon 3 (TCG ATC GAA TGC TAC GCT GCT GCG CAC AGA CCT TCC AAA GAC ACA TG) (SEQ ID NO:3) and the 3' primer contains the BsrD I site for ligation to the mouse exon 5 (AAC AGC AAT GAT TAC TGT GTT AGT CTT GGT GGA TGA AGG AGG CTC CCA TCT CAG GGT 2s GAG) (SEQ ID NO:4). The resulting PCR product was digested with Bsm I and BsrD I. The fragment was then ligated into the linearized H2Dd/pGEM-7Zf vector. The presence of exon 4 of pig in this vector added a new restriction site (Bgl II) that was used for screening. A clone containing the pig insert was sequenced and the new chimeric gene was transferred to the expression vector pcDNA3.TK (Invitrogen corporation, Carlsbad, 3 0 CA) at the Xho I and Hind III sites. The PPM construct: H2Dd/pGEM-7Zf was linearized with Not I and partially digested with BstYI to excise exons 2-3 of H2Dd.

WO 00/51630 PCT/USOO/05647 - 54 Exons 2-3 of PD 1 corresponding to the al and a2 domains were amplified by PCR using the 5' primer representing the junction mouse exon 1/pig exon 2 and containing the Not I site for insertion into H2Dd/pGEM-7Zf (GAT CGA TCG CGG CCG CCC TGG GTC CGA CTC AGA CCC GCG CTG GTC CCC ACT CCC TGA) (SEQ ID NO:5) and the 5 3' primer representing the junction pig exon 3/mouse exon 4 containing the site BstY I for ligation (TCG ATC GGA TCT GCG CGC TGC AGC GTG TCC TTC CCC) (SEQ ID NO:6). The PCR product was treated in the same manner as for the MMP construct. Table 1 shows the structure of the chimeric proteins. MHC class I on mouse cells Pig PBL (Balb/c fibro or C1498) (haplotype) Ab PPP MMM MMP PPM cc dd 34-2-12 - - ++ N/A N/A (ax3) 34-5-8s ++ +++ ++ - N/A N/A (al/ca 2) 9-3 +++- ++- ++ PT85 +++ +++ +++ +++ 74-11-10 ++ - - ++ ++ +++ 2.12.3 - - +/ 2.27.3 ++ - - ++ ++ +++ 2.28.1 +- - - - +/ 2.32.1 -+ +++ 1158A - -. ++.+++ 7-34-1 - - - 10 PPP: Pig PD1 alpha 1,2 and 3 MMM: Mouse H2Dd alpha 1,2 and 3 MMP: Mouse alpha 1 and 2; Pig alpha 3 PPM: Pig alpha 1 and 2; Mouse alpha 3 15 N/A: non applicable or not performed WO 00/51630 PCT/USOO/05647 - 55 Expression of chimeric MHC class I: Native H2Dd and PDl as well as chimeric MMP and PPM were transfected by electroporation into Balb/c fibroblasts and C 1498, a mouse lymphoma cell line (ATCC, Rockville, MD) using 290 volts in serum free RPMI. Stable transfectants were selected with G418 (Gibco/BRL, Grand Island, NY) at a 5 concentration of 1,200 pig/ml for Balb/c fibroblasts and 800 ptg/ml for C1498 for 2 weeks. Magnetic bead separation was performed on the PD I and MMP transfectants using monoclonal antibody 9-3 as the primary Ab, previously determined to be pig MHC Class I a3 domain specific. Dynabeads M-450 coated with goat antimouse IgG (Dynal, Oslo, Norway) were used to select cells expressing either protein. Magnetic bead io separation was performed as well on the H2Dd and PPM transfectants by using 34-2-12 (PharMingen, San Diego, CA) as the primary Ab. Clones were selected by limiting dilution in a 96-well plate. The selected cells were analyzed by flow cytometry using the Becton Dickinson FACScan. A variety of primary Abs were analyzed for their domain specificity on MHC class I using FITC conjugated goat anti-mouse IgG (H+L) is secondary reagent for detection (Jackson Immunoresearch laboratory, Bar Harbor, ME). Establishing porcine smooth muscle cells (SMC): SMC were derived from porcine aorta. Aorta from SLAaa and SLAdd miniature pigs were provided by David H. Sachs (Massachusetts General Hospital, Boston, MA). SMC were isolated according to 20 previously described procedure (McMahon et al. 1985. In Vitro Cell Dev Biol. 21:674). Briefly, endothelial cell layer was removed and small pieces of tissue containing the SMC layer was cut and digested with equal volumes of collagenase-P (0.8 mg/ml) and trypsin-versene (BioWhittaker) at 37'C for 60 minutes. The cell layer was washed several times and cultured with DMEM supplemented with 10% FCS in 100 mm tissue 25 culture dishes. Before reaching confluence, cells were harvested and replated at a 1 to 3 dilution. Based on morphology, greater than 98% of the cells used were SMC. Experiments were conducted using cells between passage 12 and 20. Purification of human peripheral blood T cell subsets: Peripheral blood was collected 3 0 from normal volunteers, and peripheral blood mononuclear cells (PBMC) were purified by the Ficoll-Paque gradient (Yamada et al. 1995. J. Immunol. 155:5249). Purified T WO 00/51630 PCT/US00/05647 - 56 cells, CD4+ and CD8+ T cells were enriched by negative selection using magnetic beads (Dynal, Oslo, Norway). To generate enriched T cells, B cells, macrophages, and NK cells were removed by incubating the PBMC with mouse anti-human CD14, CD 19, CD56, CD 16 followed by goat anti mIg magnetic beads. For further purification, T 5 cells were incubated with anti-CD4 or anti-CD8 mAbs (5 pg/ml) at 4'C for 45 minutes. Goat anti mouse IgG magnetic beads (Dynal, Oslo, Norway) were added at 10: 1 (bead:cell) ratio and discarded unwanted cells. After three rounds of magnetic bead separation, the negatively enriched T cells, CD4+ or CD8+ T cell subsets were greater than 95% pure as determined by FACS analysis. 10 Human anti-porcine (SMC) proliferation assay: SMC were plated in 96-well flat bottom microtiter plate at a density of 3x104 cells/well at 37 0 C. Cells were incubated with media alone or with the blocking antibody (1 Otg/ml) 1 hour at 4'C. Hybridomas producing the anti-porcine MHC class I antibodies PT-85 were purchased from VMRD, 15 Inc. (Pullman WA; Davis et al. 1987. Vet. Immunol. Immunopathol. 15:337). Monoclonal antibody 9.3 was raised against porcine PBL as previously described (Oettinger et al. 1997. Xenotransplantation 4:252). Hybridoma producing the anti human MHC class I monoclonal antibody, W6/32, was obtained from ATCC (Barnstable. 1978. Cell 14:9). Human PBMC were then added at 3x10 5 cells/well, 20 whereas human CD4 or CD8 T cells were added at lx 105 cells/well. All experiments were done in AIM V media (Gibco) supplemented with 5% heat inactivated FCS. For secondary stimulation, cells were harvested and restimulated with fresh SMC or immobilized anti human CD3 antibody (5 ptg/ml) in 96-well flat bottom plates for the times indicated in text. Supernatants were harvested from appropriate cultures at 25 indicated times and frozen for cytokine detection. To determine the proliferative response, cells were pulsed with 3 H-thymidine (1 ICi/well) for 20 hours, and harvested with a cell harvester (Packard Instruments, Meriden, CT)at indicated time points. The thymidine incorporation was determined by counting the filter plate using a microplate scintillation counter (Packard Instrument, Model #B9906). 30 WO 00/51630 PCT/USOO/05647 - 57 FACS analysis. Fluorescein isothiocyanate (FITC) or R-phycoerythrin (PE) conjugated monoclonal antibodies (mAb) to CD4, CD8, CD 14 and CD 19 were purchased from PharMingen (San Diego, CA). Cells (5x10 5 ) were incubated with FITC or PE conjugated mAb for 60 minutes at 4'C. Cells were washed and analyzed by two-color flow 5 cytometric analysis using a FACScan (Becton Dickson, Mountain View, CA). Viable cells were gated using propidium iodide (PI) at 2.5 ng/ml. ELISA. The antibodies used in the IFN-y, IL-2, IL-4 and IL- 10 assays were purchased from PharMingen (San Diego, CA). All assays were performed according to the 10 manufacturer's protocol. Briefly, plates were coated with respective anti-cytokine mAb and culture samples were captured on plates, and detected by the respective biotin labeled second anti-IFN-y anti-IL-2, anti-IL-4 or anti-IL- 10 mAbs. The assay was developed by using strepavidin-horseradish peroxidase and substrate. The color reaction was stopped by addition of equal volume of 1M H 2

