WO2019165319A1 - The use of 4-methylumbelliferone to prevent immune rejection in cases of tissue transplantation - Google Patents

Abstract

Compositions for inhibiting an adverse immune response in a transplant recipient comprising a compound that inhibits hyaluronan synthesis and a pharmaceutically acceptable carrier are described. In some embodiments, the compound that inhibits hyaluronan synthesis is 4-methylumbelliferone or a metabolite of 4-methylumbelliferone. Methods for inhibiting an adverse immune response in a mammalian transplant recipient, including administering to the mammalian transplant recipient a composition having a compound in an amount effective to inhibit hyaluronan synthesis, are also described.

Description

THE USE OF 4-METHYLUMBELLIFERONE TO PREVENT IMMUNE REJECTION IN CASES OF TISSUE TRANSPLANTATION

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of United States Provisional Application No. 62/634439, filed February 23, 2018, the entire disclosure of which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under contracts All 01984 and DK096087 awarded by the United States Department of Health and Human Services, National Institutes of Health. The United States government has certain rights in the invention.

BACKGROUND

The function of the immune system is to eliminate foreign bodies that may contain pathogens and to maintain unresponsiveness or tolerance against self-antigen. Unfortunately, the immune system does not distinguish beneficial intruders, such as transplanted tissue, from those that are harmful and thus the immune system rejects transplanted tissue or organs. Transplant rejection, which represents a major limitation to the availability and success of cell, tissue, and organ transplants, can be significantly mediated by allo-reactive T-cells present in the host which recognize donor allo-antigens.

At present, in order to prevent or reduce an immune response against a transplant, patients are treated with powerful immunosuppressive drugs. The infusion of individuals with drugs that prevent or suppress T-cell immune response does inhibit transplant rejection but can also result in general immune suppression, toxicity, and even death due to opportunistic infections. Because of the toxicity and incomplete response rate to conventional treatment of donor tissue rejection, alternative approaches are needed to treat patients who cannot withstand or do not respond to current modes of drug therapy.

In healthy individuals (i.e., those without an autoimmune disease or disorder), immune tolerance is maintained by populations of regulatory T-cells including FoxP3+ regulatory T-cells (Treg) (Sakaguchi, S., et al., Nat. Rev. Immunol. 10, 490-500 (2010)). Treg absence or depletion leads to multi-systemic autoimmunity in mice and humans (Wildin, R.S., et al., Nat. Genet. 27, 18-20 (2001)) whereas adoptive transfer of Treg can abrogate autoimmunity.

In some autoimmune disease models, Treg present in the CNS are known to limit the extent of neuroinflammation and to facilitate clinical recovery in the mouse model of multiple sclerosis, or experimental autoimmune encephalomyelitis (EAE), such that multiple investigative therapeutic strategies intended to treat autoimmune demyelination are directed at promoting the number and/or function of Treg. However, existing therapies have not managed to induce stable, functional FoxP3+ Treg, in part because Treg in vivo are a population in flux. Natural Treg (nTreg) continually emerge through thymic selection, whereas induced Treg (iTreg) originate in peripheral tissues in response to inflammatory stimuli and can revert into effector T-cells. This variability in the number and function of local Treg at sites of inflammation can impact the durability of immune tolerance in peripheral tissues.

Despite the fact that the inflammatory milieu is known to have decisive effects on immune tolerance, little is known about how the tissue micro-environment influences the function and number of Treg. Therefore, there is increasing interest in the role of the extracellular matrix (ECM) at the interface between lymphocytes and local cells in autoimmunity (Bollyky, P.L., et al., Curr. Dial·. Rep. 12, 471-480 (2012); Irving- Rodgers, H.F., et al., Diabetologia 51, 1680-1688 (2008); Hull, R.L., et al., J Biol. Chem. 287, 37154-37164 (2012); Bitan, M., et al., Diabetes. Metab. Res. Rev. 24, 413- 421 (2008); Ziolkowski, A.F., et al., J. Clin. Invest. 122, 132-141 (2012)).

One tissue component that is abundant at sites of inflammation is hyaluronan (HA), an ECM polysaccharide. HA has many functions such as providing support and anchorage for cells, segregating tissues from one another, and facilitating cell to cell signaling, development, migration and function (Bollyky, P.L., et al. (2012), supra). HA is a polymer of disaccharides composed of glucuronic acid and N-acetylglucosamine and linked via alternating b-1, 4 and b-1, 3 glycosidic bonds. HA can be 25,000 disaccharide repeats in length. In vivo polymers of HA can range in size from 5,000 to 20,000,000 Da. HA is synthesized by a class of integral membrane proteins called hyaluronan synthases (HAS), of which vertebrates have three types: HAS1, HAS2, and HAS3. These enzymes lengthen HA by repeatedly adding glucuronic acid and N-acetylglucosamine to the nascent polysaccharide as it is extruded through the cell membrane into the extracellular space (Laurent, T.C., et al., Immunol. Cell Biol. 74, Al-7 (1996)). HA is a key mediator of inflammation with roles in lymphocyte trafficking, proliferation, and antigen presentation (Laurent, T.C., and Fraser, J.R., FASEB J 6, 2397-2404 (1992); Bollyky, P.L., et ah, Cell Mol Immunol. 3, 211-220 (2010)). HA is increased in lesions associated with human autoimmune diseases including multiple sclerosis, Sjogrens disease, and autoimmune thyroiditis (Back, S.A., et ah, Nat. Med. 11, 966-972 (2005); Engstrom -Laurent, A., The Biology of Hyaluronan , CIBA Foundation Symposium, 143, 233-47 (1989); Gianoukakis, A., et ak, Endocrinology 148, 54-62 (2007). HA is also increased in the serum of individuals with lupus, rheumatoid arthritis, psoriasis, and autoimmune thyroiditis (Engstrom-Laurent, supra ; Pitsillides et ak, Rheumatol. 33, 5-10 (1994); Hansen, C., et ak, Clin. Exp. Rheumatol. 14 Suppl. 15, S59-67 (1996); Torsteinsdottir et ak, Clin. Exp. Immunol. 115, 554-560 (1999); Elkayam, O., et ak, Clin. Rheumatol. 19, 455-457 (2000); Kubo, M., et ak, Arch. Dermatol. Res. 290, 579-581 (1998).

