Hybrid thymus, methods of making, and methods of using to induce xenograft tolerance, restore immunocompetence and thymic function

Abstract

To provide a method to generate a human / pig hybrid thymus to achieve immune tolerance to pig antigens with optimal immune function of the human T-cell repertoire generated.SOLUTION: A method of inducing tolerance in a recipient mammal of a first species to a graft obtained from a donor mammal of a second species comprises the steps of: (a) introducing the hybrid thymic tissue which is thymic tissue from the second type and comprises thymic epithelial cells from the first species into the recipient mammal; and (b) implanting the graft from the donor mammal in the recipient mammal.SELECTED DRAWING: Figure 1A-B

Description

[Technical field]

[0001] CROSS-REFERENCE TO OTHER APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 734,019, filed Sep. 20, 2018, which is incorporated by reference in its entirety.

[0002] Government Interest Statement This invention was made with Government support under Grants AI084903, AI045897, and AI106697 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] FIELD OF THEINVENTION The present disclosure relates to generating a pig-human hybrid thymus and using the hybrid thymus to induce tolerance in xenotransplantation. [Background technology]

[0004] 2. Background of the Invention A severe shortage of allogeneic donors limits the number of organ transplants currently performed. This supply-demand discrepancy could be rectified by the use of organs from other species (xenografts). Given the ethical issues and impracticalities associated with the use of non-human primates, pigs are considered the most suitable donor species for humans. Their organ size and physiological similarity to humans, plus their ability to be rapidly bred and inbred, make them particularly suitable for genetic modification that could improve their ability to serve as transplant donors for humans (Sachs, Path. Biol. 42:217-219, 1994; Piedrahita et al., Am. J. Transplant, 4 Suppl. 6:43-50, 2004).

[0005] Despite recent advances, the immune response against xenografts is strong, limiting their clinical use. Transplants combined with non-specific immunosuppressive therapy are associated with high initial graft tolerance, but the main limitation to the success of clinical organ transplantation has been late graft loss, mostly due to chronic rejection of the transplant. Furthermore, immunosuppressive therapy often leads to adverse side effects or increases the risk of infection. Thus, a method to control the immune response against xenografts would greatly improve their applicability.

[0006] Genetically engineered pigs lacking the Gal gene avoid common rejection in non-human primates due to anti-Galα1-3Gal (Gal) natural antibodies. Cooper D. A brief history of cross-species organ transplantation. Proc (Bayl Univ Med Cent). 2012 Jan; 25(1): 49-57. Despite this progress, T cell-dependent antibodies can recognize other pig specificities that cause rejection. Yang YG, Sykes M. Xenotransplantation: current status and a perspective on the future. Nat Rev Immunol. 2007 Jul; 7(7): 519-31. Extended T cell suppression. Pig xenografts survive in non-human primates, but such treatment is highly toxic. Yamada K, Sykes M, Sachs DH. Tolerance in xenotransplantation. Curr Opin Organ Transplant. 2017 Dec;22(6):522-528.

[0007] Another approach to T cell suppression is tolerance induction. Xenograft tolerance approaches include mixed chimerism induction and pig thymus transplantation.

[0008] Mixed chimerism can induce tolerance to the donor at the level of T cells, B cells and natural killer (NK) cells in the recipient. Griesemer A., ​​Yamada K. and Sykes M., Xenotransplantation: Immunological hurdles and progress toward tolerance, Immunol Rev. 2014;258(1):241-258. Sachs DH, Kawai T. and Sykes M., Cold Spring Harb. Perspect. Med. 2014;4:a015529.

[0009] Thymic xenografts can also induce robust tolerance. Kalscheuer H, Onoe T, Dahmani A, Li HW, Holzl M, Yamada K, Sykes M. Xenograft Tolerance and Immune Function of Human T Cells Developing in Pig Thymus Xenografts. J Immunol. 2014 Apr 1;192(7):3442-50. Pig thymus grafts can generate diverse and functional human T cell repertoires in mice that are specifically unresponsive to the donor pig in vitro. Shimizu I, Fudaba Y, Shimizu A, Yang Y, and Sykes M. Comparison of human T cell repertoire generated in xenogeneic porcine and human thymus grafts. Transplantation. 2008 Aug 27;86(4):601-610. Nikolic B., J.P. Gardner, D.T. Scadden, J.S. Arn, D.H. Schachs, M. Sykes. Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC. J.Immunol. 1999 Mar 15;162:3402-3407.Habiro K, Sykes M, Yang YG Induction of human T-cell tolerance to pig xenoantigens via thymus transplantation in mice with an established human immune system Am J Transplant.2009 Jun;9(6):1324-9. Extension of this technique from a pig to a baboon model achieved sustained pig kidney xenograft survival in non-human primates. Yamada K,Yazawa K,Shimizu A,Iwanaga T,Hisashi Y,Nuhn M,O'Malley P,Nobori S,Vagefi PA,Patience C,Fishman J,Cooper DK,Hawley RJ,Greenstein J,Schuurman HJ,Awwad M,Sykes M,Sachs DH.Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue.Nat Med.2005 Jan;11(1):32-4.

[0010] However, remaining limitations include suboptimal recipient immune function unable to efficiently recognize foreign antigens; suboptimal survival, homeostasis and inefficient elimination of autoreactive T cells; and lack of positive selection of regulatory T cells to prevent autoimmunity. Summary of the Invention

[0011] The present disclosure provides a method of inducing tolerance in a recipient mammal of a first species to a graft obtained from a donor mammal of a second species, the method comprising: (a) introducing hybrid thymic tissue from a second species, the hybrid thymic tissue comprising thymic epithelial cells from a first species into a recipient mammal; and (b) transplanting the graft from the donor mammal into the recipient mammal. The present invention provides a method comprising: [Means for solving the problem]

[0012] In some embodiments, thymic function is essentially absent in the recipient mammal prior to performing step (a). In some embodiments, the recipient mammal is a primate, and in some embodiments, it is a human. In some embodiments, the donor mammal is a pig, and in some embodiments, it is a minipig.

[0013] In some embodiments, the thymus tissue from the donor mammal is fetal thymus tissue, hi some embodiments, the thymus tissue from the donor mammal is neonatal thymus tissue.

[0014] In some embodiments, the thymic epithelial cells from the recipient mammal are obtained from the thymus of the recipient mammal. In some embodiments, the thymic epithelial cells from the recipient mammal are generated from induced pluripotent stem cells (iPSCs) of the recipient mammal. In some embodiments, the thymic epithelial cells from the recipient mammal species are generated from embryonic stem cells that share HLA alleles with the recipient mammal. In some embodiments, the embryonic stem cells are genetically engineered to share HLA alleles with the recipient mammal.

[0015] In some embodiments, the hybrid thymic tissue is transplanted into a recipient mammal in step (a). In some embodiments, step (a) is performed prior to or simultaneously with step (b).

[0016] In some embodiments, the hybrid thymic tissue is generated by introducing thymic epithelial cells from a recipient mammal into thymic tissue from a donor mammal. In some embodiments, the hybrid thymic tissue is generated by injecting thymic epithelial cells from a first species of recipient mammal into thymic tissue from a second species of donor mammal. In some embodiments, the method further comprises administering hematopoietic stem cells (HSCs) to the recipient mammal. In some embodiments, the graft comprises cells, tissues, or organs.

[0017] In a further embodiment, the hybrid thymus tissue is prepared by the following steps: (i) treating thymus tissue from a second donor mammal with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from a first type of recipient mammal into the 2DG-treated thymic tissue; The method includes:

[0018] In some embodiments, thymic epithelial cells from a first species are suspended in Matrigel prior to injection into the 2DG-treated thymic tissue.

[0019] The disclosure also provides a method of restoring or inducing immune competence in a recipient mammal of a first species, comprising introducing thymic tissue from a donor mammal of a second species, hybrid thymic tissue comprising thymic epithelial cells from the first species, into the recipient mammal of the first species.

[0020] The disclosure also provides a method of restoring or enhancing thymic dependency of T cell precursors that develop into mature functional T cells in a recipient mammal of a first species, comprising introducing thymic tissue from a donor mammal of a second species, hybrid thymic tissue comprising thymic epithelial cells from the first species, into the recipient mammal of the first species.

[0021] In some embodiments of these methods, thymic function is essentially absent in the recipient mammal prior to the introducing step. In some embodiments, the recipient mammal is thymectomized prior to the introducing step. In some embodiments, the recipient mammal has an immune disorder.

[0022] In some embodiments, the donor mammal is a pig, and in some embodiments, the pig is a minipig. In some embodiments, the recipient mammal is a primate. In some embodiments, the recipient mammal is a human.

[0023] In some embodiments, the thymus tissue from the donor mammal is fetal thymus tissue, hi some embodiments, the thymus tissue from the donor mammal is neonatal thymus tissue.

[0024] In some embodiments, the thymic epithelial cells are obtained from the thymus of the recipient mammal. In some embodiments, the thymic epithelial cells are generated from induced pluripotent stem cells (iPSCs) of the recipient mammal. In some embodiments, the thymic epithelial cells are generated from embryonic stem cells that share HLA alleles with the recipient mammal. In some embodiments, the embryonic stem cells are genetically engineered to share HLA alleles with the recipient mammal.

[0025] In some embodiments, the hybrid thymus tissue is transplanted into a recipient mammal.

[0026] In some embodiments, the hybrid thymic tissue is generated by introducing thymic epithelial cells from a first species into thymic tissue from a donor mammal of a second species, hi some embodiments, the hybrid thymic tissue is generated by injecting thymic epithelial cells from a first species into thymic tissue from a donor mammal of the second species.

[0027] In some embodiments, the hybrid thymus tissue is prepared by the following steps: (i) treating thymus tissue from a second donor mammal with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from the first species into the 2DG-treated thymic tissue; The method includes:

[0028] In some embodiments, thymic epithelial cells from a first species are suspended in Matrigel prior to injection into the 2DG-treated thymic tissue.

[0029] The present disclosure also provides isolated hybrid thymic tissue comprising thymic epithelial cells from a first mammalian species and thymic tissue from a second mammalian species, as well as methods of making the hybrid thymic tissue.

[0030] In some embodiments, the second mammalian species is a pig, and in some embodiments, the pig is a miniature pig. In some embodiments, the first mammalian species is a primate. In some embodiments, the recipient mammal is a human.

[0031] In some embodiments, the thymus tissue from the second mammalian species is fetal thymus tissue. In some embodiments, the thymus tissue from the second mammalian species is neonatal thymus tissue.

[0032] In some embodiments, the thymic epithelial cells are obtained from a thymus from a first mammalian species. In some embodiments, the thymic epithelial cells from a first mammalian species are obtained from fetal thymic tissue. In some embodiments, the thymic epithelial cells from a first mammalian species are obtained from neonatal thymic tissue. In some embodiments, the thymic epithelial cells are generated from induced pluripotent stem cells (iPSCs) from a first mammalian species. In some embodiments, the thymic epithelial cells are generated from embryonic stem cells that share HLA alleles with the first mammalian species. In some embodiments, the embryonic stem cells are genetically engineered to share HLA alleles with the first mammalian species.

