Methods for inducing allogeneic tissue antigen tolerance

Abstract

Provided are methods of making a chimeric thymus in a subject, including implanting a donor thymic functional unit tissue graft, wherein the donor thymic functional unit tissue graft includes thymic epithelial cells (TEC) and dendritic cells. The chimeric thymus is useful to induce a dual immunologic tolerance to both donor tissue antigens and tissue antigens of the subject.

Description

Attorney Docket No. 9865.241.WOMethods for Inducing Allogeneic Tissue Antigen ToleranceSTATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No.63 / 720,976, filed November 15, 2024, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. W81XWH-19-1-0011 awarded by the Department of Defense. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] The thymus is a primary lymphoid organ responsible for the development and education of the T-cells. Thus, the thymus plays a major role in the development of self-tolerance and is crucial for induction of acquired tolerance to exogenous antigens. Morphologically, the thymus has multiple lobules, divided by fibrous septa, and every thymus lobule is composed of a cortex region and a medulla region. The cortex region includes immature T lymphocytes, and the medulla region contains mature T cells. In addition to T cells, the thymus also includes dendritic cells, macrophages, and B cells in the medulla. The cortical thymic epithelial cells (cTEC) play a role in T cell development and the medullary thymic epithelial cells (mTEC) play a role in presenting self-antigens to developing T cells and causing deletion of self-reactive T cells (Rezzani, R., et al., L. F. Thymus and aging: morphological, radiological, and functional overview. Age (Dordr) 36, 313-351 (2014)).

[0004] T-cell differentiation in the thymus generates a peripheral repertoire of mature T cells that mounts strong responses to foreign antigens but is largely unresponsive to self-antigens. This state of specific immunological tolerance to self-components involves both central and peripheral mechanisms. During thymic maturation, T cells with potential reactivity for selfantigens are eliminated in the thymus during early T-cell differentiation. This process of central tolerance (negative selection) reflects apoptosis and is a consequence of immature T cellsAttorney Docket No. 9865.241.WOreceiving strong intracellular signaling through T-cell receptor (TCR) recognition of peptides bound to major histocompatibility complex (MHC) molecules. Central tolerance occurs mainly in the medullary region of the thymus and depends upon contact with peptide-MHC complexes expressed on bone-marrow-derived antigen-presenting cells (APCs) (Cosway, E.J. et al., Redefining thymus medulla specialization for central tolerance. J Exp Med 214, 3183-3195 (2017)).

[0005] Studies have reported that thymus-dependent tolerance can be achieved via the presence of donor derived MHC Class II antigens in a recipient thymus (Bozkurt, M. et al., Composite osseomusculocutaneous sternum, ribs, thymus, pectoralis muscles, and skin allotransplantation model of bone marrow transplantation. Microsurgery 33, 43-50 (2013); Zor, F. et al., The effect of thymus transplantation on donor-specific chimerism in the rat model of composite osseomusculocutaneous sternum, ribs, thymus, pectoralis muscles, and skin allotransplantation. Microsurgery (2020)). In this way, deletion of alloreactive T cells can be achieved and transplantation from the same donor would be tolerated (Alfadil, S. et al., 2558: Optimization of intra-thymic transplantation of donor-derived thymic epithelial cells to promote lasting regulation of anti-donor reactivity. Vascularized Composite Allotransplantation 3, 46-46 (2016)). Thymus transplantation enables education of the host T-cells in the donor thymus followed by selection based on the donor thymic epithelial cells. Once T-cells that are educated in the donor thymus migrate to the periphery, they develop specific, dual-restrictive and dual tolerogenic effects to both the donor and the recipient. In addition, peripheral naive T-cells of the recipient can return to the thymus for re-education, which may facilitate transplant tolerance (Bosco, N., et al., Peripheral T cells in the thymus: have they just lost their way or do they do something? Immunol Cell Biol 87, 50-57 (2009)).

[0006] Several models of vascular transplantation of thymus in rats, mice and swine have been reported (Bozkurt, M., supra; Vagefi, P. A. et al., Role of the thymus in transplantation tolerance in miniature Swine: IV. The thymus is required during the induction phase, but not the maintenance phase, of renal allograft tolerance. Transplantation 77, 979-985 (2004)). These animal studies showed that the presence of both donor and recipient antigen within the thymus mediates clonal deletion of both donor- and recipient-reactive thymocytes and may provide for the education of T-regs. Moreover, peripheral T cells in the recipient can also undergo re-education in the donor thymus and contribute to tolerance induction (Bosco, N., et al.); (Vagefi, P. A. et al.); (Zhao, D. et al., Vascularized whole thymus transplantation in Rowlett nude rats: effect of thymus allograft volume on tolerance induction. Transpl Immunol 23, 40-44 (2010)).Attorney Docket No. 9865.241.WO

[0007] However, vascularized transplantation of thymus is not clinically applicable. Accordingly, such thymus transplantation is not considered to be a viable option. Other techniques of thymic transplantation in combination with solid organ transplantation or vascularized composite allotransplantation have been described (Chen, L. et al., Construction and functional evaluation of an autologous thymokidney model in the rat. Transplant Proc 39, 3409-3414 (2007)). Moreover, existing strategies in intra-thymic bone marrow transplantations have not been able to generate donor thymic epithelial cells (TEC), which have a major contribution to central selection, within recipient thymus.