HA is highly abundant within chronically inflamed tissues including, for example, MS lesions (Bollyky, P.L., et ak (2012), supra ; Back, S.A., et ak, supra). For example, in one study HA was shown to accumulate in demyelinated lesions in MS and EAE. Immunostaining for proteolipid protein (PLP) of a chronic MS lesion showed complete loss of myelin in the center of the lesions. CD44 staining revealed high levels of CD44 in the lesions, and elevated CD44 expression in GFAP-expressing reactive astrocytes were also found. HA staining showed high levels of HA in demyelinated regions of the lesions but at lower levels in the lesion borders (Back S.A., et ak, supra).

Typically, HA present within chronically inflamed tissues takes the form of short, highly catabolized fragments (reviewed in Bollyky, P.L., et ak (2012), supra) that are pro-inflammatory agonists of Toll-like receptor (TLR) signalling (Laurent, T.C., et ak, Immunol. Cell Biol. 74, Al-7 (1996); Jiang, D., et ak, Physiol. Rev. 91, 221-264 (2011)), and drive dendritic cell maturation and promote phagocytosis (Jiang, D., et ak, Nat. Med. 11, 1173-1179 (2005); Termeer, C., et ak, J. Exp. Med. 195, 99-111 (2002)). HA overexpression tends to drive inflammation (Olsson, M. et ak, PLoS Genet. 7, el00l332 (2011)), presumably through production of increased HA fragments, while inhibition of HA synthesis, including treatment with 4-methylumbelliferone (4-MU, Hymecromone), tends to reduce inflammation (Yoshioka, Y., et ak, Arthritis Rheum. 65, 1160-1170 (2013); McKallip, R.J., et ak, Toxins (Basel) 5, 1814-1826 (2013); Colombaro, V. et ak, Nephrol. Dial. Transplant 28, 2484-2493 (2013); Saito, T., et ak, Oncol. Lett. 5, 1068-1074 (2013)). With respect to the role of HA in local immune modulation, it is known that low molecular weight HA (LMW-HA) fragments inhibit the function of FoxP3+ Treg (Bollyky, P.L., et ah, J Immunol. 179, 744-747 (2007); Bollyky, P.L., et ah, J. Immunol. 183, 2232-2241 (2009)). These effects are mediated via TLR signaling and via interactions with the HA receptor CD44.

In the healthy CNS, astrocytes are the main producers of low levels of HA, depositing it as ECM complexes in the space between myelinated axons and in the space between myelin sheaths and astrocyte processes (Asher, R., et ah, J. Neurosci. Res. 28, 410-421 (1991)). Upon injury, however, reactive astrocytes produce abundant amounts of HA that accumulate in damaged areas (Back, S.A., et ah, supra ; Struve, J., et ah, Glia 52, 16-24 (2005); Bugiani, M., et ah, Brain 136, 209-222 (2013)). As such, HA is present at high levels in demyelinating lesions in MS patients and in mice with EAE (Back, S.A., et ah, supra).

4-MU is a selective inhibitor of HA synthesis. The compound was first used in vitro in 1995 by Nakamura et al. to inhibit HA-synthesis in skin fibroblasts. Nakamura, T., et al., Biochem. Biophys. Res. Commun. 208, 470-475 (1995). In 2004, the mechanism of 4-MU was discovered by Kakizaki et al., and since then it has been used in in vivo studies in mice and rats to investigate the 4-MU influence, mainly in cancer studies (Kakizaki, T, et al., J. Biol. Chem. 279, 33281-33289 (2004); see also, e.g., Yoshihara, S., et al., FEBS Letters 579, 2722-2726 (2005); Lokeshwar, V.B., et al., Cancer Res. 70, 2613-2623 (2010)) and in atherosclerosis studies (Nagy, N., et ak, Circulation 122, 2313-2322 (2010)). 4-MU is also already used in humans. It is available without a prescription as Heparvit(TM), a nutraceutical product for cancer patients. Furthermore, it is available with a prescription in Europe and Asia to treat biliary cholestasis under the name Hymecromone. In that setting, the drug has an excellent safety profile and has been used for several years.

Although it is known that HA deposits are abundant in chronically inflamed tissues and that 4-MU is a selective inhibitor of HA synthesis, there remains a need to develop a safe and effective therapy for inhibiting an adverse immune response in a transplant recipient by providing a well-founded understanding of the role of HA and 4-MU in alloimmunity.

Further, there is a need for the prevention and/or reduction of an unwanted immune response by a host to a transplant by immune effector cells as a method to avert host rejection of donor tissue. Also advantageous would be a method to eliminate or reduce an unwanted immune response by a donor tissue against a recipient tissue, or graft-versus-host disease.

SUMMARY

In one aspect, the present disclosure provides a composition for inhibiting an adverse immune response in a transplant recipient comprising (i) a compound that inhibits hyaluronan synthesis, and (ii) a pharmaceutically acceptable carrier.

In one embodiment, the compound is a UDP glycosyltransferase inhibitor. In one embodiment, the compound is a UDP glucuronyltransferase inhibitor.

In one embodiment, the compound is 4-methylumbelliferone. In one embodiment, the compound is a metabolite of 4-methylumbelliferone. In one embodiment, the compound is 4-methylumbelliferyl-glucuronide or a sulfated 4- methylumbelliferone. In one embodiment, the compound is an uncleavable form of 4-methylumbelliferyl-glucuronide or sulfated 4-methylumbelliferone.

In one embodiment, the compound is effective to induce a regulatory T-cell response. In one embodiment, the compound is effective to increase FoxP3+ regulatory T-cells.

In one embodiment, the adverse immune response is selected from the group consisting of alloimmunity, cell transplant rejection, tissue transplant rejection, solid organ transplant rejection, and graft-versus-host disease.

In one aspect, the present disclosure provides a method for inhibiting an adverse immune response in a mammalian transplant recipient, the method comprising administering to the mammalian transplant recipient a composition comprising a compound in an amount effective to inhibit hyaluronan synthesis in the mammalian transplant recipient.

In one embodiment, the compound is a UDP glycosyltransferase inhibitor. In one embodiment, the compound is a UDP glucuronyltransferase inhibitor.