[0033] The present disclosure also provides an isolated hybrid thymic tissue comprising thymic epithelial cells from a first mammalian species and thymic tissue from a second mammalian species, and a method of making the hybrid thymic tissue, comprising: (i) treating thymus tissue from a second mammalian species with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from the first mammalian species into the 2DG-treated thymic tissue; The present invention provides a method comprising:

[0034] In some embodiments, thymic epithelial cells from a first mammalian species are suspended in Matrigel prior to injection into the 2DG-treated thymic tissue.

[0035] For the purpose of illustrating the invention, there are shown in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0036] [Figure 1]Generation of human / pig hybrid thymus. At 12–20 weeks post-transplant, humanized mice generated with hybrid porcine / human thymus and human CD34+ cells were euthanized and transplanted thymuses were excised, sectioned, and stained to detect human TECs using two-photon confocal microscopy. Figures 1A, 1B, and 1C show images of transplanted porcine thymi that were not injected (Figure 1A), injected with human fetal thymic stromal cells (20 weeks gestational age) (Figure 1B), and injected with pediatric human thymic stromal cells (from a 4-month-old thymus) (Figure 1C). Arrows in B and C point to CK14+HLA-DR+ cells, which represent human TECs within the porcine thymus. Quantitative analysis of the entire sections shown in Figures 1A–1C is shown in Figure 1D. Additional controls of human fetal and pediatric thymi and porcine thymus were also included in the quantitative analysis. The analysis was performed by ImarisColoc software, which allows the calculation of the localization of CK14+HLA-DR+ cells among all CK14+ cells in the entire thymic section. The numbers indicated at the top of the bars are the percentage of CK14+HLA-DR+ cells among CK14+ cells. Figures 1E and 1F show representative images of human thymic mesenchymal cells (TMCs) (Figure 1E) and TECs (Figure 1F) grown in 3-D Matrigel culture system for 3 weeks from huCD45-depleted human thymocytes after digestion of a 17-year-old child thymus with Liberase. Figures 1G and 1H show representative flow cytometry characteristics of the grown TMCs and TECs, respectively. CD105-CD326+ are considered TECs, whereas CD105+CD326- cells are considered TMCs. [Diagram 2] CK14+HLA-DR+ cells were detected in hybrid thymuses generated by injecting human thymic stromal cells (derived from fetal thymus) (TECs) into pig thymuses. Figure 2A: No human TEC injection. Figure 2B: Human TEC injection. Figure 2C: 2-DG-treated pig thymus + human fetal TEC injection. [Diagram 3]FIG. 3 shows results showing the development of a method for use in generating hybrid thymus. FIG. 3A shows flow cytometry results showing the number of cells released after injection of cells into fetal porcine thymus fragments. PBMC cells were resuspended in matrigel to prevent leakage from the porcine thymus after injection. Three different methods were used to inject cells into thawed fetal porcine thymus fragments. Method A: Place the pieces in the wells of a V-bottom 96-well plate and inject using a Hamilton syringe. Method B: Inject using PE50 tubing. Method C: Inject using a Hamilton syringe while holding the pieces outside the wells with forceps until the matrigel solidifies. The number of released cells was determined by flow cytometry and followed injected and CFSE stained PBMC. FIG. 3B and 3C show the results of different reagents for ex vivo depletion of thymocytes in porcine thymus fragments. Figure 3B is a graph of the total viable cell count for cells treated with each reagent (top panel) and the percentage of viable cells for cells treated with each reagent (bottom panel). Figure 3C is a graph of the ratio of single positive (SP) CD4 (SP-CD4) or SP-CD8 cells over remaining viable double positive CD4 and CD8 cells (DP) or double negative (DN) cells containing stromal cells used as readout. Treatment with 100 nM 2DG for 12 hours resulted in the lowest ratio and was therefore the best strategy to eliminate thymocytes while preserving stromal cells. [Figure 4] FIG. 4 shows the experimental design for preparing hybrid thymus and transplanting the hybrid thymus into recipient mice. [Diagram 5]FIG. 5 shows graphs of the levels of human immune cell reconstitution following hybrid thymic transplantation of either fetal pig thymus treated with 2DG and injected with human thymic stromal cells (represented by circles on the graph), fetal pig thymus not treated with 2DG and injected with human thymic stromal cells (represented by squares on the graph), or fetal pig thymus not treated with 2DG and injected with human thymic stromal cells (represented by triangles on the graph). FIG. 5A shows the percentage of hCD45+ cells among white blood cells. FIG. 5B shows the percentage of CD3+ cells among hCD45+. FIG. 5C shows the number of hCD45+ cells per μl of blood. FIG. 5D shows the number of hCD3+ cells per μl of blood. FIG. 5E shows the percentage of CD4+ cells among hCD3+. FIG. 5F shows the number of hCD4+ cells per μl of blood. FIG. 5G shows the percentage of CD19+ cells among hCD45+. Figure 5H shows the percentage of CD14+ cells in hCD45+. Figure 5I shows the number of hCD19+ cells per μl of blood. Figure 5J shows the number of hCD14+ cells per μl of blood. Figure 5K shows the percentage of naive cells in hCD3+. Figure 5L shows the percentage of effector memory cells in hCD3+. [Figure 6] Figure 6 shows images of detection of injected human TECs mixed with porcine TECs in transplanted thymuses. An uninjected porcine thymus transplanted into a humanized mouse is shown on the left, and a hybrid thymus transplanted into a humanized mouse is shown on the top right. [Figure 7] Figure 7 shows a graph of in vitro T cell proliferation results suggesting that peripheral T cells of mice with hybrid thymus are partially tolerant to the human TEC donor. Figure 7A shows the T cell proliferation response of mice with various transplanted thymus to pig dendritic cells. Figure 7B shows the T cell proliferation response of mice with various transplanted thymus to human pig dendritic cells. Fetal pig thymus not injected with human thymic stromal cells is represented by a circle, fetal pig thymus injected with human thymic stromal cells and not treated with 2-DG is represented by a square, and fetal pig thymus injected with human thymic stromal cells treated with 2-DG is represented by a triangle. [Figure 8]Figure 8 shows increased responses to human tissue restricted antigens (TRAs) (MART-1, NYESO1 and islet antigen IA-2) in human T cells developing in the pig thymus (SW / HU mice) compared to T cells developing in the human thymus (HU / HU mice). Figure 8A is a schematic of the mouse model. Figure 8B is a graph showing the proliferative response of human peripheral T cells (18 weeks post-transplant) from mice to human TRAs (IA-2, MART-1 and NYESO1) presented by human HSC donor DCs. [Figure 9]FIG. 9 shows lower survival of human Treg and CD8 T cells developed in porcine thymus (SW / HU mice) compared to those developed in human thymus (HU / HU mice). FIG. 9 is a graph showing various cells in transplanted thymocytes in HU / HU mice compared to SW / HU mice, and various cells in spleen / lymph nodes in HU / HU mice compared to SW / HU mice. HU / HU mouse numbers are shown as circles and SW / HU mouse numbers are shown as squares. FIG. 9A shows the total number of thymocytes in transplanted thymus. FIG. 9B shows the percentage of thymocyte subsets in transplanted thymus. FIG. 9C shows the percentage of Tregs within SP-CD4+ in transplanted thymus. FIG. 9D shows the percentage of Ki67+ cells in transplanted thymus. FIG. 9E shows the percentage of CD45RO+ cells in transplanted thymus. FIG. 9F shows the percentage of CTLA-4+ cells in transplanted thymus. Figure 9G shows the total cell counts in the spleen and lymph nodes (LN) in each subset of mice. Figure 9H shows the percentage of hCD45+ cells in the spleen and lymph nodes (LN) in each subset of mice. Figure 9I shows the percentage of T cells in the spleen and lymph nodes (LN) in each subset of mice. Figure 9J shows the percentage of CD4 and CD8 cells in T cells in the spleen and lymph nodes (LN) in each subset of mice. Figure 9K shows the percentage of Tregs in the spleen and lymph nodes (LN) in each subset of mice. Figure 9L shows the percentage of naive cells in the spleen and lymph nodes (LN) in each subset of mice. Figure 9M shows the percentage of EM cells in the spleen and lymph nodes (LN) in each subset of mice. Figure 9N shows the percentage of HLA-DR+ cells in the spleen and lymph nodes (LN) in each subset of mice. Figure 9O shows the percentage of Ki67+ cells in the spleen and lymph nodes (LN) in each subset of mice. Figure 9P shows the percentage of hCD45RO+ cells in the spleen and lymph nodes (LN) in each subset of mice. Figure 9Q shows the percentage of CTLA-4+ cells in the spleen and lymph nodes (LN) in each subset of mice. [Figure 10]FIG. 10 shows long-term (>20 weeks) persistence of human TECs in "hybrid thymuses." FIG. 10A shows images of transplanted porcine thymi including those not injected with human TECs (SW / THY, top left panel), porcine thymi injected with human fetal TECs (SW / fetal hu-TES THY, top right panel), porcine thymi injected with human pediatric TECs (SW / pediatric hu-TEC THY, bottom left panel), and porcine thymi injected with human hPSCs (SW / hPSC-TEC THY, bottom right panel). FIG. 10B shows representative flow cytometry staining of gated CD45-negative cells in digested stroma from various long-term thymic grafts showing the presence of EPCAM+, CD105-negative hu-TECs only in human thymi injected with hPSC-TEC precursors (bottom left panel) (top right) and SW grafts, but not in uninjected SW THY grafts (top left). FIG. 10C is a graph of quantification of the percentage of CD45-HLA-ABC+EpCAM+ (injected human TEC) from multiple mice receiving SW thymus injected with human ES-TEC versus uninjected SW thymus. [Figure 11]FIG. 11 shows that injection of hES-TECs into pig thymus promotes enhanced T cell development and expansion of peripheral CD4+ and CD8+ T cells. Pig thymuses injected with 1-2×105 hES-TECs (green bars) or not injected (clear bars) were transplanted under the renal capsule of thymectomized NSG mice that were intravenously injected with 2×105 human fetal liver-derived CD34+ cells. Splenocytes and thymocytes from the thymic grafts were analyzed by flow cytometry at 18-22 weeks after transplantation. FIG. 11A is a graph of the absolute number of human CD3+ T cells in each group of mice. FIG. 11B is a graph of the absolute number of human CD8+ T cells in each group of mice. FIG. 11C is a graph of the absolute number of human CD4+ T cells in each group of mice. Figure 11D is a graph of the absolute number of recent thymic emigrant CD31+CD4+ naive cells defined as CD45RA+CCR7+ cells in splenic mononuclear cells in each group of mice. Figure 11E is a graph of the number of indicated cells in thymocytes in each group of mice stained for HuCD45, CD19, CD14, CD4, CD8, CD45RA and CD45RO expression. Thymocytes were gated as huCD45+CD19-CD14- cells. Absolute numbers of thymocytes from half of the thymic graft gated as total human CD45 cells, double positive CD4+CD8+, single positive CD4+CD8- and CD4-CD8+ are shown. SP CD4 and CD8 cells were further sub-gated to immature CD45RO+ compared to more mature CD45RA+ thymocytes. Means + SEM are shown for pig thymi injected with hES-TECs (n=6, squares) and pig thymi alone (n=5, triangles), representative of two independent experiments. Thymic grafts with fewer than 6x105 (n=1 each from SwTHY and SwTHY+TEC) cells were excluded from the analysis. p-values ​​were determined comparing groups injected with SwTHY hES-TECs to groups injected with SwTHY alone using the Mann-Whitney test, with p<0.05 considered significant. *p<0.05, +p=0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description of the Invention Abbreviation SW-Pig HU-Human TEC-thymic epithelial cells TMC-thymic mesenchymal cells WBC-white blood cells DP-double positive cells (both CD4+ and CD8+) SP-single positive cells (either CD4+ or CD8+) treg - regulatory T cells LN - Lymph Node TRA-tissue-specific autoantigen 2DG - 2D glucose MACS-magnetic activated cell sorting HSC - Human hematopoietic cells

[0038] The present disclosure provides a method for generating a human / pig hybrid thymus to achieve immune tolerance to pig antigens with optimal immune function of the generated human T cell repertoire. The method overcomes limitations encountered when using a single pig thymus by improving human T cell function and self-tolerance in the pig thymus while developing tolerance to pig xenografts.

