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Keywords:

  • cell cycle;
  • rodent;
  • transplantation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Previously we have shown that indoleamine 2,3-dioxygenase (IDO) and the tryptophan metabolite, 3-hydroxykynurenine (3HK) can prolong corneal allograft survival. IDO modulates the immune response by depletion of the essential amino acid tryptophan by breakdown to kynurenines, which themselves act directly on T lymphocytes. The tryptophan metabolite analogue N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA, ‘Tranilast’) shares the anthranilic acid core with 3HK. Systemic administration of DAA to mice receiving a fully MHC-mismatched allograft of cornea or skin resulted in significant delay in rejection (median survival of controls 12 days, 13 days for cornea and skin grafts, respectively, and of treated mice 24 days (< 0·0001) and 17 days (< 0·03), respectively). We provide evidence that DAA-induced suppression of the allogeneic response, in contrast to that induced by tryptophan metabolites, was a result of cell cycle arrest rather than T-cell death. Cell cycle arrest was mediated by up-regulation of the cell cycle-specific inhibitors p21 and p15, and associated with a significant reduction in interleukin-2 production, allowing us to characterize a novel mechanism for DAA-induced T-cell anergy. Currently licensed as an anti-allergy drug, the oral bioavailability and safe therapeutic profile of DAA make it a candidate for the prevention of rejection of transplanted cornea and other tissues.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The tryptophan metabolite analogue N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA, ‘Tranilast’) is prepared from the constituents of the leaves of the Nandina plant[1] and is currently licensed for clinical use in allergy on account of its inhibition of mast cell degranulation and histamine release.[2] It has other biological effects including inhibition of leukotrienes, cytokines, prostaglandins, oxygen radicals, cyclo-oxygenase-2 expression [3, 4] and has been shown to have anti-proliferative effects as well as inhibiting collagen deposition in several models of inflammatory disease.[5-7] It has also been shown to inhibit murine cardiac allograft vasculopathy by inhibiting neo-intimal smooth muscle cell proliferation.[8] Inhibition of vascular inflammation induced by DAA is believed to be as a result of its inhibition of vascular chymase, platelet-derived growth factor-induced and transforming growth factor β1-induced smooth muscle cell proliferation as well as a result of up-regulation of p21, a universal inhibitor of cyclin-dependent kinases.[8] Furthermore, it has been shown to inhibit nuclear factor-κB-dependent up-regulation of endothelial cell adhesion molecules.[9]

Structurally, DAA shares the anthranilic acid core with the tryptophan metabolites 3-hydroxyanthranilic acid and 3-hydroxykynurenine. It suppresses antigen-specific T-cell proliferation and interferon-γ and tumour necrosis factor-α production and increases production of interleukin-4 (IL-4) and IL-10 in a manner similar to the structurally related kynurenines.[10] In the same report DAA was shown to reverse paralysis in mice with established experimental autoimmune encephalomyelitis at least partly by inhibiting the activation of myelin-specific T cells.[10] Similarly, DAA has been shown to ameliorate the clinical and histological manifestations of arthritis in a mouse model of collagen-induced arthritis, a T-cell-mediated disease that displays many pathological, genetic and immunological similarities to human rheumatoid arthritis.[11]

In summary, DAA has anti-inflammatory, anti-proliferative and immunomodulatory effects. The exact mechanisms for its immunosuppressive activities remain unknown and its possible effect in allogeneic rejection has not been investigated.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

DAA (Tranilast)

The DAA was obtained as a kind gift from Nuon Therapeutics (San Mateo, CA) and also purchased from Sigma Aldrich (Poole, UK). For in vivo studies, DAA was dissolved at a maximum concentration of 10 mg/ml in 1% sodium bicarbonate by heating for 1 hr at 70°. Upon cooling, an emulsion was formed. Animals received 400 mg/kg of DAA, administered by intraperitoneal (i.p.) injections, on days 1–16 following corneal transplants; days 1–15 and from day 1 until rejection were scored. Control animals received the same volume of vehicle.

For in vitro studies, DAA was dissolved in DMSO. Stock DAA was dissolved in RPMI-1640 medium (Gibco-BRL, Paisley, UK) and added to cell cultures at concentrations ranging from 0 to 200 μm.