In one embodiment, the compound is 4-methylumbelliferone. In one embodiment, the compound is a metabolite of 4-methylumbelliferone. In one embodiment, the compound is 4-methylumbelliferyl-glucuronide or a sulfated 4-methylumbelliferone. In one embodiment, the compound is an uncleavable form of 4-methylumbelliferyl-glucuronide or sulfated 4-methylumbelliferone. In one embodiment, the compound is effective to induce a regulatory T-cell response. In one embodiment, the compound is effective to increase FoxP3+ regulatory T-cells.

In one embodiment, the mammalian transplant recipient is a human transplant recipient. In one embodiment, the adverse immune response is selected from the group consisting of alloimmunity, cell transplant rejection, tissue transplant rejection, solid organ transplant rejection, and graft-versus-host disease.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGEIRE 1 shows a mixture of activated antigen presenting cells (APC) and T lymphocytes cultured in the presence of dimethyl sulfoxide (DMSO) or 4-MU.

FIGURE 2 is a three-dimensional heat map that shows cell nuclei and HA/CD44 in CD3+ T-cells stimulated by activated APC.

FIGURE 3 is a graph that shows the amount of tritiated glucosamine in CPM in activated human mDC cultured in the presence of DMSO or 4-MU.

FIGURE 4 is a graph that shows the percentage of 7-AAD Annexin V negative cells in activated human T-cells (TC) and antigen presenting cells (DC) cultured in the presence of DMSO or 4-MU.

FIGURE 5 is a graph that shows the average number of activated T-cells bound to APC at 5 hours post culture start.

FIGURE 6 is a graph that shows the percentage of activated T-cells positive for CD69+.

FIGURE 7 is a graph that shows the amount of tritiated thymidine at 72 hours in counts per minute (CPM) in activated T-cells cultured in the presence of DMSO or 4- MU.

FIGURE 8 is a scatter plot that shows DC and T-cells cultured with anti-CD3 and anti-CD28 antibodies and then stained for intracellular cytokines.

FIGURE 9 A is a graph that shows the percentage of FoxP3+ cells of all CD3+/CD4+ T-cells after culturing in the presence of vehicle or 4-MU. FIGURE 9B is a graph that shows the mean fluorescence activity (MFI) of CD3+/CD4+ T-cells after culturing in the presence of vehicle or 4-MU.

FIGURE 10A is a graph that shows the amount of tritiated thymidine in CPM in CD3/CD28 activated T-cells isolated from mice fed a diet containing 4-MU or control chow.

FIGURE 1 OB is a graph that shows the amount of tritiated thymidine in CPM in CD3/CD28 activated T-cells isolated from mice fed a diet containing 4-MU or control chow.

FIGURE 10C is a graph that shows the amount of tritiated thymidine in CPM in OVA peptide activated T-cells isolated from mice fed a diet containing 4-MU or control chow.

FIGURE 10D is a graph that shows the amount of tritiated thymidine in CPM in OVA peptide activated T-cells isolated from mice fed a diet containing 4-MU or control chow.

FIGURE 11A graphically represents the average number of proliferation peaks or divisions in T-cells transferred into mice treated with 4-MU or a control and immunized with OVA protein and alum adjuvant.

FIGURE 11B is a graph that shows the average number of proliferations or the division index in T-cells transferred into mice treated with 4-MU or a control and immunized with ovalbumin (OVA) protein and alum adjuvant.

FIGURE 12A graphically represents the average percentage of activation. Cells were isolated from immunized animals treated with 4-MU and control. Mice were restimulated to that antigen ex vivo. CD25, CD69, CD95, and CD44 are shown.

FIGURE 12B is a graph that shows the proliferation response ex vivo to mice immunized on control and 4-MU treatment.

FIGURE 13 A is a graph that shows the amount of tritiated thymidine in CPM in a mixed population of activated T-cells and dendritic cells cultured in the presence of DMSO or 4-MU.

FIGURE 13B is a graph that shows the normalized proliferation in a mixed population of activated T-cells and dendritic cells cultured in the presence of DMSO or 4- MU. FIGURE 14A is a graph that shows the percentage FoxP3+ of total CD4+ T-cells in spleen and peripheral lymph nodes five days after transplant in mice treated with 4-MU (white bar) or a control (black bar).

FIGURE 14B is a graph that shows the time to transplant rejection in mice treated with 4-MU or a control.

FIGURE 15A is a graph that shows the amount of tritiated thymidine in CPM in a mixed population of activated T-cells and dendritic cells cultured in the presence of DMSO or 4-MU.

FIGURE 15B is a graph that shows the normalized proliferation in a mixed population of activated T-cells and dendritic cells cultured in the presence of DMSO and 4-MU.

FIGURE 16A is a graph that shows the time to transplant rejection by days in mice treated with 4-MU, 4-MUG, or a control.

FIGURE 16B is a graph that shows the survival percentage in mice treated with 4-MU (dotted line), 4-MUG (dashed line), or a control (solid line) by days post-transplant surgery.

FIGURE 17A shows a culture of human Dc and allogeneic T-cells in a scatter plot that shows the percentage of CD25+/FoxP3+ Treg in live, proliferated CD4+ T-cells cultured in the presence of DMSO or 4-MU.

FIGURE 17B is a graph that shows the percentage CD25+/FoxP3+ Treg in live, proliferated CD4+ T-cells cultured in the presence of DMSO or 4-MU.

FIGURE 17C is a graph that shows the MFI of CD25+/FoxP3+ Treg in all CD4+ T-cells cultured in the presence of DMSO or 4-MU.

FIGURE 17D is a graph that shows the FoxP3 MFI of only Treg cultured in the presence of DMSO or 4-MU.

DETAILED DESCRIPTION

The present disclosure describes a drug that inhibits HA synthesis as a novel therapeutic to selectively attenuate the immune system in cases of allo-transplantation and/or alloimmunity and transplant rejection. For example, a drug that inhibits HA synthesis can be used to inhibit an adverse immune response in a transplant recipient, for example, in cases of cell transplantation, tissue transplantation, or solid organ transplantation. In addition, a drug that inhibits HA synthesis can be used to limit the negative effects of graft-versus-host disease in a transplant recipient. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present disclosure. The following definitions are provided in order to provide clarity with respect to the terms as they are used in the specification and claims to describe the claimed subject matter.

As used herein, the term“regulatory T-cells” or“Treg” cells refers to T-cells which express the cell surface markers CD4+ and CD25+, which express FoxP3 protein as measured by a Western blot and/or FoxP3 mRNA transcript.