[0039] The method creates a hybrid pig-human thymus containing patient-specific thymic epithelial cells (TECs). The patient-specific TECs can be either obtained directly from the patient's thymus, created from patient-specific induced pluripotent stem cells, or created from embryonic stem cells that naturally or artificially share human leukocyte antigen (HLA) alleles with the patient. The patient-specific TECs can participate in positive selection, resulting in T cells that more readily recognize foreign antigens presented by recipient HLA molecules in the periphery. Furthermore, since many tissue-specific antigens (TSAs) differ between humans and pigs, adding human TECs to make human TSAs in the thymus helps ensure protection from autoimmunity. Thus, using a hybrid thymus instead of a pig thymus can improve the function and self-tolerance of the created human T cell repertoire and allow for the induction of xenograft tolerance.

[0040] The hybrid thymus / thymus tissue of the present invention (e.g., hybrid porcine-human thymus / thymus tissue) may also be used for immune reconstitution in patients who lack proper thymus function or have T cell immunodeficiency (e.g., senescent thymus in adults). Applications of hybrid thymus / thymus tissue (e.g., hybrid porcine-human thymus / thymus tissue) include pharmacology and drug screening, medical xenograft tolerance testing and preparation, and xenogeneic models for tolerance induction to therapeutic molecules that can cause immunogenicity (e.g., mAbs) used during transplantation or immune disorder treatment.

[0041] As used herein, hybrid thymic tissue refers to thymic tissue from a donor mammal of a second species and which contains thymic epithelial cells from a recipient mammal of a first species.

[0042] In one embodiment, a hybrid thymus / thymus tissue is constructed in which pig thymus / thymus tissue (e.g., fetal thymus / thymus tissue) containing human (e.g., patient-specific) thymic epithelial cells (TECs) is obtained from human thymus / thymus tissue (e.g., patient thymus / thymus tissue). In another embodiment, a hybrid thymus / thymus tissue is constructed in which pig thymus / thymus tissue (e.g., fetal thymus / thymus tissue) containing human (e.g., patient-specific) TECs is made from human (e.g., patient-specific) induced pluripotent stem cells. In yet another embodiment, a hybrid thymus / thymus tissue is constructed in which pig thymus / thymus tissue (e.g., fetal thymus / thymus tissue) containing human TECs is made from embryonic stem cells that naturally share HLA alleles with the patient or have been engineered to do so.

[0043] Inclusion of human TECs into pig thymus grafts can have functional effects on antigen recognition.

[0044] The present disclosure provides a method of inducing tolerance in a recipient mammal of a first species to a graft obtained from a donor mammal of a second species. The method may include the steps of: (a) introducing hybrid thymic tissue from the second species, the hybrid thymic tissue comprising thymic epithelial cells from the first species, into the recipient mammal; and (b) transplanting the graft from the donor mammal into the recipient mammal. The donor may be a pig, including a miniature pig. The recipient may be a human.

[0045] The present disclosure also provides a method for generating primate-pig hybrid thymus / thymic tissue to achieve immune tolerance to porcine antigens with optimal immune function of the generated primate T cell repertoire.One embodiment of the present disclosure provides a hybrid thymus / thymic tissue in pig with baboon.

[0046] The hybrid thymus / thymic tissue can be transplanted primarily as vascularized thymic lobes or as composite thymus-kidney grafts.

[0047] The hybrid thymus / thymus tissue can be transplanted intramuscularly into the recipient. The hybrid thymus / thymus tissue can be transplanted either into the quadriceps muscle alone or into additional transplantation sites (e.g., renal capsule and omentum) in the recipient. Wu et al., Xenogeneic Thymus Transplantation in A Pig-to-baboon Model, Transplantation, 2003, 75(3):282-291.

[0048] A xenograft recipient is a mammal of a first mammalian species. A xenograft donor refers to a mammal of a second mammalian species. A donor mammal is a donor of cells, tissues, and / or organs for the xenograft.

[0049] The present disclosure provides a method of inducing tolerance in a recipient mammal of a first species to a graft obtained from a donor mammal of a second species, the method comprising the steps of (a) introducing hybrid thymic tissue from the second species, the hybrid thymic tissue comprising thymic epithelial cells from the first species, into the recipient mammal, and (b) engrafting the graft from the donor mammal in the recipient mammal. Step (a) may be performed prior to or simultaneously with step (b).

[0050] The disclosure also provides a method of restoring or inducing immune competence in a recipient mammal of a first species, comprising introducing into the recipient mammal thymic tissue from a donor mammal of a second species, the hybrid thymic tissue comprising thymic epithelial cells from the first species.

[0051] Also encompassed by the disclosure is a method of restoring or promoting thymic dependency of T cell precursors that develop into mature functional T cells in a recipient mammal of a first species, the method comprising introducing into the recipient mammal thymic tissue from a donor mammal of a second species, the hybrid thymic tissue comprising thymic epithelial cells from the first species.

[0052] In one embodiment, thymic function is essentially absent in the recipient mammal prior to the introduction of the hybrid thymic tissue. In another embodiment, the recipient mammal is thymectomized prior to the introduction of the hybrid thymic tissue. In yet another embodiment, the recipient mammal has an immune disorder.

[0053] The second species may be a pig, such as a miniature pig.

[0054] The first species can be a primate, such as a non-human primate or a human.

[0055] In one embodiment, the recipient mammal is a human and the donor mammal is a minipig.

[0056] The thymus tissue from the second species can be fetal thymus tissue or neonatal thymus tissue.

[0057] The thymic epithelial cells from the first species can be obtained from the thymus of the recipient mammal. The thymic epithelial cells from the first species can be generated from induced pluripotent stem cells (iPSCs) of the recipient mammal. The thymic epithelial cells from the first species can be generated from embryonic stem cells that share HLA alleles with the recipient mammal. For example, the embryonic stem cells can naturally share HLA alleles with the recipient mammal or are genetically engineered to share HLA alleles with the recipient mammal.

[0058] In one embodiment, the hybrid thymic tissue is transplanted into a recipient mammal. For example, the hybrid thymic tissue may be transplanted primarily as vascularized thymic lobes or as composite thymus-kidney grafts.

[0059] Hybrid thymic tissue can be created by introducing thymic epithelial cells from a first species into thymic tissue from a second species.Hybrid thymic tissue can be created by injecting thymic epithelial cells from a first species into thymic tissue from a second species.

[0060] Hybrid thymic tissue may be produced by a method comprising the steps of: (i) treating thymic tissue from a second species with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from a first species into the 2DG-treated thymic tissue. In step (ii), the thymic epithelial cells may be suspended in a biomaterial, such as Matrigel, before being injected into the 2DG-treated thymic tissue.

[0061] The thymic epithelial cells can be suspended in a biomaterial (eg, Matrigel) before being injected into the thymic tissue from the second species.

[0062] In some embodiments, thymic epithelial cells from a first species may be combined (e.g., suspended) with a biomaterial prior to being introduced into the thymic tissue from a second species. The biomaterial may be a sol-gel, a hydrogel containing proteins, matrigel, an artificially constructed scaffold with cells, and combinations thereof. Non-limiting examples of biomaterials may include polyethylene-imine and dextran sulfate, poly(vinylsiloxane) copolymer polyethyleneimine, phosphorylcholine, poly(ethylene glycol), poly(lactic-co-glycolic acid), poly(lactic acid), polyhydroxyvaleric acid and copolymers, polyhydroxybutyrate and copolymers, polydioxanone, polyanhydrides, poly(amino acids), poly(orthoesters), polyesters, collagen, gelatin, cellulose polymers, chitosan, alginate, fibronectin, extracellular matrix proteins, vinculin, agar, agarose, hyaluronic acid, matrigel, and combinations thereof.

[0063] The method may further comprise administering hematopoietic stem cells (HSCs) to the recipient mammal.

[0064] The graft may comprise cells, tissues, or organs. In one embodiment, the graft comprises hematopoietic stem cells. In another embodiment, the graft comprises bone marrow. In yet another embodiment, the graft comprises heart, kidney, liver, pancreas, lung, intestine, skin, small intestine, trachea, cornea, or a combination thereof.

[0065] The present disclosure provides isolated hybrid thymic tissue comprising thymic epithelial cells from a first mammalian species and thymic tissue from a second mammalian species.