SO

4 . The absorbance of the assay 15 plate was read at 450 nm using a microplate reader (Model 3550, Bio-Rad Labs, Hercules, CA). Recombinant human IFN-y, IL-2, IL-4 and IL-10 cytokines were used as standards, respectively. The sensitivity of the assay was determined to be between 1 pg/ml to 10 ng/ml for all cytokines. 20 RNase Protection Assay. Total RNA was prepared using Trizol reagent according to the manufacturer's protocol (Gibco). RNase protection assay was performed using the RiboQuant multi-probe RNase protection assay kit according to the manufacturer's protocol (PharMingen). Briefly, the multi template probe was labeled with (aX-32P)UTP (3000 Ci/mmol, NEN) for 1 hour at 37 0 C. After DNase treatment and phenol-chloroform 25 extraction, the probe was hybridized with sample RNAs overnight at 56'C. After RNase and proteinase K treatment and phenol-chloroform extraction, samples were precipitated with ammonium acetate and ethanol, and run on a 5% acrylamide gel (19:1 acrylamide/bis). The protected probes was resolved by exposing the gel to a X-ray film for an optimal time, and developed by a film processor. 30 WO 00/51630 PCTIUSOO/05647 - 58 RESULTS: Proliferation of human PBMC to class I positive cells: Inhibition with SLA-class I reactive PT85 antibody. In order to assess the role and mechanism of PT85 masking in the human antiporcine xenogeneic response with relevance to xeno cell transplantation, a 5 proliferation assay was established that measured human T cell response to porcine cells that expressed only MHC class I antigens. Human T cell responses were measured using primary SMC from two partially inbred miniature pigs and isolated embryonic brain cells (EBC) from outbred pigs. Proliferation of human T cells against porcine SMC or EBC showed similar primary (day 7) profiles that were inhibited with the anti-class I 10 PT85 antibody (Figure la). For example, the human PBMC responses to SMC from the SLAaa and SLAdd and to EBC from outbred pigs were inhibited with PT85 by more than 50% compared to no antibody, control antibody or anti-human HLA class I W6/32 antibody. Moreover, restimulation as measured in a 3 day assay without additional antibody added, showed hyporesponsiveness in the previously PT85 blocked cell group is relative to controls (Figure lb). The HLA class I specific W6/32 mAb group was included as control in our model to help delineate anti-class I reactivity directly effecting the responding human T cells. Consistent inhibition was observed in proliferation studies with 8 different human blood donors in 12 experiments. Primary and secondary proliferation studies 20 were expanded to assess the role of PT85 blocking during the primary stimulation. PBMC from a number of different donors were stimulated with porcine SMC with or without the blocking antibodies. Primary stimulation was consistently inhibited by more than 50% in the human anti-porcine T cell response when blocked with the anti-class I PT85 F(ab') 2 antibody but not with the W6/32 or mouse IgG F(ab') 2 control antibodies 25 in all twelve human blood donors (Figure 2a). In a secondary stimulation with porcine SMC in the absence of any blocking antibody, there was greater than 60% inhibition in proliferation by day 3 for the initially masked group (Figure 2b). These results suggested that blocking the MHC class I molecule with the PT85 antibody may lead to subsequent T cell unresponsiveness. Interestingly, blocking with a second anti-porcine class I 3o antibody 9-3 had no detectable effect on the human anti-porcine T cell response.