[0008] There is a critical need for a clinically applicable strategy for inducing a dual tolerance in an allograft transplant recipient to both donor and recipient antigens and reduce the need for chronic administration of immunosuppressants.SUMMARY OF THE INVENTION

[0009] Provided herein according to some embodiments is a method of making a chimeric thymus in a subject, comprising providing a donor thymic functional unit tissue graft, wherein the donor thymic functional unit tissue graft comprises thymic cortex and medullary structures, including thymic epithelial cells (TEC) and dendritic cells; and implanting the donor thymic functional unit tissue graft by orthotopic intra-thymic transplantation into the thymus of the subject, thereby providing the chimeric thymus, wherein the donor thymic functional unit tissue graft is from a pediatric donor, and wherein the chimeric thymus induces a dual immunologic tolerance to both donor tissue antigens and tissue antigens of the subject.

[0010] In some embodiments, the implanting is carried out by implanting the donor thymic functional unit tissue graft into a dissected pocket in the thymus of the subject.

[0011] In some embodiments, the donor thymic functional unit tissue graft is provided as a thymus hemi-lobe of the donor.

[0012] Also provided is a method of reducing tissue rejection following transplant of an allogeneic tissue graft in a subject, comprising: (a) making a chimeric thymus in the subject by a method taught herein, wherein the donor thymic functional unit tissue graft is from the same donor as the allogeneic tissue graft; and (b) transplanting the allogeneic tissue graft into the subject, thereby reducing tissue graft rejection in the subject.

[0013] In some embodiments, the method further comprises inducing clonal depletion of alloreactive T cells in the subject by administering a composition comprising lympho-depleting antibodies before and / or after transplanting the allogeneic tissue graft.Attorney Docket No. 9865.241.WO

[0014] In some embodiments, the allogeneic tissue graft comprises a vascularized tissue graft such as a vascularized composite allotransplantation (VC A) graft (e.g., a hand, foot, joint, or limb transplant), a facial tissue graft, a solid organ (such as kidney, liver, heart, lung, etc.), or a uterine graft.

[0015] In some embodiments, the allogeneic tissue graft comprises a non-vascularized tissue graft such as a skin graft, a nerve graft, a cartilage graft, or a bone graft.

[0016] In some embodiments, the transplanting step (b) is carried out simultaneously with the making the chimeric thymus of step (a).

[0017] In some embodiments, the method further includes: (c) administering an immunosuppressant to the subject.

[0018] In some embodiments, the immunosuppressant is administered for an initial time (e.g., of from 1, 5, or 10 to 15, 20, 25 or 30 days) following the transplanting step (b), and then administered subsequently at a low dose to the subject. In some embodiments, the low dose is about 5, 10, 20, 50, or 100 times lower than the dosage of immunosuppressant administered for the initial time following the transplanting step (b).

[0019] Further provided is use of a chimeric thymus as taught herein in a method of reducing tissue rejection following transplant of an allogeneic tissue graft in a subject. In addition, provided is use of a chimeric thymus as taught herein in a method of inducing a dual immunologic tolerance to both donor tissue antigens and tissue antigens in a subject.

[0020] Also provided is use of an immunosuppressant for reducing tissue rejection in a subject having a chimeric thymus as taught herein, or for the manufacture of a medicament for reducing tissue rejection in a subject having a chimeric thymus as taught herein. In some embodiments, the immunosuppressant is administered at a low dose to the subject. In some embodiments, the low dose is about 5, 10, 20, 50, or 100 times lower than the dose of immunosuppressant that would be used in a subject that does not have a chimeric thymus.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 is a graph depicting the trough tacrolimus levels (ng / ml) of subjects in experimental groups A3 and A4 throughout the follow up of each subject after groin flap transplantation. Group A3 is labeled on the graph as "Groin flap only." Group A4 is labeled on the graph as "Groin flap + ITT." Tacrolimus was administered to the subjects of both groups at a rate of 1 mg / kg / day for the first 21 days after groin flap transplantation, and then the subjects were weaned from tacrolimus treatment thereafter. Venous blood samples wereAttorney Docket No. 9865.241.WOcollected from each subject and then tacrolimus levels (ng / ml) were measured using standard LCMS technique.

[0022] FIG. 2 is a graph depicting a Kaplan Meier survival curve comparing the probability of survival of groin flap allografts between experimental groups A3 and A4 subjects. Group A3 is labeled on the graph as "Groin flap only." Group A4 is labeled on the graph as "Groin flap + ITT." The subjects were followed up daily by clinical observation. The allograft groin flap survival is measured by the number of days the allograft tissue survives until Banff Grade II rejection is observed.

[0023] FIG. 3 is a graph depicting a Kaplan Meier survival curve comparing the probability of survival of groin flap allografts between experimental groups A5 and A6 subjects. Group A5 is labeled on the graph as "ALS+TAC." Group A6 is labeled on the graph as "ALS+TAC+ITT." The subjects were followed up daily by clinical observation. The allograft groin flap survival is measured by the number of days the allograft tissue survives until Banff Grade II rejection is observed.

[0024] FIG. 4 is a graph depicting the systemic tacrolimus levels (ng / ml) of subjects in experimental groups A5 and A6 throughout the follow up of each subject after groin flap transplantation. Group A5 is labeled on the graph as "ALS + Tac (Control)." Group A6 is labeled on the graph as "ALS + Tac + ITT." Tacrolimus was administered to the subjects of both groups at a rate of 1 mg / kg / day for the first 21 days after groin flap transplantation, and then the subjects were weaned from tacrolimus treatment thereafter. Jugular vein blood samples were collected from each subject and then tacrolimus levels (ng / ml) were measured using standard LCMS technique.

[0025] FIG. 5 is a graph depicting a Kaplan Meier survival curve comparing the probability of survival of groin flap allografts between experimental groups A8 and A9 subjects. Group A8 is labeled on the graph as "ALS + TAC with ultralow dose." Group A9 is labeled on the graph as "ITT + AT ,S + TAC with ultralow dose." The subjects were followed up daily by clinical observation. The allograft groin flap survival is measured by the number of days the allograft tissue survives until Banff Grade II rejection is observed.