As used herein, the term“antigen-specific regulatory T-cells” or“antigen-specific Treg” refers to Treg cells that were induced in the presence of an antigen and which express the cell surface markers CD4+ and CD25+, which express FoxP3 protein as measured by a Western blot and/or FoxP3 mRNA transcript.

As used herein, the term“derived from” or“a derivative thereof,” in the context of peptide or polypeptide sequences, means that the peptide or polypeptide is not limited to the specific sequence described, but also includes variations in that sequence, which can include amino acid additions, deletions, substitutions, or modifications to the extent that the variations in the listed sequence retain the ability to modulate an immune response.

As used herein, the term“peptide” or“polypeptide” is a linked sequence of amino acids and can be natural, recombinant, synthetic, or a modification or combination of natural, synthetic, and recombinant.

As used herein, an“adverse immune response in a transplant recipient” occurs when transplanted tissue is rejected by the recipient’s immune system, which affects the transplanted tissue. Examples of an adverse immune response in a transplant recipient include, but are not limited to, alloimmunity, cell transplant rejection, tissue transplant rejection, solid organ transplant rejection, and graft-versus-host disease.

As used herein, “cell transplant” or “cell transplantation” can include, for example, transplant of the beta islets of the pancreas.

As used herein,“tissue transplant” or“tissue transplantation” can include, for example, transplant of skin tissue.

As used herein,“organ transplant” or“organ transplantation’ can include, for example, transplant of a solid organ such as a heart.

As used herein, graft-versus-host disease can refer to graft-versus-host disease in cases of bone marrow transplantation. As used herein the term“treating” or“treatment” means the administration of a compound according to the disclosure to effectively prevent, repress, or eliminate at least one symptom associated with adverse immune response in a transplant recipient. Preventing at least one symptom associated with adverse immune response in a transplant recipient involves administering a treatment to a subject prior to onset of the symptoms associated with transplant rejection. Repressing an adverse immune response in a transplant recipient involves administering a treatment to a subject after clinical appearance of the disease.

As described herein, 4-MU is an immunomodulatory drug whose mechanism is the inhibition of HA synthesis. HA is a component of the extracellular matrix that has implications on the ability of immune cells to interact. The inventors have discovered that 4-MU limits the ability of foreign proteins from the body to be recognized and presented to the effector immune cells of the body. These findings demonstrate that immune reactivity can be limited in at least some, but not all, cells of the immune system. By limiting immune reactivity, it is possible to limit tissue rejection without fully compromising the immune system. As further described herein, modulation of the ECM can influence cellular maturity and interaction, thus limiting activity of the effector cells of the immune system.

4-MU has been shown to have a strong inhibitory effect on hyaluronic acid synthase (HAS) molecules of the immune system and, without wishing to be bound by theory, it is thought that this mechanism causes 4-MU to be protective against foreign cell and tissue rejection. Accordingly, 4-MU has strong potential as a therapeutic agent directed to the pathogenesis of foreign tissue rejection, or transplantation, due to its strong inhibitory effect on the activation of necessary immune cell components. Because 4-MU has been shown to have an excellent safety profile, 4-MU is strongly suited to treat transplantation, where current therapies have narrow therapeutic indices and dangerous immune suppressive side effects that put patients at constant risk while undergoing treatment.

As noted above, HA is a polymer of disaccharides composed of glucuronic acid and N-acetylglucosamine and linked via alternating b-1, 4 and b-1, 3 glycosidic bonds. 4-MU functions as a competitive substrate for UGT, an enzyme involved in the synthesis of HA as well as bile (Kakizaki, T, et ah, J Biol. Chem. 279, 33281-33289 (2004)). 4-MU functions as a competitive substrate for UGT, an enzyme involved in the synthesis of HA as well as bile (Kakizaki, L, et ak, J. Biol. Chem. 279, 33281-33289 (2004)). The relationship between HA and 4-MU is described in further detail in PCT/US2014/050770, which is hereby incorporated in its entirety.

HA appears to be important in promoting early T-cell activation. Because of this, inhibition of HA synthesis shows efficacy in treatment of autoimmunity. The inventors have discovered that 4-MU can be used in transplantation due to similar cell types being relevant in both pathogenesis mechanisms. The viability of this strategy was originally suggested due to the abundance of hyaluronan that is present at sites of T-cell activation.

FIGURE 1 is an image that shows a mixture of activated antigen presenting cells (APC) and T lymphocytes cultured in the presence of dimethyl sulfoxide (DMSO) or 4-MU. Referring to FIGURE 1, treating a mixture of activating APC and T lymphocytes with 4-MU in vitro limits their interactions, which is necessary for mounting a strong immune response. Human monocyte-derived dendritic cells (mDC) are used in conjunction with activating antibodies (anti-CD3) to stimulate T-cells. When the cells are additionally cultured with 4-MU, their interactions are limited and less HA appears surrounding clusters of activating cells. When APC present foreign peptides to lymphocytes to activate them, HA appears in large networks surrounding the clusters.

FIGURE 2 is a three-dimensional heat map that shows cell nuclei and HA/CD44 in CD3+ T-cells stimulated by activated APC. Referring to FIGURE 2, there is an enrichment of HA and the predominant receptor it binds to (CD44) in between interacting cells as they activate. Human CD3+ T-cells are stimulated by activated APC and imaged with a confocal microscope. Cell nuclei are shown for reference and HA/CD44 is shown on two axes. The highest signal of these two molecules co-localizes between the interacting cells, suggesting they serve a role in early stimulation of lymphocytes. Further, the enrichment of HA in between the APC and T-cells during activation suggests its importance in this event.

FIGURE 3 is a graph that shows the amount of tritiated glucosamine in CPM in activated human mDC cultured in the presence of DMSO or 4-MU. Referring to FIGURE 3, activated human mDC were cultured for 24 hours in the presence 4-MU or DMSO. To measure cellular associated HA, radioactive glucosamine was used. Post incubation, cells were washed and the digestive enzyme hyaluronidase was used to liberate cell-associated HA, which is quantified via the tritiated glucosamine present post digestion. As shown in FIGURE 3, 4-MU efficiently inhibits HA production from APC, thereby demonstrating that administration of 4-MU can be an efficacious therapy in inhibiting this activation.

