Shijie Qi

Modulation of T-cell responses to alloantigens by TR6/DcR3

TR6 (DcR3) is a new member of the TNF receptor (TNFR) family that lacks a transmembrane domain in its sequence, indicating that it is a secreted molecule. TR6 can bind to FasL and prevent FasL-induced apoptosis; it can also associate with LIGHT, another TNF family member. The role of TR6 in immune responses was investigated in this study. According to flow cytometry, recombinant human TR6-Fc binds to human LIGHT expressed on 293 cells or on activated human T cells and competes with the LIGHT receptor TR2 for the binding to LIGHT on these cells. Human TR6 could cross-react with mouse LIGHT in immunoprecipitation. TR6-Fc also downregulates cytotoxic T lymphocyte activity in vitro and graft-versus-host responses in mice. Moreover, TR6-Fc modulates lymphokine production by alloantigen-stimulated mouse T cells. TR6-Fc ameliorated rejection response to mouse heart allograft. These results indicate that TR6 can dampen T-cell responses to alloantigens. Such regulatory effects of TR6 probably occur via interference with interaction between pairs of related TNF and TNFR family members, LIGHT/TR2 being one of the possible candidate pairs.

Tacrolimus (FK506) and sirolimus (rapamycin) in combination are not antagonistic but produce extended graft survival in cardiac transplantation in the rat

Combined use of tacrolimus (FK506) with sirolimus (rapamycin [RAPA]) was examined in a model of vascularized heart allograft in the rat. For prevention of acute rejection, three different combinations of low doses of FK506 and RAPA from day 1 up to day 14 after transplantation produced significantly longer cardiac allograft survival than each agent alone (P<0.05). Identical results were observed in a model of reversal of ongoing acute rejection, where two combinations of low doses of FK506 and RAPA from day 4 up to day 18 after surgery also demonstrated significantly longer graft survival than each immunosuppressant alone (P<0.05). All the low-dose-treated groups in these two models presented significantly longer heart graft survival than naive controls (P<0.05), confirming that both agents are potent immunosuppressants in the models chosen. These results also indicate that, in contrast with in vitro studies, the combined use of FK506 and RAPA in vivo did not produce antagonism, but rather had synergistic effect in prolonging the allograft survival as compared with each agent alone. It appears likely that the abundance of FKBP-12 available for binding in vivo prevents inhibitive competition of the two agents for their receptor.

Effect of tacrolimus (FK506) and sirolimus (rapamycin) mono- and combination therapy in prolongation of renal allograft survival in the monkey

BACKGROUND Our previous studies confirmed that tacrolimus (FK506) and sirolimus [rapamycin (RAPA)], in combination, are not antagonistic but are synergistic in the prolongation of heart and small bowel grafts in the rodent. The aim of this study was to confirm further the synergistic effect of combined FK506 and RAPA in the more clinically relevant model, kidney transplantation in monkeys. METHODS A total of 60 male Vervet monkeys were randomly assigned to 10 groups (n> or =5). Monkeys with renal allografts were treated with different doses of FK506 and/or RAPA orally for 60 days. Graft survival, body weight, clinical biochemistry determinations, oral glucose tolerance test, trough levels of the two drugs, and histopathology were investigated. RESULTS Low doses of FK506 (1 or 4 mg/kg) combined with RAPA (0.5 mg/kg) produced synergistic effect in the prolongation of renal graft survival [combination index (CI) = 0.292, 0.565]. There were no additive or synergistic drug-associated toxicities such as hyperglycemia, nephrotoxicity, and hyperlipidemia. There also was no pharmacological antagonism. CONCLUSION Concomitant therapy of low-dose (drug-optimal) FK506 and RAPA produced a synergistic effect in the prolongation of kidney allograft survival in Vervet monkeys without additive drug-associated toxicities.