WO 00/51630 PCT/IUSOO/05647 - 59 The direct human anti-porcine response is initiated by CD8+ T cells and inhibited by anti-MHC class I and anti-CD8 Abs: Experiments were designed to evaluate the role of anti-class I PT85 F(ab') 2 antibody masking in the human anti-porcine T cell response. Proliferation of purified CD4+ or CD8+ T cells were tested against MHC class I positive s porcine SMC. While, human PBMC gave consistent robust proliferation against the MHC class II-negative porcine SMC, number of different anti-class I antibodies were able to show inhibition (Figure 3). The PBMC response was inhibited with PT85 by 50% and less so with 9.3 and 74-11-10 mAbs. When isolated CD8+ T cells from different donors were tested, consistent proliferation was observed in response to porcine io SMC that were inhibited with anti-class I mAb. For example, the CD8+ T cell response was completely inhibited with PT85 and 9.3 F(ab')2 as well as several other anti-porcine SLA class IIgG antibodies (Figure 3). In contrast, the human HLA class I specific W6/32 F(ab')2 antibody did not inhibit the CD8+ T cell response. In order to assess whether CD4+ T cells were dependent on the CD8+ T cells for a direct response to the 15 class I+ SMC we conducted a mixing experiment using purified CD4+ and CD8+ T cells. No increase above the CD8 response was evident indicating that CD4 cells if contributing to the PBMC response were responding to antigen presented on human APCs by the indirect pathway. The CD8+ T cell response was completely inhibited with PT-85 and 9.3 but not 20 with W6/32 mAb F(ab')2. Likewise, several other mAbs, including the 74-11-10 IgG completely inhibited the CD8+ T cell response. Moreover, this proliferation was completely inhibited with the anti-CD8 (OKT8), but not with the anti-CD4 (OKT4)mAb. While the purified CD8+ T cells(1x10 5 ) responded to porcine MHC class I+ SMC, in most experiments, the proliferation was lower than that seen with PBMC 25 (3x10 5 ). None of the primary PBMC response were inhibited completely. In contrast, purified CD4+ T cells showed no detectable proliferation. The ability of the purified CD4+ T cells to proliferate in culture was confirmed using mitogenic stimulation. Moreover, mixing purified CD4+ T cells and CD8+ T cells did not increase or decrease the response shown by the CD8+ T cells alone. These findings indicate that CD8+ T 30 cells may be the initial responders and that blocking these cells with anti-porcine MHC class I antibody prevents further stimulation mediated by the indirect pathway.