[0026] FIG. 6 is a graph depicting the systemic tacrolimus levels (ng / ml) of subjects in experimental groups A8 and A9 throughout the follow up of each subject after groin flap transplantation. Group A8 is labeled on the graph as "ALS + Ultra-low Tac (Control)." Group A9 is labeled on the graph as "ALS + Ultra-low Tac + ITT." Tacrolimus was administered to the subjects of both groups at a rate of 1 mg / kg / day for the first 21 days after groin flap transplantation, and then the subjects were administered a subtherapeutic ultralow dosage ofAttorney Docket No. 9865.241.WO0.1 mg / kg / day continuously afterward. Jugular vein blood samples were collected from each subject and then tacrolimus levels (ng / ml) were measured using standard LCMS technique.DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will now be described in further detail and embodiments. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0029] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a structure or composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0030] As used herein in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0031] Furthermore, the term "about," as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, is meant to encompass variations of 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0032] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").Attorney Docket No. 9865.241.WO

[0033] As used herein, the transitional phrase "consisting essentially of is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of' as used herein should not be interpreted as equivalent to "comprising."

[0034] An "immunologic tolerance" refers to the reduction or inhibition of an adaptive immune response in a subject to antigen(s) that would otherwise trigger an immune response after previous exposure to the antigen.

[0035] The term "inhibit" or "reduce" or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0036] The term "enhance" or "increase" refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold.

[0037] A "therapeutically effective" amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a "therapeutically effective" amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g., in the case of allogeneic tissue graft transplantation, a decrease in donor tissue rejection). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0038] A "pediatric" donor refers to an infant, child or adolescent animal donor. For a human donor, a pediatric donor is less than 18 years of age. In some embodiments, the human pediatric donor is less than 17 year of age. In some embodiments, the human pediatric donor is less than 16 year of age. In some embodiments, the human pediatric donor is less than 15 year of age.

[0039] The term "thymic functional unit tissue graft" as used herein refers to a solid tissue piece or portion of a donor thymus organ (also referred to as a "minigraft" herein) that comprises both thymus cortex and medullary structures, and is preferably morphologically organized similar to native thymus tissue, such structures including both thymic epithelial cells (TEC) and dendritic cells. Such graft may be produced by excising a portion of the thymus from a donor, for example by incision or punch biopsy. In some embodiments, the portion is a hemi-lobe of the thymus. Preferably, the tissue graft is capable of educating immature T cells to be tolerable of donor antigens, re-educating mature recipient T cells to be tolerable of donor antigens, and depleting alloreactive T cells, upon reconstituting a recipient subject thymusAttorney Docket No. 9865.241.WOmicroenvironment, thus sustaining central tolerance of the recipient subject to the donor tissue graft.

[0040] The term "hemi-lobe" as used herein refers to a lobed thymic solid tissue graft derived from a bilobed thymus organ and comprising the thymic subcomponents of the cortex and the medulla, which contain thymic functional unit tissue.

[0041] The term "dissected pocket" as used herein refers to a pocket created in a recipient thymus organ by incising the recipient thymus organ.

[0042] "Immunosuppressants" are agents capable of slowing or stopping an immune system of a subject from recognizing and / or targeting foreign and / or self-antigens. Examples of immunosuppressants include, but are not limited to, tacrolimus, rapamycin (sirolimus), everolimus, cyclosporine, my cophenolate mofetil, OKT3, basiliximab, daclizumab, belatacept, a corticosteroid (e.g., prednisone, methylprednisolone), azathioprine (e.g., Azasan, Imuran), mercaptopurine (e.g., Purinethol, Purixan) and / or methotrexate (e.g., Trexall).

[0043] The term "depletional induction" or "clonal depletion" as used herein refers to inducing a depletion of T cells and B cells in a subject for a period of time to help prevent alloreactive T or B cells from attacking an allogeneic tissue graft transplant, often by use of a composition comprising lympho-depleting antibodies. Examples of compositions with lympho-depleting antibodies include, but are not limited to, antilymphocyte serum (ALS), an antithymocyte globulin (ATG), alemtuzumab, and rituximab.

[0044] Examples of allogeneic tissue grafts that may be used for transplanting include vascularized and non-vascularized tissues. Vascularized tissues include, but are not limited to, a vascularized composite allotransplantation (VCA) graft (e.g., a hand, foot, joint, or limb transplant), a facial tissue graft, a solid organ (such as kidney, liver, heart, lung, etc.), and a uterine graft. Non-vascularized tissues include, but are not limited to, a skin graft, a nerve graft, a cartilage graft, and a bone graft.Chimeric Thymus

[0045] One aspect of the invention is a method of making a chimeric thymus in a subject in need thereof, the method comprising providing a donor thymic functional unit tissue graft, and implanting the donor thymic functional unit tissue graft, optionally via an orthotopic intra-thymic thymus transplantation, into a recipient thymus. Preferably, the donor of the donor thymic functional unit tissue graft is a pediatric subject. Preferably, the chimeric thymus induces a dual immunologic tolerance to both donor and recipient tissue antigens.Attorney Docket No. 9865.241.WO

[0046] In some embodiments, the implanting step is carried out by creating a dissected pocket in the recipient thymus. Further, in some embodiments, the donor thymic functional unit tissue graft is provided as a hemi-lobe of a donor thymus.