T-cell proliferation assays

Splenocytes from BALB/c mice were treated with a mixture of anti-CD45R/B220, anti-CD8 and anti-MHC class II supernatants (RA3-3A1, M5/114, 53.6.7 and 2.4G2) for 30 min. After antibody treatment, cells were washed and incubated with goat anti-mouse IgG-coated and goat anti-rat IgG-coated beads (Dynal, Bromborough, UK) for 30 min, bound cells were removed with a magnet. Responder CD4+ T cells (1 × 105 cells/well, purity > 90%) were stimulated with both anti-mouse CD3 and CD28 beads (Dynabeads Mouse CD3/CD28 T-cell expander: 1 bead/cell) in the presence of DAA (0–200 μm) in 96-well plates for 3 days. Proliferation was measured by a 16-hr pulse with [3H]thymidine (Amersham, Little Chalfont, UK).

Detection of cell death

CD4+ T cells (1 × 105 cells/well) were stimulated with CD3/CD28 beads (1 bead/cell) in the presence or absence of DAA for 3 days. Cells were then stained with FITC-labelled annexin V and 7-amino-actinomycin D (BD Bioscience, Oxford, UK) according to the manufacturer's instructions and analysed by flow cytometry.

Detection of regulatory T cells

CD4+ T cells were activated by CD3/CD28 beads, in the presence or absence of DAA for 7 days. Cells were stained with the APC anti-mouse/rat Foxp3 staining set (eBioscience, Hatfield, UK) after permeabilization and analysed by flow cytometry.

RNA extraction, reverse transcription and quantitative PCR

CD4+ T cells were washed after culture and RNA extraction and quantification were performed as previously described.[12] Quantitative PCR was carried out as previously described [13] and p15 and p21 mRNA quantification was carried out using the paired primers 5′-CCCTGCCACCCTTACCAGA-3′ (forward) and 5′-CAGATACCTCGCAATGTCACG-3′ (reverse) spanning 169 bp of the p15 gene and 5′-CCTGGTGATGTCCGACCTG-3′ (forward) and 5′-CCATGAGCGCATCGCAATC-3′ (reverse) spanning 103 bp of the p21 gene, respectively.

Transcripts were normalized to levels of hypoxanthine phosphoribosyl transferase (HPRT) mRNA as previously described.[14]

Western blotting

Cells ready for extraction of proteins were harvested and washed three times in cold PBS before counting. Cell lysates were prepared by resuspending 1 × 106–2 × 106 cells in 130 μl lysis buffer (1% nonidet P-40, 150 mm NaCl, 5 mm MgCl2, 10 mm HEPES buffer) supplemented with protease inhibitor cocktail (Sigma-Aldrich).

After centrifugation, supernatant was mixed with an equal volume of 2 × concentrated Laemmli sample buffer (125 mm Tris–HCl pH 6·8, 10% 2-mercaptoethanol, 4% SDS, 0·004% bromophenol blue, 20% glycerol; Sigma-Aldrich) and boiled for 5 min. Protein samples were separated on 10% SDS–PAGE and then transferred to a nitrocellulose membrane using standard electrophoretic transfer methods. Membranes were probed using rabbit anti-mouse Cyclin E antibody (Upstate/Millipore, Billerica, MA), followed by goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (Dakocytomation, Cambridge, UK). Blots were developed using the ECL plus system (Amersham Biosciences-GE Healthcare, Little Chalfont, UK).[13]

Preparation of splenocytes

After red cell lysis of the spleen and lymph node suspension, RPMI-1640 medium was added, and the cells were centrifuged and then resuspended in 1·5 ml medium. When used in mixed lymphocyte reactions, the cells were activated by incubating them with lipopolysaccharide at 10 μg/ml for 48 hr and irradiated (60 Gray) before co-culture.

Corneal transplantation

Orthotopic corneal transplantation of fully MHC-mismatched C3H strain or syngeneic BALB/c donor corneas was undertaken in inbred BALB/c (6–10 weeks old) mouse recipients (Harlan Olac UK, Bicester, UK). Animals were maintained in a specific pathogen-free facility and treated in accordance with UK Government regulations for care of experimental animals. Transplantation was performed in the right eye of all animals as previously described.[15] Corneal graft transparency was graded on a scale of 0–4 as described elsewhere.[15] Rejection was diagnosed when the opacity score reached or exceeded three in a previously transparent donor cornea.