Importantly, however, 4-MU is not carrying out its activity via an inherent toxicity, as shown by the lack of direct killing on either of these component T-cells (the T-cell or APC). FIGURE 4 is a graph that shows the percentage of 7-AAD Annexin V negative cells in activated human T-cells (TC) and antigen presenting cells (DC) cultured in the presence of DMSO or 4-MU. 4-MU is not toxic to antigen presenting cells or lymphocytes. Referring to FIGURE 4, activated human cells and antigen presenting cells, in this case dendritic cells (DC), were cultured in the presence of DMSO or 4-MU for 24 hours. A combination of 7AAD and Annexin V was used to quantify the percent “viable” cells using a flow cytometer. The results, shown in FIGURE 4, demonstrate that 4-MU’s mechanism of immune activity is not toxicity related. The results are consistent with the previously seen phenomenon that very few side effects are seen in humans administered 4-MU for the treatment of biliary cholestasis.

As one way of quantifying the activation of T-cells by APC, the average number of T-cells bound to an activated, presenting APC after a 2 hour co-culture was imaged and calculated. FIGURE 5 is a graph that shows the average number of activated T-cells bound to APC at 5 hours post culture start. Referring to FIGURE 5, mouse bone marrow derived dendritic cells (BMDC) were activated overnight in the presence of lipopolysaccharide (LPS) and either DMSO or 4-MU. The following day, anti-CD3 antibodies were incubated for a short period of time with the BMDC so that they were capable of activating T-cells. Mouse CD4+ T-cells were added for 5 hours and allowed to interact. Following incubation, cultures were washed and imaged, then quantified for the number of T-cells that appeared bound to each APC (n>400). As shown in FIGURE 5, by treating the APC with 4-MU overnight prior to co-culture, each DC bound fewer T-cells in the time period, suggesting that early activation is inhibited and subsequent T-cell activation is also limited by 4-MU. Thus, FIGURE 5 shows that 4-MU attenuates early T-cell activation. What is clearly shown is that at 5 hours, there is a blunting of T-cell activation, suggesting the induction of an adaptive CD4+ T-cell response has been limited.

The inhibition of early T-cell activation is also shown by looking at canonical surface markers following treatment with 4-MU during co-culture with activating DC or activating antibodies. FIGURE 6 is a graph that shows the percentage of activated T-cells positive for CD69. Referring to FIGURE 6, T-cells were activated by OVA peptide or with activating anti-CD3 and anti-CD28 antibodies. Mouse BMDC were matured with LPS and pulsed with OVA peptide. The cells were co-cultured for five hours with T-cells isolated from an OT II mouse (Charles River, Wilmington, Massachusetts USA), an OVA-specific TCR transgenic mouse on a B6 background, whose majority of T-cells respond to OVA. At the end of the culture, cells were stained for flow cytometry and the T-cells were analyzed for %CD69+, a marker of early activation. In addition, a group of T-cells were activated non-discriminately with activating antibodies towards CD3 and CD28.

The most potent effects of 4-MU are seen when it is present at the time of induction, as opposed to when it is given after inflammation is induced. If T-cells are stimulated in the presence of 4-MU, proliferation is greatly decreased, but only if 4-MU is present at the start of when APC and T-cells are cultured together. FIGURE 7 is a graph that shows the amount of tritiated thymidine at 72 hours in counts per minute (CPM) in activated T-cells cultured in the presence of DMSO or 4-MU. Referring to FIGURE 7, mouse T-cells were activated with CD3 and CD28 and allowed to culture for two days before tritiated thymidine was added to the culture. At 72 hours post-culture start, the wells were washed and analyzed on a scintillator. Increased thymidine reflects increased proliferation and division. 4-MU was added either at the beginning of the experiment (time=0h) or after 24 hours. As shown in FIGURE 7, if 4-MU is given after 24 hours, minimal effect is seen as compared to when 4-MU is added at the beginning of the experiment (time=0h). Thus, the effect of 4-MU is most potent during the time of antigen presentation and there is a much lessened effect if 4-MU is added after cell activation.

Decreased proliferation of T-cells coincides with decreased expression of inflammatory cytokines by these T-cells, suggesting 4-MU specifically limits the induction and proliferation of inflammatory T-cells. FIGURE 8 is a scatter plot that shows human DC and T-cells cultured with anti-CD3 and anti-CD28 antibodies and then stained for intracellular cytokines. Referring to FIGURE 8, autologous DC and T-cells were cultured with anti-CD3/28 antibodies for 72 hours and then stained for intracellular cytokines as shown. The CD4+ gate is shown to highlight cytokine production by activated T-cells. These data show that 4-MU prevents antigen presentation and T-cell activation as measured by cytokine production. The effect on anti-inflammatory Treg compartments while inflammatory T-cells are decreased due to 4-MU treatment was examined. FIGURE 9A is a graph that shows the percentage of FoxP3+ cells of all CD3+/CD4+ T-cells after culturing in the presence of vehicle or 4-MU. 4-MU promotes FoxP3+ Treg induction. Referring to FIGURE 9A, autologous CD4+ FoxP3- T-cells were cultured with anti-CD3/28 antibodies, IL-2 and TGFb for 72 hours and then stained for FoxP3. FIGURE 9B is a graph that shows the mean fluorescence activity of CD3+/CD4+ T-cells after culturing in the presence of vehicle or 4-MU. Referring to FIGURE 9B, the CD4+ gate is shown to highlight FoxP3 production by activated T-cells. These data indicate that 4-MU promotes FoxP3 induction.

When Treg inductions were performed in vitro there was an increase both the percentage of Treg as well as the functionality. To observe these effects in vivo a series of immunization models was performed. In the first, the effect of the antigen-specific T- cell response within a mouse was examined. FIGURES 10A-10D show the amount of tritiated thymidine in CPM in variously activated T-cells isolated from mice fed a diet containing 4-MU or control chow. Using the DOl 1.10 mouse model, a strain that carries a T-cell receptor transgene specific for OVA, emulating auto reactive CD4+ T-cells, mice were fed either 5% 4-MU pressed chow or control chow for two weeks. Next, an immunization of OVA protein (50 pg) was injected with alum as an adjuvant intraperitoneally into the mice. Two weeks after the sensitization injection, the mice were boosted with a similar injection. One week later, the mice were sacrificed. Spleens and draining lymph nodes were isolated. The cells were processed into a single cell suspension and re-stimulated with either a titration of CD3/CD28 (FIGURES 10A and 10B) or OVA peptide (FIGURES 10C and 10D). Proliferation was quantified with tritiated thymidine. While the polyclonal response to CD3 and CD28 was uninhibited, suggesting no generalized immune deficit, there was a significant reduction in the recall response to OVA, suggesting that the antigen-specific response was impaired due to 4- MU treatment without compromising the viability of the T-cells. The results demonstrate that in a mouse model of immunization, 4-MU inhibits the antigen specific T-cell response but does not impair normal T-cell function in these mice.