[0047] In some embodiments, the subject is a human subject in need thereof. In some embodiments, the subject is a non-human animal subject (e.g., for veterinary or research purposes).Methods of Reducing Tissue Rejection

[0048] Another aspect of the invention is a method of reducing tissue rejection following an allogeneic tissue graft transplant in a subject, the method comprising making the chimeric thymus as described herein and transplanting the allogeneic tissue graft into the recipient subject; thereby reducing tissue rejection of the allogeneic tissue graft in the subject. Preferably, the donor thymic functional unit tissue graft is from the same donor as the allogeneic tissue graft.

[0049] In some embodiments, the tissue rejection in the subject is reduced by approximately 20% to about 100% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%) compared to subjects without the chimeric thymus.

[0050] In some embodiments, the method further comprises inducing clonal depletion of alloreactive T cells in the subject by administering a composition comprising lympho-depleting antibodies before and / or after transplanting the allogeneic tissue graft. In some embodiments, the composition comprising lympho-depleting antibodies is an antilymphocyte serum (ALS), an antithymocyte globulin (ATG), alemtuzumab, and / or rituximab.

[0051] In some embodiments, the allogeneic tissue graft comprises a vascularized tissue graft or a non-vascularized tissue graft.

[0052] In some embodiments, transplanting the allogeneic tissue graft to the subject in need thereof is carried out simultaneously with making the chimeric thymus in the subject, e.g., on the same day and / or within the same surgery.

[0053] In some embodiments, the method further includes administering a therapeutically effective amount of an immunosuppressant to the subject. In some embodiments, the immunosuppressant is administered for an initial time of from 1, 5, or 10 to 15, 20, 25 or 30 days following the transplanting, and then administered subsequently at a low dose to the subject. In some embodiments, the low dose (also referred to herein as an "ultralow dose" or "subtherapeutic dose") is about 5, 10, 20, 50, or 100 times lower than the dosage of immunosuppressant administered for the initial time following the transplanting.Attorney Docket No. 9865.241.WO

[0054] In some embodiments, the immunosuppressant is tacrolimus, rapamycin (sirolimus), everolimus, cyclosporine, my cophenolate mofetil, OKT3, basiliximab, daclizumab, belatacept, a corticosteroid (e.g., prednisone, methylprednisolone), azathioprine (e.g., Azasan, Imuran), mercaptopurine (e.g., Purinethol, Purixan) and / or methotrexate (e.g., Trexall). In some embodiments, the immunosuppressant administered is tacrolimus. In some embodiments, the immunosuppressant administered is rapamycin.

[0055] In some embodiments, the immunosuppressant is administered at a low dose to the subject after the short course administration of the immunosuppressant at a therapeutically effective amount. Such low dose of the immunosuppressant may be, for example, about 5 times, about 10 times, about 20 times, about 50 times, or about 100 times lower than the standard dosage of immunosuppressant.

[0056] In some embodiments, the administering of the immunosuppressant step further comprises depletional induction of alloreactive T cells before and / or after transplanting the allogeneic tissue graft(s) by introducing a lympho-depleting antibody, thereby reducing tissue rejection in the subject. In some embodiments, the lympho-depleting antibody includes, but is not limited to, an antilymphocyte serum (ALS), an antithymocyte globulin (ATG), alemtuzumab, and / or rituximab. In some embodiments, the lympho-depleting antibody introduced to the subject is an antilymphocyte serum (ALS). In some embodiments, the lympho-depleting antibody introduced to the subject is an antithymocyte globulin (ATG).

[0057] In some embodiments, the method further comprises immunomodulating the peripheral and / or central immunoregulatory mechanisms by inducing a dual tolerogenic effect through reeducating T cells of the subject to recognize both donor and recipient tissue antigens and facilitating clonal depletional induction of alloreactive T cells.

[0058] The present invention is explained below in further detail with reference to the following non-limiting examples.EXAMPLESExample 1: Establishing a syngeneic rodent intra-thymic thymus transplantation model

[0059] In performing the orthotopic intra-thymic thymus transplantation (ITT), preserving the vascularization of the recipient thymus during the transplantation procedure is important to making the functional chimeric thymus. Ex vivo anatomical dissections revealed that a small branch of the internal mammary artery is the vascular supply to the subjects' thymus. Furthermore, the dissection demonstrated that one viable method of ITT comprises using a 2Attorney Docket No. 9865.241.WOmm punch biopsy as the donor thymus graft to make the chimeric thymus. The 2 mm punch biopsy was placed parallel to the caudal-cranial axis of the recipient thymus solid tissue graft. Thus, this placement for the donor thymus provided a blood supply for the donor thymus tissue graft.

[0060] Next, the thymus injection technique was refined to better establish a successful syngeneic rodent ITT model. First, thymus tissue, including both cortical and medullary thymic structures, was harvested from Lewis rats. Second, an incision was cut into the recipient Lewis rats using a mini-sternotomy incision. The mini-sternotomy was chosen to allow the harvested donor thymus tissue to be injected without opening the thoracis cavity of the recipient rat subject and cause minimal complication. Finally, the donor thymus tissue is injected into the thymus tissue of the recipient rats, thereby transplanting the donor thymus tissue to the recipient thymus.