WO 00/51630 PCT/USOO/05647 - 60 Domain specificity of the anti-MHC class I antibodies. In order to delineate differences between several anti-MHC class I mAbs, porcine and murine "exon-shuffled" MHC class I transfected mouse cell lines were established to examine domain specificities of these antibodies. The porcine and mouse MHC class I chimeric molecules were 5 generated using cd, cx2 and a3 domains from pig PD1 (Sullivan et al. 1997. J. Immunol. 159:2318) and mouse H-2 D genes. The recombinant genes were transfected and expressed in C1498 cells and Balb/c fibroblasts. Stable transfected lines were tested for reactivity with anti-porcine MHC class I mAbs. Only mAb 9.3 required the PD1 a3 domain for reactivity, while mAb PT85, 74.11.10 and 2.27.3 did not require the o3 1o domain for reactivity. These three Abs may require both the al and/or aX2 domains for reactivities. In contrast, mAb 7.34.1 required the pig P2 microglobulin for proper recognition of pig MHC class I molecules since it only reacted with PD 1 transfected mouse cells in the presence of pig, p2-microglobulin found in pig serum. 15 Blocking with PT85 F(Ab') 2 antibody inhibits IL-2 and IFN-y and induces IL-4 and IL 10 production: To assess whether inhibition of proliferation in the primary and secondary human anti-porcine T cell responses were correlated with changes in cytokine production, the culture supernatants were examined for IL-2, IFN-y, IL-4 and IL- 10 cytokine levels. Primary and secondary stimulation conditions were set up to harvest and 20 measure the cytokine production profile of the different cultures. Tissue culture supernatants generated from human anti-porcine SMC stimulation assays from day 2 to day 6 were collected. Supernatants from at least five different donor PBMC were monitored for cytokine production by ELISA. IL-2 production was evident in the supernatants of the unmasked human anti-porcine responses between days 2 to 5 (Figure 25 4). The level of IL-2 was decreased by days 6 and 7 during culture primarily due to consumption by T cell proliferation. However, supernatants from PT85 masked cell cultures showed decreased levels of IL-2 from day 2 to day 5. While there were differences in the levels of IL-2 detected from the different PBMC donors, all had a striking decrease in IL-2 levels. Likewise, the level of IFN-y production was inhibited in 30 cultures that were PT85 masked (Figure 5). Five individual donor PBMC were tested in WO 00/51630 PCT/USOO/05647 - 61 response to porcine SMC with or without the PT85 blocking. Supernatants from day 2 to day 7 were harvested and tested for IFN-gamma production. While all made substantial levels of the cytokine in the absence of masking, little IFN-gamma was present in cultures containing the masking PT85 antibody (Figure 5; open box). The actual s detected levels in the absence of the PT85 masking varied between 20 pg/ml to 400 pg/ml for IL-2 and between 800 pg/ml to 4500 pg/ml for IFN-gamma, the inhibition of both Type 1 cytokines was consistent in all of the donors tested (n=12). Since PT85 mAb cross-reacts with human HLA class I antigens, PBMC were stimulated with immobilized anti-CD3/CD28 mAbs with or without the masking lo antibody to assess whether PT85 had any direct effect on the responding human T cells. The results from these experiments show that the level of IFN-gamma was not altered in the presence of PT85, suggesting no direct effect on responding cells. Thus, inhibiting the production of IFN-y and IL-2 in a human anti-porcine T cell response in the presence of anti-MHC class I antibody blocking suggested a deviation from the type I pathway. is In contrast to type 1 cytokine inhibition, the production of IL- 10 in the presence of masking antibody did not decrease relative to control conditions (Figure 6). For example, while the IL- 10 levels in the day 6 supernatant ranged between 15 pg/ml to 100 pg/ml depending on donor PBMC, no decrease in levels was observed. In fact, two of the samples showed moderate IL- 10 increases during the primary stimulation (Figure 20 6; top 2 panels). IL-4 levels were also tested in supernatants but found very low or undetectable levels in these primary cultures. In contrast to no antibody conditions, the control mouse IgG F(ab')2 or the anti-HLA class I W6/32 F(ab')2 antibodies showed little or no effect on all cytokines measured. Since the persistent levels of the type 2 cytokine, IL- 10, in masked conditions 25 suggested a shift in the type 1/type 