To confirm that the antigen specific recall deficit seen in the previous immunization was due to decreased T-cell activation, an adoptive transfer model was performed. In this model, wild-type B6 mice were put on either control or 4-MU chow for 2 weeks. Fourteen days after the chow began, OT II mouse CD4+ T-cells were isolated. These cells were stained with a proliferation dye and injected retro-orbitally into the pre-treated B6 mice. After 18 hours, the mice were immunized intraperitoneal with OVA protein and Alum. Three days after this injection, the mice were sacrificed and lymphoid organs were isolated, stained, and run on a flow cytometer to analyze how much the adoptively transferred T-cells had proliferated. It was observed that 4-MU significantly decreased the proliferation of the labeled T-cells as measured by division index, or the average number of divisions that occurred. FIGURE 11A graphically represents the average number of proliferation peaks or divisions in T-cells transferred into mice treated with 4-MU or a control and immunized with OVA protein and alum adjuvant. As shown in FIGURE 11 A, the primary T-cell response in vivo is attenuated when mice are given 4-MU at the time of immunization. B6 mice were fed 4-MU or control chow for two weeks. After two weeks, 7.5 million OT II mouse CD4+ T-cells were labeled with an efluor450 proliferation dye and injected retro-orbital into the study mice on chow. One day after the adoptive transfer, the mice were immunized with 50 pg OVA protein and alum adjuvant. Three days after the immunization, mice were sacrificed and a flow cytometer was used to look at the average number of proliferation peaks or divisions that the adoptively transferred cells underwent.

FIGURE 11B is a graph that shows the average number of proliferations or the division index in T-cells transferred into mice treated with 4-MU or a control and immunized with OVA protein and alum adjuvant. The average number of proliferations or the division index is plotted for the n=4 mice. As shown in FIGURE 11B, early T-cell induction and proliferation are inhibited by 4-MU treatment.

Following this experiment, two more immunization models were used to show that 4-MU is important at the time of antigen presentation and that this decrease in recall response is due to impairment in the generation of antigen specific T-cell memory. FIGURE 12A graphically represents the average percentage of activation. Cells were isolated from immunized animals treated with 4-MU and control. Mice were restimulated to that antigen ex vivo. CD25, CD69, CD95, and CD44 are shown. To evaluate the timing of 4-MU’s action, D.Ol l mice were divided into three groups: 1) Control chow for the duration of the experiment (solid, bold line); 2) Control chow until the immunization, 4-MU chow post immunization (dotted line); and 3) 4-MU for the duration of the experiment (solid, non-bold line). The mice were immunized once with 50 pg OVA and alum on day 15. Two weeks after immunization, T-cells were isolated from these animals and re-stimulated using congenic BMDC and 1 pg/ml OVA protein for three days before a flow cytometer was used to analyze CD4+ T-cells for canonical markers of activation: CD25, CD69, CD95, and CD44. As shown in FIGURE 12A, the only group of mice that showed impairment of T-cell activation was the group that had been on 4-MU since the beginning of the experiment.

FIGURE 12B is a graph that shows the proliferation response ex vivo to mice immunized on control and 4-MU treatment. To observe the T-cell specific recall impairment for mice treated with 4-MU, D.Ol 1 mice were fed 4-MU or control chow for two weeks. After this time period, mice were immunized with OVA and the alum adjuvant. Two weeks after immunization, mice were sacrificed and CD4+ T-cells were isolated from these mice and re-stimulated with either CD3/CD28 or BMDC loaded with OVA peptide. Instead of looking at activation markers, two days after the culture began, tritiated thymidine was introduced into the co-culture (or polygenic stimulation with CD3/CD28 activating antibodies) at time=48 hours and at time=72 hours. One day after that, cells were washed and wells were analyzed on a scintillator, giving proliferation via tritiated thymidine uptake. For the same experiment, each mouse’s average proliferation response to OVA was normalized to its average response to polyclonal stimulation and this normalized value is shown.

As shown in FIGURE 12B, there was no impairment in polyclonal responsiveness while the OVA recall response was greatly attenuated, suggesting again that only the antigen specific response was impaired in these groups. This model system and the results described herein demonstrate the inhibition of antigen presentation and activation of inflammatory T-cells without overall impairment of the immune system.

To show that this model applies to allo-reactivity (transplantation), a mixed leukocyte reaction was performed. FIGURE 15A is a graph that shows the amount of tritiated thymidine in CPM in a mixed population of activated T-cells and dendritic cells cultured in the presence of DMSO or 4-MU. Mixed human CD3 T-cells and B cells were combined from different donors in culture with increasing concentrations of 4-MU as indicated. The results show that in a mixed lymphocyte assay 4-MU prevents responses against allogeneic cells. FIGURE 15B is a graph that shows the normalized proliferation in a mixed population of activated T-cells and dendritic cells cultured in the presence of DMSO and 4-MU. Pooled data from multiple replicates are shown. The results show that the allo-response is greatly inhibited by 4-MU.

Thus, in one aspect, the present disclosure provides a composition for inhibiting an adverse immune response in a transplant recipient comprising (i) a compound that inhibits hyaluronan synthesis, and (ii) a pharmaceutically acceptable carrier.

In one embodiment, the compound is a UDP glycosyltransf erase inhibitor. In one embodiment, the compound is a UDP glucuronyltransferase inhibitor. In one embodiment, the compound is 4-methylumbelliferone. In one embodiment, the compound is a metabolite of 4-methylumbelliferone. In one embodiment, the compound is 4-methylumbelliferyl-glucuronide or a sulfated 4-methylumbelliferone, or an uncleavable form of the compound.

In one embodiment, the compound is effective to induce a regulatory T-cell response. In one embodiment, the compound is effective to increase FoxP3+ regulatory T-cells.