[0061] After refining the thymus injection technique, six ITT procedures were performed on rat subjects. For this aspect of the study, two rat groups, Green Florescent Protein (GFP)-Lewis rats and non-GFP Lewis rats, were operated on and subsequentially observed as subjects. The procedures utilized cross-transplantation, so that each type of rat subject served as both a donor and a recipient of a thymus tissue graft. Three ITT procedures were performed using a GFP-Lewis rat as the donor subject and a non-GFP rat as the recipient subject, and the remaining three procedures were performed using a non-GFP Lewis rat as the donor subject and a GFP-Lewis rat as the recipient subject. In performing the ITT procedures, the donor GFP-Lewis rat subject was placed under general anesthesia and laid in the supine position. To extract donor thymus tissue grafts, first, a midline incision was cut at the anterior neck until the mid-thoracic region of the rat subject. Second, a mini-sternotomy was cut to the cranial half of the sternum at the midline of the rat subject, thus exposing the thymus. Additionally, while performing the mini-sternotomy, extensive care was taken to prevent injury to the parietal pleura of the rat subject. After exposing the donor thymus, a 2 mm biopsy punch was used to harvest two thymus minigrafts from each lobe of the donor thymus. This yielded two through and through defects in each thymus lobe of the donor rat subject and two harvested thymus minigrafts. These harvesting steps were performed on a non-GFP Lewis rat subject as well, so that each respective donor thymus minigraft could be switched and inserted into a recipient rat from the opposite group (e.g., a donor thymus minigraft extracted from a GFP-Lewis rat was inserted into a recipient non-GFP Lewis rat's thymus.). Each rat subject received a donor thymus mini graft by inserting a donor thymus mini graft into the defects of thymus lobes of the subjects. Then, the recipient rat subject thymuses with the donor thymus mini grafts were secured withAttorney Docket No. 9865.241.WOsix interrupted 10-0 sutures. Thus, the ITT procedure resulted in a chimeric thymus comprising full thickness donor thymus tissue in the recipient thymus tissue.

[0062] After the grafting ITT procedure was completed, hemostasis was conducted to stop bleeding from the operation performed. Next, the sternotomy line was sutured with 3-0 Prolene sutures. Then, the subcutaneous tissue was sutured with absorbable sutures and the skin was sutured with nonabsorbable sutures. Each rat subject received the appropriate postoperative follow up and pain medications as indicated by the Institutional Animal Care and Use Committees (IACUC) protocol. In order to evaluate the engraftment of the thymic tissue and the chimeric thymus's short- and long-term functioning, each rat subject was euthanized at postoperative day (POD) 2, 4, and 8 weeks.

[0063] At the 14thday post syngeneic ITT mark, histologic evaluation was conducted and showed that the tissue architecture of the allografted chimeric thymus was well preserved and that the graft was viable. Additionally, the GFP characteristics of the tissue were well preserved in the chimeric thymus models. The T cell count for the chimeric thymus tissue was observed to be very small. However, thymic structural cells were observed in the chimeric thymus tissue, potentially being thymic epithelial cells.

[0064] At the 30thday post syngeneic ITT mark, histologic evaluation was conducted and showed that the same structural characteristics found at the 14thday post-transplant were observed to still be preserved in the chimeric thymus tissue. Notably, a re-population of recipient T cells in the chimeric thymus tissue was observed, potentially being naive T cells from the recipient bone marrow for education in the thymus.Example 2: Allogeneic intra-thymic thymus transplantation as a model for chimeric thymus between full MHC mismatched donor-recipient pairs

[0065] Following successful completion of producing a syngeneic ITT in Lewis rats, allogeneic intra-thymic thymus transplantations were performed using Brown Norway rat subjects as donors, and Lewis rat subjects as recipients. In performing the allogeneic transplantation, the ITT technique was modified by grafting a larger amount of donor thymus using a hemi-lobe of the thymus as a minigraft from the Brown Norway rat subjects. The thymus hemi-lobe was harvested from the Brown Norway donor rat subjects to produce the allograft tissue. Further, the thymus lobes of the Lewis rat recipient subjects were incised longitudinally, incising from the superior pole to the inferior pole. The micro-forceps was inserted into the superior pole, and the blunt dissection created a pocket in the recipient thymus lobe for placing the donor thymus tissue graft. The recipient thymus pocket was flushed withAttorney Docket No. 9865.241.WOsaline to remove possible blood clots. Then, the donor thymus allograft is inserted into the recipient thymus lobe pocket by placing a suture to one pole of the donor thymus allograft, and then using the suture to pull the donor thymus allograft into the recipient thymus lobe pocket. Following the insertion of the donor allograft, both holes at the caudal and cranial end of the thymus were sutured. This method to produce the chimeric thymus was used throughout the rest of the study reported in the following examples.Example 3: Efficacy of Chimeric Thymus in Establishing Central Tolerance to VCA between Full MHC Mismatched Donor-Recipient Pairs

[0066] Following the creation of the chimeric thymus model, 22 ITT procedures were performed with different combinations of administering tacrolimus, an immunosuppressant, and transplanting an allogeneic groin flap. For this portion of the study, rat subjects were split into four different experimental groups to observe the long-term survival of the chimeric thymus and the functionality of the ITT. Group Al was comprised of five Lewis rat subjects with allogeneic Brown Norway rat ITT, and these subjects received neither any immunosuppression nor a groin flap transplant. Group A2 was comprised of five Lewis rat subjects with allogeneic Brown Norway rat ITT, who received 1 mg / kg / day of systemic tacrolimus for 21 days but did not receive a groin flap transplant. Group A3 was comprised of six Lewis rat subjects who received 1 mg / kg / day of systemic tacrolimus for 21 days and received a groin flap transplant from the same donor rat subject, but did not receive an allogeneic Brown Norway rat ITT. Group A4 was comprised of six Lewis rat subjects with allogeneic Brown Norway rat ITT, who received 1 mg / kg / day of systemic tacrolimus for 21 days and received a groin flap transplant from a Brown Norway donor to the Lewis recipient rat subject. These groups are reflected in Table 1.TABLE 1 : Experimental groups demonstrating the effects of ITT on long term survival and tolerance inductionAttorney Docket No. 9865.241.WO

[0067] The first column of Table 1 details the experimental group designation and the number of recipient rat subjects in that experimental group. The second column details whether the subjects in that experimental group have undergone an ITT procedure as described above. The third column details whether the subjects in that experimental group were administered tacrolimus (an immunosuppressant) for 21 days at a rate and concentration of 1 mg / kg / day. Additionally, the third column details whether the subj ects in the respective experimental group were administered an ultralow dosage of tacrolimus after the initial short course administration of the specified timeframe and dosage of tacrolimus. The fourth column details whether the subjects of the respective experimental groups were induced with 0.5 ml of antilymphocyte serum (ALS). ALS induction was carried out by first inducing the respective donor Brown Norway rats with 0.5 ml of ALS four days prior to the ITT procedure and / or allogeneic transplantation and then second inducing the respective Lewis rat recipient subjects with 0.5 ml of ALS on POD 0.