[0066] Alternative approaches to achieve central T-cell tolerance of highly dissimilar xenogeneic donors have been developed, including transplantation of the porcine thymus into immunocompetent, T-cell-depleted and thymectomized recipients. These studies were initiated in mice and demonstrated a remarkable and specific in vitro non-responsiveness and extended donor-specific skin graft survival (Lee LA, Gritsch HA, Sergio JJ, et al. Specific tolerance across a discordant xenogeneic transplantation barrier. Proc Natl Acad Sci USA. 1994; 91: 10864-10867. Zhao Y, Swenson K, Sergio JJ, Arn JS, Sachs DH, Sykes M Skin graft tolerance across a discordant xenogeneic barrier Nature Med. 1996; 2: 1211-1216). The mouse model allowed extensive investigation of the mechanisms of tolerance and immune function conferred by T cell reconstitution in xenogeneic thymic grafts. Intrathymic clonal deletion is the primary mechanism that tolerizes newly developed thymocytes to xenogeneic donors and recipients (Zhao Y, Sergio JJ, Swenson KA, Arn JS, Sachs DH, Sykes M. Positive and negative selection of functional mouse CD4 cells by porcine MHC in pig thymus grafts. J Immunol. 1997;159:2100-2107. Zhao Y, Rodriguez-Barbosa JI, Shimizu A, Swenson K, Sachs DH, Sykes M.).Despite efficient intrathymic negative selection of host-reactive T cells, autoimmune disease can develop in porcine thymus-grafted athymic mice: Evidence for failure of regulatory mechanisms suppressing autoimmunity (Transplantation. 2002;75:1832-1840). Additional studies have implicated Tregs developing in porcine thymus grafts in suppressing residual mouse anti-pig responses. Zhao et al. The induction of specific pig skin graft tolerance by grafting with neonatal pig thymus in thymectomized mice. Transplantation. 2000; 69:1447-1451. Rodriguez-Barbosa et al. Enhanced CD4 reconstitution by grafting neonatal porcine tissue in alternative locations is associated with donor-specific tolerance and suppression of pre-existing xenoreactive T cells. Transplantation. 2001;72:1223-1231. Using T cell receptor (TCR) transgenic recipient mice of different MHC haplotypes and TCRs for which positive and negative selection mouse MHC alleles had been previously identified, it was demonstrated that positive selection in pig thymus grafts was mediated exclusively by pig thymic MHC, with no contribution from mouse hematopoietic cells, whereas negative selection was mediated by both pig and mouse MHC, consistent with the presence of class II MHC+ APCs from both species in the donor pig thymus grafts.Zhao Y, Rodriguez-Barbosa JI, Zhao G, Shaffer J, Arn JS, Sykes M. Maturation and function of mouse T cells with a transgeneic TCR positively selected by highly disparate xenogeneic porcine MHC. Cell Mol Biol. 2000; 47: 217-228. Zhao Y, Swenson K, Sergio JJ, Sykes M. Pig MHC mediates positive selection of mouse CD4+T cells with A mouse MHC-restricted TCR in pig thymus grafts. J Immunol. 1998; 161: 1320-1326. Despite the lack of mouse MHC involvement in positive selection and the complete MHC disparity of the pig thymus and mouse recipient, these T cells were able to respond to immunization with protein antigens presented by mouse MHC molecules and, most importantly, protect mice against opportunistic pathogens whose clearance is dependent on CD4+ T cells. Zhao et al. Immune restoration by fetal pig thymus grafts in T cell-depleted,thymectomized mice J Immunol.1997;158:1641-1649. These results are interpreted as demonstrating that sufficient cross-reactivity for recognition of foreign antigens on the recipient MHC can occur when a diverse T cell repertoire is selected in a xenogeneic thymus graft.

[0067] The porcine thymus transplantation method for tolerance has been extended to a humanized mouse model to provide proof of principle that human T cells can develop normally and be centrally tolerized to porcine xenoantigens in the porcine thymus graft. Nikolic B,Gardner JP,Scadden DT,Arn JS,Sachs DH,Sykes M. Normal development in porcine thymus grafts and specific tolerance of human t cells to porcine donor MHC. J Immunol. 1999;162:3402-3407.Kalscheuer HO,T.;Dahmani,A.;Li,H.;Holzl,M.;Yamada,K.;Sykes,M.Xenograft tolerance and immune function of human T cell developing in pig thymus xenografts. J Immunology.2014;192(7):3442-3450. Both thymic and peripheral human T cells developing in porcine thymic grafts show specific non-responsiveness to the donor pig with intact responses to a third group of pigs and allogeneic humans in the mixed lymphocyte reaction (MLR). These T cells also show no responsiveness to the human hematopoietic stem cell (HSC) donor and mouse recipient in the MLR, both reflecting the contribution of human donor APCs and mouse APCs to the negative selection detected in the thymic xenografts. Kalscheuer et al. A model for personalized in vivo analysis of human immune responsiveness Science Translational Medicine.2012;4(125):125ra130. Importantly, donor-specific skin graft tolerance is observed for human T cells developing in porcine thymic grafts.

[0068] Based on the results of the mouse model, the thymus xenotransplantation technique has been extended to the large animal species combination of pig and baboon. Early studies using pig thymus fragments placed under the baboon kidney capsule demonstrated some degree of T cell recovery, donor-specific hyporesponsiveness in vitro, and extended survival of donor skin grafts compared to controls. However, the amount of transplanted and vascularized pig thymus tissue was very limited. Wu et al., Xenogeneic thymus transplantation in a pig-to-baboon model. Transplantation.2003;75(3):282-291. To achieve more robust thymic function, and in light of the mouse data above, it was expected that donor-specific Tregs developed in the pig thymus would be required to suppress pre-existing T cells that were not depleted by transplant conditioning, and subsequent studies utilized primarily vascularized pig thymus, which had already shown efficacy in tolerance induction in an allogeneic pig kidney transplantation model. Yamada K, Shimizu A, Utsugi R, et al. Thymic transplantation in miniature swine. II.Induction of tolerance by transplantation of composite thymokidneys to thymectomized recipients. J Immunol.2000;164:3079-3086. The thymus was transplanted as part of a composite "thymokidney" graft prepared in donor pigs several months earlier by placing autologous thymic fragments under the pig's renal capsule, or by direct vascular anastomosis of the pig thymic lobe in baboons. Yamada K, Yazawa K, Shimizu A, et al.Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nature medicine. 2005;11(1):32-34. Both approaches resulted in the first long-term survival of GalT knockout porcine kidneys in baboons. Tasaki et al., Rituximab treatment prevents the early development of proteinuria following pig-to-baboon xeno-kidney transplantation. Journal of the American Society of Nephrology:JASN.2014;25(4):737-744. Survival of animals receiving this treatment is limited by thrombotic complications of anti-CD40L and by proteinuria due to minimal change disease-like glomerulopathy, which can be avoided by the use of non-thrombotic anti-CD40 and by administering rituximab and CTLA 4Ig, respectively. Yamada et al., Xenotransplantation: Where Are We with Potential Kidney Recipients? Recent Progress And Potential Future Clinical Trials. Curr Transplant Rep.2017;4(2):101-109.

[0069] Baboons receiving porcine thymic-kidney grafts showed evidence of de novo recipient (baboon) thymic lymphopoiesis in the porcine thymic graft, the appearance of recent thymic emigrants in the periphery and donor-specific unresponsiveness in Elispot and MLR assays, and a decline in non-Gal natural antibodies. Tanabe et al. Role of Intrinsic (Graft) Versus Extrinsic (Host) Factors in the Growth of Transplanted Organs Following Allogeneic and Xenogeneic Transplantation, Am J Transplant. 2017 Jul;17(7):1778-1790. Minimal IgM binding was detected in these xenografts, and there was no complement fixation or significant pathology, although the latter may reflect uptake by the porcine kidney. Therefore, the results obtained in this model demonstrate the potential of composite thymic-kidney xenografts to induce tolerance in primates.

[0070] Limitations of the generation of human T cell repertoires in xenogeneic porcine thymus include preferential recognition of microbial antigens on porcine MHC, which is useful for protecting the graft but does not optimize protection against microbial pathogens infecting the host, poorly selecting for conventional T cells, and failing to actively select Tregs that recognize human tissue-specific self-antigens (TRAs). Indeed, studies in humanized mice showed reduced responses to peptides presented by human APCs following immunization when human T cells were developed in pigs rather than in human thymic grafts.

[0071] Approaches to overcome this limitation include the creation of a "hybrid thymus" in which recipient thymic epithelial cells, either obtained from a thymectomy specimen or generated from stem cells, are injected into pig thymic tissue. A hybrid thymus from a postnatal thymic donor is created, which promotes tolerance to human TRAs among human T cells.

[0072] Pig thymus grafts have been shown to support the development of normal and diverse mouse or human T cell repertoires, and these T cells have been shown to be specifically tolerant to the xenogeneic pig donor. However, recognition of foreign antigens presented by recipient HLA molecules in the periphery is suboptimal. Therefore, immune function may not be optimal. As shown herein, this can be overcome by providing recipient TECs in a pig-human hybrid thymus graft, as these TECs are involved in positive selection, resulting in T cells that can more easily recognize foreign antigens presented by recipient HLA molecules in the periphery. In the case of pig thymus grafts, survival, homeostasis and function of T cells without their "positive selection" ligands in the periphery are suboptimal. The ligands for positive selection are MHC / peptide complexes on TECs that rescue thymocytes from programmed cell death if the thymocytes have low affinity T cell receptors that recognize the complex. Providing recipient TECs in a pig-human hybrid thymus allows positive selection of T cells with the same ligand on the recipient cells in the periphery, conferring normal survival, homeostasis and function. In the case of pig thymus grafts, TECs produce antigens that are otherwise only expressed in very specific tissues in the surroundings (i.e., tissue-specific antigens, TSAs). Two important consequences of this expression of TSAs by TECs are a) clonal deletion of thymocytes that strongly recognize them, to deplete these autoreactive T cells from the repertoire, and b) positive selection of regulatory T cells that recognize them, to add a safety net to prevent autoimmunity in the surroundings. Since many TSAs differ between humans and pigs, adding human TECs to generate human TSAs in pig-human hybrid thymus grafts would help ensure protection from autoimmunity.

[0073] In summary, by using a hybrid thymus instead of a single pig thymus, it is possible to improve the function and self-tolerance of the human T cell repertoire generated in the pig thymus while developing tolerance to the pig.

[0074] In one embodiment, the recipient is thymectomized. In another embodiment, the recipient is not thymectomized. In yet another embodiment, the recipient has a slower rate of thymus formation due to age. In yet another embodiment, the recipient has a senescent thymus.

[0075] Thymic xenotransplantation using the hybrid thymus / thymus tissue of the present invention may or may not be combined with mixed chimerism induction. For example, when thymic xenotransplantation is combined with durable mixed porcine-human chimerism and mixed chimerism induction, both porcine and human APCs are present in the natural human thymus and in the porcine thymus xenograft, ensuring lifelong negative selection of thymocytes that recognize either porcine or human antigens expressed on hematopoietic cells. Furthermore, conventional T cells that recognize porcine or human TRAs are deleted in the thymus of the relevant species, and deletion escape due to development in the thymus of the opposite species is appropriately suppressed by TRA-specific Tregs that develop in the other thymus. Mixed porcine chimerism ensures tolerance of natural antibodies that recognize unknown xenogeneic targets, and NK cells are tolerized as well.

[0076] Xenotransplantation lends itself more readily to tolerance induction than allotransplantation from deceased donors, as the ability to perform xenotransplantation selectively allows for the application of tolerance protocols (e.g., mixed chimerism induction) prior to organ xenografts. In one embodiment, the method involves first tolerizing the recipient's immune system to ensure that tolerance is achieved, followed by performing the organ transplant without or with a shortened course of immunosuppression.