Skin transplantation

Skin grafting was conducted using a method previously described by Billingham and Medawar using tail skin grafted onto the lateral thorax. Male 6- to 8-week-old BALB/c mice were used as recipients and male 6- to 8-week-old C57BL/6 mice were used as donors. Plasters were removed after 8 days and grafts were examined every 2 days. Rejection was registered when < 10% of the original graft remained viable.

Statistical analyses

The means of triplicate values and associated standard deviations (SD) were calculated for all T-cell proliferation data. Statistical differences were calculated using a two-tailed t-test. Differences in graft survival were analysed using a log rank test and plotted according to the Kaplan–Meier method. A value of < 0·05 was regarded as statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

DAA inhibits murine T cell proliferation in vitro

Before analysing the effect of DAA on allograft survival, its inhibitory effect on CD4+ T-cell proliferation was confirmed. We concentrated on this subset because published evidence indicates that corneal transplantation rejection is mediated by CD4+ T cells, and depletion of CD8+ cells has no effect on graft survival.[16] DAA was added to purified CD4+ T cells that were stimulated with anti-CD3/CD28 beads and proliferation was determined using [3H]thymidine incorporation. As shown in Fig. 1, addition of DAA significantly inhibited CD4+ T-cell proliferation in a dose-dependent manner when compared with T cells incubated with vehicle alone.

image

Figure 1. Effect of N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA) on CD4+ T-cell proliferation. CD3/CD28 bead-stimulated CD4+ T cells were incubated for 72 hr in the presence of DAA or vehicle alone (Ctrl) at concentrations varying from 0 to 200 μm. Proliferation was measured by [3H]thymidine incorporation. Data represent mean ± SD of triplicate cultures. Results shown are of one experiment representative of three. *< 0·005, versus control.

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DAA does not induce T-cell death

In contrast to the kynurenines, which inhibited T-cell proliferation as a result of T-cell death[14]; DAA was found not to induce T-cell death. CD4+ T cells were stimulated with anti-CD3/CD28 beads in the presence of varying concentrations of DAA (0–200 μm) as well as vehicle control for 72 hr and cell death was assessed by flow cytometry following staining with 7-amino-actinomycin D and annexin V. DAA did not induce T-cell death in comparison to controls and increasing the concentration of DAA had no influence on T-cell viability (Fig. 2). This suggests that although structurally similar to kynurenines,[10] DAA has a different underlying molecular mechanism of action.

image

Figure 2. Effect of N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA) on T-cell viability. CD3/CD28 bead-activated CD4+ T cells were incubated with or without DAA or with vehicle for 72 hr. Cytotoxicity was evaluated by flow cytometry following vital staining with 7-amino-actinomycin D and annexin V. The bar graph summarizes the percentage of cell death detected by annexin V and 7-amino-actinomycin D. Data represent mean ± SD of triplicate samples.

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DAA can induce regulatory T-cell development

Having established that it inhibits CD4+ T-cell proliferation and that this inhibition is not as a result of T-cell death, we went on to investigate the effect of DAA on regulatory T-cell development by examining the effect of DAA on the number of FoxP3+ cells from the whole CD4+ T-cell population.

CD4+ T cells were stimulated with anti-CD3/CD28 beads in the presence or absence of DAA at varying concentrations for 7 days. There was a statistically significant increase in the percentage of CD4 cells expressing FoxP3 at low concentrations of DAA (10 μm) (Fig. 3) in comparison to vehicle alone, which was reproducible on repeated experiments.

image

Figure 3. FoxP3 expression in CD4+ T cells cultured with N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA). CD4+ T cells were activated with anti-CD3/CD28 beads with or without DAA for 7 days, cells were recovered and the percentage of CD4+ T cells that were FoxP3 positive was quantified by intracellular flow cytometry. Data represent mean ± SD of triplicate samples. *< 0·005, versus control.