[0068] The fifth column of Table 1 details whether the recipient subjects received a groin flap allogeneic transplantation. For experimental groups who received a groin flap and an ITT, with the exception of group A7, the subjects received their respective groin flap transplants and donor thymic functional unit tissue grafts from their same respective Brown Norway donor. The subjects of A7 received a bilateral groin flap transplant, and thus received a groin flap from a Brown Norway rat donor and a groin flap from an ACI rat donor. The sixth column details the mean survival of groin flap transplants measured in days after transplantation occurred. ByAttorney Docket No. 9865.241.WOobserving experimental groups Al and A2, the study aimed to demonstrate successful ITT and compare the ITT graft outcomes in the presence and the absence of immunosuppression administration to the subjects. By observing experimental groups A3 and A4, the study aimed to demonstrate the effects of ITT on allogeneic groin flap survival. By observing experimental groups A5 and A6, the study aimed to demonstrate the effects of ITT under different immunosuppressive protocols and to observe the effect of ALS on the tolerogenic potential of ITT. By observing experimental group A7, the study aimed to demonstrate the donor specificity for the tolerogenic environment created by ITT. Finally, by observing experimental groups A8 and A9, the study aimed to compare the long-term survival of allogeneic groin flap transplants with ITT and without ITT when the recipients are receiving a continuous, ultralow, sub therapeutic level dosage of tacrolimus.

[0069] The Group Al subjects were followed up for 21 days after their allogeneic ITT procedure, and then the subjects were sacrificed with perfusion fixation. The thymus tissue of the Al subjects was then collected and histologically analyzed. Histological analysis of Group Al showed successful ITT with incorporation of allogeneic thymus tissue into the recipient thymus tissue. Furthermore, the engrafted thymus tissue showed repopulation of T-cells. Notably, even in the absence of administering any systemic immunosuppression, the allografted thymus tissue survived for 21 days. Thus, this shows the immune privilege of thymus and central tolerance to the grafted thymus. However, multiple brown pigments (lipofuscin) were observed in the allogeneic thymus tissue, but not present in the recipient thymus tissue. The brown pigments may be remnants of involuted apoptotic cells, possibly caused by the lack of immunosuppression.

[0070] For Group A2, the subjects were followed up for 21 days after the 21 days of postoperative systemic tacrolimus administration. The A2 subjects were then sacrificed with perfusion fixation and histologically analyzed just as the Al subjects were. The histologic analysis of the A2 thymus samples showed engraftment of the allogeneic thymus tissue and repopulation of the tissue with T-cells. Markedly, the A2 allogeneic thymus tissue was not observed to have the same brown pigment present in the Al allogeneic thymus tissue. This indicated that the grafted thymus tissue was a better quality in the presence of short-term systemic immunosuppression administration.

[0071] For Group A3, the subjects were followed up by daily clinical observation after the 21 days of postoperative systemic tacrolimus administration. The subjects were weaned after the 21 days of immunosuppression administration, and no immunosuppression was given afterward. For Group A4, the subjects were followed up in the same manner as Group A3. TheAttorney Docket No. 9865.241.WOsubjects of Groups A3 and A4 underwent clinical observation until Banff Grade II rejection was observed. Clinical Banff Grade II rejection is evidenced by mild epidermal loss with moderate inflammation and edema. During clinical observation, venous blood samples were collected for measuring the trough tacrolimus levels. Venous tacrolimus levels were measured using standard LCMS. The purpose of measuring the venous blood tacrolimus levels was to determine whether therapeutic levels of tacrolimus were present or absent in the subjects throughout the follow up period.

[0072] The LCMS results indicated that tacrolimus levels in both Groups A3 and A4 was similarly at therapeutic levels during the 21 days of daily tacrolimus administration (FIG. 1).The tacrolimus levels for both groups declined following the weaning of the tacrolimus administration after day 21, and the blood levels reduced to non-detectable levels at day 24 for all the subjects in both groups (FIG. 1).

[0073] Once clinical Banff Grade II rejection is observed in the subject, the subject was both euthanized and perfusion fixation performed so that the groin flaps and thymus tissues could be collected for histologic evaluation. Upon evaluation of Groups A1-A4, the histological results showed that ITT significantly improved the survival of an allogeneic groin flap after short course tacrolimus treatment. For the Group A4 subjects, the mean survival time of the groin flaps were 41.5 days, of which 21 days were without any immunosuppressive treatment (FIG. 2). In comparison, the subjects of Group A3, the control group, had a mean survival of 28 days (FIG. 2). The difference in mean survival times of the groin flaps transplants between Groups A3 and A4 shows significantly prolonged survival of groin flaps transplanted to subjects with a chimeric thymus over those groin flaps transplanted to the control subjects lacking a chimeric thymus. After conducting a Gehan-Breslow-Wilcoxon test, the survival time difference between Groups A3 and A4 was found to be statistically significant.