[0077] The present disclosure provides a method of inducing tolerance in a recipient mammal of a first species (e.g., a primate such as a human) to a graft obtained from a mammal of a second species, e.g., a pig. The method comprises introducing a hybrid thymus / thymic tissue into the recipient mammal prior to or simultaneously with transplantation of the graft, and (optionally) transplanting the graft into the recipient. The hybrid thymus / thymic tissue prepares the recipient for the subsequent graft by inducing immune tolerance at the T cell level.

[0078] The present disclosure provides a method for inducing xenograft tolerance in a recipient comprising introducing a hybrid thymus / thymic tissue into the recipient.

[0079] In one embodiment, host T cells of an athymic, T cell depleted recipient transplanted with hybrid thymus / thymic tissue are allowed to mature in the hybrid thymus / thymic tissue. The host T cells that mature in the transplanted hybrid thymus / thymic tissue are immunocompetent.

[0080] The present disclosure provides a method of restoring or inducing immune competence (or restoring or promoting thymic dependency of T cell precursor cells to mature or develop into functional mature T cells) in a host or recipient, e.g., a primate host or recipient, that is capable of producing T cell precursors but is unable to produce sufficient numbers of mature, functional T cells for a normal immune response due to thymic insufficiency. The method includes introducing a hybrid thymus / thymic tissue into the recipient such that host T cells can mature in the transplanted hybrid thymus / thymic tissue.

[0081] In one embodiment, the recipient / host is a primate, e.g., a human, and the donor is a pig, e.g., a minipig.

[0082] The method may include other steps that facilitate acceptance of the hybrid thymus / thymic tissue or otherwise optimize the method. In an embodiment, liver or spleen tissue, such as fetal or neonatal liver or spleen tissue, is transplanted with the thymic tissue, and donor hematopoietic cells, such as umbilical cord blood stem cells or fetal or neonatal liver or spleen cells, are administered to the recipient, e.g., a suspension of fetal liver cells is administered intraperitoneally or intravenously. The recipient may be thymectomized, such as before or at the time the hybrid thymus / thymic tissue is introduced.

[0083] In certain embodiments, the methods include depleting, inactivating or inhibiting natural killer (NK) cells to prevent NK-mediated rejection of the thymic tissue (preferably before or at the time of introducing thymic tissue into the recipient), e.g., by introducing into the recipient an antibody capable of binding to the recipient's NK cells; depleting, inactivating or inhibiting host T cell function (preferably before or at the time of introducing thymic tissue into the recipient), e.g., by introducing into the recipient an antibody capable of binding to the recipient's T cells; depleting, inactivating or inhibiting host CD4+ cell function (preferably before or at the time of introducing thymic tissue into the recipient), e.g., by introducing into the recipient an antibody capable of binding to the recipient's CD4 or CD4+ cells.

[0084] Certain embodiments include creating hematopoietic space by, for example, one or more of low dose irradiation, e.g., about 100-400 rads, total body irradiation, administration of a bone marrow suppressant, or administration of hematopoietic stem cells to inactivating or depleting antibodies to the recipient mammal (preferably prior to transplantation of thymic tissue or hematopoietic stem cells) to deplete or partially deplete the recipient's bone marrow.

[0085] Certain embodiments include inactivating thymic T cells by one or more of irradiating the host with, for example, about 700 rads of thymic irradiation (preferably prior to transplantation of thymic tissue or hematopoietic stem cells), administering to the recipient one or more doses of an anti-T cell antibody, e.g., anti-CD4 and / or anti-CD8 monoclonal antibody, or administering to the recipient an immunosuppressant for a short period of time.

[0086] Certain embodiments include depleting or otherwise inactivating natural antibodies by one or more of administration of a drug that depletes or inactivates natural antibodies, e.g., deoxyspergualin, administration of an anti-IgM antibody, or adsorption of natural antibodies from the host's blood, e.g., by contacting the host blood with donor antigens, e.g., by hemoperfusion of a donor organ, e.g., kidney or liver, from the donor species.

[0087] Other methods may be combined with the methods disclosed herein to promote acceptance of the graft by the recipient. For example, tolerance to thymic tissue can also be induced by inserting a nucleic acid expressing a donor antigen, e.g., a donor MHC gene, into the recipient's cells, e.g., hematopoietic stem cells, and introducing the genetically engineered cells into the recipient. For example, human recipient stem cells can be engineered to express porcine MHC genes, e.g., porcine class I or class II MHC genes, or both class I and class II genes, and the cells are transplanted into a human recipient that receives the hybrid thymic tissue. When inserted into a recipient primate, e.g., a human, expression of the donor MHC genes can provide tolerance to subsequent exposure to the donor antigen, thereby inducing tolerance to thymic tissue.

[0088] For example, methods of inducing tolerance by transplantation of hematopoietic stem cells can be combined with the methods disclosed herein.

[0089] Other methods of inducing tolerance may be used to promote thymic tissue acceptance. For example, suppression of helper T cells, which can be induced by administering short-term high doses of immunosuppressants, such as cyclosporine, has been found to induce tolerance. In these methods, helper T cells are suppressed for a relatively short period immediately after transplantation of the graft and do not require or include chronic immunosuppression.

[0090] Other methods of promoting tolerance or acceptance of donor tissue, for example by altering the levels of cytokine activity or inhibiting graft-versus-recipient disease, may also be used in combination with the methods of the invention.

[0091] In another aspect, the disclosure provides a method of reducing or inhibiting T cell activity, preferably thymic or lymph node T cell activity, in a recipient mammal, e.g., a primate, e.g., a human, receiving a graft from a donor mammal, the method comprising inducing tolerance to the graft and administering an immunosuppressant, e.g., cyclosporine, to the recipient for a short period of time sufficient to inactivate T cells, preferably thymic or lymph node T cells.

[0092] "Thymic deficiency," as used herein, refers to a condition in which the ability of an individual's thymus to support the maturation of T cells is impaired compared to a normal individual. Thymic deficiency conditions include those in which the thymus or thymic function is essentially absent.

[0093] "Tolerance" as used herein refers to the inhibition or reduction of the ability of a transplant recipient to mount an immune response, e.g., against donor antigens, that would otherwise occur, e.g., in response to the introduction of non-self MHC antigens to the recipient. Tolerance can include humoral, cellular, or both humoral and cellular responses. The concept of tolerance includes both complete and partial tolerance. In other words, as used herein, tolerance includes any degree of inhibition of the ability of a transplant recipient to mount an immune response, e.g., against donor antigens.

[0094] As used herein, "hematopoietic stem cells" refer to cells that can develop into mature myeloid and / or lymphoid cells. Preferably, hematopoietic stem cells allow for long-term repopulation of myeloid and / or lymphoid lineages. Stem cells from the umbilical cord blood of the recipient or donor can be used in the methods of the present disclosure.

[0095] As used herein, "minipigs" refers to fully or partially inbred minipigs.

[0096] As used herein, "graft" refers to a body part, organ, tissue, cell, or portion thereof.

[0097] "Interstitial tissue" as used herein refers to the supporting tissue or matrix of an organ as distinguished from its functional elements or parenchyma.

[0098] As used herein, restoring, inducing, or promoting immune competence means one or both of: (1) increasing the number of mature functional T cells in the recipient (beyond that seen in the absence of treatment by the methods of the disclosure), either by increasing the number of recipient mature functional T cells or by providing mature mature functional donor T cells in the recipient; or (2) improving the immune responsiveness of the recipient, e.g., as measured by the ability to mount a cutaneous response to a recall antigen, or improving the responsiveness of the recipient's T cells, e.g., by improved proliferative response to antigen, e.g., improved response to tetanus antigen or alloantigen, as measured, e.g., by in vitro testing.

[0099] As used herein, restoring or inducing thymic dependency of T cell precursors that mature into mature T cells means either or both of increasing the number of functional mature T cells of recipient origin in the recipient, or providing mature functional donor T cells to the recipient by providing donor thymic tissue in which the T cells can mature. The increase can be partial, an increase that does not result in levels of mature functional T cells at levels that result in an essentially normal immune response, or partial, e.g., an increase that does not result in levels of mature functional T cells in the recipient at levels that result in an essentially normal immune response.

[0100] In certain embodiments, preparation of a recipient for either an organ transplant or thymus replacement comprises any or all of the following steps, which may be performed in the following order:

[0101] First, a preparation of equine anti-human thymocyte globulin (ATG) is injected intravenously into the recipient. The antibody preparation excludes mature T cells and natural killer cells. If not excluded, mature T cells may promote the rejection of both the thymus transplant and the xenograft organ after sensitization. The ATG preparation also excludes natural killer (NK) cells. NK cells probably have no effect on the transplanted organ but may act immediately to reject the newly introduced thymic tissue. Anti-human ATG obtained from any mammalian host, e.g., ATG produced in pigs, can also be used, although previous preparations of porcine ATG were of lower titer than horse-derived ATG. ATG is superior to anti-NK monoclonal antibodies, as the latter generally do not lyse all host NK cells, whereas polyclonal mixtures in ATG can lyse all host NK cells. However, anti-NK monoclonal antibodies can be used. In relatively severely immunocompromised individuals, this step is not necessary. As host (or donor) T cells mature in the xenogeneic thymus, they will become tolerant to the thymic tissue. Alternatively, as the host immune system gradually recovers, it may be desirable to treat the host to induce tolerance to the thymic tissue.

[0102] Optimally, the recipient's thymus can be removed. In a thymectomized recipient, the recipient T cells do not have the opportunity to differentiate in the recipient's thymus, but must differentiate in the hybrid thymus tissue. In some cases, the recipient may need to be splenectomized to avoid anemia.

[0103] Second, the recipient can be given low doses of radiation. This step is thought to be beneficial in bone marrow transplants (by creating hematopoietic space for the newly infused bone marrow cells), but is less important in thymus grafts without bone marrow transplants. However, sublethal doses, e.g., doses approximately equal to or greater than 100 but less than about 400 rads, total body radiation, and local thymus radiation of 700 rads can be used.

[0104] Third, natural antibodies can be adsorbed from the recipient's blood. Antibody removal can be accomplished by exposing the recipient's blood to antigens of the donor or donor species, for example, by hemoperfusion of the donor species' liver to adsorb the recipient's natural antibodies. Preformed natural antibodies (nAbs) are the primary agents of graft rejection. Natural antibodies bind to xenogeneic endothelial cells and are primarily of the IgM class. These antibodies are independent of any known prior exposure to xenogeneic donor antigens. The B cells that generate these natural antibodies tend to be T cell independent and are usually tolerized to self-antigens by exposure to these antigens during development. Again, this step may not be necessary, at least initially, in relatively severely immunocompromised patients.

[0105] The hybrid thymus tissue is transplanted into a recipient, which may include fetal or neonatal liver or spleen tissue.

[0106] Any combination including one or all of these procedures may aid in the survival of transplanted thymus tissue or another xenogeneic organ.

[0107] The methods of the disclosure can be used to confer tolerance to a xenograft, for example, where the transplant donor is a non-human animal, e.g., a pig, e.g., a minipig, and the transplant recipient is a primate, e.g., a human.