A proteasome inhibitor effectively prevents mouse heart allograft rejection

BACKGROUND We have previously demonstrated in vitro that proteasome inhibitors could suppress proliferation and induce apoptosis of activated T cells. This finding suggests that such inhibitors could be used as a novel category of immunosuppressants in blocking allograft rejection. METHODS The proteasome inhibitor dipeptide boronic acid (DPBA) was tested in vitro for its inhibitory effect on mouse T-cell proliferation and lymphokine secretion. DPBA was also used in vivo to treat mouse heterotopic heart allograft rejection. Possible side effects of this compound were examined according to blood chemistry of mice treated with DPBA. RESULTS DPBA suppressed the T-cell proliferation and potently inhibited interleukin (IL)-2, IL-6, IL-10, IL-13, and IFN-gamma produced by anti-CD3-activated T cells. Given i.p. starting 1 day after transplantation at 0.66 mg/kg per day for 16 days, or at 1 mg/kg per day for 4 days followed by 0.5 mg/kg per day for 12 days, DPBA could prolong heart allograft survival to 35.5 days (mean survival time, MST) and to 36.2 days, respectively. The control group had MST of 7.3 days. When administrated 72 hr post operation at 1 mg/kg per day for 4 days, DPBA could prolong the graft survival to 19.8 days. During the course of these effective dosages, DPBA had no apparent toxicity in the liver, kidney, pancreas, or heart, according to analysis of blood chemistry. CONCLUSIONS The proteasome inhibitor could repress allograft rejection in mice without apparent side-effects at the effective dosages. This finding has opened a new dimension in development of novel immunosuppressants for organ transplantation.

Synergistic effects of mycophenolate mofetil and sirolimus in prevention of acute heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat

BACKGROUND The effect of mycophenolate mofetil (MMF) and sirolimus (rapamycin, RAPA) mono- and combination-therapy was examined in prevention of acute heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat. METHODS Both drugs were administered orally for up to 30 days. Eleven groups (n=6) were involved in the first part of the heart allografting model. Brown Norway (RT1n) to Lewis (RT1(1)) combination was used in the heart and pancreas transplantation models, whereas Buffalo (RT1b) to Wistar Furth (RT1u) was used in the kidney transplantation model. RESULTS The naive control group showed a mean survival time of 6.5+/-0.6 days. There were graded dose-responses to monotherapy of MMF 10 and 20 mg(kg/ day (12.5+/-2.6 days; 19.3+/-9.0 days) and RAPA 0.2, 0.4, 0.8, and 1.8 mg/kg/day (19.2+/-2.0 days; 30.0+/-7.3 days; 50.8+/-12.5 days; 51.2+/-2.6 days), respectively (P=0.001). Results with the combined use of drugs indicate that a synergistic or very strong synergistic interaction was produced when compared with monotherapy of MMF or RAPA: MMF 10 mg(kg/day+RAPA 0.2 mg/kg(day (52.7+/-5.7 days, combination index [CI] =0.189), MMF 20 mg(kg/day+RAPA 0.2 mg/kg/day (57.7+/-5.7 days, CI=0.084), MMF 10 mg/kg/day+RAPA 0.4 mg(kg/day (50.2+/-13.5 days, CI=0.453), and MMF 20 mg/kg(day+ RAPA 0.4 mg/kg(day (51.5+/-6.8 days, CI=0.439), respectively. These results were repeatable in the prevention of acute pancreas and kidney allograft rejection in the rat. In the second part of the study of reversal of ongoing acute heart allograft rejection model, the combined treatment of MMF 10 mg/kg(day+RAPA 0.2 mg/ kg(day (35.5+/-16.0 days, CI=0.794) and MMF 20 mg/kg day+RAPA 0.2 mg(kg/day (57.2+/-4.7 days, CI=0.310) represented synergistic interaction compared with monotherapy of MMF or RAPA. CONCLUSIONS Concomitant therapy of MMF and RAPA produces a synergistic effect in prevention of heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat.

Dysfunction of IL-10-producing type 1 regulatory T cells and CD4+CD25+ regulatory T cells in a mimic model of human multiple sclerosis in Cynomolgus monkeys