[0074] The study then turned to observe the effects of ITT on allograft survival when administering different immunosuppressive protocols. For this portion of the study, five more experimental groups were observed (Table 1) to compare to the four previous experimental groups above. Group A5 was comprised of six Lewis rat subjects, and these subjects did not undergo an ITT procedure. The respective donor Brown Norway rats were induced with 0.5 ml of antilymphocyte serum (ALS) four days (POD -4) prior to the transplant. Then, the Lewis rat recipient subjects of A5 were induced with 0.5 ml of ALS on POD 0, followed by administering to the Lewis rat subjects tacrolimus at a concentration and rate of 1 mg / kg / day for the first 21 days. Antilymphocyte serum (ALS) was administered for this portion of the study because previous unpublished data showed that ALS induction possibly contributed to better toleranceAttorney Docket No. 9865.241.WOinduction in VC As. The subjects of A5 received a groin flap transplant from a donor Brown Norway rat subject. Group A6 was comprised of six Lewis rat subjects who received an ITT from a donor Brown Norway rat subject. The respective donor Brown Norway rats were induced with 0.5 ml of ALS four days (POD -4) prior to the transplant. Then, the Lewis rat recipient subjects of A6 were induced with 0.5 ml of ALS on POD 0, followed by administering to the A6 Lewis rat subjects tacrolimus in the same dosage, concentration, and rate as the A5 subj ects detailed above. The subj ects of A6 received a groin flap transplant from a donor Brown Norway rat subject. Group A7 was comprised of two Lewis rat subjects who received an ITT from a donor Brown Norway rat subject. The subjects of A7 were administered tacrolimus at a concentration and rate of 1 mg / kg / day for the first 21 days but were not administered any ALS. The subjects of A7 received bilateral groin flap transplants from both the Brown Norway rat donor and a third party ACI rat donor simultaneously with the ITT. The groin flap from the ACI donor was transplanted to the left groin of the subject, while the groin flap from the Brown Norway donor was transplanted to the right groin of the subject. Group A8 was comprised of six Lewis rat subjects, and these subjects did not receive an ITT. The subjects of A8 received a groin flap transplant from a donor Brown Norway rat subject. The respective donor Brown Norway rats were induced with 0.5 ml of ALS four days (POD -4) prior to the transplant. Then, the Lewis rat recipient subjects of A8 were induced with 0.5 ml of ALS on POD 0, followed by administering to the Lewis rat subjects tacrolimus at a concentration and rate of 1 mg / kg / day for the first 21 days. After the 21 days of tacrolimus administration, the subjects of A8 were administered an ultralow dose of tacrolimus at concentration of 0.1 mg / kg for the duration of the experiment. Finally, Group A9 was comprised of six Lewis rat subjects who received an ITT from a donor Brown Norway rat subject. The respective donor Brown Norway rats were induced with 0.5 ml of ALS four days (POD -4) prior to the transplant. Then, the Lewis rat recipient subjects of A9 were induced with 0.5 ml of ALS on POD 0, followed by administering to the A9 Lewis rat subjects tacrolimus at a concentration and rate of 1 mg / kg / day for the first 21 days. After the 21 days of tacrolimus administration, the subjects of A9 were administered an ultralow dose of tacrolimus at concentration of 0.1 mg / kg for the duration of the experiment. The subjects of A9 received a groin flap transplant from a donor Brown Norway rat subject.

[0075] For Group A5, the mean survival time of the groin flaps was 32.5 days, whereas the mean survival time of the Group A6 groin flaps 69.5 days (FIG.3). This difference in survival time was found to be statistically significant by performing a Gehan-Breslow-Wilcoxon test. This difference shows that ITT significantly enhances the survival time for donor transplanted tissues in recipients compared to recipients receiving a donor transplant without ITT. Further,Attorney Docket No. 9865.241.WOand without wishing to be bound by theory, it is believed that the addition of ALS aids in the deletion of all mature T cells from the bloodstream which may be alloreactive. Thus, the combination of ITT with ALS induction and tacrolimus short course treatment allows for previous alloreactive T cells to be deleted and for the donor antigens to be recognized as self in the chimeric thymus by the new T cells. The systemic tacrolimus levels for Groups A5 and A6 were measured by taking jugular vein blood samples at certain timepoints using standard LCMS technique. The LCMS results showed that during the first seven days, systemic tacrolimus levels fluctuated (FIG. 4). After the first week, LCMS results showed that the systemic tacrolimus levels held at a steady target therapeutic level around 6-8 ng / ml until weaning the subjects from tacrolimus treatment on day 21 (FIG. 4). Following the weaning, systemic tacrolimus levels decreased immediately to undetectable levels by day 23 (FIG. 4).

[0076] The Group A7 subjects were studied to determine the donor specificity of the tolerogenic environment generated by ITT. After the tacrolimus treatment was weaned from the A7 subjects, the ACI groin flaps were rejected, showing severe inflammation and epidermal loss at days 26 and 27. Notably, the Brown Norway groin flaps were able to survive without any signs of rejection. This showed that the tolerogenic environment created by ITT is donor specific.

[0077] In comparing the mean survival times of Groups A8 and A9, Group A8's groin flaps survived an average of 44.5 days, whereas Group A9's groin flaps survived an average of 90.5 days (FIG. 5). Furthermore, two of the groin flaps in Group A9 survived over 100 days until the end of the study (FIG. 5). This difference in survival time was found to be statistically significant based on the Gehan-Breslow-Wilcoxon test. Like the earlier discussed groups, the systemic tacrolimus levels fluctuated for the first seven days until reaching a steady therapeutic level between 6-8 ng / ml until day 21 (FIG. 6). After day 21, when the tacrolimus treatment was reduced to a dose of 0.1 mg / kg / day, systemic tacrolimus levels decreased significantly to sub therapeutic levels by day 23 (below 1 ng / ml) (FIG. 6). The subtherapeutic levels of systemic tacrolimus remained steady for the duration of the study and was a similar for both Groups A8 and A9 (FIG. 6).