2 cytokine ratio, levels of the cytokines IL-2, IFN gamma, IL-4 and IL-10 in day 2 culture conditions in a secondary stimulation assay using either 1) fresh porcine SMC, 2) anti-CD3 antibody or 3) anti-CD3/CD28 antibodies. These conditions were selected to examine both antigen specific and antigen non-specific secondary responses using anti-CD3 or anti-CD3 and anti-CD28 mAbs. 3 0 PBMC from four different donors were stimulated for 7 days (primary) with porcine SMC in the presence or absence of the masking PT85 antibody. Cells were harvested WO 00/51630 PCT/USOO/05647 - 62 and washed and restimulated with identical fresh SMC without additional antibody and cultured for several more days. Production of IFN-gamma by 48 hours in the group that was previously masked with PT85 was inhibited relative to the control groups (Figure 7). For example, 2 day culture supernatants from donor 1 showed 6000 pg/ml of IFN 5 gamma in the control (no Ab, mIgG, or W6/32) groups but less than 10 pg/ml was detected for the masked group (Figure 7). The same profile was also seen for human IL 2 production. In contrast, IL- 10 and IL-4 levels in the secondary stimulation increased in the previously PT85 masked group relative to the control groups. Levels of IL- 10 in 48 hour lo culture supernatants using fresh identical porcine SMC were increased in 3 of 4 donors tested (Figure 8). Moreover, increased levels of IL- 10 and IL-4 were detected when secondary cultures were generated using anti-CD3/CD28 stimulatory antibodies. For example, IL-10 levels for donor 1 PT85 relative to control group increased from 200 pg/ml to 800 pg/ml with anti-CD3 restimulation and 600 pg/ml to 1200 pg/ml with anti 15 CD3/CD28 restimulation (Figure 9). Interestingly, 2 of 4 donors also showed increased levels of IL-4 when restimulated with the anti-CD3/CD28 mAbs (Figure 9). For example, the level of IL-4 production increased from 300 pg/ml to 550 pg/ml with control versus the PT85 treatment. The second donor also showed increase only when restimulation was conducted with the anti-CD3/CD28 mAbs but not with the 20 immobilized anti-CD3 mAb alone. The other donors showed very little difference in IL 4 production between the masked and control treatments. Together, the results from the secondary stimulation more strongly suggest a shift in the type 1/type 2 cytokine ratio shift since IL-2 and IFN-gamma decreased whereas IL-4 and IL- 10 both maintained or increased their levels. Thus, the levels of IL-4 were also increased in 3 of 3 donors when 25 restimulated with anti-CD3/CD28 mAbs and to a lesser degree in 4 of 4 donors when restimulated with anti-CD3 mAb alone (Figure 10). All donors showed differences in IL-4 production between the masked and control treatments. Together, the results from secondary stimulation strongly suggest a shift in the type 1 /type2 cytokine ratio since 11-2 and IFN-gamma decreased whereas levels of IL- 10 and IL-4 were maintained or 30 increased. In fact, when IFN-gamma and IL- 10 levels were compared in the same donor, PT-85 treated samples consistently showed a decrease in the ratio of type 1 to WO 00/51630 PCT/USOO/05647 - 63 type 2 cytokines (Table 2). For example, as much as a 100-fold decrease in the IFNg, IL-10 ratio was detected (donor 4). Table 2. Masking with PT-85 leads to decreased type 1 to type 2 cytokine ratio. 5 Primary No Antibody PT-85 Stimulation with (IFNy: IL-10) (IFNy: IL-10) SMC Donor 1 166.7 2.2 Donor 2 6.4 .05 Donor 3 34.5 6.3 Donor 4 31.3 .03 Donor 5 62.5 14.3 Donor 6 39.9 4.6 Donor 7 60.4 3.75 Donor 8 76.4 11.1 RNA protection assay confirms cytokine production profiles: RNA was isolated from primary and secondary cultures that were initially stimulated with SMC in the presence or absence of PT85. While RNA protection assay revealed significantly reduced levels lo of IL-2 and IFN-gamma message in the PT85 masking conditions, the change in the message levels of IL-4, IL-5 and IL-10 were not detected in the 48 hr time point tested. However, the level of IL-15 was reduced in the PT85 treatment conditions. Since IL-15 is a strong T cell growth factor and produced only by activated myeloid cells these data indicate that human antigen presenting cells (macrophages and dendritic cells) may be is prevented from being activated in the presence of the PT85 masking. These findings suggest an important additional role for PT85 masking in the human anti-porcine response preventing the activation of the responding (host) professional APC.