In one embodiment, the adverse immune response is selected from the group consisting of alloimmunity, cell transplant rejection, tissue transplant rejection, solid organ transplant rejection, and graft-versus-host disease.

As used herein, the expression“effective amount” or“therapeutically effective amount” refers to an amount of the compound of the present disclosure that is effective to achieve a desired therapeutic result, such as, for example, inhibiting an adverse immune response in a transplant recipient. The compound of the present disclosure can be administered as a pharmaceutical composition comprising a therapeutically effective amount of the compound together with a pharmaceutically acceptable carrier. In the context of the present disclosure, a“therapeutically effective amount” is understood as the amount of a compound inhibiting the synthesis, expression, and/or activity of an identified HA polymer that is necessary to achieve the desired effect which, in this specific case, is inhibiting an adverse immune response in a transplant recipient. Generally, the therapeutically effective amount of the compound according to the present disclosure to be administered will depend, among other factors, on the individual to be treated, on the severity of the disease the individual suffers, on the chosen dosage form, and the like. For this reason, the doses mentioned in the present disclosure must be considered only as a guideline for a person skilled in the art, and the skilled person must adjust the doses according to the previously mentioned variables. Nonetheless, a compound according to the present disclosure can be administered one or more times a day, for example, 1, 2, 3 or 4 times a day, in a typical total daily amount comprised between 0.1 pg to 10,000 mg/day, typically 100 to 1,500 mg/day. The subject can be a human or non-human animal, a vertebrate, and is typically an animal, including but not limited to, cows, pigs, horses, chickens, cats, dogs, and the like. More typically, the subject is a mammal, and in a particular embodiment, human.

As used herein, the term“pharmaceutically acceptable carrier” means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a subject. The components of the pharmaceutical compositions also are capable of being commingled with each other, in a manner such that there is no interaction, which would substantially impair the desired pharmaceutical efficiency. Such preparations can routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes (including pH-dependent release formulations), lipidoids, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of the compositions, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249, 1527-1533 (1990) and Langer and Tirrell, Nature 428, 487-492 (2004). In addition, the compositions described herein can be formulated as a depot preparation, time-release, delayed release or sustained release delivery system.

The mode of administration can be any medically acceptable mode including oral administration, sublingual administration, intranasal administration, intratracheal administration, inhalation, ocular administration, topical administration, transdermal administration, intradermal administration, rectal administration, vaginal administration, subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, intrasternal, administration, or via transmucosal administration. In addition, modes of administration can be via an extracorporeal device and/or tissue-penetrating electro-magnetic device.

The particular mode selected will depend upon the particular compound selected, the desired results, the particular condition being treated and the dosage required for therapeutic efficacy. The methods described herein, generally speaking, can be practiced using any mode of administration that is medically acceptable, for example, any mode that produces effective levels of inflammatory response alteration without causing clinically unacceptable adverse effects.

The compositions can be provided in different vessels, vehicles or formulations depending upon the disorder and mode of administration. For example, for oral application, the compositions can be administered as sublingual tablets, gums, mouth washes, toothpaste, candy, gels, films, etc.; for ocular application, as eye drops in eye droppers, eye ointments, eye gels, eye packs, as a coating on a contact lens or an intraocular lens, in contacts lens storage or cleansing solutions, etc.; for topical application, as lotions, ointments, gels, creams, sprays, tissues, swabs, wipes, etc.; for vaginal or rectal application, as an ointment, a tampon, a suppository, a mucoadhesive formulation, and the like.

The compositions can be administered by injection, e.g., by bolus injection or continuous infusion, via intravenous, subcutaneous, intramuscular, intraperitoneal, intrasternal routes. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. For oral administration, the compositions can be formulated readily by combining the compositions with pharmaceutically acceptable carriers well known in the art, e.g., as a sublingual tablet, a liquid formulation, or an oral gel.

For administration by inhalation, the compositions can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator can be formulated containing a powder mix of the compositions and a suitable powder base such as lactose or starch. Medical devices for the inhalation of therapeutics are known in the art. In some embodiments the medical device is an inhaler. In other embodiments the medical device is a metered dose inhaler.

The compositions can also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

The compounds described herein are comparable to generic immunosuppressive drugs such as the calcineurin inhibitor cyclosporine. Because the described compounds act at the stage of antigen presentation, the rest of the immune system is left intact. 4-methylumblliferone has been shown to have a very good safety profile, and side effects are usually limited and benign. Thus, the drug can be used alone or, for example, in conjunction with generic immunosuppressive compounds to be able to allow patients to lower dosages of drugs that possess extensive side effects.

In order to test the model system described herein in solid organ transplantation, cardiac allo-transplants were performed. C57BL/6 mice were treated with 4-MU or control chow for one month. At that time, the heart from a BALB/c mouse was transferred and grafted onto the renal artery or inferior vena cava of the C57BL/6 mouse. The time to rejection is measured as the day the recipient heart stops beating as measured by palpation. FIGURE 14A is a graph that shows the percentage FoxP3+ of total CD4+ T-cells in spleen and peripheral lymph nodes five days after transplant in mice treated with 4 MU (white bar) or a control (black bar). A cohort of mice was sacrificed on day 5 and the peripheral lymph nodes (LN) as well as spleen were analyzed for %Treg of CD4+ T-cells. The results show that in a mouse cardiac allo-transplantation, the time to rejection is increased and there are an increased number of protective Treg found in mice treated with 4-MU.

FIGURE 14B is a graph that shows the time to transplant rejection in mice treated with 4-MU or a control. As shown in FIGURE 14B, a significant increase in time to rejection and an enrichment of regulatory T-cells were observed, demonstrating that 4-MU limits the induction of inflammatory T-cells that induce rejection while enriching for the regulatory Treg cells that appear to be protective. In order to demonstrate the effect of 4-MUG, male C57BL/6 mice were fed control chow, 5% 4-MU pressed chow, or 2 mg/ml 4-MUG dissolved in water for one month prior to heterotropic cardiac allo-transplantation (n=5-8 per group). Age and sex- matched BALB/c donor hearts were isolated and grafted onto the aorta of treated animals. Graft rejection was measured by abdominal palpitation once per day, and rejection day was reported as the day that the recipient heart stopped beating. FIGURE 16A is a graph that shows the time to transplant rejection by days in mice treated with 4-MU, 4-MUG, or a control. FIGURE 16B is a graph that shows the survival percentage in mice treated with 4-MU (dotted line), 4-MUG (dashed line), or a control (solid line) by days post-transplant surgery. The results demonstrate that 4-MU as well as 4-MUG increase the rejection time in a heterotropic cardiac allo-transplant.