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Cell cycle arrest in T cells cultured with DAA

Experiments indicated that DAA inhibited T-cell proliferation not as a result of cell death but possibly through regulatory mechanisms. One additional possibility was suggested by the earlier reports that DAA inhibits vascular smooth muscle proliferation in association with enhanced p21 expression on neointimal cells[8] and that suppression of T-cell proliferation in response to DAA was associated with a G1-S phase arrest in CD4+ T cells.[13] The protein product of the Waf1/Cip1 gene, p21, is identified as a universal inhibitor of cyclin-dependent kinases,[17, 18] which are essential for cell progression through the G1-S phase check point. We postulated that the DAA-induced inhibition of T-cell proliferation was a result of G1-S phase cell cycle arrest secondary to up-regulation of specific cell cycle inhibitors, in particular p21.

The role of the cell cycle inhibitors p15 and p21 was investigated in CD4+ T cells incubated with varying concentrations of DAA. CD4+ T cells were stimulated with anti-CD3/CD28 beads in the presence or absence of DAA at varying concentrations for 72 hr. Expression of p15 and p21 at the mRNA level (normalized to HPRT) was determined by real-time PCR. DAA induced up-regulation of p21 and p15 expression in comparison to vehicle alone and the increasing DAA concentration resulted in a dose-dependent increase in expression of the cell cycle inhibitors (Fig. 4a,b). Expression of cyclin E by CD4+ T cells incubated with DAA was also investigated. Increasing the DAA concentration resulted in a dose-dependent reduction in cyclin E protein expression (Fig. 4c,d). Taken together these data indicate that T cells incubated with DAA fail to progress through the restriction point into late G1 and are therefore arrested at earlier stages of the G1 phase.

image

Figure 4. Cell cycle arrest in CD4+ T cells cultured with N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA). p21(a) and p15 (b) mRNA expression in DAA-treated CD4+ T cells. CD4+ T cells were stimulated with anti-CD3/CD28 beads in the presence or absence of DAA at varying concentrations for 72 hr, cells were washed, and RNA was extracted and subsequently converted to cDNA; then real-time PCR was carried out. The levels of p15 and p21 mRNA are normalized to those of hypoxanthine phosphoribosyl transferase mRNA and shown as the mean ± SD of triplicate determinations. (c) Expression of the 52 000 molecular weight Cyclin E protein in the lysates of CD4+ T cells incubated with DAA and vehicle alone (relative to T-cell expression of the 42 000 molecular weight β-actin protein) was determined by Western blotting on day 3 of culture. (d) Densitometry data of Cyclin E blots. Results shown are from one experiment, representative of three.

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Cell cycle arrest in CD4+ T cells cultured with DAA is associated with reduced IL-2 production

Anergy of helper T cells is induced by ligation of the T-cell receptor by antigen alone and is overcome by co-stimulation or IL-2. Having shown that DAA inhibits T-cell proliferation through cell cycle arrest in the G1-S phase, the levels of IL-2 in supernatants of T cells cultured with DAA were analysed. CD4+ T cells were stimulated with anti-CD3/CD28 beads in the presence or absence of DAA at varying concentrations for 72 hr. Culture supernatants from the assays were harvested for detection of the IL-2 cytokine by ELISA. Relative to concentrations of IL-2 in the culture supernatants of CD4+ T cells incubated with vehicle alone, there was a significant dose-dependent reduction in IL-2 levels in DAA-treated CD4+ T cells (Fig. 5).

image

Figure 5. Production of interleukin-2 (IL-2) in N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA) -treated T-cell assays. CD4+ T cells were stimulated with anti-CD3/CD28 beads in the presence or absence of DAA at various concentrations for 72 hr. Culture supernatants from the assays were harvested for detection of the IL-2 cytokine by ELISA. Data represent mean ± SD of triplicate samples, *< 0·05, versus control. Results shown are of one experiment representative of two.