WO 00/51630 PCTIUSO0/05647 - 64 In these studies monoclonal antibodies against porcine MHC class I are shown to block proliferation of human T cells in an in vitro mixed culture of porcine smooth muscle cells with human PBMC. Antibodies directed to the cc/aX2 domains as well as to the a3 domain of class I on the class I+/class II-SMC inhibited proliferation of the s human cells in a primary response, and human cells that were cultured with the antibody treated porcine cells in the primary incubation retained their functional phenotype in a secondary incubation with porcine SMC. Human cells harvested from the PT-85 treated cultures showed a marked decrease in proliferation as compared to human cells obtained from untreated primary cultures. 10 Human CD8+ T cells proliferated in response to the porcine SMC when the coculture was performed without the blocking antibody. These in vitro experiments showed that blocking the porcine MHC class I antigens on the class I+/class II-SMC inhibited CD8+ T cell proliferation. Although CD8+ cells are normally thought of as effector cells while CD4+ cells play a regulatory role, CD8+ cells have been implicated 15 as immunoregulatory in a number of systems (Klarnet, J.P. et al. 1989. Jlmmunol. 142:2187-91; Srikiatkhachorn, A et al. 1997. JExp Med 186:421-32). CD8+ cells can secret low levels of IL-2 in addition to high levels of IFN-y (Fong, T.A. et al. 1990. J Immunol. 144:1744-52). It has also been demonstrated that CD8+ cells are capable of producing other regulatory cytokines including IL-4 and IL-10. Thus, if the early CD8+ 20 T cell response is blocked in cellular xenotransplantation, it may influence the overall immune response and graft survival by altering the cytokine profile. This is of particular interest in cellular transplantation in which an ability to block a response initially directed to MHC class I on the transplanted cells could have subsequent effect on the immune response via CD8+ T cells. 25 Since most activated CD8+ T cells secret a type 1 pattern of cytokines (Fong, T.A. et al. 1990. JImmunol. 144:1744-52), it is reasonable that they can skew the cytokine profile of simultaneously activated CD4+ T cells toward type 1 phenotype (Fitch, F.W. et al. 1993. Annu Rev Immunol. 11:29-48). Further support for this conclusion from in vivo data show that removal of CD8+ T cells inhibit IFN-y 30 production by CD4+ T cells (Kemeny, D.M. et al. 1994. Immunol Today. 15:107-10; WO 00/51630 PCT/USOO/05647 - 65 Rus, V. et al. 1995. Jlmmunol. 155:2396-406). Moreover, preventing or blocking CD8 activation may have profound effect on the subsequent CD4+ T cell activation towards a type 2 phenotype (Williams, N.S. et al. JImmunol. 159:2091-9). The alterations in cytokine profile seen in this study were comprised of a decrease in IFN-y and IL-2 s production and an increased or sustained IL- 10 secretion in the human anti-porcine response when PT-85, an anti-porcine MHC class I antibody, was used to block the response. Such a change is consistent with a type 1 to type 2 shift, including an increase in IL-4 production although this was less easily measured than IL- 10. lo Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for promoting graft acceptance in a subject comprising: administering a cell, tissue, or organ and a cytokine profile altering agent to the subject s to thereby promote acceptance of the cell, tissue, or organ in the subject.