[0108] The donor of the xenograft and the individual providing the tolerance-inducing thymic tissue may be the same individual or may be as closely related as possible, for example, it is preferable to derive the xenograft from a highly or completely inbred donor colony.

[0109] The second mammalian species (i.e., the donor) can be a non-human mammalian species, such as a porcine species (e.g., a miniature pig species) or a non-human primate species. Non-limiting examples of the first mammalian species include pigs, rodents, non-human primates, cows, goats, and horses.

[0110] In one embodiment, the second mammalian species (i.e., the donor) is an at least partially inbred miniature pig (e.g., the pig is homozygous at the swine leukocyte antigen (SLA) locus and / or homozygous at at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of all other loci). Genetic engineering can be performed on fully or partially inbred pigs (e.g., miniature pigs, transgenic pigs, etc.). For example, inbred Massachusetts General Hospital (MGH) miniature pigs may be used in the methods of the invention. These include MGH miniature pigs that have been inbred for 40 years and are homozygous at all loci. In one embodiment, inbred SLA dd Miniature swine may be used. Mezrich et al. and Sachs, Histocompatible miniature swine: An inbred large-animal model. Transplantation, 2003;75:904-907. Pigs from the National Swine Resources and Research Center (NSRRC, RADIL, University of Missouri, Columbia MO) may be used in the methods of the invention.

[0111] The first mammalian species (i.e., the recipient) can be a primate, such as a non-human primate (e.g., a baboon or a cynomolgus monkey), or a human. In one embodiment, the second species is human.

[0112] In various embodiments, the donor (second species) and the recipient (first species) are of different species, for example, the donor is a non-human animal, such as a miniature pig, and the recipient is a human.

[0113] Methods of transplanting a graft from such a donor animal of a second mammalian species into a recipient mammal of a first mammalian species (eg, human) are also encompassed by the present disclosure.

[0114] The cells, tissues, organs or fluids of the transgenic donor animals may be used for transplantation (e.g., xenotransplantation). Grafts taken from the donor animals for transplantation may include, but are not limited to, heart, kidney, liver, pancreas, lung transplants, intestine, skin, thyroid, bone marrow, small intestine, trachea, cornea, limbs, bone, endocrine glands, blood vessels, connective tissue, progenitor stem cells, blood cells, hematopoietic cells, pancreatic islets, brain cells, and cells, fluids, and combinations thereof from endocrine and other organs.

[0115] The cell can be any type of cell, hi some embodiments, the cell is a hematopoietic cell (e.g., a hematopoietic stem cell, a lymphocyte, a bone marrow cell), a pancreatic cell (e.g., a beta islet cell), a kidney cell, a cardiac cell, or a liver cell.

[0116] Bone marrow cells (BMC) from the donor animal, or hematopoietic stem cells (eg, fetal liver suspension or mobilized peripheral blood stem cells) may be infused into the recipient.

[0117] The methods may also include one or more of the following treatments: treatments that inhibit T cells, block complement, or otherwise downregulate the recipient's immune response to the graft.

[0118] Treatments to promote graft tolerance and / or reduce immune recognition include the use of immunosuppressants (e.g., cyclosporine, FK506), antibodies (e.g., anti-T cell antibodies such as polyclonal antithymocyte antiserum (ATG) and / or monoclonal anti-human T cell antibodies such as LoCD2b), irradiation, and methods to induce mixed chimerism. U.S. Patent Nos. 6,911,220, 6,306,651, 6,412,492, 6,514,513, 6,558,663, and 6,296,846. Kuwaki et al., Nature Med., 11(1):29-31, 2005. Yamada et al., Nature Med., 11(1):32-34, 2005.

[0119] In some embodiments, the recipient is thymectomized and / or splenectomized. Thymic irradiation can be used.

[0120] In some embodiments, the recipient is administered a low dose of radiation (eg, a sublethal dose of 100 rads to 400 rads of total body irradiation). Local thymic radiation may also be used.

[0121] The recipient can be treated with drugs that deplete complement, such as cobra venom factor.

[0122] Natural antibodies can be eliminated by organ perfusion and / or transplantation of tolerance-inducing bone marrow. Natural antibodies can be absorbed from the recipient's blood by hemoperfusion of the donor species' liver. Cells, tissues, or organs used for transplantation can be genetically modified so that they are not recognized by the host's natural antibodies (e.g., the cells are a-1,3-galactosyltransferase deficient).

[0123] In some embodiments, the methods include treatment with human anti-human CD154 mAb, mycophenolate mofetil, and / or methylprednisolone. The methods can also include agents useful for supportive care, such as anti-inflammatory agents (e.g., prostacyclin, dopamine, ganciclovir, levofloxacin, cimetidine, heparin, antithrombin, erythropoietin, and aspirin).

[0124] In some embodiments, donor stromal tissue is administered.

[0125] Immunosuppressants, also referred to as immunosuppressant drugs, can be any compound that reduces the function or activity of one or more aspects of the immune system, such as the humoral or cellular immune system or components of the complement system.

[0126] Non-limiting examples of immunosuppressants include: (1) antimetabolites such as purine synthesis inhibitors (inosine monophosphate dehydrogenase (IMPDH) inhibitors, such as azathioprine, mycophenolic acid, and mycophenolate mofetil), pyrimidine synthesis inhibitors (e.g., leflunomide and teriflunomide), and antifolates (e.g., methotrexate); (2) calcineurin inhibitors such as tacrolimus, cyclosporine A, pimecrolimus, and voclosporin. (3) TNF-α inhibitors such as thalidomide and lenalidomide; (4) IL-1 receptor antagonists such as anakinra; (5) mammalian target of rapamycin (mTOR) inhibitors such as rapamycin (sirolimus), deforolimus, everolimus, temsirolimus, zotarolimus, and biolimus A9; (6) corticosteroids such as prednisone; and (7) antibodies against any one of a number of cellular or serum targets, including antilymphocyte globulin and antithymocyte globulin.

[0127] Non-limiting exemplary cellular targets and their respective inhibitor compounds include complement component 5 (e.g., eculizumab); tumor necrosis factor (TNF) (e.g., infliximab, adalimumab, certolizumab pegol, afelimomab, and golimumab); IL-5 (e.g., mepolizumab); IgE (e.g., omalizumab); BAYX (e.g., nerelimomab); interferons (e.g., faralimomab); IL-6 (e.g., ercilimomab); IL-12 and IL-13 (e.g., lebrikizumab and ustekinumab); CD3 (e.g., muromonab-CD3, otelixizumab, teplizumab, visilizumab); CD4 (e.g., clenoliximab, keliximab, and zanolimumab); CD11a (e.g., efalizumab); CD18 (e.g., erlizumab); CD 20 (e.g., afutuzumab, ocrelizumab, pascolizumab); CD23 (e.g., lumiliximab); CD40 (e.g., teneliximab, toralizumab); CD62L / L-selectin (e.g., acelizumab); CD80 (e.g., galiximab); CD147 / basigin (e.g., gabilimomab); CD154 (e.g., ruplizumab); BLyS (e.g., belimumab); These include, but are not limited to, CTLA-4 (e.g., ipilimumab, tremelimumab); CAT (e.g., bertilimumab, lerdelimumab, metelimumab); integrins (e.g., natalizumab); IL-6 receptor (e.g., tocilizumab); LFA-1 (e.g., odulimomab); and IL-2 receptor / CD25 (e.g., basiliximab, daclizumab, inolimomab).

[0128] The recipient's natural antibodies can be eliminated by organ perfusion and / or tolerance-inducing bone marrow transplantation.

[0129] In one embodiment, the recipient is treated with a preparation of horse anti-human thymocyte globulin (ATG) injected intravenously (e.g., about 25-100 mg / kg, e.g., 50 mg / kg, e.g., 3, 2, 1 days prior to transplantation). The antibody preparation eliminates mature T cells and natural killer cells. The ATG preparation also eliminates natural killer (NK) cells. Anti-human ATG obtained from any mammalian host can also be used. In addition, if further T cell depletion is indicated, the recipient can be treated with a monoclonal anti-human T cell antibody, such as LoCD2b (Immerge BioTherapeutics, Inc., Cambridge, Mass.). For bone marrow transplantation, the recipient can be administered a low dose of radiation. Optionally, the recipient can be treated (e.g., 1 day prior) with a complement-depleting agent, such as cobra toxin factor.

[0130] In some embodiments, maintenance therapy (e.g., beginning immediately prior to transplant and continuing for at least several days after transplant) includes treatment with human anti-human CD154 mAb. Mycophenolate mofetil (MMF) can be administered to maintain whole blood levels. Methylprednisolone may be administered beginning the day of transplant and then tapered over the next 3-4 weeks.

[0131] Various agents useful for supportive care (eg, days 0-14) include anti-inflammatory agents such as prostacyclin, dopamine, ganciclovir, levofloxacin, cimetidine, heparin, antithrombin, erythropoietin, and aspirin.

[0132] In some embodiments, donor stromal tissue is administered. It may be obtained from fetal liver, thymus, and / or fetal spleen and may be transplanted to the recipient, for example, in a renal capsule. Thymus tissue can be prepared for transplantation by embedding under the autologous renal capsule for revascularization. Stem cell engraftment and hematopoiesis across different species barriers can be enhanced by providing a hematopoietic stromal environment from the donor species. The stromal matrix provides species-specific factors, such as hematopoietic growth factors, adhesion molecules, and their ligands, necessary for the interaction between hematopoietic cells and their stromal environment.

[0133] Since the liver is the primary site of hematopoiesis in the fetus, fetal liver can also serve as an alternative to bone marrow as a source of hematopoietic stem cells. Each organ contains an organ-specific stromal matrix that can support the differentiation of the respective undifferentiated stem cells transplanted into the host. As an alternative or supplement to implantation, fetal liver cells can be administered in a fluid suspension.

[0134] Bone marrow cells (BMC), or another source of donor hematopoietic stem cells, such as fetal liver suspension, can be infused into the recipient. The donor BMC will have the appropriate site in the recipient to grow and proliferate in succession with the remaining host cells, forming a chimeric lymphohematopoietic population. This process exposes newly forming B cells (and the antibodies they produce) to donor antigens, so that the graft will be recognized as self. Tolerance to the donor is also observed at the T cell level in animals in which engraftment of hematopoietic stem cells, such as BMC, has been achieved. The use of xenogeneic donors allows the feasibility of using bone marrow cells and organs from the same animal or from genetically matched animals.

[0135] Working Example The present invention will be better understood from the following Experimental Details, however, those skilled in the art will readily appreciate that the specific methods and results described are merely illustrative of the invention, as more fully described in the claims which follow.