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Having demonstrated up-regulation of markers consistent with T-cell anergy in response to DAA and shown that low concentrations are associated with a minor increase in regulatory cell development, we looked for induction of donor alloantigen-specific tolerance in vivo using re-challenge assays. In these, we administered DAA (400 mg/kg/day) to animals for 5 days. On day 1 they were immunized with C3H lipopolysaccharide-stimulated splenocytes (8 × 106), and on day 10 spleen-purified CD4+ T cells were challenged with allogeneic dendritic cells or third-party dendritic cells and the proliferation was noted. There was no evidence of any decrease in the allogeneic response in vitro, indicating that DAA had not induced effective T-cell regulation or hyporesponsiveness in vivo (data not shown).

Systemic administration of DAA results in prolongation of corneal allograft survival

Given the ability of DAA to inhibit allogeneic T-cell responses in vitro we went on to determine whether systemic administration of DAA resulted in prolonged allograft survival of fully MHC-mismatched donor corneas. DAA was administered at 400 mg/kg i.p. in BALB/c corneal allograft recipients on a daily basis on days 1–16 post-transplant. This dose was based on previous published studies,[11] and results in a plasma concentration that is approximately 10 times that obtained in patients on standard doses of the drug.[19] Control animals were given the same volume of vehicle on days 1–16 post-transplant (n = 10/group). DAA administration resulted in significant prolongation in graft survival [median survival time (MST) 24 days] in comparison to controls (MST 12 days, < 0·0001, Fig. 6).

image

Figure 6. Effect of systemic N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA) administration on corneal allograft survival. BALB/c (H2d) mice received unilateral C3H (H2k) donor corneal allografts and intraperitoneal injections of DAA were administered at a dose of 400 mg/kg/day on days 1–16 post-transplantation (n = 10/group). There was significant prolongation of graft survival in the DAA-treated group (median survival time 23·5 days, < 0·0001) in comparison with controls (median survival time 11·5 days).

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Systemic administration of DAA results in prolongation of skin allograft survival

The significant prolongation in corneal allograft survival after DAA treatment led us to investigate whether this effect would be observed in transplant tissues that induce a stronger allogeneic response. Corneal allograft rejection is predominantly mediated by the indirect pathway of allorecognition. Skin allograft rejection, however, is also mediated by the direct pathway. Skin donors were C57BL/6 strain, fully MHC mismatched with multiple minor mismatches with respect to BALB/c recipients. This donor–recipient strain combination has similar rejection kinetics for skin as for cornea in the C3H/He–BALB/c combination (a fast-rejecting donor–recipient strain combination, with an MST of approximately 12 days). Animals were treated with DAA at a dose of 400 mg/kg/day i.p. on days 1–15 and from day 1 until rejection was scored to compare the effect of short as well as continuous treatment on allograft survival. Control animals were given the same volume of vehicle on days 1–15 post-transplant (n = 5/group). DAA administration resulted in a significant prolongation in skin allograft survival in both the short and continuous treatment groups (MST 17 and 21 days, respectively) in comparison to controls (MST 13 days, < 0·03 and < 0·001, respectively) (Fig. 7). The difference between short and continuing treatment on skin allograft survival was not significant.

image

Figure 7. Effect of systemic N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA) administration on skin allograft survival. Skin transplants were carried out in C57BL/6 male donors to BALB/c male recipients, a strain combination, which has similar rejection kinetics to the C3H/He to BALB/c corneal transplant strain combination. Animals were treated with DAA at a dose of 400 mg/kg/day intraperitoneally on days 1–15 and from day 1 until rejection was scored to compare the effect of short as well as continuous treatment on allograft survival. Control animals were given the same volume of vehicle on days 1–15 post-transplant (n = 5/group). Systemic DAA administration resulted in a significant prolongation in skin allograft survival in both the short and continuous treatment groups (median survival time = 17, < 0·03 and 21 days, < 0·001 respectively) in comparison to controls (median survival time = 13 days).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The anthranilic acid derivative DAA was initially found to inhibit mast cell degranulation and allergen-induced release of histamine,[2] and was subsequently developed for the treatment of allergic diseases. More recently, it has been shown to be effective in mouse models of experimental autoimmune encephalomyelitis[10] and collagen-induced arthritis,[11] both studies demonstrating its immunosuppressive effects on T-cell proliferation in a manner similar to that of the structurally related kynurenines. Having earlier shown the effectiveness of kynurenines in prolonging allograft survival,[14] we set out to examine whether DAA would be effective in preventing allograft rejection and its mechanisms of activity.