[0078] For this portion of the study, the subjects were sacrificed once Grade III rejection was observed, and then thymus biopsy was performed. The thymus tissues were embedded and sectioned. Then, immunofluorescence (IF) staining was performed using three different stains. First, the 5 mm thick histologic sections are stained with Rat RTl.Ac FITC (Clone OX-27). Rat RTl.Ac FITC only stains Brown Norway Class I antigens. After this staining step, the tissue specimen was washed and stained with Anti -Rat RT 1.A (Clone OX- 18). Anti -Rat RT 1.AAttorney Docket No. 9865.241.WOstained the remaining Class I antigens in the tissue specimen. Finally, the tissue specimen is stained with DAPI to stain the nucleus of the cells. The IF results of the chimeric thymus showed its structural integrity, containing both cortex and medulla thymic sections. Brown Norway donor Class I antigens were shown to be expressed in both the thymic cortex and medulla in coexistence with Lewis Class I recipient antigens. Further, the donor Class I antigens were expressed extensively, especially expressed in the thymic medulla, which aids in donor tolerance induction.

[0079] The study concluded that the method of making the chimeric thymus enabled central tolerance through negative selection of donor specific T cell clones during the T cell maturation process, as well as potentially through the elimination of alloreactive T cells.

[0080] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

Attorney Docket No. 9865.241.WOWHAT IS CLAIMED IS:

1. A method of making a chimeric thymus in a subject comprising:providing a donor thymic functional unit tissue graft, wherein the donor thymic functional unit tissue graft comprises thymic cortex and medullary structures, including thymic epithelial cells (TEC) and dendritic cells; andimplanting the donor thymic functional unit tissue graft by orthotopic intra-thymic transplantation into the thymus of the subject, thereby providing the chimeric thymus in the subject,wherein the donor thymic functional unit tissue graft is from a pediatric donor, and wherein the chimeric thymus induces a dual immunologic tolerance to both donor tissue antigens and tissue antigens of the subject.

2. The method of claim 1, wherein the implanting is carried out by implanting the donor thymic functional unit tissue graft into a dissected pocket in the thymus of the subject.

3. The method of claim 1 or claim 2, wherein the donor thymic functional unit tissue graft is a thymus hemi-lobe of the donor.

4. The method of any one of claims 1-3, wherein the subject is a human subject.

5. The method of any one of claims 1-3, wherein the subject is a non-human animal subject.

6. A method of reducing tissue rejection following transplant of an allogeneic tissue graft in a subject, comprising:(a) making a chimeric thymus in the subject by the method of any one of claims 1-5, wherein the donor thymic functional unit tissue graft is from the same donor as the allogeneic tissue graft; and(b) transplanting the allogeneic tissue graft into the subject,thereby reducing tissue graft rejection in the subject.Attorney Docket No. 9865.241.WO7. The method of claim 6, further comprising inducing clonal depletion of alloreactive T cells in the subject by administering a composition comprising lympho-depleting antibodies before and / or after transplanting the allogeneic tissue graft.

8. The method of claim 7, wherein the composition comprising lympho-depleting antibodies is an antilymphocyte serum (ALS), an antithymocyte globulin (ATG), alemtuzumab, and / or rituximab.

9. The method of claim 7, wherein the composition comprising lympho-depleting antibodies is an antilymphocyte serum (ALS).

10. The method of any one of claims 6-9, wherein the allogeneic tissue graft comprises a vascularized tissue graft such as a vascularized composite allotransplantation (VCA) graft (e.g., a hand, foot, joint, or limb transplant), a facial tissue graft, a solid organ (such as kidney, liver, heart, lung, etc.), or a uterine graft.

11. The method of any one of claims 6-9, wherein the allogeneic tissue graft comprises a non-vascularized tissue graft such as a skin graft, a nerve graft, a cartilage graft, or a bone graft.

12. The method of any one of claims 6-11, wherein the transplanting step (b) is carried out simultaneously with the making the chimeric thymus of step (a).

13. The method of any one of claims 6-12, further comprising:(c) administering an immunosuppressant to the subject.

14. The method of claim 13, wherein the immunosuppressant is administered for an initial time (e.g., of from 1, 5, or 10 to 15, 20, 25 or 30 days) following the transplanting step (b), and then administered subsequently at a second time at a low dose to the subject.

15. The method of claim 14, wherein the low dose is about 5, 10, 20, 50, or 100 times lower than the dosage of immunosuppressant administered for the initial time following the transplanting step (b).Attorney Docket No. 9865.241.WO16. The method of any one of claims 13-15, wherein the immunosuppressant comprises tacrolimus, rapamycin (sirolimus), everolimus, cyclosporine, my cophenolate mofetil, 0KT3, basiliximab, daclizumab, belatacept, a corticosteroid (e.g., prednisone, methylprednisolone), azathioprine, mercaptopurine, and / or methotrexate.

17. The method of any one of claims 13-15, wherein the immunosuppressant is tacrolimus.

18. A chimeric thymus produced by a method of any one of claims 1-5.

19. Use of the chimeric thymus of claim 18 in a method of reducing tissue rejection following transplant of an allogeneic tissue graft in a subject.

20. Use of an immunosuppressant for reducing tissue rejection in a subject having the chimeric thymus of claim 18.

21. Use of an immunosuppressant for the manufacture of a medicament for reducing tissue rejection in a subject having the chimeric thymus of claim 18.

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