[0136] Example 1 - Creation of a human / pig hybrid thymus to achieve immune tolerance to porcine antigens with optimal immune function The potent immune response to xenografts is difficult to control with conventional immunosuppression without excessive toxicity. Thymus transplantation is a promising approach to induce T cell tolerance for xenotransplantation. Humanized mice generated with human hematopoietic stem cells (HSC) and swine (SW) thymus grafts have previously been shown to be tolerant to both species. However, this approach still poses several challenges. First, T cells selected on SW MHC in the swine thymus may not optimally recognize antigens presented by the surrounding human MHC (HLA). Second, SW thymic epithelial cells (TECs) do not present human tissue-specific self-antigens (TRAs), thus compromising negative selection and potentially lacking Tregs specific for human TRAs. These issues were overcome by generating the human / swine hybrid thymus described herein.

[0137] method To generate human / pig hybrid thymocytes, thymic stromal cells were isolated by digestion of human fetal (20 weeks gestation) and pediatric (4 months) thymuses with Liberase, followed by magnetic removal of human CD45+ cells. Human CD45 cells were resuspended in Matrigel, treated with 2-deoxyglucose to inhibit glycolysis, and injected into frozen / thawed fetal SW thymus tissue, resulting in reduced numbers of porcine thymocytes in the fetal SW thymus (Figure 2A-C).

[0138] These injected SW thymi were then transplanted into irradiated NOD scid common γ-chain knockout (NSG) mice, followed by injection of human fetal liver-derived CD34+ HSCs from the same huTEC donor or allogeneic donors. At 12–20 weeks post-transplant, transplanted thymi were excised, sectioned, and stained to detect human TECs using two-photon confocal microscopy (Figure 1A–1D). Because the number of TECs in the adult thymus is reduced due to thymic atrophy, huTECs and thymic mesenchymal cells (TMCs) derived from the thymus from a 17-year-old donor were grown on a 2D Matrigel matrix and the cells were injected into fetal pig thymus tissue, followed by transplantation into humanized mice (Figure 1E–1H).

[0139] result huTEC-injected SW thymi were functional and supported human thymus formation in humanized mice. Cytokeratin (CK) 14+HLA-DR+ and CK8+HLA-DR+ cells as well as CK8+CK14+HLA-DR+ cells were detected in hybrid thymi generated by both human fetal and pediatric donors. These TECs were widely distributed and intermingled with porcine TECs (Figures 1A-1D). 17-year-old thymic EpCAM+TECs were expanded 5-fold in one passage. Hybrid thymi generated with in vitro expanded human TECs and mesenchymal cells contained human TECs (Figures 1E-1H).

[0140] conclusion Injection of human thymic stromal cells into the porcine thymus is an effective technique for generating human / pig hybrid thymuses. Human TECs from old thymi could be expanded in vitro using the protocol described herein and were detectable long-term in porcine explants.

[0141] Example 2 - Development of a method to generate hybrid thymus Injection method The cells were resuspended in Matrigel to prevent them from leaking out of the pig thymus after injection. In principle, in this experiment, human PBMCs were used instead of human thymic epithelial cells. First, 10 million human PBMCs were stained with CFSE (2.5 μM) as a tracking dye. CFSE-stained PBMCs (8 million cells) were resuspended in 140 μl Matrigel on ice to a cell concentration of 50,000 cells per μl. Three different methods of injecting cells into thawed fetal pig thymus fragments: Method A: Placing the pieces into wells of a V-bottom 96-well plate (5-8 μl) and injecting using a Hamilton syringe; Method B: Injection using PE50 tube (20 μl) Method C: Injection (4-6 μl) using a Hamilton syringe while holding the piece outside the well with forceps until the Matrigel solidifies.

[0142] All injected pieces were transferred to different wells of a 96-well plate containing DMEM / F12 medium supplemented with 10% FBS. After 3 h, the pieces were digested with Liberase. The number of released cells was determined by flow cytometry and CFSE-stained injected PBMCs were tracked. As shown in Figure 3A, at least a portion of the injected cells was recovered for all three injection methods.

[0143] In summary, injected cells were retained in pig thymus tissue when they were resuspended in Matrigel prior to injection. Matrigel is the trade name for a gel-like protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and produced and marketed by Corning Life Sciences and BD Biosciences.

[0144] A method for depleting porcine thymocytes to prevent rejection of human thymic stromal cells There are different strategies to deplete thymocytes in porcine thymic fragments ex vivo, including anti-porcine CD3 immunotoxin and complement-mediated toxicity (complement + rabbit serum as a source of anti-porcine CD2). However, these two strategies were not effective. The former strategy induces apoptosis in porcine thymocytes at higher concentrations, killing SP and DP T cells as well as non-T cells, leaving a large population of viable thymocytes in the thymus. The latter strategy was not effective in depleting porcine thymocytes in thymic fragments ex vivo. Results not shown.

[0145] Given these results, the following reagents were tested to deplete porcine thymocytes ex vivo using the following method: Day 0: Thaw porcine thymus pieces and incubate with one of the following (in triplicate): a) Cyclosporine A b) Hydrocortisone c) Notch inhibitors (γ-secretase inhibitors = GSi) d) ABT-737 e) 2-deoxyguanosine f) 2D glucose g) Untreated Tissues were digested at different time points and examined for the percentage of dead and apoptotic cells, see Figure 3B.

[0146] Ex vivo conditioning with 2-deoxyglucose (2DG, 100 mM) for 12 hours was the best approach to deplete thymocytes while leaving stromal cells alive. The ratio of remaining live double positive CD4 and CD8 cells (DP) or single positive (SP) CD4 (SP-CD4) or SP-CD8 cells to double negative (DN) cells, including stromal cells, was used as readout. Treatment with 100 nM 2DG for 12 hours resulted in the lowest ratio and was therefore the best strategy to eliminate thymocytes while preserving stromal cells. See Figure 3C.

[0147] Generation of hybrid thymus Using 2-DG treatment and a Hamilton syringe to deplete porcine thymocytes for injection of human thymic stromal cells (Method C above), hybrid thymi were generated and tested in vivo. Specifically, two vials of fetal porcine thymus were thawed. After pipetting up and down to release as many thymocytes as possible, half of the pieces were treated with 100 mM 2DG for 12 hours, while the other half was left untreated.

[0148] The next day, six vials of human fetal thymus were thawed. After pipetting up and down to release as many thymocytes as possible, Liberase was used to digest and release the stromal cells to create a single cell suspension. After depletion of human CD45+ cells using magnetic activated cell sorting (MACS), 10 million thymic stromal cells were dissolved in 150 μl of cold Matrigel at a concentration of 66,000 cells per μl. Approximately 10 μl of cells (approximately 660,000 thymic stromal cells) were injected into each fetal pig thymus piece (either 2DG-treated or untreated). The pieces were held with fine forceps for approximately 2 minutes at room temperature until the Matrigel solidified. Each piece was then transferred to a well of a 96-well plate containing medium with 10% human serum. As a control, some pieces were not injected with human thymic stromal cells. After 10 min in the incubator, the plates were transferred, still on ice, to the mouse facility for transplantation into immunodeficient NSG mice. The recipient mice had previously been thymectomized, so they did not have a native mouse thymus and the only site for thymus formation was the transplanted thymus fragment. First, the recipient NSG mice were irradiated (100cG) and subsequently injected with human fetal liver-derived CD34+ hematopoietic stem cells (HSCs). Then, one thymus fragment was transplanted under the kidney capsule of each recipient mouse.

[0149] The experimental design is shown in FIG.

[0150] Every 2-3 weeks after transplantation, blood was collected from the mice to assess the level of human immune cell reconstitution. As shown in Figure 5, treatment of the porcine thymus fragments with 2-DG did not affect human thymus formation in the transplanted thymus.

[0151] At 20 weeks after transplantation, mice were euthanized and transplanted thymuses were removed and frozen in OCT. After cryosectioning, slides were stained with antibodies against human HLA-DR, cytokeratin 8 (CK8, as a marker for cortical thymic epithelial cells (TECs)), and CK14 (as a marker for medullary TECs). Since the CK antibody is cross-reactive to both human and pig, HLA-DR was used to differentiate human and pig TECs. As shown in Figure 6, injected human TECs mixed with pig TECs were detected in the transplanted thymus. HLA-DR+ cells that were negative for CK were HSC-derived antigen-presenting cells migrating from bone marrow to the transplanted thymus.

[0152] The in vitro T cell expansion results suggested that peripheral T cells from mice with hybrid thymuses were partially tolerant to the human TEC donor (Figure 7B).

[0153] Human T cells from all mice were tolerant to porcine donor dendritic cells (DCs), but not to the porcine DCs of the third group in SLA-CC (FIG. 7A).

[0154] Human T cells from all mice were tolerant to human HSC-donor DCs (FIG. 7B).

[0155] Human T cells from mice with a pig thymus proliferated at similar levels in response to both human thymic donor-DCs (allogeneic with human HSCs) and human allogeneic-DCs. Human T cells from mice with a hybrid thymus proliferated at reduced levels in response to human thymic donor-DCs compared to allogeneic-DCs (Figure 7B).

[0156] Example 3 - Increased responsiveness to human tissue-specific autoantigens (TRAs) (MART-1, NYESO1 and islet antigen IA-2) in human T cells developing in the porcine thymus (SW / HU mice) compared to cells developing in the human thymus (HU / HU mice) method To evaluate the hypothesis of the lack of negative selection of human TRA-specific T cells developing in the pig thymus, two groups of humanized mice were generated using the same human fetal liver CD34+ HSCs and either autologous human fetal thymus (HU / HU) or pig fetal thymus (SW / HU). See Figure 8A. In both groups, the native mouse thymus was removed to ensure that thymus formation occurred exclusively in either the human or pig thymus. Approximately 22 weeks after transplantation, mice were euthanized and pooled lymph node (LN) and splenocytes were depleted of mouse CD45+ cells using MACS. The remaining cells were co-cultured with autologous HSC-derived dendritic cells with different human TRA proteins to measure T cell proliferation in response to these TRAs.

[0157] result As shown in Figure 8B, human peripheral T cells in SW / HU mice showed significantly increased proliferative responses to human TRAs (IA-2, MART-1, and NYESO1) presented by autologous human DCs. The amino acid sequences of these TRAs differ significantly between humans and pigs. This finding supported the lack of negative selection of human TRA-specific T cells in the pig thymus and demonstrated the necessity of using a hybrid thymus.

[0158] Example 4 - Lower survival of human Treg and CD8 T cells developed in the pig thymus (SW / HU mice) compared to cells developed in the human thymus (HU / HU mice) method Mice (SW / HU and HU / HU mice) prepared in Example 3 were euthanized at approximately 24 weeks after transplantation. The transplanted thymus and pooled spleen and lymph nodes (cervical, axillary, brachial and mesenteric LN) were collected. Thymocytes, splenocytes, and LN cells were isolated by physical force (crushing the thymus tissue between two slides and crushing the spleen and LN through a 70 μm cell strainer using a syringe plunger). RBCs in splenocytes were lysed using ACK lysis buffer (Gibco). The isolated cells were counted using a hemocytometer. After counting the total cell number, 0.2 to 1 million cells from each thymus and pooled spleen and LN were stained with the antibodies shown in Figure 9 for flow cytometry analysis. The cells were read on a BD Fortessa flow cytometry machine, and the data were analyzed with Flowjo software.