2. The method of claim 1, wherein the cell is a xenogeneic cell.

3. A method for promoting xenograft acceptance in a subject comprising: 1o administering an isolated xenogeneic cell and a cytokine profile altering agent to the subject to thereby promote acceptance of the xenogeneic cell in the subject.

4. The method of claim 3 wherein the xenogeneic cell is a porcine cell. is

5. The method of claim 4, wherein the cell is selected from the group consisting of: a fetal cell, a stem cell, and a progenitor cell.

6. The method of claim 3 wherein the xenogeneic cell is obtained from a pig which is predetermined to be free from at least one organism selected from the group 20 consisting of: zoonotic, cross-placental and organotropic organisms.

7. The method of claim 3, wherein the cytokine profile altering agent is a cytokine selected from the group consisting of IL-4 and IL- 10. 25

8. The method of claim 3, wherein the cytokine profile altering agent is a cytokine fusion protein selected from the group consisting of IL-41g and IL-I OIg

9. The method of claim 3, wherein the cytokine profile altering agent is an antibody. 30 WO 00/51630 PCT/USOO/05647 - 67

10. The method of claim 9, wherein the antibody is selected from the group consisting of: an antibody which binds to IL-2, IFN-gamma, and IL-12.

11. The method of claim 3, wherein the cell is selected from the group 5 consisting of a pancreatic islet cell, a kidney cell, a cardiac cell, a muscle cell, a liver cell, a lung cell, an endothelial cell, a central nervous system cell, a peripheral nervous system cell, an epithelial cell, an eye cell, a skin cell, an ear cell, and a hair follicle cell.