CD4(+)CD25(+) Treg and IL-10(+) Tr1 cells play a major role in controlling autoimmunity by suppressing self-reactive T cells. Dysfunction of Tregs appears to be a critical factor in the pathogenesis of autoimmune diseases. Multiple sclerosis (MS) is an inflammatory demyelinating disorder of CNS, where CD4(+) T cells result in nervous tissue damage. The aim of this study was to investigate the protective role of Treg and Tr1 cells in a mimic model of human MS in Cynomolgus monkeys. This study indicated the suppressive capacity of Tregs from MS monkeys was impaired compared with naive controls. The population of CD4(+)CD25(+) Tregs was decreased in acute stage of MS. However, they showed a restored function and percentage in remitting monkeys. In stable phase, CD4(+)CD25(+) Tregs differentially expressed elevated level of CD62P cell adhesion molecule which contributes to the mechanism by which Treg cells inhibit CD4(+) T cell responses. On the other hand, the percentage of CD4(+)IL-10(+) Tr1 and suppressive function of Tr1 cells were found reduced in MS monkeys. IL-10 secretion was diminished almost 9-fold in active MS, and recovered in active MS. This deficit in IL-10 secretion was specific to CD3/CD46, but not to CD3/CD28 stimulation. The concentrations of IFN-gamma secreted by CD3/CD46-activated T cells were also not affected. These results demonstrate that Tregs are dysfunctional in Cynomolgus monkey with MS. Loss of regulatory function appears to be an important factor in the pathogenesis of MS. Hence, to develop new approaches for induction of Tregs in vivo may be beneficial for the clinical treatment in autoimmune diseases.

Rapamycin graft pretreatment in small bowel and kidney transplantation in the rat.

The effect of rapamycin (RAPA) as graft pretreatment was evaluated in orthotopic small bowel and kidney allotransplantation (Tx) in the rat. In the small bowel Tx model, six groups were involved, each including three combinations for evaluation of host-ver-sus-graft (HVG) [Lewis (LEW) × Brown Norway (BN) (LBN)-F1 → Lewis], graft-versus-host (GVH) (LEW → F1), and combined HVG and GVH immune responses (BN → LEW). RAPA graft pretreatment alone (16 μg/ml × 3 ml) was able to induce a modest but significant prolongation of survival in all three combination models compared with controls (P<0.05). The same was observed for low dose CsA treatment (2 mg/kg/day × 14 days) of the recipient only (P<0.05). Combination of graft pretreatment with RAPA and CsA recipient treatment produced a marked prolongation of survival especially in HVG response. Recipients treatment with one 48-μg bolus of RAPA i.v. immediately after graft revascularization failed to achieve any prolongation of survival for the GVH or combined HVG and GVH responses. This seems to exclude a “carryover” effect of RAPA from graft to recipient. RAPA efficacy was also clearly confirmed in the kidney graft pretreatment model as compared to recipient treatment with an equivalent RAPA dose. These results demonstrate that graft RAPA pretreatment prolongs SB survival after Tx in the rat for HVG, GVH, and bidirectional immune responses. Intragraft interaction with passenger leukocytes or APC function appears as one of the possible mechanisms. RAPA graft pretreatment potentiates low dose CsA recipient treatment suggesting a possible use in clinical organ Tx.

Adoptive transfer of CD4+CD25+ regulatory cells combined with low-dose sirolimus and anti-thymocyte globulin delays acute rejection of renal allografts in Cynomolgus monkeys

Although donor alloantigen specific Treg cells play an important role in transplant tolerance, therapeutic applications are limited by their low frequency. In this study, isolated Tregs from Cynomolgus monkeys were efficiently expanded by a co-culture system, and maintained suppressive function on the proliferation of CD4(+) effector cells in vitro. Adoptive transfer of expanded donor alloantigen specific Tregs without any immunosuppressants could prolong survival of MHC-mismatched allografts in Cynomolgus monkeys. To reach the feasibility of clinical transplantation, our objectives focused on whether exposure of monkey Tregs to immunosuppressants could preserve suppressive function in vitro and in vivo. The results showed that low-dose sirolimus selectively expanded Tregs, increased the expression of CD25(bright) and Foxp3 markers, and suppressed TCR- or allo-antigens induced CD4(+) T cell proliferation in vitro. In vivo, after pre-treated with anti-thymocyte globulin (ATG) for consecutive 3days, a 14-day therapy of adoptive infusion of donor alloantigen-specific Tregs combined with low-dose sirolimus delayed acute rejection of renal allografts in Cynomolgus monkeys, showing an MST of 48.5days as compared with those of untreated and sirolimus-treated monkeys (7days and 22days). The frequencies of CD4(+)CD25(bright) T cells were significantly elevated in mesenteric lymph nodes vs. those in inguino lymph nodes and peripheral blood. In summary, expanded donor alloantigen specific Tregs exposed to sirolimus can preserve inhibition in vitro and in vivo. Tregs are more resistant to sirolimus than other T cells. Expanded donor alloantigen specific Tregs combined with sirolimus and ATG prolongs renal allograft survival in monkeys, suggesting that sirolimus might be one of the best synergists for Tregs therapy.