Referring generally to FIGURES 17A, 17B, 17C, and 17D, human monocyte derived dendritic cells from one human subject were cultured with allogeneic naive CD4+ T-cells from a separate subject. After five days of culture, 4-MU treated samples showed increased percentage of CD25+/FoxP3+ Treg. FIGURE 17A shows a culture of human Dc and allogeneic T-cells in a scatter plot that shows the percentage of CD25+/FoxP3+ Treg in live, proliferated CD4+ T-cells cultured in the presence of DMSO or 4-MU. FIGURE 17B is a graph that shows the percentage CD25+/FoxP3+ Treg in live, proliferated CD4+ T-cells cultured in the presence of DMSO or 4-MU, with combined data for n=3 replicates (representative data of three different human subject crosses). As shown in FIGURES 17A and 17B, there is an increase in the percentage of Treg of live, proliferated CD4+ T-cells cultured in the presence of 4-MU as compared to the control.

FIGURE 17C is a graph that shows the MFI of CD25+/FoxP3+ Treg in all CD4+ T-cells cultured in the presence of DMSO or 4-MU. Referring to FIGURE 17C, FoxP3 geometric MFI shows no overall increase when all live, proliferated CD4+ T-cells are analyzed. FIGURE 17D is a graph that shows the FoxP3 MFI of only Treg cultured in the presence of DMSO or 4-MU. As shown in FIGURE 17D, FoxP3 geometric MFI for only live Treg shows an increase in the 4-MU treated samples compared to DMSO for n=3 technical replicates (representative data of three different human subject crosses). Overall, the data shown in FIGURES 17A, 17B, 17C, and 17D show that 4-MU induces FoxP3+ Treg in a human allogeneic mixed leukocyte reaction.

Thus, in one aspect, the present disclosure provides a method for inhibiting an adverse immune response in a mammalian transplant recipient, the method comprising administering to the mammalian transplant recipient a composition comprising a compound in an amount effective to inhibit hyaluronan synthesis in the mammalian transplant recipient.

In one embodiment, the compound is a UDP glycosyltransf erase inhibitor. In one embodiment, the compound is a UDP glucuronyltransferase inhibitor. In one embodiment, the compound is 4-methylumbelliferone. In one embodiment, the compound is a metabolite of 4-methylumbelliferone. In one embodiment, the compound is 4-methylumbelliferyl-glucuronide or a sulfated 4-methylumbelliferone, or an uncleavable form of the compound.

In one embodiment, the compound is effective to induce a regulatory T-cell response. In one embodiment, the compound is effective to increase FoxP3+ regulatory T-cells.

In one embodiment, the mammalian transplant recipient is a human transplant recipient. In one embodiment, the adverse immune response is selected from the group consisting of alloimmunity, cell transplant rejection, tissue transplant rejection, solid organ transplant rejection, and graft-versus-host disease.

Thus, it has been discovered that compounds that inhibit HA synthesis, for example 4-MU, can be used as a mono or adjunct therapy for patients undergoing transplant due to the compounds’ ability to induce Foxp3+ regulatory T-cells. These compounds are desirable because of a good safety profile and limited side effects, where most immunosuppressive compounds carry significant morbidity. Due to the described compounds’ ability to limit antigen presentation without compromising the rest of the immune system, the compounds can be used, for example, as a safe maintenance drug for patients on long term immunosuppression.

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition for inhibiting an adverse immune response in a transplant recipient comprising (i) a compound that inhibits hyaluronan synthesis, and (ii) a pharmaceutically acceptable carrier.

2. The composition of Claim 1, wherein the compound is a

UDP-glycosyltransferase inhibitor.

3. The composition of Claim 2, wherein the compound is a

UDP-glucuronyltransferase inhibitor.

4. The composition of Claim 3, wherein the compound is

4-methylumbelliferone.

5. The composition of Claim 4, wherein the compound is a metabolite of 4-methylumbelliferone.

6. The composition of Claim 5, wherein the compound is

4-methylumbelliferyl-glucuronide or a sulfated 4-methylumbelliferone.

7. The composition of Claim 6, wherein the compound is an uncleavable form of 4-methylumbelliferyl-glucuronide or sulfated 4-methylumbelliferone

8. The composition of Claim 1, wherein the compound is effective to induce a regulatory T-cell response.

9. The composition of Claim 8, wherein the compound is effective to increase FoxP3+ regulatory T-cells.

10. The composition of Claim 1, wherein the adverse immune response is selected from the group consisting of alloimmunity, cell transplant rejection, tissue transplant rejection, solid organ transplant rejection, and graft-versus-host disease.

11. A method for inhibiting an adverse immune response in a mammalian transplant recipient, the method comprising administering to the mammalian transplant recipient a composition comprising a compound in an amount effective to inhibit hyaluronan synthesis in the mammalian transplant recipient.

12. The method of Claim 11, wherein the compound is a

UDP-glycosyltransferase inhibitor.

13. The method of Claim 12, wherein the compound is a

UDP-glucuronyltransferase inhibitor.

14. The method of Claim 13, wherein the compound is

4-methylumbelliferone.

15. The method of Claim 14, wherein the compound is a metabolite of 4-methylumbelliferone.

16. The method of Claim 15, wherein the compound is

4-methylumbelliferyl-glucuronide or a sulfated 4-methylumbelliferone.

17. The method of Claim 15, wherein the compound is an uncleavable form of 4-methylumbelliferyl glucuronide or sulfated 4-methylumbelliferone.

18. The method of Claim 11, wherein the compound is effective to induce a regulatory T-cell response.

19. The method of Claim 18, wherein the compound is effective to increase FoxP3+ regulatory T-cells.

20. The method of Claim 11, wherein the mammalian transplant recipient is a human transplant recipient.

21. The method of Claim 20, wherein the adverse immune response is selected from the group consisting of alloimmunity, cell transplant rejection, tissue transplant rejection, solid organ transplant rejection, and graft-versus-host disease.