We confirmed that DAA inhibited T-cell proliferation, not through inducing cell death and probably not primarily through immunoregulatory mechanisms, but by inducing cell cycle arrest. In keeping with the earlier demonstration that DAA-induced T-cell cycle arrest is at the G1-S phase,[10] we demonstrated a novel action in the up-regulation of p21 and p15, cell-cycle-specific inhibitors that bind and inhibit cyclin E–Cdk2 complexes in late G1 phase. The finding of a decrease in cyclin E protein expression with increasing DAA concentration further supports cell cycle arrest to be at the G1-S phase, as the irreversible progression of a cell to S phase entry occurs at the G1 restriction point and is characterized by activation of Cdk2 and synthesis of cyclin E.[20] Mechanisms by which p21 blocks cell cycle progression include (i) binding and inactivation of cyclin D–Cdk4, 6 and cyclin E–Cdk2 complexes, resulting in hypophosphorylation of pRB and subsequent cell cycle arrest,[21] (ii) inhibition of proliferating cell nuclear antigen, which can inhibit DNA polymerase δ, the principal replicative DNA polymerase, earlier reported to inhibit the cell cycle in both G1 and G2 phases in Jurkat T cells,[22] and (iii) interaction with members of the mitogen-activated protein kinase pathway, specifically p-Jun N-terminal kinase and p-c-Jun, resulting in an inhibition in proliferation and IL-2 secretion in anergic T helper type 1 cells.[23] Another striking observation not reported previously is the dose-dependent decrease in the pro-inflammatory cytokine IL-2 that was observed following DAA treatment. This reduction in IL-2 secretion with the up-regulation of cell cycle inhibitors p21 and p15, allowed us to define a novel mechanism of action of DAA-induced T-cell anergy, possibly working through triggering of the mitogen-activated protein kinase pathway as described above.

Having characterized a novel mechanism for the in vitro DAA-induced suppression of T-cell activity, we showed a significant prolongation in both corneal and skin allograft survival following systemic administration. Our findings do not support the induction of regulatory T cells as a major contributory factor: certainly at the doses administered we did not find indefinite transplant survival or tolerance. However, we did not look at the expression of FoxP3 in CD25 cells, which may have been helpful. DAA induction of regulatory T-cell development, to the extent to that found here, is a novel finding in itself and warrants further investigation, particularly with the emerging evidence on kynurenines,[24] DAA[25] and additional data from our laboratory (Zaher SS and George AJT, unpublished data) indicating these to be agonists of the aryl-hydrocarbon receptor (AhR) and demonstrating kynurenines to induce the generation of FoxP3+ regulatory T cells in an AhR-dependent manner.

It is possible that the absence of long-term allograft survival in these high responder donor–recipient strain combinations may result from the short duration of DAA treatment. In corneal allograft recipients it was evident that continued therapy with DAA was associated with continued graft acceptance, rejection (grade 3) only occurring on cessation of therapy. We did not design long-term treatment experiments to investigate possible extended allograft survival or tolerance. Shorter survival of skin allografts in DAA-treated recipients is likely to be explained by the greater allogenicity of this tissue. The use of DAA in other disease models such as collagen-induced arthritis showed that disease severity increased upon cessation of therapy, indicating that continued treatment is necessary for therapeutic effect.[11] However, it has only recently been shown that treatment with a higher dose of DAA, 650 mg/kg, for 30 days in a rat cardiac allograft model resulted in allograft tolerance in 45% of recipients.[26]

In conclusion, we have shown that DAA is effective in prolonging allograft survival. We have confirmed that it inhibits T-cell proliferation through cell cycle arrest and characterize a novel mechanism for the in vitro DAA-induced T-cell anergy. As the doses used in this study (400 mg/kg/day) are equivalent to the doses that are known to be safe and effective in treatment of allergy in humans,[11] this is a novel candidate for the prevention of allogeneic rejection. The additional properties of DAA mentioned previously including anti-allergy and reduced scar formation may be of particular benefit in the context of corneal transplantation.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

This work was supported by a project grant and research training fellowship from Fight for Sight (SSZ).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References
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