[0159] result The numbers of spleen and LN cells and transplanted thymocytes were significantly higher in HU / HU mice compared to SW / HU mice (Figures 9A and 9G). The ratio of DP (double positive CD4+CD8+) to SP cells (either single positive SP-CD4 or SP-CD8) in the transplanted thymus was similar between HU / HU and SW / HU mice (Figure 9B). A functional thymus should have a higher ratio of DP than SP cells.

[0160] Additionally, the fraction of Treg (regulatory T cells) in SP-CD4 cells was similar (Figure 9C). The levels of proliferating (Ki67+) SP-CD4 and Treg cells were higher in SW / HU thymus compared to HU / HU thymus (Figures 9D and 9F). Additionally, a higher fraction of SW / HU thymic Treg expressed CD45RO (Figure 9E).

[0161] In contrast to similar levels of SP-CD4 and SP-CD8 cells in the thymus, SW / HU mice had lower proportions of CD8 cells among T cells and Treg among CD4 cells in the periphery (spleen and lymph nodes, Figures 9I-9K). This finding indicated that both of these cell subsets need to interact with the same MHC for which they were selected to survive. A higher rate of naive to memory conversion was evident in both peripheral CD4 and CD8 cell subsets of SW / HU mice (Figures 9L and 9M). Ki67+ (proliferation) (Figure 9O), HLA-DR+ (activation) (Figure 9N), CD45RO+ (Figure 9P), and CTLA-4+ cells (Figure 9Q) within peripheral CD4, CD8, and Treg cells did not differ between HU / HU and SW / HU.

[0162] These results further demonstrate the need for the use of hybrid thymuses.

[0163] Example 5 - Generation of hybrid thymus with embryonic stem cell-derived TECs (ES-TECs) and long-term persistence of human TECs in the hybrid thymus method 2×10 5 Human fetal or pediatric thymic stromal cells (hu-TECs) or hPSC-TEC precursors (1–2 × 10) were injected intravenously with human fetal liver-derived CD34 cells before transplantation under the renal capsule of thymectomized immunodeficient NSG (Nod / Scid / Ilr2g- / -) mice. 5 We injected human TECs into fetal pig thymus tissue (SW THY). As a control, we transplanted fetal pig thymus tissue that had not been injected with human TECs.

[0164] Approximately 20 weeks after transplantation, the transplanted thymus was removed, sectioned, and stained to detect human TECs using two-photon confocal microscopy.

[0165] The cells were then released from the stroma and the number of cells was determined by flow cytometry.

[0166] result As shown in Figure 10, human TECs in the "hybrid thymus" were long-term (>20 weeks) persistent. As shown in Figure 10A, all grafts (top left panel), including pig thymuses that were not injected with human TECs, have human HSC-derived HLA-DR+ APCs (green) and pig / human CK14+ TECs (red). However, only the hybrid thymus had detectable HLA-DR+CK14+ human TECs (yellow, see arrow).

[0167] Flow cytometric staining of gated CD45-negative cells in digested stroma from long-term thymic grafts showed the presence of EPCAM+, CD105-negative hu-TECs only in human thymus (Fig. 10B, top right) and SW grafts injected with hPSC-TEC precursors (Fig. 10B, bottom left panel), but not in uninjected SW THY grafts (Fig. 10B, top left). See Fig. 10C.

[0168] Example 6 - Injection of hES-TECs into the porcine thymus promotes T cell development and peripheral CD4 + and CD8 + Increased T cells method 2×10 5 hES-TECs (1–2 × 10) were injected intravenously with human fetal liver-derived CD34 cells prior to transplantation under the renal capsule of thymectomized immunodeficient NSG (Nod / Scid / Ilr2g) mice. 5 We injected human TECs into fetal pig thymus tissue (SW THY). As a control, fetal pig thymus tissue was transplanted without human TECs.

[0169] Splenocytes and thymocytes from thymic grafts were analyzed by flow cytometry at 18-22 weeks after transplantation.

[0170] result As shown in Figure 11A-D, thymic grafts injected with hES-TECs showed higher absolute numbers of human splenic CD3 + T cells, CD8 + T cells, CD4 + T cells and CD45RA+ CCR7 + Recent thymic emigrated CD31 cells defined + CD4 + with untreated cells.

[0171] To assess the final stage of terminal differentiation, thymocytes were stained for expression of HuCD45, CD19, CD14, CD4, CD8, CD45RA, and CD45RO. As shown in Figure 11E, thymic grafts injected with hES-TECs had higher numbers of total thymocytes, as well as higher numbers of human CD45 cells, double positive CD4+CD8+, single positive CD4+CD8-, CD4-CD8+, and immature CD45RO+.

[0172] The scope of the present invention is not limited to what has been specifically shown and described above. Those skilled in the art will recognize that there are suitable alternatives to the examples of materials, configurations, constructions and dimensions shown. Numerous references, including patents and various publications, have been cited and described in the description of the present invention. The citation and description of such references are provided merely to clarify the description of the present invention and are not an admission that the references are prior art to the invention described herein. All references cited and described herein are incorporated herein by reference in their entirety. Those skilled in the art will recognize variations, modifications and other embodiments of what is described herein without departing from the spirit and scope of the present invention. While certain embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the present invention. The foregoing is provided by way of example only and not by way of limitation.

Claims

1. Use of isolated hybrid thymic tissue for the manufacture of a medicament for inducing tolerance in a recipient mammal of a first mammalian species to a graft obtained from a donor mammal of a second mammalian species, said isolated hybrid thymic tissue comprising thymic epithelial cells from the first mammalian species and thymic tissue from the second mammalian species.

2. The use described in claim 1, wherein the second mammalian species is a pig.

3. The use described in claim 2, wherein the pig is a miniature pig.

4. The use described in claim 1, wherein the first mammalian species is a primate or a human.

5. The use described in claim 1, wherein the thymic tissue from the second mammalian species is fetal thymic tissue or neonatal thymic tissue.

6. The use described in claim 1, wherein the thymic epithelial cells are obtained from the thymus of a first mammalian species.

7. The use described in claim 1, wherein the thymic epithelial cells are produced from induced pluripotent stem cells (iPSCs) or embryonic stem cells.

8. The use described in claim 7, wherein the thymic epithelial cells are generated from embryonic stem cells that share HLA alleles with a first mammalian species.

9. The use described in claim 7, wherein the embryonic stem cells are genetically engineered to share HLA alleles with the first mammalian species.

10. The use described in claim 1, wherein the hybrid thymic tissue is produced by introducing thymic epithelial cells from the first mammalian species into the thymic tissue from the second mammalian species.

11. The use described in claim 1, wherein the hybrid thymic tissue is produced by injecting thymic epithelial cells from the first mammalian species into thymic tissue from the second mammalian species.

12. The hybrid thymus tissue according to claim 11, further comprising: (i) treating thymus tissue from the second mammalian species with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from the first mammalian species into the 2DG-treated thymic tissue. The use according to claim 1, which is produced by an in vitro or ex vivo method comprising:

13. The use described in claim 12, wherein in step (ii), the thymic epithelial cells are suspended in Matrigel before being injected into the 2DG-treated thymic tissue.

14. Use of isolated hybrid thymic tissue for the manufacture of a medicament for restoring or inducing immune competence in a recipient mammal of a first mammal, said isolated hybrid thymic tissue comprising thymic epithelial cells from a first mammalian species and thymic tissue from a second mammalian species.

15. The use of claim 14, wherein the second mammalian species is a pig.

16. The use described in claim 15, wherein the pig is a miniature pig.

17. The use of claim 14, wherein the first mammalian species is a primate or a human.

18. The use of claim 14, wherein the thymic tissue from the second mammalian species is fetal thymic tissue or neonatal thymic tissue.

19. The use described in claim 14, wherein the thymic epithelial cells are obtained from the thymus of the first mammalian species.

20. The use of claim 14, wherein the thymic epithelial cells are produced from induced pluripotent stem cells (iPSCs) or embryonic stem cells.

21. The use of claim 20, wherein the thymic epithelial cells are generated from embryonic stem cells that share an HLA allele with a first mammalian species.

22. The use of claim 20, wherein the embryonic stem cells are genetically engineered to share HLA alleles with the first mammalian species.

23. The use of claim 14, wherein the hybrid thymic tissue is produced by introducing thymic epithelial cells from the first mammalian species into thymic tissue from the second mammalian species.

24. The use of claim 14, wherein the hybrid thymic tissue is produced by injecting thymic epithelial cells from the first mammalian species into thymic tissue from the second mammalian species.

25. The hybrid thymus tissue according to claim 24, further comprising: (i) treating thymus tissue from the second mammalian species with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from the first mammalian species into the 2DG-treated thymic tissue. The use according to claim 14, which is produced by an in vitro or ex vivo method comprising:

26. The use of claim 25, wherein in step (ii), the thymic epithelial cells are suspended in Matrigel before being injected into the 2DG-treated thymic tissue.

27. ​​Use of isolated hybrid thymic tissue for the manufacture of a medicament for restoring or promoting thymic dependency for the development of T cell precursor cells into mature functional T cells in a recipient mammal of a first mammalian species, said isolated hybrid thymic tissue comprising thymic epithelial cells from the first mammalian species and thymic tissue from a second mammalian species.

28. The use of claim 27, wherein the second mammalian species is a pig.

29. The use described in claim 28, wherein the pig is a miniature pig.

30. The use of claim 27, wherein the first mammalian species is a primate or a human.

31. The use of claim 27, wherein the thymic tissue from the second mammalian species is fetal thymic tissue or neonatal thymic tissue.

32. The use of claim 27, wherein the thymic epithelial cells are obtained from the thymus of a first mammalian species.

33. The use of claim 27, wherein the thymic epithelial cells are produced from induced pluripotent stem cells (iPSCs) or embryonic stem cells.

34. The use of claim 33, wherein the thymic epithelial cells are generated from embryonic stem cells that share HLA alleles with a first mammalian species.

35. 34. The use of claim 33, wherein the embryonic stem cells are genetically engineered to share HLA alleles with the first mammalian species.

36. The use of claim 27, wherein the hybrid thymic tissue is produced by introducing thymic epithelial cells from the first mammalian species into the thymic tissue from the second mammalian species.

37. The use of claim 27, wherein the hybrid thymic tissue is produced by injecting thymic epithelial cells from the first mammalian species into thymic tissue from the second mammalian species.

38. The hybrid thymus tissue, comprising: (i) treating thymus tissue from the second mammalian species with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from the first mammalian species into the 2DG-treated thymic tissue.

28. The use according to claim 27, produced by an in vitro or ex vivo method comprising:

39. The use of claim 38, wherein in step (ii), the thymic epithelial cells are suspended in Matrigel before being injected into the 2DG-treated thymic tissue.

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