12. A method for promoting xenograft acceptance in a subject comprising: io i) modifying a xenogeneic cell to express an exogenous polypeptide comprising a cytokine profile altering agent to produce a modified xenogeneic cell; ii) administering the modified xenogeneic cell to a subject in order to promote xenograft acceptance in the subject. is

13. The method of claim 12, wherein the xenogeneic cell is a porcine cell.

14. The method of claim 13, wherein the cell is selected from the group consisting of: a fetal cell, a stem cell, and a progenitor cell. 20

15. The method of claim 12, wherein the xenogeneic cell is obtained from a pig which is predetermined to be free from at least one organism selected from the group consisting of: zoonotic, cross-placental and organotropic organisms

16. The method of claim 12, wherein the heterologous gene encodes a 25 cytokine.

17. The method of claim 14, wherein the heterologous gene encodes 11-4 or IL-10 WO 00/51630 PCT/USOO/05647 - 68

18. The method of claim 12, wherein the cell is selected from the group consisting of a pancreatic islet cell, a kidney cell, a cardiac cell, a muscle cell, a liver cell, a lung cell, an endothelial cell, a central nervous system cell, a peripheral nervous system cell, an epithelial cell, an eye cell, a skin cell, an ear cell, and a hair follicle cell. 5

19. The method of claim 3 or 12, further comprising administering xenogeneic lymphoid cells to the subject.

20. The method of claim 18, wherein the xenogeneic lymphoid cells are 1o administered intravenously.

21. The method of claim 20, wherein the lymphoid cell are administered into the portal vasculature of the subject. 15

22. The method of claim 3 or 12, wherein the xenogeneic cell is administered intravenously.

23. The method of claim 3 or 12, further comprising administering an immunosuppressive agent to the subject. 20

24. The method of claim 23, wherein the immunosuppressive agent is selected from the group consisting of methylprednisolone, cyclosporin A, and FK506.

25. A method for promoting acceptance of a xenogeneic cell or tissue in a 2s human subject comprising: i) administering an isolated xenogeneic cell to the subject wherein the cell or tissue bears a surface antigen capable of causing an immune response against the cell or tissue in the subject, said antigen being modified, masked, or eliminated to decrease the immune response; and 30 ii) administering a cytokine profile altering agent to the subject such that acceptance of the xenogeneic cell is promoted in the subject. WO 00/51630 PCT/USOO/05647 - 69

26. The method of claim 25, wherein the xenogeneic cell is a porcine cell.

27. The method of claim 26, wherein the cell is selected from the group consisting of: a fetal cell, a stem cell, and a progenitor cell. 5

28. The method of claim 25, wherein the xenogeneic cell is obtained from a pig which is predetermined to be free from at least one organism selected from the group consisting of zoonotic, cross-placental and organotropic organisms 10

29. The method of claim 25, wherein the antigen is an MHC class I antigen or an MHC class II antigen.

30. The method of claim 25, wherein the masking agent is a non-lytic anti MHC class I antibody fragment or an anti-MHC class II antibody or fragment thereof. 15

31. The method of claim 25, wherein the anti-MHC class I antibody fragment is an anti-MHC class I F(ab')2 fragment.

32. The method of claim 25, wherein the cell is selected from the group 20 consisting of a pancreatic islet cell, a kidney cell, a cardiac cell, a muscle cell, a liver cell, a lung cell, an endothelial cell; a central nervous system cell, a peripheral nervous system cell, an epithelial cell, an eye cell, a skin cell, an ear cell, and a hair follicle cell.

33. The method of claim 25, further comprising administering a soluble form 25 of a costimulatory molecule to the subject. WO 00/51630 PCT/USOO/05647 - 70

34. A method for promoting Th2 cytokine production in a subject comprising: i) administering a xenogeneic cell or tissue to the subject wherein the cell or tissue bears a surface antigen capable of causing an immune response against the cell or 5 tissue in the subject, said antigen being modified, masked, or eliminated to decrease the immune response, such that upon introduction of the composition into the subject lysis of the cell or tissue is prevented; and ii) determining that Th2 cytokine production is promoted in the subject.