The immunosuppressive effect of rapamycin on mouse small bowel transplantation

The efficacy of rapamycin (RAPA) was tested on small bowel transplantation in the mouse and compared with cyclosporine (CsA). Four groups were involved, each one included three combinations (n > or = 6) for evaluation of host-versus-graft (HVG, C57BL/6 X BALB/c F1 (CB6F1)-to-BALB/c), graft-versus-host (GVH, BALB/c-to-CB6F1), and combined HVG and GVH responses (C57BL/6-to-BALB/c). Grafts were transplanted to recipients heterotopically. Groups were as follows: group 1: naive controls; groups 2 and 3: recipient mice treated with RAPA 2 mg/kg/day and 4 mg/kg/day orally for 14 days, respectively; group 4: recipient mouse treated with CsA 4 mg/kg/day orally for 14 days. In the HVG model, the mean survival time (MST) of recipients was significantly longer in group 2 (32.9 +/- 17.7 days, P=0.006), group 3 (32.7 +/- 10.4 days, P=0.0001), and group 4 (37.9 +/- 11.8 days, P=0.0001), compared with naive controls in group 1 (8.5 +/- 1.6 days). In the GHV model, the MST of recipients in group 2 (41.8 +/- 19.9 days, P=0.002), group 3 (48.2 +/- 21.4 days, P=0.001) and group 4 (56.5 +/- 30.6 days, P=0.003) were significantly prolonged compared with control group 1 (8.5 +/- 1.6 days). In combined HVG and GVH responses, MST of recipient in group 2 (20.9 +/- 4.9 days, P=0.0001), group 3 (27.0 +/- 4.3 days, P=0.008), and group 4 (35.2 +/- 23.9 days, P=0.0001) were also significantly longer than that in controls (6.9 +/- 1.4 days), but in all three combinations, there were no statistically significant differences between groups 2 and 3, groups 2 and 4, or groups 3 and 4 (P>0.05). RAPA is a potent immunosuppressant able to significantly prolong small bowel allograft survival in mice using a short-term treatment. There is no statistically significant difference in recipient survival between low and high doses of RAPA treatment and the CsA standard dose used in this study.

Combined therapy of CD4(+)CD25(+) regulatory T cells with low-dose sirolimus, but not calcineurin inhibitors, preserves suppressive function of regulatory T cells and prolongs allograft survival in mice.

Our previous study proved that sirolimus is a potent immunosuppressant which induces long-term allograft survival depends on persistence of alloantigens. CD4(+)CD25(+) regulatory T (Treg) cells are potent suppressors in transplantation. Our objectives focus on whether combined-therapy of Tregs with immunosuppressants could prolong allograft survival in mice. The study showed that inhibition of Tregs was maintained by co-cultured with sirolimus (1 nM) in vitro, but not tacrolimus (1 nM) or CsA (1 nM). When the concentration was increased >100 nM, suppression was fallen. Based on the ability of sirolimus to target effector T cells, but retaining the inhibition of Tregs, an adoptive infusion of donor alloantigen specific Tregs combined with 30-day sirolimus (1 mg/kg) and 3-day ATG (20 mg/kg) was found to prolong heart allograft survival in mice. Even though the cell numbers of CD4(+) T cells were found to decrease in sirolimus-treated mice, sirolimus selectively enhanced the numbers of CD4(+)CD25(+) cells and increased the expression of Foxp3 in spleens and lymph nodes, respectively, in recipients. However, combined therapy with low-dose CsA (5 mg/kg) or tacrolimus (1 mg/kg) reduced significantly the expression of Foxp3 and failed to prolong the allograft survival. In summary, expanded Tregs exposed to sirolimus can survive, proliferate, and preserve inhibition in vitro. Tregs are more resistant to sirolimus than other T cells. Combined with Tregs, sirolimus rather than calcineurin inhibitors, prolongs the allograft survival. Sirolimus may be the best copartner for Tregs therapy. It also suggests calcineurin-dependent signals may be required in the development of Tregs.